WO2023142873A1 - 一种修饰的棒状杆菌属微生物及其生产苏氨酸的应用和构建方法 - Google Patents
一种修饰的棒状杆菌属微生物及其生产苏氨酸的应用和构建方法 Download PDFInfo
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Definitions
- the invention relates to the technical field of microbial engineering, in particular to a modified microorganism of the genus Corynebacterium and its application and construction method for producing threonine.
- L-Threonine (L-Threonine), the chemical name is ⁇ -hydroxy- ⁇ -aminobutyric acid, the molecular formula is C 4 H 9 NO 3 , and the relative molecular mass is 119.12.
- L-threonine is an essential amino acid. Threonine is mainly used in medicine, chemical reagents, food fortifiers, feed additives, etc.
- threonine from oxaloacetate requires five steps of catalytic reactions, which are aspartate kinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), and homoserine dehydrogenase (encoded by asd). Hydrogenase (encoded by hom), homoserine kinase (encoded by thrB) and threonine synthase (encoded by thrC). Hermann Sahm et al.
- Corynebacterium glutamicum hom gene coding for a feedback-resistant homoserine dehydrogenase.[J].Journal of Bacteriology,1991,173(10):3228-3230.), lysC gene (Eikmanns B J,Eggeling L,Sahm H.Molecular aspects of lysine,threonine, and isoleucine biosynthesis in Corynebacterium glutamicum.[J].Antonie Van Leeuwenhoek,1993,64(2):145-163.).
- the purpose of the present invention is to improve the ability of the strain to produce threonine by reducing the expression of the putative membrane protein, thereby providing a threonine (L-threonine) producing strain and its construction method and application.
- the present invention provides a modified microorganism of the genus Corynebacterium, which, compared with an unmodified microorganism, has reduced or no expression of a presumed membrane protein, and the microorganism compared with Due to the enhanced threonine production capacity of unmodified microorganisms.
- the reference sequence number of the putative membrane protein on NCBI is CAF21557, or an amino acid sequence with 90% similarity thereto.
- the reduction or non-expression of the putative membrane protein in the microorganism is achieved by reducing the expression of the gene encoding the putative membrane protein or knocking out the endogenous gene encoding the putative membrane protein.
- Mutagenesis, site-directed mutagenesis or homologous recombination can be used to reduce the expression of genes encoding putative membrane proteins or to knock out endogenous genes encoding putative membrane proteins.
- the activity of enzymes related to threonine synthesis in the microorganism is enhanced and/or reduced; wherein, the enzymes related to the synthesis of threonine with enhanced activity are selected from Asparagus At least one of aminotransferase, phosphoenolpyruvate carboxylase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and homoserine dehydrogenase from Streptococcus mutans; Enzymes related to threonine synthesis are selected from at least one of 4-hydroxydipicolinate synthase and citrate synthase; preferably, their reference sequence numbers on NCBI are WP_011013497.1, WP_011014465, respectively. 1. FOB93_04945, WP_003854900.1, WP_011014792.1, WP_011013914.1, or an amino acid sequence with a similarity of 90% to the above
- the microorganism is any one of the following 1 ⁇ 6:
- the enhancement of the activity of enzymes related to threonine synthesis in the microorganism is achieved by being selected from the following 1) to 5), or an optional combination:
- the reduction of the activity of enzymes related to threonine synthesis in the microorganism is achieved by being selected from the following 6) to 10), or an optional combination:
- the Corynebacterium glutamicum described in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (see NCBI Corunebacterium glutamicum evolutionary tree https:/ /www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
- the present invention provides a method for constructing a threonine-producing strain, the method comprising:
- A weakening the gene encoding the hypothetical membrane protein in corynebacteria with amino acid production ability, and obtaining a gene weakened strain; the attenuation includes knocking out or reducing the expression of the hypothetical membrane protein encoding gene; and/or
- the upstream homology arm up was obtained by PCR amplification with the P53/P54 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P59/P60 primer pair, which was amplified with the P55/P56 primer pair
- the Ptuf fragment was obtained, and the ppc D299N fragment was obtained by PCR amplification with the P57/P58 primer pair.
- the upstream homology arm up was obtained by PCR amplification with the P75/P76 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P77/P78 primer pair.
