BIOSYNTHETIC PRODUCTION OF UDP-RHAMNOSE
20220090158 · 2022-03-24
Assignee
Inventors
- Guohong Mao (Burlington, MA, US)
- Phillip James Hunt (Tewksbury, MA, US)
- Michael James Batten (Westford, MA, US)
- Timothy Joseph Hanley (Waltham, MA, US)
- Oliver Yu (Lexington, MA, US)
Cpc classification
C12Y501/03002
CHEMISTRY; METALLURGY
C12P19/56
CHEMISTRY; METALLURGY
C12Y101/01133
CHEMISTRY; METALLURGY
International classification
C12P19/56
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to the biosynthesis of UDP-Rhamnose and recombinant polypeptides having enzymatic activity useful in the relevant biosynthetic pathways for producing UDP-Rhamnose. The present invention also provides a method for preparing a steviol glycoside composition comprising at least one rhamnose-containing steviol glycoside.
Claims
1. A biosynthetic method of preparing uridine diphosphate-rhamnose (UDP-rhamnose) from uridine diphosphate-glucose (UDP-glucose), the method comprising incubating UDP-glucose with one or more recombinant polypeptides having UDP-rhamnose synthase activity in the presence of NAD+ and a source of NADPH for a sufficient time to produce UDP-rhamnose.
2. The method of claim 1, wherein the one or more recombinant polypeptides comprise a first recombinant polypeptide that is a trifunctional enzyme having UDP-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-glucose 3,5-epimerase, and UDP-4-keto-rhamnose 4-keto-reductase activities.
3. The method of claim 1, wherein the one or more recombinant polypeptides comprise a first recombinant polypeptide that is a fusion enzyme comprising a first domain having UDP-glucose 4,6-dehydratase activity and a second domain having UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-keto-rhamnose 4-keto-reductase activities.
4. The method of claim 1, wherein the one or more recombinant polypeptides comprise a first recombinant polypeptide having UDP-glucose 4,6-dehydratase activity and a second recombinant polypeptide having UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-keto-rhamnose 4-keto-reductase activities.
5. The method of claim 3, wherein the first domain of the fusion enzyme comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7 or SEQ ID NO: 31.
6. The method of claim 5, wherein the second domain of the fusion enzyme comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 61, or SEQ ID NO: 63.
7. The method of claim 6, wherein the fusion enzyme comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, SEQ ID NO: 83, SEQ ID NO: 85, or SEQ ID NO: 87.
8.-12. (canceled)
13. The method of claim 1, wherein the one or more recombinant polypeptides comprise a first recombinant polypeptide that is a fusion polypeptide coded by a nucleotide resulting from the fusion between a first nucleotide coding for a UDP-glucose 4,6-dehydratase enzyme and a second nucleotide coding for a bifunctional enzyme having UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-keto-rhamnose 4-keto-reductase activities.
14.-17. (canceled)
18. The method of claim 2, wherein the trifunctional enzyme comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 5.
19. The method of claim 4, wherein the first recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, or SEQ ID NO: 37, and/or wherein the second recombinant polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO: 63, or SEQ ID NO: 71.
20. (canceled)
21. The method of claim 1, comprising expressing said one or more recombinant polypeptides in a transformed cellular system.
22.-29. (canceled)
30. The method of claim 1, wherein the uridine diphosphate-glucose and the one or more recombinant polypeptides are incubated with sucrose and a third recombinant polypeptide having sucrose synthase activity.
31. (canceled)
32. A biosynthetic method of preparing a steviol glycoside composition comprising at least one rhamnose-containing steviol glycoside, the method comprising: (a) incubating a substrate selected from the group consisting of sucrose, uridine diphosphate and uridine diphosphate-glucose, with one or more recombinant polypeptides having UDP-rhamnose synthase activity in the presence of NAD+ and a source of NADPH to produce uridine diphosphate-rhamnose; and (b) reacting the uridine diphosphate-rhamnose with a steviol glycoside substrate in the presence of a recombinant polypeptide having rhamnosyltransferase activity, so that a rhamnose moiety is coupled to the steviol glycoside substrate to produce at least one rhamnose-containing steviol glycoside.
33. The method of claim 32, wherein the steviol glycoside substrate is rebaudioside A.
34. The method of claim 32, wherein the steviol glycoside composition comprises rebaudioside N, rebaudioside J, or both.
35. The method of claim 32, further comprises reacting the rhamnose-containing steviol glycoside in the presence of a recombinant polypeptide having glycosyltransferase activity, so that a glucose moiety is coupled to the rhamnose-containing steviol glycoside.
36. The method of claim 32, wherein the substrate comprises uridine diphosphate-glucose.
37. The method of claim 36, wherein the uridine diphosphate-glucose substrate is provided in situ by reacting sucrose and uridine diphosphate in the presence of a sucrose synthase.
38. A nucleic acid comprising a sequence encoding a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO. 13, SEQ ID NO. 83, SEQ ID NO. 85 or SEQ ID NO. 87.
39. A cell comprising the nucleic acid of claim 38.
40.-42. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0043] As used herein, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.
[0044] To the extent that the term “include,” “have,” or the like is used in the description or the claims, such term is intended to be inclusive in a manner similar to the term “comprise” as “comprise” is interpreted when employed as a transitional word in a claim.
