Enhanced processes and reagents for host engineering

Abstract

Nonnaturally occurring host cells altered to increase their ability to transfer genetic molecules into the host cells as compared to an unaltered host cell are provided. Also provided are methods for identifying endogenous loci of a host cell which inhibit transformation efficiency and/or electroporation of genetic molecules into the cell as well as methods for producing nonnaturally occurring host cells with enhanced transformation efficiency and/or the modified ability to allow for genomic integration of an exogenous DNA sequence via electroporation. Methods for producing biochemicals and products produced with the nonnaturally occurring host cells are also provided.

Claims

1. A nonnaturally occurring organism having at least one hereditary alteration which increases the ability to transfer genetic molecules into the organism as compared to an unaltered organism, wherein the nonnaturally occurring organism is a strain of Cupriavidus necator and wherein the alteration is deletion of an endogenous target locus H16_A0006-9.

2. The nonnaturally occurring organism of claim 1 wherein the alteration increases transformation efficiency.

3. The nonnaturally occurring organism of claim 1 wherein the alteration allows for genomic integration of an exogenous DNA sequence via electroporation into the organism.

4. The nonnaturally occurring organism of claim 1 wherein the alteration increases transformation efficiency and allows for genomic integration of an exogenous DNA sequence via electroporation into the organism.

5. The nonnaturally occurring organism of claim 1 wherein the genetic molecule comprises an antibiotic resistance gene, a Km-based, Tc-based, and/or Cm-based plasmid, or any combination thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 provides a comparison demonstrating more efficient transformation of C. necator H16 with plasmid DNA purified from itself as compared to plasmid DNA purified from E. coli.

(2) FIG. 2 compares transformation efficiency of C. necator H16 WT (left) and H16_A0006-9 (right) transformed with 75 ng pMOL28-tetA (top panel) or pMOL28-tetAR (bottom panel). Each spot represents a 10-fold dilution. N=neat, 1=10.sup.1 etc.

DETAILED DESCRIPTION

(3) Disclosed herein are nonnaturally occurring hosts altered to increase their ability to transfer genetic molecules into the host cells and methods for their production.

(4) The nonnaturally occurring hosts of the present invention are produced in accordance with the present invention by introducing at least one alteration into the host which increases the ability to transfer genetic molecules into the host as compared to an unaltered host.

(5) In one nonlimiting embodiment, the alteration is a hereditary alteration. By hereditary alteration as used herein, it is meant a change, modification, variation or transformation in a genetic factor of a host including, but in no way limited to, an alteration in the genome of the host or an alteration in levels or types of protein expressed by the host. In one nonlimiting embodiment, the alteration increases the transformation efficiency of the altered nonnaturally occurring host as compared to an unaltered host. In one nonlimiting embodiment, transformation efficiency is increased for exogenous genetic molecules. In one nonlimiting embodiment, the alteration allows for genomic integration of an exogenous nucleic acid sequence via electroporation into the host. In one nonlimiting embodiment, the alteration increases the transformation efficiency of the altered nonnaturally occurring host as compared to an unaltered host and allows for genomic integration of an exogenous DNA sequence via electroporation into the altered nonnaturally occurring host cell.

(6) Various modifications and/or alterations to the host can be made. In one nonlimiting embodiment, the alteration is a hereditary alteration. In one nonlimiting embodiment, the alteration is a genetic alteration. In one nonlimiting embodiment, the alteration comprises deletion of an endogenous target locus. In one nonlimiting embodiment, the alteration comprises deletion of an endogenous target locus which interferes with transformation of an exogenous nucleic acid sequences. In one nonlimiting embodiment, the alteration is deletion of an endogenous endonuclease locus which cleaves any exogenous nucleic acid sequences. Endogenous genes of the nonnaturally occurring host may be further disrupted to prevent the formation of undesirable metabolites or prevent the loss of intermediates in the metabolic engineering pathway for which the cells will be used. Nonnaturally occurring hosts of the present invention may also be referred to as recombinant hosts, to as recombinant host cells, engineered cells, or engineered hosts.

(7) By genetic molecules as used herein it is meant to include, but is not limited to, nucleic acid sequences such as DNA, RNA, cDNA, as well as expression vectors, plasmids and the like as well as amino acid sequences, polypeptides and proteins. In one nonlimiting embodiment, the genetic molecule may comprise an antibiotic resistance gene, a Kanamycin-based, Tetracycline-based, and/or Chloramphenicol-based plasmid, or any combination thereof.

