WO2023142848A1 - 启动子、产苏氨酸重组微生物及其应用 - Google Patents

启动子、产苏氨酸重组微生物及其应用 Download PDF

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WO2023142848A1
WO2023142848A1 PCT/CN2022/142851 CN2022142851W WO2023142848A1 WO 2023142848 A1 WO2023142848 A1 WO 2023142848A1 CN 2022142851 W CN2022142851 W CN 2022142851W WO 2023142848 A1 WO2023142848 A1 WO 2023142848A1
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promoter
threonine
enzyme
enhanced
gene
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French (fr)
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康培
王治权
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 promoter, threonine-producing recombinant microorganisms and applications thereof.
  • 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.
  • catalytic enzymes involved in these five-step reactions are aspartokinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), homoserine dehydrogenase (encoded by hom), homoserine kinase (encoded by thrB), and threonine synthase (encoded by thrC).
  • the purpose of the present invention is to provide a mutant dapA gene promoter.
  • Another object of the present invention is to provide a recombinant microorganism constructed by using the promoter for gene expression regulation and its application.
  • the present invention provides the following technical solutions:
  • the present invention provides a kind of promotor, and it has the nucleotide sequence shown in SEQ ID NO.1.
  • the above-mentioned promoter is a mutant dapA gene (4-hydroxy-tetrahydrodipicolinate synthase gene) promoter (mPdapA), and this mutant promoter has the activity of initiating gene transcription, but, unlike the dapA gene wild type Compared with the promoter, the activity of the promoter to initiate gene transcription was significantly reduced.
  • nucleotide sequence of the above-mentioned mutated dapA gene promoter is shown in SEQ ID NO.1.
  • the present invention also provides a biological material, which contains the above-mentioned mutated dapA gene promoter; the biological material is recombinant DNA, vector or host cell.
  • the above-mentioned recombinant DNA can be obtained by operably linking the target gene downstream of the promoter, or can be obtained by operably linking the target gene downstream of the promoter, upstream or downstream operably
  • the above-mentioned vectors may be plasmid vectors, viral vectors or transposons.
  • the above-mentioned host cells are preferably microbial cells.
  • the microbial cells are preferably bacteria of the genus Escherichia or Corynebacterium.
  • the bacterium of the genus Escherichia is preferably Escherichia coli (Escherichia coli); Corynebacterium crenatum, Corynebacterium thermoaminogenes or Corynebacterium aminogenes.
  • the present invention provides the application of the above-mentioned mutated dapA gene promoter in promoting the expression of the target gene.
  • the above application specifically includes: operably linking the target gene with the mutated dapA gene promoter to obtain recombinant DNA, introducing the recombinant DNA into host cells, and expressing the target gene.
  • the above mutated dapA gene promoter can be used to drive the expression of metabolism-related genes of target metabolites, especially for reducing the expression of genes related to competing pathways of target metabolites, so as to increase the accumulation of target metabolites.
  • the present invention also provides the application of the above-mentioned mutated dapA gene promoter in improving the yield or transformation rate of microbial metabolites, or in constructing production strains of microbial metabolites.
  • the metabolite is threonine or a derivative thereof.
  • lysine and isoleucine are the main by-products of threonine fermentative production, and the synthetic pathway of lysine and isoleucine is the main competitive pathway of threonine synthetic pathway. Therefore, it is necessary to block or weaken by-product pathways such as the lysine synthesis pathway, so that more carbon metabolism flows to the threonine synthesis pathway.
  • directly knocking out the lysine synthesis gene dapA and other genes will lead to a sharp decrease in the growth rate of the strain or cause auxotrophy in the cells, which is not conducive to the fermentative production of threonine.
  • the present invention found that using the above-mentioned mutated dapA gene promoter to weaken the lysine and isoleucine synthesis genes can avoid auxotrophy and significantly increase the flow of carbon metabolism to the threonine synthesis pathway under the condition of ensuring the normal growth of the strain. Distribution, enhance the strain's threonine synthesis ability, reduce the accumulation of lysine and isoleucine.
  • the present invention provides a recombinant microorganism, in which the gene encoding at least one enzyme selected from the following (1) to (4) is transcribed by the above-mentioned mutated dapA gene promoter:
  • the genes encoding 4-hydroxy-tetrahydrodipicolinate synthase and diaminopimelate dehydrogenase are transcribed by the above-mentioned mutated dapA gene promoter.
  • the coding genes of 4-hydroxy-tetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, and threonine dehydratase are composed of the above-mentioned mutated
  • the dapA gene promoter drives transcription.
  • the encoding of 4-hydroxy-tetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase and citrate synthase The genes were all transcribed by the above-mentioned mutated dapA gene promoter.
  • the reference sequence numbers of the above-mentioned 4-hydroxy-tetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase and citrate synthase on NCBI are respectively WP_011014792.1, WP_011015254.1, WP_011014022.1, WP_003862033.1, or an amino acid sequence having 90% similarity to the above reference sequence and having equivalent functions.
  • the above-mentioned transcription driven by the mutated dapA gene promoter is specifically replacing the original promoter of the gene with the mutated dapA gene promoter.
  • aspartokinase and homoserine dehydrogenase in the threonine synthesis pathway are strictly regulated by intracellular threonine concentration. Therefore, the starting strain capable of accumulating threonine must first open up its synthesis pathway, which mainly includes the release of feedback inhibition of aspartokinase and homoserine dehydrogenase, and the enhancement of enzyme activity.
  • the enzymatic activity of aspartokinase and/or homoserine dehydrogenase is enhanced and/or feedback inhibition is relieved.