[0045] The word “exemplary” is used herein to mean serving as an example, instance, or illustration. Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.
[0046] Cellular system is any cells that provide for the expression of ectopic proteins. It included bacteria, yeast, plant cells and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes the in vitro expression of proteins based on cellular components, such as ribosomes.
[0047] Coding sequence is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to refer to a DNA sequence that encodes for a specific amino acid sequence.
[0048] The term “growing the cellular system” means providing an appropriate medium that would allow cells to multiply and divide. It also includes providing resources so that cells or cellular components can translate and make recombinant proteins.
[0049] Protein expression can occur after gene expression. It consists of the stages after DNA has been transcribed to messenger RNA (mRNA). The mRNA is then translated into polypeptide chains, which are ultimately folded into proteins. DNA is present in the cells through transfection—a process of deliberately introducing nucleic acids into cells. The term is often used for non-viral methods in eukaryotic cells. It may also refer to other methods and cell types, although other terms are preferred: “transformation” is more often used to describe non-viral DNA transfer in bacteria, non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated DNA transfer. Transformation, transduction, and viral infection are included under the definition of transfection for this application.
[0050] According to the current disclosure, a yeast as claimed herein are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. Yeasts are unicellular organisms which evolved from multicellular ancestors but with some species useful for the current disclosure being those that have the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudo hyphae or false hyphae.
[0051] The names of the UGT enzymes used in the present disclosure are consistent with the nomenclature system adopted by the UGT Nomenclature Committee (Mackenzie et al., “The UDP glycosyltransferase gene super family: recommended nomenclature updated based on evolutionary divergence,” P
[0052] The term “complementary” is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the subjection technology also includes isolated nucleic acid fragments that are complementary to the complete sequences as reported in the accompanying Sequence Listing as well as those substantially similar nucleic acid sequences.
[0053] The terms “nucleic acid” and “nucleotide” are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
[0054] The term “isolated” is to be given its ordinary and customary meaning to a person of ordinary skill in the art, and when used in the context of an isolated nucleic acid or an isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid or polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a transgenic host cell.
[0055] The terms “incubating” and “incubation” as used herein means a process of mixing two or more chemical or biological entities (such as a chemical compound and an enzyme) and allowing them to interact under conditions favorable for producing one or more chemical or biological entities which are distinctly different from the initial starting entities.
[0056] The term “degenerate variant” refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxy inosine residues. A nucleic acid sequence and all of its degenerate variants will express the same amino acid or polypeptide.
[0057] The terms “polypeptide,” “protein,” and “peptide” are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art; the three terms are sometimes used interchangeably and are used without limitation to refer to a polymer of amino acids, or amino acid analogs, regardless of its size or function. Although the term “protein” is often used in reference to relatively large polypeptides, and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term “polypeptide” as used herein refers to peptides, polypeptides, and proteins, unless otherwise noted. The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein when referring to a polynucleotide product. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing.
[0058] The terms “polypeptide fragment” and “fragment,” when used in reference to a reference polypeptide, are to be given their ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both.
[0059] The term “functional fragment” of a polypeptide or protein refers to a peptide fragment that is a portion of the full-length polypeptide or protein, and has substantially the same biological activity, or carries out substantially the same function as the full-length polypeptide or protein (e.g., carrying out the same enzymatic reaction).
[0060] The terms “variant polypeptide,” “modified amino acid sequence” or “modified polypeptide,” which are used interchangeably, refer to an amino acid sequence that is different from the reference polypeptide by one or more amino acids, e.g., by one or more amino acid substitutions, deletions, and/or additions. In an aspect, a variant is a “functional variant” which retains some or all of the ability of the reference polypeptide.
[0061] The term “functional variant” further includes conservatively substituted variants. The term “conservatively substituted variant” refers to a peptide having an amino acid sequence that differs from a reference peptide by one or more conservative amino acid substitutions and maintains some or all of the activity of the reference peptide. A “conservative amino acid substitution” is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one charged or polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; the substitution of one basic residue such as lysine or arginine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another; or the substitution of one aromatic residue, such as phenylalanine, tyrosine, or tryptophan for another. Such substitutions are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. The phrase “conservatively substituted variant” also includes peptides wherein a residue is replaced with a chemically-derivatized residue, provided that the resulting peptide maintains some or all of the activity of the reference peptide as described herein.
[0062] The term “variant,” in connection with the polypeptides of the subject technology, further includes a functionally active polypeptide having an amino acid sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identical to the amino acid sequence of a reference polypeptide.
[0063] The term “homologous” in all its grammatical forms and spelling variations refers to the relationship between polynucleotides or polypeptides that possess a common evolutionary origin, including polynucleotides or polypeptides from super families and homologous polynucleotides or proteins from different species (Reeck et al., C
[0064] “Suitable regulatory sequences” is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
[0065] “Promoter” is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. 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 comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters, which cause a gene to be expressed in most cell types at most times, are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
[0066] The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it can affect the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
[0067] The term “expression” as used herein, is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is used without limitation to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the subject technology. “Over-expression” refers to the production of a gene product in transgenic or recombinant organisms that exceeds levels of production in normal or non-transformed organisms.
[0068] “Transformation” is to be given its ordinary and customary meaning to a person of reasonable skill in the craft and is used without limitation to refer to the transfer of a polynucleotide into a target cell. The transferred polynucleotide can be incorporated into the genome or chromosomal DNA of a target cell, resulting in genetically stable inheritance, or it can replicate independent of the host chromosomal. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “transformed”.
