WO2023142854A1 - 一种苏氨酸生产菌株及其应用 - Google Patents

一种苏氨酸生产菌株及其应用 Download PDF

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WO2023142854A1
WO2023142854A1 PCT/CN2022/142935 CN2022142935W WO2023142854A1 WO 2023142854 A1 WO2023142854 A1 WO 2023142854A1 CN 2022142935 W CN2022142935 W CN 2022142935W WO 2023142854 A1 WO2023142854 A1 WO 2023142854A1
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enhanced
enzyme
activity
threonine
microorganism
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French (fr)
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康培
薛婷莉
宫卫波
何君
李岩
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廊坊梅花生物技术开发有限公司
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    • C12R2001/15—Corynebacterium

Definitions

  • the invention relates to the technical field of microbial engineering, in particular to a threonine producing strain and application thereof.
  • L-Threonine (L-Threonin), the chemical name is ⁇ -hydroxy- ⁇ -aminobutyric acid, the molecular formula is C 4 H 9 NO 3 , the relative molecular weight is 119.12, and it is white orthorhombic or crystalline powder. Odorless, slightly sweet taste, melts and decomposes at 253°C, soluble in water at high temperature, solubility at 25°C is 20.5g/100ml, isoelectric point 5.6, insoluble in ethanol, ether and chloroform.
  • L-threonine is an essential amino acid, mainly used in medicine, chemical reagents, food fortifiers, feed additives, etc. Threonine has an important application in the field of feed additives and its usage is growing rapidly. Threonine is often added to the feed of juvenile piglets and poultry, and is the second limiting amino acid in pig feed and the third limiting amino acid in poultry feed. Adding L-threonine to the compound feed can adjust the amino acid balance of the feed, promote the growth of poultry and livestock, improve the meat quality, improve the nutritional value of feed with low amino acid digestibility, and reduce the cost of feed raw materials. Therefore, it is widely used in EU countries (mainly Germany) , Belgium, Denmark, etc.) and American countries, threonine has been widely used in the feed industry.
  • threonine from oxaloacetate requires a five-step catalytic reaction, and the catalytic enzymes are aspartate kinase (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).
  • lysC aspartate kinase
  • aspartate semialdehyde dehydrogenase encoded by asd
  • Homoserine dehydrogenase encoded by hom
  • homoserine kinase encoded by thrB
  • threonine synthase encoded by thrC
  • the object of the present invention is to improve the threonine-producing ability of the strain by enhancing the activity of transketolase, thereby providing a threonine-producing strain and its application.
  • the present invention provides a modified Corynebacterium microorganism, which has an enhanced transketolase activity compared to an unmodified microorganism, and the microorganism has an enhanced activity compared to an unmodified microorganism.
  • the modified microorganism has enhanced threonine production capacity.
  • the reference sequence number of the transketolase on NCBI is NP_600788.1, or an amino acid sequence with a similarity of 90% and equivalent functions.
  • the activity enhancement described above is achieved by being selected from the following 1) to 6), or an optional combination:
  • transketolase can be enhanced by mutagenesis, site-directed mutation or homologous recombination.
  • the activity of transketolase is enhanced in the following manner: the transcription of the original transketolase encoding gene on the chromosome is initiated by using the Psod promoter, and at the same time, the transketolase coding of a copy is initiated by the Psod promoter on the chromosome. Gene.
  • the microorganisms have enhanced activity of 6-phosphogluconate dehydrogenase and/or NADP-dependent glyceraldehyde 3-phosphate dehydrogenase and/or release feedback inhibition.
  • the reference sequence numbers of 6-phosphogluconate dehydrogenase and NADP-dependent glyceraldehyde 3-phosphate dehydrogenase on NCBI are NP_600669.1, FOB93_04945, or 90% similarity and equivalent functions amino acid sequence.
  • the enzymes related to the threonine synthesis pathway in the microorganism have enhanced activity and/or relieve feedback inhibition; wherein, the enzymes related to the threonine synthesis pathway are selected from At least one of aspartokinase and homoserine dehydrogenase.
  • the reference sequence numbers of aspartokinase and homoserine dehydrogenase on NCBI are respectively WP_003855724.1 and WP_003855724.1, or amino acid sequences with 90% similarity and equivalent functions.
