WO2021248890A1 - 产l-赖氨酸的重组菌株及其构建方法与应用 - Google Patents
产l-赖氨酸的重组菌株及其构建方法与应用 Download PDFInfo
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- C12R2001/15—Corynebacterium
Definitions
- the yield of lysine is related to the enzyme activity in the biosynthetic pathway, and the activity can generally be enhanced by amplifying one or more genes in the lysine biosynthetic pathway or by using a modified promoter of the gene.
- amino acid sequence of SEQ ID NO: 3 is the protein encoded by the gene NCgl2176.
- the microorganism or recombinant strain has an enhanced L-lysine production capacity compared with the wild-type or parent strain.
- the mutation includes the mutation of base 528 of the polynucleotide sequence shown in SEQ ID NO:1 from adenine (A) to cytosine (C).
- Selection markers may include markers that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface proteins. In an environment treated with such selective agents, since cells expressing only the selection marker can survive or display different phenotypic traits, transformed cells can be selected.
- the term "transformation” refers to the introduction of a polynucleotide into a host cell so that the polynucleotide can be used as an extra-genomic element or inserted into the genome of the host cell to replicate.
- the method of transforming the vector used in the present invention may include a method of introducing nucleic acid into a cell.
- the electric pulse method can be implemented according to the host cell.
- the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum YP97158, preservation number: CGMCC No. 12856, preservation date: August 16, 2016, preservation unit: Chinese microorganism strains General Microbiology Center of the Preservation Management Committee, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355, which has been recorded in Chinese Patent Application CN106367432A (application date September 1, 2016, publication date February 1, 2017 ).
- the microorganism or recombinant strain may also have other improvements related to increasing the production of L-lysine, for example, genes related to the production of NADPH (such as genes encoding glucose dehydrogenase, genes encoding gluconate kinase Gene, gene encoding glyceraldehyde-3-phosphate dehydrogenase, gene encoding glucose-6-phosphate dehydrogenase, or gene encoding 6-phosphogluconate dehydrogenase) and/or with L-lysine
- genes related to the production of NADPH such as genes encoding glucose dehydrogenase, genes encoding gluconate kinase Gene, gene encoding glyceraldehyde-3-phosphate dehydrogenase, gene encoding glucose-6-phosphate dehydrogenase, or gene encoding 6-phosphogluconate dehydrogenase
- Other genes related to biosynthesis or secretion e.g.
- the second aspect of the present invention provides a polynucleotide sequence, an amino acid sequence encoded by the polynucleotide sequence, a recombinant vector including the polynucleotide sequence, and a recombinant strain containing the polynucleotide sequence.
- the polynucleotide sequence includes a polynucleotide encoding a polypeptide containing the amino acid sequence shown in SEQ ID NO: 3, wherein the lysine at position 176 is replaced by a different amino acid.
- amino acid sequence shown in SEQ ID NO: 3 wherein the amino acid sequence after lysine (K) at position 176 is replaced by asparagine (N) is shown in SEQ ID NO: 4.
- the polynucleotide sequence encoding the polypeptide containing the amino acid sequence shown in SEQ ID NO: 3 contains the polynucleotide sequence shown in SEQ ID NO: 1.
- the mutation refers to a change in the base/nucleotide of the site
- the mutation method can be selected from at least one of methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination. kind.
- PCR site-directed mutagenesis and/or homologous recombination are preferably used.
- the amino acid sequence includes the amino acid sequence shown in SEQ ID NO:4.
- the plasmid is pK18mobsacB plasmid.
- the plasmid is pXMJ19 plasmid.
- the recombinant strain contains the polynucleotide sequence.
- the polynucleotide sequence of wild-type NCgl2176 shown in SEQ ID NO:1 in the host strain is modified to make the 528th base mutated to obtain a recombinant strain of Corynebacterium containing the gene encoding the mutant NCgl2176.
- the modification includes at least one of methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
- the mutation refers to the mutation of the 528th base in SEQ ID NO:1 from adenine (A) to cytosine (C); specifically, the polynucleoside containing the gene encoding the mutation NCgl2176
- the acid sequence is shown in SEQ ID NO: 2.
- construction method includes the following steps:
- the recombinant vector is introduced into the host strain to obtain the recombinant coryneform strain containing the mutant NCgl2176 encoding gene.
