WO2023151421A1 - Micro-organisme corynebacterium modifié, son utilisation et son procédé de construction - Google Patents

Micro-organisme corynebacterium modifié, son utilisation et son procédé de construction Download PDF

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WO2023151421A1
WO2023151421A1 PCT/CN2022/143761 CN2022143761W WO2023151421A1 WO 2023151421 A1 WO2023151421 A1 WO 2023151421A1 CN 2022143761 W CN2022143761 W CN 2022143761W WO 2023151421 A1 WO2023151421 A1 WO 2023151421A1
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enhanced
gene
microorganism
threonine
enzyme
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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 relates to the technical field of microbial engineering, in particular to a modified microorganism of the genus Corynebacterium and its application and construction method.
  • L-threonine (L-Threonin), the chemical name is ⁇ -hydroxy- ⁇ -aminobutyric acid, the molecular formula is C 4 H 9 NO 3 , and the relative molecular mass is 119.12. L-threonine is an essential amino acid. Threonine is mainly used in medicine, chemical reagents, food fortifiers, feed additives, etc.
  • 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 (encoded by hom), homoserine kinase (encoded by thrB) and threonine synthase (encoded by thrC). Hermann Sahm et al.
  • 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 or weakening the expression of the Cgl0978 gene, thereby providing a threonine (L-threonine) producing strain and its construction method and application.
  • the present invention provides a modified microorganism of the genus Corynebacterium, the expression of the Cgl0978 gene of the microorganism is reduced or lost compared with the unmodified microorganism, and the The modified microorganism has enhanced threonine production capacity.
  • the same genes as the Cgl0978 gene also have genes numbered NCgl0939 and cg1116.
  • Threonine is catalyzed by threonine dehydratase to generate isoleucine.
  • the gene encoding this enzyme is ilvA, and the mainstream is to reduce the by-product of isoleucine.
  • the method is to reduce the expression level of ilvA, or inactivate ilvA.
  • Cgl0978 has the function of threonine dehydratase, and the production of isoleucine is reduced by inactivating Cgl0978, and the ability of the bacterial strain to synthesize threonine is improved.
  • Mutagenesis, site-directed mutation or homologous recombination can be used to reduce or inactivate the expression of the Cgl0978 gene (such as knocking out the endogenous Cgl0978 gene).
  • the activity of enzymes related to the threonine synthesis pathway and/or precursor supply pathway in the microorganism is enhanced; wherein, the threonine synthesis pathway and/or precursor Enzymes related to the supply pathway are selected from at least one of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase; preferably, their reference sequences on NCBI
  • the numbers are respectively WP_003855724.1, WP_003854900.1, WP_011013816.1, WP_011014465.1, or amino acid sequences with a similarity of 90% to the above reference sequence.
  • the microorganism is any one of the following 1 ⁇ 4:
  • the enhancement of the activity of enzymes related to the threonine synthesis pathway and/or precursor supply pathway in the microorganism is achieved by being selected from the following 1) to 5), 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:
  • step B Enhancing the enzymes related to the threonine synthesis pathway and/or precursor supply pathway in the gene-weakened strain of step A to obtain a strain with enhanced enzyme activity;
  • the enhanced approach is selected from the following 1) to 5), or an optional combination:
  • the enzymes related to the threonine synthesis pathway and/or precursor supply pathway are selected from aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, phosphoenol pyruvate carboxylase at least one of .
  • 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 Cgl0978 gene attenuation or inactivation in threonine fermentation production or improvement of threonine fermentation yield.
  • the fermentation yield of threonine is improved by inactivating the Cgl0978 gene 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 Cgl0978 coding region is inactivated by removing it from the genome.
  • lysC By mutating the gene lysC encoding aspartokinase, its start codon is mutated from GTG to ATG, the 311th amino acid encoded by it is changed from threonine to isoleucine, and the lysC gene is changed from Psod Initiates transcription, culminating in expression enhancement and deregulation of aspartokinase.
  • the nucleotide sequence of Psod is shown in SEQ ID NO.37.
  • the Cgl0978 inactivated strain is applied to threonine production, and the threonine yield can be increased by 20.8-51.2% compared with that before the modification, and the isoleucine content can be reduced from 0.8g/L to 0.2g/L.
  • the inactivated Cgl0978 is further enhanced and reconciled with the expression of at least one of aspartokinase, homoserine dehydrogenase, pyruvate carboxylase, and phosphoenolpyruvate carboxylase in the threonine synthesis pathway.
  • the yield of threonine is increased, and the yield of isoleucine, a downstream product of threonine, is decreased, which provides a new idea for improving the production capacity of threonine.
  • Aspartokinase encoded gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247;
  • Threonine synthase encoding gene name thrC, NCBI number: cg2437, Cgl2220, NCgl2139;
  • Phosphoenolpyruvate carboxylase encoding gene ppc, NCBI number: cg1787, Cgl1585, NCgl1523.
  • the model strain ATCC13032 was used as the starting strain to construct the inactivated strain of Cgl0978, and it was found that the inactivation of Cgl0978 had no effect on the strain. Since the model strain ATCC13032 is not a threonine-producing bacterium, it is foreseeable that the inactivation of Cgl0978 has no obvious effect on the strain of.
  • the present invention firstly constructed a strain with threonine production capacity, first deregulated and strengthened the expression of aspartokinase, and then By deregulating and enhancing the expression of homoserine dehydrogenase, the modified strain SMCT363 with threonine production capacity was obtained.