[0069] The terms “transformed,” “transgenic,” and “recombinant,” when used herein in connection with host cells, are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to a cell of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host cell, or the nucleic acid molecule can be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or subjects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
[0070] The terms “recombinant,” “heterologous,” and “exogenous,” when used herein in connection with polynucleotides, are to be given their ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to a polynucleotide (e.g., a DNA sequence or a gene) that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of site-directed mutagenesis or other recombinant techniques. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position or form within the host cell in which the element is not ordinarily found.
[0071] Similarly, the terms “recombinant,” “heterologous,” and “exogenous,” when used herein in connection with a polypeptide or amino acid sequence, means a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, recombinant DNA segments can be expressed in a host cell to produce a recombinant polypeptide.
[0072] The terms “plasmid,” “vector,” and “cassette” are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art and are used without limitation to refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell. “Transformation cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. “Expression cassette” refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
[0073] The present disclosure relates, in some embodiments, to the biosynthetic production of UDP-rhamnose. In a preferred embodiment, the present invention relates to the production of UDP-L-rhamnose, the chemical structure of which is shown in
[0074] Referring to
[0075] UDP-rhamnose biosynthetic pathway in plants and fungi involves three different enzymes. In the first step of this biosynthetic pathway, UDP-glucose 4,6 dehydratase (“DH”) converts UDP-glucose into UDP-4-keto-6-deoxy glucose (UDP4K6G). In the second step of this biosynthetic pathway, the enzyme UDP-4-keto-6-deoxy-glucose 3,5 epimerase converts UDP-4-keto-6-deoxy glucose into UDP-4-keto rhamnose. At the third enzymatic step in this biosynthetic pathway, UDP-4-keto rhamnose-4-ketoreductase convert UDP-4-keto rhamnose in to UDP-rhamnose. In various embodiments, the present invention provides trifunctional recombinant polypeptides having UDP-glucose 4,6-dehydratase, UDP-4-keto-6-deoxy-glucose 3,5-epimerase, and UDP-4-keto-rhamnose 4-keto-reductase activities. Such a trifunctional polypeptide is also referred as RHM enzyme. Since the trifunctional recombinant polypeptides exhibit three different enzyme functions, this trifunctional recombinant protein is also referred as multi-enzyme protein.
[0076] In certain embodiments, the present invention provides recombinant polypeptide having only the activity of the UDP-glucose 4,6-dehydratase enzyme and that recombinant polypeptide is referred herein as the “DH” (dehydratase) polypeptide. In another embodiment, the present invention provides bifunctional recombinant polypeptide having both UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-keto-rhamnose 4-keto-reductase activities. This bifunctional recombinant polypeptide is referred herein as the “ER” (the letter “E” standing for epimerase activity and the letter “R” standing for reductase activity). In yet another embodiment, the present invention provides a recombinant fusion polypeptide wherein an enzyme having UDP-glucose 4,6-dehydratase activity (the DH polypeptide) is fused with a bifunctional ER polypeptide having both UDP-4-keto-6-deoxy-glucose 3,5-epimerase and UDP-4-keto-rhamnose 4-keto-reductase activities. Such a fusion polypeptide is found to have the capacity to catalyze the conversion of UDP-glucose to UDP-rhamnose.
[0077] The cofactor NAD.sup.+ is needed in the DH-catalyzed step and the cofactor NADPH is needed in the second of the ER-catalyzed step.
[0078] Referring to Table 1, the inventors have identified various trifunctional UDP-rhamnose synthase for the bioconversion of UDP-glucose to UDP-rhamnose. As shown in
[0079] In some embodiments, the present disclosure relates to a biosynthetic method for preparing UDP-rhamnose by incubating a substrate such as UDP-glucose with an artificial fusion enzyme obtained from the fusion of a high activity DH enzyme and a high activity ER enzyme. DH and ER enzymes can be obtained from a variety of sources as shown in the Examples below and their activities can be determined using biochemical assays. The nucleic acid sequence coding for a selected DH enzyme can be fused with the nucleic acid coding for a selected ER enzyme using the recombinant technologies well-known to a person skilled in the art to generate a recombinant fusion peptide catalyzing the synthesis of UDP-rhamnose from UDP-glucose. The DH enzyme and the ER enzyme can be coupled via a peptide linker. In various embodiments, the peptide linker can comprise 2-15 amino acids. Exemplary linkers include those comprising glycine and serine. In preferred embodiments, the DH enzyme and the ER enzyme can be coupled via a GSG linker (Table 3).
[0080] In various embodiments, UDP-glucose can be prepared in situ from UDP and sucrose in the presence of a sucrose synthase (SUS). For example, the SUS can have an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 15.
[0081] As shown in
[0082] With continued reference to
[0083]
[0084]
[0085] Part of the present disclosure relates to the production of rhamnose-containing steviol glycosides using UDP-Rhamnose as the rhamnose donor moiety. Referring back to
EXAMPLES
Example 1
Enzymatic Activity Screening of UDP-Rhamnose Synthase Enzymes
[0086] Phylogenetic, gene cluster, and protein BLAST analyses were used to identify candidate UDP-rhamnose synthase (“RHM”) genes for producing UDP-Rhamnose from UDP-glucose. Full-length DNA fragments of all candidate RHM genes were optimized and synthesized according to the codon preference of E. coli (Gene Universal, DE). The synthesized DNA fragments were cloned into a bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen).
