WO2023142859A1 - 修饰的棒状杆菌属微生物及其构建方法和应用 - Google Patents

修饰的棒状杆菌属微生物及其构建方法和应用 Download PDF

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WO2023142859A1
WO2023142859A1 PCT/CN2022/143100 CN2022143100W WO2023142859A1 WO 2023142859 A1 WO2023142859 A1 WO 2023142859A1 CN 2022143100 W CN2022143100 W CN 2022143100W WO 2023142859 A1 WO2023142859 A1 WO 2023142859A1
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threonine
enzyme
enhanced
microorganism
gene encoding
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French (fr)
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康培
刘涛
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 belonging to the genus Corynebacterium and its construction method and application.
  • L-threonine 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, mainly used in medicine, chemical reagents, food fortifiers, feed additives and other fields.
  • Corynebacterium glutamicum is an important industrial microorganism. In Corynebacterium glutamicum, five-step catalytic reactions are required to generate threonine from oxaloacetate, and the catalytic enzymes of these five-step reactions are aspartokinase (encoded by lysC), aspartate semialdehyde dehydrogenase ( asd code), homoserine dehydrogenase (hom code), homoserine kinase (thrB code) and threonine synthase (thrC code).
  • asd code aspartate semialdehyde dehydrogenase
  • homoserine dehydrogenase homoserine dehydrogenase
  • thrB code homoserine kinase
  • thrC code threonine synthase
  • Lothar Eggling et al increased the production of threonine by weakening the coding gene glyA in the threonine utilization pathway and overexpressing the threonine export protein ThrE (Simic P, Willuhn J, Sahm H, et al. Identification of glyA (Encoding Serine Hydroxymethyltransferase) and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum[J].Applied and Environmental Microbiology,2002,68(7):3321-3327. ).
  • cg1507-cg1524 are genes encoding putative membrane proteins, non-essential genes of Corynebacterium glutamicum (Baumgart, M.; Unthan, S.; Ruckert, C.; Sivalingam, J.; Grunberger, A.; Kalinowski, J.
  • the purpose of the present invention is to improve the threonine-producing ability of the bacterial strain by inactivating non-essential genes, thereby providing a modified Corynebacterium microorganism and its construction method and application.
  • the present invention aims at developing threonine-producing microorganisms of the genus Corynebacterium. During the research and development process, it is found that knocking out the non-essential gene cg1507-cg1524 (Cgl1336-Cgl1352 or NCgl1281-NCgl1298) in Corynebacterium glutamicum can significantly improve the threonine production while improving the growth performance of the strain.
  • the invention provides the application of attenuation or inactivation of non-essential genes in improving threonine production of microorganisms or constructing threonine-producing microorganisms; the non-essential genes are cg1507-cg1524, Cgl1336-Cgl1352 or NCgl1281-NCgl1298.
  • cg1507-cg1524 is a chromosome segment composed of multiple genes corresponding to NCBI numbers cg1507 to cg1524.
  • the weakening mentioned above can be achieved by any one or both of the following (1)-(2):
  • having lower activity means that the activity is lower than that of the primary transcription or translation element of a non-essential gene.
  • transcription elements include promoters, enhancers, etc.
  • translation elements include ribosome binding sites, 5'-UTR, etc.
  • the mutation may be to mutate the start codon of the non-essential gene to other start codons except ATG (for example: GTG or TTG).
  • the inactivation mentioned above can be performed by deleting, inserting or replacing one or more nucleotides of non-essential genes, so that the non-essential genes are no longer expressed.
  • the above-mentioned applications are realized by knocking out the non-essential genes.
  • the microorganism is a bacterium of the genus Corynebacterium, preferably Corynebacterium glutamicum.
  • the present invention provides a modified microorganism of the genus Corynebacterium, the non-essential gene of the microorganism is weakened or inactivated compared with the unmodified microorganism; the non-essential gene is cg1507-cg1524, Cgl1336-Cgl1352 or NCgl1281-NCgl1298.
  • the weakening mentioned above can be achieved by any one or both of the following (1)-(2):
  • having lower activity means that the activity is lower than that of the primary transcription or translation element of a non-essential gene.
  • transcription elements include promoters, enhancers, etc.
  • translation elements include ribosome binding sites, 5'-UTR, etc.
  • the mutation may be to mutate the start codon of the non-essential gene to other start codons except ATG (for example: GTG or TTG).
  • the inactivation mentioned above can be performed by deleting, inserting or replacing one or more nucleotides of non-essential genes, so that the non-essential genes are no longer expressed.
  • said non-essential genes in said microorganism are knocked out or inactivated.
  • the microorganism has an enhanced threonine production capacity compared to an unmodified microorganism.
  • the present invention finds that in the wild-type strain or the strain capable of accumulating threonine, inactivating the non-essential gene can improve the synthesis ability of threonine. In comparison, in strains capable of accumulating threonine, the increase in threonine was greater.
  • the activity of any one or more enzymes in the following (1) to (7) is enhanced and/or feedback inhibition is relieved:
  • the above-mentioned activity enhancement is achieved by being selected from the following 1) to 6), or an optional combination:
  • the reference sequence numbers of the above-mentioned aspartate kinase, homoserine dehydrogenase, aspartate semialdehyde dehydrogenase, homoserine kinase, pyruvate carboxylase, and glucose-6-phosphate dehydrogenase on NCBI are respectively WP_003855724.1, WP_003854900.1, WP_011013506.1, WP_011014183.1, WP_011013816.1, NP_600790.1, or an amino acid sequence with 90% similarity to the above reference sequence and having equivalent functions.
  • the above-mentioned threonine export protein is preferably a threonine export protein derived from Escherichia coli, and its reference sequence number on NCBI is YP_026264.1, or an amino acid sequence with 90% similarity to the above reference sequence and having equivalent functions .
  • the activity of any one or more enzymes in the following (1) to (3) is reduced or lost:
  • the reduction or loss of activity is achieved by reducing the expression of the gene encoding the enzyme or knocking out an endogenous gene encoding the enzyme.
  • the reference sequence numbers of the above-mentioned diaminopimelate dehydrogenase, 4-hydroxytetrahydrodipicolinate synthase, and citrate synthase on NCBI are respectively WP_011015254.1, WP_011014792.1, WP_011013914.1, or the above-mentioned Amino acid sequences with a reference sequence similarity of 90% and equivalent functions.
  • the microorganism is any of the following:
  • Non-essential genes are inactivated, and the enzymatic activity of at least one of aspartokinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, and homoserine kinase is enhanced and/or feedback inhibition is relieved microorganism;
  • Non-essential genes are inactivated, and the enzymatic activity of at least one of aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, and threonine exporter is enhanced and/or microorganisms that relieve feedback inhibition;
  • Non-essential gene inactivation, and aspartate kinase, aspartate semialdehyde dehydrogenase, homoserine dehydrogenase, homoserine kinase, threonine export protein and pyruvate carboxylase at least one microorganism whose enzymatic activity is enhanced and/or feedback inhibition relieved;
  • the enzymatic activity enhancement described above is achieved by any one or more of the following methods:
  • the strong promoter includes Psod or PcspB.
  • the nucleotide sequences of the promoters Psod and PcspB are shown in SEQ ID NO.1 and 2, respectively.
  • the enzymatic activity enhancement of aspartate kinase, aspartate semialdehyde dehydrogenase, pyruvate carboxylase and glucose-6-phosphate dehydrogenase is achieved by replacing their original promoters with Psod promoters;
  • Enzyme activity enhancement of homoserine dehydrogenase and homoserine kinase is achieved by replacing their original promoters with PcspB promoters;
  • Enzyme activity of the threonine exporter derived from Escherichia coli is enhanced by inserting a copy of the threonine exporter gene rhtC derived from Escherichia coli into the genome.
