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WO2008119009A2 - Matériaux et procédés pour une production efficace d'alanine - Google Patents

Matériaux et procédés pour une production efficace d'alanine Download PDF

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WO2008119009A2
WO2008119009A2 PCT/US2008/058410 US2008058410W WO2008119009A2 WO 2008119009 A2 WO2008119009 A2 WO 2008119009A2 US 2008058410 W US2008058410 W US 2008058410W WO 2008119009 A2 WO2008119009 A2 WO 2008119009A2
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Prior art keywords
alanine
genetically modified
gene
modified microorganism
brevibacterium
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PCT/US2008/058410
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WO2008119009A3 (fr
WO2008119009A4 (fr
Inventor
Xueli Zhang Zhang
Kaemwich Jantama
Jonathan C. Moore
Keelnatham T. Shanmugam
Lonnie O'neal Ingram
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University Of Florida Research Foundation, Inc.
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Publication of WO2008119009A3 publication Critical patent/WO2008119009A3/fr
Publication of WO2008119009A4 publication Critical patent/WO2008119009A4/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)

Definitions

  • L-alanine is produced commercially by the enzymatic decarboxylation of L-aspartic acid using immobilized cells or cell suspensions of Pseudomonas dacunhae as a biocatalyst with a yield >90% (Shibatani et ah, 1979).
  • L- alanine is used with other L-amino acids as a pre- and post-operative nutrition therapy (Hols el ah, 1999).
  • Alanine is also used as a food additive because of its sweet taste (Lee et ah, 2004).
  • Alanine is a central intermediate and an essential component of cellular proteins. Most microorganisms produce alanine for biosynthesis using a glutamate-pyruvate transaminase (Hashimoto and Katsumata, 1998). Some organisms such as Arthrobacter oxydans (Hashimoto and Katsumata, 1993; Hashimoto and Katsumata, 1998; Hashimoto and Katsumata, 1999), Bacillus sphaericus (Ohashima and Soda, 1979), and Clostridium sp. P2 (Orlygsson et ah, 1995) produce alanine from pyruvate and ammonia using an NADH-linked alanine dehydrogenase. However, fermentations are slow and yields from the best natural producers are typically 60% or less due to co-product formation (Hashimoto and Katsumata, 1998; Table 1).
  • Plasmid-borne genes encoding NADH-linked alanine dehydrogenase have been tested as an approach to develop improved biocatalysts with varying degrees of success (Table 1).
  • Engineered strains of Zymomonas mobilis CP4 expressing the B. sphaericus cilaD gene produced low levels of racemic alanine during the anaerobic fermentation of 5% glucose (Uhlenbusch et ah, 1991).
  • An WM-deleted strain of Lactococcus lactis containing a mutation in alanine racemase was engineered in a similar fashion and produced 12.6 g 1 -1 L-alanine from 1.8% glucose (Hols et ah, 1999).
  • coli aceF ldhA double mutant containing pTrc99A- ⁇ 7/ ⁇ Z plasmid produced 32 g 1 -1 racemic alanine in 27 h during a two-stage (aerobic and anaerobic) fermentation with a yield of 0.63 g alanine g "1 glucose (Lee et ah, 2004).
  • the racemic alanine titer was increased to 88 g1 -1 in a more complex process with yields approaching the theoretical maximum (Smith et ah, 2006).
  • this strain produced only racemic alanine, utilized multi-copy plasmids requiring antibiotic selection, and required complex media with a complex multi-stage fermentation process (Smith et ah, 2006; Table 1).
  • the subject invention provides novel microorganisms useful in the production of alanine. Additionally, the subject invention provides novel constructs for use in transforming any of numerous host organisms, for example, Escherichia coli, to express and/or suppress certain genes to produce alanine when the host organism is cultivated in a fermentable medium. Accordingly, the materials and methods of the subject invention can be used to enhance alanine production in host organisms thereby providing an increased supply of alanine for use in food and industrial applications.
  • E. coli derivatives of Escherichia coli
  • E. coli W e.g., ATCC 27325. Additional advantages of this invention will become readily apparent from the ensuing description.
  • Figure 1 Integration vector used for chromosomal insertion of G. stear other mophilus alaD into the E. coli idhA. Sequence encoding the N-terminal and C- terminal regions are designated alaA ' and alaA ", respectively.
  • FIGS. 2 A and 2B Plasmids used to delete mgsA. Plasmid pLOI4229 (left) was used to delete the mgsA gene and insert the cat-sacB cassette in the first recombination step.
  • Plasmid pLOI4230 (right) was used to remove the cat-sacB cassette to create a deletion devoid of foreign sequence. Sequence encoding the N-terminal and C-terminal regions are designated mgsA ' and mgsA ", respectively.
  • Plasmids used to delete dadX Plasmid pLOI4218 (left) was used to delete the dadX gene and insert the cat-sacB cassette in the first recombination step.
  • Plasmid pLOI4220 (right) was used to remove the cat-sacB cassette to create a deletion devoid of foreign sequence. Sequence encoding the N-terminal and C-terminal regions are designated dadX ' and dadX", respectively.
