CN116083329A - Method for producing gamma-butyrolactone or 1, 4-butanediol by fermentation - Google Patents

Method for producing gamma-butyrolactone or 1, 4-butanediol by fermentation Download PDF

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CN116083329A
CN116083329A CN202211176997.7A CN202211176997A CN116083329A CN 116083329 A CN116083329 A CN 116083329A CN 202211176997 A CN202211176997 A CN 202211176997A CN 116083329 A CN116083329 A CN 116083329A
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corynebacterium glutamicum
fermentation
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孙宇
杨令霞
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Beijing Green Kangcheng Biotechnology Co ltd
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Priority to PCT/CN2023/114599 priority patent/WO2024066822A1/en
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Abstract

The invention provides a method for producing gamma-butyrolactone or 1, 4-butanediol by fermentation, which takes corynebacterium glutamicum as a chassis through a synthetic biological technology, and obtains GBL by introducing exogenous glutamate decarboxylase gene gadB mutant, alcohol dehydrogenase gene yqhD and endogenous or exogenous gamma-aminobutyric acid transaminase gene gabT, realizing the one-step fermentation production of 4-hydroxybutyric acid by fermenting renewable raw materials such as glucose, sucrose and the like by utilizing recombinant microorganisms, and simply heating and decarboxylating fermentation liquor. Further, by introducing a carboxylic acid reductase CAR gene and a phosphopantetheinyl transferase gene sfp into the microorganism, BDO can be produced by directly fermenting glucose, sucrose, or the like with the recombinant microorganism. The invention realizes the direct fermentation production from renewable raw materials to important chemicals GBL and BDO, the production process is clean and safe, the carbon emission is low, and the invention has important industrial application value.

Description

Method for producing gamma-butyrolactone or 1, 4-butanediol by fermentation
Technical Field
The invention belongs to the technical field of genetic engineering and biological fermentation, and particularly relates to a method for producing gamma-butyrolactone or 1, 4-butanediol by fermentation.
Background
1, 4-Butanediol (BDO) is an important organic chemical and fine chemical raw material, and is widely used for manufacturing various high molecular materials such as polyester, polyurethane, polyether polyol and the like, for example, the BDO is a key monomer for synthesizing engineering plastics such as polybutylene terephthalate (PBT), and is also a key monomer for synthesizing biodegradable materials such as polybutylene succinate (PBS) and PBAT. In recent years, with the advent of plastic-limiting and carbon-neutralizing related policies, the demands of biodegradable materials PBS and PBAT have been developed in a blowout manner, thereby driving the rapid growth of BDO demands in the global market. BDO is also an important precursor for synthesizing chemicals such as tetrahydrofuran, gamma-butyrolactone and the like. Gamma-butyrolactone (GBL) is also an important organic and medical intermediate, has wide application in medicines, pesticides, petrochemical industry and the like, is one of the main components of lithium battery electrolyte, and is also an important raw material for synthesizing chemicals such as 2-pyrrolidone, N-methyl-2-pyrrolidone (NMP), polyvinylpyrrolidone (PVP) and the like.
At present, BDO is synthesized mainly by a chemical method and by taking calcium carbide and the like as raw materials through a complex chemical conversion process, which is an extremely high-energy-consumption process, so that an environment-friendly, energy-saving and consumption-reducing biological method technology is developed, and the BDO produced by directly fermenting renewable biomass raw materials has important industrial application value. The production of GBL is mainly produced by taking BDO as a raw material through a chemical method, and no report on direct synthesis of GBL through a biological method is found at present.
Disclosure of Invention
The invention aims to provide a method for producing gamma-butyrolactone or 1, 4-butanediol by fermentation.
The invention is characterized in that: according to the synthetic biology technology, corynebacterium glutamicum is used as a chassis, exogenous glutamate decarboxylase gene gadB mutant, alcohol dehydrogenase gene yqhD and endogenous or exogenous gamma-aminobutyric acid transaminase gene gabT are enhanced by introducing the chassis, the first realization of one-step fermentation production of 4-hydroxybutyric acid by fermenting renewable raw materials such as glucose, sucrose and the like by utilizing recombinant microorganisms is achieved, and GBL can be obtained by simply heating and dehydrating fermentation liquor. Further, by introducing a carboxylic acid reductase CAR gene and a phosphopantetheinyl transferase gene sfp into the microorganism, BDO can be produced by directly fermenting glucose, sucrose and the like by using the recombinant microorganism.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a recombinant corynebacterium glutamicum constructed by overexpressing a glutamate decarboxylase mutant gene gadB and an alcohol dehydrogenase gene yqhD in corynebacterium glutamicum.
