WO2023142871A1 - Micro-organisme modifié du genre corynebacterium, son procédé de construction et son utilisation - Google Patents

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

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WO2023142871A1
WO2023142871A1 PCT/CN2022/143517 CN2022143517W WO2023142871A1 WO 2023142871 A1 WO2023142871 A1 WO 2023142871A1 CN 2022143517 W CN2022143517 W CN 2022143517W WO 2023142871 A1 WO2023142871 A1 WO 2023142871A1
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enhanced
microorganism
threonine
enzyme
gene encoding
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康培
程江红
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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    • C12R2001/15—Corynebacterium

Definitions

  • the invention relates to the technical field of microbial engineering, in particular to a modified microorganism of the genus Corynebacterium and its construction method and application.
  • L-Threonine (L-Threonine), 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 oxaloacetate decarboxylase, 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 microorganism has reduced or lost oxaloacetate decarboxylase activity compared with an unmodified microorganism, and the microorganism is relatively Enhanced threonine production capacity compared to unmodified microorganisms.
  • the reference sequence number of oxaloacetate decarboxylase on NCBI is WP_003861462.1, or an amino acid sequence with 90% similarity thereto.
  • the reduction or loss of the activity of oxaloacetate decarboxylase in the microorganism is achieved by reducing the expression of the gene encoding oxaloacetate decarboxylase or knocking out the endogenous gene encoding oxaloacetate decarboxylase.
  • Mutagenesis, site-directed mutation or homologous recombination can be used to reduce the expression of the gene encoding oxaloacetate decarboxylase or to knock out the endogenous gene encoding oxaloacetate decarboxylase.
  • the activity of enzymes related to the threonine synthesis pathway and/or reducing power supply pathway in the microorganism is enhanced; wherein, the enzymes related to the threonine synthesis pathway and/or reducing power Enzymes related to the supply pathway are selected from at least one of aspartokinase, homoserine dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase; preferably, their names on NCBI
  • the reference sequence numbers are respectively WP_003855724.1, WP_003854900.1, NP_600790.1, NP_600669.1, or amino acid sequences with a similarity of 90% to the above reference sequences.
  • the microorganism is any one of the following 1 ⁇ 4:
  • Microorganisms with reduced or lost oxaloacetate decarboxylase activity and enhanced aspartokinase, homoserine dehydrogenase, glucose-6-phosphate dehydrogenase and/or 6-phosphogluconate dehydrogenase activities are produced by Microorganisms with reduced or lost oxaloacetate decarboxylase activity and enhanced aspartokinase, homoserine dehydrogenase, glucose-6-phosphate dehydrogenase and/or 6-phosphogluconate dehydrogenase activities.
  • the enhancement of the activity of enzymes related to the threonine synthesis pathway and/or reducing power supply 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:
  • step B Enhancing the enzymes related to the threonine synthesis pathway and/or reducing power supply pathway in the gene-weakened strain of step A to obtain a strain with enhanced enzyme activity;
  • the enhanced pathway is selected from the following 1) to 6), or an optional combination:
  • the enzymes related to the threonine synthesis pathway and/or reducing power supply pathway are selected from aspartokinase, homoserine dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase at least one of the enzymes.
  • 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 the gene encoding oxaloacetate decarboxylase in threonine fermentation production or improvement of threonine fermentation yield.
  • the fermentation yield of threonine was improved by inactivating oxaloacetate decarboxylase 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 oxaloacetate decarboxylase is inactivated by deleting the bases of the open reading frame of the odx gene.
  • lysC By mutating the gene lysC encoding aspartokinase, its start codon is mutated from GTG to ATG, the 311th amino acid of its encoded amino acid is mutated from threonine to isoleucine, and the lysC gene is mutated from Psod initiates transcription, ultimately leading to expression enhancement and deregulation of aspartokinase.