[0087] Each expression construct was transformed into E. coli BL21 (DE3), which was subsequently grown in LB media containing 50 μg/mL kanamycin at 37° C. until reaching an OD.sub.600 of 0.8-1.0. Protein expression was induced by adding 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture was incubated further at 16° C. for 22 hours. Cells were harvested by centrifugation (3,000×g; 10 min; 4° C.). The cell pellets were collected and were either used immediately or stored at −80° C.
[0088] The cell pellets typically were re-suspended in lysis buffer (50 mM potassium phosphate buffer, pH 7.2, 25 μg/ml lysozyme, 5 μg/ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol, and 0.4% Triton X-100). The cells were disrupted by sonication at 4° C., and the cell debris was clarified by centrifugation (18,000×g; 30 min). The supernatant was loaded to an equilibrated (equilibration buffer: 50 mM potassium phosphate buffer, pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen) affinity column. After loading of the protein samples, the column was washed with equilibration buffer to remove unbound contaminant proteins. The His-tagged RHM recombinant polypeptides were eluted with an equilibration buffer containing 250 mM of imidazole.
[0089] The purified candidate RHM recombinant polypeptides were assayed for UDP-rhamnose synthase activity by using UDP-glucose as substrate. Typically, the recombinant polypeptide (20-50 μg) was tested in a 200 μl in vitro reaction system. The reaction system contains 50 mM potassium phosphate buffer, pH 8.0, 3 mM MgCl.sub.2, 3-6 mM UDP-glucose, 1-3 mM NAD.sup.+, 1 mM DTT and 1-3 mM NADPH. The reaction was performed at 30-37° C. and reaction was terminated by adding 200 μL chloroform. The samples were extracted with same volume chloroform by vertex for 10 mins. The supernatant was collected for high-performance liquid chromatography (HPLC) analysis after 10 mins centrifugation.
[0090] HPLC analysis was then performed using an Agilent 1200 system (Agilent Technologies, CA), including a quaternary pump, a temperature-controlled column compartment, an auto sampler and a UV absorbance detector. The chromatographic separation was performed using Dionex Carbo PA10 column (4×120 mm, Thermo Scientific) with mobile phase delivered at a flow rate of 1 ml/min. The mobile phase was H.sub.2O (MPA) and 700 mM ammonium acetate (pH 5.2) (MPB). The gradient concentration of MPB was programmed for sample analysis. The detection wavelength used in the HPLC analysis was 261 nm. After activity screening, three RHM enzymes (NR12, NR32 and NR33) were identified as candidates for bioconversion of UDP-glucose to UDP-rhamnose (Table 1).
[0091] The activities of three different RHM enzymes namely NR12, NR32 and NR33 were studied for three different time period (3 hours, 6 hours and 18 hours). The enzyme activities at the end of three hours are shown in the top panel (A) of
[0092] With continued reference to
[0093] In addition, the inventors also found that a two-step cofactor addition approach can enhance the conversion efficiency, indicating that later NADPH addition can avoid the negative feedback regulation of UDP-rhamnose on DH enzyme. In the two-step cofactors addition process, NAD.sup.+ was added in the initial reaction and NADPH was added in the reaction after 3 hr. As shown in
[0094] These results showed that a two-step cofactor addition approach may be used to enhance the conversion efficiency from UDP-glucose to UDP-rhamnose.
Example 2
Two Step Addition of Cofactors
[0095]
Example 3
Optimization of One-Pot Multi-Enzyme System for In Vitro Synthesis of UDP-Rhamnose
[0096] Sucrose synthase (SUS) can break down a molecule of sucrose to yield a molecule of fructose and a molecule of glucose. In addition, SUS can transfer one glucose to UDP to form UDP-glucose. Therefore, by including sucrose, UDP, and SUS in the feedstock, the required UDP-glucose component in the UDP-rhamnose synthesis pathway disclosed herein can be replenished in the presence of sucrose synthase.
[0097] In addition, NADPH is a critical cofactor of ER activity. In the course of the ER-catalyzed reaction, NADPH is oxidized to NADP.sup.+. By incorporating an NADP.sup.+-dependent oxidation reaction as part of the UDP-rhamnose synthesis disclosed herein, NADPH can be regenerated. Exemplary NADP.sup.+-dependent oxidation reactions include the oxidation of malate into pyruvate, the oxidation of formate into CO.sub.2, and the oxidation of phosphite into phosphate. By including malate, formate, or phosphite and the corresponding enzyme (MaeB, FDH, and PTDH, respectively) that can catalyze each of these oxidation reactions in the feedstock, NADPH is continuously regenerated, further optimizing the overall UDP-rhamnose production yield. Tables 1 provides information about the sequences of various enzymes.
[0098] In this example, six different experiments were performed with varying combinations of starting materials in a one-pot multi-enzyme reaction system using the two-step cofactor addition approach. Table 2 provides the composition of six different reaction systems tested int this experiment.