  • Figures 4 A and 4B Alignment of the nucleotide and translated amino acid sequences of alanine dehydrogenase genes from G stearotheromphillus XL-65-6 and B. sphaericus IFO3525.
  • Figure 4A Nucleotide sequence alignment (65% identity).
  • Figure 4B Alignment of translated amino acid sequences (73% identity).
  • Figures 5A and 5B Alanine pathway in recombinant E. coli.
  • Figure 5A Native and recombinant fermentation pathways. The G. stearothermophilus alaD coding region and transcriptional terminator were integrated into the native IdhA gene under transcriptional control of the IdhA promoter. Solid stars represent gene deletions present in XZ 132. Note that the native biosynthetic route for alanine production is omitted for simplicity.
  • Figure 5B Coupling of ATP production and growth to NADH oxidation and L-alanine production.
  • Figures 6A and 6B Metabolic evolution of XZl 11 to develop XZl 12. Strain XZl 12 was isolated after 10 serial transfers at 24 h intervals in NBS mineral salts medium containing 20 g1 -1 glucose and 1 niM betaine (inoculum of 0.03 CDW1 -1 ; pH controlled with 2N potassium hydroxide). Fermentation broths were sampled daily for 4 days.
  • Figure 6 A Cell mass (g1 -1 ).
  • Figure 6B Alanine production (4 days).
  • Figures 7 A and 7B Effects of different bases for pH control on growth and alanine production of strain XZl 12 (NBS mineral salts medium containing 50 g 1 -1 glucose and 1 mM betaine; inoculum of 0.03 CDW 1 -1 ). Bases were automatically added to control pH.
  • Figure 7A Cell mass (g 1 -1 ).
  • Figure 7B Alanine production (4 days). Symbols: ⁇ , 2N potassium hydroxide; A, 2N potassium hydroxide and IN ammonia bicarbonate; T, 5N ammonia hydroxide.
  • Figures 8A and 8B Metabolic evolution of XZl 12 to select XZl 13.
  • Strain XZl 13 was isolated after 12 serial transfers at 24 h intervals in NBS mineral salts medium containing 50 g1 -1 glucose and 1 mM betaine (inoculum of 0.017 CDW 1 -1 ; pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days.
  • Figure 8A Cell mass (g1 -1 ).
  • Figure 8B Alanine production (3 days).
  • Strain XZl 15 was isolated after 25 serial transfers at 24 h intervals in NBS mineral salts medium containing 80 g1 -1 glucose and 1 mM betaine (inoculum of 0.017 CDW 1 -1 ; pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown.
  • Figure 9 A Cell mass (g 1 " ).
  • Figure 9B Alanine production (3 days).
  • Figures 1OA and 1OB Metabolic evolution of XZ121 to select XZ123. Strain XZ123 was isolated after 40 serial transfers at 24 h intervals in NBS mineral salts medium containing 80 g 1 -1 glucose and 1 mM betaine (inoculum of 0.017 CDW1 -1 ; pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown.
  • Figure 1OA Cell mass (g 1 -1 ).
  • Figure 1OB Alanine production (3 days).
  • Figures HA and HB Effect of pH on cell growth and alanine production by strain XZl 23 (NBS mineral salts medium containing 80 g 1 -1 glucose and 1 mM betaine; inoculum of 0.017 CDW 1 -1 ). Broth pH was automatically controlled by the addition of 5N ammonium hydroxide.
  • Figure HA Cell Mass.
  • Figure HB Alanine production. Symbols: D, pH 6.5; ⁇ , pH 7.0; V, pH 7.5; and o pH 8.0.
  • Figures 12A and 12B Effect of inoculum level on growth and alanine production by strain XZ 123 (NBS mineral salts medium containing 80 g1 -1 glucose and 1 mM betaine; pH controlled with 5 N ammonium hydroxide.
  • Figure 12 A Cell mass.
  • Figure 12B Alanine production. Symbols: D, 0.005 g CDW 1 -1 ; ⁇ , 0.008 g CDW 1-; 1 V, 0.017 g CDW 1; -1 o, 0.035 g CDW r 1 ; O, 0.08 g CDW 1; -1 X, 0.17 g CDW . 1 -1
  • Figures 13A and 13B Metabolic evolution of XZ126 to develop XZ129.
  • Strain XZl 29 was isolated after 30 serial transfers (1 :100 dilution) at 24 h intervals in NBS mineral salts medium containing 80 g 1 -1 glucose and 1 mM betaine (pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown.
  • Figure 13 A Cell mass (g 1-) 1 .
  • Figure 13B Alanine production (3 days).
  • Figures 14A and 14B Metabolic evolution of XZ129 to develop XZ130. Strain
  • XZl 30 was isolated after 7 serial transfers at a 1 :100 dilution and 7 transfers at 1:300 dilution. Cells were transferred at 24 h intervals in AMI mineral salts medium containing 80 g1 -1 glucose and 1 mM betaine (pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days.
  • Figure 14A Cell mass (g 1-) 1 .
  • Figure 14B Alanine production (3 days).