  • the release of feedback inhibition of aspartokinase is achieved by mutating the gene encoding aspartate kinase, so that the encoded aspartokinase undergoes a T311I mutation;
  • the release of feedback inhibition of homoserine dehydrogenase is achieved by mutating the gene encoding homoserine dehydrogenase so that the homoserine dehydrogenase undergoes a G378E mutation;
  • the release of feedback inhibition of pyruvate carboxylase is achieved by mutating the gene encoding pyruvate carboxylase so that the coded pyruvate carboxylase undergoes a P458S mutation;
  • the release of feedback inhibition of glucose-6-phosphate dehydrogenase is achieved by mutating the gene encoding glucose-6-phosphate dehydrogenase so that the encoded glucose-6-phosphate dehydrogenase undergoes an A243T mutation.
  • the inactivation mentioned above is achieved by deleting, inserting or replacing one or more nucleotides of the target gene, so that the target gene is no longer expressed.
  • the gene encoding diaminopimelate dehydrogenase is inactivated, and the start codons of the gene encoding 4-hydroxytetrahydrodipicolinate synthase synthase and the gene encoding citrate synthase are mutated to GTG.
  • Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. Corynebacterium acid ATCC 13032.
  • the present invention also provides a method for constructing a threonine-producing strain, the method comprising: weakening or inactivating a non-essential gene in a Corynebacterium bacterium with amino acid production capacity to obtain a gene weakened strain; the non-essential gene is cg1507-cg1524, Cgl1336-Cgl1352 or NCgl1281-NCgl1298.
  • the method further includes: enhancing the activity of any one or more enzymes in (1) to (7) below and/or removing feedback inhibition:
  • the enhancement of the activity is achieved by being selected from the following 1) to 6), or an optional combination:
  • the method also includes: reducing the activity of any one or more enzymes in the following (1)-(3):
  • the reduction or loss of the activity is achieved by reducing the expression of the gene encoding the enzyme or knocking out the endogenous gene encoding the enzyme.
  • the present invention provides any of the following applications of the microorganisms described above:
  • threonine derivatives described in the present invention can be compounds synthesized from threonine as a precursor, including isoleucine, glycine and the like.
  • the present invention also provides a method for fermentatively producing threonine or its derivatives, which includes the steps of culturing the above-mentioned microorganisms and isolating threonine or its derivatives from the culture.
  • the above method includes: inoculating the microorganisms in a seed medium for seed cultivation to obtain a seed liquid, inoculating the seed liquid in a fermentation medium for cultivation to obtain a fermentation liquid, and separating and extracting the fermentation liquid to obtain threonine or its derivatives.
  • the fermentation medium comprises the following components: corn steep liquor 45-55mL/L, glucose 25-35g/L, ammonium sulfate 3-5g/L, MOPS 25-35g/L, potassium dihydrogen phosphate 8-12g /L, urea 15-25g/L, biotin 8-12mg/L, magnesium sulfate 5-7g/L, ferrous sulfate 0.5-1.5g/L, VB1 ⁇ HCl 35-45mg/L, calcium pantothenate 45-55mg /L, Niacinamide 35-45mg/L, Manganese Sulfate 0.5-1.5g/L, Zinc Sulfate 15-25mg/L, Copper Sulfate 15-25mg/L, pH 7.0-7.2.
  • the beneficial effect of the present invention is that: the present invention significantly improves the threonine-producing ability of the bacterial strain by inactivating non-essential genes cg1507-cg1524, and strengthening or weakening enzymes such as aspartokinase and homoserine dehydrogenase , the threonine production of the strain was significantly improved compared with that before the modification, and the growth performance of the strain was improved at the same time.
  • the transformation of non-essential gene cg1507-cg1524 can be used in the fermentative production of threonine and has good application value.
  • Non-essential genes NCBI number: cg1507-cg1524, Cgl1336-Cgl1352, NCgl1281-NCgl1298;
  • Aspartokinase encoded gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247;
  • Aspartate semialdehyde dehydrogenase encoding gene name asd, NCBI number: cg0307, Cgl0252, NCgl0248;
  • Homoserine dehydrogenase encoding gene name hom, NCBI number: cg1337, Cgl1183, NCgl1136;
  • Diaminopimelate dehydrogenase encoded gene name ddh, NCBI number: cg2900, Cgl2617, NCgl2528;
  • Glucose-6-phosphate dehydrogenase encoding gene name zwf, NCBI number: cg1778, Cgl1576, NCgl1514;
  • Homoserine kinase encoding gene name thrB, NCBI number: cg1338, Cgl1184, NCgl1137;
  • Threonine export protein derived from Escherichia coli, encoded gene name rhtC, NCBI number b3823;
  • Citrate synthase encoded gene name gltA, NCBI number: cg0949, Cgl0829, NCgl0795.
  • Embodiment 1 strain genome transformation plasmid construction
  • the upstream homology arm up was obtained by PCR amplification with the PCT72/PCT73 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the PCT74/PCT75 primer pair.
  • PCT72/PCT75 primers to perform fusion PCR with up and dn fragments as templates to obtain the full-length fragment up-dn.
  • pK18mobsacB was digested with BamHI/HindIII.
  • the digested up-dn and pK18mobsacB were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB- ⁇ cg1507-cg1524.
  • 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-lysCV1M-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-Psod-lysC g1a-T311I .
  • the primers used are P1, P2, P3, P4, P5, and P6.
  • the primers used are P29, P30, P31, P32, P33, P34, P35, and P36.
  • the primers used are P7, P8, P9, P10, P11, and P12.
  • the upstream homology arm up was obtained by PCR amplification with the P157/P158 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P159/P160 primer pair
  • the P161/P162 The primer pair was used for PCR amplification to obtain rhtC
  • the ATCC13032 genome was used as a template to perform PCR amplification for the P163/P164 primer pair to obtain the downstream homology arm dn. Fusion PCR was carried out with P157/P160 primer pair and up and Psod as the template to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-rhtC-dn was obtained by fusion PCR using the P157/P164 primer pair and up-Psod, rhtC, 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-Psod-rhtC.
  • the plasmid construction method refers to the above 2, and the primers used are P13, P14, P15, P16, P17, P18, P19, and P20.
  • the primers used are P129, P130, P131, P132, P133, P134, P135, and P136.
  • the plasmid construction method refers to the above 1, and the primers used are P99, P100, P101, and P102.
  • the primers used are P75, P76, P77, and P78.
  • the primers used are P153, P154, P155, and P156.
  • Embodiment 2 Construction of Genome Modification Strain
  • ATCC13032 competent cells were prepared according to the classic method of Corynebacterium glutamicum (C. glutamicum Handbook, Chapter 23). Transform the competent cells with the recombinant plasmid pK18mobsacB-Psod-lysC g1a-T311I by electroporation, and select transformants on the selection medium containing 15 mg/L kanamycin, wherein the gene of interest is 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.
  • 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. Colonies grown on sucrose media did not carry the inserted vector sequence in their genomes.
  • the target fragment was amplified by PCR and subjected to nucleotide sequencing analysis, and the target mutant strain was named SMCT196.
  • the start codon of the lysC gene was mutated from GTG to ATG, the 311th amino acid encoded was changed from threonine to isoleucine, and the promoter of the lysC gene was replaced by the Psod promoter. .