- the full-length fragment up-dn was obtained by fusion PCR using the P75/P78 primer pair and the up and dn fragments 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-dapA a1g .
- the upstream homology arm up was obtained by PCR amplification with the P153/P154 primer pair, and the downstream homology arm dn was obtained by PCR amplification with the P155/P156 primer pair.
- P153/P156 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 two were assembled with a seamless cloning kit, and Trans1T1 competent cells were transformed to obtain the recombinant plasmid pK18mobsacB-gltA a1g .
- Embodiment 2 Construction of wild-type strain genome transformation strain
- the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. Strains grown on sucrose media do not carry the inserted vector sequence in their genome.
- the target sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT211.
- SMCT212 was used as the starting strain, and the modification of aspartate aminotransferase expression enhancement was carried out (pK18mobsacB-Psod-aspB was introduced into SMCT212), and the obtained modified strain was named SMCT213.
- the strain construction method refers to the above 1), using SMCT213 as the starting bacterium, and carrying out the transformation of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase expression enhancement derived from Streptococcus mutans (introducing pK18mobsacB-Ptuf-gapN into SMCT213), the obtained transformation
- the strain was named SMCT214.
- SMCT214 was used as the starting strain to modify the expression of 4-hydroxydipicolinate synthase (introducing pK18mobsacB-dapA a1g into SMCT214), and the obtained modified strain was named SMCT215.
- SMCT215 was used as the starting strain to modify the expression of citrate synthase attenuation (pK18mobsacB-gltA a1g was introduced into SMCT215), and the obtained modified strain was named SMCT216.
- strain name genotype SMCT211 ATCC13032, PcspB-hom G378E SMCT212 ATCC13032, PcspB-hom G378E , Ptuf-ppc D299N SMCT213 ATCC13032, PcspB-hom G378E , Ptuf-ppc D299N , Psod-aspB SMCT214 ATCC13032, PcspB-hom G378E , Ptuf-ppc D299N , Psod-aspB, Ptuf-gapN SMCT215 ATCC13032, PcspB-hom G378E , Ptuf-ppc D299N , Psod-aspB, Ptuf-gapN, dapA a1g SMCT216 ATCC13032, PcspB-hom G378E , Ptuf-ppc D299N ,
- ATCC13032 competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Charpter 23).
- the recombinant plasmid pK18mobsacB- ⁇ cg1746 was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the target gene was inserted into the chromosome due to homology.
- the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. During this culture, the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
- SMCT217 was used as the starting strain, and homoserine dehydrogenase expression was enhanced (pK18mobsacB-PcspB-hom G378E was introduced into SMCT217), and the obtained modified strain was named SMCT218.
- SMCT218 For the strain construction method, refer to the above 1), using SMCT218 as the starting bacteria, carry out the modification of enhancing the expression of phosphoenolpyruvate carboxylase (pK18mobsacB-Ptuf-ppc D299N is introduced into SMCT218), and the obtained modified strain is named SMCT219.
- SMCT219 was used as the starting strain, and the modification of aspartate aminotransferase expression enhancement was carried out (pK18mobsacB-Psod-aspB was introduced into SMCT219), and the obtained modified strain was named SMCT220.
- SMCT221 was used as the starting strain to modify the expression of 4-hydroxydipicolinate synthase (pK18mobsacB-dapA a1g was introduced into SMCT221), and the obtained modified strain was named SMCT222.
- SMCT222 was used as the starting strain to modify the expression of citrate synthase attenuation (pK18mobsacB-gltA a1g was introduced into SMCT222), and the obtained modified strain was named SMCT223.
- Seed activation medium BHI 3.7%, agar 2%, pH7.
- Seed medium peptone 5/L, yeast extract 5g/L, sodium chloride 10g/L, ammonium sulfate 16g/L, urea 8g/L, potassium dihydrogen phosphate 10.4g/L, dipotassium hydrogen phosphate 21.4g /L, biotin 5mg/L, magnesium sulfate 3g/L. Glucose 50g/L, pH 7.2.