  • the reference sequence numbers of aspartokinase and homoserine dehydrogenase on NCBI are respectively WP_003855724.1 and WP_003854900.1, or amino acid sequences with 90% similarity to the above reference sequences and equivalent functions.
  • the microorganism is any one of the following 1 ⁇ 7:
  • the gene encoding threonine dehydratase is transcribed by the above-mentioned mutated dapA gene promoter, and the enzymatic activity of aspartokinase and/or homoserine dehydrogenase is enhanced and/or feedback inhibition is relieved;
  • citrate synthase is transcribed by the above-mentioned mutated dapA gene promoter, and the enzymatic activity of aspartokinase and/or homoserine dehydrogenase is enhanced and/or feedback inhibition is relieved;
  • the enhancement of the enzyme activity is achieved by being selected from the following 1) to 6), or an optional combination:
  • the threonine dehydratase is encoded by the ilvA and tdcB genes.
  • the enhancement of the enzyme activity is achieved by replacing the original promoter of the gene encoding the enzyme with a strong promoter.
  • the strong promoter is preferably Psod or PcspB.
  • the enhancement of the enzymatic activity of aspartokinase is preferably achieved by replacing the original promoter of the gene encoding it with a strong Psod promoter, and mutating its start codon to ATG.
  • homoserine dehydrogenase is enhanced preferably by replacing the original promoter of the coding gene with the strong PcspB promoter.
  • Defeedback inhibition of aspartokinase is preferably achieved by its T311I mutation.
  • Defeedback inhibition of homoserine dehydrogenase is preferably achieved by subjecting it to the G378E mutation.
  • the recombinant microorganism described in the present invention is preferably a bacterium of the genus Corynebacterium; more preferably Corynebancterium glutamicum.
  • Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc.
  • Corynebacterium acid ATCC13032.
  • the present invention also provides a method for constructing the above-mentioned recombinant microorganism, which method comprises at least one of 4-hydroxy-tetrahydrodipicolinate synthase, diaminopimelate dehydrogenase, threonine dehydratase and citrate synthase
  • the promoter of the gene encoding the enzyme is replaced by the above-mentioned mutated dapA gene promoter.
  • the method further comprises: enhancing the enzymatic activity of aspartokinase and/or homoserine dehydrogenase and/or releasing feedback inhibition thereof;
  • the enhancement of the enzyme activity is achieved by being selected from the following 1) to 6), or an optional combination:
  • the present invention also provides any of the following applications of the recombinant microorganism:
  • the microbial metabolite is threonine.
  • the present invention also provides a method for fermentatively producing threonine or its derivatives, which includes the steps of cultivating the recombinant microorganism and isolating threonine or its derivatives from the culture.
  • the above method includes: inoculating the recombinant microorganism 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 fermented liquid, and separating and extracting the fermented liquid to obtain threonine acid 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, V B1 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 obtains a mutated dapA gene promoter with significantly reduced transcriptional activity by rationally designing the promoter of the dapA gene, and the promoter can be used for weakened expression of microbial genes.
  • Aspartokinase encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247.
  • Homoserine dehydrogenase encoding gene name hom, NCBI number: cg1337, Cgl1183, NCgl1136.
  • Diaminopimelate dehydrogenase encoding gene name ddh, NCBI number: cg2900, Cgl2617, NCgl2528.
  • Threonine dehydratase encoding gene name ilvA, NCBI number: cg2334, Cgl2127, NCgl2046.
  • Threonine dehydratase encoding gene name tdcB, NCBI number: cg1116, Cgl0978, NCgl0939.
  • Citrate synthase encoding gene name gltA, NCBI number: cg0949, Cgl0829, NCgl0795.
  • the present invention is based on the Corynebacterium glutamicum wild-type bacterial strain ATCC13032, constructs threonine production bacterial strain through metabolic engineering transformation.
  • threonine mainly including aspartokinase (lysC), homoserine dehydrogenase (hom) to relieve feedback inhibition and enhance expression.
  • lysC aspartokinase
  • homoserine dehydrogenase homoserine dehydrogenase
  • lysine and isoleucine are the main by-products of threonine fermentation, and the synthesis pathway of lysine and isoleucine is the main competitive pathway of threonine synthesis pathway. Therefore, it is necessary to block or weaken by-product pathways such as the lysine synthesis pathway, so that more carbon metabolism flows to the threonine synthesis pathway.
  • directly knocking out lysine synthesis genes such as dapA will lead to a sharp decrease in the growth rate of the strain or cause auxotrophy in the cells, which is not conducive to the fermentative production of threonine.
  • the present invention adopts the method of rationally transforming the dapA gene promoter, rationally designs and screens the mutated dapA gene promoter (mPdapA), in order to weaken the expression level of 4-hydroxy-tetrahydrodipicolinate synthase, so as to achieve Take into account the purpose of cell growth and optimize carbon flow.
  • mPdapA mutated dapA gene promoter
  • the present invention carries out site-directed mutation (replacing the original dapA gene promoter with the mutated dapA promoter) to the promoter of the 4-hydroxyl-tetrahydrodipicolinate synthase coding gene dapA ), obtain bacterial strain SMCT138, the expression level of this bacterial strain 4-hydroxyl-tetrahydrodipicolinate synthase declines, the ability to produce threonine improves to 3.0g/L by 2.4g/L, the content of by-product lysine From 1.1g/L to 0.8g/L.
  • the present invention also uses SMCT137 as the starting strain, and replaces ddh, ilvA, tdcB, and gltA promoters with mutated dapA promoters to obtain modified bacteria SMCT139, SMCT140, SMCT141, and SMCT142.