- the Corynebacterium glutamicum genome can be derived from strain ATCC13032, and its genome sequence can be obtained from the NCBI website.
- the primer is:
- P2 5'CCGGGGACTGGTTTTCGGGTGTTGGTGTGC 3'(SEQ ID NO: 6)
- the overlap PCR amplification is performed as follows: denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 90s (30 cycles).
- the step (2) includes the construction of recombinant plasmids, including: assembling the isolated and purified NCgl2176 A528C and pK18mobsacB plasmids through the NEBuider recombination system to obtain the recombinant plasmid pK18-NCgl2176 A528C .
- the host strain is YP97158.
- the primers for amplifying upstream homology arm fragments are:
- the primers for amplifying the downstream homology arm fragments are:
- P12 5'CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCTATGACACCTTCAACGGATC 3'(SEQ ID NO: 16).
- the construction method includes the following steps:
- NCgl 2176 gene coding region and promoter region sequence or NCgl 2176 A528C gene coding region and promoter region sequence, construct an overexpression plasmid vector, and transfer the vector into the host strain to achieve the strain overexpressing NCgl 2176 or NCgl 2176 A528C gene.
- the primers for amplifying the sequence of the coding region of the gene and its promoter region are:
- the recombinant strain obtained by the present invention can be used alone in the fermentation production of L-lysine, or can be mixed with other L-lysine-producing bacteria to produce L-lysine by fermentation.
- nucleotide sequence that has more than 90%, preferably more than 95%, or more than 98% identity with the nucleotide sequence shown in SEQ ID NO: 30, and it retains the promoter described in (a) Enhance the activity of, and the -49th nucleotide is kept as adenine (A), the -51th nucleotide is kept as thymine (T), the -54 ⁇ -58th nucleotide is kept as GGTGT .
- the present invention also provides an expression cassette comprising the above-mentioned promoter, the expression cassette comprising the promoter and a coding sequence operably linked to the promoter.
- the coding sequence is the coding sequence of the dapB gene.
- the present invention also provides a recombinant strain containing the above-mentioned promoter nucleotide sequence or the above-mentioned recombinant vector.
- the recombinant strain according to the present invention may further include other modifications.
- the primer in the step (1) is:
- the obtained DNA fragment contains EcoR I and Sph I restriction sites at both ends of the obtained DNA fragment through overlap PCR amplification (Overlap PCR).
- the step (2) includes: separating and purifying the amplified products of overlapping PCR reactions, and after double-enzyme digestion (EcoR I/Sph I), and the same double-enzyme digestion (EcoR I/Sph I) After the shuttle plasmid is connected, the allelic replacement recombinant vector is obtained.
- SEQ ID NO 4 NCgl2176 K176N encoded protein amino acid sequence
- the dapB gene promoter region (SEQ ID NO:29) introduced a point mutation, the -49bp position C of the nucleotide sequence of the dapB gene promoter region changed to A, the -51bp position G changed to T, and -54--58bp CTGCA changed to GGTGT (SEQ ID NO: 30).
