WO2023142881A1 - Procédé de construction d'une souche produisant de la thréonine - Google Patents

Procédé de construction d'une souche produisant de la thréonine Download PDF

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WO2023142881A1
WO2023142881A1 PCT/CN2022/143762 CN2022143762W WO2023142881A1 WO 2023142881 A1 WO2023142881 A1 WO 2023142881A1 CN 2022143762 W CN2022143762 W CN 2022143762W WO 2023142881 A1 WO2023142881 A1 WO 2023142881A1
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dehydrogenase
enhanced
threonine
pyruvate
activity
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康培
宫卫波
何君
李岩
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廊坊梅花生物技术开发有限公司
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Definitions

  • the invention belongs to the technical field of microbial engineering, and in particular relates to a method for constructing a threonine-producing bacterial strain.
  • threonine from oxaloacetate requires five steps of catalytic reactions, which are aspartate kinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), and homoserine dehydrogenase (encoded by asd). Hydrogenase (hom coded), homoserine kinase (thrB) and threonine synthase (thrC) coded.
  • Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.[J].Journal of Bacteriology,1991,173(10):3228-3230), lysC gene (Eikmanns B J,Eggeling L,Sahm H.Molecular aspects of lysine,threonine,and isoleucine biosynthesis in Corynebacterium glutamicum.[J].Antonie Van Leeuwenhoek,1993,64(2):145-163).
  • the purpose of the present invention is to improve the ability of the strain to produce threonine by inactivating pyruvate quinone dehydrogenase, thereby providing a method for constructing a threonine (L-threonine) producing strain.
  • the present invention provides a modified microorganism of the genus Corynebacterium, which has a reduced or lost activity of pyruvate quinone dehydrogenase compared with an unmodified microorganism, and the microorganism Enhanced threonine production capacity compared to unmodified microorganisms.
  • the reference sequence number of pyruvate quinone dehydrogenase on NCBI is WP_011015247.1, or an amino acid sequence with 90% similarity thereto.
  • the reduction or loss of the activity of pyruvate quinone dehydrogenase in the microorganism is achieved by reducing the expression of the gene encoding pyruvate quinone dehydrogenase or knocking out the endogenous gene encoding pyruvate quinone dehydrogenase.
  • the microorganism is any one of the following 1 ⁇ 5:
  • the enhancement of the activity of enzymes related to the threonine synthesis pathway in the microorganism is achieved by being selected from the following 1) to 6), or an optional combination:
  • the Corynebacterium glutamicum described in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (see NCBI Corunebacterium glutamicum evolutionary tree https:/ /www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
  • the present invention provides a method for constructing a threonine-producing strain, the method comprising:
  • the enhanced approach is selected from the following 1) to 5), or an optional combination:
  • the present invention provides a method for producing threonine, the method comprising the steps of:
  • step b) collecting the threonine produced from said culture obtained in step a).
  • the present invention provides the application of knockout or reduced expression of gene encoding pyruvate quinone dehydrogenase in threonine fermentation production or improvement of threonine fermentation yield.
  • the fermentation yield of threonine is improved by inactivating pyruvate quinone dehydrogenase in Corynebacterium having amino acid production ability.
  • the Corynebacterium glutamicum described in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (see NCBI Corunebacterium glutamicum evolutionary tree https:/ /www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
  • the present invention provides the use of the modified Corynebacterium genus microorganism or the threonine-producing strain constructed according to the above-mentioned method in the fermentative production of threonine or in improving the fermentative yield of threonine.
  • transformation methods of the above-mentioned related strains are transformation methods known to those skilled in the art.
  • the present invention has at least the following advantages and beneficial effects:
  • the present invention focuses on the influence of the inactivation of pyruvate quinone dehydrogenase on the production of threonine, and inactivates the coding gene of pyruvate quinone dehydrogenase on the basis of the Corynebacterium glutamicum ATCC 13032 strain to obtain bacterial strain SMCT322, Threonine
  • the amino acid content is 0.2g/L. It can be seen that the loss of pyruvate quinone dehydrogenase activity is beneficial to the production of threonine.