  • the microorganism is any one of the following 1 ⁇ 4:
  • the enhancement of the activity of the above-mentioned enzymes is achieved by being selected from the following 1) to 6), or an optional combination:
  • the enhancement of the enzymatic activity is achieved by using a strong promoter that is more active than the original promoter of the gene to promote the transcription of the gene, and/or by mutating the start codon of the gene to ATG.
  • the strong promoter is preferably the promoters PcspB, Psod or Ptuf.
  • the nucleotide sequences of the promoters Psod, PcspB, and Ptuf are shown in SEQ ID NO.1, 2, and 3, respectively.
  • the enhancement of the activity of aspartokinase is achieved by inserting the promoter Psod before its start codon, and mutating its start codon from GTG to ATG.
  • 6-phosphogluconate dehydrogenase was enhanced by inserting the promoter Psod before its initiation codon.
  • the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase is preferably the NADP-dependent glyceraldehyde 3-phosphate dehydrogenase derived from Streptococcus mutans, and the expression thereof preferably uses Ptuf as a promoter.
  • the release of feedback inhibition of the above-mentioned aspartokinase is achieved by mutating aspartokinase to cause T311I mutation; the release of feedback inhibition of homoserine dehydrogenase is achieved by mutating homoserine dehydrogenase to cause G378E mutation .
  • the microorganism described in the present invention is Corynebacterium glutamicum.
  • Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc.
  • Corynebacterium acid ATCC 13032.
  • the present invention provides a method for constructing a threonine-producing strain, the method comprising:
  • the pathway for enhancing the activity is selected from the following 1) to 6), or an optional combination:
  • the present invention provides a method for producing threonine, the method comprising the steps of:
  • step b) collecting the threonine produced from said culture obtained in step a).
  • the present invention provides the application of the present invention to enhance the activity of transketolase in the fermentative production of threonine or increase the fermentative yield of threonine.
  • the fermentative yield of threonine is improved by enhancing the expression of transketolase in Corynebacterium with amino acid production ability.
  • the corynebacterium described in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum includes ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (see NCBI Corunebacterium glutamicum evolutionary tree https:/ /www.ncbi.nlm.nih.gov/genome/469), more preferably Corynebacterium glutamicum ATCC 13032.
  • the present invention provides the use of the modified Corynebacterium genus microorganism or the threonine-producing strain constructed according to the above-mentioned method in the fermentative production of threonine or in improving the fermentative yield of threonine.
  • transformation methods of the above-mentioned related strains are transformation methods known to those skilled in the art.
  • the beneficial effect of the present invention is that: the present invention significantly improves the supply of reducing power in the threonine synthesis process through the enhanced expression of the transketolase of the pentose phosphate pathway, thereby improving the threonine synthesis ability of the bacterial strain, threonine
  • the acid production was significantly improved compared with the unmodified strain; combined with the enhanced activities of 6-phosphogluconate dehydrogenase, NADP-dependent glyceraldehyde 3-phosphate dehydrogenase and enzymes related to the threonine synthesis pathway, the threonine production further improvement.
  • the above modification can be applied to the fermentative production of threonine, and has good application value.
  • Transketolase encoding gene name tkt, NCBI number: cg1774, Cgl1574, NCgl1512.
  • Aspartokinase encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247.
  • Homoserine dehydrogenase encoding gene name hom, NCBI number: cg1337, Cgl1183, NCgl1136.
  • 6-phosphogluconate dehydrogenase encoding gene name gnd, NCBI number: cg1643, Cgl1452, NCgl1396.
  • NADP-dependent glyceraldehyde 3-phosphate dehydrogenase encoding gene name gapN, NCBI number: FOB93_04945.
  • Embodiment 1 strain genome transformation plasmid construction
  • transketolase promoter enhanced expression plasmid pK18mobsacB-P sod -tkt
  • the upstream homology arm up was obtained by PCR amplification with the P175/P176 primer pair
  • the promoter fragment P sod was obtained by PCR amplification with the P177/P178 primer pair
  • PCR amplification was performed with the P179/P180 primer pair
  • the downstream homology arm down was obtained, and fusion PCR was carried out with the P175/P180 primer pair and up, P sod and down as templates to obtain the fragment tkt-up-P sod -down.
