METHODS FOR GENE AMPLIFICATION
20250297266 ยท 2025-09-25
Inventors
Cpc classification
C12N2710/20022
CHEMISTRY; METALLURGY
C12N15/67
CHEMISTRY; METALLURGY
C12N15/63
CHEMISTRY; METALLURGY
International classification
C12N15/67
CHEMISTRY; METALLURGY
Abstract
Disclosed are methods of genetic engineering to manipulate gene copy number in vivo, as well genetic constructs for amplifying gene copy number in vivo, and recombinant cells that comprise amplified genes. The methods of increasing gene copy number involve reducing expression levels of a haploinsufficient gene in the genome of recombinant cells, such as through replacing the endogenous promoter with a weaker promoter.
Claims
1-25. (canceled)
26. A method for increasing copy number of a nucleic acid construct in the genome of a yeast cell, wherein the nucleic acid construct comprises a heterologous nucleic acid sequence and a recombinant polynucleotide, the method comprising: introducing the nucleic acid construct into the genome, wherein the heterologous nucleic acid sequence is introduced in operable connection with a endogenous haploinsufficient gene of the genome; and reducing expression of the endogenous haploinsufficient gene, wherein the recombinant polynucleotide reduces expression of the endogenous haploinsufficient gene and the reduced expression of the endogenous haploinsufficient gene increases copy number in the genome of the nucleic acid construct and the endogenous haploinsufficient gene, thereby increasing the copy number of the heterologous nucleic acid sequence in the genome of the cell.
27. The method of claim 26, wherein the heterologous nucleic sequence comprises at least one coding sequence in operable connection with a promoter that is operable in the cell.
28. The method of claim 26, wherein the nucleic acid construct comprises an origin of replication.
29. The method of claim 26, wherein the recombinant polynucleotide of the nucleic acid construct is selected from the group consisting of: (a) a polynucleotide that comprises a promoter that is weaker than the promoter of the endogenous haploinsufficient gene, which when introduced into the genome of the cell, is operably connected to the endogenous haploinsufficient gene; (b) a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by replacement of the endogenous promoter of the endogenous haploinsufficient gene with a weaker promoter; (c) a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by replacement of at least one codon of the haploinsufficient gene with a codon that has a lower translational efficiency in the cell than the codon it replaces: (d) a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by disruption of endogenous haploinsufficient gene; (e) a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by operably connecting a nucleotide sequence encoding an RNA destabilizing element to the endogenous haploinsufficient gene; and (f) a polynucleotide that reduces the level of an expression product of the haploinsufficient gene.
30. The method of claim 29, wherein the recombinant polynucleotide of the nucleic acid construct is a polynucleotide that comprises a promoter that is weaker than the promoter of the endogenous haploinsufficient gene, which when introduced into the genome of the cell, is operably connected to the endogenous haploinsufficient gene, or a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by replacement of the endogenous promoter of the endogenous haploinsufficient gene with a weaker promoter.
31. The method of claim 26, wherein the increased copy number of the endogenous haploinsufficient gene or the nucleic acid construct is from 2 to 200 copies, suitably 3 to 100 copies, suitably 3 to 70 copies, suitably 3 to 60 copies.
32. The method of claim 26, wherein the endogenous haploinsufficient gene is selected from the group consisting of RPL25, SEC23, RPL33A, RPS15, RPC10, RPS5, ACT1, NIP1, RPS13, NUS1, SMC1, RNA14, RPB7, SPC97, STH1, ARP7, TAF61 and RPN11.
33. The method of claim 30, wherein the weaker promoter is selected from the group consisting of ERG1 promoter, PDA1 promoter, BTS1 promoter, GLO2 promoter and COG7 promoter.
34. The method of claim 32, wherein the endogenous haploinsufficient gene is operably connected to an origin of replication, wherein the origin of replication is ARS306 or ARS1max.
35. A genetically modified yeast cell, comprising a nucleic acid construct in its genome, wherein the nucleic acid construct comprises: (1) a recombinant polynucleotide that reduces expression of a haploinsufficient gene that is endogenous to the cell of interest; and (2) a heterologous nucleic acid sequence in operable connection with the haploinsufficient gene, wherein the heterologous nucleic sequence comprises at least one coding sequence in operable connection with a promoter that is operable in the cell.
36. The genetically modified yeast cell of claim 35, wherein the nucleic acid construct further comprises an origin of replication.
37. The genetically modified yeast cell of claim 36, wherein the recombinant polynucleotide of the nucleic acid construct is selected from the group consisting of: (a) a polynucleotide that comprises a promoter that is weaker than the promoter of the endogenous haploinsufficient gene, which when introduced into the genome of the cell, is operably connected to the endogenous haploinsufficient gene; (b) a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by replacement of the endogenous promoter of the endogenous haploinsufficient gene with a weaker promoter; (c) a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by replacement of at least one codon of the haploinsufficient gene with a codon that has a lower translational efficiency in the cell than the codon it replaces: (d) a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by disruption of endogenous haploinsufficient gene; (e) a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by operably connecting a nucleotide sequence encoding an RNA destabilizing element to the endogenous haploinsufficient gene; and (f) a polynucleotide that reduces the level of an expression product of the haploinsufficient gene.
38. The genetically modified yeast cell of claim 37, wherein: the haploinsufficient gene is ribosomal 60S subunit protein L25 or GTPase-activating protein SEC23; the weaker promoter is selected from the group consisting of ERG1 promoter, PDA1 promoter, BTS1 promoter, GLO2 promoter and COG7 promoter; and the origin of replication is the autonomous replicating sequence ARS306 or ARS1max.
39. A nucleic acid construct comprising a recombinant polynucleotide that reduces expression of a haploinsufficient gene that is endogenous to a yeast cell of interest.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Embodiments of the disclosure are described herein, by way of non-limiting example only, with reference to the following drawings.
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DETAILED DESCRIPTION
1. Definitions
[0050] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described. For the purposes of the present disclosure, the following terms are defined below.
[0051] The present description uses numerical ranges to quantify certain parameters relating to this disclosure. It should be understood that when numerical ranges are provided, such ranges are to be construed as providing support for claim limitations that recite the lower value of the range as well as claim limitations that recite the upper value of the range. For example, a disclosed numerical range of 10 to 100 provides support for a claim reciting greater than 10 (with no upper bounds) and a claim reciting less than 100 (with no lower bounds) and provided support for and includes the end points of 10 and 100.
[0052] The articles a and an are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, an element means one element or more than one element.
[0053] As used herein, the term about refers to a quantity, level, value, number, dimension, size, percentage or amount that varies by as much as 10% (e.g., by 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%) to a reference quantity, level, value, number, dimension, size, percentage or amount.
[0054] As used herein, the term amplicon refers to a piece of DNA or RNA that is the source and/or product of amplification or replication events.
[0055] The term amplification as used herein, for example in relation to gene amplification or transgene amplification, refers to an increase in copy number of a single copy gene or transgene to at least 2 copies. The increase in copy number is preferably 2 to 100 copies, preferably 2 to 90 copies, preferably 2 to 80 copies, preferably 2 to 70 copies, more preferably 2 to 60 copies, more preferably 4 to 60 copies, more preferably 4 to 50 copies, or any integer copy number between these ranges.
[0056] As used herein, and/or refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).
[0057] By coding sequence it is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene or for the final mRNA product of a gene (e.g. the mRNA product of a gene following splicing). By contrast, the term non-coding sequence refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene or for the final mRNA product of a gene.
[0058] The terms complementary and complementarity refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence A-G-T, is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be complete or total complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
[0059] Throughout this specification, unless the context requires otherwise, the words comprise, comprises and comprising will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term comprising and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By consisting of is meant including, and limited to, whatever follows the phrase consisting of. Thus, the phrase consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
[0060] The terms construct, nucleic acid construct and the like refer to a recombinant genetic molecule including one or more nucleic acid sequences from different sources. Thus, constructs are chimeric molecules in which two or more nucleic acid sequences of different origin are assembled into a single nucleic acid molecule and include any construct that contains (1) nucleic acid sequences, including regulatory and coding sequences that are not found together in nature (i.e., at least one of the nucleotide sequences is heterologous with respect to at least one of its other nucleotide sequences), or (2) sequences encoding parts of functional RNA molecules or proteins not naturally adjoined, or (3) parts of promoters that are not naturally adjoined. Representative constructs include any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or linear or circular single stranded or double stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecules have been operably linked. Constructs of the present disclosure will generally include the necessary elements to direct expression of a nucleic acid sequence of interest that is also contained in the construct. Such elements may include control elements such as a promoter that is operably linked to (so as to direct transcription of) the nucleic acid sequence of interest, and often includes a polyadenylation sequence as well. In certain embodiments of the disclosure, the construct may be contained within a vector. In addition to the components of the construct, the vector may include, for example, one or more selectable markers, one or more origins of replication, such as prokaryotic and eukaryotic origins, at least one multiple cloning site, and/or elements to facilitate stable integration of the construct into the genome of a host cell. Two or more constructs can be contained within a single nucleic acid molecule, such as a single vector, or can be containing within two or more separate nucleic acid molecules, such as two or more separate vectors. An expression construct (also referred to herein as an expression cassette) generally includes at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, for example, promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.
[0061] The term corresponding as used herein in reference to a particular gene is intended to mean an analogous or equivalent or comparable gene. For example, where reference is made to a corresponding endogenous gene, it is intended to mean the analogous, equivalent or comparable naturally-occurring gene. Where reference is made to a corresponding exogenous gene, it is intended to mean an analogous, equivalent or comparable exogenous gene. In some embodiments, the corresponding gene has analogous or equivalent function or having sequence similarity. In one embodiment, the corresponding gene may be identical in function and/or sequence. In another embodiment, the corresponding gene may have about the same function or activity. In another embodiment, the corresponding gene may have reduced function or activity. In some embodiments, the phrase corresponds to or corresponding to is meant a nucleic acid sequence that displays substantial sequence identity to a reference nucleic acid sequence. In general the nucleic acid sequence will display at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence identity to the reference nucleic acid sequence.
[0062] The terms disruption and disrupted, as applied to a nucleic acid, are used interchangeably herein to refer to any genetic modification that decreases or eliminates expression and/or the functional activity of the nucleic acid or an expression product thereof. For example, disruption of a gene includes within its scope any genetic modification that decreases or eliminates expression of the gene and/or the functional activity of a corresponding gene product (e.g., mRNA and/or protein). Genetic modifications include complete or partial inactivation, suppression, deletion, interruption, blockage, or down-regulation of a nucleic acid (e.g., a gene). Illustrative genetic modifications include, but are not limited to, gene knock-out, inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or use of inhibitory nucleic acids (e.g., inhibitory RNAs such as sense or antisense RNAs, molecules that mediate RNA interference such as siRNA, shRNA, miRNA; etc.), inhibitory polypeptides (e.g., antibodies, polypeptide-binding partners, dominant negative polypeptides, enzymes etc.) or any other molecule that inhibits the activity of a haploinsufficient gene or level or functional activity of an expression product of a haploinsufficient gene.
[0063] As used herein, the terms encode, encoding and the like refer to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide. For example, a nucleic acid sequence is said to encode a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide. Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence. Thus, the terms encode, encoding and the like include an RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of an RNA molecule, a protein resulting from transcription of a DNA molecule to form an RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide an RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.
[0064] The terms endogenous and native are used interchangeably herein to refer to a nucleic acid or protein, or part thereof, that is naturally present and/or expressed in an organism or cell thereof. For example, an endogenous haploinsufficient gene refers to a haploinsufficient gene that is naturally expressed in an organism or cell thereof. The term may also be used to refer to the naturally occurring genomic location of a given gene or genetic element of a particular organism. In contrast, the term exogenous refers to material or things such as polynucleotide or polypeptide sequences having an external origin, or is outside of an organism. A vector, plasmid, or other artificial construct that includes an endogenous polynucleotide sequence combined with polynucleotide sequences of the unmodified vector etc. is, as a whole, an exogenous polynucleotide and may also be referred to as an exogenous polynucleotide including an endogenous polynucleotide sequence. Also, a particular polynucleotide sequence that is isolated from a first organism and transferred to second organism by molecular biological techniques is typically considered an exogenous polynucleotide with respect to the second organism.
[0065] The term expression, as used herein, typically refers to any step involved in the production of an RNA molecule or a polypeptide, such as by transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
[0066] The term gene is used herein to refer to a unit of inheritance that comprises a coding sequence and optionally transcriptional and/or translational regulatory sequences and/or non-translated sequences (i.e., introns, 5 and 3 untranslated sequences) whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene may include or encode promoter sequences, signal peptides, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions. In some embodiments the gene may comprise only coding sequence. In other embodiments, the gene may comprise coding sequences and non-coding sequences.
[0067] The term gene product or expression product as used herein refers to an RNA or protein that results from expression of a gene. For example, the gene product may be an RNA, such as mRNA, rRNA, tRNA, miRNA or siRNA, or may be a polypeptide product.
[0068] As used herein, the term haploinsufficiency refers to a state in which the total level and/or activity of a gene product (e.g., a particular protein) is insufficient for normal cellular function. For example, haploinsufficiency arises where one allele at a heterozygous locus provides little or no gene product, and a single copy of the wild-type allele at a locus in heterozygous combination with a variant allele is insufficient for normal cellular function. In haploids, haploinsufficiency arises when a single copy of a gene is insufficient to maintain normal cellular function. A haploinsufficient gene is therefore a gene that needs more than one allele to be functional in order to maintain normal cell function or express the wild type phenotype, or when a single functional copy of a gene is insufficient to maintain normal cellular function. Consequently, haploinsufficient genes exhibit extreme sensitivity to decreased gene expression.
[0069] The term homologous is used herein in a comparative sense to indicate that a nucleotide or polypeptide sequence being referred to as having the same origin or structure.
[0070] The term heterologous is used herein in a comparative sense to indicate that a nucleotide or polypeptide sequence being referred to is from a different source, position or structure from the source or the origin, or is linked to a second nucleotide sequence (or polypeptide) with which it is not normally associated, or is modified such that it is in a form that is not normally associated with the original material. Therefore the term heterologous nucleic acid sequence is used herein to indicate a nucleic acid is from a different source, position or structure from the source or the origin, or is linked to a second nucleotide sequence (or polypeptide) with which it is not normally associated, or is modified such that it is in a form that is not normally associated with the original material. The term heterologous nucleic acid sequence is used interchangeably herein with the term transgene.
[0071] The term homologous recombination as used herein in relation to genetic manipulation and genetic engineering techniques, has the same meaning as would be understood by the person skilled in the art; that is, a method of introducing exogenous DNA sequences in a targeted controlled fashion, at a specific, pre-determined genomic region or loci. The pre-determined genomic loci will largely depend on the genomic region that is being targeted for integration of the polynucleotide construct.
[0072] The terms mutant and variant and modified may be used interchangeably herein, to refer to a non-wild-type organism, strain, expression pattern or expression level, gene/polynucleotide sequence or amino acid sequence. The terms modification, alteration, substitution and the like, as used herein in relation to an amino acid residue/position or a nucleotide, typically mean that the amino acid or nucleotide in the particular position has been modified compared to the amino acid of the wild-type or parent polypeptide.
[0073] As used herein, the term nucleic acid, nucleic sequence, polynucleotide, oligonucleotide and nucleotide sequence as used herein refers to mRNA, RNA, CRNA, rRNA, cDNA, or DNA, or a combination thereof. The term typically refers to polymeric form of nucleotides, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single-, double- or triple-stranded forms of DNA and RNA. It can be of recombinant, artificial and/or synthetic origin and it can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar. The nucleic acids of the present disclosure can be in isolated or purified form, and made, isolated and/or manipulated by techniques known per se in the art, e.g., cloning and expression of cDNA libraries, amplification, enzymatic synthesis or recombinant technology. The nucleic acids can also be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Belousov (1997) Nucleic Acids Res. 25:3440-3444.
[0074] As used herein, the term operably connected or operably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a regulatory sequence (e.g., a promoter) operably linked to a nucleotide sequence of interest (e.g., a coding and/or non-coding sequence) refers to positioning and/or orientation of the control sequence relative to the nucleotide sequence of interest to permit expression of that sequence under conditions compatible with the control sequence. The control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct its expression. Thus, for example, intervening non-coding sequences (e.g., untranslated, yet transcribed, sequences) can be present between a promoter and a coding sequence, and the promoter sequence can still be considered operably linked to the coding sequence. Likewise, in the present disclosure, operable connection in a nucleic acid construct of a heterologous nucleic acid sequence with a recombinant polynucleotide that reduces expression of a haploinsufficient gene that is endogenous to a cell of interest, encompasses positioning and/or orientation of the heterologous nucleic acid sequence and haploinsufficient gene in such a way so that reduced expression of the haploinsufficient gene increases copy number in the genome of the nucleic acid construct.
[0075] The terms origin of replication and replication origin are used interchangeably to refer to a particular sequence or genomic location at which replication is initiated on a chromosome, genome, plasmid or virus.
[0076] The terms peptide, polypeptide and protein are to be understood as referring to a chain of amino acids linked by peptide bonds, irrespective of the number of amino acids forming said chain. Amino acids are typically represented by their one-letter or three-letters code, according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (Ile); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gln); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val); W: tryptophan (Trp) and Y: tyrosine (Tyr).
[0077] A promoter refers to one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter may include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter may optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Promoter includes a minimal promoter that is a short nucleic acid sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which control elements (e.g., cis-acting elements) are added for control of expression. Promoter also refers to a nucleotide sequence that includes a minimal promoter plus control elements (e.g., cis-acting elements) that are capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an enhancer is a nucleic acid sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific nucleic acid-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic nucleic acid segments. A promoter may also contain nucleic acid sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions. Promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as minimal or core promoters. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. A minimal or core promoter thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.
[0078] The term tandemly repeated amplicon as used herein, refers to a stretch of nucleic acids that comprises two or more DNA amplicons that are repeated in such a way that the repeats lie adjacent or neighboring to each other.
[0079] The term transgene as used herein refers to any nucleotide sequence used in the transformation of an organism. Thus, a transgene can be a coding sequence, a non-coding sequence, a cDNA, a gene or fragment or portion thereof, a genomic sequence, a regulatory element and the like. A transgenic organism, such as a transgenic animal, transgenic plant, transgenic yeast, or transgenic bacterium, is an organism into which a transgene has been delivered or introduced and the transgene can be expressed in the transgenic organism to produce a product, the presence of which can impart an effect and/or a phenotype in the organism.
[0080] The term vector typically refers to a DNA or RNA molecule used as a vehicle to transfer recombinant genetic material, such as a heterologous nucleic acid construct of the present disclosure, into a host cell. The vector may be a linear or circular double stranded nucleic acid molecule. Suitable vectors include plasmids, bacteriophages, viruses, fosmids, cosmids, and artificial chromosomes. A vector typically comprises an insert (a heterologous nucleic acid sequence or transgene) and a larger sequence that serves as the backbone of the vector. The purpose of a vector which transfers genetic information to the host is typically to isolate, multiply, or express the insert in the target cell. Vectors can be episomal, i.e., do not integrate into the genome of a host cell, or can integrate into the host cell genome. The vectors may also be replication competent or replication-deficient. Exemplary polynucleotide vectors include, but are not limited to, plasmids, yeast artificial chromosomes (YACs), cosmids, transposons, synthetic DNA fragments. Exemplary viral vectors include, for example, AAV, lentiviral, retroviral, adenoviral, herpes viral and hepatitis viral vectors. Selection of the vectors to be used will take into consideration the size of the insert, the host cell to be transfected and the desired transformation efficiency or outcome, and would be readily known to the persons skilled in the art.