  • the strain construction method refers to the above 1, using SMCT196 as the starting bacterium, the plasmid pK18mobsacB-Psod-asd is introduced into the strain SMCT196, and the transformation of aspartate semialdehyde dehydrogenase expression enhancement is carried out, and the obtained modified strain is named SMCT197, and the strain Compared with SMCT196, the promoter of the asd gene of this strain was replaced by the Psod promoter.
  • the strain construction method refers to the above 1, using SMCT197 as the starting bacterium, the plasmid pK18mobsacB-PcspB-hom G378E is introduced into the strain SMCT197, and the transformation of homoserine dehydrogenase expression enhancement is carried out, and the obtained modified strain is named SMCT198, compared with the strain SMCT197 , the mutation of the hom gene of the strain resulted in the G378E mutation of its encoded protein, and the promoter of the hom gene was replaced by the PcspB promoter derived from the strain ATCC14067.
  • SMCT198 As the starting bacterium, the plasmid pK18mobsacB-PcspB-thrB was introduced into the strain SMCT198, and the transformation of homoserine kinase expression enhancement was carried out.
  • the obtained modified strain was named SMCT199.
  • the strain In the promoter of the thrB gene was replaced by the PcspB promoter.
  • the strain construction method refers to the above 1, using SMCT199 as the starting bacterium, the plasmid pK18mobsacB-cg2899::rhtC is introduced into the strain SMCT199, and the expression of the threonine export protein derived from Escherichia coli is modified to weaken the expression, and the obtained modified strain is named SMCT200.
  • this strain inserted a copy of rhtC gene after the last base of cg2899 gene.
  • the strain construction method refers to the above 1, using SMCT200 as the starting bacterium, the plasmid pK18mobsacB-Psod-pyc P458S is introduced into the strain SMCT200, and the transformation of pyruvate carboxylase enhancement is carried out.
  • the obtained modified strain is named SMCT201.
  • the mutation of the pyc gene of the strain resulted in the P458S mutation of its encoded protein, and the promoter of the pyc gene was replaced by the Psod promoter.
  • SMCT201 For the strain construction method, refer to the above 1, using SMCT201 as the starting bacterium, introduce the plasmid pK18mobsacB-Psod-zwf A243T into the strain SMCT201, and carry out the transformation of enhancing the expression of glucose-6-phosphate dehydrogenase, and the obtained modified strain is named SMCT202, and the strain Compared with SMCT201, the amino acid sequence encoded by the zwf gene of this strain had an A243T mutation, and the promoter of the zwf gene was replaced by the Psod promoter.
  • SMCT202 For the method of strain construction, refer to the above 1, using SMCT202 as the starting bacterium, introduce the plasmid pK18mobsacB- ⁇ ddh into the strain SMCT202, and carry out the modification of weakening the expression of diaminopimelate dehydrogenase, and the obtained modified strain is named SMCT203, compared with the strain SMCT202 , the ddh gene of the strain was knocked out.
  • SMCT203 For the strain construction method, refer to the above 1, using SMCT203 as the starting bacterium, introduce the plasmid pK18mobsacB-dapA a1g into the strain SMCT203, and carry out the transformation of weakening the expression of 4-hydroxytetrahydrodipicolinate synthase synthase, and the obtained transformed strain is named SMCT204 , compared with strain SMCT203, the start codon of the dapA gene of this strain was mutated to GTG.
  • SMCT204 As the starting bacterium, the plasmid pK18mobsacB-gltA a1g was introduced into the strain SMCT204, and the expression of citrate synthase was weakened.
  • the modified strain obtained was named SMCT205.
  • the strain The start codon of the gltA gene was mutated to GTG.
  • Embodiment 3 Shake flask fermentation verification of constructing bacterial strain
  • Each bacterial strain constructed in embodiment 2 is carried out shake flask fermentation verification, specifically as follows:
  • Seed activation medium BHI 3.7%, agar 2%, pH 7.
  • 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, V B1 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, SMCT196, SMCT197, SMCT198, SMCT199, SMCT200, SMCT201, SMCT202, SMCT203, SMCT204, SMCT205, SMCT206, SMCT207, SMCT208, SMCT209, SMCT210, SMCT211, SMCT212, SMCT21 3.
  • SMCT214, SMCT215, SMCT216 slant seeds 1 were looped into a 500mL Erlenmeyer flask containing 20mL of seed medium, and cultured with shaking at 30°C and 220r/min for 16h to obtain seed liquid.
  • Fermentation culture Inoculate 2 mL of seed liquid into a 500 mL Erlenmeyer flask containing 20 mL of fermentation medium, and culture with shaking at 33° C. and 220 r/min for 24 hours to obtain a fermentation liquid.
  • Table 3 shows the detection results of threonine production ability of each strain.
  • coli-derived threonine exporter pyruvate decarboxylation Enhancing the expression of at least one enzyme, glucose-6-phosphate dehydrogenase, releasing feedback inhibition, and weakening the expression of at least one diaminopimelate dehydrogenase, 4-hydroxytetrahydrodipicolinate synthase, and citrate synthase
  • the production of threonine is further improved, namely: the combination of the inactivation of cg1507-cg1524 and the modification of the above sites is also conducive to the production of threonine; in addition, aspartokinase, aspartate half At least one of aldehyde dehydrogenase enzymes, homoserine dehydrogenase, homoserine kinase, threonine export protein derived from E.
  • coli pyruvate decarboxylase
  • glucose-6-phosphate dehydrogenase is enhanced, feedback inhibition is relieved, and two Attenuation of expression of at least one of aminopimelate dehydrogenase, 4-hydroxytetrahydrodipicolinate synthase, and citrate synthase is beneficial to the threonine production of the strain.