(8) The term exogenous as used herein with reference to a nucleic acid (or a protein) and a host refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid. Thus, a non-naturally-occurring nucleic acid is considered to be exogenous to a host once in the host. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular host microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a cell of yeast y once that chromosome is introduced into a cell of yeast y.

(9) In contrast, the term endogenous as used herein with reference to a nucleic acid (e.g., a gene) (or a protein) and a host refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular host as it is found in nature. Moreover, a cell endogenously expressing a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a host of the same particular type as it is found in nature. Moreover, a host endogenously producing or that endogenously produces a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a host of the same particular type as it is found in nature.

(10) The methodologies disclosed herein were used to produce a nonnaturally occurring host cell with an approximate 10000-fold increase its ability to transfer genetic molecules as compared to the wild-type progenitor cells. In this nonlimiting example, the altered host was C. necator H16. As shown in FIG. 1, C. necator H16 can be transformed more efficiently with plasmid DNA purified from itself rather than E. coli. While not being bound to any particular theory, it is believed that C. necator has specific DNA endonuclease(s) that cleave exogenous DNA. The DNA of C. necator is believed to be protected by methylation.

(11) Accordingly, seven endonuclease loci were identified in the genome of C. necator H16. These target loci were then deleted individually and transformation efficiency was tested for each modified strain.

(12) Results are shown in Table 1.

(13) TABLE-US-00001 TABLE 1 Colonies recovered following electroporation Target Locus (75 ng plasmid DNA) None (WT) 92 H16_A0006-9 Lawn (4 repeats) H16_A0014 127 H16_A1147 28 H16_A3579 128 H16_B0176 40 H16_PHG170 52 H16_PHG327 101
As shown in Table 1, C. necator H16_A0006-9 strain displayed a notable increase in transformation efficiency. In these experiments, C. necator H16_A0006-9 was transformed with 75 ng plasmid DNA.

(14) Ten-fold serial dilutions were then plated to enumerate the number of colonies with the plasmid. Results are shown in Table 2.

(15) TABLE-US-00002 TABLE 2 Wild-type control/Parental H16_A0006-9 strain Strain Dilution Rep 1 Rep 2 Rep3 Single replicate 100 l Neat Lawn Lawn Lawn 50 10.sup.1 Lawn Lawn Lawn 2 10.sup.2 ~1000 ~1000 ~1000 1 10.sup.3 ~200 ~1000 ~800 10.sup.4 120 ~800 ~500 10.sup.5 90 ~400 66 10.sup.6 55 200 25 10.sup.7 110 63
The transformation efficiency upon alteration in accordance with the methodologies of the present invention was increased by 4 orders of magnitude. This is comparable to efficiencies routinely observed for laboratory E. coli strains (i.e., approx. 110.sup.6-7 transformants per g DNA).

(16) Seven different plasmids were then electroporated into the C. necator H16_A0006-9 strain. Plasmids included both replicons and suicide vectors involving 2 different antibiotic markers, Km and Tc, and various inserts. pBBR1MCS cloning vectors used have been described by Kovach et al. (Gene 1995 166:175-176). Results are depicted in Table 3.

(17) TABLE-US-00003 TABLE 3 Single colonies Cloning Purified Colony visible at Plasmid from Marker Insert Count dilution Repeats pBBR1-Km E. coli Kan None Lawn 10.sup.7 >10 pBBR1-MCS2- E. coli Kan IPA Lawn 10.sup.4 2 IPA pathway (7 colonies) pBBR1-Km- E. coli Kan Lycopene Lawn 10.sup.4 1 lycopene pathway (62 colonies) pathway (Erwinia herbicola) pBBR1-MCS3- E. coli Tet None 50 1 mob- PBBR1-MCS3 E. coli Tet None 0 1 pTc-INT- E. coli Kan Kanamycin 5-25 3 phaC::kan cassette from pBBR1- MCS2 pTc-INT- E. coli Tet Kanamycin 0 2 phaC::kan cassette from pBBR1- MCS2

(18) Additional modifications which further improved transformation efficiency by approximately 3-fold included re-growth and recovery of cells in Super Optimal Broth, Catabolite Repression (SOC) broth, rather than Tryptone Soya Broth, DNA of 50-100 ng, and a two hour recovery following electroporation. With these improvements, transformation of wild-type H16 with 75 ng pBBR1-based kanamycin vector yielded 500-1000 colonies.

