WO2008119009A4 - Materials and methods for efficient alanine production - Google Patents
Materials and methods for efficient alanine production Download PDFInfo
- Publication number
- WO2008119009A4 WO2008119009A4 PCT/US2008/058410 US2008058410W WO2008119009A4 WO 2008119009 A4 WO2008119009 A4 WO 2008119009A4 US 2008058410 W US2008058410 W US 2008058410W WO 2008119009 A4 WO2008119009 A4 WO 2008119009A4
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- genetically modified
- alanine
- brevibacterium
- modified microorganism
- gene
- Prior art date
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Classifications
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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
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- 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
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- 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)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The subject application provides genetically engineered microorganisms that produce L-alanine as the primary fermentation product from sugars. Pentose sugars, such as xylose, and hexose sugars, such as glucose, can be effectively fermented to L-alanine. The strains described herein have the ability to metabolize all sugars that are constituents of lignocellulosic biomass and a variety of disaccharides, including lactose, maltose, sucrose and others.
Claims
50
AMENDED CLAIMS [received by the International Bureau on 26 January 2009 (26.01.2009) (3 pages)]
9. The genetically modified microorganism according to claim 1, wherein said genetically modified microorganism is metabolically evolved.
10. The genetically modified microorganism according to claim 1, wherein any antibiotic resistance genes are deleted with FLP recombinase.
11. A genetically modified microorganism that comprises the following genetic modifications: a) the optional insertion of a Klebsiella oxytoca casΛB gene or other genes encoding cellobiose utilizing enzyme II and phospho-β-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; h) inactivation or deletion of a methylglyoxal synthase gene; and i) inactivation or deletion of an alanine racemase gene.
12. The genetically modified microorganism according to claim 11, wherein the integrated alanine dehydrogenase gene is a heterologous alanine dehydrogenase gene.
13. The genetically modified microorganism according to claim 11 or 12, wherein the genetic ally modified microorganism is Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacϊer viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescent, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevibacterium ammoniagenes, divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fiiscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusillum, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum,
51
gene have not been inserted into said microorganism and one or more of the ftjmarate reductase subunit genes is inactivated by insertion, or deletion.
18. The genetically modified microorganism according to claim 13, wherein the 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 claim 13, wherein a single alanine dehydrogenase gene is inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.
20. The genetically modified organism according to claim 13, wherein multiple copies of an alanine dehydrogenase gene are inserted into the chromosome of said microorganism or the lactate dehydrogenase gene of said microorganism.
21. The genetically modified microorganism according to claim 11, wherein said genetically modified microorganism is metabolically evolved.
22. The genetically modified microorganism according to claim 11, wherein antibiotic genes are deleted with FLP recombinase.
23. A method of culturing or growing a genetically modified microorganism comprising inoculating a culture medium with one or more genetically modified microorganism according to claims 1 or 11 and culturing or growing said genetically modified microorganism.
24. A method of producing L-alanine comprising culturing one or more genetically modified microorganism according to claim 23 under conditions that allow for the production of alanine.
52
25. The method according to claim 23, wherein said genetically modified microorganism is cultured in a mineral salts medium.
26. The method according to claim 25, wherein the mineral salts medium comprises between 2% and 20% (w/v) of a sugar.
27. The method according to claim 26, wherein 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.
28. The method according to claim 26, wherein the sugar is glucose or sucrose or a combination of glucose, sucrose, pentose and xylose.
29. The method according to claim 24, wherein chiraHy pure alanine is produced.
30. The method according to claim 24, wherein the yield of alanine is at least 90%.
31. The method according to claim 30, wherein the yield is at least 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, or 99%.
32. The method according to claim 24, wherein there is no detectable contamination of one stereoisomeric form of alanine with the other stereoisomeric form or the chiral purity of L-alanine is at least 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.