  • the transformation of the above-mentioned sites was gradually integrated into SMCT138 to obtain the modified bacteria SMCT143, SMCT144, SMCT145, and SMCT146.
  • the threonine output of the modified strain SMCT143 was increased by 20%, and the by-product lysine was reduced from 0.8g/L to 0.4g/L.
  • the threonine output of the modified strain SMCT144 was increased by 67%, and the by-product isoleucine was reduced from 1.3 g/L is reduced to 0.6g/L; the threonine output of the modified bacteria SMCT145 is increased by 113%, and the by-product isoleucine is reduced from 0.6g/L to 0.3g/L; the threonine output of the modified bacteria SMCT146 has been improved 203%.
  • 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-Psod-lysC g1a-T311I .
  • Plasmid construction method reference 1 the primers used are P29, P30, P31, P32, P33, P34, P35, P36.
  • the artificially synthesized dapA promoter mPdapA (sequence shown in SEQ ID NO.1) with a mutated sequence.
  • PCR amplification was performed with the PW21/22 primer pair to obtain the mPdapA promoter fragment.
  • the upstream homology arm up was obtained by PCR amplification with the PW1/2 primer pair
  • the upstream homology arm dn was obtained by PCR amplification with the PW3/4 primer pair
  • the up, mPdapA and dn were used as templates for fusion PCR to obtain the fragment up-mPdapA-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- ⁇ PdapA::mPdapA.
  • the primers used are PW5, PW6, PW7, PW8, PW21, and PW22.
  • the primers used are PW9, PW10, PW11, PW12, PW21, and PW22.
  • tdcB threonine dehydratase promoter replacement plasmid pK18mobsacB- ⁇ PtdcB::mPdapA
  • the primers used are PW13, PW14, PW15, PW16, PW21, and PW22.
  • the primers used are PW17, PW18, PW19, PW20, PW21, and PW22.
  • ATCC13032 competent cells were prepared according to the classic method of Corynebacterium 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.
  • Transformants obtained by screening were cultured overnight in common liquid brain heart infusion medium at a temperature of 30° C. and 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 fragment was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT136.
  • the lysC gene was mutated, and the corresponding start codon was mutated from GTG to ATG, and the 311th amino acid encoded by it Threonine was mutated to isoleucine, and the promoter of the lysC gene was replaced by the strong promoter Psod.
  • SMCT136 pK18mobsacB-PcspB-hom GapG378E was introduced into the starting bacterium, and the transformation of homoserine dehydrogenase expression enhancement was carried out.
  • the obtained modified strain was named SMCT137.
  • the hom gene was mutated, the corresponding amino acid mutation site was G378E, and the promoter of the hom gene was replaced by the strong promoter PcspB.
  • SMCT137 As the starting bacterium, import pK18mobsacB- ⁇ Pddh::mPdapA into the starting bacterium, and carry out the transformation of diaminopimelate dehydrogenase promoter replacement, and the obtained modified strain is named SMCT139. , the promoter of the diaminopimelate dehydrogenase gene was replaced with the mutated dapA gene promoter (mPdapA).
  • SMCT143 pK18mobsacB- ⁇ Pddh::mPdapA was introduced into the starting strain, and the transformation of diaminopimelate dehydrogenase promoter replacement was carried out, and the obtained modified strain was named SMCT143.
  • SMCT137 as the starting bacterium, import pK18mobsacB- ⁇ PilvA::mPdapA into the starting bacterium, and carry out the transformation of threonine dehydratase (ilvA) promoter replacement, and the obtained modified strain is named SMCT140.
  • the promoter of the threonine dehydratase gene ilvA was replaced with the mutated dapA gene promoter (mPdapA).
  • SMCT143 pK18mobsacB- ⁇ PilvA::mPdapA was introduced into the starting bacterium, and the threonine dehydratase (ilvA) promoter was replaced for transformation, and the obtained modified strain was named SMCT144.
  • SMCT141 The promoter of the threonine dehydratase gene tdcB was replaced with the mutated dapA gene promoter (mPdapA).
  • SMCT145 pK18mobsacB- ⁇ PtdcB::mPdapA was introduced into the starting bacterium to carry out the transformation of threonine dehydratase (tdcB) promoter replacement, and the obtained modified strain was named SMCT145.
  • SMCT137 As the starting bacterium, import pK18mobsacB- ⁇ PgltA::mPdapA into the starting bacterium, and carry out the transformation of citrate synthase promoter replacement.
  • the obtained modified strain is named SMCT142.
  • the citrate synthase in this strain The promoter of the gene was replaced with the mutated dapA gene promoter (mPdapA).
  • SMCT145 As the starting bacterium, pK18mobsacB- ⁇ PgltA::mPdapA was introduced into the starting bacterium, and the transformation of the citrate synthase promoter was carried out, and the obtained modified strain was named SMCT146.
  • 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 slant seeds of strains SMCT136, SMCT137, SMCT138, SMCT139, SMCT140, SMCT141, SMCT142, SMCT143, SMCT144, SMCT145, SMCT146 and connect them to a 500mL Erlenmeyer flask containing 20mL of seed medium for 30 Cultivate with shaking at 220r/min for 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.
  • Table 4 shows the threonine synthesis of the strains with individually weakened dapA, ddh, ilvA, tdcB and gltA.
  • Table 5 shows the fermentation results of strains SMCT138, SMCT143, SMC144, SMCT145 and SMCT146 with superposition and attenuation of the five genes dapA, ddh, ilvA, tdcB and gltA.