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Abstract

本发明涉及微生物工程技术领域，具体涉及修饰的棒状杆菌属微生物及其构建方法和应用。本发明提供非必需基因的弱化或失活在提高微生物的苏氨酸产量或构建生产苏氨酸的微生物中的应用；所述非必需基因为cg1507-cg1524、Cgl1336-Cgl1352或NCgl1281-NCgl1298。本发明通过失活非必需基因，显著提高了菌株生产苏氨酸的能力，同时提升了菌株的生长性能。上述非必需基因的改造可用于苏氨酸的发酵生产中，具有较好的应用价值。

Description

修饰的棒状杆菌属微生物及其构建方法和应用 技术领域
本发明涉及微生物工程技术领域，具体涉及修饰的棒状杆菌属微生物及其构建方法和应用。
背景技术
L-苏氨酸(L-Threonine)的化学名称为β-羟基-α-氨基丁酸，分子式为C 4H 9NO 3，相对分子质量为119.12。L-苏氨酸是一种必需氨基酸，主要用于医药、化学试剂、食品强化剂、饲料添加剂等领域。
谷氨酸棒状杆菌是一种重要的工业微生物。谷氨酸棒状杆菌中，由草酰乙酸生成苏氨酸需要五步催化反应，这五步反应的催化酶分别为天冬氨酸激酶(lysC编码)、天冬氨酸半醛脱氢酶(asd编码)、高丝氨酸脱氢酶(hom编码)、高丝氨酸激酶(thrB编码)以及苏氨酸合酶(thrC编码)。在产苏氨酸的谷氨酸棒状杆菌的开发中，Hermann Sahm等获得了抗反馈抑制的hom基因(Reinscheid D J,Eikmanns B J,Sahm H.Analysis of a Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.[J].Journal of Bacteriology,1991,173(10):3228-3230.)以及lysC基因(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.)。Lothar Eggling等通过弱化苏氨酸利用途径中的编码基因glyA，同时过表达苏氨酸外运蛋白ThrE，提高了苏氨酸的产量(Simic P,Willuhn J,Sahm H,et al.Identification of glyA(Encoding Serine Hydroxymethyltransferase)and Its Use Together with the Exporter ThrE To Increase l-Threonine Accumulation by Corynebacterium glutamicum[J].Applied and Environmental Microbiology,2002,68(7):3321-3327.)。
目前利用谷氨酸棒状杆菌生产苏氨酸的报道主要集中在其合成路径的改造，关于非必需基因敲除的研究主要集中在敲除后菌株生长变化、是否会产生营养缺陷以及是否有利于基因改造等方面，对苏氨酸合成的影响少有报道。cg1507-cg1524是编码假定膜蛋白的基因，是谷氨酸棒状杆菌的非必需基因(Baumgart,M.；Unthan,S.；Ruckert,C.；Sivalingam,J.；Grunberger,A.；Kalinowski,J.；Bott,M.；Noack,S.；Frunzke,J.Construction of a prophage-free variant of Corynebacterium glutamicum ATCC 13032for use as a platform strain for basic research and industrial biotechnology.Appl.Environ.Microbiol.2013,79,6006-6015.)。
发明内容
本发明的目的是通过失活非必需基因使菌株生产苏氨酸的能力得到提升，从而提供一种修饰的棒状杆菌属微生物及其构建方法和应用。
本发明以开发产苏氨酸的棒状杆菌属微生物为目的，在研发过程中发现敲除谷氨酸棒状杆菌中的非必需基因cg1507-cg1524(Cgl1336-Cgl1352或NCgl1281-NCgl1298)能够显著提高菌株的苏氨酸产量，同时改善菌株的生长性能。
基于上述发现，本发明提供以下技术方案：
本发明提供非必需基因的弱化或失活在提高微生物的苏氨酸产量或构建生产苏氨酸的微生物中的应用；所述非必需基因为cg1507-cg1524、Cgl1336-Cgl1352或NCgl1281-NCgl1298。
以cg1507-cg1524为例，以上所述的cg1507-cg1524为NCBI编号cg1507至cg1524对应的多个基因组成的染色体片段。
以上所述的弱化可通过以下(1)～(2)中的任意一种或两种方式实现：
(1)采用具有更低转录或翻译启动、调控活性的元件调控非必需基因的转录或翻译，使其表达量降低；
(2)对非必需基因进行突变以使其表达量降低。
上述(1)中，具有更低活性是指与非必需基因的原始转录或翻译元件相比，活性更低。
其中，转录元件包括启动子、增强子等；翻译元件包括核糖体结合位点、5’-UTR等。
上述(2)中，所述突变可为将非必需基因的起始密码子突变为除ATG以外的其它起始密码子(例如：GTG或TTG)。
以上所述的失活可通过对非必需基因进行一个或多个核苷酸的缺失、插入或替换，使得非必需基因不再表达。
优选地，以上所述的应用为通过敲除所述非必需基因实现。
以上所述的应用中，所述微生物为棒状杆菌属细菌，优选为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
进一步地，本发明提供一种修饰的棒状杆菌属微生物，所述微生物相比于未修饰的微生物，其非必需基因被弱化或失活；所述非必需基因为cg1507-cg1524、Cgl1336-Cgl1352或NCgl1281-NCgl1298。
以上所述的弱化可通过以下(1)～(2)中的任意一种或两种方式实现：
(1)采用具有更低转录或翻译启动、调控活性的元件调控非必需基因的转录或翻译，使其表达量降低；
(2)对非必需基因进行突变以使其表达量降低。
上述(1)中，具有更低活性是指与非必需基因的原始转录或翻译元件相比，活性更低。
其中，转录元件包括启动子、增强子等；翻译元件包括核糖体结合位点、5’-UTR等。
上述(2)中，所述突变可为将非必需基因的起始密码子突变为除ATG以外的其它起始密码子(例如：GTG或TTG)。
以上所述的失活可通过对非必需基因进行一个或多个核苷酸的缺失、插入或替换，使得非必需基因不再表达。
优选地，所述微生物中的所述非必需基因被敲除或失活。
优选地，所述微生物相比于未修饰的微生物具有增强的苏氨酸生产能力。
本发明发现，在野生型菌株或能够积累苏氨酸的菌株中，失活所述非必需基因均能够提升苏氨酸的合成能力。相比较而言，在能够积累苏氨酸的菌株中，苏氨酸的提升幅度更大。
优选地，所述微生物与未修饰的微生物相比，以下(1)～(7)中的任意一个或多个酶的活性增强和/或解除反馈抑制：
(1)天冬氨酸激酶；
(2)天冬氨酸半醛脱氢酶；
(3)高丝氨酸脱氢酶；
(4)高丝氨酸激酶；
(5)苏氨酸外运蛋白；
(6)丙酮酸羧化酶；
(7)葡萄糖-6-磷酸脱氢酶。
上述活性增强是由选自以下1)～6)，或任选的组合实现的：
1)通过导入具有所述酶的编码基因的质粒而增强；
2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
5)通过对酶的氨基酸序列进行改变而增强；
6)通过对编码酶的核苷酸序列进行改变而增强。