[0099] In each of the six systems, UDP-glucose was not included. Instead, UDP, sucrose and SUS were provided to produce the required UDP-glucose. Referring to
[0100] The experiments also confirmed the effect of NADPH regeneration in UDP-rhamnose production. With continued reference to
Example 4
Enzymatic Activity Screening of UDP-glucose 4,6-dehydratase
[0101] UDP-glucose 4,6-dehydratase (DH) can catalyze the enzymatic reaction for bioconversion of UDP-glucose (UDPG) to UDP-4-keto-6-deoxy-glucose (UDP4K6G). In order to identify specific DH enzymes, enzyme candidates were selected based on polygenetic and Blast analysis.
[0102] Full length DNA fragments of all candidate DH genes were commercially synthesized. Almost all codons of the cDNA were changed to those preferred for E. coli (Gene Universal, DE). The synthesized DNA was cloned into a bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen).
[0103] Each expression construct was transformed into E. coli BL21 (DE3), which was subsequently grown in LB media containing 50 μg/mL kanamycin at 37° C. until reaching an OD600 of 0.8-1.0. Protein expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the culture was further grown at 16° C. for 22 hr. Cells were harvested by centrifugation (3,000×g; 10 min; 4° C.). The cell pellets were collected and were either used immediately or stored at −80° C.
[0104] The cell pellets typically were re-suspended in lysis buffer (50 mM potassium phosphate buffer, pH 7.2, 25 ug/ml lysozyme, 5 ug/ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol, and 0.4% Triton X-100). The cells were disrupted by sonication under 4° C., and the cell debris was clarified by centrifugation (18,000×g; 30 min). Supernatant was loaded to an equilibrated (equilibration buffer: 50 mM potassium phosphate buffer, pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen) affinity column. After loading of protein sample, the column was washed with equilibration buffer to remove unbound contaminant proteins. The His-tagged DH recombinant polypeptides were eluted by equilibration buffer containing 250 mM imidazole.
[0105] The purified candidate DH recombinant polypeptides were assayed for UDP-4-keto-6-deoxy-glucose synthesis by using UDPG as substrate. Typically, the recombinant polypeptide (20 μg) was tested in a 200 μl in vitro reaction system. The reaction system contains 50 mM potassium phosphate buffer, pH 8.0, 3 mM MgCl.sub.2, 3 mM UDPG, 3 mM NAD.sup.+ and 1 mM DTT. The reaction was performed at 30-37° C. and reaction was terminated by adding 200 μL chloroform. The samples were extracted with same volume chloroform by vertex for 10 mins. The supernatant was collected for high-performance liquid chromatography (HPLC) analysis after 10 mins centrifugation.
[0106] HPLC analysis was then performed using an Agilent 1200 system (Agilent Technologies, CA), including a quaternary pump, a temperature-controlled column compartment, an auto sampler and a UV absorbance detector. The chromatographic separation was performed using Dionex Carbo PA10 column (4×120 mm, Thermo Scientific) with mobile phase delivered at a flow rate of 1 ml/min. The mobile phase was H.sub.2O (MPA) and 700 mM ammonium acetic (pH 5.2) (MPB). The gradient concentration of MPB was programmed for sample analysis. The detection wavelength used in the HPLC analysis was 261 nm.
[0107] After activity screening, 12 novel DH enzymes were identified for bioconversion of UDPG to UDP4K6G (Table 1). As shown in
Example 5
Enzymatic Activity Screening of Bifunctional UDP-4-keto-6-deoxy-Glucose 3,5-epimerase/UDP-4-keto Rhamnose 4-keto Reductase
[0108] Bifunctional UDP-4-keto-6-deoxy-glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) enzymes can convert UDP-4-keto-6-deoxy-glucose to UDP-β-L-rhamnose. In order to identify specific ER enzymes, certain enzyme candidates were selected based on polygenetic and Blast analysis.
[0109] Full length DNA fragments of all candidate ER genes were commercially synthesized. Almost all codons of the cDNA were changed to those preferred for E. coli (Gene Universal, DE). The synthesized DNA was cloned into a bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen).
[0110] Each expression construct was transformed into E. coli BL21 (DE3), which was subsequently grown in LB media containing 50 μg/mL kanamycin at 37° C. until reaching an OD600 of 0.8-1.0. Protein expression was induced by addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the culture was further grown at 16° C. for 22 hr. Cells were harvested by centrifugation (3,000×g; 10 min; 4° C.). The cell pellets were collected and were either used immediately or stored at −80° C.
[0111] The cell pellets typically were re-suspended in lysis buffer (50 mM potassium phosphate buffer, pH 7.2, 25 ug/ml lysozyme, 5 ug/ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol, and 0.4% Triton X-100). The cells were disrupted by sonication under 4° C., and the cell debris was clarified by centrifugation (18,000×g; 30 min). Supernatant was loaded to an equilibrated (equilibration buffer: 50 mM potassium phosphate buffer, pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen) affinity column. After loading of protein sample, the column was washed with equilibration buffer to remove unbound contaminant proteins. The His-tagged ER recombinant polypeptides were eluted by equilibration buffer containing 250 mM imidazole.