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Abstract

提供了一种启动子，其具有如SEQ ID NO.1所示的核苷酸序列。该启动子通过对dapA 基因启动子进行理性设计获得，转录活性明显降低，可用于微生物基因的弱化表达。利用该启动子替换谷氨酸棒杆菌中dapA 基因、ddh 基因、ilvA 基因、tdcB 基因和 gltA 基因的原始启动子，使得这些基因的表达水平明显降低，由此构建的重组微生物中碳代谢流更多地流向苏氨酸合成代谢途径，苏氨酸的产量较出发菌株显著提高，同时具有较好的生长性能。

Description

启动子、产苏氨酸重组微生物及其应用 技术领域
本发明涉及微生物工程技术领域，具体涉及启动子、产苏氨酸重组微生物及其应用。
背景技术
L-苏氨酸(L-Threonin)，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，苏氨酸主要用于医药、化学试剂、食品强化剂、饲料添加剂等方面。
谷氨酸棒杆菌中，由草酰乙酸生成苏氨酸需要五步催化反应，这五步反应涉及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)。针对lysC基因和hom基因，目前已有抗反馈抑制的hom基因和lysC基因的报道(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；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.)。目前已有一些利用谷氨酸棒杆菌发酵生产苏氨酸的工程菌构建的报道，例如：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，使得苏氨酸的产量由49mM提高到67mM(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苏氨酸的合成路径的代谢工程改造，而关于碳代谢流优化的报道较少。此外，在谷氨酸棒状杆菌中进行基因表达调控时，基因强化表达主要采用人工合成的强启动子、内源基因的强启动子或引入异源强启动子；而对基因进行弱化表达的启动子的报道则相对较少，基因的弱化主要依赖起始密码子的替换以及基因敲除技术。
发明内容
本发明的目的是提供一种突变的dapA基因启动子。本发明的另一目的是提供利用该启动子进行基因表达调控构建的重组微生物及其应用。
具体地，本发明提供以下技术方案：
本发明提供一种启动子，其具有如SEQ ID NO.1所示的核苷酸序列。
以上所述的启动子为突变的dapA基因(4-羟基-四氢吡啶二羧酸合酶基因)启动子(mPdapA)，该突变启动子具有启动基因转录的活性，但是，与dapA基因野生型启动子相比，该启动子启动基因转录的活性明显降低。
优选地，上述突变的dapA基因启动子的核苷酸序列如SEQ ID NO.1所示。
本发明还提供一种生物材料，其含有上述突变的dapA基因启动子；所述生物材料为重组DNA、载体或宿主细胞。
以上所述的重组DNA可为在所述启动子的下游可操作性地连接目的基因得到的重组DNA，或者为在所述启动子的下游可操作性地连接目的基因、上游或下游可操作性地连接其他转录调控元件、翻译调控元件得到的重组DNA，或者为在所述启动子的上游和/或下游可操作性地连接用于同源重组的同源臂片段得到的重组DNA。
以上所述的载体可为质粒载体、病毒载体或转座子。
以上所述的宿主细胞优选为微生物细胞。
所述微生物细胞优选为埃希氏菌属或棒状杆菌属细菌。其中，埃希氏菌属细菌优选为大肠杆菌(Escherichia coli)；棒状杆菌属细菌优选为谷氨酸 棒状杆菌(Corynebacterium glutamicum)、北京棒杆菌(Corynebacterium pekinense)、有效棒杆菌(Corynebacterium efficiens)、钝齿棒杆菌(Corynebacterium crenatum)、嗜热产氨棒杆菌(Corynebacterium thermoaminogenes)或产氨棒杆菌(Corynebacterium aminogenes)。
本发明提供上述突变的dapA基因启动子在启动目的基因表达中的应用。
上述应用具体为：将目的基因与突变的dapA基因启动子可操作性地连接，得到重组DNA，将重组DNA导入宿主细胞中，表达目的基因。
上述突变的dapA基因启动子可用于驱动目标代谢产物的代谢相关基因的表达，尤其可用于降低目标代谢产物的竞争途径相关基因的表达，以提高目标代谢产物的积累。
本发明还提供上述突变的dapA基因启动子在提高微生物代谢产物的产量或转化率，或在构建微生物代谢产物的生产菌株中的应用。
优选地，所述代谢产物为苏氨酸或其衍生物。
在苏氨酸发酵生产过程中，赖氨酸和异亮氨酸是苏氨酸发酵生产的主要副产物，赖氨酸和异亮氨酸合成途径是苏氨酸合成途径的主要竞争途径。因此，需要对赖氨酸合成途径等副产物途径进行阻断或弱化，使碳代谢流更多地流向苏氨酸合成途径。然而，直接敲除赖氨酸合成基因dapA等基因会导致菌株生长速度急剧下降或造成细胞营养缺陷，不利于苏氨酸的发酵生产。本发明发现，利用上述突变的dapA基因启动子弱化赖氨酸和异亮氨酸合成基因，能够避免产生营养缺陷，在保证菌株正常生长的情况下，显著提高碳代谢流向苏氨酸合成途径的分配，增强菌株苏氨酸的合成能力，减少赖氨酸和异亮氨酸的积累。
基于上述发现，本发明提供一种重组微生物，所述重组微生物中，选自以下(1)～(4)中至少一个酶的编码基因由上述突变的dapA基因启动子驱动转录：
(1)4-羟基-四氢吡啶二羧酸合酶；
(2)二氨基庚二酸脱氢酶；