上述天冬氨酸激酶、高丝氨酸脱氢酶、天冬氨酸半醛脱氢酶、高丝氨酸激酶、丙酮酸羧化酶、葡 萄糖-6-磷酸脱氢酶在NCBI上的参考序列编号分别为WP_003855724.1、WP_003854900.1、WP_011013506.1、WP_011014183.1、WP_011013816.1、NP_600790.1，或与上述参考序列相似性为90％且具有同等功能的氨基酸序列。
上述苏氨酸外运蛋白优选为大肠杆菌来源的苏氨酸外运蛋白，其在NCBI上的参考序列编号为YP_026264.1，或与上述参考序列相似性为90％且具有同等功能的氨基酸序列。
优选地，所述微生物与未修饰的微生物相比，以下(1)～(3)中的任意一个或多个酶的活性降低或丧失：
(1)二氨基庚二酸脱氢酶；
(2)4-羟基四氢吡啶二羧酸合酶；
(3)柠檬酸合成酶。
优选地，所述活性降低或丧失是通过降低编码所述酶的基因的表达或敲除内源的编码所述酶的基因来实现的。
上述二氨基庚二酸脱氢酶、4-羟基四氢吡啶二羧酸合酶、柠檬酸合成酶在NCBI上的参考序列编号分别为WP_011015254.1、WP_011014792.1、WP_011013914.1，或与上述参考序列相似性为90％且具有同等功能的氨基酸序列。
以上增强酶活性、解除反馈抑制的改造可与降低酶活性或失活的改造的基因靶点分别单独作用或联合作用，由此得到的菌株均可以不同程度地积累苏氨酸。在这些菌株中失活非必需基因均可以不同程度地提升苏氨酸的产量。
作为本发明的优选方案，所述微生物为以下任一种：
(1)非必需基因失活，且天冬氨酸激酶的酶活性增强和/或解除反馈抑制的微生物；
(2)非必需基因失活，且天冬氨酸激酶和/或天冬氨酸半醛脱氢酶的酶活性增强和/或解除反馈抑制的微生物；
(3)非必需基因失活，且天冬氨酸激酶、天冬氨酸半醛脱氢酶和高丝氨酸脱氢酶中的至少一个的酶活性增强和/或解除反馈抑制的微生物；
(4)非必需基因失活，且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶和高丝氨酸激酶中的至少一个的酶活性增强和/或解除反馈抑制的微生物；
(5)非必需基因失活，且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶以及苏氨酸外运蛋白中的至少一个的酶活性增强和/或解除反馈抑制的微生物；
(6)非必需基因失活，且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、苏氨酸外运蛋白以及丙酮酸羧化酶中的至少一个的酶活性增强和/或解除反馈抑制的微生物；
(7)非必需基因失活，且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、苏氨酸外运蛋白、丙酮酸羧化酶以及葡萄糖-6-磷酸脱氢酶中的至少一个的酶活性增强和/或解除反馈抑制的微生物；
(8)非必需基因失活，且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、苏氨酸外运蛋白、丙酮酸羧化酶以及葡萄糖-6-磷酸脱氢酶中的至少一个的酶活性增强和/或解除反馈抑制，同时，二氨基庚二酸脱氢酶的酶活性降低或丧失的微生物；
(9)非必需基因失活，且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、苏氨酸外运蛋白、丙酮酸羧化酶以及葡萄糖-6-磷酸脱氢酶中的至少一个的酶活性增强和/或解除反馈抑制，同时，二氨基庚二酸脱氢酶和/或4-羟基四氢吡啶二羧酸合酶合成酶的酶活性降低或丧失的微生物；
(10)非必需基因失活，且天冬氨酸激酶、天冬氨酸半醛脱氢酶、高丝氨酸脱氢酶、高丝氨酸激酶、苏氨酸外运蛋白、丙酮酸羧化酶以及葡萄糖-6-磷酸脱氢酶中的至少一个的酶活性增强和/或解除反馈抑制，同时，二氨基庚二酸脱氢酶、4-羟基四氢吡啶二羧酸合酶合成酶和/或柠檬酸合成酶的酶 活性降低或丧失的微生物。
优选地，以上所述的酶活性增强通过以下任意一种或多种方式实现：
(1)将目标基因的原始启动子替换为强启动子；
(2)将目标基因的起始密码子突变为ATG；
(3)在染色体上插入一个或多个拷贝的目标基因。
其中，所述强启动子包括Psod或PcspB。
启动子Psod、PcspB的核苷酸序列分别如SEQ ID NO.1、2所示。
优选地，天冬氨酸激酶、天冬氨酸半醛脱氢酶、丙酮酸羧化酶以及葡萄糖-6-磷酸脱氢酶的酶活性增强通过将其原始启动子替换为Psod启动子实现；
高丝氨酸脱氢酶、高丝氨酸激酶的酶活性增强通过将其原始启动子替换为PcspB启动子实现；
大肠杆菌来源的苏氨酸外运蛋白的酶活性增强通过在基因组上插入一个拷贝的大肠杆菌来源的苏氨酸外运蛋白编码基因rhtC实现。
优选地，天冬氨酸激酶的解除反馈抑制通过将天冬氨酸激酶编码基因突变，使得其编码的天冬氨酸激酶发生T311I突变实现；
高丝氨酸脱氢酶的解除反馈抑制通过将高丝氨酸脱氢酶编码基因突变，使得高丝氨酸脱氢酶发生G378E突变实现；
丙酮酸羧化酶的解除反馈抑制通过将丙酮酸羧化酶编码基因突变，使得其编码的丙酮酸羧化酶发生P458S突变实现；
葡萄糖-6-磷酸脱氢酶的解除反馈抑制通过将葡萄糖-6-磷酸脱氢酶编码基因进行突变，使得其编码的葡萄糖-6-磷酸脱氢酶发生A243T突变实现。
以上所述的失活通过将目标基因经一个或多个核苷酸的缺失、插入或替换，使得目标基因不再表达实现。
优选地，二氨基庚二酸脱氢酶编码基因被失活，4-羟基四氢吡啶二羧酸合酶合成酶编码基因和柠檬酸合成酶编码基因的起始密码子被突变为GTG。
本发明所述的微生物优选为谷氨酸棒状杆菌(Corynebacterium glutamicum)。谷氨酸棒状杆菌包括ATCC13032、ATCC13870、ATCC13869、ATCC21799、ATCC21831、ATCC14067、ATCC13287等(参见NCBI Corunebacterium glutamicum进化树https://www.ncbi.nlm.nih.gov/genome/469)，更优选谷氨酸棒状杆菌ATCC 13032。
本发明还提供一种产苏氨酸菌株的构建方法，所述方法包括：弱化或失活具有氨基酸生产能力的棒状杆菌属细菌中的非必需基因，获得基因弱化菌株；所述非必需基因为cg1507-cg1524、Cgl1336-Cgl1352或NCgl1281-NCgl1298。
优选地，所述方法还包括：增强以下(1)～(7)中的任意一个或多个酶的活性和/或将其解除反馈抑制：
(1)天冬氨酸激酶；
(2)天冬氨酸半醛脱氢酶；
(3)高丝氨酸脱氢酶；
(4)高丝氨酸激酶；
(5)苏氨酸外运蛋白；
(6)丙酮酸羧化酶；
(7)葡萄糖-6-磷酸脱氢酶；
所述活性的增强是由选自以下1)～6)，或任选的组合实现的：
1)通过导入具有所述酶的编码基因的质粒而增强；
2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
5)通过对酶的氨基酸序列进行改变而增强；
6)通过对编码酶的核苷酸序列进行改变而增强；
和/或，所述方法还包括：降低以下(1)～(3)中的任意一个或多个酶的活性或使其酶活性丧失：
(1)二氨基庚二酸脱氢酶；
(2)4-羟基四氢吡啶二羧酸合酶合成酶；
(3)柠檬酸合成酶；
优选地，所述活性的降低或丧失是通过降低编码所述酶的基因的表达或敲除内源的编码所述酶的基因来实现的。
上述有关菌株的改造方法包括基因的强化等均为本领域技术人员可知的改造方式，参见满在伟.高产L-精氨酸钝齿棒杆菌的系统途径工程改造[D].江南大学,2016；崔毅.代谢工程改造谷氨酸棒杆菌生产L-亮氨酸[D].天津科技大学.；徐国栋.L-异亮氨酸生产菌株的构建及发酵条件优化.天津科技大学,2015.