[0081] The term recombinant, as used herein, refer to a biomolecule, e.g., a gene or protein, or to a cell or microorganism. The term recombinant may be used in reference to cloned DNA isolates, chemically synthesized polynucleotides, or polynucleotides that are biologically synthesized by heterologous systems, as well as proteins or polypeptides encoded by such nucleic acids, e.g. enzymes. A recombinant nucleic acid is a nucleic acid linked to a nucleotide or polynucleotide to which it is not linked in nature. For example, the recombinant polynucleotide may be in the form of an expression vector. As use herein, a recombinant cell refers to a cell that has introduced into it exogenous nucleic acid, typically exogenous DNA, such as a vector or other polynucleotides. The term includes the progeny of the original cell into which the exogenous DNA has been introduced. Thus, a recombinant cell as used herein generally refers to a cell that has been transformed, transfected or transduced with exogenous DNA. The host cell may be transformed, transfected or transduced in a transient or stable manner. The exogenous nucleic acid is typically introduced into a host cell so that it is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector. The term recombinant cell encompasses any progeny of a parent host cell that is not identical to the parent host cell due to the alterations introduced.
[0082] As used herein, RNA destabilizing element refers to a nucleic acid sequence in an RNA that is bound by proteins and which protein binding changes the stability and/or translation of the RNA. Examples of RNA destabilizing elements include Class I AU rich elements (ARE), Class II ARE, Class III ARE, U rich elements, GU rich elements, and stem-loop destabilizing elements (SLDE).
[0083] The term sequence identity as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison (e.g. over 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200 or more nucleotides or amino acids residues). Thus, a percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present disclosure, sequence identity will be understood to mean the match percentage calculated by an appropriate method. For example, sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Sequences may be aligned using a global alignment algorithms (e.g., Needleman and Wunsch algorithm; Needleman and Wunsch, 1970), which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for the purposes of determining percent amino acid sequence identity can be achieved by any means available to persons skilled in the art, illustrative examples of which include publicly available computer software, such as is available at http://blast.ncbi.nim.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Persons skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. As used herein, % sequence identity typically refers to values generated using pair wise sequence alignment that creates an optimal global alignment of two sequences (e.g., using the Needleman-Wunsch algorithm).
[0084] In regard to the term variants and derivatives, these terms are taken to refer to a biological equivalent of the sequence from which it was derived.
[0085] The term wild-type is used herein to denote an organism, gene, or gene product, or the expression pattern or expression level of the gene or gene product in a non-modified organism; that is, as it appears in nature, or that which is most frequently observed in a population and is thus arbitrarily designed the normal or wild-type form.
[0086] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.
[0087] It is to be understood that this disclosure is not limited to the particular methodology, protocols, proteins, organisms, vectors, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
2. Methods for Increasing Copy Number of a Gene
[0088] The present disclosure provides a method for increasing copy number of a haploinsufficient gene in the genome of a cell. This method generally comprises, consists or consists essentially of reducing expression of the haploinsufficient gene to thereby increase the copy number of the haploinsufficient gene in the genome of the cell. Also provided is a method for increasing copy number of a heterologous nucleic acid sequence in the genome of a cell, driven by amplification (increasing the copy number) of an operably connected haploinsufficient gene.
[0089] Reducing the expression of the haploinsufficient gene product can be achieved in many ways. For example, the expression level of the of haploinsufficient gene product can be reduced by reducing the level of transcription and/or translation of the haploinsufficient gene. This may include means to reduce the rate of transcription or translation, or by reducing the number of transcripts or protein products produced from the haploinsufficient gene. This may include means that degrades, inactivates or destabilizes the haploinsufficient gene transcript or expression product as defined herein. For example, this may include the provision of siRNA, miRNA, an antisense DNA or antisense RNA molecules that ultimately results in a reduction in the level of the haploinsufficient gene product.
[0090] Reduced expression level provides an evolutionary and selection force that drives an increase in the copy number of the haploinsufficient gene, so that cells are viable, or maintain growth fitness. This selective pressure driving the increase in copy number of the haploinsufficient gene can be advantageously exploited to effect bystander amplification of an operably connected heterologous nucleic acid sequence. In other words, the evolutionary and selection force exerted by the haploinsufficient gene typically encompasses additional bystander regions situated around or neighboring the haploinsufficient gene, resulting in concomitant increase in the copy number of neighboring sequences.
2.1 Haploinsufficient Genes
[0091] In mammals, about 300 genes are known to be haploinsufficient (Dang et al. Eur J Human Genet. 16(11): 1350-7), including IFNGR2 (Interferon gamma receptor 2), PTEN, BRCA1 and 2, and p53, TERC, and RUNX genes. In the yeast Saccharomyces cerevisiae, more than 180 haploinsufficient genes have been identified by fitness profiling of heterozygous deletion strains. Examples of haploinsufficient genes in yeast include: RPL25 (ribosomal 60S subunit protein L25), SEC23 (component of the Sec23p-Sec24p heterodimer of the COPII vesicle coat), RPL33A, RPS15, RPC10, RPS5, ACT1, NIP1, RPS13, NUS1, SMC1, RNA14, RPB7, SPC97, STH1, ARP7, TAF61, RPN11, YPL142C, SEC23, RPL18A, act1, RPL17A, nip1, rpb8, CCT7, CCT2, RPL5, RPS13, RPO26, YDL193W, YLR076C, RRP4, RPL30, RPS20, YBR190W, sui2, YNL313C, rpb5, smc1, RPB3, TUB1, RVB2, SEC34, CCT3, RNA14, YHR083W, NMD3, YPR136C, RRP45, rpb7, YHR196W, DYS1, SPC97, CCT4, RPS2, SUI3, TAF145, RRP9, TIF35, YDR449C, YNL110C, TIF6, TSC10, ndc1, RPS3, DIS3, esp1, prp11, YNL114C, NOG1, SMD2, CDC47, MEX67, YJL009W, RRP43, PAN1, CCT5, YHR085W, MTR3, IMP3, SIK1, YMR093W, SPC98, CFT2, YDR367W, TAF90, PAB1, MOB1, ENP1, SPT6, RPP0, RIM2, YDL221W, IMP4, YJL069C, YLR339C, ARP9, RPC53, YDR355C, YGL047W, YML093W, YCL053C, NOP1, UTR5, YGR115C, TID3, NSP1, YDL152W, RPT3, GCD10, SPB1, YDR365C, GNA1, SEC53, YIR010W, YML127W, DCP2, HXT12, ORC4, mcm2, RSC6, RPC11, TFB1, HYP2, YGR277C, GP18, TLG1, NUP145, YLR033W, RLP7, pol1, RPB10, RRP42, RPN5, YDR060W, YDR396W, GLC7, RPP1, SEC24, yef3, rpc19, rap1, RPN2, DNA43, DIP2, cdc25, CSL4, ACC1, NOP58, BFR2, YDR339C, spp41, ECO1, YIL083C, RHO3, SFH1, YNR046W, YOL022C, YOL134C, ipl1, ATP16, SEC31, YDR013W, FAL1, YRA1, YFR003C, SLN1, YKR071C, SEC14, SEC21, cdc13, BCP1, TRS120, YDR412W, YDR437W, PUP3, EPL1, TAF67, NHP2, YDL209C, STS1, SQT1, sec11, YKR081C, RFC4, YPL251W, MED8, tub2, PRE5, BRX1, YPL233W, MRS5, POP4, ses1, YFL035C, YGR128C, PUP2, PRI1, EXO70, YNL132W, rpc34, MAS6, ARC40, NUP192, SEC65, YNL038W, top2, alg1, RPN6, TIM22, TFC6, prp3, SKI6, YHR188C, ERG9, GCD14, kre9, NOP4, YBR070C, pgi1, YIL003W, NUP159, RPL15A, prp4, alg7, YDL015C, COP1, DAD1, SSS1, PCF11, YFL018W-A, ERG1, MET30, YJL011C, MTR4, NUP82, SMC4, HRT1, NAN1, SHR3, PDS1, YDR434W, PRE4, CRM1, DNA2, YLR243W, ROT1, POP3, SRB6, TRS20, rib5, rpo21, HEM3, DBF4, RSC8, ERG7, YHR186C, cdc6, RAM2, STU2, TUB4, YCS4, DBP9, TAF65, YNL026W, YNL260C, RPB11, pet9, YDL148C, YDR053W, SLU7, SRP101, FRQ1, YDR413C, cdc4, YPT1, YGR280C, ARP4, ARP3, YKL195W, GCD7, FOL3, Rsa2, fol1, MED7, NIP29, REB1, cdc53, YDL196W, GLE1, TRR1, NCB2, YDR527W, RRN7, YJL072C, NET1, PRP19, CDC46, sis1, SEC12, RPA43, rpa190, SRP68, PRE2, mak5, cdc2, SAS10, YPD1, HEM13, RRP1, YDR489W, pre1, FRS2, hip1, SEC6, YJL097W, YLR002C, PIK1, CDC33, ORC2, EXO84, YFH1, ARH1, TFB3, SPC105, TOM20, YIL104C, TAO3, TRL1, MPP10, GRC3, YLR022C, STT4, RPM2, LST8, sec2, PRE6, RER2, PDI1, cdc7, KRS1, DOP1, TRS31, rib3, YGR265W, YHR070W, YRB2, PRE3, SMC3, YJL195C, YLR101C, YLR323C, AFG2, MPT1, YNL247W, RFC3, cdc31, idi1, spt14, SEC8, rib7, cdc28, RPT2, kin28, LCB2, pdc2, SMT3, YDR531W, CBF2, fol2, cdc12, PRP21, DRS1, BOS1, TAF19, NUF2, YOL146W, pup1, YTM1, PRE7, AME1, YDL016C, YRB1, RVB1, RPN9, SNM1, PMI40, RPT6, UFD1, ZPR1, cdc8, ACP1, YKR038C, YKR079C, YLR007W, TOM22, YNL306W, YOL078W, RIO1, prt1, NUD1, rad53, RPL32, ira1, sup45, NFS1, PGK1, SRP14, SNU23, GUK1, YGR190C, RRP3, QNS1, BIG1, YJL091C, HYS2, YLL034C, YSH1, YML125C, YNL245C, TBF1, STN1, WBP1, YGR156W, TYS1, gpi1, YJL010C, YJL086C, YKL059C, ECM9, RRN5, ADE13, SEC61, YML023C, ERG13, YNL124W, sui1, DBP6, RPO31, RPT5, MYO2, ALA1, SEC62, SRP72, MYO1, MLC1, and MYO2. Further examples of haploinsufficiency genes have been described elsewhere (see for example, Deutschbauer et al. (2005) Genetics 169:1915-1925). In some embodiments of the disclosure, the haploinsufficient gene is selected from the group consisting of RPL25, SEC23, RPL33A, RPS15, RPC10, RPS5, ACT1, NIP1, RPS13, NUS1, SMC1, RNA14, RPB7, SPC97, STH1, ARP7, TAF61 and RPN11. In one embodiment of the disclosure, the haploinsufficient gene is RPL25. In another embodiment of the disclosure, the haploinsufficient gene is SEC23.
[0092] Haploinsufficient genes can also be identified by comparative genomics and their suitability confirmed by testing growth fitness in association with expression dosage of a gene. Means and method for identifying haploinsufficient genes would be known to the persons skilled in the art. For diploid organisms, haploinsufficiency can also be achieved by disrupting one allele and integrating the amplifiable nucleic acid construct at the other allele locus, or by simultaneously integrating the amplifiable constructs at both alleles, to give rise to reduced gene dosage of the haploinsufficient gene. Established genetic recombination or genetic engineering techniques can be used for targeted allele disruption and integration of genetic construct. For example, site directed mutagenesis for targeted allele disruption, and nuclease-mediated DNA double-chain break like CRISPR systems for the integration of the amplifiable construct.
2.2 Reducing the Level of the Haploinsufficient Gene Product
[0093] Reducing the expression of the haploinsufficient gene can be achieved in many ways. For example, expression of the haploinsufficient gene can be reduced by reducing the transcription and/or translational efficiency of the haploinsufficient gene.
[0094] Alternatively, or in addition, the expression of the haploinsufficient gene product may be reduced by replacing the endogenous promoter of an endogenous haploinsufficient gene with a weaker promoter. The weaker promoter as described herein is to be understood in a comparative sense; that is the, the weaker promoter controlling the expression of the haploinsufficient gene is weaker relative to the native or endogenous promoter of the haploinsufficient gene. Driving expression through a weaker promoter attenuates the transcription level of the haploinsufficient gene.
[0095] Alternatively, or in addition, the level of the haploinsufficient gene product is reduced by modulating transcriptional and/or translational activity (i.e. rate of transcription, or production of mRNA) through the use of non-preferred codons (i.e., codons that have a lower transcriptional and/or translation efficiency than the codons they replace), whereby for example, replacement or addition of one or more codons in the haploinsufficient gene coding sequence with alternative codons that have a lower transcriptional and/or transcriptional efficiency functions to reduce the expression of the haploinsufficient gene.
[0096] In some embodiments, the level of the haploinsufficient gene product is reduced by driving expression of the haploinsufficient gene through a weaker promoter and the use of a variant haploinsufficient gene comprising non-preferred codons.
[0097] Expression of the haploinsufficient gene may also be reduced through disruption of the haploinsufficient gene. For example, the haploinsufficient gene may be disrupted by means that degrades, inactivates or destabilizes the haploinsufficient gene transcript or expression product as defined herein. For example, this may include the provision or expression of siRNA, miRNA, an antisense DNA or antisense RNA molecules that results in reduced expression of the haploinsufficient gene. Reducing expression of the haploinsufficient gene product can comprise modifying the haploinsufficient gene to include a nucleotide sequence encoding an RNA destabilizing element.
[0098] Disrupting the haploinsufficient gene may include replacing the endogenous gene with a variant haploinsufficient gene that has reduced expression and/or function. This variant haploinsufficient gene may comprise mutations that affect gene function, or comprise protein degradation motifs. This may include the modification of the haploinsufficient gene to include ubiquitin molecules that targets the expression product for degradation. For example, the haploinsufficient gene may be modified to include synthetic protease sites that results in targeted protein degradation, which ultimately results in a reduction in the level of the haploinsufficient gene product.
2.3 Weaker Promoter
[0099] In some embodiments, the expression of the haploinsufficient gene product is reduced by modulating transcriptional activity (i.e. rate of transcription, or production of mRNA) by replacing the endogenous promoter of the haploinsufficient gene with a weaker promoter.
[0100] The identification of suitable weaker promoters must be determined relative to the endogenous promoter of the native haploinsufficient gene. Standard methods of testing and assays for comparing promoter strength using reporter gene assays, including those disclosed herein, will be known to persons skilled in the art. By the way of an example, promoters that have been shown to drive a range of expression levels include promoters of RPL33A, RPS15, RPC10, ACT1, NIP1, RPS13, NUS1, SMC1, RNA14, RPB7, SPC97, STH1, ARP7 and TAF61 genes. The weak promoters can be from the promoters controlling the expression of a transcriptional factor, including GLN3, TOR1, DAL80, GCR1, GCR2, YNF1, YPK2, ADR1, NRG1, MIG1, ROX1, HAP4, HAC1, and UPC2 (Peng et al. Communication Biology). In one embodiment of the disclosure, the weaker promoter is selected from the ERG1 promoter, the PDA1 promoter, the BTS1 promoter, the GLO2 promoter, or the COG7 promoter as means of controlling expression of the haploinsufficient gene. Examples of promoter strength characterization will be known to be persons skilled in art, and have been previously disclosed, including in Peng et al. Microbial cell factories 14, 91 (2015).
[0101] The weak or weaker promoter can drive expression of the haploinsufficient gene at a level that is no more than 99% to 1% (and all integer percentages in between, including 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% 1%) or even less, of the level of the haploinsufficient gene driven by the native promoter.
[0102] The weaker promoter controlling the expression of the haploinsufficient gene may be 1-20 times weaker than the native or endogenous promoter. In other embodiments, the weaker promoter controlling the expression of the haploinsufficient gene is 1-10 times weaker than the native promoter. In other embodiments, the weaker promoter controlling the expression of the haploinsufficient gene is 2-8 times weaker than the native promoter. In other embodiments, the weaker promoter controlling the expression of the haploinsufficient gene is 2-5 times weaker than the native promoter. In other embodiments, the weak promoter controlling the expression of the haploinsufficient gene that is 2-4 times weaker than the native promoter. Standard methods for comparing and testing promoter strength using reporter gene assays in the host cell of interest can be easily performed by the skilled person. For example, the strength of the native promoter of the haploinsufficient gene in driving reporter gene expression can be compared to a range of known promoters to identify a promoter that is suitably weaker (i.e. comparing transcriptional efficiency/amount of transcript or polypeptide gene product produced). Non-preferred codons have lower translational efficiency.
[0103] Although exploitation of codon usage bias has been previously used to optimize translation, inclusion of non-optimal, less preferred or rare codons (collectively referred to herein as non-preferred codons) that have lower transcriptional and/or translational efficiency can also attenuate transcription and translation. Examples of non-preferred codons would be known to the person skilled in the art (e.g. Sharp et al. (1988) Nucleic Acids Research 16(17):8207; Athey et al. (2017) BMC Informatics 18:391). For example, in yeast, the non-preferred glycine codon GGA has lower translational efficiency. Codons with lower translational efficiency and codon usage bias for different organisms will be known to the person skilled in the art.
[0104] Thus, in some embodiments, the expression of the haploinsufficient gene product is reduced by replacing at least one codon of the haploinsufficient gene with a codon that has a lower transcriptional or translational efficiency in the cell, and/or by adding to the haploinsufficient gene at least one codon that has a lower transcriptional or translational efficiency in the cell. Non-preferred codon with lower transcriptional or translational efficiency can be added upstream or downstream of the gene (e.g., in an untranslated region of the gene), or within the coding sequence of the gene.
[0105] In some embodiments, 1, 2, 3, 4, 5 or more non-preferred codon(s) is (are) introduced into the haploinsufficient gene. In embodiments in which codons of the haploinsufficient gene are replaced with non-preferred codons, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the codons of the of the haploinsufficient gene may be replaced with non-preferred codons.
[0106] In some embodiments, introduction of the non-preferred codon does not result in a modification in the amino acid sequence of the haploinsufficient gene product. In other embodiments, the non-preferred codon that is introduced results in a modification in the amino acid sequence of the haploinsufficient gene product, to give rise to a variant polypeptide of the haploinsufficient gene product. The modification in the amino acid sequence of the haploinsufficient gene product maybe an amino acid insertion. The modification in the amino acid sequence of the haploinsufficient gene product may be an amino acid substitution. The modification in the amino acid sequence of the haploinsufficient gene product may be an amino acid deletion. It will be appreciated, that the modification in the amino acid sequence by incorporation of a non-preferred codon should not result in a non-functional haploinsufficient gene product. In some embodiments, the modification results in reduced expression of the haploinsufficient gene.