(3)苏氨酸脱水酶；
(4)柠檬酸合酶。
作为本发明的一种实施方式，所述重组微生物中，4-羟基-四氢吡啶二羧酸合酶和二氨基庚二酸脱氢酶的编码基因由上述突变的dapA基因启动子驱动转录。
作为本发明的另一种实施方式，所述重组微生物中，4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶、苏氨酸脱水酶的编码基因由上述突变的dapA基因启动子驱动转录。
作为本发明的另一种实施方式，所述重组微生物中，4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶、苏氨酸脱水酶以及柠檬酸合酶的编码基因均由上述突变的dapA基因启动子驱动转录。
上述4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶、苏氨酸脱水酶以及柠檬酸合酶在NCBI上的参考序列编号分别为WP_011014792.1、WP_011015254.1、WP_011014022.1、WP_003862033.1，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。
以上所述的由突变的dapA基因启动子驱动转录具体为将所述基因的原始启动子替换为突变的dapA基因启动子。
以上所述的重组微生物的4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶、苏氨酸脱水酶、柠檬酸合酶的表达量显著降低，其苏氨酸及其衍生物的合成能力明显增强，具有较出发菌株明显更高的苏氨酸产量和转化率，且副产物的积累明显减少。
由于微生物存在严谨的代谢调控，其苏氨酸合成路径中的天冬氨酸激酶和高丝氨酸脱氢酶受到胞内苏氨酸浓度的严格调控。因此，能够积累苏氨酸的出发菌株首先要打通其合成路径，主要包括天冬氨酸激酶、高丝氨酸脱氢酶的解除反馈抑制、酶活性增强等。
具体地，所述重组微生物中，天冬氨酸激酶和/或高丝氨酸脱氢酶的酶 活性被增强和/或解除反馈抑制。
天冬氨酸激酶、高丝氨酸脱氢酶在NCBI上的参考序列编号分别为WP_003855724.1、WP_003854900.1，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，所述微生物为如下①～⑦中的任一种：
①4-羟基-四氢吡啶二羧酸合酶的编码基因由上述突变的dapA基因启动子驱动转录，且天冬氨酸激酶和/或高丝氨酸脱氢酶的酶活性被增强和/或解除反馈抑制的微生物；
②二氨基庚二酸脱氢酶的编码基因由上述突变的dapA基因启动子驱动转录，且天冬氨酸激酶和/或高丝氨酸脱氢酶的酶活性被增强和/或解除反馈抑制的微生物；
③苏氨酸脱水酶的编码基因由上述突变的dapA基因启动子驱动转录，且天冬氨酸激酶和/或高丝氨酸脱氢酶的酶活性被增强和/或解除反馈抑制的微生物；
④柠檬酸合酶的编码基因由上述突变的dapA基因启动子驱动转录，且天冬氨酸激酶和/或高丝氨酸脱氢酶的酶活性被增强和/或解除反馈抑制的微生物；
⑤4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶的编码基因由上述突变的dapA基因启动子驱动转录，且天冬氨酸激酶和/或高丝氨酸脱氢酶的酶活性被增强和/或解除反馈抑制的微生物；
⑥4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶、苏氨酸脱水酶的编码基因由上述突变的dapA基因启动子驱动转录，且天冬氨酸激酶和/或高丝氨酸脱氢酶的酶活性被增强和/或解除反馈抑制的微生物；
⑦4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶、苏氨酸脱水酶、柠檬酸合酶的编码基因由上述突变的dapA基因启动子驱动转录，且天冬氨酸激酶和/或高丝氨酸脱氢酶的酶活性被增强和/或解除反馈抑制的微生物。
优选地，所述酶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。
对于谷氨酸棒状杆菌而言，所述苏氨酸脱水酶由ilvA和tdcB基因编码。
进一步优选地，所述酶活性增强通过将所述酶的编码基因的原始启动子替换为强启动子实现。
所述强启动子优选为Psod或PcspB。
其中，天冬氨酸激酶的酶活性增强优选通过将其编码基因的原始启动子替换为Psod强启动子，并将其起始密码子突变为ATG实现。
高丝氨酸脱氢酶的酶活性增强优选通过将编码基因的原始启动子替换为PcspB强启动子。
天冬氨酸激酶的解除反馈抑制优选通过使其发生T311I突变实现。
高丝氨酸脱氢酶的解除反馈抑制优选通过使其发生G378E突变实现。
本发明所述的重组微生物优选为棒状杆菌属细菌；更优选为谷氨酸棒状杆菌(Corynebancterium 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ATCC13032。
本发明还提供上述重组微生物的构建方法，该方法包括将4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶、苏氨酸脱水酶和柠檬酸合酶中至少一个酶的编码基因的启动子替换为上述突变的dapA基因启动子。
优选地，所述方法还包括：增强天冬氨酸激酶和/或高丝氨酸脱氢酶的 酶活性和/或将其解除反馈抑制；
其中，所述酶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。
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.