  • the nucleotide sequence of Psod is shown in SEQ ID NO.35.
  • the present invention improves the yield of threonine produced by the strain by inactivating oxaloacetate decarboxylase, and can efficiently produce threonine after the threonine synthesis pathway/or reducing power supply pathway in the strain is opened up through further transformation , providing a new idea for the production of threonine.
  • Oxaloacetate decarboxylase encoding gene name odx, NCBI number: Cgl1290, NCgl1241, cg1458
  • Aspartokinase encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247.
  • Homoserine dehydrogenase encoding gene name hom, NCBI number: cg1337, Cgl1183, NCgl1136.
  • Glucose-6-phosphate dehydrogenase encoding gene name zwf, NCBI number: cg1778, Cgl1576, NCgl1514.
  • 6-phosphogluconate dehydrogenase encoding gene name gnd, NCBI number: cg1643, Cgl1452, NCgl1396.
  • the oxaloacetate decarboxylase is inactivated on the wild bacterium ATCC13032, and the threonine yield of the obtained modified bacterium SMCT345 is 0.2g/L. It is speculated that this site is beneficial to the synthesis of threonine, but due to the strict metabolic regulation in the bacterium , the aspartokinase and homoserine dehydrogenase in the threonine synthesis pathway are strictly regulated by the intracellular threonine concentration.
  • the first thing to do is to open up its synthesis pathway, which mainly includes the deregulation and expression enhancement of aspartokinase and homoserine dehydrogenase, and the acquisition of the transformed strain SMCT346 enables the strain to have preliminary threonine synthesis ability , and its threonine yield is 2.5g/L.
  • the ability of the strain to produce threonine increased from 2.5g/L to 3.4g/L.
  • the present invention further strengthens the expression of 6-phosphogluconate dehydrogenase and at least one enzyme in glucose-6-phosphate dehydrogenase in the SMCT346 strain, and performs odx inactivation to obtain a series of bacterial strains SMCT349, SMCT351 and SMCT353.
  • the yields of amino acids were increased by 40%, 46.7%, and 50%, respectively. It shows that the increase of threonine production is caused by the inactivation of oxaloacetate decarboxylase.
  • the expression enhancement during the transformation process includes promoter replacement, ribosome binding site change, copy number increase, plasmid overexpression and other means, all of which are known to researchers in the field.
  • the above means cannot be exhausted by examples, so the embodiments of the present invention are only illustrated with promoter enhancement as a representative.
  • the means of weakening expression include promoter replacement, change of ribosome binding site, start codon replacement, open reading frame base deletion and other means, which cannot be exhausted by examples. Therefore, the embodiments of the present invention adopt the method of gene inactivation Open reading frame base deletion means are illustrated as representative.
  • Embodiment 1 strain genome transformation plasmid construction
  • 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 Psod was obtained by PCR amplification with the P25/P26 primer pair.
  • lysC g1a-T311I was amplified by PCR with the P27/P28 primer pair to obtain the dn of the downstream homology arm. Fusion PCR was performed with P21/P24 primer pair and up and Psod as the template 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 .
  • the upstream homology arm up was obtained by PCR amplification with the P29/P30 primer pair, and the promoter fragment PcspB was obtained by PCR amplification using the ATCC14067 genome as a template and the P31/P32 primer pair, and the ATCC13032 genome was used as a template to obtain
  • the P33/P34 primer pair was used for PCR amplification to obtain hom G378E
  • the P35/P36 primer pair was used for PCR amplification to obtain the downstream homology arm dn.
  • 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 P123/P124 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P125/P126 primer pair
  • the Psod was obtained by PCR amplification with the P127/P128 primer pair.
  • Downstream homology arm dn Fusion PCR was carried out with P123/P126 primer pair and up and Psod as templates to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-dn was obtained by fusion PCR using the P123/P128 primer pair and up-Psod and dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with a seamless cloning kit, transformed into Trans1 T1 competent cells, and obtained the recombinant plasmid pK18mobsacB-Psod-gnd.