  • Figures 15A and 15B Metabolic evolution of XZ130 to develop XZ131. Strain XZl 31 was isolated after 16 serial transfers (1 :100 dilution) at 24 h intervals in AMI mineral salts medium containing 100 g 1 -1 glucose and 1 mM betaine (pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown.
  • Figure 15A Cell mass (g 1 -1 ).
  • Figure 15B Alanine production (3 days).
  • Figures 16A and 16B Metabolic evolution of XZ131 to develop XZ 132. Strain XZ 132 was isolated after 30 serial transfers (1 :100 dilution) at 24 h intervals in AMI mineral salts medium containing 12O g 1 -1 glucose and 1 mM betaine (pH controlled with 5N ammonium hydroxide). Fermentation broths were analyzed daily for 3 days. For clarity, graphs from every third transfer are shown.
  • Figure 16A Cell mass (g 1 -1 ).
  • Figure 16B Alanine production (3 days).
  • FIG. 17 Diagram summarizing the construction of XZ132 for L-alanine production.
  • Figure 18. Pathway for pyruvate production from L-alanine.
  • L-alanine is first converted to D-alanine by an alanine racemase such as DADX (dadX) or ALR (air) and oxidized to pyruvate by a D-amino acid dehydrogenase such as DADA (dadA).
  • FAD + serves as an intermediate (FADH) and electron carrier.
  • FADH is oxidized through the electron transport system (dotted arrow) to regenerate FAD + with molecular oxygen serving as the ultimate electron acceptor.
  • SEQ ID NO: 1 is the ⁇ / ⁇ D-forward primer; including the ribosomal binding region
  • SEQ ID NO: 2 is the ⁇ / ⁇ D-reverse primer; downstream from the putative transcriptional terminator region.
  • SEQ ID NO: 3 is the idhA-forward primer.
  • SEQ ID NO: 4 is the MhA -reverse primer.
  • SEQ ID NO: 5 is the ldhA-up (ydbH) primer.
  • SEQ ID NO: 6 is the idhA-down (hslj) primer.
  • SEQ ID NO: 7 is the JMc ⁇ ts ⁇ cBupNhel primer.
  • SEQ ID NO: 8 is the Jmc ⁇ tsacBdownNhel primer.
  • SEQ ID NO: 9 is the c ⁇ t-up2 primer.
  • SEQ ID NO: 10 is the s ⁇ cB-down2 primer.
  • SEQ ID NO: 11 is the mgsA-up primer.
  • SEQ ID NO: 12 is the mgsA-down primer.
  • SEQ ID NO: 13 is the mgsA-1 primer.
  • SEQ ID NO: 14 is the mgsA-2 primer.
  • SEQ ID NO: 15 is the d ⁇ dX-up primer.
  • SEQ ID NO: 16 is the d ⁇ dX-down primer.
  • SEQ ID NO: 17 is the d ⁇ dX-4 primer.
  • SEQ ID NO: 18 is the d ⁇ dX-5 primer.
  • the subject invention provides materials and methods wherein unique and advantageous combinations of gene mutations are used to direct carbon flow to a desired product, e.g., alanine.
  • a desired product e.g., alanine.
  • the techniques of the subject invention can be used to obtain products from native pathways as well as from recombinant pathways.
  • the subject invention provides a versatile platform for the production of these products with only mineral salts and sugar as nutrients.
  • bacterium that can be used in the present invention include, but are not limited to, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter lumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium laclofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglut
  • the subject invention provides strains of E. coli suitable for the production of alanine.
  • the microorganisms of the subject invention can employ a single step process using sugars as substrates, high rates of production, high yields, simple nutrient requirements (e.g., mineral salts medium), and a robust metabolism permitting the bioconversion of hexoses, pentoses, and many dissacharides.
  • microorganisms according to the instant disclosure can have one or more target genes inactivated by various methods known in the art.
  • target genes can be inactivated by the introduction of insertions, deletions, or random mutations into the target gene.
  • certain aspects of the invention provide for the insertion of at least one stop codon (e.g., one to ten or more stop codons) into the target gene.
  • Some aspects of the invention provide for the introduction or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more bases in order to introduce a frame shift mutation in a target gene.
  • Other aspects of the invention provide for the insertion or of 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29 or more bases in order to introduce a frame shift mutation in a target gene.
  • Yet other embodiments of the subject application provide for the introduction of one or more point mutations (e.g., 1 to 30 or more) within a target gene.
  • aspects of the invention provide for the total or complete deletion of a target gene from the microorganisms of the invention.
  • metabolic pathways are inactivated and the enzymatic activity of the target gene is eliminated.
  • Deletions provide maximum stability as there is no opportunity for a reverse mutation to restore function.
  • Target gene(s) refer(s) to one or more of the fumarate reductase subunit genes (e.g., the frdA, frdB, frdC, frdD, various combinations of the subunit genes or all of the subunit genes (e.g., frdABCD)), the alcohol dehydrogenase gene (e.g., adhE), the pyruvate formatelyase gene (e.g., pflB), the acetate kinase gene (e.g., ackA); the lactate dehydrogenase gene (e.g., idhA), the methylglyoxal synthase gene (e.g., mgsA); and/or the alanine racemase gene (e.g., dadX).