In the invention, the glutamate decarboxylase mutant gene gadB and the alcohol dehydrogenase gene yqhD are derived from escherichia coli; the amino acid sequences encoded by the gene galB and the gene yqhD are respectively shown in SEQ ID NO. 1 and SEQ ID NO. 2.
Further, an endogenous or exogenous gamma-aminobutyric acid transaminase gene gabT is expressed in the recombinant corynebacterium glutamicum.
The gamma-aminobutyric acid transaminase gene gabT can be derived from corynebacterium glutamicum, escherichia coli or pseudomonas putida, and the coded amino acid sequences are respectively shown as SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5.
In a second aspect, the invention provides the use of said recombinant corynebacterium glutamicum in the fermentative production of gamma-butyrolactone.
Further, the recombinant corynebacterium glutamicum is utilized to ferment and produce gamma-butyrolactone by taking an inexpensive carbon source as a raw material.
The inexpensive carbon source may be at least one selected from glucose, sucrose, maltose, cellobiose, and the like.
In a third aspect, the invention provides a corynebacterium glutamicum engineering bacterium, which is constructed by expressing a carboxylic acid reductase CAR gene and a phosphopantetheinyl transferase gene sfp in the recombinant corynebacterium glutamicum.
The CAR gene can be derived from Mycobacterium marinum, nocardia iowensis, mycolicibacterium smegmatis or Mycobacteroides abscessus, and the coded amino acid sequences are respectively shown as SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9.
The phosphopantetheinyl transferase gene sfp can be derived from bacillus subtilis, and the coded amino acid sequence is shown in SEQ ID NO. 10.
In a fourth aspect, the invention provides an application of the corynebacterium glutamicum engineering bacterium in fermentation production of 1, 4-butanediol.
Further, the corynebacterium glutamicum engineering bacteria are utilized to ferment and produce gamma-butyrolactone by taking a cheap carbon source as a raw material.
The inexpensive carbon source may be at least one selected from glucose, sucrose, maltose, cellobiose, and the like.
In a fifth aspect, the invention provides a glutamic acid decarboxylase mutant, the amino acid sequence of which is shown as SEQ ID NO. 1.
In a sixth aspect, the present invention provides a method for constructing the recombinant corynebacterium glutamicum and the engineering bacterium of corynebacterium glutamicum, wherein the genes can be modified or altered by conventional genetic engineering methods.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
the invention mainly uses corynebacterium glutamicum as a chassis by a synthetic biology technology, introduces exogenous glutamate decarboxylase gene gadB mutant, alcohol dehydrogenase gene yqhD and enhanced endogenous or exogenous gamma-aminobutyric acid transaminase gene gabT into the chassis, initially realizes the one-step fermentation production of 4-hydroxybutyric acid by fermenting renewable raw materials such as glucose, sucrose and the like by recombinant microorganisms, and obtains GBL by simply heating and decarboxylating fermentation liquor. Further, by introducing a carboxylic acid reductase CAR gene and a phosphopantetheinyl transferase gene sfp into the microorganism, BDO can be produced by directly fermenting glucose, sucrose and the like by using the recombinant microorganism. The invention realizes the direct fermentation production from renewable raw materials to important chemicals GBL and BDO, the production process is clean and safe, the carbon emission is reduced by more than 60% compared with a chemical method, and the invention has important industrial application value.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular Cloning: a Laboratory Manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 construction method of recombinant Corynebacterium glutamicum producing GBL
No microorganism can naturally synthesize GBL in nature. According to the invention, glutamate produced by corynebacterium glutamicum is firstly converted into gamma-aminobutyric acid by introducing glutamate decarboxylase gabB into corynebacterium glutamicum, the gamma-aminobutyric acid aminotransferase gabT is further converted into 4-hydroxybutyric acid under the action of self or exogenously introduced gamma-aminobutyric acid aminotransferase gabT and exogenously introduced alcohol dehydrogenase yqhD, and the 4-hydroxybutyric acid in fermentation liquor generates GBL under acid catalysis.