  • the threonine synthesis pathway of the strain was further strengthened, mainly including aspartokinase, homoserine dehydrogenase, The expression of at least one of aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase, and threonine synthase is enhanced or deregulated.
  • Inactivation or weakening during the transformation process includes promoter replacement, ribosome binding site changes, point mutations, and sequence deletions.
  • Expression enhancement during the transformation process includes promoter replacement and ribosome binding site changes. , copy number increase, plasmid overexpression and other means, and the above means are well known to researchers in the field. The above means cannot be exhausted by examples, and the specific examples only use promoter enhancement as a representative for illustration.
  • the present invention adopts following technical scheme:
  • One of the technical schemes of the present invention provides a method for producing threonine by using a strain that loses activity of pyruvate quinone dehydrogenase.
  • the second technical solution of the present invention provides a method for inactivating pyruvate quinone dehydrogenase and aspartokinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, homoserine kinase, threonine Synthase at least one expression-enhanced or deregulated expression-enhanced strain to produce threonine.
  • the third technical solution of the present invention is to provide a method for producing threonine using a bacterial strain in which pyruvate quinone dehydrogenase is inactivated and the expression of aspartokinase and homoserine dehydrogenase is enhanced.
  • the fourth technical solution of the present invention is to provide a method for producing threonine using strains with inactivated pyruvate quinone dehydrogenase and enhanced expression of aspartate kinase, homoserine dehydrogenase, and aspartate aminotransferase .
  • the seventh technical solution of the present invention is to provide a method for producing threonine using a bacterial strain in which the pyruvate quinone dehydrogenase is inactivated and the expression of aspartokinase, homoserine dehydrogenase, and threonine synthase is enhanced.
  • Aspartokinase encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247.
  • Aspartate semialdehyde dehydrogenase encoding gene name asd, NCBI number: Cgl0252, Cg0307, NCgl0248.
  • Homoserine dehydrogenase encoding gene name hom, NCBI number: Cg1337, Cgl1183, NCgl1136.
  • Threonine synthase encoding gene name thrC, NCBI number: cg2437, Cgl2220, NCgl2139.
  • Homoserine kinase encoding gene thrB, NCBI number: cg1338, Cgl1184, NCgl1137.
  • Aspartate aminotransferase encoding gene aspB, NCBI number: cg0294, Cgl0240, NCgl0237.
  • the PCR amplification system is as follows:
  • the PCR amplification procedure is as follows:
  • Transformation method refer to the instructions of Trans1-T1 Phage Resistant Chemically Competent Cell.
  • the upstream homology arm up was obtained by PCR amplification with the P21/P22 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P23/P24 primer pair
  • the P25/P26 primer lysCg1a-T311I was obtained by PCR amplification
  • the downstream homology arm dn was obtained by PCR amplification with the P27/P28 primer pair. Fusion PCR was carried out with P21/P24 primer pair and up and Psod as templates to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-lysCg1a-T311I-dn was obtained by fusion PCR with P21/P28 primer pair and up-Psod, lysCg1a-T311I, dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I .
  • g1a means that the first base of the start codon of the lysC gene (see SEQ ID NO: 1 for the wild-type gene sequence of lysC) is mutated from g to a
  • T311I means that the 311th base of the aspartokinase encoded by the lysC gene is The amino acid is mutated from T to I.
  • PCR amplification was performed with the P29/P30 primer pair to obtain the upstream homology arm up, and the ATCC14067 genome was used as a template to perform PCR amplification with the P31/P32 primer pair to obtain the promoter fragment PcspB
  • the ATCC13032 genome was used as a template to obtain hom G378E by PCR amplification with P33/P34 primer pair, and the downstream homology arm dn was obtained by PCR amplification with P35/P36 primer pair.
  • the fragment up-PcspB was obtained.
  • the full-length fragment up-PcspB-hom G378E -dn was obtained by fusion PCR with P29/P36 primer pair and up-PcspB, hom G378E , dn as template.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P cspB -hom G378E .
  • the upstream homology arm up was obtained by PCR amplification with the P1/P2 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P3/P4 primer pair
  • the P5/P6 primer Perform PCR amplification to obtain the downstream homology arm dn.
  • the full-length fragment up-Psod-dn was obtained by fusion PCR with P1/P6 primer pair and up, Psod, dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, transformed into Trans1T1 competent cells, and obtained the recombinant plasmid pK18mobsacB-P sod -asd.