  • the upstream homology arm up was obtained by PCR amplification with the P129/P130 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P131/P132 primer pair
  • the Psod was obtained by PCR amplification with the P133/P134 primer pair.
  • zwf A243T PCR amplification with P135/P136 primer pair to obtain the downstream homology arm dn. Fusion PCR was carried out with P129/P132 primer pair and up and Psod as the template to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-zwf A243T -dn was obtained by fusion PCR with P129/P136 primer pair and up-Psod, zwf A243T , dn as templates.
  • pK18mobsacB was digested with BamHI/HindIII. The two were assembled with the seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-Psod-zwf A243T .
  • PCR amplification was performed with the P185-odx-up-1F/P186-odx-up-1R primer pair to obtain the upstream homology arm up, and the P187-odx-dn-2F/P188-odx-dn- The 2R primer pair was used for PCR amplification to obtain the dn of the downstream homology arm.
  • pK18mobsacB was digested with BamHI/HindIII.
  • the two were assembled with a seamless cloning kit, transformed into Trans1 T1 competent cells, and obtained the recombinant plasmid pK18mobsacB- ⁇ odx.
  • the primers used in the plasmid construction process are shown in Table 1 below:
  • Embodiment 2 Construction of Genome Modification Strain
  • ATCC13032 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -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 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 is amplified by PCR and analyzed by nucleotide sequencing to obtain the target mutant strain.
  • the lysC gene was mutated, its start codon was mutated from GTG to ATG, the 311th threonine in the encoded amino acid sequence was mutated to isoleucine, and the promoter of lysC gene was replaced with a strong promoter Sub-Psod.
  • the enhanced expression of homoserine dehydrogenase was modified (pK18mobsacB-P cspB -hom G378E was introduced into the above modified bacteria), and the obtained modified strain was named SMCT346.
  • the strain construction method refers to the above.
  • 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.
  • SMCT346 was used as the starting strain, and the enhanced expression of 6-phosphogluconate dehydrogenase was modified (pK18mobsacB-Psod-gnd was introduced into SMCT346), and the obtained modified strain was named SMCT348.
  • the promoter of the gnd gene was replaced by the strong promoter Psod.
  • the strain construction method refers to the above 1), using SMCT346 and SMCT348 as the starting bacteria, the enhanced expression of glucose-6-phosphate dehydrogenase is carried out (pK18mobsacB-Psod-zwf A243T is introduced into SMCT346 and SMCT348), and the obtained modified strains are named SMCT350 and SMCT352.
  • the zwf gene was mutated, the corresponding amino acid mutation site was A243T, and the promoter of the zwf gene was replaced by the strong promoter Psod.
  • SMCT351 ATCC13032 Psod-lysC g1a-T311I , PcspB-hom G378E , Psod-zwf A243T , ⁇ odx SMCT352 ATCC13032, Psod-lysC g1a-T311I , PcspB-hom G378E , Psod-gnd, Psod-zwf A243T SMCT353 ATCC13032, Psod-lysC g1a-T311I , PcspB-hom G378E , Psod-gnd, Psod-zwf A243T , ⁇ odx
  • Seed activation medium BHI 3.7%, agar 2%, pH 7.0.
  • 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, SMCT345, SMCT346, SMCT347, SMCT348, SMCT349, SMCT350, SMCT351, SMCT352, SMCT353 slant seeds 1 loop 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.

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Abstract

La présente invention se rapporte au domaine technique du génie génétique. La divulgation concerne spécifiquement un micro-organisme modifié du genre Corynebacterium, son procédé de construction et son utilisation. L'activité de l'oxaloacétate décarboxylase dans le micro-organisme modifié du genre corynebacterium de la présente invention est réduite ou perdue par rapport à celle dans un micro-organisme non modifié, et le micro-organisme modifié a une capacité de production de thréonine améliorée par rapport au micro-organisme non modifié. Selon la présente invention, au moyen de l'inactivation de l'oxaloacétate décarboxylase, l'alimentation en oxaloacétate, un précurseur synthétique de thréonine, est améliorée, de telle sorte que la capacité de production de thréonine de la souche est améliorée, fournissant une nouvelle approche pour la production de thréonine.
PCT/CN2022/143517 2022-01-30 2022-12-29 Micro-organisme modifié du genre corynebacterium, son procédé de construction et son utilisation WO2023142871A1 (fr)

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