  • pK18mobsacB was digested with BamHI/HindIII.
  • the digested pK18mobsacB and tkt-up-P sod -down were assembled with a seamless cloning kit, transformed into Trans1T1 competent cells, and the recombinant plasmid pK18mobsacB-P sod -tkt was obtained.
  • tkt gene can be inserted into the chromosome by using the recombinant plasmid, and the transcription of the tkt gene is initiated by P sod , and the nucleotide sequence of P sod -tkt is shown in SEQ ID NO.4.
  • the primers used are P21-P28.
  • the plasmid construction method refers to the above 1, and the primers used are P29-P36.
  • the plasmid construction method refers to the above 1, and the primers used are P123-P128.
  • the template for the amplification of the gapN gene uses the Streptococcus mutans genome, and the primers used are P137-P144.
  • the pK18mobsacB-P tuf -gapN plasmid can be used to insert the heterologous gapN gene into the chromosome.
  • 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).
  • the recombinant plasmid pK18mobsacB-P sod -tkt was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15 mg/L kanamycin, wherein the 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.
  • 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 obtained and named SMCT151. Compared with the strain ATCC13032, the P sod promoter was inserted before the start codon TTG of the tkt gene.
  • the strain construction method refers to the above 1, using SMCT151 as the starting bacteria, transforming the pK18mobsacB-P sod -tkt 2nd plasmid, and carrying out two-copy enhanced expression transformation of transketolase, and the obtained modified strain is named SMCT152.
  • SMCT152 After the stop codon of the pgl gene (cgl1578) of the chromosome, a copy of the tkt gene transcription initiated by P sod was added.
  • the obtained modified strain was named SMCT153.
  • the lysC gene of this strain was mutated, causing its start codon to be mutated from GTG to ATG, and the 311th position of the encoded amino acid was mutated from threonine to isoleucine.
  • the P sod promoter was inserted before the start codon of the lysC gene, the mutation of the hom gene resulted in the G378E mutation of its encoded protein, and the P cspB promoter was inserted before the ATG of the hom gene.
  • SMCT153 As the starting bacterium, transform the pK18mobsacB-P sod -gnd plasmid, and carry out the enhanced expression transformation of 6-phosphogluconate dehydrogenase.
  • the obtained modified strain is named SMCT154.
  • SMCT154 Compared with the strain SMCT153 , Insert the P sod promoter before the start codon ATG of the gnd gene.
  • SMCT153 and SMCT154 as the starting bacteria respectively, transform the pK18mobsacB-P tuf -gapN plasmid, and carry out the enhanced expression transformation of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase, and the obtained modified strains are respectively named SMCT155, SMCT156, compared with their corresponding origin strains, the gapN gene transcribed by P tuf was inserted after the stop codon of the cgl1705 gene of the chromosome.
  • SMCT153, SMCT154, SMCT155, and SMCT156 as starting bacteria, pK18mobsacB-P sod -tkt and pK18mobsacB-P sod -tkt 2nd were superimposedly transformed, and the transformed strains were named SMCT157, SMCT158, SMCT159, SMCT160 respectively.
  • the Psod promoter was inserted before the start codon TTG of the tkt gene, and at the same time, a copy of the tkt gene transcribed by Psod was added after the stop codon of the pgl gene of the chromosome.
  • Example 2 Each modified bacterial strain constructed in Example 2 was verified by shake flask fermentation, 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, 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 1 of SMCT151, SMCT152, SMCT153, SMCT154, SMCT155, SMCT156, SMCT157, SMCT158, SMCT159, SMCT160 and put them in a 500mL Erlenmeyer flask containing 20mL of seed medium, at 30°C, Shake culture at 220r/min for 16h to obtain seed liquid.
  • 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 to obtain a fermentation liquid.