The gene fragment of glutamic acid decarboxylase gabB of the escherichia coli is artificially synthesized (the amino acid sequence is shown as SEQ ID NO:1, and the gene sequence is shown as SEQ ID NO: 11). Compared with wild-type glutamate decarboxylase, the glutamate decarboxylase has higher activity under neutral pH conditions. PCR was performed using the gene fragment as a template and the primers of gapB-F (5'-attaagcttgcatgcctgcactttaagaaggagatataccatggataagaagcaagtaacg-3') and gapB-R (5'-ggtatatctccttcttaaagttagtgatcgctgagatatt-3'), to obtain a gapB fragment of about 1.4kb and to perform PCR purification. The genome of Escherichia coli MG1655 was used as a template, and yqhD-F (5'-ctttaagaaggagatataccatgaacaactttaatctgcac-3') and yqhD-R (5'-ggtacccggggatcctctagttagcgggcggcttcgtata-3') were used as primers to carry out PCR, whereby a yqhD fragment of about 1.2kb was obtained and PCR purification was carried out. The pXMJ19 plasmid (purchased from Addgene) was digested with PstI and XbaI, and the purified gabB fragment and yqhD fragment were ligated to pXMJ19 in one step using Gibson Assembly kit (NEB), and the obtained recombinant plasmid was named pXMJ-gabB-yqhD.
PCR was performed using the genome of Corynebacterium glutamicum as a template and the primers gabT_cg-F (5'-ggatccccgggtaccgagctctttaagaaggagatataccgtggaagatctctcataccg-3') and gabT_cg-R (5'-caaaacagccaagctgaattttagcccaccttctggtgcg-3'), to obtain a gabT_cg fragment of about 1.35kb and PCR purification was performed. PCR was performed using the genome of Escherichia coli MG1655 as a template and the primers gabT_ec-F (5'-ggatccccgggtaccgagctctttaagaaggagatataccatgaacagcaataaagagtt-3') and gabT_ec-R (5'-caaaacagccaagctgaattctactgcttcgcctcatcaa-3'), to obtain a gabT_ec fragment of about 1.35kb, and PCR purification was performed. PCR was performed using the genome of Pseudomonas putida as a template and the primers gabT_pp-F (5'-ggatccccgggtaccgagctctttaagaaggagatataccatgagcaagaccaacgaatc-3') and gabT_pp-R (5'-caaaacagccaagctgaatttcaggcaagttcagcgaagc-3') as primers, to obtain a gabT_pp fragment of about 1.35kb and PCR purification was performed. The plasmid pXMJ-gabB-yqhD was digested with KpnI and EcoRI, and the above-purified gabT_cg fragment, gabT_ec fragment and gabT_pp fragment were ligated to pXMJ-gabB-yqhD using the Gibson Assembly kit (NEB), respectively, and the obtained recombinant plasmids were named pXMJ-gabB-yqhD-gabT_cg, pXMJ-gabB-yqhD-gabT_ec and pXMJ-gabB-yqhD-gabT_pp, respectively.
Plasmids pXMJ-gabB-yqhD, pXMJ-gabB-yqhD-gabT_cg, pXMJ-gabB-yqhD-gabT_ec, and pXMJ-gabB-yqhD-gabT_pp were transferred to Corynebacterium glutamicum S9114 by electrotransformation, and recombinant bacteria were obtained by screening on chloramphenicol LB plates containing 10mg/L, and designated S9114/pXMJ-gabB-yqhD, S9114/pXMJ-gabB-yqhD-gabT_cg, S9114/pXMJ-gabB-yqhD-gabT_ec, and S9114/pXMJ-gabB-yqhD-gabT_pp, respectively. Simultaneously, the pXMJ19 empty plasmid was also transferred into Corynebacterium glutamicum S9114 to obtain control strain S9114/pXMJ.
Example 2 fermentation production of GBL Using inexpensive sugar feedstock
Strains S9114/pXMJ-gabB-yqhD, S9114/pXMJ-gabB-yqhD-gabT_cg, S9114/pXMJ-gabB-yqhD-gabT_ec, S9114/pXMJ-gabB-yqhD-gabT_pp and a control strain S9114/pXMJ were inoculated into a 5L fermenter to conduct cultivation, the initial volume of the broth was 2L, the fermentation temperature was 30℃and the ventilation amount was 1vvm, the dissolved oxygen value of the fermentation process was maintained at 20% by adjusting the rotation speed, the pH of the broth was controlled at 7.0 by automatic feeding of 25% ammonia, and the fermentation was conducted for 4 hours by adding 0.1mM IPTG and the fermentation time was 48 hours.