- Fermentation medium corn steep liquor 50mL/L, glucose 30g/L, ammonium sulfate 4g/L, MOPS 30g/L, potassium dihydrogen phosphate 10g/L, urea 20g/L, biotin 10mg/L, magnesium sulfate 6g/L , ferrous sulfate 1g/L, VB1 ⁇ HCl 40mg/L, calcium pantothenate 50mg/L, nicotinamide 40mg/L, manganese sulfate 1g/L, zinc sulfate 20mg/L, copper sulfate 20mg/L, pH 7.2.
- Seed culture pick ATCC 13032, SMCT211, SMCT212, SMCT213, SMCT214, SMCT215, SMCT216, SMCT217, SMCT218, SMCT219, SMCT220, SMCT221, SMCT222, SMCT223 slant seeds 1 loop and connect to 500mL with 20mL seed medium In the Erlenmeyer flask, shake culture at 30°C and 220r/min for 16h.
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Abstract
本发明涉及微生物工程技术领域,具体公开了一种修饰的棒状杆菌属微生物及其生产苏氨酸的应用和构建方法。本发明的修饰的棒状杆菌属微生物相比于未修饰的微生物,其假定膜蛋白的表达降低或不表达,且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。本发明通过使假定膜蛋白失活,提高了菌株生产苏氨酸的产量。为苏氨酸的生产提供了一个新的思路。
Description
本发明涉及微生物工程技术领域,具体地说,涉及一种修饰的棒状杆菌属微生物及其生产苏氨酸的应用和构建方法。
L-苏氨酸(L-Threonine),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-苏氨酸是一种必需的氨基酸,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,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)。Hermann Sahm等人一直致力于高产苏氨酸的谷棒菌株的开发,并取得一定突破,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.)、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.)。继Hermann Sahm之后,Lothar Eggling在该领域进行了进一步的探索,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.)。
但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合成路径中,关于非必需基因敲除等方面的报道较少。关于基因组最小化的研究主要集中在基因组简化是否会造成菌株生长缓慢、营养缺陷以及简化后是否有利于进行基因改造等方面。目前尚未有关于基因组简化与苏氨酸产量相关的报道。因此,有必要对谷氨酸棒状杆菌生产苏 氨酸进行进一步研究。
发明内容
本发明的目的是通过降低假定膜蛋白的表达使菌株生产苏氨酸的能力得到提升,从而提供一种产苏氨酸(L-苏氨酸)菌株及其构建方法与应用。
本发明的技术方案如下:
为了实现本发明目的,第一方面,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,其假定膜蛋白的表达降低或不表达,且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。优选地,假定膜蛋白在NCBI上的参考序列编号为CAF21557,或与其相似性为90%的氨基酸序列。
进一步地,所述微生物体内假定膜蛋白的表达降低或不表达是通过降低编码假定膜蛋白基因的表达或敲除内源的编码假定膜蛋白的基因来实现的。
可以采用诱变、定点突变或同源重组的方法来降低编码假定膜蛋白基因的表达或敲除内源的编码假定膜蛋白的基因。
进一步地,所述微生物与未修饰的微生物相比,其体内苏氨酸合成相关的酶的活性增强和/或降低;其中,活性增强的与所述苏氨酸合成相关的酶选自天冬氨酸氨基转移酶、磷酸烯醇丙酮酸羧化酶、变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶和高丝氨酸脱氢酶中的至少一种;活性降低的与所述苏氨酸合成相关的酶选自4-羟基四氢吡啶二羧酸合酶和柠檬酸合成酶中的至少一种;优选地,它们在NCBI上的参考序列编号分别为WP_011013497.1、WP_011014465.1、FOB93_04945、WP_003854900.1、WP_011014792.1、WP_011013914.1,或与上述参考序列相似度为90%的氨基酸序列。