2.4 Bystander Amplification
[0107] Without wishing to be bound by any one theory or mode of operation, it is proposed that genetic manipulations that lead to reduced expression of a haploinsufficient gene result in selective pressure that drives an increase in the copy number of the haploinsufficient gene to maintain growth fitness of the cell. In accordance with the present disclosure, this increase in copy number not only amplifies the haploinsufficient gene but extends to neighboring genomic regions upstream or downstream of the haploinsufficient gene, which are referred to herein as bystander regions. This phenomenon can be exploited advantageously to effect bystander amplification of any heterologous nucleic acid sequences or transgenes that are situated adjacent and operably connected to the haploinsufficient gene.
[0108] The heterologous nucleic acid sequence can be positioned at any suitable position relative to the haploinsufficiency gene, which permits bystander amplification of the heterologous nucleic acid sequence when the genetically manipulated haploinsufficient gene is amplified. Such positioning can be determined through routine procedures known in the art. In representative examples, the heterologous nucleic acid sequence may be separated from the haploinsufficient gene by about 1 to about 4000 bp (and all integer base pairs in between), by about 1 to about 2000 bp (and all integer base pairs in between), by about 1 to about 1000 bp (and all integer base pairs in between), by about 1 to about 500 bp (and all integer base pairs in between), by about 1 to about 300 bp (and all integer base pairs in between), by about 1 to about 200 bp (and all integer base pairs in between), or by about 1 to about 100 bp (and all integer base pairs in between). In some embodiments, the heterologous nucleic acid sequence may be separated from the haploinsufficient gene by no more than 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 150 bp, 200 bp, 250 bp or 300 bp. The skilled person would also understand that the distance the heterologous nucleic acid sequence is separated from the haploinsufficient gene may be influenced by the size of the heterologous nucleic acid sequence that flanks the haploinsufficient gene, but this is well within the ordinary skill in the art.
[0109] Expression of the haploinsufficient gene may also be reduced by targeted modification. For example, the haploinsufficient gene may be modified by disrupting the endogenous haploinsufficient gene (e.g., by knock-out) and integrating an exogenous haploinsufficient gene into the genome, wherein the exogenous haploinsufficient gene is expressed at a lower level than the endogenous haploinsufficient gene before disruption.
[0110] Disruption of the haploinsufficient gene can be achieved by deleting the endogenous haploinsufficient gene. The entire haploinsufficient gene, or only part of the gene can be deleted, so that the haploinsufficient gene is no longer functional; and an exogenous haploinsufficient gene can be integrated into the genome, wherein the exogenous haploinsufficient gene is expressed at a lower level than the endogenous haploinsufficient gene before disruption. Alternatively, the haploinsufficient gene can be disrupted by insertion of an exogenous sequence into the haploinsufficient gene, resulting in gene inactivation, either by producing a non-functional gene product, or by targeting the gene product for destruction or silencing; for example, the introduction of a stop codon, retrotransposons, anti-sense sequences, or siRNA sequences.
[0111] The haploinsufficient gene knock out strategies can be achieved using gene targeting strategies such as homologous recombination. The knock-out strategies may also be targeted at pre-determined, or a specified genome location using other targeted, site-specific genome integration strategies such as CRISPR-Cas9, Zinc Finger nucleases and TALEN genome editing techniques, application of which would be known to the person skilled in the art.
[0112] Insertion of the nucleic acid construct can be targeted to a pre-determined, or a specified genome locus. Methods of targeted, site-specific genome integration include using homologous recombination and CRISPR-Cas9, Zinc Finger nucleases and TALEN genome editing techniques, application of which would be known to the person skilled in the art. The nucleic acid construct can be targeted to the endogenous genomic location of the haploinsufficient gene, such that integration of the nucleic acid construct results in substitution of the native promoter of the haploinsufficient gene with the weaker promoter. Alternatively, the nucleic acid construct is targeted to the endogenous genomic location of the haploinsufficient gene, such that integration results in substitution of the entire endogenous haploinsufficient gene.
[0113] In another scenario, the endogenous haploinsufficient gene is disrupted and the nucleic acid construct comprising an exogenous haploinsufficient gene that is expressed at a lower level than the endogenous haploinsufficient gene before disruption, can be targeted for integration at a genomic location away from the endogenous haploinsufficient gene, or can be randomly integrated (i.e. not targeted to a specific genomic location).
[0114] In methods where the reducing the expression of the haploinsufficient gene comprises replacing the endogenous promoter of the haploinsufficient gene with a weaker promoter, or replacing or adding at least one codon of the haploinsufficient gene with a codon that has a lower translational efficiency in the cell, the integration of the polynucleotide construct is targeted. That is, the integration of the nucleic construct is targeted to the genomic loci comprising the endogenous promoter of the endogenous haploinsufficient gene or the endogenous haploinsufficient gene. The nucleic acid construct can be targeted for integration in the genome of the cell through homologous recombination, methods of which would be known to persons skilled in the art.
[0115] Targeting the genetic modifications, such as incorporation of non-preferred codons at a pre-determined, or a specified genome location can be performed using other targeted, site-specific genome integration strategies such as CRISPR-Cas9, Zinc Finger nucleases and TALEN genome editing techniques, application of which would be known to the person skilled in the art.
3. Nucleic Acid Constructs
[0116] Provided herein is a nucleic acid construct comprising a recombinant polynucleotide that reduces expression of a haploinsufficient gene that is endogenous to a cell of interest.
[0117] The nucleic acid construct, when introduced into the cell may be amplified in the cell to form a tandemly repeated amplicon in the genome of the cell. This tandemly amplified region comprises multiple copies of the nucleic acid construct.
[0118] The tandem repeated amplicon may contain 2-200 copies or repeats of the DNA segments or nucleic acid constructs. The tandem amplified region may contain 2 to 100 copies or repeats of the DNA segments or nucleic acid constructs. The tandem amplified region may contain 2 to 80 copies or repeats of the DNA segments or nucleic acid constructs. The tandem amplified region may contain 2 to 70 copies or repeats of the DNA segments or nucleic acid constructs. The tandem amplified region may contain 2 to 60 copies or repeats of the DNA segments of nucleic acid constructs, more preferably 4 to 60 copies or repeats of the DNA segments nucleic or acid constructs, more preferably 4 to 50 copies or repeats of the DNA segments nucleic or acid constructs, or any integer copies or repeats between these ranges.
[0119] In some embodiments, the nucleic acid construct further comprises a heterologous nucleic acid sequence in operable connection with the haploinsufficient gene.
[0120] The recombinant polynucleotides described herein may comprise a native sequence (e.g., an wild-type or native sequence that encodes a wild-type protein) of the haploinsufficient gene, or a variant, a derivative of the haploinsufficient gene, or a part or a fragment thereof of the haploinsufficient gene. Recombinant polynucleotide variants or derivatives may contain one or more substitutions, additions, deletions and/or insertions, as further described herein.
[0121] The polynucleotide variant may result in altered efficiency in transcriptional and translational regulation of the polynucleotide, such that the polynucleotide is capable of elevated or reduced expression. The polynucleotide variant may encode a polypeptide that has the amino acid sequence of the native or wild type polypeptide of the haploinsufficient gene. The polynucleotide may encode a polypeptide that has a variant polypeptide, such that the encoded polypeptide retains functional activity. The activity of the encoded polypeptide may be partially or substantially diminished relative to the unmodified or reference polypeptide. The activity of the encoded polypeptide may be partially or substantially augmented relative to the unmodified or reference polypeptide. The effect on the enzymatic activity of the encoded polypeptide may generally be assessed as described herein and known in the art.
[0122] The recombinant polynucleotide may comprise a polynucleotide that comprises a weaker promoter that has a lower transcriptional activity than the native promoter that is operably connected to the haploinsufficient gene such that when it is inserted upstream of the haploinsufficient gene, it will drive expression of the haploinsufficient gene at reduced levels when compared to the native promoter.
[0123] The nucleic acid construct of the present disclosure further comprises a heterologous nucleic acid sequence in operable connection with the haploinsufficient gene.
[0124] The heterologous nucleic acid sequence comprises at least one coding sequence in operable connection with a promoter that is operable in the cell. This allows expression of the coding sequence. The coding sequence can be a gene that encodes for a heterologous protein. The coding sequence can encode for heterologous gene products, which may be valuable in the industrial production of biofuels, proteins, biochemicals, chemicals, enzymes, pharmaceuticals and biopharmaceuticals. The coding sequence can encode for genes or polypeptides for producing products such as terpenoids, flavonoids, fatty acids, RNAi, nanobodies, phenolics, isoprenoids, alkaloids, and polyketides. Biopharmaceuticals include vaccines, insulin, antibodies, erythropoietin, hormones, blood factors, interferons, interleukins, growth factors, fusion proteins, recombinant enzymes. In some embodiments, the coding sequence encodes for sesquiterpene nerolidol, monoterpene limonene, or tetraterpene lycopene.
[0125] A nucleic acid construct as disclosed herein may comprise homologous arms for targeted homologous recombination mediated integration into the genome. Design (i.e., length, nucleotide sequence) of the homologous arms would be known to the persons skilled in the art. The homologous arms of the nucleic acid construct are situated flanking the heterologous nucleic acid sequence and the exogenous haploinsufficient gene.
[0126] The nucleic acid construct as disclosed herein may include an origin of replication that can be situated anywhere in the region between the homologous arms of the nucleic acid construct. The origin of replication may be situated adjacent to the heterologous nucleic acid sequence. The origin of replication may be situated adjacent to the haploinsufficient gene or portions thereof. The origin of replication may be situated between the heterologous nucleic acid sequence and haploinsufficient gene. The coding sequences and heterologous nucleic acid sequences described herein may be suitably deduced or derived from the amino acid sequence of the polypeptides described herein and codon usage may be adapted according to the host cell in which the nucleic acid shall be transcribed.
[0127] As will be understood by those skilled in the art, the nucleic acid constructs, the heterologous nucleic acids and coding sequences of this disclosure can include genomic sequences, extra-genomic, and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present disclosure, and a polynucleotide may, but need not, be linked or conjugated to other molecules and/or support materials.
[0128] The nucleic acid construct of the present disclosure can be up to about 10000 base pairs in length. The nucleic acid construct of the present disclosure can be up to about 9000 base pairs in length, up to about 8000 base pairs in length, up to about 7000 base pairs in length, up to about 6000 base pairs in length, up to about 5000 base pairs in length, up to about 4000 base pairs in length, up to about 3000 base pairs in length, up to about 2000 base pairs in length up to about 1000 base pairs in length, or from about 500 to about 10000 bases pairs in length (and all integer base pairs in between). The size of the nucleic acid construct that can be accommodated by a selected vector can be readily determined by the skilled person.
[0129] The heterologous nucleic acid sequences disclosed herein may be codon optimized to improve expression in the cell. Suitable methods for codon optimization will be familiar to persons skilled in the art, illustrative examples of which are described in the reference manual Sambrook et al. (Sambrook et al., 2001). Codon usage bias for different organisms will be known to the person skilled in the art.
3.1 Homologous Arms
[0130] The nucleic acid construct may further comprise homologous arms that facilitate targeted genomic integration. In some embodiments, replacement of the endogenous promoter or the endogenous haploinsufficient gene can be achieved by homologous recombination at a pre-determined genomic locus.
[0131] The homologous arms of the nucleic acid construct are homologous to DNA sequences of the host cell genome which are adjacent or flanking the targeted locus. The sequence of the homologous arms may be identical or similar (which include homologous identical sequences and homologous non-identical sequences) to the regions of the host cell genome to which the homologous arms are complementary. Homologous non-identical sequences refer to a first sequence which shares a degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence. For example, a polynucleotide comprising the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene. As used herein, the degree of homology between the two homologous, non-identical sequences is sufficient to allow homologous recombination there between, utilizing normal cellular mechanisms. Two homologous non-identical sequences can be any length and their degree of non-homology can be as small as a single nucleotide (e.g., for a genomic point mutation introduced targeted homologous recombination) or as large as 10 or more kilobases (e.g., for insertion of a gene at a predetermined locus in a chromosome). Two polynucleotides comprising homologous non-identical sequences need not be the same length. For example, an exogenous polynucleotide (i.e., vector polynucleotide) of between 20 and 4,000 nucleotides or nucleotide pairs can be used.
[0132] The characterization of two sequences as homologous, identical sequences or homologous, non-identical sequences may be determined by comparing the percent identity between the two sequences (polynucleotide or amino acid). Homologous, identical sequences have 100% sequence identity. Homologous, non-identical sequences may have sequence identity greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.
[0133] The homologous arms may be any length that allows for site-specific homologous recombination. A homologous arm may be any length between about 2000 bp and 500 bp including all integer values between. For example, a homologous arm may be about 2000 bp, about 1500 bp, about 1000 bp, or about 500 bp. In embodiments having two homologous arms, the homologous arms may be the same or different length. Thus, each of the two homologous arms may be any length between about 2000 bp and 500 bp including all integer values between. For example each of the two homologous arms may be about 2000 bp, about 1500 bp, about 1000 bp, or about 500 bp. A portion of the polynucleotide arm adjacent to one or both (i.e., between) homologous arms modifies the targeted locus in the host cell genome by homologous recombination. Techniques for homologous recombination in other organisms are generally known (see, e.g., Kriegler, 1990, Gene transfer and expression: a laboratory manual, Stockton Press). The modification may change a length of the targeted locus including a deletion of nucleotides or addition of nucleotides. The addition or deletion may be of any length. The modification may also change a sequence of the nucleotides in the targeted locus without changing the length. The targeted locus may be any portion of the host cell genome including coding regions, non-coding regions, and regulatory sequences. In an embodiment the modification may ablate a gene thereby creating a knock-out organism. In another embodiment, the modification may modulate the expression of the gene. In an embodiment the modification may add a gene that functions as a reporter or marker (e.g., GFP or antibiotic resistance). In an embodiment, the modification may add an exogenous gene. In an embodiment, the modification may add an endogenous gene under control of an exogenous promoter (e.g., a strong promoter, a weak promoter, an inducible promoter, etc.).
3.2 Origins of Replication
[0134] In some embodiments, the nucleic acid construct may include addition of exogenous protein domains including post-translational modification sites, protein-stabilizing domains, cellular localization signals, and protein-protein interaction domains. In other embodiments, the nucleic acid construct may comprise addition of nucleic acid sequences that are not translated into a protein including, but not limited to, a non-coding RNA molecule, a gene regulatory element, a promoter, a regulatory protein binding site, a RNA binding site, a ribosome binding site, a transcriptional terminator, or a RNA-stabilizing element. In an embodiment, the polynucleotide construct may include an origin of replication.
[0135] In eukaryotes, the origin of replication is where the hexameric protein complex, origin recognition complex (ORC) is recruited to initiate and control replication.
[0136] In S. cerevisiae, replication origins are defined by consensus DNA sequence elements, called autonomously replicating sequences (ARS) that support efficient DNA replication initiation of extrachromosomal DNA. ARS are about 100-200 base pairs long, and comprises a conserved ARS consensus sequence (ACS). The ARS serves as the primary binding site for the hexameric origin recognition complex (ORC).
[0137] In some embodiments, the genetic construct comprises an origin of replication. In some embodiments, the origin of replication is a strong replication origin. In some embodiments, the origin of replication is an early-firing autonomously replicating sequence. In another embodiment, the origin of replication is an ARS. There are many known ARSs, and suitable ARS would be known to the person skilled in the art (see for example, Liachko et al. (2011) BMC Genomics 12:633). In some embodiments, the ARS can be an artificial ARS. In a preferred embodiment, the origin of replication is ARS306 or ARS1max.
3.3 Gene Transfer/Introduction
[0138] The nucleic acid construct, expression cassette or expression vector according to the present disclosure may be transferred into a cell by any suitable method known to persons skilled in the art, illustrative examples of which include electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic gene gun transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation and liposome-mediated transformation.
[0139] Transformation allows uptake and incorporation of the exogenous genetic material, to effect stable, heritable alteration in the cell genome. Exogenous nucleotides may include gene foreign to the target organism or addition of a nucleotide sequence present in the wild-type organism. The results of a stable genetic modification caused by transformation is maintained in at least a portion of a population of cells for ten or more generations or for a length of time equal or greater to ten times the average generation time for the modified organism.
3.4 Cells
[0140] Also provided herein is a cell comprising the nucleic acid construct as described herein.
[0141] The cell of the present disclosure is a cell that comprises haploinsufficient genes. The cell may be a prokaryote or a eukaryote or an archaean cell. The prokaryotic cell may be any Gram-positive or Gram-negative bacterium. In some embodiments the bacterial cell is selected from the group of Escherichia coli, Pseudomonas, Bacillus, and Streptomyces. In one embodiment, the bacteria may be Bacillus subtilis. In another embodiment, the bacteria may be Clostridium saccharoperbutylacetonicum. In one embodiment, the cell is a cyanobacteria cell. In some embodiments the cyanobacteria is a Synechocystis spp., Cyanothece spp., Nostoc spp., Scytonema spp., Arthrospira spp. such as Arthrospira platensis, Arthrospira fusiformis and Arthrospira maxima, or Microcystis aeruginosa. The cell may also be a eukaryotic cell, such as a yeast, fungal, algal, microalgal, mammalian, insect or plant cell. In some embodiments, the cell is an algae or a microalgae. In some embodiments, the algae or microalgae is a kelp or seaweed or sea lettuce (Ulva spp.), such as brown algae or Sargassum spp. including Sargassum fusiforme. In some embodiments, the algae or microalgae is Chlorella spp., Dunaliella spp., Gracilaria spp., Eucheuma spp., Saccharina japonica, Gracilaria spp., Pyropia spp., Chlamydomonas spp., Haematococcus spp., Kappaphycus alvarezii or Undaria pinnatifida. In some embodiments the algae or microalgae is Ankistrodesmus spp., Botryococcus braunii, Crypthecodinium cohnii, Cyclotella spp., Hantzschia spp., Nannochloris spp., Nannochloropsis spp., Neochloris oleoabundans, Nitzschia spp., Phaeodactylum tricornutum, Scenedesmus spp., Schizochytrium spp., Stichococcus spp., Tetraselmis suecica or Thalassiosira pseudonana. In a particular embodiment, the cell is a yeast cell. In a further particular embodiment, the yeast cell is selected from the group of Trichoderma, Aspergillus, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Torulaspora, Pichia, Thermus, Hansenula, Torulopsis, Komagataella, Candida, Karwinskia or Yarrowia. In representative embodiments, the yeast is selected from Saccharomyces species (e.g., Saccharomyces cerevisiae), Kluyveromyces species (e.g., Kluyveromyces lactis), Torulaspora species, Yarrowia species (e.g., Yarrowia lipolitica), Schizosaccharomyces species (e.g., Schizosaccharomyces pombe), Pichia species (e.g., Pichia pastoris or Pichia methanolica), Hansenula species (e.g., Hansenula polymorpha), Torulopsis species, Komagataella species, Candida species (e.g., Candida boidinii), and Karwinskia species. In another embodiment, the cell is S. cerevisiae or S. pombe or a Pichia species. The cell may be any cell useful in the production heterologous gene products. The cell may be any cell that is suitable for function as cell factories, which will be known or easily recognised by the person skilled in the art.
[0142] In some embodiments, the cell of the present disclosure is a cell that is produced by any of the methods disclosed herein.