33. A composition comprising one or more genetically modified microorganism according to claims 1 or 11 and medium.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US90823407P | 2007-03-27 | 2007-03-27 | |
| US60/908,234 | 2007-03-27 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| WO2008119009A2 WO2008119009A2 (en) | 2008-10-02 |
| WO2008119009A3 WO2008119009A3 (en) | 2009-02-26 |
| WO2008119009A4 true WO2008119009A4 (en) | 2009-04-16 |
Family
ID=39789270
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US2008/058410 WO2008119009A2 (en) | 2007-03-27 | 2008-03-27 | Materials and methods for efficient alanine production |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2008119009A2 (en) |
Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004044210A2 (en) | 2002-11-06 | 2004-05-27 | University Of Florida | Materials and methods for the efficient production of acetate and other products |
| CA2688938C (en) | 2007-03-20 | 2018-01-16 | University Of Florida Research Foundation, Inc. | Materials and methods for efficient succinate and malate production |
| US8993287B2 (en) | 2008-11-18 | 2015-03-31 | University Of Florida Research Foundation, Inc. | Biocatalysts and methods for conversion of hemicellulose hydrolysates to biobased products |
| AU2010232533A1 (en) | 2009-04-02 | 2011-10-13 | University Of Florida Research Foundation, Inc. | Engineering the pathway for succinate production |
| CN101974476A (en) * | 2010-08-31 | 2011-02-16 | 安徽华恒生物工程有限公司 | XZ-A26 bacterial strain for producing L-alanine with high yield as well as construction method and application thereof |
| JP2016503650A (en) * | 2012-12-28 | 2016-02-08 | アンホエ ファホン バイオエンジニアリング カンパニー リミテッド | Genetically engineered bacteria that produce DL-alanine, and methods for producing DL-alanine by using the genetically engineered bacteria |
| CN103045528B (en) * | 2012-12-28 | 2014-05-07 | 安徽华恒生物工程有限公司 | Engineering bacteria producing DL-alanine and method of producing DL-alanine by using engineering bacteria |
| BR112016002105A2 (en) * | 2013-08-30 | 2017-08-29 | Basf Se | MODIFIED MICROORGANISM, METHOD FOR PRODUCING ALANINE AND USE OF A MODIFIED MICROORGANISM |
| US10208324B2 (en) * | 2013-12-13 | 2019-02-19 | Basf Se | Recombinant microorganism for improved production of fine chemicals |
| CN106574254A (en) * | 2014-04-30 | 2017-04-19 | 赢创德固赛有限公司 | Method for producing L-amino acids using alkaliphilic bacteria |
| US10717998B2 (en) * | 2015-03-18 | 2020-07-21 | Basf Se | Recombinant microorganism for improved production of fine chemicals |
| CN104774790B (en) * | 2015-04-03 | 2020-11-06 | 江南大学 | Escherichia coli for efficiently fermenting and producing L-alanine |
| CN108350040B (en) * | 2015-06-04 | 2022-03-25 | 巴斯夫欧洲公司 | Recombinant microorganisms for improved production of fine chemicals |
| MX2017016109A (en) * | 2015-06-12 | 2018-04-20 | Basf Se | Recombinant microorganism for improved production of alanine. |
| CN110904062B (en) * | 2018-09-18 | 2022-03-15 | 安徽华恒生物科技股份有限公司 | Strain capable of producing L-alanine at high yield |
| CN110982857A (en) * | 2019-09-23 | 2020-04-10 | 安徽丰原生物化学股份有限公司 | Fermentation production method of L-alanine |
| CN114457123B (en) * | 2020-05-13 | 2023-06-20 | 安徽华恒生物科技股份有限公司 | Recombinant microorganism for producing L-valine as well as construction method and application thereof |
| CN115960942A (en) * | 2021-10-08 | 2023-04-14 | 安徽丰原生物技术股份有限公司 | Genetic engineering strain for producing L-alanine and construction method and application thereof |
| CN115960941A (en) * | 2021-10-08 | 2023-04-14 | 安徽丰原生物技术股份有限公司 | Genetic engineering strain for producing DL-alanine and construction method and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6372457B1 (en) * | 1997-01-14 | 2002-04-16 | Arkion Life Sciences Llc | Process and materials for production of glucosamine |
| US7052883B2 (en) * | 2001-04-03 | 2006-05-30 | Degussa Ag | Process for the production of L-amino acids using strains of the family Enterobacteriaceae that contain an attenuated fruR gene |
| US7037504B2 (en) * | 2001-10-23 | 2006-05-02 | Waratah Pharmaceuticals, Inc. | Epidermal growth factor protein and gene, and methods of use therefor |
| DE10210527A1 (en) * | 2002-03-09 | 2003-09-18 | Degussa | Alleles of the aceA gene from coryneform bacteria |
-
2008
- 2008-03-27 WO PCT/US2008/058410 patent/WO2008119009A2/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2008119009A3 (en) | 2009-02-26 |
| WO2008119009A2 (en) | 2008-10-02 |
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