优选地,所述微生物为如下①~⑥中的任一种:
①假定膜蛋白的表达降低或不表达且高丝氨酸脱氢酶活性增强的微生物;
②假定膜蛋白的表达降低或不表达且高丝氨酸脱氢酶和/或磷酸烯醇丙酮酸羧化酶活性增强的微生物;
③假定膜蛋白的表达降低或不表达且高丝氨酸脱氢酶、磷酸烯醇丙酮酸羧化酶和/或天冬氨酸氨基转移酶活性增强的微生物;
④假定膜蛋白的表达降低或不表达且高丝氨酸脱氢酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸氨基转移酶和/或变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶活性增强的微生物;
⑤假定膜蛋白的表达降低或不表达,高丝氨酸脱氢酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸氨基转移酶和/或变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶活性增强且4-羟基四氢吡啶二羧酸合酶活性降低的微生物;
⑥假定膜蛋白的表达降低或不表达,高丝氨酸脱氢酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸氨基转移酶和/或变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶活性增强且4-羟基四氢吡啶二羧酸合酶和/或柠檬酸合成酶活性降低的微生物。
所述微生物体内苏氨酸合成相关的酶的活性的增强是由选自以下1)~5),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;
所述微生物体内苏氨酸合成相关的酶的活性的降低是由选自以下6)~10),或任选的组合实现的:
6)通过导入具有所述酶的突变编码基因的质粒而降低;
7)通过减少染色体上所述酶的编码基因的拷贝数而降低;
8)通过改变染色体上所述酶的编码基因的启动子序列而降低;
9)通过将弱启动子与所述酶的编码基因可操作地连接而降低;
10)通过对酶的氨基酸序列进行改变而降低。
优选地,本发明所述棒杆菌为谷氨酸棒状杆菌(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。
第二方面,本发明提供产苏氨酸菌株的构建方法,所述方法包括:
A、弱化具有氨基酸生产能力的棒杆菌中编码假定膜蛋白的基因,获得基因弱化菌株;所述弱化包括敲除或降低假定膜蛋白编码基因的表达;和/或
B、增强和/或降低步骤A基因弱化菌株中与苏氨酸合成相关的酶的活性,获得酶活增强和/或降低的菌株;
所述增强的途径选自以下1)~5),或任选的组合:
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;
所述降低的途径选自以下6)~10),或任选的组合:
6)通过导入具有所述酶的突变编码基因的质粒而降低;
7)通过减少染色体上所述酶的编码基因的拷贝数而降低;
8)通过改变染色体上所述酶的编码基因的启动子序列而降低;
9)通过将弱启动子与所述酶的编码基因可操作地连接而降低;
10)通过对酶的氨基酸序列进行改变而降低;
其中,活性增强的与所述苏氨酸合成相关的酶选自天冬氨酸氨基转移酶、磷酸烯醇丙酮酸羧化酶、变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶和高丝氨酸脱氢酶中的至少一种;活性降低的与所述苏氨酸合成相关的酶选自4-羟基四氢吡啶二羧酸合酶和柠檬酸合成酶中的至少一种。
第三方面,本发明提供一种生产苏氨酸的方法,所述方法包括如下步骤:
a)培养上述微生物,以获得所述微生物的培养物;
b)从步骤a)中获得的所述培养物中收集所产生的苏氨酸。
第四方面,本发明提供编码假定膜蛋白的基因的敲除或降低表达在苏氨酸发酵生产或提高苏氨酸发酵产量中的应用。
进一步地,通过失活具有氨基酸生产能力的棒杆菌(Corynebacterium)中的假定膜蛋白来提高苏氨酸的发酵产量。
优选地,本发明所述棒杆菌为谷氨酸棒状杆菌(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。
第五方面,本发明提供所述修饰的棒状杆菌属微生物或按照上述方法构建得到的产苏氨酸菌株在苏氨酸发酵生产或提高苏氨酸发酵产量中的应用。
上述有关菌株的改造方法包括基因的强化和弱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。
优选,本发明修饰的棒状杆菌属微生物中,天冬氨酸氨基转移酶、磷酸烯醇丙酮酸羧化酶和高丝氨酸脱氢酶中的至少一种的表达和/或酶活性的提高,通过使天冬氨酸氨基转移酶、磷酸烯醇丙酮酸羧化酶和高丝氨酸脱氢酶中的至少一种的编码基因由较其天然启动子活性更强的强启动子启动转录实现。变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶的表达和/或酶活性的提高,通过在出发菌株染色体Cgl1705位置插入由Ptuf启动转录的gapN基因实现。