[0143] The cell may be any cell useful in the production heterologous gene products. The cell may be a prokaryote or a eukaryote. The prokaryotic cell may be any Gram-positive or Gram-negative bacterium. The cell may also be a eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell. In particular embodiments, the cell is selected from the group of Escherichia coli, Pseudomonas, Bacillus, Streptomyces, Trichoderma, Aspergillus, Saccharomyces, Pichia, Thermus or Yarrowia. Any cell that is suitable for function as cell factories will be known or easily recognized by the person skilled in the art.
[0144] As used herein, the cell has introduced into it exogenous nucleic acids, such as a vector or other polynucleotides. The cell may be transformed, transfected or transduced in a transient or stable manner. The polynucleotide construct, expression cassette or vector is introduced into a host cell so that the polynucleotide, cassette or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector.
[0145] The cell may comprise one copy of the nucleic acid construct in its genome. The cell of the present disclosure may comprise 2 to 200 copies, suitably 3 to 100 copies, suitably 3 to 70 copies, suitably 3 to 60 copies of the nucleic acid construct. The nucleic acid construct may be amplified to form a transgenic tandem amplified region in the genome of the cell, wherein the transgenic tandem amplified region comprises multiple copies of the nucleic acid construct. In one embodiment, the recombinant cell may comprise of more than one transgenic tandem amplified region in its genome.
[0146] In some embodiments, the nucleic acid construct that is amplified in the cell comprises origin of replications, in preferred embodiments, the nucleic acid construct that is amplified in the recombinant yeast cell comprises the autonomous replicating sequences ARS306 or ARS1max.
4. Expression of Heterologous Nucleic Acids and/or Proteins
[0147] The methods, nucleic acid constructs and cells disclosed herein are useful for increasing expression of introduced genes, transgenes and heterologous proteins in cells, such as in the industrial production of biofuels, proteins, biochemicals, chemicals, enzymes, pharmaceuticals and biopharmaceuticals. Genes and products that can be expressed using the present disclosure can also be used in the synthesis of other products, including phenolics, isoprenoids, alkaloids, and polyketides. Biopharmaceuticals include vaccines, insulin, antibodies, erythropoietin, hormones, blood factors, interferons, interleukins, growth factors, fusion proteins, recombinant enzymes. Other useful products that can be expressed in the cell of the present invention, for example, include flavor and fragrance compositions for use in food, medicine and cosmetic preparations.
[0148] Thus provided herein is a method of expressing a nucleic acid in a cell, the method comprising culturing the cell disclosed herein or a cell produced by any one of the methods disclosed herein, to express the nucleic acid construct comprising the corresponding nucleic acid.
[0149] The cell comprising the nucleic acid construct of the present disclosure may be cultivated in a nutrient medium suitable for production of the gene product (i.e. a polypeptide or nucleic acid) encoded by the heterologous nucleic acid. The cell can be cultivated or cultured for a period of time and/or under the appropriate conditions to allow expression of the gene product or synthesis of a related product, using methods that will be known to persons skilled in the art. Suitable examples include cultivating the cell by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the gene product/product to be expressed and/or isolated. The cultivation will typically take place in a suitable nutrient medium, from commercial suppliers or prepared according to published compositions or any other culture medium suitable for cell growth.
[0150] Where the expressed gene product or related product is secreted into the nutrient medium, it can be recovered directly from the culture supernatant. Optionally, the gene product or related product can be recovered or purified from cell lysates or after permeabilization of the host cell membrane. The gene product or product may be recovered purified using any suitable method known to persons skilled in the art, illustrative examples of which include collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. Optionally, the gene product or related product may be partially or totally purified by a variety of procedures known in the art including, but not limited to, thermal shock, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain substantially pure fractions of the gene product or related product.
[0151] The gene product or related product may be used, in crude or purified form, either alone or in combination with additional products. The present disclosure also extends to compositions comprising the gene product or related product, the nucleic acid construct or the cell described herein.
[0152] The composition may be liquid or dry, for instance in the form of a powder. In some embodiments, the composition is a lyophilizate. For instance, the composition may comprise the gene product, nucleic acid construct and/or cells and optionally excipients and/or reagents etc. Suitable excipients may include buffers commonly used in biochemistry, agents for adjusting pH, preservatives such as sodium benzoate, sodium sorbate or sodium ascorbate, conservatives, protective or stabilizing agents such as starch, dextrin, arabic gum, salts, sugars e.g., sorbitol, trehalose or lactose, glycerol, polyethyleneglycol, polyethene glycol, polypropylene glycol, propylene glycol, divalent ions such as calcium, sequestering agent such as EDTA, reducing agents (e.g., beta-mercaptoethanol, dithiothreitol, ascorbic acid, tris(2-carboxyethyl)phosphine), amino acids, a carrier such as a solvent or an aqueous solution, and the like. The excipient may be polyvinylalcohol (PVA) and co-polymers thereof with PVP or with other polymers, polyacrylates, urea, chitosan and chitosan glutamate, sorbitol or other polyols such as mannitol. The excipient may be PVPK30, cellulose derivatives, such as, but not limited to, polyvinylpyrrolidone, polyethylene-/polypropylene-/polyethylene-oxide block copolymers such as Pluronic F68, polymethacrylates, sodium dodecyl sulfate, polyoxyethylene sorbitan fatty acid esters such as Tween 80, bile salts such as sodium deoxycholate, polyoxyethylene mono esters of a saturated fatty acid such as Solutol HS 15, water soluble tocopheryl polyethylene glycol succinic acid esters such as Vitamin E TPGS, hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), hydroxypropylmethylcellulose acetate succinate (HPMC-AS), hydroxypropylcellulose phthalate (HPMC-P), methylcellulose (MC), polyethyleneglycols, and earth alkali metal silicas and silicates, e.g. fumed silicas, precipitated silicas, calcium silicates, such as Zeopharm600, or magnesium aluminometasilicates such as Neusilin US2. The gene product as described herein is solubilized together with one or more excipients, such as excipients that may suitably stabilize or protect the gene product from degradation.
[0153] The excipients may function as a carrier or a diluent to preserve or alter a particular quality of the composition such as the effectiveness, stability, dispersiveness, miscibility wettability, texture, taste or aroma. The excipient may be a bulking agent, or an anti-fouling agent, or an anti-caking agent. Examples of appropriate excipients include, but not limited to bonding agents (for example, microcrystalline cellulose, tragacanth or bright Glue), coatings, disintegrants, fillers, diluents, softening agents, sweeteners, emulsifying agents, natural flavoring, artificial flavor enhancements (e.g. NaCl, KCl, MSG, guanosine monophosphate (GMP), inosin monophospahte (IMP), ribonucleotides such as disodium inosinate, disodium guanylate, N-(2-hydroxyethyl)-lactamide, N-lactoyl-GMP, N-lactoyl tyramine, gamma amino butyric acid, allyl cysteine, 1-(2-hydroxy-4-methoxylphenyl)-3-(pyridine-2-yl) propan-1-one, arginine, potassium chloride, ammonium chloride, succinic acid, N-(2-methoxy-4-methyl benzyl)-N-(2-(pyridin-2-yl)ethyl)oxalamide, N-(heptan-4-yl)benzo(D)(1,3)dioxole-5-carboxamide, N-(2,4-dimethoxybenzyl)-N-(2-(pyridin-2-yl)ethyl)oxalamide, N-(2-methoxy-4-methyl benzyl)-N-2(2-(5-methyl pyridin-2-yl)ethyl)oxalamide, cyclopropyl-E,Z-2,6-nonadienamide), colouring agents, lubricants, functional agent (for example, nutrients), viscosity modifiers, fillers, glidants (for example, cataloid), surfactants or infiltration agents. Other examples of excipients include silicon dioxide (silica, silica gel), carbohydrates and/or carbohydrate polymers (polysaccharides), cyclodextrins, starches, degraded starches (starch hydrolysates), chemically or physically modified starches, modified celluloses, pectin, inulin, maltodextrins and dextrins. The excipient may be a acetin, magnesium stearate, hydrogenated vegetable oil, essential oil, plant extracts, fruit essence, spices, extracts, oils, gelatin, alcohols, triacetine, glycerol, miglycol, acetaldehyde, dimethyl sulfide, ethyl acetate, ethyl propionate, methyl butyrate, and ethyl butyrate.
[0154] The carrier or excipient may function as a processing aid or to shield or protect the other components from the effects of moisture, light, or oxygen or any other aggressive media. The carrier material might also act as a means of controlling the release of flavor or aroma from the composition, or control the degradation or release of the active compound. Further examples of carriers and excipients include sucrose, glucose, lactose, levulose, fructose, maltose, ribose, dextrose, isomalt, sorbitol, mannitol, xylitol, lactitol, maltitol, pentatol, arabinose, pentose, xylose, galactose, maltodextrin, dextrin, chemically modified starch, hydrogenated starch hydrolysate, succinylated or hydrolysed starch, agar, carrageenan, gum arabic, gum acacia, tragacanth, alginates, methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose, derivatives and mixtures thereof.
[0155] Suitable excipients would depend on the composition and its intended use, therefore selection of the appropriate excipient would be known to the skilled person. The skilled person will appreciate that the cited materials are hereby given by way of example and are not to be interpreted as limiting the invention.
[0156] It will be appreciated that the above described terms and associated definitions are used for the purpose of explanation only and are not intended to be limiting.
[0157] In order that the disclosure may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting example.
Representative Embodiments of the Disclosure
[0158] 1. A method for increasing copy number of a haploinsufficient gene in the genome of a cell, the method comprising, consisting or consisting essentially of reducing expression of the haploinsufficient gene to thereby increase the copy number of the haploinsufficient gene in the genome of the cell.
[0159] 2. The method of embodiment 1, wherein the haploinsufficient gene is operably connected to an origin of replication.
[0160] 3. A method for increasing copy number of a heterologous nucleic acid sequence in the genome of a cell, the method comprising, consisting or consisting essentially of: introducing the heterologous nucleic acid sequence into the genome, wherein the heterologous nucleic acid sequence is introduced in operable connection with a haploinsufficient gene of the genome; and reducing expression of the haploinsufficient gene, wherein the reduced expression of the haploinsufficient gene increases copy number in the genome of a nucleic acid construct comprising the heterologous nucleic acid sequence and the haploinsufficient gene, thereby increasing the copy number of the heterologous nucleic acid sequence in the genome of the cell.
[0161] 4. The method of embodiment 3, wherein the heterologous nucleic sequence comprises at least one coding sequence in operable connection with a promoter that is operable in the cell.
[0162] 5. The method of embodiment 3 or embodiment 4, wherein the heterologous nucleic sequence is located upstream or downstream of the haploinsufficient gene.
[0163] 6. The method of any one of embodiments 1 to 5, wherein the nucleic acid construct comprises an origin of replication.
[0164] 7. The method of any one of embodiments 1 to 6, wherein the method excludes rescuing expression of the haploinsufficient gene through use of a separate rescuing agent.
[0165] 8. The method of any one of embodiments 1 to 7, wherein expression of the haploinsufficient gene is reduced by any one or more of the following: [0166] a. replacing the endogenous promoter of the haploinsufficient gene with a weaker promoter; [0167] b. replacing or adding at least one codon of the haploinsufficient gene with a codon that has a lower translational efficiency in the cell; [0168] c. disrupting the haploinsufficient gene; [0169] d. modifying the haploinsufficient gene to include a nucleotide sequence encoding an RNA destabilizing element; and [0170] e. expressing a nucleic acid molecule in the cell, which reduces the level of an expression product of the haploinsufficient gene.
[0171] 9. The method of any one of embodiments 1 to 8, wherein the increased copy number of the haploinsufficient gene or the heterologous nucleic acid sequence is from 2 to 200 copies, suitably 3 to 100 copies, suitably 3 to 70 copies, suitably 3 to 60 copies.
[0172] 10. The method of any one of embodiments 1 to 9, wherein the cell is a yeast, fungal, bacterial, algal, microalgae, cyanobacterial, insect or mammalian cell, suitably a yeast cell.
[0173] 11. The method of any one of embodiments 1 to 10, wherein the haploinsufficient gene is selected from the group consisting of RPL25, SEC23, RPL33A, RPS15, RPC10, RPS5, ACT1, NIP1, RPS13, NUS1, SMC1, RNA14, RPB7, SPC97, STH1, ARP7, TAF61 and RPN11.
[0174] 12. The method of any one of embodiments 1 to 11, wherein expression of the haploinsufficient gene is reduced by replacing the endogenous promoter of the haploinsufficient gene with a weaker promoter, wherein the weaker promoter is selected from the group consisting of ERG1 promoter, PDA1 promoter, BTS1 promoter, GLO2 promoter and COG7 promoter.
[0175] 13. The method of any one of embodiments 1 to 12, wherein the haploinsufficient gene is operably connected to an origin of replication, wherein the origin of replication is ARS306 or ARS1max.
[0176] 14. A cell that is produced by any one of the methods of embodiments 1 to 13.
[0177] 15. A nucleic acid construct comprising a recombinant polynucleotide that reduces expression of a haploinsufficient gene that is endogenous to a cell of interest.
[0178] 16. The nucleic acid construct of embodiment 15, further comprising a heterologous nucleic acid sequence in operable connection with the haploinsufficient gene.
[0179] 17. The nucleic acid construct of embodiment 16, wherein the heterologous nucleic sequence comprises at least one coding sequence in operable connection with a promoter that is operable in the cell.
[0180] 18. The nucleic acid construct of embodiment 16 or embodiment 17, wherein the heterologous nucleic sequence is located upstream or downstream of the recombinant polynucleotide.
[0181] 19. The nucleic acid construct of any one of embodiments 15 to 18, further comprising an origin of replication.
[0182] 20. The nucleic acid construct of any one of embodiments 15 to 19, wherein the recombinant polynucleotide is selected from: [0183] a. a polynucleotide that comprises a promoter that is weaker than the endogenous promoter of the endogenous haploinsufficient gene, which when introduced into the genome of the cell, is operably connected to the haploinsufficient gene; [0184] b. a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by replacement of the endogenous promoter of the endogenous haploinsufficient gene with a weaker promoter, and/or replacement or addition of at least one codon of the endogenous haploinsufficient gene with a codon that has a lower translational efficiency in the cell; [0185] c. a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by disruption of endogenous haploinsufficient gene; [0186] d. a modified haploinsufficient gene that is distinguished from the endogenous haploinsufficient gene by operably connecting a nucleotide sequence encoding an RNA destabilizing element to the endogenous haploinsufficient gene; and [0187] e. a polynucleotide that reduces the level of an expression product of the haploinsufficient gene.
[0188] 21. The nucleic acid construct of any one of embodiments 15 to 20, wherein the recombinant polynucleotide is distinguished from the endogenous haploinsufficient gene by replacement of the endogenous promoter of the endogenous haploinsufficient gene with a weaker promoter, wherein the weaker promoter is selected from the group consisting of ERG1 promoter, PDA1 promoter, BTS1 promoter, GLO2 promoter and COG7 promoter.
[0189] 22. The nucleic acid construct of any one of embodiments 15 to 21, wherein the haploinsufficient gene is a gene is selected from the group consisting of RPL25, SEC23, RPL33A, RPS15, RPC10, RPS5, ACT1, NIP1, RPS13, NUS1, SMC1, RNA14, RPB7, SPC97, STH1, ARP7, TAF61 and RPN11.
[0190] 23. The nucleic acid construct of any one of embodiments 19 to 22, wherein the origin of replication is an autonomous replicating sequence, where in the autonomous replicating sequence is ARS306 or ARS1max.
[0191] 24. The nucleic acid construct of any one of embodiments 17 to 23, wherein the coding sequence encodes an expression product selected from a polypeptide, (e.g. a polypeptide for producing a terpenoid, a flavonoid or a fatty acid, an antibody, a nanobody) or a functional RNA molecule (e.g., RNAi that inhibits expression of a target gene).
[0192] 25. A cell comprising the nucleic acid construct of any one of claims 15 to 24.
[0193] 26. The cell of embodiment 25, wherein the cell comprises 2 to 200 copies, suitably 3 to 100 copies, suitably 3 to 70 copies, suitably 3 to 60 copies.
[0194] 27. The cell of embodiment 25 or embodiment 26, wherein the cell is a yeast, bacterial, archaean, algal, microalgae, cyanobacterial, insect or mammalian cell, suitably a yeast cell.
[0195] 28. A method for expressing nucleic acid, the method comprising: [0196] culturing the cell of any one of embodiments 25 to 27 to express the nucleic acid construct of any one of embodiments 15 to 24.
[0197] 29. The cell of any one of embodiments 25 to 27, wherein the nucleic acid construct comprises the haploinsufficient gene ribosomal 60S subunit protein L25, wherein the haploinsufficient gene ribosomal 60S subunit protein L25 is operably connected to a weaker promoter that is weaker that the native ribosomal 60S subunit protein L25, wherein the weaker promoter is selected from ERG1 promoter, PDA1 promoter, BTS1 promoter, GLO2 promoter and COG7 promoter.
[0198] 30. The cell of embodiment 29, wherein the haploinsufficient gene ribosomal 60S subunit protein L25 is operably connected to the ERG1 promoter.
[0199] 31. The cell of embodiment 29, wherein the haploinsufficient gene ribosomal 60S subunit protein L25 is operably connected to the PDA1 promoter.
[0200] 32. The cell of embodiment 29, wherein the haploinsufficient gene ribosomal 60S subunit protein L25 is operably connected to the BTS1 promoter.
[0201] 33. The cell of any one of embodiments 25 to 27, wherein the nucleic acid construct comprises the haploinsufficient gene GTPase-activating protein SEC23, wherein the haploinsufficient gene GTPase-activating protein SEC23 is operably connected to a weaker promoter that is weaker that the native GTPase-activating protein SEC23, wherein the weaker promoter is selected from ERG1 promoter, PDA1 promoter, BTS1 promoter, GLO2 promoter and COG7 promoter.
[0202] 34. The cell of embodiment 33, wherein the haploinsufficient gene GTPase-activating protein SEC23 is operably connected to the ERG1 promoter.
[0203] 35. The cell of embodiment 33, wherein the haploinsufficient gene ribosomal 60S subunit protein L25 is operably connected to the PDA1 promoter.
[0204] 36. The cell of embodiment 33, wherein the haploinsufficient gene ribosomal 60S subunit protein L25 is operably connected to the BTS1 promoter.
[0205] 37. The cell of embodiment 33, wherein the haploinsufficient gene ribosomal 60S subunit protein L25 is operably connected to the GLO2 promoter.
[0206] 38. The cell of embodiment 33, wherein the haploinsufficient gene ribosomal 60S subunit protein L25 is operably connected to the COG7 promoter.
[0207] 39. The cell of any one of embodiments 25 to 38, wherein the haploinsufficient gene comprises at least one codon that has a lower translational efficiency.
EXAMPLES
Example 1
Materials and Methods
Construct Design for In Vivo Gene Amplification (HapAmp)
[0208] The likelihood of gene amplification is increased when there is: (1) a gene linked to cell fitness, and (2) homologous DNA sequences to support recombination. In addition, a strong replication origin can promote amplification. These three elements exist in tandem repeat in the rDNA region and the CUP1 region in the yeast genome (
[0209] A genetic construct was designed to enable gene amplification in yeast (
[0210] The promoter element of the genetic construct is weaker than the native promoter of the haploinsufficient gene and positioned such that integration results in substitution of the native promoter of the haploinsufficient gene with the weaker promoter. Genes of interest or transgenes to be amplified and/or expressed heterologously, can be inserted between Arm 3 and the weaker promoter.