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Abstract
Description
成分 | 配方 |
蔗糖 | 10g/L |
多聚蛋白胨 | 10g/L |
牛肉膏 | 10g/L |
酵母粉 | 5g/L |
尿素 | 2g/L |
氯化钠 | 2.5g/L |
琼脂粉 | 20g/L |
pH | 7.0 |
培养温度 | 32度 |
成分 | 配方 |
淀粉水解糖 | 30g/L |
硫酸铵 | 12g/L |
硫酸镁 | 0.87g/L |
糖蜜 | 20g/L |
酸化玉米浆 | 3mL/L |
磷酸 | 0.4mL/L |
氯化钾 | 0.53g/L |
消泡剂(2%泡敌) | 4mL/L |
硫酸亚铁 | 120mg/L |
硫酸锰 | 120mg/L |
烟酰胺 | 42mg/L |
泛酸钙 | 6.3mg/L |
维生素B1 | 6.3mg/L |
铜、锌盐溶液 | 0.6g/L |
生物素 | 0.88mg/L |
菌株 | L-赖氨酸产量(g/100ml) | OD(660nm) |
YP97158 | 18.8 | 37.3 |
YPL-4-011 | 19.0 | 37.2 |
YPL-4-012 | 19.3 | 36.5 |
YPL-4-013 | 19.8 | 37.0 |
YPL-4-014 | 20.1 | 36.8 |
YPL-4-015 | 20.3 | 35.9 |
YPL-4-016 | 18.2 | 36.7 |
Claims (16)
- 一种生成L-赖氨酸的属于棒杆菌属的微生物,其特征在于,具有编码SEQ ID NO:3的氨基酸序列的多核苷酸的改善的表达,和/或,SEQ ID NO:29所示的启动子区域第-49位、第-51位、第-54~-58位的碱基发生突变;优选,所述改善的表达是编码SEQ ID NO:3的氨基酸序列的多核苷酸的表达增强,或者编码SEQ ID NO:3的氨基酸序列的多核苷酸具有点突变,或者编码SEQ ID NO:3的氨基酸序列的多核苷酸具有点突变且表达是增强的。
- 如权利要求1所述的微生物,其特征在于,编码SEQ ID NO:3的氨基酸序列的多核苷酸的点突变,使得SEQ ID NO:3的氨基酸序列的第176位赖氨酸被不同的氨基酸所取代;优选第176位赖氨酸被天冬酰胺所取代。
- 如权利要求1-2任一项所述的微生物,其特征在于,编码SEQ ID NO:3的氨基酸序列的多核苷酸包含SEQ ID NO:1的核苷酸序列。
- 如权利要求1-3任一项所述的微生物,其特征在于,所述具有点突变的多核苷酸序列是由SEQ ID NO:1所示多核苷酸序列第528位碱基发生突变而形成的;优选,所述突变包括SEQ ID NO:1所示多核苷酸序列第528位碱基由腺嘌呤(A)突变为胞嘧啶(C);优选,所述具有点突变的多核苷酸序列包括SEQ ID NO:2所示的多核苷酸序列。
- 如权利要求1-4任一项所述的微生物,其特征在于,SEQ ID NO:29所示的启动子区域的第-49位核苷酸胞嘧啶(C)突变为腺嘌呤(A)、第-51位的核苷酸鸟嘌呤(G)突变为胸腺嘧啶(T)、第-54~-58位的核苷酸CTGCA突变为GGTGT;优选,所述启动子核苷酸序列如下所述:(a)SEQ ID NO:30所示的核苷酸序列;或者是,(b)与SEQ ID NO:30所示的核苷酸序列具有90%以上,优选的具有95%以上、或98%以上同一性的核苷酸序列,且其保留(a)所述启动子的增强活性,并且第-49位核苷酸保持为腺嘌呤(A)、第-51位的核苷酸保持为胸腺嘧啶(T)、第-54~-58位的核苷酸保持为GGTGT。
- 如权利要求1-5任一项所述的微生物,其特征在于,所述微生物是谷氨酸棒杆菌(Corynebacterium glutamicum);优选为YP97158。
- 一种多核苷酸序列,其特征在于,包括编码含有SEQ ID NO:3所示的氨基酸序列的多核苷酸,其第176位赖氨酸被不同的氨基酸所取代;优选第176位赖氨酸被天冬酰胺所取代;优选所述多核苷酸序列包括编码含有SEQ ID NO:4所示的氨基酸序列的多核苷酸;优选所述多核苷酸序列是由SEQ ID NO:1所示多核苷酸序列第528位碱基发生突变而形成的;优选所述突变是SEQ ID NO:1所示多核苷酸序列第528位碱基由腺嘌呤(A)突变为胞嘧啶(C);优选所述多核苷酸序列包括SEQ ID NO:2所示的多核苷酸序列。
- 一种氨基酸序列,其特征在于,所述序列如SEQ ID NO:4所示。
- 一种重组载体,其特征在于,包含权利要求7所述的多核苷酸序列。
- 一种重组菌株,其特征在于,包含权利要求7所述的多核苷酸序列。
- 一种启动子核苷酸序列,其包括SEQ ID NO:29所示的启动子区域第-49位、第-51位、第-54~-58位的碱基发生突变形成的核苷酸序列;优选,SEQ ID NO:29所示的启动子区域的第-49位核苷酸胞嘧啶(C)突变为腺嘌呤(A)、第-51位的核苷酸鸟嘌呤(G)突变为胸腺嘧啶(T)、第-54~-58位的核苷酸CTGCA突变为GGTGT。
- 如权利要求11所述的启动子核苷酸序列,其特征在于,所述启动子核苷酸序列如下所述:(a)SEQ ID NO:30所示的核苷酸序列;或者是,(b)与SEQ ID NO:30所示的核苷酸序列具有90%以上,优选的具有95%以上、或98%以上同一性的核苷酸序列,且其保留(a)所述启动子的增强活性,并且第-49位核苷酸保持为腺嘌呤(A)、第-51位的核苷酸保持为胸腺嘧啶(T)、第-54~-58位的核苷酸保持为GGTGT。
- 一种启动子的表达盒,其特征在于,所述表达盒包含权利要求11-12任一项所述的启动子核苷酸序列,和可操纵地连接在所述启动子后的编码序列;优选,所述编码序列为dapB基因的编码序列。