  • the threonine yield of SMCT363 was 2.4g/L, and the isoleucine content was 0.3g/L.
  • the threonine yield of the modified strain SMCT364 obtained by inactivating Cgl0978 of SMCT363 was 2.9 g/L, the isoleucine content was 0.1 g/L, the isoleucine content was reduced, and the threonine yield was increased by 20.8%.
  • threonine-producing strains SMCT365 and SMCT366 were further constructed, which respectively express enhanced and deregulated pyruvate carboxylase and phosphoenolpyruvate on the basis of SMCT363 carboxylase. And further inactivated Cgl0978 to obtain modified strains SMCT367 and SMCT368, the threonine yields of the modified strains increased by 30% and 41.2% respectively compared with those without inactivation of Cgl0978.
  • the modified strain SMCT369 was obtained by strengthening and deregulating phosphoenolpyruvate carboxylase, and then Cgl0978 was inactivated to obtain SMCT370. g/L.
  • Inactivation or weakening during the transformation process includes promoter replacement, ribosome binding site changes, point mutations, and sequence deletions.
  • Expression enhancement during transformation 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, so the embodiments of the present invention only use promoter enhancement and point mutations as representatives for illustration.
  • the upstream homology arm up was obtained by PCR amplification with the P145/P146 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P147/P148 primer pair. Fusion PCR was performed as a template to obtain the full-length fragment ⁇ Cgl0978.
  • 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- ⁇ Cgl0978
  • 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 The pair was amplified by PCR to obtain lysC g1a-T311I
  • 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-lysC g1a-T311I -dn was obtained by fusion PCR with P21/P28 primer pair and up-Psod, lysC g1a -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 .
  • 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.
  • Fusion PCR was carried out with P29/P32 primer pair and up and PcspB as templates to obtain the fragment up-PcspB.
  • 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 P13/P14 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P15/P16 primer pair
  • the P17/P18 primer The pair was amplified by PCR to obtain pyc P458S
  • the downstream homology arm dn was obtained by PCR amplification with the P19/P20 primer pair.
  • the fragment up-Psod was obtained.
  • Fusion PCR was performed with P13/P20 primer pair and up-Psod, pyc P458S and dn as templates to obtain the full-length fragment up-Psod-pyc P458S -dn.
  • 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 -pyc P458S .
  • the upstream homology arm up was obtained by PCR amplification with the P53/P54 primer pair
  • the promoter fragment Ptuf was obtained by PCR amplification with the P55/P56 primer pair
  • the P57/P58 primer The ppc D299N was obtained by PCR amplification
  • the downstream homology arm dn was obtained by PCR amplification with the P59/P60 primer pair. Fusion PCR was performed with P53/P56 primer pair and up and Ptuf as templates to obtain the fragment up-Ptuf.
  • the full-length fragment up-Ptuf-ppc D299N -dn was obtained by fusion PCR with P53/P60 primer pair and up-Ptuf, ppc D299N and 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 tuf -ppc D299N .
  • ATCC13032 competent cells were prepared according to the classical method of glutamicum (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, in which the target gene 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.
  • 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 obtained and named SMCT362 respectively.
  • the lysC gene was mutated, its start codon was mutated from GTG to ATG, the 311th amino acid encoded by it was changed from threonine to isoleucine, and the promoter of lysC gene was replaced with a strong promoter Psod.
  • SMCT362 For the strain construction method, refer to the above 1), using SMCT362 as the starting strain, carry out the modification of homoserine dehydrogenase expression enhancement and deregulation (pK18mobsacB-P cspB -hom G378E is introduced into SMCT362), and the obtained modified strain is named SMCT363.
  • the hom gene was further mutated, and the corresponding amino acid mutation site was G378E, and the promoter of the hom gene was replaced with a strong promoter PcspB.
  • SMCT363 was used as the starting bacteria to carry out the modification of pyruvate carboxylase expression enhancement and deregulation (pK18mobsacB-P sod -pyc P458S was introduced into SMCT363), and the obtained modified strain was named SMCT365.
  • the pyc gene is further mutated, the corresponding amino acid mutation site is P458S, and the promoter of the pyc gene is replaced by a strong promoter P sod .
  • the strain construction method refers to the above 1), using SMCT363 and SMCT365 as the starting bacteria, the transformation of phosphoenolpyruvate carboxylase expression enhancement and deregulation (introducing pK18mobsacB-P tuf -ppc D299N into SMCT363 and SMCT365), the obtained transformation
  • the strains were named SMCT366, SMCT369.
  • the ppc gene was further mutated, the corresponding amino acid mutation site was D299N, and the promoter of the ppc gene was replaced with a strong promoter P tuf .
  • strain genotype SMCT361 ATCC13032, ⁇ Cgl0978 SMCT362 ATCC13032, P sod -lysC g1a-T311I SMCT363 ATCC13032, P sod -lysC g1a-T311I , P cspB -hom G378E SMCT364 ATCC13032, P sod -lysC g1a-T311I , P cspB -hom G378E , ⁇ Cgl0978 SMCT365 ATCC13032, P sod -lysC g1a-T311I , P cspB -hom G378E , P sod -pyc P458S SMCT366 ATCC13032, P sod -lysC g1a-T311I , P cspB -hom G378E , P tuf -ppc D299N SMCT3
  • 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 culture Pick ATCC13032, SMCT361, SMCT363, SMCT364, SMCT365, SMCT366, SMCT367, SMCT368, SMCT369, SMCT370 slant seeds 1 ring and connect them to a 500mL Erlenmeyer flask containing 20mL seed medium, 30°C, 220r/ Min shake culture 16h.
  • 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 Cgl0978 inactivation strains are different, ranging from 0.9g/L to 3.6g/L, indicating that the inactivation of Cgl0978 and the combination of different sites have different effects, and when it is combined with
  • the threonine The yields were all increased by 34% to 124%.