本发明还提供所述重组微生物的如下任一种应用：
(1)在发酵生产微生物代谢产物或其衍生物中的应用；
(2)在选育微生物代谢产物或其衍生物的生产菌株中的应用；
(3)在提高微生物代谢产物的产量和/或转化率中的应用。
优选地，所述微生物代谢产物为苏氨酸。
本发明还提供一种发酵生产苏氨酸或其衍生物的方法，该方法包括培养所述重组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：将所述重组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，V B1·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：本发明通过对dapA基因的启动子进行理性设计，获得一个转录活性明显降低的突变的dapA基因启动子，该启动子可用于微生物基因的弱化表达。
利用该启动子替换谷氨酸棒状杆菌中4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶、苏氨酸脱水酶、柠檬酸合酶的编码基因的原始启动子，使得这些基因的表达水平明显降低，由此构建的重组微生物中碳代谢流更多地流向苏氨酸合成代谢途径，菌株生产苏氨酸的能力得到显著提高，苏氨酸的产量较出发菌株提高了279％，同时具有较好的生长性能。
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。
以下实施例涉及的基因和酶的详细信息如下：
天冬氨酸激酶，编码基因名称lysC，NCBI编号：cg0306、Cgl0251、NCgl0247。
高丝氨酸脱氢酶，编码基因名称hom，NCBI编号：cg1337、Cgl1183、NCgl1136。
4-羟基-四氢吡啶二羧酸合酶，编码基因名称dapA，NCBI编号：cg2161、Cgl1971、NCgl1896。
二氨基庚二酸脱氢酶，编码基因名称ddh，NCBI编号：cg2900、Cgl2617、NCgl2528。
苏氨酸脱水酶，编码基因名称ilvA，NCBI编号：cg2334、Cgl2127、NCgl2046。
苏氨酸脱水酶，编码基因名称tdcB，NCBI编号：cg1116、Cgl0978、NCgl0939。
柠檬酸合酶，编码基因名称gltA，NCBI编号：cg0949、Cgl0829、NCgl0795。
本发明在谷氨酸棒杆菌野生型菌株ATCC13032的基础上，通过代谢工程 改造构建苏氨酸生产菌株。首先，打通苏氨酸的合成路径，主要包括天冬氨酸激酶(lysC)、高丝氨酸脱氢酶(hom)解除反馈抑制和表达强化。具体地，在谷氨酸棒杆菌ATCC13032的基础上强化天冬氨酸激酶的表达并通过突变解除该酶的反馈抑制，获得改造菌SMCT136。在此基础上强化高丝氨酸脱氢酶的表达并通过突变解除该酶的反馈抑制，获得改造菌SMCT137，该菌株具备初步的苏氨酸合成能力，其苏氨酸产量为2.4g/L。
对于改造菌SMCT137，赖氨酸和异亮氨酸是苏氨酸发酵生产的主要副产物，赖氨酸和异亮氨酸合成途径是苏氨酸合成途径的主要竞争途径。因此，需要对赖氨酸合成途径等副产物途径进行阻断或弱化，使碳代谢流更多地流向苏氨酸合成途径。然而，直接敲除dapA等赖氨酸合成基因会导致菌株生长速度急剧下降或造成细胞营养缺陷，不利于苏氨酸发酵生产。因此，本发明采用理性改造dapA基因启动子的方法，理性设计并筛选获得了突变的dapA基因启动子(mPdapA)，以期减弱4-羟基-四氢吡啶二羧酸合酶的表达水平，从而达到兼顾细胞生长与优化碳流的目的。在SMCT137的基础上，本发明对赖氨酸合成途径的4-羟基-四氢吡啶二羧酸合酶编码基因dapA的启动子进行了定点突变(以突变的dapA启动子替换原始dapA基因启动子)，获得菌株SMCT138，该菌株4-羟基-四氢吡啶二羧酸合酶的表达水平下降，生产苏氨酸的能力由2.4g/L提高至3.0g/L，副产物赖氨酸的含量由1.1g/L降低到0.8g/L。
此外，本发明还以SMCT137为出发菌株，采用突变的dapA启动子分别替换ddh、ilvA、tdcB、gltA的启动子，获得改造菌SMCT139、SMCT140、SMCT141、SMCT142。经验证，相应的蛋白二氨基庚二酸脱氢酶、苏氨酸脱水酶(ilvA)、苏氨酸脱水酶(tdcB)、柠檬酸合酶的表达量降低，ddh启动子替换使得赖氨酸含量由1.1g/L降低到0.9g/L，苏氨酸的产量提高了0.4g/L，ilvA、tdcB启动子替换分别使得异亮氨酸的含量降低了0.6g/L和0.2g/L，苏氨酸产量分别提高了0.8g/L和0.6g/L，gltA启动子替换后苏氨酸产量提高了0.8g/L。
进一步地，在上述单独弱化的基础上，逐步将上述位点的改造整合到SMCT138中获得改造菌SMCT143、SMCT144、SMCT145、SMCT146。改造菌SMCT143的苏氨酸产量提高了20％，副产物赖氨酸由0.8g/L降低到0.4g/L，改造菌SMCT144的苏氨酸产量提高67％，副产物异亮氨酸由1.3g/L降低到0.6g/L；改造菌SMCT145的苏氨酸产量提高113％，副产物异亮氨酸由0.6g/L降低到0.3g/L；改造菌SMCT146的苏氨酸产量提高了203％。
以下借由实施例具体说明改造菌的构建过程和菌株发酵生产苏氨酸的性能。
实施例1菌株基因组改造质粒构建
1、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-lysC g1a-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。
2、高丝氨酸脱氢酶表达强化质粒pK18mobsacB-PcspB-hom GapG378E的构建
质粒构建方法参考1，所用引物为P29、P30、P31、P32、P33、P34、P35、P36。
3、4-羟基-四氢吡啶二羧酸合酶启动子替换质粒pK18mobsacB-ΔPdapA::mPdapA的构建
人工合成序列突变的dapA启动子mPdapA(序列如SEQ ID NO.1所示)。以人工合成的mPdapA序列为模板，以PW21/22引物对进行PCR扩增，得到mPdapA启动子片段。以ATCC13032基因组为模板，以PW1/2引物对进行PCR 扩增得到上游同源臂up，以PW3/4引物对进行PCR扩增得到上游同源臂dn，采用PW1/4引物对以up、mPdapA、dn为模板进行融合PCR，获得片段up-mPdapA-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-ΔPdapA::mPdapA。