[0211] Driving expression through a weaker promoter attenuates the protein yield from haploinsufficient gene immediately downstream of the promoter. This, in turn, is expected to decrease the cell fitness in yeast. Native amplification of the region between homologous Arm 3 in the construct and Arm 2 (or Arm3 naturally existing in genome) will then occur as yeast evolves to recover fitness.
Plasmid and Strain Construction
[0212] Plasmids used in this work are listed in Table 2, and strains are listed in Table 3. Primers used in polymerase chain reaction (PCR) and PCR performed in this work are listed in Table 4. Plasmid construction processes are listed in Table 5. Yeast strain construction processes are listed in Table 6. A LiAc/SS carrier DNA/PEG method (Gietz, R. D. & Schiestl, Nature Protocols 2, 38-41 (2007)) was used for yeast transformation.
Yeast Cultivation
[0213] For characterization of yEGFP-expressing strains, yeast cells from glycerol stocks were streaked on YNB-glucose agar, which comprised of 6.9 g L.sup.1 yeast nitrogen base without amino acids (YNB, FORMEDIUM #CYN0402) with pH adjusted to 6.0 using sodium hydroxide solution, 20 g L.sup.1 glucose, and 20 g L.sup.1 agar. MES-buffered YNB-glucose medium was used in following cultivation, which comprised of 19.5 g L.sup.1 2-(N-morpholino) ethanesulfonic acid (MES), 6.9 g L.sup.1 YNB, 20 g L.sup.1 glucose, and its pH was adjusted to 6.0 with ammonia hydroxide solution. For the growth in flask, seed cultures grown to the exponential phase (OD6004) were inoculated into 20 ml MES-buffered YNB-glucose medium in 125 ml Erlenmeyer flasks to start the cultivation in a 200 rpm 30 C. incubator. For the growth in 96-well microplate, yeast cells were grown in YNB-glucose medium (6.9 g L.sup.1 YNB, 20 g L.sup.1 glucose, pH 6.0) for about 20 hour to stationary phase in a 350 rpm 30 C. incubator to prepare seed culture. Seed culture (5 l) was inoculated into 100 l MES-buffered YNB-glucose medium to prepare Culture 1. Culture 1 (2 l) was inoculated into 100 l MES-buffered YNB-glucose medium to prepare Culture 2. Culture 2 was incubated in a 350 rpm 30 C. incubator overnight for analysis of yEGFP fluorescent in the cells grown to the exponential growth phase, and Culture 1 for two nights for analysis in the cells grown to the ethanol growth phase.
[0214] For characterization of nerolidol/limonene-producing strains, dodecane-overlayed two-phase flask cultivation was used. Yeast cells from glycerol stocks were streaked on YNB-high-glucose agar, which contained 6.9 g L.sup.1 YNB (pH 6.0), 200 g L.sup.1 glucose, and 20 g L.sup.1 agar. Before initiating the two-phase flask cultivation, cells were pre-cultured in MES-buffered YNB-20 g L.sup.1 glucose to exponential phase (OD600 between 1 to 4) and collected by centrifugation. Collected cells were then resuspended in fresh fermentation medium. To initiate the cultivation, appropriate volumes of pre-cultured cells were transferred to MES-buffered YNB medium with 20 g L.sup.1 glucose to an initial OD600 of 0.2 in a total volume of 23 mL medium in a 250 ml flask, and 2 mL sterile dodecane was added after inoculation. In the first 12 hours of cultivation, 3 ml culture was sampled for growth curve measurement. Dodecane was sampled and stored at 80 C. for terpene analysis.
[0215] Flask cultivations for lycopene-producing strains were prepared as the flask cultivation used for yEGFP-expressing strains.
[0216] For chromoprotein/HPV-expressing strains, yeast cells grown overnight in 5 ml MES-buffered YNB-glucose medium were inoculated into 20 ml fresh MES-buffered YNB-glucose medium or 20 ml YP-galactose (20 g L.sup.1 peptone, 10 g L.sup.1 yeast extract, and 20 g L.sup.1 galactose) to start characterization cultures.
Flow Cytometry
[0217] Fluorescence in single cells was analyzed using a BD Accuri C6 flow cytometer (BD Biosciences, USA). For analysis of yEGFP fluorescence, cells sampled from characterizations were directly used for flow cytometry analysis. For analysis of Y-FAST fluorescence, 100-time-concentrated HMBR, synthesized as reported previously and dissolved in dimethyl sulfoxide, was added to the samples to 20 M final concentration and the sample was mixed before analysis. FSC.H threshold was set at the value of 250,000 for exclusion of debris particles. GFP and/or Y-FAST fluorescence was excited by a 488 nm laser and monitored through a 530/20 nm bandpass filter (FL1.A), with 10,000 events recorded per sample. Mean values of FSC.A, SSC.A, and FL1.A for all detected events were extracted using a BD Csampler software (BD Accuri C6 software version 1.0.264.21). GFP or Y-FAST fluorescence level was expressed as the percentage of the average background auto-fluorescence from the exponential-phase cells of GFP-negative reference strain GH4 as described previously.
Metabolite Analysis
[0218] The Metabolomics Australia Queensland Node analyzed extracellular metabolites. Sesquiterpenes and monoterpenes in dodecane samples were analyzed as previously described (Peng, B. et al. Metabolic engineering 39, 209-219 (2017)). Dodecane samples (in some cases, diluted with dodecane) were diluted in 40-fold volume of ethanol. The ethanol-diluted samples (20 L) were injected. A Zorbax Extend C18 column (4.6150 mm, 3.5 m, Agilent PN: 763953-902) equipped with a guard column (SecurityGuard Gemini C18, Phenomenex PN: AJO-7597) was used. Analytes were eluted at 35 C. at 0.9 mL/min using the mixture of solvent A (water) and solvent B (45% acetonitrile, 45% methanol, and 10% water), with a linear gradient of 5-100% solvent B from 0-24 min, then 100% from 24-30 min, and finally 5% from 30.1-35 min. Analytes of interest were monitored using a diode array detector (Agilent DAD SL, G1315C) at 202 nm wavelength. Analytical standards were used to prepare the standard curve for quantification.
[0219] For lycopene measurement, yeast cells were collected and resuspended in 200 L 2 M L.sup.1 sodium hydroxide and vortexed with 200 mg glass bead and 1 mL hexane for at least 10 min. Lycopene concentration was calculated from the absorbance of hexane extracts at 471 nm. Dilution was performed to make absorbance reading <0.6. Lycopene molar extinction coefficient (18210.sup.3) was used to calculate lycopene concentration (Takehara, M. et al. Journal of agricultural and food chemistry 62, 264-269 (2014)).
Protein Purification
[0220] Yeast cells were homogenized by vortexing with glass beads for 15 min in phosphate-buffered saline (PBS) buffer plus 2 mM ethylenediaminetetraacetic acid (EDTA). Whole-cell lysates, lysate supernatants, and lysate pellets were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis on Mini-PROTEAN Precast Gels (Bio-rad).
[0221] The lysis was followed by centrifugation at 18000g for 30 minutes to pellet the cellular debris. The soluble fraction was then loaded on top of a gradient made of 1 mL of 20% Iodixanol/PBS buffer, 1 mL of 30% Iodixanol/PBS and 1 mL of 40% Iodixanol/PBS in a Thinwall Ultra-Clear Tube (Beckman Coulter, Indianapolis, USA) and subjected to ultracentrifugation for 2 hours 30 minutes at 150,000 g on a SW41 Ti rotor or a using a Beckman Optima L-100XP ultracentrifuge (Beckman Coulter, Indianapolis, USA). A band containing the virus-like particles encapsulating protein was extracted using a 1 ml syringe by poking a whole through the tube. Bradford was used to measure protein concentration and sample was further examined on TEM and purity confirmed on Mini-PROTEAN Precast Gels (Bio-Rad).
Transmission Electron Microscopy
[0222] Samples containing purified VLPs of 0.1 mg mL-1 were applied to formvar/carbon coated grids (ProSciTech Pty Ltd, Australia) and incubated for 2 minutes. Grids were then washed with 40 L of distilled water for 30 sec twice, and then stained with 20 g L.sup.1 uranyl acetate for 1 minute, after being blotted on filter paper. Images were taken on a HITACHI HT7700 transmission electron microscope at accelerating voltage of 80 keV at the Centre for Microscopy and Microanalysis.
Genome Sequencing
[0223] Yeast genomic DNA was extracted using MagAttract HMW DNA Kit (Qiangen) with a modified protocol. Yeast cells (20 ml, OD.sub.600 around 10) were washed once using phosphate-buffered saline (PBS) buffer and resuspend in 2 ml 1M sorbitol solution. Yeast cell walls were digested by adding 30 U Zymolyase-20T (nacalai, Japan; 1 U per l in 1*PBS containing 100 mM DTT and 50% v/v glycerol) at 30 C. for 30 minutes. Yeast protoplast cells were collected and resuspended in 300 l Buffer AL (MagAttract HMW DNA Kit) by pipetting using wide bore pipette tips, and then 360 buffer ATL (MagAttract HMW DNA Kit) was added and mixed. Following this, protocol provided in MagAttract HMW DNA Kit (Qiangen) was adopted including digestion by Proteinase K and Rnase A and purification using magnetic beads. Genomic DNA was eluted using 400 l Buffer AE (MagAttract HMW DNA Kit) and treated using 100 l tris-saturated phenol (pH 8.0, Ameresco) by flickering and 100 l chloroform was added and mixed. Upper-layer water phase was collected after centrifuging at 17,000 g for 5 minutes and mixed with 1 ml ethanol. Magnetic beads (MagAttract HMW DNA Kit) was used to purify genomic DNA with twice 70% ethanol wash and elution in 50 l water. Concentration of genomic DNA was quantified using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Thermo Fisher). Genomic DNA (500 ng) was used to prepare genome sequencing library using Rapid Barcoding Kit (SQK-RBK004, Oxford Nanopore) and sequenced using R9 flowcell MIN106D and MinION Mk1C (Oxford Nanopore). High-accurate basecalling was performed using Guppy ( ) installed MinION Mk1C. Galaxy Australia online server was used for data processing. Collapse Collection (Galaxy Version 5.1.0) was used to combine fastq dataset into a single file. Nanoplot was used for statistical analysis of MinION reads. Canu assembler was used for genome sequence assembly. Maker (Galaxy Version 2.31.11) was used to collect annotation evidence with input of S. cerevisiae gene sequences and heterologous gene sequences as ESTs input file. miniMap2 was used to align trimmed reads outputted by Canu assembler against contigs outputted Canu assembler. JBrowse (version 1.16.10-desktop) and Integrative Genomics Viewer (version 2.8.13) were used to illustrate genome structure and read alignment.
Example 2
Using RPL25 or SEC23 Haploinsufficient Gene Loci and Promoter Substitution to Drive Gene Amplification
[0224] Ribosomal 60S subunit protein L25 (RPL25) and the SEC23-encoding component of the Sec23p-Sec24p heterodimer of the COPII vesicle coat are two haploinsufficient genes shown to have an effect on growth fitness (Deutschbauer et al. (2005) Genetics, 169, 1915-1925). These two genes have the strongest fitness effect in rich medium and in minimal mineral medium.
[0225] Four constructs were designed with RPL25 as the haploinsufficient gene that acts as the driving gene (i.e. gene that drives amplification), LEU2 as selection marker, and an early-firing autonomously replicating sequence (ARS) ARS306; and three constructs with SEC23 as the driving gene, hygromycin B resistant gene hphMX as selection marker, and the strong ARS1max ARS.
[0226] To identify promoters with suitable expression strengths, a wide variety of yeast promoters were tested (see Table 1 below, and
TABLE-US-00001 TABLE 1 Yeast Promoters Promoter Linked gene RPL33A 60S ribosomal protein L33-A RPS15 40S ribosomal protein S15 RPC10 DNA-directed RNA polymerases I, II, and III subunit RPABC4 ACT1 Actin NIP1 Eukaryotic translation initiation factor 3 subunit C RPS13 40S ribosomal protein S13 NUS1 Dehydrodolichyl diphosphate synthase complex subunit NUS1 SMC1 Structural maintenance of chromosomes protein 1 RNA14 mRNA 3-end-processing protein RNA14 RPB7 DNA-directed RNA polymerase II subunit RPB7 SPC97 Spindle pole body component SPC97 STH1 Nuclear protein STH1/NPS1 ARP7 Actin-related protein 7 TAF61 Transcription initiation factor TFIID subunit 12 RPN11 Ubiquitin carboxyl-terminal hydrolase RPN11
[0227] For the RPL25 constructs we used the YEF3 promoter (which has similar strength to the RPL25 promoter; Construct 1 in
[0228] The constructs were transformed into the S. cerevisiae CEN.PK strain. Transformation plates were screened by imaging yEGFP fluorescence under blue light, with imaging of the transformation plates showed fluorescing clones for the 8 constructs tested. Construct 3 without the ARS also lead to the formation of very fluorescent colonies after transformation (
[0229] Where promoter strength was similar or greater than the native promoter, yEGFP was found at a single copy on the genome (
[0230] However, where the native promoter was substituted for weaker promoters, yEGFP gene copy number and fluorescence both increased (
[0231] The most remarkable result was where the RPL25 promoter was substituted for the BTS1 promoter; this resulted in 47 copies of yEGFP per genome and a 92-fold increase yEGFP fluorescence (
[0232] The stability of the expression of the yEGFP gene can be maintained long term. The strain comprising construct 4 was cultured for at least 48 generations, to measure the GFP fluorescence levels in the cells over time. For each transferring subculture, cells was inoculated in Yeast extract-Peptone-Glucose (YPD) medium to OD600 equaling to 0.004, grown overnight to OD6001 for flow cytometry analysis, and further grown to 24 h to start the next subculture. GFP fluorescence analyses and population homogeneity also did not show significant changes over time (up to at least 48 generations).
Example 3
Translational Downregulation Using Non-Preferred Codons to Drive Gene Amplification
[0233] To further increase copy number at the SEC23 locus, we attenuated translation by making a construct with three non-preferred glycine codons (GGA) inserted following the start codon of SEC23 under the control of the COG7 promoter (
[0234] A further increase in gene copy and fluorescence was obtained (
Example 4
Growth Rates of Clones with Increased Copy Number
[0235] Increased copy number did not negatively impact the growth rate of any of the strains with the exception of clones with the PBTS1-PL25 construct (
[0236] Long-read sequencing on strains containing Construct 3 and Construct 4 confirmed that the constructs were integrated into the RPL25 (YOL127W) locus and that yEGFP-RPL25 sequences were amplified in tandem repeat structures (
Example 5
Improving Heterologous Production of the Sesquiterpene Trans-Nerolidol
[0237] The performance of the presently described genetic amplification strategy/method for C.sub.15 sesquiterpene (trans-nerolidol) production was assessed. A background strain with upregulated mevalonate pathway for production of terpene precursors was used for these experiments. In this strain, the GAL80 repressor gene is disrupted allowing diauxic induction of GAL promoters, which are used to control transgene expression.
[0238] We constructed a reference strain N401-1 harboring a multi-copy 2 plasmid pJT9RFR 38 (
[0239] The nerolidol synthase expression cassette (Y-FAST-2A-Ac.NES1) was cloned into the RPL25 insertion vector in the amplification region with three different promoters for replacement of the RPL25 promoter; the ERG20 expression cassette was cloned at the non-amplification region (
[0240] Compared to the reference strain N401-1, these three strains exhibited faster growth (
[0241] The reference 2 plasmid strain harbored 14 copies of the Y-FAST-2A-AcNES1 construct-similar to strain N401-3, and higher than that in strain N401-2. However, N401-1 had the lowest Y-FAST fluorescence (
[0242] In contrast with the 2 plasmid strain, the strains harboring the integrated in vivo amplification constructs showed better synchronicity for Y-FAST induction (
Example 6
Improving Heterologous Production of the Monoterpene Limonene
[0243] The performance of the presently described genetic amplification strategy/method was tested with the production of C.sub.10 monoterpenes. Monoterpene production requires introduction of a dedicated C.sub.10 geranyl pyrophosphate (GPP) synthase (Ignea, C. et al. ACS synthetic biology (2013)). A previously used Erg20p.sup.N127W mutant, which excludes the C15 chain from the active site to generate a GPP pool, in combination with targeted degradation of the endogenous C.sub.15 synthase Erg20p via protein degron tags to decrease competition at the C.sub.10 node by Erg20p and redirect GPP towards monoterpene production, was used. In mevalonate pathway-enhanced strains, this approach delivered less than 100 mg L.sup.1; an order of magnitude below the levels achieved for sesquiterpene engineering.
[0244] In these experiments, a mevalonate pathway-enhanced strain with the endogenous Erg20p under an auxin-inducible protein degradation mechanism (Lu, Z. et al. Nature communications 12, 1051 (2021)) was used as a background strain.
[0245] Two different promoter constructs were developed for amplification of the limonene synthetic module (
[0246] Strain LIM141MH showed a slower exponential growth and the lower levels of Y-FAST fluorescence compared to strain LIM141M, despite having more copies of the limonene synthase module (
[0247] Both strains produced an order of magnitude more limonene than over previous efforts using 2 plasmids, producing 0.95 g L.sup.1 limonene at 96 hr, by strain LIM141M (
Example 7
Improving Heterologous Triterpenoid Lycopene Production in Yeast
[0248] A three-gene lycopene synthetic module controlled by GAL promoters was previously constructed in a 2 plasmid (
[0249] The lycopene synthetic module was sub-cloned into both the PDA1 and BTS1 promoter RPL25-driving HapAmp vectors (
[0250] Strain LYC4 (P.sub.PDA1-RPL25) accumulated slightly more lycopene than strain LYC1, although the increase was not significant (
Example 8
High-Level Expression of Heterologous Proteins in Yeast
[0251] Yeast is commonly used as a platform organism for protein production, including production of pharmaceutical proteins, with the advantage of the lack of endotoxins. However, a notorious disadvantage is that heterologous proteins production is not as high as what is achievable with E. coli expression systems. The high-level expression in E. coli can be attributed to the usage of high-copy-number plasmids (such as the common pET vectors with copy number about 1520) and the use of a very strong inducible promoter.
[0252] In the following experiments, the PBTS1-RPL25-driving genetic construct was used to introduce the AeBlue chromoprotein gene (
[0253] Having confirmed that the chromoproteins were effective markers, human papillomavirus (HPV) 16 major capsid protein L1 gene was inserted after the AeBlue expression cassette (
[0254] Cells with multi-copy integration of the AeBlue expression cassette showed a strong Tibetan blue color, while cells with an empty cassette were milky white color (
[0255] SDS-PAGE analysis of whole cell and soluble protein extracts showed bands at 25 kD (AeBlue molecular weight) in all samples, with much stronger bands observed in the multi-copy integration strain samples than in the 2 plasmid strain samples (
[0256] A second strong band at 50 kD band (HPV16-L1 molecular weight) was observed in samples from cells expressing HPV-L1, although it was not as distinct at the putative AeBlue band (
[0257] Disclosed herein is a novel genetic engineering method to integrate multiple copies of heterologous gene(s) into the yeast genome using in vivo gene amplification driven by a haploinsufficient gene. The functional strength per copy of a haploinsufficient gene is strongly associated with growth fitness, which can be exploited as an evolutionary force to drive gene amplification. Decreased expression level provides an evolutionary force that drives amplification of linked haploinsufficient and heterologous genes, so that cells are growth-competitive.