The fermentation medium formulation included (g/L): glucose 100, (NH) 4 ) 2 SO 4 20，K 2 HPO 4 1.0，MgSO 4 0.5，MnSO 4 0.2，FeSO 4 0.2, corn steep liquor 20, pyridoxal phosphate 0.01, thiamine hydrochloride 0.001 and chloramphenicol 0.005.
After 48 hours of fermentation, the fermentation was stopped, the pH was adjusted to 0.8 by adding concentrated HCl to the fermentation broth, and the reaction was carried out at room temperature for 24 hours, followed by detection of the strain product by High Performance Liquid Chromatography (HPLC). The strains S9114/pXMJ-gabB-yqhD, S9114/pXMJ-gabB-yqhD-gabT_cg, S9114/pXMJ-gabB-yqhD-gabT_ec, S9114/pXMJ-gabB-yqhD-gabT_pp can produce GBL of 7.2g/L, 11.3g/L, 14.1g/L, 13.7g/L, respectively, whereas the control strain S9114/pXMJ does not produce GBL. It is demonstrated that the introduction of an artificial route in Corynebacterium glutamicum can successfully achieve efficient production of glucose to GBL.
The carbon source of the fermentation medium is changed from 100g/L glucose to 100g/L sucrose, other components are unchanged, and the fermentation process is kept completely consistent. The strains S9114/pXMJ-gabB-yqhD, S9114/pXMJ-gabB-yqhD-gabT_cg, S9114/pXMJ-gabB-yqhD-gabT_ec, S9114/pXMJ-gabB-yqhD-gabT_pp can produce GBL of 7.4g/L, 10.2g/L, 13.3g/L, 12.9g/L, respectively, whereas the control strain S9114/pXMJ does not produce GBL. The introduction of an artificial pathway into Corynebacterium glutamicum has been shown to successfully achieve efficient production of sucrose to GBL.
The carbon source of the fermentation medium is changed from 100g/L glucose to molasses (the sucrose content is 100 g/L), other components are unchanged, and the fermentation process is kept completely consistent. The strains S9114/pXMJ-gabB-yqhD, S9114/pXMJ-gabB-yqhD-gabT_cg, S9114/pXMJ-gabB-yqhD-gabT_ec, S9114/pXMJ-gabB-yqhD-gabT_pp can produce GBL of 6.7g/L, 9.9g/L, 12.1g/L, 11.7g/L, respectively, whereas the control strain S9114/pXMJ does not produce GBL. It is demonstrated that the introduction of an artificial route in Corynebacterium glutamicum can successfully achieve efficient production of molasses to GBL.
EXAMPLE 3 construction method of recombinant Corynebacterium glutamicum producing BDO
The strain constructed by the method can efficiently convert low-cost carbon sources such as glucose, sucrose, molasses and the like to produce GBL. The invention further converts the precursor 4-hydroxybutyric acid of GBL into BDO by introducing carboxylic acid reductase, thereby realizing the direct fermentation production of BDO by using cheap carbon sources.
The main function of the phosphopantetheinyl transferase gene sfp (the amino acid sequence is shown as SEQ ID NO:10, the gene sequence is shown as SEQ ID NO: 20) of the artificially synthesized bacillus subtilis is to activate carboxylic acid reductase. PCR was performed using the gene fragment as a template and sfp-F (5'-atcctctagagtcgacctgcactttaagaaggagatataccaatgaaaatttacggcatcta-3') and sfp-R (5'-cagtgccaagcttgcatgcctcaaagtaactcctcgtagg-3') as primers, to obtain an sfp fragment of about 0.7kb, and PCR purification was performed. The pEC-K18mob plasmid (from Addgene) was digested singly with PstI and the sfp fragment obtained by the above purification was ligated in one step to pEC-K18mob using Gibson Assembly kit (NEB), and the recombinant plasmid obtained was designated pEC-sfp.