- 一种重组载体,其特征在于,包含权利要求11-12任一项所述的启动子核苷酸序列;优选,所述启动子核苷酸序列与pK18mobsacB质粒相连接来构建所述重组载体。
- 一种重组菌株,其特征在于,包含权利要求11-12任一项所述的启动子核苷酸序列,或包含权利要求13所述的表达盒,或包含权利要求14所述的重组载体;优选,所述重组菌株的宿主菌株是YP97158。
- 一种生产L-赖氨酸的方法,所述方法包括:培养权利要求1-6任一项所述的微生物或者权利要求10或15所述的重组菌株,并从所述培养物中回收L-赖氨酸。
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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KR1020227032897A KR20230002331A (ko) | 2020-06-08 | 2020-12-30 | L-라이신을 생산하는 재조합 균주 및 이의 구축 방법과 응용 |
EP20939893.2A EP4148120A1 (en) | 2020-06-08 | 2020-12-30 | Recombinant strain producing l-lysine and construction methods therefor and use thereof |
BR112022017298A BR112022017298A2 (pt) | 2020-06-08 | 2020-12-30 | Microrganismo produtor de l-lisina, sequência polinucleotídica, sequência de aminoácidos, vetores recombinantes, cepas recombinantes, sequência nucleotídica promotora, cassete de expressão e método para produzir l-lisina |
US18/001,070 US20230295645A1 (en) | 2020-06-08 | 2020-12-30 | Recombinant strain producing l-lysine and construction methods therefor and use thereof |
JP2022552166A JP2023527951A (ja) | 2020-06-08 | 2020-12-30 | L-リジン産生組換え菌株、その構築方法及び使用 |
MX2022013618A MX2022013618A (es) | 2020-06-08 | 2020-12-30 | Cepa recombinante que produce l-lisina y metodos de construccion para la misma y uso de la misma. |
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CN202010514023.XA CN111850010B (zh) | 2020-06-08 | 2020-06-08 | 一种dapB基因改造的重组菌株及其构建方法与应用 |
CN202010514023.X | 2020-06-08 | ||
CN202010790877.0A CN111979165B (zh) | 2020-08-07 | 2020-08-07 | 一种产l-赖氨酸的重组菌株及其构建方法与应用 |
CN202010790877.0 | 2020-08-07 |
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2020
- 2020-12-30 KR KR1020227032897A patent/KR20230002331A/ko not_active Application Discontinuation
- 2020-12-30 BR BR112022017298A patent/BR112022017298A2/pt unknown
- 2020-12-30 JP JP2022552166A patent/JP2023527951A/ja active Pending
- 2020-12-30 US US18/001,070 patent/US20230295645A1/en active Pending
- 2020-12-30 MX MX2022013618A patent/MX2022013618A/es unknown
- 2020-12-30 EP EP20939893.2A patent/EP4148120A1/en active Pending
- 2020-12-30 WO PCT/CN2020/141539 patent/WO2021248890A1/zh unknown
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JP2023527951A (ja) | 2023-07-03 |
EP4148120A1 (en) | 2023-03-15 |
BR112022017298A2 (pt) | 2023-02-07 |
KR20230002331A (ko) | 2023-01-05 |
US20230295645A1 (en) | 2023-09-21 |
MX2022013618A (es) | 2022-11-16 |
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