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Abstract

提供了一种苏氨酸生产菌株的构建方法，通过构建转酮酶活性增强的棒状杆菌，提高了所述菌株生产苏氨酸的能力；结合增强6-磷酸葡萄糖脱氢酶、NADP依赖性甘油醛3-磷酸脱氢酶以及苏氨酸合成途径相关的酶的活性，可进一步提升苏氨酸的产量，应用于苏氨酸的大规模生产。

Description

一种苏氨酸生产菌株及其应用 技术领域
本发明涉及微生物工程技术领域，具体涉及一种苏氨酸生产菌株及其应用。
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，为白色斜方晶系或结晶性粉末，无臭，味微甜，253℃熔化并分解，高温下溶于水，25℃溶解度为20.5g/100ml，等电点5.6，不溶于乙醇、乙醚和氯仿。
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、饲料添加剂等方面。苏氨酸在饲料添加剂领域具有重要的应用且用量增长迅速。苏氨酸常被添加至未成年仔猪和家禽的饲料中，是猪饲料的第二限制氨基酸和家禽饲料的第三限制氨基酸。在配合饲料中添加L-苏氨酸，可以调整饲料的氨基酸平衡，促进禽畜生长，改善肉质，改善氨基酸消化率低的饲料的营养价值，降低饲料原料成本等因此在欧盟国家(主要是德国、比利时、丹麦等)和美洲国家，苏氨酸已广泛地应用于饲料行业。
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，其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)。目前利用谷氨酸棒状杆菌生产苏氨酸的报道主要集中在对苏氨酸的合成代谢路径的改造，其中包括：抗反馈抑制的hom基因和lysC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；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.)；以及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(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.)等。
为提高苏氨酸的发酵生产效率，降低生产成本，构建能够高效生产苏氨酸的生产菌株具有重要意义。
发明内容
本发明的目的是通过增强转酮酶的活性使菌株生产苏氨酸的能力得到提升，从而提供一种苏氨酸生产菌株及其应用。
苏氨酸合成过程中需要消耗还原力，但是，谷氨酸棒状杆菌中的还原力合成受到严格的调控，还原力的合成和消耗的平衡是维持谷氨酸棒状杆菌的正常生长和保证代谢产物生产的关键。本发明在苏氨酸的代谢工程研究中发现，与其它还原力合成相关基因的改造相比，强化菌株的转酮酶，能够更有效地提升苏氨酸合成过程中的还原力供应，显著提高菌株的苏氨酸合成能力。
为实现本发明的目的，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，其转酮酶的活性增强，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，转酮酶在NCBI上的参考序列编号为NP_600788.1，或与其相似性为90％且具有同等功能的氨基酸序列。
以上所述的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。
可以采用诱变、定点突变或同源重组的方法来增强转酮酶的活性。
优选地，通过以下方式增强转酮酶的活性：采用Psod启动子启动染色体上原始的转酮酶编码基因的转录，同时，在染色体上增加一个拷贝的由Psod启动子启动转录的转酮酶编码基因。
进一步地，所述微生物相比于未修饰的微生物，其6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶的活性增强和/或解除反馈抑制。
优选地，6-磷酸葡萄糖酸脱氢酶、NADP依赖性甘油醛3-磷酸脱氢酶在NCBI上的参考序列编号分别为NP_600669.1、FOB93_04945，或与其相似性为90％且具有同等功能的氨基酸序列。
进一步地，所述微生物相比于未修饰的微生物，其体内与苏氨酸合成途径相关的酶的活性增强和/或解除反馈抑制；其中，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶中的至少一种。
优选地，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_003855724.1，或与其相似性为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①～④中的任一种：
①转酮酶活性增强且天冬氨酸激酶和/或高丝氨酸脱氢酶活性增强和/或解除反馈抑制的微生物；
②转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶和/或6-磷酸葡萄糖酸脱氢酶活性增强和/或解除反馈抑制的微生物；
③转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物；
④转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶、6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物。
上述酶的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。
优选地，所述酶活性的增强通过采用较基因的原始启动子活性更强的强启动子启动基因的转录实现，和/或，通过将基因的起始密码子突变为ATG实现。
所述强启动子优选为启动子PcspB、Psod或Ptuf。其中，启动子Psod、PcspB、Ptuf的核苷酸序列分别如SEQ ID NO.1、2、3所示。
优选地，天冬氨酸激酶的活性增强通过在其起始密码子前插入启动子Psod，并将其起始密码子由GTG突变为ATG实现。
高丝氨酸脱氢酶的活性增强通过将其原始启动子替换为启动子PcspB实现。
6-磷酸葡萄糖酸脱氢酶的活性增强通过在其起始密码子前插入启动子Psod实现。
NADP依赖性甘油醛3-磷酸脱氢酶优选为变异链球菌来源的NADP依赖性甘油醛3-磷酸脱氢酶，其表达优选以Ptuf作为启动子。
上述天冬氨酸激酶的解除反馈抑制通过将天冬氨酸激酶突变，使其发生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突变，使其发生G378E突变实现。
优选地，本发明所述微生物为谷氨酸棒状杆菌(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、强化具有氨基酸生产能力的棒状杆菌中编码转酮酶的基因的表达，获得基因强化菌株；和/或
B、增强6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶的活性和/或将其解除反馈抑制；和/或
C、增强与苏氨酸合成途径相关的酶的活性和/或将其解除反馈抑制，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶中的至少一种；
所述活性增强的途径选自以下1)～6)，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；
6)通过对编码酶的核苷酸序列进行改变而增强。
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)培养所述微生物，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，本发明提供本发明提供转酮酶的活性增强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，通过强化具有氨基酸生产能力的棒状杆菌(Corynebacterium)中的转酮酶的表达来提高苏氨酸的发酵产量。
优选地，本发明所述棒状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.