本发明提供以上所述的微生物的以下任一种应用：
(1)在发酵生产苏氨酸或其衍生物中的应用；
(2)在作为出发菌株用于构建苏氨酸或其衍生物的生产菌株中的应用；
(3)在提高苏氨酸或其衍生物的产量和/或转化率中的应用。
本发明所述的苏氨酸衍生物可为以苏氨酸为前体合成的化合物，包括异亮氨酸、甘氨酸等。
本发明还提供一种发酵生产苏氨酸或其衍生物的方法，其包括培养以上所述的微生物并从培养物中分离得到苏氨酸或其衍生物的步骤。
具体地，上述方法包括：将所述微生物接种于种子培养基中进行种子培养，得到种子液，将种子液接种于发酵培养基中培养，得到发酵液，将发酵液经分离提取得到苏氨酸或其衍生物。
优选地，所述发酵培养基包含如下组分：玉米浆45-55mL/L，葡萄糖25-35g/L，硫酸铵3-5g/L，MOPS 25-35g/L，磷酸二氢钾8-12g/L，尿素15-25g/L，生物素8-12mg/L，硫酸镁5-7g/L，硫酸亚铁0.5-1.5g/L，VB1·HCl 35-45mg/L，泛酸钙45-55mg/L，烟酰胺35-45mg/L，硫酸锰0.5-1.5g/L，硫酸锌15-25mg/L，硫酸铜15-25mg/L，pH 7.0-7.2。
本发明的有益效果在于：本发明通过失活非必需基因cg1507-cg1524，以及对天冬氨酸激酶和高丝氨酸脱氢酶等酶的强化或弱化共同作用显著提高了菌株生产苏氨酸的能力，菌株的苏氨酸产量较未经改造之前显著提高，同时提升了菌株的生长性能。非必需基因cg1507-cg1524的改造可用于苏氨酸的发酵生产中，具有较好的应用价值。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
本发明所涉及的蛋白及其编码基因的信息如下：
非必需基因，NCBI编号：cg1507-cg1524、Cgl1336-Cgl1352、NCgl1281-NCgl1298；
天冬氨酸激酶，编码基因名称lysC，NCBI编号：cg0306、Cgl0251、NCgl0247；
天冬氨酸半醛脱氢酶，编码基因名称asd，NCBI编号：cg0307、Cgl0252、NCgl0248；
高丝氨酸脱氢酶，编码基因名称hom，NCBI编号：cg1337、Cgl1183、NCgl1136；
二氨基庚二酸脱氢酶，编码基因名称ddh，NCBI编号：cg2900、Cgl2617、NCgl2528；
丙酮酸羧化酶，编码基因名称pyc，NCBI编号：cg0791、Cgl0689、NCgl0659；
葡萄糖-6-磷酸脱氢酶，编码基因名称zwf，NCBI编号：cg1778、Cgl1576、NCgl1514；
高丝氨酸激酶，编码基因名称thrB，NCBI编号：cg1338、Cgl1184、NCgl1137；
大肠杆菌来源的苏氨酸外运蛋白，编码基因名称rhtC，NCBI编号为b3823；
4-羟基四氢吡啶二羧酸合酶，编码基因名称dapA，NCBI编号：cg2161、Cgl1971、NCgl1895；
柠檬酸合成酶，编码基因名称gltA，NCBI编号：cg0949、Cgl0829、NCgl0795。
实施例1 菌株基因组改造质粒构建
1、cg1507-cg1524敲除质粒pK18mobsacB-Δcg1507-cg1524的构建
以ATCC13032基因组为模板，以PCT72/PCT73引物对进行PCR扩增得到上游同源臂up，以PCT74/PCT75引物对进行PCR扩增得到下游同源臂dn。用PCT72/PCT75引物对以up、dn片段为模板进行融合PCR获得全长片段up-dn。pK18mobsacB用BamHI/HindIII酶切。将酶切后的up-dn和pK18mobsacB用无缝克隆试剂盒进行组装，转化Trans1T1感受态细胞，获得重组质粒pK18mobsacB-Δcg1507-cg1524。
2、天冬氨酸激酶表达强化质粒pK18mobsacB-Psod-lysC g1a-T311I的构建
以ATCC13032基因组为模板，以P21/P22引物对进行PCR扩增得到上游同源臂up，以P23/P24引物对进行PCR扩增得到启动子片段Psod，以P25/P26引物对进行PCR扩增得到lysC g1a-T311I，以P27/P28引物对进行PCR扩增得到下游同源臂dn。以P21/P24引物对以up、Psod为模版进行融合PCR，获得片段up-Psod。以P21/P28引物对以up-Psod、lysC g1a-T311I、dn为模板进行融合PCR获得全长片段up-Psod-lysCV1M-T311I-dn。pK18mobsacB用BamHI/HindIII酶切。两者用无缝克隆试剂盒进行组装，转化Trans1T1感受态细胞，获得重组质粒pK18mobsacB-Psod-lysC g1a-T311I。
3、天冬氨酸氨基转移酶表达强化质粒pK18mobsacB-Psod-asd的构建
质粒构建方法参考上述2，所用引物为P1、P2、P3、P4、P5、P6。
4、高丝氨酸脱氢酶表达强化质粒pK18mobsacB-PcspB-hom G378E的构建
质粒构建方法参考上述2，所用引物为P29、P30、P31、P32、P33、P34、P35、P36。
5、高丝氨酸激酶表达强化质粒pK18mobsacB-PcspB-thrB的构建
质粒构建方法参考上述2，所用引物为P7、P8、P9、P10、P11、P12。
6、大肠杆菌来源的苏氨酸外运蛋白表达强化质粒pK18mobsacB-cg2899::rhtC的构建
以ATCC13032基因组为模板，以P157/P158引物对进行PCR扩增得到上游同源臂up，以P159/P160引物对进行PCR扩增得到启动子片段Psod，以大肠杆菌MG1655基因组为模板以P161/P162引物对进行PCR扩增得到rhtC，以ATCC13032基因组为模板P163/P164引物对进行PCR扩增得到下游同源臂dn。以P157/P160引物对以up、Psod为模版进行融合PCR，获得片段up-Psod。以P157/P164引物对以up-Psod、rhtC、dn为模板进行融合PCR获得全长片段up-Psod-rhtC-dn。pK18mobsacB用BamHI/HindIII酶切。两者用无缝克隆试剂盒进行组装，转化Trans1T1感受态细胞，获得重组质粒pK18mobsacB-Psod-rhtC。
7、丙酮酸羧化酶表达强化质粒pK18mobsacB-Psod-pyc P458S的构建
质粒构建方法参考上述2，所用引物为P13、P14、P15、P16、P17、P18、P19、P20。
8、葡萄糖-6-磷酸脱氢酶表达强化质粒pK18mobsacB-Psod-zwf A243T的构建
质粒构建方法参考上述2，所用引物为P129、P130、P131、P132、P133、P134、P135、P136。
9、二氨基脱氢酶表达弱化质粒pK18mobsacB-Δddh的构建
质粒构建方法参考上述1，所用引物为P99、P100、P101、P102。
10、4-羟基四氢吡啶二羧酸合酶表达弱化质粒pK18mobsacB-dapA a1g的构建
质粒构建方法参考上述1，所用引物为P75、P76、P77、P78。
11、柠檬酸合成酶表达弱化质粒pK18mobsacB-gltA a1g的构建
质粒构建方法参考上述1，所用引物为P153、P154、P155、P156。
以上质粒构建过程中所用的引物如表1所示。
表1 引物序列
Figure PCTCN2022143100-appb-000001
Figure PCTCN2022143100-appb-000002
Figure PCTCN2022143100-appb-000003
实施例2 基因组改造菌株的构建
1、天冬氨酸激酶强化表达菌株的构建
按照谷氨酸棒状杆菌经典方法(C.glutamicum Handbook,Charpter 23)制备ATCC13032感受态细胞。将重组质粒pK18mobsacB-Psod-lysC g1a-T311I以电穿孔方法转化该感受态细胞，并在含有15mg/L卡那霉素的选择培养基上筛选转化子，其中目的基因由于同源性被插入到染色体中。将筛得的转化子过夜培养于普通液体脑心浸液培养基中，培养温度为30℃，回转摇床220rpm振荡培养。此培养过程中，转化子发生第二次重组，通过基因交换将载体序列从基因组中除去。将培养物做连续梯度稀释(10 -2连续稀释至10 -4)，稀释液涂布在含有10％蔗糖的普通固体脑心浸液培养基上，33℃静置培养48h。蔗糖培养基上长出的菌落的基因组中不携带插入的载体序列。通过PCR扩增目的片段并进行核苷酸测序分析，获得目的突变菌株命名为SMCT196。该菌株与ATCC13032菌株相比，lysC基因起始密码子由GTG突变为ATG，其编码氨基酸的第311位由苏氨酸变为异亮氨酸，同时lysC基因的启动子被替换为Psod启动子。