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Abstract

L'invention concerne un micro-organisme Corynebacterium modifié, son procédé de construction et son utilisation dans la production de thréonine. Le micro-organisme Corynebacterium modifié a une expression affaiblie ou inactivée du gène Cgl0978 par comparaison avec des micro-organismes non modifiés, et a une capacité de production améliorée pour la thréonine et un rendement réduit d'isoleucine par rapport aux micro-organismes non modifiés.
PCT/CN2022/143761 2022-02-08 2022-12-30 Micro-organisme corynebacterium modifié, son utilisation et son procédé de construction WO2023151421A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1355295A (zh) * 2000-08-11 2002-06-26 味之素株式会社 生产苏氨酸和异亮氨酸的方法
CN1446914A (zh) * 2002-03-21 2003-10-08 第一制糖株式会社 L－苏氨酸生产方法
CN1768132A (zh) * 2003-04-04 2006-05-03 希杰株式会社 tdcBC/pckA基因失活的微生物和利用该微生物生产L－苏氨酸的方法
CN105505969A (zh) * 2014-09-26 2016-04-20 中国科学院天津工业生物技术研究所 一种提高l-苏氨酸转化率的方法及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1355295A (zh) * 2000-08-11 2002-06-26 味之素株式会社 生产苏氨酸和异亮氨酸的方法
CN1446914A (zh) * 2002-03-21 2003-10-08 第一制糖株式会社 L－苏氨酸生产方法
CN1768132A (zh) * 2003-04-04 2006-05-03 希杰株式会社 tdcBC/pckA基因失活的微生物和利用该微生物生产L－苏氨酸的方法
CN105505969A (zh) * 2014-09-26 2016-04-20 中国科学院天津工业生物技术研究所 一种提高l-苏氨酸转化率的方法及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Protein 7 October 2016 (2016-10-07), ANONYMOUS : "Threonine dehydratase [Corynebacterium glutamicum ATCC 13032]", XP093083886, retrieved from NCBI Database accession no. BAB98371.1 *
DONG, XUNYAN: "Metabolic Engineering of Escherichia Coli and Corynebacterium Glutamicum for the Production of l-threonine", BIOTECHNOLOGY ADVANCES, vol. 29, no. 1, 28 February 2011 (2011-02-28) *

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