  • the frdA the frdA, frdB, frdC, frdD, various combinations of the subunit genes or all of the subunit genes (e.g., frdABCD)
  • a “heterologous alanine dehydrogenase gene” is to be understood to be a gene obtained from any microorganism other than E. coli.
  • the alanine dehydrogenase gene from Geobacillus stearothermophilus or any other microorganism (e.g., thermophilic microorganisms) can be used.
  • Non-limiting embodiments of the invention include: 1.
  • a genetically modified microorganism that comprises the inactivation or deletion of a lactate dehydrogenase gene and the integration of an alanine dehydrogenase gene and one or more of the following genetic modifications: a) the optional inactivation or deletion of a pyruvate formatelyase gene; b) the optional inactivation or deletion of an alcohol dehydrogenase gene; c) the optional inactivation or deletion of an acetate kinase gene; d) the optional inactivation of one or more fumarate reductase subunit genes by insertion or deletion; e) the optional inactivation or deletion of an alanine racemase gene; and/or f) the optional inactivation or deletion of a methylglyoxal synthase gene.
  • a genetically modified microorganism that comprises the following genetic modifications: a) the optional insertion of a Klebsiella oxyloca casAB gene or other genes encoding cellobiose utilizing enzyme II and phospho- ⁇ -glucosidase behind the stop codon of lacY; b) integration of an Erwinia chrysanthemi celY gene or any other endoglucanase or cellulase encoding gene into one or more fumarate reductase subunit genes or inactivation of one or more fumarate reductase subunit genes by insertion or deletion; c) inactivation or deletion of an alcohol dehydrogenase gene; d) inactivation or deletion of a pyruvate formatelyase gene; e) inactivation or deletion of an acetate kinase gene; f) inactivation or deletion of a lactate dehydrogenase gene; g) integration of an alanine dehydrogenase gene
  • microorganisms comprises the following additional genetic modifications set forth in embodiment Ia though If: Ia only; Ib only; Ic only; Id only; Ie only; Ia and Ib only; Ia and Ic only; Ia and Id only; Ia and Ie only; Ia and If only; Ib and Ic only; Ib and Id only; Ib and Ie only; Ib and If only; Ic and Id only; Ic and Ie only; Ic and If only; Id and Ie only; Id and If only; Ie and If only; Ia, Ib, Ic, Id, and Ie only; Ia, Ib, Ic, Id and If only; Ia, Ib, Ic, Ie and If only; Ia, Ib, Ic, Ie and If only; Ia, Ib, Id, Ie, If and only; Ia, Ib, Id, Ie, If and only; Ia, Ic, Id, Ie and If only; Ia, Ib
  • a method of culturing or growing a genetically modified microorganism comprising inoculating a culture medium with one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 and culturing or growing said genetically modified microorganism.
  • a method of producing L-alanine comprising culturing one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 under conditions that allow for the production of alanine.
  • the mineral salts medium contains 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20% (w/v) of a sugar.
  • composition comprising one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 and medium.
  • a genetically modified microorganism that comprises the inactivation or deletion of a lactate dehydrogenase gene and the integration of an alanine dehydrogenase gene and the following genetic modifications: a) the inactivation or deletion of a pyruvate formatelyase gene; b) the inactivation or deletion of an alcohol dehydrogenase gene; c) the inactivation or deletion of an acetate kinase gene; d) the inactivation of one or more fumarate reductase subunit genes by insertion or deletion; e) the inactivation or deletion of an alanine racemase gene; and f) the inactivation or deletion of a methylglyoxal synthase gene.
  • Nocardia rugosa Planococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas fluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp.
  • a genetically modified microorganism that comprises the following genetic modifications: a) the optional insertion of a Klebsiella oxytoca casAB gene or other genes encoding cellobiose utilizing enzyme II and phospho- ⁇ -glucosidase behind the stop codon of lacY; b) integration of an Erwinia chrysanthemi celY gene or any other endoglucanase or cellulase encoding gene into one or more fumarate reductase subunit genes or inactivation of one or more fumarate reductase subunit genes by insertion or deletion; c) inactivation or deletion of an alcohol dehydrogenase gene; d) inactivation or deletion of a pyruvate formatelyase gene; e) inactivation or deletion of an acetate kinase gene; f) inactivation or deletion of a lactate dehydrogenase gene; g) integration of an alanine dehydrogenase gene
  • cellobiose utilizing enzyme II, phospho- ⁇ -glucosidase and Erwinia chrysanthemi celY gene or any other endoglucanase or cellulase encoding gene are inactivated or deleted in said genetically modified microorganism after insertion.
  • lactate dehydrogenase gene is inactivated by the insertion of an alanine dehydrogenase gene into the lactate dehydrogenase gene.
  • 19. The genetically modified organism according to embodiment 11, 12, 13, 14, 15, 16, 17 or 18, wherein a single alanine dehydrogenase gene is inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.
  • a method of culturing or growing a genetically modified microorganism comprising inoculating a culture medium with one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • a method of producing L-alanine comprising culturing one or more genetically modified microorganism according to any one of embodiments 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 under conditions that allow for the production of alanine.
  • composition comprising one or more genetically modified microorganism according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 and medium.