[0112] The purified candidate ER recombinant polypeptides were assayed for UDP-rhamnose synthesis by using UDP-4-keto-6-deoxy-glucose (UDP4K6G) as substrate. Typically, the recombinant polypeptide (20 μg) was tested in a 200 μl in vitro reaction system. The reaction system contains 50 mM potassium phosphate buffer, pH 8.0, 3 mM MgCl.sub.2, 3 mM UDP-4-keto-6-deoxy glucose, 3 mM NADPH and 1 mM DTT. The reaction was performed at 30-37° C. and reaction was terminated by adding 200 μL chloroform. The samples were extracted with same volume chloroform by vertex for 10 mins. The supernatant was collected for high-performance liquid chromatography (HPLC) analysis after 10 mins centrifugation.
[0113] HPLC analysis was then performed using an Agilent 1200 system (Agilent Technologies, CA), including a quaternary pump, a temperature-controlled column compartment, an auto sampler and a UV absorbance detector. The chromatographic separation was performed using Dionex Carbo PA10 column (4×120 mm, Thermo Scientific) with mobile phase delivered at a flow rate of 1 ml/min. The mobile phase was H.sub.2O (MPA) and 700 mM ammonium acetic (pH 5.2) (MPB). The gradient concentration of MPB was programmed for sample analysis. The detection wavelength used in the HPLC analysis was 261 nm.
[0114] After activity screening, 17 novel ER enzymes were identified for bioconversion of UDP-4-keto-6-deoxy-glucose to UDP-L-rhamnose (Table 1). As shown in
Example 6
Identify Novel Fusion Enzyme for UDP-Rhamnose Production
[0115] Construction of fusion enzymes by recombinant DNA technology could be useful in obtaining new trifunctional enzymes with UDP-rhamnose synthase activity. However, the fusion of two functional enzymes do not necessarily provide an active fusion enzyme having the activity of both enzyme components. In addition, suitable linkers are often identified only empirically.
[0116] Based on extensive screening of various DH and ER enzyme candidates as well as N-terminal and C-terminal domains of trifunctional RHM enzymes, a series of fusion enzymes with specific DH and ER domains were identified and screened.
[0117] After such further screening, six fusion enzymes were found to have trifunctional activity for bioconversion of UDP-glucose to UDP-rhamnose (Table 3).
[0118] Specifically, five of these fusions enzymes are based on high activity DH enzyme NX10 fused with different ER enzymes (NX5C, NX13, NR5C, NR40C, and NR41C), namely, NRF1 (NX10-NX5C), NRF2 (NX10-NX13), NRF3 (NX10-NR5C), NRF4 (NX10-NR40C), and NRF5 (NX10-NR41C). An additional fusion enzyme with trifunctional activity, NRF7 (NR66N-NR41C), is based on high activity DH enzyme NR66N fused with high activity ER enzyme NR41C. As shown in
Example 7
Combination of UDP-Rhamnose and Steviol Glycoside Production
[0119] As described in commonly-owned International Application No. PCT/US2019/021876, now published as WO2019/178116A1, the inventors have identified various UDP-rhamnosyltransferases (1,2 RhaT) for the biosynthesis of rhamnose-containing steviol glycosides such as Reb J and Reb N. Specifically, Reb J and Reb N can be synthesized from Reb A and UDP-rhamnose.
[0120] Referring to
[0121] In the first step, UDP-glucose was converted to UDP-rhamnose by an RHM enzyme such as NRF1 (SEQ ID NO: 9) through a two-step cofactor addition process. UDP-glucose (6 mM) was fully converted to UDP-4-keto-6-deoxyglucose at 3 hour (
[0122] In the second step, Reb A and a UDP-rhamnosyltransferase such as EUCP1 (SEQ ID NO: 23) were added into the reaction system. The UDP-rhamnosyltransferase enzyme transfers one rhamnose moiety from UDP-rhamnose to the C-2′ of the 19-O-glucose of the Reb A substrate, thereby converting Reb A to Reb J. The level of Reb J was measured at 22-hr. The activity of EUCP1 was confirmed by HPLC, which shows the presence of Reb J (
[0123] In the third step, a UDP-glycosyltransferase enzyme such as CP1 (SEQ ID NO: 25), a sucrose synthase enzyme such as SUS (SEQ ID NO: 15) and sucrose was added into the reaction mixture. The SUS enzyme catalyzed the reaction that produces UDP-glucose and fructose from UDP and sucrose. The CP1 enzyme catalyzed the conversion of Reb J to Reb N, specifically, by transferring one glucosyl moiety from UDP-glucose to the C-3′ of the 19-O-glucose of Reb J to produce Reb N and UDP. The UDP produced was converted back to UDP-glucose by the SUS enzyme in the presence of sucrose for UDP-rhamnose and Reb N production. HPLC analysis confirmed that Reb N was produced from Reb J at 25-hr (
[0124] Based on these results, and referring again to