Car_mm, car_ni, car_ms and Car_ma (the amino acid sequences are shown as SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively) of carboxylic acid reductase genes Car_mm, car_ni, car_ms and Car_ma derived from Mycobacterium marinum, nocardia iowensis, mycolicibacterium smegmatis and Mycobacteroides abscessus are artificially synthesized, and the nucleotide sequences are shown as SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19 respectively. The plasmid pEC-sfp was digested with EcoRI and KpnI, and the four gene fragments synthesized as described above were inserted into the plasmids pEC-sfp, respectively, and the obtained plasmids were designated pEC-Car_mm-sfp, pEC-Car_ni-sfp, pEC-Car_ms-sfp, pEC-Car_ma-sfp, respectively. Plasmids pEC-Car_mm-sfp, pEC-Car_ni-sfp, pEC-Car_ms-sfp, pEC-Car_ma-sfp were transformed into Corynebacterium glutamicum strain S9114/pXMJ-gabB-yqhD-gabT_ec, respectively, and the obtained recombinant strains were designated as S9114-X-MM, S9114-X-NI, S9114-X-MS, S9114-X-MA, respectively.
Strains S9114-X-MM, S9114-X-NI, S9114-X-MS, S9114-X-MA and control strains S9114/pXMJ-gabB-yqhD-gabT_ec and S9114/pXMJ are inoculated into a 5L fermentation tank for culture, the initial volume of fermentation liquor is 2L, the fermentation temperature is 30 ℃, the ventilation rate is 1vvm, the dissolved oxygen value in the fermentation process is maintained at 20% by adjusting the rotation speed, the pH of the fermentation liquor is controlled to be 7.0 by automatically feeding 25% ammonia water, and 0.1m MIPTG is added for induction during 4h of fermentation, and the fermentation time is 48h.
The fermentation medium formulation included (g/L): glucose 100, (NH) 4 ) 2 SO 4 20,K 2 HPO 4 1.0,MgSO 4 0.5,MnSO 4 0.2,FeSO 4 0.2, corn steep liquor 20, pyridoxal phosphate 0.01, thiamine hydrochloride 0.001 and chloramphenicol 0.005.
Fermentation was stopped after 48 hours of fermentation, and then the products of the strain were detected by High Performance Liquid Chromatography (HPLC). Strains S9114-X-MM, S9114-X-NI, S9114-X-MS, S9114-X-MA produced 7.8g/L, 6.9g/L, 7.2g/L, 7.7g/L BDO, respectively, whereas neither of the control strains S9114/pXMJ-gabB-yqhD-gabT_ec nor S9114/pXMJ produced BDO. It was demonstrated that the introduction of an artificial route in Corynebacterium glutamicum could successfully achieve efficient production of glucose to BDO.
The carbon source of the fermentation medium is changed from 100g/L glucose to 100g/L sucrose, other components are unchanged, and the fermentation process is kept completely consistent. Strains S9114-X-MM, S9114-X-NI, S9114-X-MS, S9114-X-MA produced BDO at 6.4g/L, 6.2g/L, 6.3g/L, 6.9g/L, respectively, whereas neither of the control strains S9114/pXMJ-gabB-yqhD-gabT_ec nor S9114/pXMJ produced BDO. The introduction of an artificial pathway in Corynebacterium glutamicum was shown to successfully achieve efficient production of sucrose to BDO.
The carbon source of the fermentation medium is changed from 100g/L glucose to molasses (the sucrose content is 100 g/L), other components are unchanged, and the fermentation process is kept completely consistent. Strains S9114-X-MM, S9114-X-NI, S9114-X-MS, S9114-X-MA produced BDO at 5.7g/L, 5.9g/L, 5.1g/L, 4.7g/L, respectively, whereas neither of the control strains S9114/pXMJ-gabB-yqhD-gabT_ec nor S9114/pXMJ produced BDO. It was demonstrated that the introduction of an artificial route in Corynebacterium glutamicum could successfully achieve efficient production of molasses to BDO.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The recombinant corynebacterium glutamicum is characterized in that the recombinant corynebacterium glutamicum is obtained by over-expressing a glutamate decarboxylase mutant gene gadB and an alcohol dehydrogenase gene yqhD in the corynebacterium glutamicum;
the glutamate decarboxylase mutant gene gadB and the alcohol dehydrogenase gene yqhD are derived from escherichia coli; the amino acid sequences encoded by the gene galB and the gene yqhD are respectively shown in SEQ ID NO. 1 and SEQ ID NO. 2.
2. The recombinant corynebacterium glutamicum according to claim 1, wherein an endogenous or exogenous gamma-aminobutyric acid transaminase gene gabT is further expressed in the recombinant corynebacterium glutamicum.
3. The recombinant corynebacterium glutamicum according to claim 2, wherein said gamma-aminobutyric acid transaminase gene gabT is derived from corynebacterium glutamicum, escherichia coli or pseudomonas putida, and the amino acid sequences encoded by the gene gabT are shown in SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5, respectively.