并且,高丝氨酸脱氢酶的表达和/或酶活性的提高,还通过使编码高丝氨酸脱氢酶的基因hom突变,从而使其编码蛋白携带G378E突变实现。磷酸烯醇丙酮酸羧化酶的表达和/或酶活性的提高,还通过使编码磷酸烯醇丙酮酸羧化酶的基因ppc突变,从而使其编码蛋白携带D299N突变实现。
优选地,高丝氨酸脱氢酶的编码基因由PcspB启动转录,PcspB的核苷酸序列如SEQ ID NO.43所示。
天冬氨酸氨基转移酶的编码基因由Psod启动转录,Psod的核苷酸序列如SEQ ID NO.44所示。
变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶的编码基因由Ptuf启动转录,磷酸烯醇丙酮酸羧化酶的编码基因由Ptuf启动转录。Ptuf的核苷酸序列如SEQ ID NO.45所示。
优选,本发明重组微生物中,4-羟基四氢吡啶二羧酸合酶和柠檬酸合成酶中的至少一种的表达和/或酶活性降低,通过使4-羟基四氢吡啶二羧酸合酶和柠檬酸合成酶中的至少一种的编码基因突变实现。
更优选地,使编码4-羟基四氢吡啶二羧酸合酶的基因dapA突变,从而使得其起始密码子由ATG突变为GTG,使编码柠檬酸合成酶的基因gltA突变,从而使得其起始密码子由ATG突变为GTG。
本发明的有益效果至少在于:
本发明通过使假定膜蛋白失活(敲除非必需基因cg1746),增强了菌株的生长速度,适应能力以及对蛋白的表达能力,较未改造之前提高了菌株生产苏氨酸的产量。将本发明的重组微生物应用于苏氨酸生产,可获得更佳的生产效率,为苏氨酸的生产提供了一个新的思路。
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明涉及的蛋白及其编码基因如下:
假定膜蛋白,NCBI编号:cg1746、Cgl1548、NCgl1490;
天冬氨酸氨基转移酶,编码基因名称aspB,NCBI编号:cg0294、Cgl0240、NCgl0237;
高丝氨酸脱氢酶,编码基因名称hom,NCBI编号:cg1337、Cgl1183、NCgl1136;
磷酸烯醇丙酮酸羧化酶,编码基因名称ppc,NCBI编号:cg1787、Cgl1588、NCgl1523;
变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶,编码基因名称gapN,NCBI编号:FOB93_04945;
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。
本发明以模式菌株ATCC 13032为出发菌株构建了一系列苏氨酸生产菌SMCT211、SMCT212、SMCT213、SMCT214、SMCT215、SMCT216,同时构建相应的cg1746失活菌株SMCT218、SMCT219、SMCT220、SMCT221、SMCT222、SMCT223,其苏氨酸产量分别提高20%、26.7%、36%、38.9%、50%、56.6%,同时cg1746失活与苏氨酸合成路径相关的蛋白改造相结合时,苏氨酸产量也会有进一步提升。
改造过程中的表达强化包括启动子的替换,核糖体结合位点的改变、拷贝数的增加、质粒过表达等手段;改造过程中的表达弱化包括启动子的替换,起始密码子改变,核糖体结合位点的改变,基因敲除,基因突变等手段。以上手段均为本领域研究人员公知手段。以上手段无法通过举例而穷尽,因此本发明中的实施例仅用启动子替换作为代表对表达强化进行说明,采用基因敲除和基因突变为代表对表达弱化进行说明。
实施例1 菌株基因组改造质粒构建
1)cg1746敲除质粒pK18mobsacB-Δcg1746
以ATCC 13032基因组为模板,以PCT84/PCT85引物对进行PCR扩增得到上游同 源臂up,以PCT86/PCT87引物对进行PCR扩增得到下游同源臂dn。用PCT84/PCT87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。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-Δcg1746。
2)高丝氨酸脱氢酶表达强化质粒pK18mobsacB-PcspB-hom
G378E
以ATCC 13032基因组为模板,以P29/P30引物对进行PCR扩增得到上游同源臂up,以P35/P36引物对进行PCR扩增得到下游同源臂dn,以P33/P34引物对进行PCR扩增得到hom
G378E片段。以ATCC 14067基因组为模板用P31/P32引物对进行扩增得到PcspB片段。用P29/P36为引物以up、dn、PcspB片段、hom
G378E片段为模板进行融合PCR获得全长片段up-PcspB-hom
G378E-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-PcspB-hom
G378E。
3)天冬氨酸氨基转移酶表达强化质粒pK18mobsacB-Psod-aspB
以ATCC 13032基因组为模板,以P103/P104引物对进行PCR扩增得到上游同源臂up,以P107/P108引物对进行PCR扩增得到下游同源臂dn,用P105/P106引物对进行扩增得到Psod片段。以up、dn、Psod片段为模板用P103/P108进行融合PCR获得全长片段up-Psod-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-aspB