  • the subject invention provides microorganisms that have been engineered to enhance cell growth and alanine production in various media.
  • the following microorganisms were deposited with the Agricultural Research Service Culture Collection, 1815 N. University Street, Peoria, Illinois, 61604 U.S.A. These cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122.
  • the deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of the deposits does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
  • the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures.
  • the depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
  • accession numbers and deposit dates are as follows;
  • strain SZ 194 was previously engineered from a derivative of E. coli W (ATCC 27325) and served as a starting point for constructions (Zhou et ah, 2006b).
  • cultures were grown aerobically at 3O 0 C, 37°C, or 39 0 C in Luria broth (1O g 1- D 1 ifco tryptone, 5 g D1i-f 1 co yeast extract and 5 g NaCl1) -1 containing 2% (w/v) glucose or 5% (w/v) arabinose.
  • Ampicillin 50 mg l "1
  • tetracycline 12.5 mg 1 -1
  • kanamycin 50 mg 1-
  • chloramphenicol 40 mg ) w1 -1 ere added as needed.
  • strains were grown without antibiotics at 37°C in NBS mineral salts medium (Causey et ah, 2004) supplemented with 100 mM ammonia sulfate, 1 mM betaine and 2% (w/v) glucose. Fermentation experiments (2%-12% sugar) were carried out in NBS medium and AMI medium (Martinez et ah, 2007; Table 4). Broth was maintained at pH 7 by the automatic addition of 5 M NH 4 OH.
  • the primers for amplifying alaD from G. stearothermophilus XL-65-6 were designed based on the alaD sequence of G. stearothermophilus strain 10.
  • the forward primer (5 '-3' GGAAAAAGGAGGAAAAAGTGATGAAGATCGGCATT) (SEQ ID NO: 1) included the ribosomal binding region (bold) and the amino terminus (underlined).
  • the reverse primer (5'- 3' GAAGGAGTTGATCATTGTTTAACGAGAGAGG) (SEQ ID NO: 2) was downstream from the putative transcriptional terminator region.
  • the filter was immersed in 1OmM potassium phosphate buffer (pH 7.2), and incubated for 20 min at 8O 0 C for lysis of the cells and denaturation of the E. coli proteins.
  • the dried filter paper was assayed in a reaction mixture containing 50 mM L-alanine, 50 mM Tris-HCl buffer (pH 9.0), 0.625 mM NAD + , 0.064 mM phenazine methosulfate, and 0.24 mM nitro blue tetrazolium.
  • the cells with alanine dehydrogenase appeared as blue spots on the filter.
  • the alaD gene was integrated into the chromosomal ldhA gene of SZl 94.
  • the fragment (Smal-Kpnl, 1.7 kb) containing a tet gene flanked by two FRT sites was isolated from pLOI2065 and cloned into pLOI4211 between a unique Bar ⁇ l site (Klenow-treated) and Kpnl site to produce plasmid pLOI4213 (6.0 kb). In this plasmid, transcription of alaD and tet are oriented in the same direction.
  • the Apal (treated with T4 DNA polymerase to produce a blunt end)- ⁇ jr ⁇ I fragment (2.2 kb) containing alaD and tet was isolated from pLOI4213 and cloned into pLOI2395 ⁇ Hindi to Kpnl sites) to produce pLOI4214 (6.5 kb).
  • pLOI4214 6.5 kb
  • idhA, alaD and let genes are transcribed in the same direction.
  • the Ascl fragment (4.3 kb) containing these three genes was isolated from pLOI4214 and cloned into the R6K integration vector pLOI2224 to produce pLOI4215 (6.2 kb).
  • Plasmid pLOI4215 contains resistance genes for both tetracycline and kanamycin ( Figure 1).
  • the Ascl fragment (4.3 kb) containing IdhA, alaD and tet genes was isolated from pLOI4215, further cut by Xmnl to eliminate any remaining uncut plasmid DNA, and electroporated into SZ 194 containing the Red recombinase plasmid pKD46 (Datsenko and Wanner, 2000). Integrants were selected for tetracycline resistance, confirmed by sensitivity to kanamycin and ampicillin and by PCR analysis using the primers of IdhA and its neighboring genes ydbH and hslJ (Table 3). Deletion ofmgsA and dadX genes
  • a modified method for deleting E. coli chromosomal genes was developed using two steps of homologous recombination (Thomason et ah, 2005). With this method, no antibiotic genes or scar sequences remain on the chromosome after gene deletion.
  • part of the target gene was replaced by a DNA cassette containing a chloramphenicol resistance gene ⁇ cat) and levansucrase gene (sacB).
  • the cat-sacB cassette was removed by selection for resistance to sucrose. Cells containing the sacB gene accumulate levan during incubation with sucrose and are killed. Surviving recombinants are highly enriched for loss of the cat-sacB cassette.
  • a new cassette was constructed as a template to facilitate gene deletions.
  • the cat- sacB region was amplified from pEL04 (Lee et ah, 2001; Thomason el ah, 2005) by PCR using the JMcatsacBwpNhel and JMcatsacBdov/nNhel primers (Table 3), digested with Nhe ⁇ , and ligated into the corresponding site in pLOI3421 to produced pLOI4151.