2、天冬氨酸半醛脱氢酶强化表达菌株的构建
菌株构建方法参考上述1，以SMCT196为出发菌，将质粒pK18mobsacB-Psod-asd导入菌株 SMCT196中，进行天冬氨酸半醛脱氢酶表达强化的改造，获得的改造菌株命名为SMCT197，与菌株SMCT196相比，该菌株的asd基因的启动子被替换为Psod启动子。
3、高丝氨酸脱氢酶强化表达菌株的构建
菌株构建方法参考上述1，以SMCT197为出发菌，将质粒pK18mobsacB-PcspB-hom G378E导入菌株SMCT197中，进行高丝氨酸脱氢酶表达强化的改造，获得的改造菌株命名为SMCT198，与菌株SMCT197相比，该菌株的hom基因发生突变导致其编码蛋白产生G378E的突变，同时hom基因的启动子被替换为菌株ATCC14067来源的PcspB启动子。
4、高丝氨酸激酶强化表达菌株的构建
菌株构建方法参考上述1，以SMCT198为出发菌，将质粒pK18mobsacB-PcspB-thrB导入菌株SMCT198中，进行高丝氨酸激酶表达强化的改造，获得的改造菌株命名为SMCT199，与菌株SMCT198相比，该菌株中，thrB基因的启动子被替换为PcspB启动子。
5、大肠杆菌来源的苏氨酸外运蛋白强化表达菌株的构建
菌株构建方法参考上述1，以SMCT199为出发菌，将质粒pK18mobsacB-cg2899::rhtC导入菌株SMCT199中，进行大肠杆菌来源的苏氨酸外运蛋白表达弱化的改造，获得的改造菌株命名为SMCT200，与菌株SMCT199相比，该菌株在cg2899基因最后一个碱基之后插入一个拷贝的rhtC基因。
6、丙酮酸羧化酶强化表达菌株的构建
菌株构建方法参考上述1，以SMCT200为出发菌，将质粒pK18mobsacB-Psod-pyc P458S导入菌株SMCT200中，进行丙酮酸羧化酶强化的改造，获得的改造菌株命名为SMCT201，与菌株SMCT200相比，菌株的pyc基因发生突变导致其编码蛋白产生P458S的突变，同时pyc基因的启动子被替换为Psod启动子。
7、葡萄糖-6-磷酸脱氢酶强化表达菌株的构建
菌株构建方法参考上述1，以SMCT201为出发菌，将质粒pK18mobsacB-Psod-zwf A243T导入菌株SMCT201中，进行葡萄糖-6-磷酸脱氢酶表达强化的改造，获得的改造菌株命名为SMCT202，与菌株SMCT201相比，该菌株的zwf基因编码的氨基酸序列发生A243T的突变，同时zwf基因的启动子被替换为Psod启动子。
8、二氨基庚二酸脱氢酶弱化表达菌株的构建
菌株构建方法参考上述1，以SMCT202为出发菌，将质粒pK18mobsacB-Δddh导入菌株SMCT202中，进行二氨基庚二酸脱氢酶表达弱化的改造，获得的改造菌株命名为SMCT203，与菌株SMCT202相比，该菌株的ddh基因被敲除。
9、4-羟基四氢吡啶二羧酸合酶弱化表达菌株的构建
菌株构建方法参考上述1，以SMCT203为出发菌，将质粒pK18mobsacB-dapA a1g导入菌株SMCT203中，进行4-羟基四氢吡啶二羧酸合酶合成酶表达弱化的改造，获得的改造菌株命名为SMCT204，与菌株SMCT203相比，该菌株的dapA基因的起始密码子突变为GTG。
10、柠檬酸合成酶弱化表达菌株的构建
菌株构建方法参考上述1，以SMCT204为出发菌，将质粒pK18mobsacB-gltA a1g导入菌株SMCT204中，进行柠檬酸合成酶表达弱化的改造，获得的改造菌株命名为SMCT205，与菌株SMCT204相比，该菌株的gltA基因的起始密码子突变为GTG。
11、cg1507-cg1527失活菌株的构建
菌株构建方法参考上述1，以ATCC13032、SMCT196、SMCT197、SMCT198、SMCT199、SMCT200、SMCT201、SMCT202、SMCT203、SMCT204、SMCT205作为出发菌，分别将质粒pK18mobsacB-Δcg1507-cg1524导入上述出发菌中，进行cg1507-cg1524基因的失活，获得改造菌株SMCT206、SMCT207、SMCT208、SMCT209、SMCT210、SMCT211、SMCT212、SMCT213、SMCT214、SMCT215、SMCT216，这些改造菌与其对应的出发菌相比，基因组上的cg1507-cg1524被敲除。 以上获得的菌株的基因型信息如表2所示。
表2 菌株基因型信息
Figure PCTCN2022143100-appb-000004
Figure PCTCN2022143100-appb-000005
实施例3 构建菌株的摇瓶发酵验证
对实施例2构建的各菌株进行摇瓶发酵验证，具体如下：
1、培养基
种子活化培养基：BHI 3.7％，琼脂2％，pH 7。
种子培养基：蛋白胨5/L，酵母抽提物5g/L，氯化钠10g/L，硫酸铵16g/L，尿素8g/L，磷酸二氢钾10.4g/L，磷酸氢二钾21.4g/L，生物素5mg/L，硫酸镁3g/L。葡萄糖50g/L，pH 7.2。
发酵培养基：玉米浆50mL/L，葡萄糖30g/L，硫酸铵4g/L，MOPS 30g/L，磷酸二氢钾10g/L，尿素20g/L，生物素10mg/L，硫酸镁6g/L，硫酸亚铁1g/L，V B1·HCl 40mg/L，泛酸钙50mg/L，烟酰胺40mg/L，硫酸锰1g/L，硫酸锌20mg/L，硫酸铜20mg/L，pH 7.2。
2.工程菌摇瓶发酵生产L-苏氨酸
(1)种子培养：挑取ATCC13032、SMCT196、SMCT197、SMCT198、SMCT199、SMCT200、SMCT201、SMCT202、SMCT203、SMCT204、SMCT205、SMCT206、SMCT207、SMCT208、SMCT209、SMCT210、SMCT211、SMCT212、SMCT213、SMCT214、SMCT215、SMCT216斜面种子1环接至装有20mL种子培养基的500mL三角瓶中，30℃、220r/min振荡培养16h，得到种子液。
(2)发酵培养：将2mL种子液接种至装有20mL发酵培养基的500mL三角瓶中，33℃、220r/min振荡培养24h，得到发酵液。
(3)取1mL发酵液离心(12000rpm，2min)，收集上清液，用HPLC检测工程菌与对照菌发酵液中的L-苏氨酸。
各菌株生产苏氨酸能力的检测结果如表3所示。
表3 发酵检测结果
菌株编号 OD 562 苏氨酸(g/L) 菌株编号 OD 562 苏氨酸(g/L)
ATCC13032 25 - SMCT206 26 0.2
SMCT196 24 1.2 SMCT207 25 1.3
SMCT197 24 1.5 SMCT208 25 1.7
SMCT198 24 2.4 SMCT209 25 2.7
SMCT199 23 2.8 SMCT210 25 3.2
SMCT200 23 3.8 SMCT211 25 4.5
SMCT201 22 4.4 SMCT212 24 5.3
SMCT202 23 5.3 SMCT213 24 6.5
SMCT203 24 6.4 SMCT214 23 7.9
SMCT204 22 8.3 SMCT215 25 10.5
SMCT205 21 9.9 SMCT216 23 12.7
注：表3中“-”代表未检测到苏氨酸。
由表3的结果可以看出，敲除cg1507-cg1524的改造菌与未失活cg1507-cg1524的菌株相比，其苏氨酸的产量有不同幅度的提升，苏氨酸产量提高在10％-28％之间，同时，菌株的生长也有一定程度的改善。此外，当敲除cg1507-cg1524与天冬氨酸激酶、天冬氨酸半醛脱氢酶酶、高丝氨酸脱氢酶、高丝氨酸激酶、大肠杆菌来源的苏氨酸外运蛋白、丙酮酸脱羧酶、葡萄糖-6-磷酸脱氢酶至少一个表达强 化、解除反馈抑制以及二氨基庚二酸脱氢酶、4-羟基四氢吡啶二羧酸合酶、柠檬酸合成酶至少一个表达弱化的改造相结合时，其苏氨酸产量有进一步提升，即：cg1507-cg1524的失活与上述位点的改造的组合同样有利于苏氨酸的生产；此外天冬氨酸激酶、天冬氨酸半醛脱氢酶酶、高丝氨酸脱氢酶、高丝氨酸激酶、大肠杆菌来源的苏氨酸外运蛋白、丙酮酸脱羧酶、葡萄糖-6-磷酸脱氢酶至少一个表达强化、解除反馈抑制以及二氨基庚二酸脱氢酶、4-羟基四氢吡啶二羧酸合酶、柠檬酸合成酶至少一个表达弱化均有利于菌株的苏氨酸生产。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Figure PCTCN2022143100-appb-000006