(19) In general, transformation efficiency with pBBR1-based-tetracycline (Tc) plasmids (pBBR1-MCS3) was less as compared to the excellent transformation efficiency observed with pBBR1-based-kanamycin (Km) plasmids.

(20) When an independent pMOL28-based vector containing the tetAR cassette was constructed and used to transform C. necator wild-type (WT) and H16_A0006-9, the presence of tetR increased transformation efficiency of H16_A0006-9 by approximately 4 orders of magnitude and of WT by approximately 100-fold, relative to plasmid containing tetA only (Example 5 and FIG. 2). Functionally, tetR controls expression of tetA which is induced only in the presence of tetracycline (Bertram & Hillen, Microb Biotechnol. 2008 January; 1(1):2-16). The tetAR cassette characterized herein originates from RK2 plasmid and is described in detail by Waters et al. (Nucleic Acids Research 1983 11(17):6089-6105). The reason for increased TE of tetAR-based vectors remains unclear, although previous reports suggest that tetR expression prevents toxic build-up of the tetA membrane protein during recovery following transformation which may reduce the osmotic sensitivity of the cell, a phenomenon which has been suggested for E. coli (Stavropoulos & Strathdee, FEMS Microbiol Lett. 2000 Sep. 1; 190(1):147-50).

(21) Further, high transformation efficiency was achieved with pBBR1-MCS2 variants carrying a range of different inserts (e.g. lycopene and IPA pathways), thus indicating that DNA sequence variations do not impact transformation efficiency/frequency.

(22) Thus, as demonstrated by these experiments, the methodologies of the present invention were useful in substantially increasing the transformation efficiency of the altered host. Further, genomic integration of exogenous DNA sequences was achieved via electroporation rather than conjugation.

(23) The significant advantages of the exemplified altered host cell demonstrated herein are indicative of host cells of the present invention being useful as metabolic engineering chassis.

(24) By metabolic engineering chassis as used herein, it is meant an organism which readily accepts new genes and new biochemistry and serves as a frame in which to tailor a specific biochemical function while maintaining the growth behavior and application range of the respective wild type organism.

(25) The significant advantages of the exemplified altered host cell presented herein are also demonstrative of the present invention providing a useful method for identifying an endogenous target locus in a host cell which inhibits transformation efficiency. In this method, single selected endogenous target loci in the host cell are modified. In one nonlimiting embodiment, the single selected endogenous target loci is deleted. In one nonlimiting embodiment, the endogenous target locus to be selected is an endonuclease locus. The transformation efficiency in these cells is then compared with the transformation efficiency in the progenitor/wild-type host cell. An increase in transformation efficiency in cells with a modified target locus is indicative of that target locus inhibiting transformation efficiently of the cell.

(26) These advantages are also indicative of the present invention providing a useful method for identifying an endogenous target locus in a host cell which inhibits transformation efficiency via electroporation. In this method, single selected endogenous target loci in the host cell are modified. In one nonlimiting embodiment, the single selected endogenous target loci is deleted. In one nonlimiting embodiment, the endogenous target locus to be selected is an endonuclease locus. Electroporation of a plasmid in the altered cell is then compared with electroporation of the same plasmid in the progenitor/wild-type host cell. An increase in transformation efficiency of the plasmid in cells with a modified target locus is indicative of that target locus inhibiting transfer of plasmids in the progenitor/wild-type host cell.

(27) In addition, these experiments demonstrate the usefulness of these methodologies in enhancing transformation efficiency of a host cell. In these methods, an endogenous target locus in a host cell which inhibits transformation efficiency can be identified as described and exemplified herein. In one nonlimiting embodiment, the endogenous target locus is an endonuclease locus. Once identified, the endogenous target locus can be modified to produce a nonnaturally occurring host cell having at least one alteration with increased ability to transfer genetic molecules into the host cell as compared to an unaltered host cell. In one nonlimiting embodiment, the endogenous target locus is deleted.

(28) The experiments also demonstrate the usefulness of these methodologies in modifying a host cell to allow for genomic integration of an exogenous DNA sequence via electroporation. In these methods, an endogenous target locus in the host cell which inhibits transformation efficiency is identified as described and exemplified herein. In one nonlimiting embodiment, the endogenous target locus is an endonuclease locus. Once identified, the endogenous target locus can be modified to produce a nonnaturally occurring host cell allowing for genomic integration of an exogenous DNA sequence via electroporation. In one nonlimiting embodiment, the endogenous target locus is deleted.