本发明的有益效果在于：本发明通过对磷酸戊糖途径的转酮酶的强化表达，显著提高了苏氨酸合成过程中的还原力供应，进而提高了菌株的苏氨酸合成能力，苏氨酸产量较未经改造的菌株显著提高；结合6-磷酸葡萄糖酸脱氢酶、NADP依赖性甘油醛3-磷酸脱氢酶以及苏氨酸合成途径相关的酶的活性增强，苏氨酸的产量进一步提升。上述改造可应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。
以下实施例中所涉及的酶和基因的详细信息如下：
转酮酶，编码基因名称tkt，NCBI编号：cg1774，Cgl1574，NCgl1512。
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。
6-磷酸葡萄糖酸脱氢酶,编码基因名称gnd,NCBI编号：cg1643，Cgl1452，NCgl1396。
NADP依赖性甘油醛3-磷酸脱氢酶,编码基因名称gapN，NCBI编号：FOB93_04945。
实施例1 菌株基因组改造质粒构建
1、转酮酶启动子强化表达质粒pK18mobsacB-P sod-tkt的构建
以ATCC13032基因组为模板，以P175/P176引物对进行PCR扩增得到上游同源臂up，以P177/P178引物对进行PCR扩增得到启动子片段P sod，以P179/P180引物对进行PCR扩增得到下游同源臂down，以P175/P180引物对以up、P sod、down为模板进行融合PCR，获得片段tkt-up-P sod-down。pK18mobsacB用BamHI/HindIII酶切。将酶切后的pK18mobsacB和tkt-up-P sod-down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-P sod-tkt。
2、转酮酶启动子强化表达二拷贝质粒pK18mobsacB-P sod-tkt 2nd的构建
质粒构建方法参考上述1，分别使用tkt-1f/1r,tkt-2f/2r,tkt-3f/3r,tkt-4f/4r四对引物扩增出4个小片段，使用tkt-1f/2r，tkt-3f/4r进行1次融合PCR，再使用tkt-1f/4r进行第二次融合PCR，获得全长片段。再按照上述1中的方法构建获得重组质粒pK18mobsacB-P sod-tkt 2nd。利用该重组质粒可在染色体上插入一个额外拷贝的tkt基因，该tkt基因由P sod启动转录，P sod-tkt的核苷酸序列如SEQ ID NO.4所示。
3、天冬氨酸激酶表达强化质粒pK18mobsacB-P sod-lysC g1a-T311I的构建
质粒构建方法参考上述1，所用引物为P21-P28。
4、高丝氨酸脱氢酶表达强化质粒pK18mobsacB-P cspB-hom G378E的构建
质粒构建方法参考上述1，所用引物为P29-P36。
5、6-磷酸葡萄糖酸脱氢酶表达强化质粒pK18mobsacB-P sod-gnd的构建
质粒构建方法参考上述1，所用引物为P123-P128。
6、NADP依赖性甘油醛3-磷酸脱氢酶表达强化质粒pK18mobsacB-P tuf-gapN的构建
质粒构建方法参考上述1，gapN基因扩增的模板使用变异链球菌基因组，所用引物为P137-P144。利用pK18mobsacB-P tuf-gapN质粒可在染色体上插入异源gapN基因。
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
名称 序列
p175 GAGCTCGGTACCCGGGGATCCACTGCGCAAAACTCGCGC
p176 GCCCGGAATAATTGGCAGCTATCCTTCCTGGGTTAAACCG
p177 CCGGTTTAACCCAGGAAGGATAGCTGCCAATTATTCCGGGCT
p178 CAGGTGACAGCGTCAAGGTGGTCAAGGGTAAAAAATCCTTTCGTAGG
p179 CCTACGAAAGGATTTTTTACCCTTGACCACCTTGACGCTGTCACCT
p180 ACGACGGCCAGTGCCAAGCTTTAGATGTGGTGGTCGAATGGG
p21 AATTCGAGCTCGGTACCCGGGGATCCAGCGACAGGACAAGCACTGG
p22 CCCGGAATAATTGGCAGCTATGTGCACCTTTCGATCTACG
p23 CGTAGATCGAAAGGTGCACATAGCTGCCAATTATTCCGGG
p24 TTTCTGTACGACCAGGGCCATGGGTAAAAAATCCTTTCGTA
p25 TACGAAAGGATTTTTTACCCATGGCCCTGGTCGTACAGAAA
p26 TCGGAACGAGGGCAGGTGAAGGTGATGTCGGTGGTGCCGTCT