Figure PCTCN2022143100-appb-000007
Figure PCTCN2022143100-appb-000008
Figure PCTCN2022143100-appb-000009
Figure PCTCN2022143100-appb-000010
Figure PCTCN2022143100-appb-000011
Figure PCTCN2022143100-appb-000012
Figure PCTCN2022143100-appb-000013
Figure PCTCN2022143100-appb-000014
Figure PCTCN2022143100-appb-000015
Figure PCTCN2022143100-appb-000016
Figure PCTCN2022143100-appb-000017

Claims (11)

  1. 非必需基因的弱化或失活在提高微生物的苏氨酸产量或构建生产苏氨酸的微生物中的应用；
    所述非必需基因为cg1507-cg1524、Cgl1336-Cgl1352或NCgl1281-NCgl1298。
  2. 根据权利要求1所述的应用，其特征在于，所述微生物为棒状杆菌属细菌，优选为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
  3. 一种修饰的棒状杆菌属微生物，其特征在于，所述微生物相比于未修饰的微生物，其非必需基因弱化或失活；
    所述非必需基因为cg1507-cg1524、Cgl1336-Cgl1352或NCgl1281-NCgl1298。
  4. 根据权利要求3所述的微生物，其特征在于，所述微生物相比于未修饰的微生物具有增强的苏氨酸生产能力。
  5. 根据权利要求3或4所述的微生物，其特征在于，所述微生物与未修饰的微生物相比，以下(1)～(7)中的任意一个或多个酶的活性增强和/或解除反馈抑制：
    (1)天冬氨酸激酶；
    (2)天冬氨酸半醛脱氢酶；
    (3)高丝氨酸脱氢酶；
    (4)高丝氨酸激酶；
    (5)苏氨酸外运蛋白；
    (6)丙酮酸羧化酶；
    (7)葡萄糖-6-磷酸脱氢酶；
    优选地，所述活性增强是由选自以下1)～6)，或任选的组合实现的：
    1)通过导入具有所述酶的编码基因的质粒而增强；
    2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
    3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
    4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
    5)通过对酶的氨基酸序列进行改变而增强；
    6)通过对编码酶的核苷酸序列进行改变而增强。
  6. 根据权利要求3～5任一项所述的微生物，其特征在于，所述微生物与未修饰的微生物相比，以下(1)～(3)中的任意一个或多个酶的活性降低或丧失：
    (1)二氨基庚二酸脱氢酶；
    (2)4-羟基四氢吡啶二羧酸合酶；
    (3)柠檬酸合成酶；
    优选地，所述活性降低或丧失是通过降低编码所述酶的基因的表达或敲除内源的编码所述酶的基因来实现的。
  7. 根据权利要求3～6任一项所述的微生物，其特征在于，所述微生物为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
  8. 产苏氨酸菌株的构建方法，其特征在于，所述方法包括：弱化或失活具有氨基酸生产能力的棒状杆菌属细菌中的非必需基因，获得基因弱化菌株；所述非必需基因为cg1507-cg1524、Cgl1336-Cgl1352或NCgl1281-NCgl1298
    优选地，所述方法还包括：增强以下(1)～(7)中的任意一个或多个酶的活性和/或将其解除反馈抑制：
    (1)天冬氨酸激酶；
    (2)天冬氨酸半醛脱氢酶；
    (3)高丝氨酸脱氢酶；
    (4)高丝氨酸激酶；
    (5)苏氨酸外运蛋白；
    (6)丙酮酸羧化酶；
    (7)葡萄糖-6-磷酸脱氢酶；
    所述活性的增强是由选自以下1)～6)，或任选的组合实现的：
    1)通过导入具有所述酶的编码基因的质粒而增强；
    2)通过增加染色体上所述酶的编码基因的拷贝数而增强；
    3)通过改变染色体上所述酶的编码基因的启动子序列而增强；
    4)通过将强启动子与所述酶的编码基因可操作地连接而增强；
    5)通过对酶的氨基酸序列进行改变而增强；
    6)通过对编码酶的核苷酸序列进行改变而增强；
    可选地，所述方法还包括：降低以下(1)～(3)中的任意一个或多个酶的活性或使其酶活性丧失：
    (1)二氨基庚二酸脱氢酶；
    (2)4-羟基四氢吡啶二羧酸合酶；
    (3)柠檬酸合成酶；
    优选地，所述活性的降低或丧失是通过降低编码所述酶的基因的表达或敲除内源的编码所述酶的基因来实现的。
  9. 根据权利要求8所述的方法，其特征在于，所述棒状杆菌为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
  10. 权利要求3～7任一项所述的微生物的以下任一种应用：
    (1)在发酵生产苏氨酸或其衍生物中的应用；
    (2)在作为出发菌用于构建苏氨酸或其衍生物的生产菌株中的应用；
    (3)在提高苏氨酸或其衍生物的产量和/或转化率中的应用。
  11. 一种发酵生产苏氨酸或其衍生物的方法，其特征在于，包括培养权利要求3～7任一项所述的微生物或用权利要求8或9所述的方法构建的产苏氨酸菌株并从培养物中分离得到苏氨酸或其衍生物的步骤。
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Publication number Priority date Publication date Assignee Title
US20200181660A1 (en) * 2017-04-27 2020-06-11 Universität Bielefeld Carotenoid and amino acid biosynthesis using recombinant corynebacterium glutamicum

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Publication number Priority date Publication date Assignee Title
US20200181660A1 (en) * 2017-04-27 2020-06-11 Universität Bielefeld Carotenoid and amino acid biosynthesis using recombinant corynebacterium glutamicum

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Title
BAUMGART, M. ET AL.: "Construction of a Prophage-Free Variant of Corynebacterium Glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 79, no. 19, 31 October 2013 (2013-10-31), XP055815382, DOI: 10.1128/AEM.01634-13 *

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