4)变异链球菌来源的NADP依赖性的甘油醛-3-磷酸脱氢酶表达强化质粒pK18mobsacB-Ptuf-gapN
以ATCC 13032基因组为模板,以P137/P138引物对进行PCR扩增得到上游同源臂up,以P143/P144引物对进行PCR扩增得到下游同源臂dn,用P139/P140引物对进行扩增得到Ptuf片段,以合成的变异链球菌来源的gapN为模板,用P141/P142引物对进行PCR扩增获得gapN片段。以up、Ptuf为模板用P137/P140进行融合PCR获得up-Ptuf,以up-Ptuf、gapN、dn片段为模板用P137/P144引物进行融合PCR获得全长片段up-Ptuf-gpaN-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-Ptuf-gapN。
5)磷酸烯醇丙酮酸羧化酶表达强化质粒pK18mobsacB-Ptuf-ppc
D299N
以ATCC 13032基因组为模板,以P53/P54引物对进行PCR扩增得到上游同源臂up,以P59/P60引物对进行PCR扩增得到下游同源臂dn,用P55/P56引物对进行扩增 得到Ptuf片段,用P57/P58引物对进行PCR扩增获得ppc
D299N片段。以up、Ptuf为模板用P53/P56进行融合PCR获得up-Ptuf,以up-Ptuf、ppc
D299N、dn片段为模板用P53/P60引物进行融合PCR获得全长片段up-Ptuf-ppc
D299N-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-Ptuf-ppc
D299N。
6)4-羟基四氢吡啶二羧酸合酶表达弱化质粒pK18mobsacB-dapA
a1g
以ATCC13032基因组为模板,以P75/P76引物对进行PCR扩增得到上游同源臂up,以P77/P78引物对进行PCR扩增得到下游同源臂dn。用P75/P78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。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-dapA
a1g。
7)柠檬酸合成酶表达弱化质粒pK18mobsacB-gltA
a1g
以ATCC13032基因组为模板,以P153/P154引物对进行PCR扩增得到上游同源臂up,以P155/P156引物对进行PCR扩增得到下游同源臂dn。用P153/P156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。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-gltA
a1g。
质粒构建过程中所用的引物如下表1所示:
表1
注:表1中引入点突变的引物相应碱基为加粗加下划线碱基。
实施例2 野生型菌株基因组改造菌株的构建
1)高丝氨酸脱氢酶强化表达菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备ATCC 13032感受态细胞。重组质粒pK18mobsacB-PcspB-hom
G378E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株在其基因组中不携带插入的载体序列。通过PCR扩增目的序列,核苷酸测序分析,获得目的突变菌株命名为SMCT211。
2)磷酸烯醇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SMCT211为出发菌,进行磷酸烯醇丙酮酸羧化酶表达强化的改造(将pK18mobsacB-Ptuf-ppc
D299N导入SMCT211),获得的改造菌株命名为SMCT212。
3)天冬氨酸氨基转移酶强化表达菌株的构建
菌株构建方法参考上述1),以SMCT212为出发菌,进行天冬氨酸氨基转移酶表达强化的改造(将pK18mobsacB-Psod-aspB导入SMCT212),获得的改造菌株命名为SMCT213。
4)变异链球菌来源的NADP依赖的甘油醛-3-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SMCT213为出发菌,进行变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶表达强化的改造(将pK18mobsacB-Ptuf-gapN导入SMCT213),获得的改造菌株命名为SMCT214。
5)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SMCT214为出发菌,进行4-羟基四氢吡啶二羧酸合酶表达弱化的改造(将pK18mobsacB-dapA
a1g导入SMCT214),获得的改造菌株命 名为SMCT215。
6)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SMCT215为出发菌,进行柠檬酸合成酶表达弱化的改造(将pK18mobsacB-gltA
a1g导入SMCT215),获得的改造菌株命名为SMCT216。