[0258] Provided here are examples of the application of this method to improve production of different types of terpene products, however the application of this method is not limited to the terpene products. Also shown is that the present method can be used to enable high-level expression of any other heterologous protein in yeast, at levels similar to that achieved in E. coli for protein production.
[0259] This method advantageous for the introduction of heterologous genes via genome integration. Firstly, integration copy number can be titrated by altering the expression dosage per copy of haploinsufficient gene. Expression level can be reduced by a variety of methods, including but not limited to (1) replacing the gene promoter with a weaker promoter, and (2) using non-preferred codons.
[0260] Amplification efficiency observed was 4 to 47 copies of the heterologous genes, with an inverse relationship between promoter strength and copy number. However, it can be easily recognized that suitable alteration of the expression dosage of the haploinsufficiency gene will drive less or more amplification.
[0261] A number of weak promoters are described herein (Table 1 and
[0262] Another advantage is that the maintenance of integration is auto-selectable: selection pressure is provided from the dosage sensitivity of the haploinsufficient gene, which is linked to the gene of interest and is maintained to support normal growth rates. This means that no antibiotics or modification of other environmental conditions in the culture are required to provide ongoing selection pressure for maintenance of the gene of interest. Compared to use of a 2 plasmid, this method provides for improved stable expression of heterologous proteins in yeast (
[0263] The presence of multiple haploinsufficient genes within a host cell genome means that many different loci are available for engineering gene amplification. Characterization of the promoter strength of fifteen additional haploinsufficient genes provided here (Table 1) can also be used to drive gene amplification.
[0264] Initial integration of the genes of interest uses standard yeast transformation procedures by selection of an auxotrophic or antibiotic marker (e.g., LEU2 or hphMax). Use of visual markers (fluorescent proteins or chromoproteins) can facilitate the selection of correct clones with amplified constructs.
[0265] The present disclosure disclosed herein successfully improved production of heterologous terpenes including the C.sub.15 sesquiterpene nerolidol (
[0266] Production of C.sub.15 terpenes in yeast is typically relatively straightforward, with g L.sup.1 titres achievable. The C.sub.15 precursor, FPP, is produced in yeast naturally to deliver sterol pathway products required for yeast growth. In addition, sesquiterpene synthases have reasonably good catalytic properties, making them more competitive to access FPP.
[0267] However production of C.sub.10 monoterpenes, however, has historically been very challenging. This is due to both a dearth of C.sub.10 precursors and the poor catalytic properties of many monoterpene synthases. These limitations have previously restricted published titers of monoterpenes to mg L.sup.1 in flask cultivation. Here, we have achieved g L.sup.1 titers (
[0268] Variation in the different systems results in variable improvement ratios, for example, limonene production improvement was 20-fold, whereas nerolidol improvement was 1.7-fold, and lycopene improvement was 5-fold. However a higher titer is seen with in vivo gene amplification. In particular, for monoterpenes, insufficient catalytic efficiency of terpene synthase is a significant bottleneck for production of heterologous terpenoids in yeast. Increasing copy number via insertion of tandem repeats at the same locus combined with screening for improved production or introduction of additional expression cassettes at separate loci has been used to overcome this bottleneck previously. However, these approaches require complex cloning and extended experimental timelines to deliver the desired improvements. The presently disclosed disclosure advantageously provides means to overcome these challenges by providing a faster and simpler method to achieve superior results.
[0269] In addition to its application in metabolic engineering, the presently disclosure can be used for increasing heterologous protein production. Using chromoprotein AeBlue and the HPV16 L1 capsid protein as examples (
[0270] The presently disclosed method is applicable to other industrially relevant chassis organisms that have haploinsufficient genes. A potential haploinsufficient gene may encode essential components of the machineries for protein synthesis and transportation or other essential cell structures. Putative haploinsufficient genes can be identified by comparative genomics and confirmed by testing growth fitness in association with expression dosage of a gene.
TABLE-US-00002 TABLE 2 Plasmids used Plasmid Properties pILGFP3 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3- yEGFP > T.sub.URA3 pILGFP1D5 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3- yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP5A3 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.YEF3 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP1A6 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.RPL25 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP1C6 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.SEC23 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP1E6 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.PDA1 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP1E7 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.ERG1 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP1G7 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.BTS1 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP4F5 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.GLO2 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP4H5 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.COG7 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP89 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3- P.sub.TEF1 > yEGFP > T.sub.URA3 pILGFP1DFB Yeast integration plasmid; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2-T.sub.RPL25(Arm 3)- ARS305-P.sub.TEF1 > yEGFP > T.sub.URA3 pILGFP3A5C Yeast integration plasmid; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2-T.sub.RPL25(Arm 2)- ARS305-P.sub.TEF1 > yEGFP > T.sub.URA3- P.sub.YEF3 > RPL25(partial; Arm3) pILGFP3AE4 Yeast integration plasmid; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2-T.sub.RPL25(Arm 3)- ARS305-P.sub.TEF1 > yEGFP > T.sub.URA3- P.sub.ERG1 > RPL25(partial; Arm2) pILGFP3AG4 Yeast integration plasmid; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2-T.sub.RPL25(Arm 3)- ARS305-P.sub.TEF1 > yEGFP > T.sub.URA3- P.sub.PDA1 > RPL25(partial; Arm2) pILGFP3AA5 Yeast integration plasmid; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2-T.sub.RPL25(Arm 3)- ARS305-P.sub.TEF1 > yEGFP > T.sub.URA3- P.sub.BTS1 > RPL25(partial; Arm2) pILGFP3AG4ARSd Yeast integration plasmid; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2-T.sub.RPL25(Arm 3)- P.sub.TEF1 > yEGFP > T.sub.URA3- P.sub.PDA1 > RPL25(partial; Arm2) pILGFP4BG6 Yeast integration plasmid; P.sub.SEC23(Arm 1) > P.sub.Ag.TEF1 > hphMX4 > T.sub.Ag.TEF1- T.sub.SEC23(Arm 3)-ARS1max-P.sub.TEF1 > yEGFP > T.sub.URA3 pILGFP5EG3 Yeast integration plasmid; P.sub.SEC23(Arm 1) > P.sub.Ag.TEF1 > hphMX4 > T.sub.Ag.TEF1- T.sub.SEC23(Arm 3)-ARS1max-P.sub.TEF1 > yEGFP > T.sub.URA3-P.sub.ERG1 > SEC23(partial; Arm2) pILGFP5EA4 Yeast integration plasmid; P.sub.SEC23(Arm 1) > P.sub.Ag.TEF1 > hphMX4 > T.sub.Ag.TEF1- T.sub.SEC23(Arm 3)-ARS1max-P.sub.TEF1 > yEGFP > T.sub.URA3-P.sub.GLO2 > SEC23(partial; Arm2) pILGFP5EC4 Yeast integration plasmid; P.sub.SEC23(Arm 1) > P.sub.Ag.TEF1 > hphMX4 > T.sub.Ag.TEF1- T.sub.SEC23(Arm 3)-ARS1max-P.sub.TEF1 > yEGFP > T.sub.URA3-P.sub.COG7 > SEC23(partial; Arm2) pILGFP5EF3 Yeast integration plasmid; P.sub.SEC23(Arm 1) > P.sub.Ag.TEF1 > hphMX4 > T.sub.Ag.TEF1- T.sub.SEC23(Arm 3)-ARS1max-P.sub.TEF1 > yEGFP > T.sub.URA3-P.sub.COG7 > ATGGGAGGAGGA- SEC23(partial; Arm2) pILGFP6G3 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.RPL33A > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6A4 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.RPS15 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6C4 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.RPC10 > yEGFP > T.sub.PGK1-T.sub.URA3 pACT1-GFP Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.ACT1 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6G4 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.NIP1 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6A5 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.RPS13 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6C5 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.NUS1 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6E5 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.SMC1 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6G5 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.RNA14 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6A6 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.RPB7 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6C6 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.SPC97 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6E6 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.STH1 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6G6 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.ARP7 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6A7 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.TAF61 > yEGFP > T.sub.PGK1-T.sub.URA3 pILGFP6C7 Yeast integration plasmid; P.sub.URA3 > KI.URA3 > T.sub.KI.URA3-P.sub.RPN11 > yEGFP > T.sub.PGK1-T.sub.URA3 pRS425 E. coli/S. cerevisiae shuttle plasmid; 2, LEU2 pIR3DH8 Yeast integration plasmid; gal80Arm1-P.sub.AgTEF1-KIURA3-T.sub.AgTEF1-gal80Arm2 pJT9RFR pRS425 derivative; T.sub.RPL3 < ScERG20 < P.sub.GAL1-P.sub.GAL2 > Y.FAST-EVBR1.2A- AcNES1 > T.sub.RPL41B pINER2R pILGFP3AE4 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2-P.sub.GAL1 > ERG20 > T.sub.RPL3- T.sub.RPL25(Arm 3)- ARS305- P.sub.GAL2 > Y.FAST-EVBR1.2A-AcNES1 > T.sub.RPL41B > RPL25(partial; Arm2) pINER3R pILGFP3AG4 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2-P.sub.GAL1 > ERG20 > T.sub.RPL3- T.sub.RPL25(Arm 3)- ARS305- P.sub.GAL2 > Y.FAST-EVBR1.2A-AcNES1 > T.sub.RPL41B -P.sub.PDA1 > RPL25(partial; Arm2) pINER4R pILGFP3AA5 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2-P.sub.GAL1 > ERG20 > T.sub.RPL3- T.sub.RPL25(Arm 3)- ARS305- P.sub.GAL2 > Y.FAST-EVBR1.2A-AcNES1 > T.sub.RPL41B - P.sub.BTS1 > RPL25(partial; Arm2) pIT6EG7m pILGFP3AG4 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2- T.sub.RPL25(Arm 3)- ARS305- P.sub.Sk.GAL2 > Y.FAST-EVBR1.2A-Ec.MBP-Linker~SacI~6*G-ERG20.sup.F96W N127W > T.sub.RPL3 -P.sub.PDA1 > RPL25(partial; Arm2) pIT6EG7ml pILGFP3AG4 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2- T.sub.RPL25(Arm 3)- ARS305- P.sub.Sk.GAL2 > Y.FAST-EVBR1.2A-Ec.MBP-Linker-LI.LS-6*G-ERG20.sup.F96W N127W > T.sub.RPL3-P.sub.PDA1 > RPL25(partial; Arm2) pIT6EG7mlh pILGFP3AA5 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2- T.sub.RPL25(Arm 3)- ARS305- P.sub.Sk.GAL2 > Y.FAST-EVBR1.2A-Ec.MBP-Linker-LI.LS-6*G-ERG20.sup.F96W N127W > T.sub.RPL3 -P.sub.BTS1 > RPL25(partial; Arm2) pPT6EG7ml pRS425 derivative; P.sub.Sk.GAL2 > Y.FAST-EVBR1.2A-Ec.MBP-Linker~SacI~6*G- ERG20.sup.F96W N127W > T.sub.RPL3 pLAC1 pRS425 derivative; P.sub.GAL1 > ERG20.sup.F96C > T.sub.EBS1-P.sub.Sk.GAL2 > Xd.CRtYB.sup.E83K > T.sub.CYC1- P.sub.Se.GAL2 > XdCrtI > T.sub.RPL41B pILAC2 pILGFP3AG4 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2- T.sub.RPL25(Arm 3)- ARS305- P.sub.GAL1 > ERG20.sup.F96C > T.sub.EBS1-P.sub.Sk.GAL2 > Xd.CRtYB.sup.E83K > T.sub.CYC1- P.sub.Se.GAL2 > XdCrtI > T.sub.RPL41B -P.sub.PDA1 > RPL25(partial; Arm2) pILAC3 pILGFP3AA5 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2- T.sub.RPL25(Arm 3)- ARS305- P.sub.GAL1 > ERG20.sup.F96C > T.sub.EBS1-P.sub.Sk.GAL2 > Xd.CRtYB.sup.E83K > T.sub.CYC1- P.sub.Se.GAL2 > XdCrtI > T.sub.RPL41B -P.sub.BTS1 > RPL25(partial; Arm2) pIAeBlue pILGFP3AA5 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2- T.sub.RPL25(Arm 3)- ARS305- P.sub.ALD6 > AeBlue > T.sub.PGK1- P.sub.BTS1 > RPL25(partial; Arm2) pIEforRed pILGFP3AA5 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2- T.sub.RPL25(Arm 3)- ARS305- P.sub.ALD6 > EforRed > T.sub.PGK1- P.sub.BTS1 > RPL25(partial; Arm2) pIR3DH8K Yeast integration plasmid; gal80Arm1-P.sub.TPI1-KanMX4-gal80Arm2 pPAeBlueHPV16LR pRS425 derivative; P.sub.ALD6 > AeBlue > T.sub.PGK1- P.sub.Se.GAL2 > HPV16-L1C-6*H > T.sub.RPL41B pIAeBlueHPV16LR pILGFP3AA5 derivative; P.sub.RPL25(Arm 1) > KI.LEU2 > T.sub.KI.LEU2- T.sub.RPL25(Arm 3)- ARS305- P.sub.ALD6 > EforRed > T.sub.PGK1- P.sub.Se.GAL2 > HPV16-L1C-6*H > T.sub.RPL41B-P.sub.BTS1 > RPL25(partial; Arm2)