4、二氨基庚二酸脱氢酶启动子替换质粒pK18mobsacB-ΔPddh::mPdapA的构建
质粒构建方法参考3，所用引物为PW5、PW6、PW7、PW8、PW21、PW22。
5、苏氨酸脱水酶(ilvA)启动子替换质粒pK18mobsacB-ΔPilvA::mPdapA的构建
质粒构建方法参考3，所用引物为PW9、PW10、PW11、PW12、PW21、PW22。
6、苏氨酸脱水酶(tdcB)启动子替换质粒pK18mobsacB-ΔPtdcB::mPdapA的构建
质粒构建方法参考3，所用引物为PW13、PW14、PW15、PW16、PW21、PW22。
7、柠檬酸合酶启动子替换质粒pK18mobsacB-ΔPgltA::mPdapA的构建
质粒构建方法参考3，所用引物为PW17、PW18、PW19、PW20、PW21、PW22。
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 PCTCN2022142851-appb-000001
Figure PCTCN2022142851-appb-000002
Figure PCTCN2022142851-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。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。将筛选获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。该培养过程中，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株在其基因组中不携带插入的载体序列。通过PCR扩增目的片段并经核苷酸测序分析，获得目的突变菌株命名为SMCT136，该菌株中，lysC基因被突变，对应的起始密码子由GTG突变为ATG，其编码的氨基酸第311位由苏氨酸突变为异亮氨酸，且lysC基因的启动子被替换为强启动子Psod。
2、高丝氨酸脱氢酶表达强化菌株的构建
菌株构建方法参考1，以SMCT136为出发菌，将pK18mobsacB-PcspB-hom GapG378E导入该出发菌中，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SMCT137，该菌株中，hom基因被突变，对应的氨基酸突变位点为G378E，且hom基因的启动子被替换为强启动子PcspB。
3、4-羟基-四氢吡啶二羧酸合酶启动子替换菌株的构建
菌株构建方法参考1，以SMCT137为出发菌，将pK18mobsacB-ΔPdapA::mPdapA导入出发菌中，进行4-羟基-四氢吡啶二羧酸合酶基因的启动子替换的改造，获得的改造菌株命名为SMCT138，该菌株中，4-羟基-四氢吡啶二羧酸合酶基因的启动子被替换为突变的dapA基因启动子(mPdapA)。
4、二氨基庚二酸脱氢酶启动子替换菌株的构建
菌株构建方法参考1，以SMCT137为出发菌，将pK18mobsacB-ΔPddh::mPdapA导入出发菌中，进行二氨基庚二酸脱氢酶启动子替换的改造，获得的改造菌株命名为SMCT139，该菌株中，二氨基庚二酸脱氢酶基因的启动子被替换为突变的dapA基因启动子(mPdapA)。
同时，以SMCT138为出发菌，将pK18mobsacB-ΔPddh::mPdapA导入出发菌中，进行二氨基庚二酸脱氢酶启动子替换的改造，获得的改造菌株命名为SMCT143。
5、苏氨酸脱水酶(ilvA)启动子替换菌株的构建
菌株构建方法参考1，以SMCT137为出发菌，将 pK18mobsacB-ΔPilvA::mPdapA导入出发菌中，进行苏氨酸脱水酶(ilvA)启动子替换的改造，获得的改造菌株命名为SMCT140，该菌株中苏氨酸脱水酶基因ilvA的启动子被替换为突变的dapA基因启动子(mPdapA)。
同时，以SMCT143为出发菌，将pK18mobsacB-ΔPilvA::mPdapA导入出发菌中，进行苏氨酸脱水酶(ilvA)启动子替换的改造，获得的改造菌株命名为SMCT144。
6、苏氨酸脱水酶(tdcB)启动子替换菌株的构建
菌株构建方法参考1，以SMCT137为出发菌，将pK18mobsacB-ΔPtdcB::mPdapA导入出发菌中，进行苏氨酸脱水酶(tdcB)启动子替换的改造，获得的改造菌株命名为SMCT141，该菌株中苏氨酸脱水酶基因tdcB的启动子被替换为突变的dapA基因启动子(mPdapA)。
同时，以SMCT144为出发菌，将pK18mobsacB-ΔPtdcB::mPdapA导入出发菌中，进行苏氨酸脱水酶(tdcB)启动子替换的改造，获得的改造菌株命名为SMCT145。
7、柠檬酸合酶启动子替换菌株的构建
菌株构建方法参考1，以SMCT137为出发菌，将pK18mobsacB-ΔPgltA::mPdapA导入出发菌中，进行柠檬酸合酶启动子替换的改造，获得的改造菌株命名为SMCT142，该菌株中柠檬酸合酶基因的启动子被替换为突变的dapA基因启动子(mPdapA)。
同时，以SMCT145为出发菌，将pK18mobsacB-ΔPgltA::mPdapA导入出发菌中，进行柠檬酸合酶启动子替换的改造，获得的改造菌株命名为SMCT146。
以上构建的菌株及其基因型信息如表2所示。
表2菌株及其基因型
Figure PCTCN2022142851-appb-000004
Figure PCTCN2022142851-appb-000005
实施例3构建菌株的摇瓶发酵实验
对实施例2构建的工程菌进行发酵验证，具体方法如下：
1、培养基
种子活化培养基：BHI 3.7％，琼脂2％，pH 7.0。