TABLE-US-00001 TABLE 1 Sequence Information Seq. ID No. Sequence Detail 1 NR12 - Predicted amino acid sequence of UDP-rhamnose synthase from Ricinus communis. 2 NR12 - Predicted nucleic acid sequence of UDP-rhamnose synthase from Ricinus communis. 3 NR32 - Predicted amino acid sequence of UDP-rhamnose synthase from Ceratopteris thalictroides. 4 NR32 - Predicted nucleic acid sequence of UDP-rhamnose synthase from Ceratopteris thalictroides. 5 NR33 - Predicted amino acid sequence of UDP-rhamnose synthase from Azolla filiculoides. 6 NR33 - Predicted nucleic acid sequence of UDP-rhamnose synthase from Azolla filiculoides. 7 NX10 - Amino acid sequence of UDP-glucose 4,6-dehydratase (DH) [Botrytis cinerea] 8 NX10 - Nucleic acid sequence of UDP-glucose 4,6-dehydratase (DH) [Botrytis cinerea] 9 Amino acid sequence of Fusion enzyme NRF1 10 Nucleic acid sequence of Fusion enzyme NRF1 11 Amino acid sequence of Fusion enzyme NRF2 12 Nucleic acid sequence of Fusion enzyme NRF2 13 Amino acid sequence of Fusion enzyme NRF3 14 Nucleic acid sequence of Fusion enzyme NRF3 15 Amino acid sequence of Sucrose synthase SUS [Arabidopsis thaliana] 16 Nucleic Acid sequence of Sucrose synthase SUS [Arabidopsis thaliana] 17 Amino acid sequence of Malic enzyme MaeB [Escherichia coli] 18 Nucleic acid sequence of Malic enzyme MaeB [Escherichia coli] 19 Amino acid sequence of Formate dehydrogenase FDH [Candida boidinii] 20 Nucleic acid sequence of Formate dehydrogenase FDH [Candida boidinii] 21 Amino acid sequence of Phosphite dehydrogenase PTDH [Pseudomonas stutzeri] 22 Nucleic acid sequence of Phosphite dehydrogenase PTDH [Pseudomonas stutzeri] 23 EUCP1 - Amino acid sequence of UDP-rhamnosyltransferase (1,2 RhaT) 24 EUCP1 - Nucleic acid sequence of UDP-rhamnosyltransferase (1,2 RhaT) 25 CP1 - Amino acid sequence of UDP-glycosyltransferase (UGT) 26 CP1 - Nucleic acid sequence of UDP-glycosyltransferase (UGT) 27 NR55N - Amino acid sequence of UDP-glucose 4,6-dehydratase (DH) Acrostichum aureum 28 NR55N - Nucleic acid sequence of UDP-glucose 4,6-dehydratase (DH) Acrostichum aureum 29 NR60N - Amino acid sequence of UDP-glucose 4,6-dehydratase (DH) Ettlia oleoabundans 30 NR60N - Nucleic acid sequence of UDP-glucose 4,6-dehydratase (DH) Ettlia oleoabundans 31 NR66N - Amino acid sequence of UDP-glucose 4,6-dehydratase (DH) Volvox carteri 32 NR66N - Nucleic acid sequence of UDP-glucose 4,6-dehydratase (DH) Volvox carteri 33 NR67N - Amino acid sequence of UDP-glucose 4,6-dehydratase (DH) Chlamydomonas reinhardtii 34 NR67N - Nucleic acid sequence of UDP-glucose 4,6-dehydratase (DH) Chlamydomonas reinhardtii 35 NR68N - Amino acid sequence of UDP-glucose 4,6-dehydratase (DH) Oophila amblystomatis 36 NR68N - Nucleic acid sequence of UDP-glucose 4,6-dehydratase (DH) Oophila amblystomatis 37 NR69N - Amino acid sequence of UDP-glucose 4,6-dehydratase (DH) Dunaliella primolecta 38 NR69N - Nucleic acid sequence of UDP-glucose 4,6-dehydratase (DH) Dunaliella primolecta 39 NR15N - Amino acid sequence of RHM Ostreococcus lucimarinus 40 NR15N - Nucleic acid sequence of RHM Ostreococcus lucimarinus 41 NR53N - Amino acid sequence of RHM Nannochloropsis oceanica 42 NR53N - Nucleic acid sequence of RHM Nannochloropsis oceanica 43 NR58N - Amino acid sequence of RHM Ulva lactuca 44 NR58N - Nucleic acid sequence of RHM Ulva lactuca 45 NR62N - Amino acid sequence of RHM Golenkinia longispicula 46 NR62N - Nucleic acid sequence of RHM Golenkinia longispicula 47 NR65N - Amino acid sequence of RHM Tetraselmis subcordiformis 48 NR65N - Nucleic acid sequence of RHM Tetraselmis subcordiformis 49 NR21C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Physcomitrella patens subsp. Patens 50 NR21C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Physcomitrella patens subsp. Patens 51 NR27C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Pyricularia oryzae 52 NR27C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Pyricularia oryzae 53 NR36C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Nannochloropsis oceanica 54 NR36C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Nannochloropsis oceanica 55 NR37C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Ulva lactuca 56 NR37C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Ulva lactuca 57 NR38C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Tetraselmis cordiformis 58 NR38C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Tetraselmis cordiformis 59 NR39C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Tetraselmis subcordiformis 60 NR39C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Tetraselmis subcordiformis 61 NR40C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Chlorella sorokiniana 62 NR40C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Chlorella sorokiniana 63 NR41C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Chlamydomonas moewusii 64 NR41C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Chlamydomonas moewusii 65 NR42C - Amino Acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Golenkinia longispicula 66 NR42C - Nucleic Acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Golenkinia longispicula 67 NR43C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Chlamydomonas reinhardtii 68 NR43C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Chlamydomonas reinhardtii 69 NR44C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Chromochloris zofingiensis 70 NR44C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Chromochloris zofingiensis 71 NR46C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Dunaliella primolecta 72 NR46C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Dunaliella primolecta 73 NR47C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Pavlova lutheri 74 NR47C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Pavlova lutheri 75 NR48C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Nitella mirabilis 76 NR48C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Nitella mirabilis 77 NR49C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Marchantia polymorpha 78 NR49C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Marchantia polymorpha 79 NR50C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Selaginella moellendorffii 80 NR50C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Selaginella moellendorffii 81 NR51C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Bryum argenteum var argenteum 82 NR51C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Bryum argenteum var argenteum 83 NRF4 - Amino acid sequence of RHM, fusion enzyme 84 NRF4 - Nucleic acid sequence of RHM, fusion enzyme 85 NRF5 - Amino acid sequence of RHM, fusion enzyme 86 NRF5 - Nucleic acid sequence of RHM, fusion enzyme 87 NRF7- Amino acid sequence of RHM, fusion enzyme 88 NRF7 - Nucleic acid sequence of RHM, fusion enzyme 89 NR64N - Amino acid sequence of RHM from Tetraselmis cordiformis 90 NR64N - Nucleic acid sequence of RHM from Tetraselmis cordiformis 91 NX5C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Arabidopsis thaliana 92 NX5C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Arabidopsis thaliana 93 NX13 - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Pyricularia oryzae 94 NX13 - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Pyricularia oryzae 95 NR5C - Amino acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Citrus clementina 96 NR5C - Nucleic acid sequence of bifunctional UDP-4-keto-6-deoxyl- glucose 3,5-epimerase/UDP-4-keto rhamnose 4-keto reductase (ER) Citrus clementina 97 EU11 - Amino acid sequence of 1,2-rhamnosyltransferase - Oryza sativa 98 EU11 - Nucleotide sequence of 1,2-rhamnosyltransferase - Oryza sativa 99 HV1 - Amino acid sequence of 1,2-rhamnosyltransferase - Hordeum vulgare 100 HV1 - cleotide sequence of 1,2-rhamnosyltransferase - Hordeum vulgare 101 UGT2E-B - Artificial Sequence - Amino acid sequence of 1,2- rhamnosyltransferase 102 UGT2E-B - Artificial Sequence - Nucleotide sequence of 1,2- rhamnosyltransferase 103 NX114 Amino acid sequence of 1,2-rhamnosyltransferase - Oryza brachyantha 104 NX114 Nucleic acid sequence of 1,2-rhamnosyltransferase - Oryza brachyantha 105 CP2 - Artificial Sequence - Amino acid sequence of UDP- glycosyltransferase 106 CP2 - Artificial Sequence - Nucleotide sequence of UDP-glycosyltransferase 107 UGT76G1 - Amino acid acid sequence of UDP-glycosyltransferase - Stevia rebaudiana 108 UGT76G1 - Nucleic acid sequence of UDP-glycosyltransferase - Stevia rebaudiana 109 GS - Amino acid sequence of fusion enzyme - UDP-glycosyltransferase + Sucrose Synthase 110 Artificial Sequence - Nucleic acid sequence of fusion enzyme - UDP- glycosyltransferase + Sucrose Synthase
TABLE-US-00002 TABLE 2 One-pot multi-enzyme in vitro synthesis of UDP-rhamnose. Reaction No. 1 2 3 4 5 6 PB pH 8.0 50 mM 50 mM 50 mM 50 mM 50 mM 50 mM UDP 3 mM 3 mM 3 mM 3 mm 3 mm 3 mM Sucrose 250 mM 250 mM 250 mM 250 mM 250 mM 250 mM NAD+ 3 mM 3 mM 3 mM 3 mM 3 mM 3 mM NADPH 3 mM 3 mM 1 mM 1 mM 0 0 NADP+ 0 0 0 0 1 Mm 1 mM DTT 1 mM 1 mM 1 mM 1 mm 1 mM 1 mM NRF1 0 0.2 g/l 0.2 g/l 0.2 g/l 0.2 g/l 0.2 g/l MaeB 0 0 0 0.1 g/l 0 0.1 g/l SUS 0.2 g/l 0.2 g/l 0.2 g/l 0.2 g/l 02. g/l 0.2 g/l Malate 5 mM 5 mM 5 mM 5 mM 5 mm 5 mM MgCl2 3 mM 3 mM 3 mM 3 mM 3 mM 3 mM
TABLE-US-00003 TABLE 3 Amino acid sequence organization in fusion enzymes Fusion N-terminal end Linker amino C-terminal end enzyme (SEQ ID NO.) acid sequence (SEQ ID NO.) NRF1 NX10 GSG NX5C (SEQ ID NO. 7) (Gly-Ser-Gly) (SEQ ID NO. 91) NRF2 NX10 GSG NX13 (SEQ ID NO. 7) (Gly-Ser-Gly) (SEQ ID NO. 93) NRF3 NX10 GSG NR5C (SEQ ID NO. 7) (Gly-Ser-Gly) (SEQ ID NO. 95) NRF4 NX10 GSG NR40C (SEQ ID NO. 7) (Gly-Ser-Gly) (SEQ ID NO. 61) NRF5 NX10 GSG NR41C (SEQ ID NO. 7) (Gly-Ser-Gly) (SEQ ID NO. 63) NRF7 NR66N GSG NR41C (SEQ ID No. 31) (Gly-Ser-Gly) (SEQ ID NO. 63)