TABLE-US-00003 TABLE 3 Saccharomyces cerevisiae strains used in this work Strain Genotype CEN.PK2-1C MATa ura3-52 trp1-289 leu2-3, 112 his3 1 CEN.PK113- MATa ura3-52 5D CEN.PK113- MATa leu2-3 16B CEN.PK113- MATa 7D ILHA series strains GH4 CEN.PK113-5D derivative; ura3(1, 704)::KI.URA3 > T.sub.KI.URA3 G5A3 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.YEF3 > yEGFP > T.sub.PGK1 (FIG. 2d) G1A6 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.RPL25 > yEGFP > T.sub.PGK1 (FIG. 2d) G1C6 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.SEC23 > yEGFP > T.sub.PGK1 (FIG. 2d) G1E6 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.PDA1 > yEGFP > T.sub.PGK1 (FIG. 2d) G1E7 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.ERG1 > yEGFP > T.sub.PGK1 (FIG. 2d) G1G7 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.BTS1 > yEGFP > T.sub.PGK1 (FIG. 2d) G4F5 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.GLO2 > yEGFP > T.sub.PGK1 (FIG. 2d) G4H5 CEN. PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.COG7 > yEGFP > T.sub.PGK1 (FIG. 2d) G3A5C CEN.PK113-16B derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2-T.sub.RPL25- ARS305-P.sub.TEF1 > yEGFP > T.sub.URA3- P.sub.YEF3-RPL25 (FIG. 2, Construct 1) G3AE4 CEN.PK113-16B derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2-{T.sub.RPL25- ARS305-P.sub.TEF1 > yEGFP > T.sub.URA3- P.sub.ERG1-RPL25}.sub.n (FIG. 2, Construct 2) G3AG4 CEN.PK113-16B derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2-{T.sub.RPL25- ARS305-P.sub.TEF1 > yEGFP > T.sub.URA3- P.sub.PDA1-RPL25}.sub.n (FIG. 2, Construct 3) G3AA5 CEN.PK113-16B derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2-{T.sub.RPL25- ARS305-P.sub.TEF1 > yEGFP > T.sub.URA3- P.sub.BTS1-RPL25}.sub.n (FIG. 2, Construct 4) G5EG3 CEN.PK113-7D derivative; SEC23:: P.sub.Ag.TEF1 > hphMX4 > T.sub.Ag.TEF1- T.sub.SEC23-ARS1max- P.sub.TEF1 > yEGFP > T.sub.URA3-P.sub.ERG1 > SEC23 (FIG. 2, Construct 5) G5EA4 CEN.PK113-7D derivative; SEC23:: P.sub.Ag.TEF1 > hphMX4 > T.sub.Ag.TEF1- {T.sub.SEC23-ARS1max- P.sub.TEF1 > yEGFP > T.sub.URA3-P.sub.GLO2 > SEC23}CT.sub.n (FIG. 2, Construct 6) G5EC4 CEN.PK113-7D derivative; SEC23:: P.sub.Ag.TEF1 > hphMX4 > T.sub.Ag.TEF1- {T.sub.SEC23-ARS1max- P.sub.TEF1 > yEGFP > T.sub.URA3-P.sub.COG7 > SEC23}.sub.n (FIG. 2, Construct 7) G5EF3 CEN.PK113-7D derivative; SEC23:: P.sub.Ag.TEF1 > hphMX4 > T.sub.Ag.TEF1- {T.sub.SEC23-ARS1max- P.sub.TEF1 > yEGFP > T.sub.URA3-P.sub.COG7 > ATGGGAGGAGGA-SEC23}.sub.n (FIG. 2, Construct 8) G6G3 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.RPL33A > yEGFP > T.sub.PGK1 (FIG. S2) G6A4 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.RPS15 > yEGFP > T.sub.PGK1 (FIG. S2) G6C4 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.RPC10 > yEGFP > T.sub.PGK1 (FIG. S2) GATC1-GFP CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.ACT1 > yEGFP > T.sub.PGK1 (FIG. S2) G6G4 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.NIP1 > yEGFP > T.sub.PGK1 (FIG. S2) G6A5 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.RPS13 > yEGFP > T.sub.PGK1 (FIG. S2) G6C5 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.NUS1 > yEGFP > T.sub.PGK1 (FIG. S2) G6E5 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.SMC1 > yEGFP > T.sub.PGK1 (FIG. S2) G6G5 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.RNA1 > yEGFP > T.sub.PGK1 (FIG. S2) G6A6 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.RPB7 > yEGFP > T.sub.PGK1 (FIG. S2) G6C6 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.SPC97 > yEGFP > T.sub.PGK1 (FIG. S2) G6E6 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.STH1 > yEGFP > T.sub.PGK1 (FIG. S2) G6G6 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.ARP7 > yEGFP > T.sub.PGK1 (FIG. S2) G6A7 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.TAF61 > yEGFP > T.sub.PGK1 (FIG. S2) G6C7 CEN.PK113-5D derivative; ura3(1, 704):: KI.URA3 > T.sub.KI.URA3- P.sub.RPN11 > yEGFP > T.sub.PGK1 (FIG. S2) o401R CEN.PK2-1C derivative; HMG2.sup.K6R(152, 1)::HIS3-T.sub.EFM1 < EfmvaS < P.sub.GAL1-P.sub.GAL10 > ACS2 > T.sub.ACS2- P.sub.GAL2 > EfmvaE > T.sub.EBS1-P.sub.GAL7 pdc5 (31, 94)::P.sub.GAL2 > ERG12 > T.sub.NAT5-P.sub.TEF2 > ERG8 > T.sub.IDP1- T.sub.PRM9 < MVD1 < P.sub.ADH2-T.sub.RPL15A < IDI1 < P.sub.TEF1-TRP1 ERG9(1333, 1335)::T.sub.URA3- P.sub.GAL7 > MVD1 > T.sub.PRM9-P.sub.GAL2 > ERG12 > T.sub.NAT5- T.sub.IDP1 < ERG8 < P.sub.GAL10-P.sub.GAL1 > IDI1 > T.sub.RPL15A-loxP-ble-loxP o401UR o401R derivative; gal80::P.sub.AgTEF1 > KI.URA3 > T.sub.AgTEF1 N401-1 o401UR derivative; [pJT9RFR] N401-2 o401UR derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2-P.sub.GAL1 > ERG20 > T.sub.RPL3-{T.sub.RPL25- ARS305- P.sub.GAL2 > Y.FAST- EVBR1.2A-AcNES1 > T.sub.RPL41B - P.sub.ERG1-RPL25}.sub.n N401-3 o401UR derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2-P.sub.GAL1 > ERG20 > T.sub.RPL3-{T.sub.RPL25- ARS305- P.sub.GAL2 > Y.FAST- EVBR1.2A-AcNES1 > T.sub.RPL41B - P.sub.PDA1-RPL25}.sub.n N401-4 o401UR derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2-P.sub.GAL1 > ERG20 > T.sub.RPL3-{T.sub.RPL25- ARS305- P.sub.GAL2 > Y.FAST- EVBR1.2A-AcNES1 > T.sub.RPL41B - P.sub.BTS1-RPL25}.sub.n o141R o401R derivative; ERG20(32, 3)::CUP1-AID* ura3(1, 704)::KI.URA3-T.sub.PGK1-P.sub.ACS2 > SKP1-OsTIR1 gal80::P.sub.AgTEF1 > KanMX4 > T.sub.AgTEF1 ura3(1, 704)::KIURA3-T.sub.PGK1-P.sub.ACS2 > SKP1-OsTIR1 LIM141M o141R derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2 -{T.sub.RPL25- ARS305- P.sub.Sk.GAL2 > Y.FAST-EVBR1.2A-Ec.MBP- Linker~SacI~6*G-ERG20.sup.F96W N127W > T.sub.RP1418 - P.sub.PDA1-RPL25}.sub.n gal80::P.sub.AgTEF1 > KanMX4 > T.sub.AgTEF1 LIM141MH o141R derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2 -{T.sub.RPL25- ARS305- P.sub.Sk.GAL2 > Y.FAST-EVBR1.2A-Ec.MBP- Linker~SacI~6*G-ERG20.sup.F95W N127W > T.sub.RP141B - P.sub.BTS1-RPL25}.sub.n gal80::P.sub.AgTEF1 > KanMX4 > T.sub.AgTEF1 LAC1 o401R derivative; [pLAC1] gal80::P.sub.AgTEF1 > KanMX4 > T.sub.AgTEF1 LAC4 o401UR derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2 -{T.sub.RPL25- ARS305- P.sub.GAL1 > ERG20.sup.F96C > T.sub.EBS1- P.sub.SK.GAL2 > Xd.CRtYB.sup.E83K > T.sub.CYC1-P.sub.Se.GAL2 > XdCrtI > T.sub.RPL41B - P.sub.PDA1-RPL25}.sub.n LAC5 o401UR derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2 -{T.sub.RPL25- ARS305- P.sub.GAL1 > ERG20.sup.F96C > T.sub.EBS1- P.sub.Sk.GAL2 > Xd.CRtYB.sup.E83K > T.sub.CYC1-P.sub.Se.GAL2 > XdCrtI > T.sub.RPL41B - P.sub.BTS1-RPL25}.sub.n 16BJ3 CEN.PK113-16B derivative; gal80::P.sub.AgTEF1 > KanMX4 > T.sub.AgTEF1 16BJ3C 16BJ3 derivative; [pRS425] (FIG. 6; Empty, 2) 16BJ3AeBlue 16BJ3 derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2-P.sub.GAL1 > ERG20 > T.sub.RPL3-{T.sub.RPL25- ARS305- P.sub.ALD6 > AeBlue > T.sub.PGK1- P.sub.BTS1-RPL25}.sub.n (FIG. 6; AeBlue, MI) HPV16LPR 16BJ3 derivative; [pPAeBlueHPV16LR] (FIG. 6; AeBlue + HPV16-L1, 2) HPV16LMR 16BJ3 derivative; RPL25:: KI.LEU2 > T.sub.KI.LEU2-P.sub.GAL1 > ERG20 > T.sub.RPL3-{T.sub.RPL25- ARS305- P.sub.ALD6 > AeBlue > T.sub.PGK1- P.sub.Se.GAL2 > HPV16-L1C-6*H > T.sub.RPL41B-P.sub.BTS1-RPL25}.sub.n (FIG. 6; AeBlue + HPV16-L1, MI)
TABLE-US-00004 TABLE4 ListofprimersandDNAfragmentsusedinthiswork.P.sub.XXXandT.sub.XXXindicatepromoterand terminatorsequenceofgeneXXX,respectively;italicizedandunderlinedindicatesequences complementarytotheDNAtemplate. SEQ Overlap ID extension PCR/gBlock No: PCRfragment fragment Primername Sequence(5.fwdarw.3) 1 T.sub.PGK1from PPGPGK1ts GGATGAATTGTACAAAAGATCTTAAATTGA SGD ATTGAATTGAAATCGATAG 2 PPGPGK1ta CCCTTTGCAAATAGTCCTACTAGT AAATAATATCCTTCTCGAAAGC 3 P.sub.YEF3from PPGYEF3ps AAGGGTTGCTCGAGAAAGAGCTC SGD ATACATAACATTTTAAGATAAGCAAGTG 4 PPGYEF3pa TGAATAATTCTTCACCTTTAGACAT CTTTTAATGTTATCGATGGATTC 5 P.sub.RPL25from PPGRPL25ps AAGGGTTGCTCGAGAAAGAGCTC SGD TCTTATCTTGTATGCCCGATAT 6 PPGRPL25pa TGAATAATTCTTCACCTTTAGACAT TTTATCTTATTGATCTTCTTTGTTTA 7 P.sub.SEC23from PPGSEC23ps AAGGGTTGCTCGAGAAAGAGCTC SGD TGTCTTGTTGTGTTGTGACG 8 PPGSEC23pa TGAATAATTCTTCACCTTTAGACAT GGCTAGAAAAGAGGAAGGG 9 P.sub.PDA1from PPGPDA1ps AAGGGTTGCTCGAGAAAGAGCTC SGD GAAATTCAAAACTCTCCAGAC 10 PPGPDA1pa TGAATAATTCTTCACCTTTAGACAT TGGCACAAATGTGGTTTCC 11 P.sub.ERG1from PPGERG1ps AAGGGTTGCTCGAGAAAGAGCTC SGD TGCGATACTGCCGTAGCG 12 PPGERG1pa TGAATAATTCTTCACCTTTAGACAT GACCCTTTTCTCGATATGTT 13 P.sub.BTS1from PPGBTS1ps AAGGGTTGCTCGAGAAAGAGCTC SGD CCGCCATCTCTACTCACTC 14 PPGBTS1pa TGAATAATTCTTCACCTTTAGACAT TGATTTTCCAGACTCGTAAAC 15 P.sub.COG7from PPGCOG7ps AAGGGTTGCTCGAGAAAGAGCTC SGD CCGGATATGAAAATGGAATGC 16 PPGCOG7pa TGAATAATTCTTCACCTTTAGACAT ATTCTGCTTAGTTTGGCCTTC 17 P.sub.GLO2from PPGGLO2ps AAGGGTTGCTCGAGAAAGAGCTC SGD AGTTCATTGATGTTGAAGAAGTG 18 PPGGLO2pa TGAATAATTCTTCACCTTTAGACAT TTTTTGTCCTCCTTTTCTTGTG 19 P.sub.RPL25- P.sub.RPL25(Arm PGRNRPL25ps AACGACGGCCAGTGAATTCAGTTTAAACA KI.LEU2- 1)fromSGD TGTACTAATCAGTCTAAC 20 T.sub.KI.LEU-T.sub.RPL25 PGRNRPL25pa TGGTATATGATTTTGTGGACATTITGCGGC CGCTTTATCTTATTGATCTTCTTTGTTTAG 21 KI.LEU2from PGRNKILEU2s GCGGCCGCAAAATGTCCACAAAATCATAT pUG73 ACCAG 22 PGRNKILEU2a TCTAGATTTGGGCCCGATCCCAATACAAC AGATCA 23 T.sub.RPL25(Arm PGRNRPL25ts CTGTTGTATTGGGATCGGGCCCAAATCTA 3)fromSGD GATCTAATTGGTTTAATTAATAAATTTAATA 24 PGRNRPL25ta CCTCACGAAGAAGTTAAGCTTGAGCATCG GACCGAAGCAT 25 ARS306 PGRNARS306s ATGCTTCGGTCCGATGCTCAAGCTTAACTT fromSGD CTTCGTGAGG 26 PGRNARS306a GTATGCTATACGAAGTTATTAGGCTCGAG CTCGAGTTAATTTATCTCATG 27 P.sub.YEF3-RPL25 P.sub.YEF3(2) PPGRPL25- GGAATCTCGGTCGTAATGATTTGCATGC (Arm2) fromSGD YEF3ps ATACATAACATTTTAAGATAAGCAAGTG 28 PPGRPL25- GCAGTTCACATACCAGATGGAGCCAT YEF3pa CTTTTAATGTTATCGATGGATTC 29 RPL25 PPGRPL25s ATGGCTCCATCTGGTATGTGAACTGC partial(Arm 2)fromSGD 30 PPGRPL25a GACCATGATTACGCCAAGCTTGTTT AAACTATGTTCCTTGATACCTC 31 P.sub.ERG1-RPL25 P.sub.ERG1(2) PPGRPL25- GGAATCTCGGTCGTAATGATTTGCATGC (Arm2) fromSGD ERG1ps TGCGATACTGCCGTAGCG 32 PPGRPL25- GCAGTTCACATACCAGATGGAGCCAT ERG1pa GACCCTTTTCTCGATATGTT RPL25 PPGRPL25s Asabove partial(Arm 2)fromSGD PPGRPL25a Asabove 33 P.sub.PDA1-RPL25 P.sub.PDA1(2) PPGRPL25- GGAATCTCGGTCGTAATGATTTGCATGC (Arm2) fromSGD PDA1ps GAAATTCAAAACTCTCCAGAC 34 PPGRPL25- GCAGTTCACATACCAGATGGAGCCAT PDA1pa TGGCACAAATGTGGTTTCC RPL25 PPGRPL25s Asabove partial(Arm 2)fromSGD PPGRPL25a Asabove 35 P.sub.BTS1-RPL25 P.sub.BTS1(2) PPGRPL25- GGAATCTCGGTCGTAATGATTTGCATGC (Arm2) fromSGD BTS1ps CCGCCATCTCTACTCACTC 36 PPGRPL25- GCAGTTCACATACCAGATGGAGCCAT BTS1pa TGATTTTCCAGACTCGTAAAC RPL25 PPGRPL25s Asabove partial(Arm 2)fromSGD PPGRPL25a Asabove 37 P.sub.SEC23- P.sub.SEC23(2) PPGSEC23p1s AACGACGGCCAGTGAATTCAGTTT hphMX- fromSGD AAACTCTTCTGCTTCGTTCAGCTG T.sub.SEC23- ARSMax1 38 PPGSEC23p1a GCACGTCAAGACTGTCAAGGAGGGTATTC GGGCCCGTATCTTTTTTTCTTTTTTCAAAC 39 G hphMX PPMLhphs GACTTAGATTGGTATATATACGCATATG pAG32 GAATACCCTCCTTGACAGTC 40 PPMLhpha ATTGATAATGATAAACTCGAACTGACTAGT CGTTAGTATCGAATCGACAG 41 T.sub.SEC23(Arm PPGSEC23ts GTCGCTATACTGCTGTCGATTCGATACTAA 3)fromSGD CGGCGGCCGCGAGCAACGGCTTTCTTTTG 42 T ACAAATGAAAAGAGATGCGGCCGTATGGT PPGSEC23ta GTGAAAATCT 43 ARS1Max AGATTTTCACACCATACGGCCGCATCTCTT (gBlock) TTCATTTGTATTTAAATCCATTTCAAATTTT ATGTTTAGTTCGAGATCCTCAGTTTTCGGC GCATAGGAACCACGTACATAATAACTAAA CATAAATCTATAATAAATAAAAAACAACGA TGGGAGCTCGAGCCTAATAACTTCGTATA GCATAC 44 PPGARS1maxa GTATGCTATACGAAGTTATTAGGCTCGAG CTCCCATCGTTGTTTTTTATTTATTATAGA 45 P.sub.ERG1-SEC23 P.sub.ERG1(3) PPGSEC23- GGAATCTCGGTCGTAATGATTT (Arm2) fromSGD ERG1ps GATATGAAGGCATGC TGCGATACTGCCGTAGCG 46 PPGSEC23- CGTTGATGTCTTCATTAGTCTCGAAGTCCA ERG1pa TGACCCTTTTCTCGATATGTT 47 SEC23 PPGSEC23s ATGGACTTCGAGACTAATGAAGACATCAA partial(Arm CG 2)fromSGD 48 PPGSEC23a GACCATGATTACGCCAAGCTTGTTTA AACGTTTCCGTAAGTGATCAAC 49 P.sub.GLO2-SEC23 P.sub.GLO2(2) PPGSEC23- GGAATCTCGGTCGTAATGATTT (Arm2) fromSGD GLO2ps GATATGAAGGCATGC AGTTCATTGATGTTGAAGAAGTG 50 PPGSEC23- CGTTGATGTCTTCATTAGTCTCGAAGTCCA GLO2pa TTTTTTGTCCTCCTTTTCTTGTG SEC23 PPGSEC23s Asabove partial(Arm 2)fromSGD PPGSEC23a Asabove 51 P.sub.COG7-SEC23 P.sub.COG7(2) PPGSEC23- GGAATCTCGGTCGTAATGATTT (Arm2) fromSGD COG7ps GATATGAAGGCATGC CCGGATATGAAAATGGAATGC 52 PPGSEC23- CGTTGATGTCTTCATTAGTCTCGAAGTCCA COG7pa TATTCTGCTTAGTTTGGCCTTC SEC23 PPGSEC23s Asabove partial(Arm 2)fromSGD PPGSEC23a Asabove P.sub.COG7-3G- P.sub.COG7-3G(2) PPGSEC23- Asabove SEC23(Arm fromSGD COG7ps 2) 53 SEC23 PPGSEC23- GTTGATGTCTTCATTAGTCTCGAAGTCTCC partial(Arm TCCTCCCAT 2)fromSGD COG7pa1 ATTCTGCTTAGTTTGGCCTTC PPGSEC23s Asabove PPGSEC23a Asabove 54 P.sub.RPL33Afrom PPGRPL33As AAGGGTTGCTCGAGAAAGAGCTC SGD GTAAAAAGAACAAGAAGAGAATAAAAC 55 PPGRPL33Aa TGAATAATTCTTCACCTTTAGACAT TTTTCAATTTATTTGATTGTTGGTTTC 56 P.sub.RPS15from PPGRPS15s AAGGGTTGCTCGAGAAAGAGCTC SGD CTCGAATAATAACGGCTCTC 57 PPGRPS15a TGAATAATTCTTCACCTTTAGACAT GATCGGTCGTGATTATCTTG 58 P.sub.RPC10from PPGRPC10s AAGGGTTGCTCGAGAAAGAGCTC SGD CCTCGTGTTGTTATAACGAC 59 PPGRPC10a TGAATAATTCTTCACCTTTAGACAT TGTTATACTTGTGGACTTTTATTC 60 P.sub.ACT1from pACT1s AAGGGTTGCTCGAGAAAGAGCTCAACCTG SGD AAGGGACAGAGTTTAAC 61 pACT1a GTGAATAATTCTTCACCTTTAGACATTGTT AATTCAGTAAATTTTCGATCTTGGG 62 P.sub.NIP1from PPGNIP1s AAGGGTTGCTCGAGAAAGAGCTC SGD CGTATCCAATTCGGACGTTG 63 PPGNIP1a TGAATAATTCTTCACCTTTAGACAT TTTCGTAGATCTCGGGCTTG 64 P.sub.RPS13from PPGRPS13s AAGGGTTGCTCGAGAAAGAGCTC SGD ACGTTGAAGAATTGAGGGAG 65 PPGRPS13a TGAATAATTCTTCACCTTTAGACAT TTTGACTGATTGTTGTTGATTG 66 P.sub.NUS1from PPGNUS1s AAGGGTTGCTCGAGAAAGAGCTC SGD AAACGCCACTAATCAACCTG 67 PPGNUS1a TGAATAATTCTTCACCTTTAGACAT CTAAGAAAAACAATGGGGAAAATAT 68 P.sub.SMC1from PPGSMC1s AAGGGTTGCTCGAGAAAGAGCTC SGD AGCTGGAAAAATGCGTAATAAC 69 PPGSMC1a TGAATAATTCTTCACCTTTAGACAT TGCGTCTCCTTGTGCCTGCT 70 P.sub.RNA14from PPGRNA14s AAGGGTTGCTCGAGAAAGAGCTC SGD CAACGTCAACATAATTCAATAG 71 PPGRNA14a TGAATAATTCTTCACCTTTAGACAT ATCTCTTGTTTGACTCTCCAG 72 P.sub.RPB7from PPGRPB7s AAGGGTTGCTCGAGAAAGAGCTC SGD ACCACTGAGGCTAGTGATCT 73 PPGRPB7a TGAATAATTCTTCACCTTTAGACAT TCTCAGAAATTGAGTTATTTATAC 74 P.sub.SPC97from PPGSPC97s AAGGGTTGCTCGAGAAAGAGCTC SGD TTGTGGTGCCACTTTCCGTA 75 PPGSPC97a TGAATAATTCTTCACCTTTAGACAT TTTTTCACGCAAGATGTGTAC 76 P.sub.STH1from PPGSTH1s AAGGGTTGCTCGAGAAAGAGCTC SGD GTTTGATAGCAGTCCATTAAC 77 PPGSTH1a TGAATAATTCTTCACCTTTAGACAT TCGCGCTTGCTCTAAACTGTG 78 P.sub.ARP7from PPGARP7s AAGGGTTGCTCGAGAAAGAGCTC SGD GTAGCGGATGACATCCTGAT 79 PPGARP7a TGAATAATTCTTCACCTTTAGACAT