4. Use of a recombinant corynebacterium glutamicum according to any one of claims 1 to 3 for the fermentative production of gamma-butyrolactone.
5. The use according to claim 4, wherein the gamma-butyrolactone is produced by fermentation using an inexpensive carbon source as a starting material;
the cheap carbon source is at least one selected from glucose, sucrose, maltose and cellobiose.
6. The corynebacterium glutamicum engineering bacterium is characterized in that the engineering bacterium is obtained by constructing a carboxylic acid reductase CAR gene and a phosphopantetheinyl transferase gene sfp expressed in the recombinant corynebacterium glutamicum according to any one of claims 1 to 3.
7. The engineering bacterium according to claim 6, wherein the carboxylic acid reductase CAR gene is derived from Mycobacterium marinum, nocardia iowensis, mycolicibacterium smegmatis or Mycobacteroides abscessus, and the amino acid sequences encoded by the carboxylic acid reductase CAR gene are shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8 or SEQ ID NO. 9, respectively; and/or
The phosphopantetheinyl transferase gene sfp is derived from bacillus subtilis, and the coded amino acid sequence is shown in SEQ ID NO. 10.
8. The use of the engineering bacteria of claim 6 or 7 in the fermentative production of 1, 4-butanediol.
9. The use according to claim 8, characterized in that 1, 4-butanediol is produced by fermentation starting from an inexpensive carbon source;
the cheap carbon source is at least one selected from glucose, sucrose, maltose and cellobiose.
10. The glutamic acid decarboxylase mutant is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117089507A (en) * 2023-09-28 2023-11-21 北京化工大学 Escherichia coli genetically engineered bacterium for synthesizing 1, 4-butanediol by using cheap carbon source and application thereof
WO2024066822A1 (en) * 2022-09-26 2024-04-04 北京绿色康成生物技术有限公司 METHOD FOR PRODUCING γ-BUTYROLACTONE OR 1,4-BUTANEDIOL BY FERMENTATION

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101668861A (en) * 2007-03-16 2010-03-10 基因组股份公司 compositions and methods for the biosynthesis of 1,4-butanediol and its precursors
US20100112654A1 (en) * 2008-09-10 2010-05-06 Burk Mark J Microorganisms for the production of 1,4-butanediol
US20110014669A1 (en) * 2009-01-23 2011-01-20 Microbia, Inc. Production of 1,4 Butanediol in a Microorganism
US20110129899A1 (en) * 2009-10-13 2011-06-02 Robert Haselbeck Microorganisms for the production of 1,4-butanediol, 4-hydroxybutanal, 4-hydroxybutyryl-coa, putrescine and related compounds, and methods related thereto
CN102498215A (en) * 2009-06-04 2012-06-13 基因组股份公司 Microorganisms for the production of 1,4-butanediol and related methods
CN102781926A (en) * 2010-02-11 2012-11-14 梅塔玻利克斯公司 Process for gamma-butyrolactone production
US20140030779A1 (en) * 2012-06-04 2014-01-30 Genomatica, Inc. Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds
US20140170714A1 (en) * 2011-08-10 2014-06-19 Metabolix, Inc. Post process purification for gamma-butyrolactone production
US20140371417A1 (en) * 2013-04-26 2014-12-18 Genomatica, Inc. Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds
CN105647912A (en) * 2014-12-01 2016-06-08 清华大学 Acid resistance expression cassette and screening method thereof
US20160264978A1 (en) * 2011-06-22 2016-09-15 Genomatica, Inc. Microorganisms for producing 1,4-butanediol and methods related thereto
US20190276863A1 (en) * 2009-11-25 2019-09-12 Genomatica, Inc. Microorganisms and methods for the coproduction 1,4-butanediol and gamma-butyrolactone

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101381002B1 (en) * 2012-04-23 2014-04-04 동국대학교 산학협력단 Method for gamma-aminobutyric acid with glycerol as substrate by biotransformation process