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，VB1·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：挑取菌株SMCT136、SMCT137、SMCT138、SMCT139、SMCT140、SMCT141、SMCT142、SMCT143、SMCT144、SMCT145、SMCT146的斜面种子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。
(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。
(3)取1mL发酵液离心(12000rpm，2min)，收集上清液，采用HPLC方法检测工程菌与对照菌发酵液中的L-苏氨酸、赖氨酸和异亮氨酸的含量。
其中，初步具备苏氨酸合成能力的菌株的发酵结果如表3所示。
表3出发菌株的苏氨酸产量
菌株编号 OD562 L-苏氨酸(g/L) 赖氨酸(g/L) 异亮氨酸(g/L)
SMCT136 24 1.2 1.3 0.8
SMCT137 23 2.4 1.1 1.1
由表3结果可知，在野生型菌株ATCC13032的基础上进行天冬氨酸激酶的改造后，菌株能够初步积累苏氨酸，随着高丝氨酸脱氢酶表达的强化，苏氨酸的产量有了进一步的提升，可积累苏氨酸2.4g/L。
dapA、ddh、ilvA、tdcB和gltA单独弱化的菌株的苏氨酸合成情况如表4所示。
表4单独弱化菌株的苏氨酸产量
菌株编号 OD562 L-苏氨酸(g/L) 赖氨酸(g/L) 异亮氨酸(g/L)
SMCT137 23 2.4 1.1 1.1
SMCT138 23 3.0 0.8 1.2
SMCT139 23 2.8 0.9 1.1
SMCT140 22 3.2 1.1 0.5
SMCT141 23 3.0 1.1 0.9
SMCT142 22 3.2 1.1 1.1
以上结果表明，菌株SMCT138、SMCT139、SMCT140、SMCT141、SMCT142与其出发菌株SMCT137相比，苏氨酸产量均有一定的提升，利用mPdapA替换与赖氨酸合成相关基因dapA和ddh的启动子，赖氨酸产量分别降低了0.3和0.2g/L，利用mPdapA替换与异亮氨酸合成相关基因ilvA和tdcB的启动子，异亮氨酸产量分别降低了0.6和0.2g/L，用mPdapA替换gltA启动子，苏 氨酸产量提升了0.8g/L。
dapA、ddh、ilvA、tdcB和gltA这五个基因叠加弱化的菌株SMCT138、SMCT143、SMC144、SMCT145和SMCT146的发酵结果如表5所示。
表5叠加弱化菌株的苏氨酸产量
菌株编号 OD562 L-苏氨酸(g/L) 赖氨酸(g/L) 异亮氨酸(g/L)
SMCT137 23 2.4 1.1 1.1
SMCT138 23 3.0 0.8 1.2
SMCT143 22 3.6 0.4 1.3
SMCT144 22 5.0 0.4 0.6
SMCT145 22 6.4 0.4 0.3
SMCT146 21 9.1 0.4 0.3
以上结果表明，经碳代谢流的逐步优化后，苏氨酸产量由2.4g/L提高至9.1g/L，提高了279％；说明在苏氨酸末端合成路径打通之后，优化碳代谢流可以明显提升菌株生产苏氨酸的能力。
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 PCTCN2022142851-appb-000006
Figure PCTCN2022142851-appb-000007
Figure PCTCN2022142851-appb-000008
Figure PCTCN2022142851-appb-000009
Figure PCTCN2022142851-appb-000010
Figure PCTCN2022142851-appb-000011
Figure PCTCN2022142851-appb-000012

Claims (10)

  1. 一种启动子，其特征在于，其具有如SEQ ID NO.1所示的核苷酸序列。
  2. 一种生物材料，其特征在于，其含有权利要求1所述的启动子；所述生物材料为重组DNA、载体或宿主细胞。
  3. 权利要求1所述的启动子在启动目的基因表达中的应用。
  4. 权利要求1所述的启动子在提高微生物代谢产物的产量或转化率，或在构建微生物代谢产物的生产菌株中的应用。
  5. 一种重组微生物，其特征在于，所述重组微生物中，选自以下(1)～(4)中的至少一个酶的编码基因由权利要求1所述的启动子驱动转录：
    (1)4-羟基-四氢吡啶二羧酸合酶；
    (2)二氨基庚二酸脱氢酶；
    (3)苏氨酸脱水酶；
    (4)柠檬酸合酶。
  6. 根据权利要求5所述的重组微生物，其特征在于，所述重组微生物中，天冬氨酸激酶和/或高丝氨酸脱氢酶的酶活性被增强和/或解除反馈抑制；
    优选地，所述酶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。
  7. 根据权利要求5或6所述的重组微生物，所述重组微生物为棒状杆 菌属细菌；优选为谷氨酸棒状杆菌(Corynebancterium glutamicum)。
  8. 权利要求5～7任一项所述的重组微生物的构建方法，其特征在于，所述方法包括：将4-羟基-四氢吡啶二羧酸合酶、二氨基庚二酸脱氢酶、苏氨酸脱水酶和柠檬酸合酶中至少一个酶的编码基因的启动子替换为权利要求1所述的启动子；
    优选地，所述方法还包括：增强天冬氨酸激酶和/或高丝氨酸脱氢酶的酶活性和/或将其解除反馈抑制；
    所述酶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。
  9. 权利要求5～7任一项所述的重组微生物的如下任一种应用：
    (1)在发酵生产微生物代谢产物或其衍生物中的应用；
    (2)在选育微生物代谢产物或其衍生物的生产菌株中的应用；
    (3)在提高微生物代谢产物的产量和/或转化率中的应用；
    优选地，所述微生物代谢产物为苏氨酸。
  10. 一种发酵生产苏氨酸或其衍生物的方法，其特征在于，所述方法包括培养权利要求5～7任一项所述的重组微生物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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