  • the upstream homology arm up was obtained by PCR amplification with the P103/P104 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P105/P106 primer pair
  • the P107/P108 primer Perform PCR amplification to obtain the downstream homology arm dn.
  • the full-length fragment up-Psod-dn was obtained by fusion PCR with P103/P108 primer pair and up, Psod, dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P sod -aspB.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18-P cspB -thrB.
  • the upstream homology arm up was obtained by PCR amplification with the P37/P38 primer pair, and the promoter fragment Psod-thrC g1a was obtained by PCR amplification with the P39/P40 primer pair.
  • the dn of the downstream homology arm was obtained by PCR amplification with the /P42 primer pair.
  • the full-length fragment up-Psod-thrC g1a -dn was obtained by fusion PCR with P37/P42 primer pair and up, Psod-thrC g1a , dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-P sod -thrC g1a .
  • g1a means that the first base of the start codon of the thrC gene (see SEQ ID NO: 2 for the wild-type gene sequence of thrC) is mutated from g to a.
  • the primers used in the construction process are shown in Table 1:
  • ATCC13032 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB- ⁇ poxB was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the chromosome due to homology.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT322.
  • ATCC13032 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-Psod-lysC g1a-T311I was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15 mg/L kanamycin, wherein the gene of interest was inserted due to homology into the chromosome.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm.
  • the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT323.
  • SMCT323 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P cspB -hom G378E was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the in the chromosome.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT324.
  • SMCT324 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -asd was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology middle.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT325.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT326.
  • SMCT324 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18-P cspB -thrB was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15 mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology middle.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT327.
  • SMCT324 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -thrC g1a was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the in the chromosome.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT328.
  • SMCT324, SMCT325, SMCT326, SMCT327, SMCT328 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Charpter 23).
  • the recombinant plasmid pK18mobsacB- ⁇ poxB was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm.
  • the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
  • the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strains were named SMCT329, SMCT330, SMCT331, SMCT332, SMCT333.
  • the strains obtained are shown in Table 2:
  • Embodiment 3 constructs bacterial strain shaking flask verification
  • Seed activation medium BHI 3.7%, agar 2%, pH7.
  • Seed medium peptone 5/L, yeast extract 5g/L, sodium chloride 10g/L, ammonium sulfate 16g/L, urea 8g/L, potassium dihydrogen phosphate 10.4g/L, dipotassium hydrogen phosphate 21.4g /L, biotin 5mg/L, magnesium sulfate 3g/L. Glucose 50g/L, pH 7.2.
  • Fermentation medium corn steep liquor 50mL/L, glucose 30g/L, ammonium sulfate 4g/L, MOPS 30g/L, potassium dihydrogen phosphate 10g/L, urea 20g/L, biotin 10mg/L, magnesium sulfate 6g/L , ferrous sulfate 1g/L, VB1 ⁇ HCl 40mg/L, calcium pantothenate 50mg/L, nicotinamide 40mg/L, manganese sulfate 1g/L, zinc sulfate 20mg/L, copper sulfate 20mg/L, pH 7.2.
  • Seed training picking SMCT181, SMCT182, SMCT183, SMCT184, SMCT185, SMCT186, SMCT187, SMCT188, SMCT189, SMCT190, SMCT192, SMCT193, SMCT194, SMCT193, SMCT194, SMCT194, SMCT194, SMCT194 , SMCT195, SMCT196 and SMCT197
  • Fermentation culture inoculate 2 mL of seed solution into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and culture at 33° C. and 220 r/min for 24 hours with shaking.
  • the threonine yields of different pyruvate quinone dehydrogenase inactivated strains were different, ranging from 0.2g/L to 1.2g/L, indicating that the inactivation of pyruvate quinone dehydrogenase was related to the Combinations have different effects, and when combined with aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, aspartate aminotransferase, homoserine kinase in the threonine synthesis pathway When combining at least one expression enhancement of threonine synthase and threonine synthase, the threonine production can be increased by 10-21 times.