TCTTGACAGATCCTTTATAATG 80 P.sub.TAF61from PPGTAF61s AAGGGTTGCTCGAGAAAGAGCTC SGD GCTTGTTCTCTCGTTGATAC 81 PPGTAF61a TGAATAATTCTTCACCTTTAGACAT TGTCGTATTTTATACACACACTG 82 P.sub.RPN11from PPGRPN11s AAGGGTTGCTCGAGAAAGAGCTC SGD CTGCGGGAACCTCTTCCACA 83 PPGRPN11a TGAATAATTCTTCACCTTTAGACAT TATGTCTCGTCTTTCTTGTTAAG 84 P.sub.GAL1-ERG20- PIJTERG20s ACAGGTTCCGGTTAGCCTGCGCTAGC P.sub.RPL3from TTATATTGAATTTTCAAAAATTCTTAC 85 pJT9RFR PIJTERG20a TTTATTAATTAAACCAATTAGATCTAG GGGCCC ATTGTAGCAAAGATTGTAAGGAAATAG 86 P.sub.GAL2- PIJTNES1s CATTACTTCATGAGATAAATTAA Y.FAST- CTCGAGTGTACTAATCCAAGGAGGTT 87 EVBR1.2A- PIJTNES1a CTTTGTCTGGAGAGTTTTGAATTTC AcNES1- GAGCTCACGCCACAGAAACCTCAGA T.sub.RPL41Bfrom pJT9RFR 88 P.sub.Sk.GAL2- P.sub.Sk.GAL2from PSYKSKGAL2ps GTATCATTACTTCATGAGATAAATTAACTC Y.FAST- pILGFP4Q GAGTAAACCAATTTTATTTGAACTTGC 89 EVBR1.2A- PSYKSKGAL2pa CTTACCTTCTTCAATTTTCATTTTGGATCCA Ec.MBP- CTGTAAAAAACTTTTTTTATTATAC 90 Linker~SacI~ Y.FAST- PTSYFASTs GTATAATAAAAAAAGTTTTTTACAGTGGAT 6*G- EVBR1.2A CCAAAATGGAACACGTTGCTTTCG ERG20.sup.F96W .sup.N127W-T.sub.RPL3 from pJT9RFR 91 PITYAFST2Aa CCAACTTACCTTCTTCAATTTTTGGACCTG GGTTAAGTTCAAC 92 PITYFAST-MBPS GCTGGTGACGTTGAACTTAACCCAGGTCC AAAAATTGAAGAAGGTAAGTTGG 93 Ec.MPB PTSMBPa ACCACCACCACCACCACCGAGCTCACCAG (codon- AACCTGGCTTAGTGATTCTAGTTTGGGCA optimized) TC 94 ERG20.sup.F96W PTSERG20s CCAGGTTCTGGTGAGCTCGGTGGTGGTG N.sup.127Wpart1 GTGGTGGTGCTTCAGAAAAAGAAATTAGG frompJT11 AG 95 Erg20F96Wa CATATCATCGGCGACCAACCAGTAAGCCT GCAACAAC 96 ERG20.sup.F96W Erg20F96Ws GTTGTTGCAGGCTTACTGGTTGGTCGCCG .sup.N127Wpart2 ATGATATG frompJT11 97 GA_RPL3t_URA3a AAATCATTACGACCGAGATTCCCGGGATT GTAGCAAAGATTGTAAGG 98 LI.LSfrom GA_MBP_LMSS ATCACTAAGCCAGGTTCTGGTTCTGGTAG pJT11 AAGATCAGCTAACTATCAACCATCC 99 GA_LMS_6Ga GAAGCACCACCACCACCACCACCACCCTT TGTACCTGGTGATGCG 100 P.sub.BTS1-RPL25 PMIRPL25BckBns TTAGCTTATTCTGAGGTTTCTGTGGCGTG (Arm2)- 101 pUC19from PMIRPL25BckBna TCCGGGGTGTTAGACTGATTAGTACATGT pILGFP3AA5 TT 102 P.sub.ALD6from PPGALD6ps AAGGGTTGCTCGAGAAAGAGCTC SGD CATATGGCGTATCCAAGCC 103 PPGALD6pa1 CACAAACACATACTATCAGAATACAGGAT CCAAAATGTCTAAAGGTGAAGAATTATTCA 104 P.sub.ALD6- PILEforReds CATTACTTCATGAGATAAATTAACTCGAG AeBlue-T.sub.PGK1 CATATGGCGTATCCAAGCC 105 (.sub.PALD6- PILEforReda AAATCATTACGACCGAGATTCCCGGG EforRed- AAATAATATCCTTCTCGAAAGC T.sub.PGK1) 106 P.sub.Se.GAL2- P.sub.Se.GAL2from PHPVSeGAL2ps GCTTTCGAGAAGGATATTATTTCCCGGGC HPV16L1AC1 pILGFP4M CACAGAGAACAGGAGATTAC 4-6*H- T.sub.RPL41B 107 PHPVSeGAL2pa AGATGGCAACCACAAAGACATTTTGTCGA CTGTAAATGTGTGTATATATTATATTATAG 108 HPV16L1AC1 PHPVHPV16Ls CTATAATATAATATATACACACATTTACAG 4-6*H TCGACAAAATGTCTTTGTGGTTGCCATCT (codon optimized) fromgBlock 109 PHPVHPV16La TCCGCCCTGCAGGTCACTATTAATGATGG TGATGGTGGTGAGCAGTTGTAGAGGTAGA AG 110 T.sub.RPL41Bfrom PHPVRPL41Bts ACTGCTCACCACCATCACCATCATTAATAG SGD TGACCTGCAGGGCGGATTGAGAGCAAATC G 111 PHPVRPL41Bta GCATGCAAATCATTACGACCGAGATTGCC GGCACGCCACAGAAACCTCAGAAT 112 P.sub.ALD6- PHPVALD6ps GGGCGAATTGGGTACCGGGCCC AeBlue- CATATGGCGTATCCAAGCCG T.sub.PGK1- PSe..sub.GAL2- HPV16L1C1 4-6*H- T.sub.RPL41B 113 PHPVRPL41Bta CACTAAAGGGAACAAAAGCTGGAGCTC CGCCACAGAAACCTCAGAAT HPV16L1C2 PHPVHPV16Ls Asabove 2-6*H 114 PHPVHPV16aad GCCCTGCAGGTCACTATTAATGATGGTGA a TGGTGGTGACCCAAAGTGAACTTTGGCTT AG 115 PHPVHPV16a GATTTGCTCTCAATCCGCCCTGCAGGTCA CTATTA 116 Removing PMIRPL25ta CCTCACGAAGAAGTTAAGCTTGAGCATCG ARSin GACCGAAGCATAAG Construct3 117 PMITEF1s ATTACTTCATGAGATAAATTAACCTGCAGG CGTATAAACAATGCATACTTTGTAC
TABLE-US-00005 TABLE 5 Construction of the plasmids used in this work. Numbers refer to DNA fragments listed in Table 4. Plasmid Construction process pILGFP1D5 Fragment T.sub.PGK1 (#1) was cloned into SpeI of pILGFP3 through Gibson Assembly to generate plasmid pILGFP1D5 pILGFP5A3 Fragment P.sub.YEF3 (#2) was cloned into BamHI site of plasmid pILGFP1D5 through Gibson Assembly to generate plasmid pILGFP5A3, and: pILGFP1A6 Fragment P.sub.RPL25 (#3) to generate plasmid pILGFP1A6 pILGFP1C6 Fragment P.sub.SEC23 (#4) to generate plasmid pILGFP1C6 pILGFP1E6 Fragment P.sub.PDA1 (#5) to generate plasmid pILGFP1E6 pILGFP1E7 Fragment P.sub.ERG1 (#6) to generate plasmid pILGFP1E7 pILGFP1G7 Fragment P.sub.BTS1 (#7) to generate plasmid pILGFP1G7 pILGFP4F5 Fragment P.sub.COG7 (#8) to generate plasmid pILGFP4F5 pILGFP4H5 Fragment P.sub.GLO2 (#9) to generate plasmid pILGFP4H5 pILGFP6G3 Fragment P.sub.RPL33A (#20) to generate plasmid pILGFP6G3 pILGFP6A4 Fragment P.sub.RPS15 (#21) to generate plasmid pILGFP6A4 pILGFP6C4 Fragment P.sub.RPC10 (#22) to generate plasmid pILGFP6C4 pACT1-GFP Fragment P.sub.ACT1 (#23) to generate plasmid pACT1-GFP pILGFP6G4 Fragment P.sub.NIP1 (#24) to generate plasmid pILGFP6G4 pILGFP6A5 Fragment P.sub.RPS13 (#25) to generate plasmid pILGFP6A5 pILGFP6C5 Fragment P.sub.NUS1 (#26) to generate plasmid pILGFP6C5 pILGFP6E5 Fragment P.sub.SMC1 (#27) to generate plasmid pILGFP6E5 pILGFP6G5 Fragment P.sub.RNA14 (#28) to generate plasmid pILGFP6G5 pILGFP6A6 Fragment P.sub.RPB7 (#29) to generate plasmid pILGFP6A6 pILGFP6C6 Fragment P.sub.SPC97 (#30) to generate plasmid pILGFP6C6 pILGFP6E6 Fragment P.sub.STH1 (#31) to generate plasmid pILGFP6E6 pILGFP6G6 Fragment P.sub.ARP7 (#32) to generate plasmid pILGFP6G6 pILGFP6A7 Fragment P.sub.TAF61 (#33) to generate plasmid pILGFP6A7 pILGFP6C7 Fragment P.sub.RPN11 (#34) to generate plasmid pILGFP6C7 pILGFP1DFB Fragment P.sub.RPL25-KI.LEU2-T.sub.KI.LEU-T.sub.RPL25 (#10) was cloned into EcoRI/XbaI sites of pILGFP89 through Gibson assembly to generate plasmid pILGFP1DFB pILGFP3A5C Fragment P.sub.YEF3-RPL25 (Arm 2) (#11) was cloned into SphI site of plasmid pILGFP1DFB through Gibson assembly to generate plasmid pILGFP3A5C, and: pILGFP3AE4 Fragment P.sub.ERG1-RPL25 (Arm 2) (#12) to generate pILGFP3AE4 pILGFP3AG4 Fragment P.sub.PDA1-RPL25 (Arm 2) (#13) to generate pILGFP3AG4 pILGFP3AA5 Fragment P.sub.PST1-RPL25 (Arm 2) (#14) to generate pILGFP3AA5 pILGFP3AG4ARSd pILGFP3AG4 was used as the template to amplify fragment #46, which was self-ligated to generate plasmid pILGFP3AG4ARSd. pILGFP4BG6 Fragment P.sub.SEC23-hphMX-T.sub.SEC23-ARSMax1 (#15) was cloned into EcoRI/XbaI sites of pILGFP89 through Gibson assembly to generate plasmid pILGFP4BG6 pILGFP5EG3 Fragment P.sub.ERG1-SEC23 (Arm 2) (#16) was cloned into SphI site of plasmid pILGFP4BG6 through Gibson assembly to generate plasmid pILGFP5EG3, and: pILGFP5EA4 Fragment P.sub.GLO2-SEC23 (Arm 2) (#17) to generate plasmid pILGFP5EA4 pILGFP5EC4 Fragment P.sub.COG7-SEC23 (Arm 2) (#18) to generate plasmid pILGFP5EC4 pILGFP5EF3 Fragment P.sub.COG7-3G-SEC23 (Arm 2) (#19) to generate plasmid pILGFP5EC4 pINER2R Step 1: Fragment P.sub.GAL1-ERG20-P.sub.RPL3 (#35) was cloned into ApaI site of plasmid pILGFP3AE4 through Gibson assembly to generate plasmid pITinter1. Step 3: Fragment P.sub.GAL2-Y.FAST-EVBR1.2A-AcNES1-T.sub.RPL41B (#36) was cloned into SacI/Xmal sites of plasmid pITinter1 through Gibson assembly to generate pINER2R pINER3R Step 1: Fragment P.sub.GAL1-ERG20-P.sub.RPL3 (#35) was cloned into ApaI site of plasmid pILGFP3AG4 through Gibson assembly to generate plasmid pITinter2. Step 3: Fragment P.sub.GAL2-Y.FAST-EVBR1.2A-AcNES1-T.sub.RPL41B (#36) was cloned into SacI/XmaI sites of plasmid pITinter2 through Gibson assembly to generate pINER3R pINER4R Step 1: Fragment P.sub.GAL1-ERG20-P.sub.RPL3 (#35) was cloned into ApaI site of plasmid pILGFP3AA5 through Gibson assembly to generate plasmid pITinter3. Step 3: Fragment P.sub.GAL2-Y.FAST-EVBR1.2A-AcNES1-T.sub.RPL41B (#36) was cloned into SacI/XmaI sites of plasmid pITinter3 through Gibson assembly to generate pINER3R pIT6EG7m Fragment P.sub.Sk.GAL2-Y.FAST-EVBR1.2A-Ec.MBP-Linker~SacI~6*G-ERG20.sup.F96W N127W- T.sub.RPL3 (#37) was cloned into XhoI/XmaI sites of pILGFP3AG4 to generate pIL6EG7m pIT6EG7ml Fragment LI.LS (#38) was cloned into XhoI/XmaI sites of pILGFP3AG4 through Gibson assembly to generate pIL6EG7ml pIT6EG7mlh Fragment P.sub.BTS1-RPL25 (Arm2)-pUC19 (#39) was assembled with the larger fragment of PmeI/SmaI-digested plasmid pIT6EG7ml to generate plasmid pIT6EG7mlh pPT6EG7ml P.sub.Sk.GAL2 > Y.FAST-EVBR1.2A-Ec.MBP-Linker~SacI~6*G-ERG20.sup.F36W N127W > T.sub.RPL3 was cut out from pIT6EG7ml with XhoI and XmaI and cloned into XhoI/XmaI sites in pRS425 to generate pPT6EG7ml. pILAC2 (or pILAC3) Step 1: plasmid pLAC1 was digested with NotI, and then mung bean nuclease; and further purified through a PCR clean-up kit. Step 2: Step 1 product was digested with EcoRI and XmaI, and the larger fragment was purified through a Gel-cutting purification kit. Step 3: plasmid pILGFP3AG4 (or pILGFP3AA5) was digested with XhoI, plasmid pLacI was digested with NotI, and then mung bean nuclease; and further purified through a PCR clean-up kit. Step 4: Step 3 product was digested with XmaI, and the larger fragment was purified through a Gel-cutting purification kit. Step 5: Step 2 product and Step 4 product were ligated to generate pILAC2 (or pILAC3). pIAeBlue (or Step 1: Fragment P.sub.ALD6 (#40) was cloned into BamHI site of plasmid pIEforRed) pILGFP1D5 through Gibson Assembly to generate plasmid pILGFP4D2. Step 2: gBlock fragment AeBlue (or EforRed) with codon usage optimized was cloned into BamHI/Bg/II sites of plasmid pILGFP4D2 through Gibson Assembly to generate plasmid pILAeBlue (or pILEforRed) Step 3: Fragment P.sub.ALD6-AeBlue-T.sub.PGK1 (#41) (or P.sub.ALD6-EforRed-T.sub.PGK1; #42) was amplified from pILAeBlue (or pILEforRed) and cloned into XhoI/XmaI sites of pILGFP3AA5 through Gibson assembly to generate pIAeBlue (or pIEforRed). pIAeBlueHPV16LR Step 1: Fragment P.sub.Se.GAL2-HPV16L1C14-6*H-T.sub.RPL41B (#43) was cloned into SmaI site of plasmid pIAeBlue to generate pIAeBlueHPV16L. Step 2: Fragment HPV16L1C22-6*H (#45) was cloned Sa/I/SbfI sites of pIAeBlueHPV16L to generate pIAeBlueHPV16LR. pPAeBlueHPV16LR Step 1: Fragment P.sub.ALD6-AeBlue-TPGK1-PSe.sub..GAL2-HPV16L1C14-6*H-T.sub.RPL41B (#44) amplified from pIAeBlueHPV16L was cloned into ApaI/SacI sites of plasmid pRS425 to generate pPAeBlueHPV16L. Step 2: Fragment HPV16L1C22-6*H (#45) was cloned Sa/I/SbfI sites of pPAeBlueHPV16L to generate pPAeBlueHPV16LR.
TABLE-US-00006 TABLE 6 Construction of the ILHA series strains used in this work. Plasmids refer to Table S1. DNA fragments refer to Table S3. Strain Construction process G5A3 Plasmid pILGFP5A3 digested with SwaI was transformed into CEN.PK113-5D to generate strain G5A3, and: G1A6 pILGFP1A6 to generate strain G1A6 G1C6 pILGFP1C6 to generate strain G1C6 G1E6 pILGFP1E6 to generate strain G1E6 G1E7 pILGFP1E7 to generate strain G1E7 G1G7 pILGFP1G7 to generate strain G1G7 G4F5 pILGFP4F5 to generate strain G4F5 G4H5 pILGFP4H5 to generate strain G4H5 G6G3 pILGFP6G3 to generate strain G6G3 G6A4 pILGFP6A4 to generate strain G6A4 G6C4 pILGFP6C4 to generate strain G6C4 G6E4 pILGFP6E4 to generate strain ACT1-GFP G6G4 pILGFP6G4 to generate strain G6G4 G6A5 pILGFP6A5 to generate strain G6A5 G6C5 pILGFP6C5 to generate strain G6C5 G6E5 pILGFP6E5 to generate strain G6E5 G6G5 pILGFP6G5 to generate strain G6G5 G6A6 pILGFP6A6 to generate strain G6A6 G6C6 pILGFP6C6 to generate strain G6C6 G6E6 pILGFP6E6 to generate strain G6E6 G6G6 pILGFP6G6 to generate strain G6G6 G6A7 pILGFP6A7 to generate strain G6A7 G6C7 pILGFP6C7 to generate strain G6C7 G3A5C pILGFP3A5C to generate strain G3A5C G3AE4 pILGFP3AE4 to generate strain G3AE4 G3AG4 pILGFP3AG4 to generate strain G3AG4 G3AA5 pILGFP3AA5 to generate strain G3AA5 G5EG3 pILGFP5EG3 to generate strain G5EG3 G5EA4 pILGFP5EA4 to generate strain G5EA4 G5EC4 pILGFP5EC4 to generate strain G5EC4 G5EF3 pILGFP5EF3 to generate strain G5EF3 o401UR Plasmid pIR3DH8 digested by PmeI was transformed into strain o401R to generate strain o401UR N401-1 Plasmid pJT9RFR was transformed into strain o401UR to generate strain N401-1 N401-2 Plasmid pINER2R digested by PmeI was transformed into strain o401UR to generate strain N401-2 N401-3 Plasmid pINER3R digested by PmeI was transformed into strain o401UR to generate strain N401-3 N401-4 Plasmid pINER4R digested by PmeI was transformed into strain o401UR to generate strain N401-4 LIM141R/ o141R derivative; LIM141R2 [pPT6EG7ml] LIM141M Plasmid pIT6EG7ml digested by PmeI was transformed intro strain o141R to generate strain N141M LIM141MH Plasmid pIT6EG7mlh digested by PmeI was transformed intro strain o141R to generate strain N141MH LAC4 Plasmid pILAC2 digested by PmeI was transformed into strain o401UR to generate strain LAC4 LAC5 Plasmid pILAC3 digested by PmeI was transformed into strain o401UR to generate strain LAC5 16BJ3 Plasmid pIR3DH8 digested by PmeI was transformed into strain CEN.PK113- 16B to generate strain 16BJ3 16BJ3C Plasmid pRS425 was transformed into strain 16BJ3 to generate strain 16BJ3C 16BJ3AeBlue Plasmid pIAeBlue digested by PmeI was transformed into strain 16BJ3 to generate strain 16BJ3AeBlue HPV16LPR Plasmid pPAeBlueHPV16L1R was transformed into strain 16BJ3 to generate strain HPV16LPR HPV16LMR Plasmid pIAeBlueHPV16L1R digested by PmeI was transformed into strain 16BJ3 to generate strain HPV16LPR
[0271] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.
[0272] The citation of any reference herein should not be construed as an admission that such reference is available as Prior Art to the instant application.
[0273] Throughout the specification the aim has been to describe the preferred embodiments of the disclosure without limiting the disclosure to any one embodiment or specific collection of features. Those of skill in the art will therefore appreciate that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present disclosure. All such modifications and changes are intended to be included within the scope of the appended claims.