p27 AGACGGCACCACCGACATCACCTTCACCTGCCCTCGTTCCGA
p28 GTAAAACGACGGCCAGTGCCAAGCTTAGCCTGGTAAGAGGAAACGT
p29 AATTCGAGCTCGGTACCCGGGGATCCCTGCGGGCAGATCCTTTTGA
p30 ATTTCTTTATAAACGCAGGTCATATCTACCAAAACTACGC
p31 GCGTAGTTTTGGTAGATATGACCTGCGTTTATAAAGAAAT
p32 GTATATCTCCTTCTGCAGGAATAGGTATCGAAAGACGAAA
p33 TTTCGTCTTTCGATACCTATTCCTGCAGAAGGAGATATAC
p34 TAGCCAATTCAGCCAAAACCCCCACGCGATCTTCCACATCC
p35 GGATGTGGAAGATCGCGTGGGGGTTTTGGCTGAATTGGCTA
p36 GTAAAACGACGGCCAGTGCCAAGCTTGCTGGCTCTTGCCGTCGATA
p123 ACGAATTCGAGCTCGGTACCCGGGGATCCCGATGTGGGTGACACATGGGGTGCCGTCA
p124 GGAAACCTACGAAAGGATTTTTTACCCATGACTAATGGAGATAATCTCGCACAG
p125 CTGTGCGAGATTATCTCCATTAGTCATGGGTAAAAAATCCTTTCGTAGGTTTCC
p126 GTAAAATCGCCACTACCCCCAAATGGTTAGCTGCCAATTATTCCGGGCTTGTGA
p127 TCACAAGCCCGGAATAATTGGCAGCTAACCATTTGGGGGTAGTGGCGATTTTAC
p128 GTTGTAAAACGACGGCCAGTGCCAAGCTTCATGGTGCGCAGTGTGGTTCGTGCGACG
p137 ACGAATTCGAGCTCGGTACCCGGGGATCCTGTTTACCTGACACTCAAGCCCCGTGCAC
p138 GCCGATTTCAAGATATCTAACAAGCCGCTTAGTCTGAGATAATCTGGGTCAGTGGT
p139 ACCACTGACCCAGATTATCTCAGACTAAGCGGCTTGTTAGATATCTTGAAATCGGC
p140 TTGACATAATTTTTATATTGTTTTGTCATTTACTGAATCCTAAGGGCAACGGCGTTGA
p141 TCAACGCCGTTGCCCTTAGGATTCAGTAAATGACAAAACAATATAAAAATTATGTCAA
p142 AGATGAAGTAGGTGGGTGAATATAGCTGTTATTTGATATCAAATACGACGGATTTA
p143 TAAATCCGTCGTATTTGATATCAAATAACAGCTATATTCACCCACCTACTTCATCT
p144 TTGTAAAACGACGGCCAGTGCCAAGCTTGATTGGAATCGGCATGGGTGTTCTGCGT
tkt2-1f GAGCTCGGTACCCGGGGATCCGATGAGCGCAATGTCCCTGT
tkt2-1r CCCGGAATAATTGGCAGCTATTAGAGATTTCCTGCAGCATCA
tkt2-2f ATGATGCTGCAGGAAATCTCTAATAGCTGCCAATTATTCCGGGCTTGT
tkt2-2r CAGGTGACAGCGTCAAGGTGGTCAAGGGTAAAAAATCCTTTCGTAGG
tkt2-3f CCTACGAAAGGATTTTTTACCCTTGACCACCTTGACGCTGTCACCTG
tkt2-3r CTTCTTGTTAGAGCTGGCGCTGCTTAACCGTTAATGGAGTCCTTGGCCGC
tkt2-4f GCGGCCAAGGACTCCATTAACGGTTAAGCAGCGCCAGCTCTAACAAGAAG
tkt2-4r ACGACGGCCAGTGCCAAGCTTGTGGATCCAGGTTAAGCGCA
实施例2 基因组改造菌株的构建
1、转酮酶启动子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-P sod-tkt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SMCT151，该菌株与菌株ATCC13032相比，在tkt基因起始密码子TTG前插入P sod启动子。
2、转酮酶多拷贝强化表达菌株的构建
菌株构建方法参考上述1，以SMCT151为出发菌，转化pK18mobsacB-P sod-tkt 2nd质粒，进行转酮酶二拷贝强化表达改造，获得的改造菌株命名为SMCT152，该菌株与菌株SMCT151相比，在染色体的pgl基因(cgl1578)终止密码子后增加了一个拷贝的由P sod启动转录的tkt基因。