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Abstract

La présente invention porte sur un procédé de construction d'une souche productrice de thréonine. Dans la présente invention, l'utilisation d'une souche (Corynebacterium) inactivée par la pyruvate quinone déshydrogénase dans la production de thréonine peut augmenter le rendement en thréonine jusqu'à 25 à 45,8 % par comparaison avec l'utilisation d'une souche non modifiée. En combinant en outre la souche inactivée par la pyruvate quinone déshydrogénase avec l'expression améliorée d'au moins l'une parmi l'aspartokinase, l'homosérine déshydrogénase, l'aspartate semi-aldéhyde déshydrogénase, l'aspartate aminotransférase, l'homosérine kinase, la thréonine synthase, etc, dans une voie de synthèse de thréonine, le rendement en thréonine est augmenté de 10 à 21 fois. La procédé présente une nouvelle approche pour la production à grande échelle de thréonine et présente une grande valeur d'application.
PCT/CN2022/143762 2022-01-29 2022-12-30 Procédé de construction d'une souche produisant de la thréonine WO2023142881A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1767616A2 (fr) * 2005-09-22 2007-03-28 Degussa GmbH Procédé de préparation de L-acides aminés ou de cétoacides en utilisant des bactéries coryneformes ayant une expression attenuée du gène aceE (pdhA)
US20100184162A1 (en) * 2006-02-02 2010-07-22 Akira Imaizumi Method for production of an l-amino acid
CN113322218A (zh) * 2020-02-28 2021-08-31 廊坊梅花生物技术开发有限公司 重组谷氨酸棒杆菌及生产l-苏氨酸的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1767616A2 (fr) * 2005-09-22 2007-03-28 Degussa GmbH Procédé de préparation de L-acides aminés ou de cétoacides en utilisant des bactéries coryneformes ayant une expression attenuée du gène aceE (pdhA)
US20100184162A1 (en) * 2006-02-02 2010-07-22 Akira Imaizumi Method for production of an l-amino acid
CN113322218A (zh) * 2020-02-28 2021-08-31 廊坊梅花生物技术开发有限公司 重组谷氨酸棒杆菌及生产l-苏氨酸的方法

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Title
BLOMBACH BASTIAN; SCHREINER MARK E.; MOCH MATTHIAS; OLDIGES MARCO; EIKMANNS BERNHARD J.: "Effect of pyruvate dehydrogenase complex deficiency on-lysine production with", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER BERLIN HEIDELBERG, BERLIN/HEIDELBERG, vol. 76, no. 3, 2 March 2007 (2007-03-02), Berlin/Heidelberg, pages 615 - 623, XP037048381, ISSN: 0175-7598, DOI: 10.1007/s00253-007-0904-1 *
MA, HONGWU; LIU, HUIJUAN; ZHU, NIANQING; CHEN, TAO: "Metabolic Engineering of Corynebacterium glutamicum for Acetoin Production", JOURNAL OF TIANJIN UNIVERSITY (SCIENCE AND TECHNOLOGY), vol. 47, no. 11, 30 November 2014 (2014-11-30), CN , pages 967 - 972, XP009548026, ISSN: 0493-2137, DOI: 10.11784/tdxbz201304053 *

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