  • the cat-sacB cassette was amplified by PCR using pLOI4151 as a template with the cat-up2 and sacB- down2 primers (EcoRV site included in each primer), digested with EcoRY, and used in subsequent ligations.
  • the mgsA gene and neighboring 500 bp regions were amplified using the mgsA-x ⁇ and mgsA-down primers and cloned into the pCR 2.1-TOPO vector (Invitrogen) to produce plasmid pLOI4228.
  • a 1000-fold diluted plasmid preparation of this plasmid served as a template for inside-out amplification using the mgsA-X and mgsA- 2 primers (both within the mgsA gene and facing outward).
  • the resulting 4958 bp fragment containing the replicon was ligated to the Zscoi?V-digested cat-sacB cassette from pLOI4151 to produce pLOI4229.
  • This 4958 bp fragment was also used to construct a second plasmid, pLOI 4230, by phosphorylation and self-ligation.
  • pLOI4230 the central region of mgsA is deleted (yccT'-mgsA '-mgsA "- helD ').
  • step 1 fragment After electroporation of the step 1 fragment into XZl 15 containing pKD46 (Red recombinase) and 2 h of incubation at 3O 0 C to allow expression and segregation, recombinants were selected for chloramphenicol (40 mg ) a1-n 1 d ampicillin (20 mg I " ) resistance in Luria broth at 3O 0 C (18 h). Three clones were selected, grown in Luria broth containing Ampicillin and arabinose (5% w/v), and prepared for electroporation.
  • step 2 fragment cells were incubated at 37 0 C for 4 h and then transferred into a 250-ml flask containing 100 ml of modified LB (100 mM MOPS buffer added and NaCl omitted) containing 10% sucrose. After overnight incubation (37 0 C), clones were selected on modified LB plates (no sodium chloride; 100 mM MOPS added) containing 6% sucrose (39 0 C, 16 h). Resulting clones were tested for loss of ampicillin and chloramphenicol resistance. Construction was confirmed by PCR using the mgsA up/down primer set. A clone with a deletion in the central region of mgsA was selected and designated XZ121.
  • dadX gene was deleted in a manner analogous to that used to delete the mgsA gene.
  • Primers for dadX deletion are shown in Table 3, and the corresponding plasmids shown in Table 2 and Figure 3.
  • NBS mineral salts medium (Causey et al., 2004) with 1 mM betaine (Zhou et al., 2006a) was used in the initial fermentation (pH 7.0).
  • Pre-inoculum was grown by inoculating three colonies into a 250 ml flask (100 ml NBS medium, 2% glucose, and 100 mM ammonium sulfate). After 16h (37 0 C, 120 rpm), this pre-inoculum was diluted into 500 ml fermentation fieakers containing 300 ml NBS medium (2-8% glucose, 100 mM ammonium sulfate and 1 mM betaine) with 33 mg cell dry wt (CDW) 1-.
  • Cell mass was estimated by measuring the optical density at 550 nm (OD550).
  • Organic acids and glucose concentrations were measured by HPLC (Underwood et al, 2002). Fermentation products were determined by using mass spectroscopy and an amino acid analyser at the University of Florida Interdisciplinary Center for Biotechnology Research. Alanine was found to be the predominant product.
  • the alanine concentration and isomeric purity were further measured by HPLC using the Chiralpak MA(+) chiral column (Chiral Technologies Inc).
  • Alanine dehydrogenase is found in Bacillus (and Geobacillus) species where it plays a pivotal role in energy generation during sporulation (Ohashima and Soda, 1979;
  • ALD from B. sphaericus IFO3525 has been widely used with varying degrees of success to engineer alanine production in heterologous bacteria (Uhlenbusch et al.,
  • IFO3525 is presumed to be due in part to the high specific activity (Ohashima and Soda,
  • thermostable ALD from the thermophile, G. stearothermophilus XL-65-6, based on our prior experience in expressing genes from this organism in recombinant E. coli (Burchhardt and Ingram, 1992; Lai and Ingram, 1993; Lai and Ingram, 1995).
  • the ribosomal-binding region, coding region and transcriptional terminator of alaD were amplified from G. stearothermophilus XL-65-6 and sequenced.
  • the deduced amino acid sequence was identical to that reported for Geobacillus kaustophilus HTA426 and very similar to G. stearothermophilus strain 10 (99% identity), and G. stearothermophilus strain
  • IFO 12550 (94% identity).
  • the nucleotide sequence (65% identity) and the deduced ALD amino acid sequence (74% identity) were quite different from the B. sphaericus IFO3525 gene ( Figure 4A and 4B), the gene previously used for alanine production in heterologous bacteria.
  • E. coli W strain SZ 194 (pflB frdBC adhE ackA) was previously constructed to produce only D-lactic acid. AU major fermentation pathways except lactate have been blocked in this strain by gene deletions.
  • part of the native ldhA coding region was replaced by a DNA fragment containing the ribosomal-binding region, coding region and transcriptional terminator of alaD from G. stear other moph ⁇ lus XL-65-6 ( Figure 5A).