3、天冬氨酸激酶和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ATCC13032为出发菌，叠加转化pK18mobsacB-P sod-lysC g1a-T311I，pK18mobsacB-P cspB-hom G378E质粒，进行天冬氨酸激酶和高丝氨酸脱氢酶的强化表达改造，获得的改造菌株命名为SMCT153，该菌株与ATCC13032菌株相比，lysC基因发生突变导致其起始密码子由GTG突变为ATG，编码氨基酸的第311位由苏氨酸突变为异亮氨酸，同时lysC基因起始密码子前插入P sod启动子，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ATG前插入P cspB启动子。
4、6-磷酸葡萄糖酸脱氢酶表达强化菌株的构建
菌株构建方法参考上述1，以SMCT153为出发菌，转化pK18mobsacB-P sod-gnd质粒，进行6-磷酸葡萄糖酸脱氢酶强化表达改造，获得的改造菌株命名为SMCT154，该菌株与菌株SMCT153相比，gnd基因起始密码子ATG前插入P sod启动子。
5、NADP依赖性甘油醛3-磷酸脱氢酶表达强化菌株的构建
菌株构建方法参考上述1，分别以SMCT153，SMCT154为出发菌，转化pK18mobsacB-P tuf-gapN质粒，进行NADP依赖性甘油醛3-磷酸脱氢酶强化表达改造，获得的改造菌株分别命名为SMCT155，SMCT156，这两株菌与其对应的出发菌相比，在染色体的cgl1705基因的终止密码子之后插入了由P tuf启动转录的gapN基因。
6、转酮酶多拷贝强化表达叠加菌株构建
分别以SMCT153，SMCT154，SMCT155，SMCT156为出发菌，叠加转化pK18mobsacB-P sod-tkt和pK18mobsacB-P sod-tkt 2nd，改造获得的菌株分别命名为SMCT157，SMCT158，SMCT159，SMCT160，这些菌株与其对应的出发菌相比，在tkt基因的起始密码子TTG前插入Psod启动子，同时，在染色体的pgl基因终止密码子之后增加了一个拷贝的由Psod启动转录的tkt基因。
上述获得的菌株基因型信息如表2所示。
表2 菌株基因型信息
菌株名称 基因型
SMCT151 ATCC13032,Psod-tkt
SMCT152 SMCT151,Psod-tkt 2nd
SMCT153 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E
SMCT154 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,Psod-gnd
SMCT155 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,Ptuf-gapN
SMCT156 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,Psod-gnd,Ptuf-gapN
SMCT157 SMCT153,Psod-tkt，Psod-tkt 2nd
SMCT158 SMCT154,Psod-tkt，Psod-tkt 2nd
SMCT159 SMCT155,Psod-tkt，Psod-tkt 2nd
SMCT160 SMCT156,Psod-tkt，Psod-tkt 2nd
实施例3 菌株的摇瓶发酵验证
对实施例2构建的各改造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，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SMCT151、SMCT152、SMCT153、SMCT154、SMCT155、SMCT156、SMCT157、SMCT158、SMCT159、SMCT160的斜面种子1环接至装有20mL种子培养基的500mL三角瓶中，于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，于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。
苏氨酸的摇瓶发酵结果如表3所示。
表3 发酵检测结果
Figure PCTCN2022142935-appb-000001
结果显示，在SMCT153、SMCT154、SMCT155、SMCT156中强化tkt基因的表达均使得菌株的苏氨酸产量显著提高，其中，在SMCT156菌株中强化tkt基因的表达后，菌株的苏氨酸产量提升了5g/L，表明增强苏氨酸合成过程中所需还原力的供应可明显提升菌株生产苏氨酸的能力，而将提升还原力供应的改造与苏氨酸末端合成路径相关基因的表达强化组合，菌株生产苏氨酸产量会进一步提升。