获得的菌株列表如下表2。
表2
菌株名称 | 基因型 |
SMCT211 | ATCC13032,PcspB-hom G378E |
SMCT212 | ATCC13032,PcspB-hom G378E,Ptuf-ppc D299N |
SMCT213 | ATCC13032,PcspB-hom G378E,Ptuf-ppc D299N,Psod-aspB |
SMCT214 | ATCC13032,PcspB-hom G378E,Ptuf-ppc D299N,Psod-aspB,Ptuf-gapN |
SMCT215 | ATCC13032,PcspB-hom G378E,Ptuf-ppc D299N,Psod-aspB,Ptuf-gapN,dapA a1g |
SMCT216 | ATCC13032,PcspB-hom G378E,Ptuf-ppc D299N,Psod-aspB,Ptuf-gapN,dapA a1g,gltA a1g |
实施例3 cg1746敲除菌株基因组改造菌株的构建
1)cg1746敲除菌株的构建
按照谷棒经典方法(C.glutamicumHandbook,Charpter23)制备ATCC13032感受态细胞。重组质粒pK18mobsacB-Δcg1746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株在其基因组中不携带插入的载体序列。通过PCR扩增目的序列,核苷酸测序分析,获得目的突变菌株命名为SMCT217。
2)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SMCT217为出发菌,进行高丝氨酸脱氢酶表达强化的改造(将pK18mobsacB-PcspB-hom
G378E导入SMCT217),获得的改造菌株命名为SMCT218。
3)磷酸烯醇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SMCT218为出发菌,进行磷酸烯醇丙酮酸羧化酶表达强化的改造(将pK18mobsacB-Ptuf-ppc
D299N导入SMCT218),获得的改造菌株命名为SMCT219。
4)天冬氨酸氨基转移酶强化表达菌株的构建
菌株构建方法参考上述1),以SMCT219为出发菌,进行天冬氨酸氨基转移酶表达强化的改造(将pK18mobsacB-Psod-aspB导入SMCT219),获得的改造菌株命名为SMCT220。
5)变异链球菌来源的NADP依赖的甘油醛-3-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SMCT220为出发菌,进行变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶表达强化的改造(将pK18mobsacB-Ptuf-gapN导入SMCT220),获得的改造菌株命名为SMCT221。
6)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SMCT221为出发菌,进行4-羟基四氢吡啶二羧酸合酶表达弱化的改造(将pK18mobsacB-dapA
a1g导入SMCT221),获得的改造菌株命名为SMCT222。
7)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SMCT222为出发菌,进行柠檬酸合成酶表达弱化的改造(将pK18mobsacB-gltA
a1g导入SMCT222),获得的改造菌株命名为SMCT223。
获得的菌株列表如下表3所示。
表3
实施例4 构建菌株摇瓶验证
1.培养基
种子活化培养基:BHI 3.7%,琼脂2%,pH7。
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ATCC 13032、SMCT211、SMCT212、SMCT213、SMCT214、SMCT215、SMCT216、SMCT217、SMCT218、SMCT219、SMCT220、SMCT221、SMCT222、SMCT223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,用HPLC检测工程菌与对照菌发酵液中的L-苏氨酸,其浓度如下表4所示。
表4谷氨酸棒状杆菌生产苏氨酸能力的比较
由上表可以看出,失活cg1746的改造菌与未失活菌株相比,其苏氨酸的产量有不同幅度的提升,苏氨酸产量提高在20%-56.6%之间。此外当cg1746与高丝氨酸脱氢酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸氨基转移酶、变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶至少一个表达强化、4-羟基四氢吡啶二羧酸合酶、柠檬酸合成酶至少一 个表达弱化的位点相结合时,其苏氨酸产量有进一步提升,由此可以看出cg1746与上述位点的组合同样有利于苏氨酸的生产。
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。
Claims (10)