  • the promoterless alaD was oriented in the same direction as ldhA to allow expression from the native ldhA promoter ( Figure 1).
  • XZ 105 was transformed with pFT-A, which contains an inducible FLP recombinase (Martinez-Morales el al., 1999; Posfai et ah, 1997).
  • the chromosomal FRT-flanked tet gene in XZ 105 was removed by inducing the FLP recombinase. After growing in 39°C to eliminate the temperature sensitive plasmid pFT-A, resulting strain was designated XZl I l. Expression of G.
  • stearolhermophilus alaD in XZl I l is transcriptionally regulated by the ldhA promoter, the same promoter that regulates the production of lactate dehydrogenase (dominant fermentation pathway) in native E. coli.
  • the maximum cell mass was 0.41 g whe1n -1 using 2 N potassium hydroxide and 0.397 g 1 -1 for 2 N potassium hydroxide with 1 N ammonia carbonate.
  • the alanine concentration was 174 mM and 170 mM respectively after 4 days.
  • the maximum cell mass was 0.767 g an1d -1 the alanine concentration after 4 days was 387 mM ( Figure 7). Thereafter, 5 N ammonia hydroxide was used in all the fermentations.
  • Strain XZl 12 was then transferred in NBS medium with 50 g 1 -1 glucose. The cell yield and alanine production increased continuously with the transfers ( Figure 8). After transferring for 12 times, one single colony was isolated and designated XZl 13. Strain XZl 13 could complete the fermentation of 50 g 1 -1 glucose in 48 h, producing 483 mM alanine. The alanine yield from glucose was 86% (g/g), and the L-alanine purity was 97.0%.
  • mgsA methylglyoxal synthase gene
  • the methylglyoxal bypass is an alternative pathway that produces lactate in E. coli
  • Strain XZ121 was constructed by deletion of the mgsA gene. After 40 rounds of metabolic evolution to co-select additional mutations that increase growth and product formation
  • XZ123 completed the fermentation of 80 g 1 " ' glucose in 48 h (832 mM alanine), 12 h faster than the parent strain XZl 15.
  • the lactate concentration was below 1 mM although the acetate and succinate levels were slightly higher than in XZl 15 (Table 5).
  • the alanine yield from glucose was 93%, and the L-alanine purity was 97.7%.
  • E. coli has two distinct alanine racemase genes, which are responsible for the conversion between L-alanine and D-alanine (Wild et al., 1985).
  • One racemase encoded by the air gene is constitutive (Wild et ah, 1985) and less abundant than the racemase encoded by dadX (Neidhardt et al., 1996).
  • DADX is a catabolic enzyme that is induced by D- or L- alanine in the absence of glucose.
  • dadX was deleted in XZ 123 to make strain XZ 126. After 30 transfers to allow metabolic evolution, strain XZ129 was isolated ( Figure 13).
  • Strain XZ129 completed the fermentation of 80 g 1 -1 glucose within 42 h, and accumulated 833 mM alanine with a yield of 93%.
  • the chiral purity of L-alanine increased to 99.4% (Table 5).
  • Cell yield and volumetric productivity of alanine were 6-fold and 11 -fold greater, respectively, than with the parent strain XZl 11 (Table 6).
  • a low salts mineral medium AMI
  • AMI low salts mineral medium
  • This medium was tested for alanine production after serially transferring strain XZ 129 for 14 times ( Figure 14).
  • the resulting strain designated XZl 30, completed the fermentation of 80 g gluc1-o 1 se within 42 h, and accumulated 847 mM alanine with the yield of 94%.
  • the cell yield and volumetric productivity was 33% and 3% greater, respectively, than XZ 129 in NBS medium (Table 6).
  • Strain XZl 30 was serially transferred in AMI medium with 100 g 1 -1 glucose for 16 times and the resulting strain designated XZl 31 ( Figure 15). This strain was serially transferred 30 times in 120 g 1 -1 glucose for 30 times to produce XZ132 ( Figure 16). Strain XZ132 completed the fermentation of 120 g 1 " glucose within 48 h, and accumulated 1279 mM alanine (114 g 1 -1 ) with a yield of 95% (Table 3). Cell yield and alanine volumetric productivity were 10 and 30 times greater, respectively than the parent strain XZl I l (Table 6).
  • strain XZl 32 Fermentation of the pentose sugar, xylose (50 g )1 w -1 as also tested in AMI medium. With xylose, strain XZl 32 produced 483 mmol of L-alanine (99.5% chiral purity) in 72 h with a yield of 86% by weight. Further improvement for xylose fermentation to L-alanine would be expected based on metabolic evolution although this was not investigated. Thus strain XZ 132 could be used as a biocatalyst for the conversion of biomass-derived hexose and pentose sugars to L-alanine.
  • This strain produced high titers and yields of alanine but required complex processes (aerobic growth phase and anaerobic production phase) and produced only racemic alanine.
  • multiple copies of the plasmid-borne B. sphaericus alaD gene are expressed from a / ⁇ c/-regulated artificial promoter (Vtrc).