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 PCTCN2022142935-appb-000002
Figure PCTCN2022142935-appb-000003
Figure PCTCN2022142935-appb-000004
Figure PCTCN2022142935-appb-000005
Figure PCTCN2022142935-appb-000006
Figure PCTCN2022142935-appb-000007
Figure PCTCN2022142935-appb-000008
Figure PCTCN2022142935-appb-000009
Figure PCTCN2022142935-appb-000010

Claims (11)

  1. 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，其转酮酶的活性增强，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。
  2. 根据权利要求1所述的微生物，其特征在于，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。
  3. 根据权利要求2所述的微生物，其特征在于，采用诱变、定点突变或同源重组的方法来增强转酮酶的活性。
  4. 根据权利要求1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，其6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶的活性增强和/或解除反馈抑制。
  5. 根据权利要求1～4任一项所述的微生物，其特征在于，所述微生物相比于未修饰的微生物，其体内与苏氨酸合成途径相关的酶的活性增强和/或解除反馈抑制；
    其中，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶中的至少一种。
  6. 根据权利要求5所述的微生物，其特征在于，所述微生物为如下①～④中的任一种：
    ①转酮酶活性增强且天冬氨酸激酶和/或高丝氨酸脱氢酶活性增强和/或解除反馈抑制的微生物；
    ②转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶和/或6-磷酸葡萄糖酸脱氢酶活性增强和/或解除反馈抑制的微生物；
    ③转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物；
    ④转酮酶活性增强且天冬氨酸激酶、高丝氨酸脱氢酶、6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶活性增强和/或解除反馈抑制的微生物。
  7. 根据权利要求4或5所述的微生物，其特征在于，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。
  8. 根据权利要求1-7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)。
  9. 产苏氨酸菌株的构建方法，其特征在于，所述方法包括：
    A、强化具有氨基酸生产能力的棒状杆菌中编码转酮酶的基因的表达，获得基因强化菌株；和/或
    B、增强6-磷酸葡萄糖酸脱氢酶和/或NADP依赖性甘油醛3-磷酸脱氢酶的活性和/或将其解除反馈抑制；和/或
    C、增强与苏氨酸合成途径相关的酶的活性和/或将其解除反馈抑制，所述与苏氨酸合成途径相关的酶选自天冬氨酸激酶、高丝氨酸脱氢酶中的至少一种；
    所述活性增强的途径选自以下1)～6)，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；
    6)通过对编码酶的核苷酸序列进行改变而增强。
  10. 根据权利要求9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)。
  11. 一种生产苏氨酸的方法，其特征在于，所述方法包括如下步骤：
    a)培养权利要求1-8任一项所述的微生物或用权利要求9或10所述的方法构建的产苏氨酸菌株，以获得所述微生物或所述产苏氨酸菌株的培养物；
    b)从步骤a)中获得的所述培养物中收集所产生的苏氨酸。
PCT/CN2022/142935 2022-01-30 2022-12-28 一种苏氨酸生产菌株及其应用 WO2023142854A1 (zh)

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