- 一种修饰的棒状杆菌属微生物,其中,所述微生物相比于未修饰的微生物,其假定膜蛋白的表达降低或不表达,且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。
- 根据权利要求1所述的微生物,其中,所述微生物体内假定膜蛋白的表达降低或不表达是通过降低编码假定膜蛋白基因的表达或敲除内源的编码假定膜蛋白的基因来实现的。
- 根据权利要求2所述的微生物,其中,采用诱变、定点突变或同源重组的方法来降低编码假定膜蛋白基因的表达或敲除内源的编码假定膜蛋白的基因。
- 根据权利要求1所述的微生物,其中,所述微生物与未修饰的微生物相比,其体内苏氨酸合成相关的酶的活性增强和/或降低;其中,活性增强的与所述苏氨酸合成相关的酶选自天冬氨酸氨基转移酶、磷酸烯醇丙酮酸羧化酶、变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶和高丝氨酸脱氢酶中的至少一种;活性降低的与所述苏氨酸合成相关的酶选自4-羟基四氢吡啶二羧酸合酶和柠檬酸合成酶中的至少一种。
- 根据权利要求4所述的微生物,其中,所述微生物为如下①至⑥中的任一种:①假定膜蛋白的表达降低或不表达且高丝氨酸脱氢酶活性增强的微生物;②假定膜蛋白的表达降低或不表达且高丝氨酸脱氢酶和/或磷酸烯醇丙酮酸羧化酶活性增强的微生物;③假定膜蛋白的表达降低或不表达且高丝氨酸脱氢酶、磷酸烯醇丙酮酸羧化酶和/或天冬氨酸氨基转移酶活性增强的微生物;④假定膜蛋白的表达降低或不表达且高丝氨酸脱氢酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸氨基转移酶和/或变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶活性增强的微生物;⑤假定膜蛋白的表达降低或不表达,高丝氨酸脱氢酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸氨基转移酶和/或变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶活性增强且4-羟基四氢吡啶二羧酸合酶活性降低的微生物;⑥假定膜蛋白的表达降低或不表达,高丝氨酸脱氢酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸氨基转移酶和/或变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶活性增强 且4-羟基四氢吡啶二羧酸合酶和/或柠檬酸合成酶活性降低的微生物。
- 根据权利要求4所述的微生物,其中,所述微生物体内苏氨酸合成相关的酶的活性的增强是由选自以下1)至5),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)通过对所述酶的氨基酸序列进行改变而增强;所述微生物体内苏氨酸合成相关的酶的活性的降低是由选自以下6)至10),或任选的组合实现的:6)通过导入具有所述酶的突变编码基因的质粒而降低;7)通过减少染色体上所述酶的编码基因的拷贝数而降低;8)通过改变染色体上所述酶的编码基因的启动子序列而降低;9)通过将弱启动子与所述酶的编码基因可操作地连接而降低;10)通过对所述酶的氨基酸序列进行改变而降低。
- 根据权利要求1至6中任一项所述的微生物,其中,所述微生物为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
- 产苏氨酸菌株的构建方法,其中,所述方法包括:A、弱化具有氨基酸生产能力的棒状杆菌中编码假定膜蛋白的基因,获得基因弱化菌株;所述弱化包括敲除或降低假定膜蛋白编码基因的表达;和任选地B、增强和/或降低步骤A基因弱化菌株中与苏氨酸合成相关的酶的活性,获得酶活增强和/或降低的菌株;所述增强的途径选自以下1)至5),或任选的组合: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)通过对所述酶的氨基酸序列进行改变而增强;所述降低的途径选自以下6)至10),或任选的组合:6)通过导入具有所述酶的突变编码基因的质粒而降低;7)通过减少染色体上所述酶的编码基因的拷贝数而降低;8)通过改变染色体上所述酶的编码基因的启动子序列而降低;9)通过将弱启动子与所述酶的编码基因可操作地连接而降低;10)通过对所述酶的氨基酸序列进行改变而降低;其中,活性增强的与所述苏氨酸合成相关的酶选自天冬氨酸氨基转移酶、磷酸烯醇丙酮酸羧化酶、变异链球菌来源的NADP依赖的甘油醛-3-磷酸脱氢酶和高丝氨酸脱氢酶中的至少一种;活性降低的与所述苏氨酸合成相关的酶选自4-羟基四氢吡啶二羧酸合酶和柠檬酸合成酶中的至少一种。
- 根据权利要求8所述的方法,其中,所述棒状杆菌为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
- 一种生产苏氨酸的方法,其中,所述方法包括如下步骤:a)培养权利要求1至7中任一项所述的微生物或通过权利要求8或9所述的方法构建的菌株,以获得所述微生物的培养物;b)从步骤a)中获得的所述培养物中收集所产生的苏氨酸。
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