  • All prior biocatalysts that produced high titers and yields of alanine used multi-copy plasmids for the high level expression of recombinant alanine dehydrogenase genes from mesophilic bacteria, antibiotics for plasmid maintenance, and required complex medium supplements such as casamino acids and yeast extract.
  • Strains that produced chirally pure L-alanine typically required supplementation with D-alanine. These media requirements increase the cost of materials, purification, chiral separations, and waste disposal. Multi-step aerobic and anaerobic (fed batch) processes may also increase capital costs.
  • thermophilic ALD ⁇ alaD thermophilic ALD ⁇ alaD
  • E. coli biocatalyst designed for lactate production as the host for this heterologous gene.
  • the deduced amino acid and nucleotide sequences of the B. sphaericus and G. stearothermophilus alaD genes are quite different.
  • the kinetic properties of the G. stearothermophilus ALD would appear less favorable than those of the B. sphaericus ALD.
  • G. stearothermophilus XL-65-6 was selected as a source of alaD based on our previous successes in stably expressing genes from this organism in E. coli with minimal effects on growth (Lai and Ingram, 1993, Lai and Ingram, 1995).
  • the alaD gene was integrated under the control of the chromosomal ldhA promoter, the promoter which controls expression of the dominant fermentative enzyme in native E. coli. In this way, alaD expression would be expected to mimic native regulation of fermentation processes.
  • ATP production for growth was coupled to NADH oxidation using alanine dehydrogenase, providing a growth-based selection for strain improvements by metabolic evolution (Figure 5B). Additional mutations were added to improve growth (mgsA deletion) and chiral purity (dadX deletion) that have not previously been employed in the development of biocatalysts for L-alanine production.
  • Figure 17 provides a summary of the steps involved in strain construction.
  • L-alanine producing strains in this study could be used as a highly efficient route to produce pyruvate using enzymatic hydrolysis.
  • L- alanine is catabolized to pyruvate by a two step process.
  • the DADX racemase interconverts the L and D forms, followed by conversion to pyruvate plus ammonia by a membrane-bound D-amino acid dehydrogenase (Denu and Fitzpatrick, 1992; Lobocka et ah, 1994).
  • This reaction could be accelerated and driven to completion by stripping with air and/or reduced pressure to facilitate removal of ammonia (Figure 18).
  • sugars would be converted to pyruvate with higher efficiencies (using alanine as an intermediate) than previously achieved during direct fermentation of sugars to pyruvate (Causey et ah, 2004).
  • NBS + 1 mM betaine NBS media amended with betaine (1 mM).
  • b Calculation includes KOH used to neutralize betaine-HCl stock.
  • c Trace metal stock (1000X) was prepared in 120 mM HCl. OO
  • Yields are based on grams of alanine produced from total glucose; the maximum theoretical yield for alanine is 0.989 gram per gram of glucose (2 mol of alanine per mol of glucose).
  • Yields in parenthesis are based on grams of alanine produced per gram of glucose consumed.
  • Average volumetric productivity was calculated based on total alanine produced per total fermentation time. d
  • the maximum volumetric productivity and specific productivity of XZl 11 in NBS medium was based on 0-24 h.
  • the maximum volumetric productivity and specific productivity of XZ129 and XZ132 was based on 24-36 h.
  • the maximum volumetric productivity of XZl 30 was based on 12-24 h. g The maximum specific productivity of XZl 30 was based on 0-12 h.
  • the maximum volumetric productivity and specific productivity of XZl 11 in AMI medium was based on 72-96 h.

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Abstract

La présente invention concerne des micro-organismes génétiquement modifiés qui produisent de la L-alanine comme produit de fermentation primaire à partir de sucres. Des sucres de pentose, tels que le xylose, et des sucres d'hexose, tels que le glucose, peuvent être efficacement fermentés en L-alanine. Les souches décrites ici ont la capacité de métaboliser tous les sucres qui constituent la biomasse lignocellulosique et une variété de disaccharides, y compris le lactose, le maltose, le saccharose et d'autres.
PCT/US2008/058410 2007-03-27 2008-03-27 Matériaux et procédés pour une production efficace d'alanine WO2008119009A2 (fr)

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WO2016198529A1 (fr) 2015-06-12 2016-12-15 Basf Se Micro-organisme de recombinaison pour la production améliorée d'alanine
CN110904062A (zh) * 2018-09-18 2020-03-24 安徽华恒生物科技股份有限公司 一株高产l-丙氨酸的菌株
CN110982857A (zh) * 2019-09-23 2020-04-10 安徽丰原生物化学股份有限公司 一种l-丙氨酸的发酵生产方法
EP3929297A4 (fr) * 2020-05-13 2022-09-21 Anhui Huaheng Biotechnology Co., Ltd. Micro-organisme recombinant pour la production de l-valine, procédé de construction s'y rapportant et application associée
WO2023056699A1 (fr) * 2021-10-08 2023-04-13 安徽丰原生物技术股份有限公司 Souche génétiquement modifiée capable de produire de la l-alanine, son procédé de construction et son application
WO2023056700A1 (fr) * 2021-10-08 2023-04-13 安徽丰原生物技术股份有限公司 Souche bactérienne génétiquement modifiée produisant de la dl-alanine, son procédé de construction et son application

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