BR112016029375A2 (en) * 2014-06-16 2017-10-17 Invista Tech Sarl methods, reagents and cells to biosynthesize compounds
US9920339B2 (en) * 2014-06-16 2018-03-20 Invista North America S.A.R.L. Methods, reagents and cells for biosynthesizing compounds
CN116083329A (en) * 2022-09-26 2023-05-09 北京绿色康成生物技术有限公司 Method for producing gamma-butyrolactone or 1, 4-butanediol by fermentation

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101668861A (en) * 2007-03-16 2010-03-10 基因组股份公司 compositions and methods for the biosynthesis of 1,4-butanediol and its precursors
US20100112654A1 (en) * 2008-09-10 2010-05-06 Burk Mark J Microorganisms for the production of 1,4-butanediol
US20110014669A1 (en) * 2009-01-23 2011-01-20 Microbia, Inc. Production of 1,4 Butanediol in a Microorganism
CN102498215A (en) * 2009-06-04 2012-06-13 基因组股份公司 Microorganisms for the production of 1,4-butanediol and related methods
CN113528417A (en) * 2009-06-04 2021-10-22 基因组股份公司 Microorganisms for producing 1, 4-butanediol and related methods
US20110129899A1 (en) * 2009-10-13 2011-06-02 Robert Haselbeck Microorganisms for the production of 1,4-butanediol, 4-hydroxybutanal, 4-hydroxybutyryl-coa, putrescine and related compounds, and methods related thereto
US20190276863A1 (en) * 2009-11-25 2019-09-12 Genomatica, Inc. Microorganisms and methods for the coproduction 1,4-butanediol and gamma-butyrolactone
CN102781926A (en) * 2010-02-11 2012-11-14 梅塔玻利克斯公司 Process for gamma-butyrolactone production
US20160264978A1 (en) * 2011-06-22 2016-09-15 Genomatica, Inc. Microorganisms for producing 1,4-butanediol and methods related thereto
US20140170714A1 (en) * 2011-08-10 2014-06-19 Metabolix, Inc. Post process purification for gamma-butyrolactone production
US20140030779A1 (en) * 2012-06-04 2014-01-30 Genomatica, Inc. Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds
US20140371417A1 (en) * 2013-04-26 2014-12-18 Genomatica, Inc. Microorganisms and methods for production of 4-hydroxybutyrate, 1,4-butanediol and related compounds
CN105647912A (en) * 2014-12-01 2016-06-08 清华大学 Acid resistance expression cassette and screening method thereof

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
JAE WOONG CHOI等: "Enhanced production of gamma-aminobutyrate(GABA) in recombinant Corynebacterium glutamicum by expressing glutamate decarboxylase active in expanded pH range", 《MICROBIAL CELL FACTORIES》, vol. 14, no. 21, 15 February 2015 (2015-02-15), pages 2, XP021211597, DOI: 10.1186/s12934-015-0205-9 *
JIAN WANG等: "Bacterial synthesis of C3-C5 diols via extending amino acid catabolism", 《PNAS》, vol. 117, no. 32, 27 July 2020 (2020-07-27), pages 19160 - 19163 *
无: "GenBank: HBP9016664.1", 《GENBANK》, 1 February 2022 (2022-02-01) *
无: "GenBank: PNG88356.1", 《GENBANK》, 18 January 2018 (2018-01-18) *
无: "NCBI Reference Sequence: WP_001058802.1", 《GENBANK》, 13 March 2021 (2021-03-13) *
无: "NCBI Reference Sequence: WP_001087611.1", 《GENBANK》, 16 December 2020 (2020-12-16) *
无: "NCBI Reference Sequence:005082584.1", 《GENBANK》, 7 January 2020 (2020-01-07) *
无: "NCBI Reference Sequence:WP_ 011013682.1", 《GENBANK》, 25 May 2020 (2020-05-25) *
无: "NCBI Reference Sequence:WP_003234549.1", 《GENBANK》, 10 July 2019 (2019-07-10) *
无: "NCBI Reference Sequence:WP_011730755.1", 《GENBANK》, 6 January 2020 (2020-01-06) *
无: "NCBI Reference Sequence:WP_012393886.1", 《GENBANK》, 15 June 2019 (2019-06-15) *
无: "UniProtKB/Swiss-Prot:Q6RKB1.1", 《GENBANK》, 25 May 2022 (2022-05-25) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024066822A1 (en) * 2022-09-26 2024-04-04 北京绿色康成生物技术有限公司 METHOD FOR PRODUCING γ-BUTYROLACTONE OR 1,4-BUTANEDIOL BY FERMENTATION
CN117089507A (en) * 2023-09-28 2023-11-21 北京化工大学 Escherichia coli genetically engineered bacterium for synthesizing 1, 4-butanediol by using cheap carbon source and application thereof

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