(29) The nonnaturally occurring host cells of the present invention are useful in methods for producing biochemicals. In these methods, a nonnaturally occurring host cell of the present invention is transformed with one or more exogenous nucleic acid sequences encoding one or more enzymes required for production of the biochemical. The cells are then subjected to selected conditions in which the biochemical is produced. For example, a fermentation strategy can be used that entails anaerobic, micro-aerobic or aerobic cultivation. A fermentation strategy can entail nutrient limitation such as nitrogen, phosphate or oxygen limitation. A cell retention strategy using a ceramic hollow fiber membrane can be employed to achieve and maintain a high cell density during fermentation. The principal carbon source fed to the fermentation can derive from a biological or non-biological feedstock. The biological feedstock can be, or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles or municipal waste. The non-biological feedstock can be, or can derive from, natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, non-volatile residue (NVR) a caustic wash waste stream from cyclohexane oxidation processes or waste stream from a chemical or petrochemical industry.

(30) In one nonlimiting embodiment, at least one of the enzymatic conversions may comprise gas fermentation within the host cell. In this embodiment, the gas fermentation may comprise at least one of natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, non-volatile residue, caustic wash from cyclohexane oxidation processes, or waste stream from a chemical or petrochemical industry. In one nonlimiting embodiment, the gas fermentation comprises CO.sub.2/H.sub.2.

(31) In addition, the present invention provides bio-derived, bio-based, or fermentation-derived products produced using the methods and/or cells disclosed herein. Examples of such products include, but are not limited to, compositions comprising at least one bio-derived, bio-based, or fermentation-derived compound or any combination thereof, as well as polymers, molded substances, formulations and semi-solid or non-semi-solid streams comprising one or more of the bio-derived, bio-based, or fermentation-derived compounds or compositions, combinations or products thereof.

(32) The following section provides further illustration of the cells and methods of the present invention. These working examples are illustrative only and are not intended to limit the scope of the invention in any way.

EXAMPLES

Example 1: Identification of Endonuclease Loci in Genome of C. necator H16

(33) The C. necator H16 genome was queried for annotated open reading frames (ORFS) containing the annotation nuclease using genome analysis software (GeneData). The list was rationalized based on protein homology to known restriction endonuclease genes to a final list of seven targets.

Example 2: Deletion of Individual Target Loci

(34) Standard allele exchange protocols were carried out. 400 to 800 base pairs upstream (LHA) and downstream (RHA) of each individual endonuclease locus of C. necator H16 along with the first five and final five codons of each gene were amplified by PCR using NEB Q5 DNA polymerase. Appropriate homologous overhangs were designed onto each primer to facilitate cloning by Gibson Assembly (see Gibson et al. Nat Methods. 2009 6(5):343-5). Purified PCR-amplified fragments were cloned into a Pvul-digested p(Tc-INT-phaC::kan) plasmid.

Example 3: Transfer of Allele Exchange Constructs

(35) Verified allele exchange constructs were transferred to the conjugative bacterial donor E. coli S17-1 by transformation and then to C. necator H16 by conjugation. C. necator trans-conjugants were selected on media supplemented with appropriate antibiotics. Plates were incubated until individual colonies were visible. Three independent clones were cultured in liquid medium with no antibiotic and then plated on appropriate media supplemented with sucrose to select for double crossover integrants. Individual colonies were screened for chromosomal deletion by colony PCR.

Example 4: Electroporation/Transformation Efficiency

(36) Electroporation/transformation efficiency was tested for each modified strain as follows. Restriction endonuclease mutants were transformed with pBBR1-based plasmid by electroporation. The cell/plasmid mixture was recovered and then plated onto medium with appropriate antibiotic. The plates were incubated until individual colonies were visible. Colonies were counted and the transformation efficiency calculated.

Example 5: Improving Transformation Efficiency of Tetracycline (Tc)-Based Plasmids in C. necator H16_A0006-9

(37) An independent pMOL28-based vector containing the tetAR cassette (as depicted in SEQ ID NO:1 with TetA at nucleotides 1-1200, the regulatory region at nucleotides 1248-1305 and TetR at nucleotides 1306-1956) was constructed by Gibson Assembly and used to transform C. necator wild-type (WT) and H16_A0006-9. The presence of tetR increased transformation efficiency of H16A0006-9 by approximately 4 orders of magnitude and of WT by approximately 100-fold, relative to plasmid containing tetA only (FIG. 2).