WO2023142861A1 - 产苏氨酸工程菌的构建方法 - Google Patents

产苏氨酸工程菌的构建方法 Download PDF

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WO2023142861A1
WO2023142861A1 PCT/CN2022/143105 CN2022143105W WO2023142861A1 WO 2023142861 A1 WO2023142861 A1 WO 2023142861A1 CN 2022143105 W CN2022143105 W CN 2022143105W WO 2023142861 A1 WO2023142861 A1 WO 2023142861A1
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threonine
enhanced
formyl
enzyme
citrate synthase
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WO2023142861A9 (zh
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康培
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 belongs to the technical field of microbial engineering, and in particular relates to a construction method of threonine-producing engineering bacteria.
  • L-threonine (L-Threonin), chemical name ⁇ -hydroxy- ⁇ -aminobutyric acid, molecular formula C 4 H 9 NO 3 , relative molecular mass 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 a five-step catalytic reaction, which are aspartate kinase (encoded by lysC), aspartate semialdehyde dehydrogenase (encoded by asd), and homoserine dehydrogenase. Hydrogenase (hom coded), homoserine kinase (thrB) and threonine synthase (thrC) coded.
  • 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 threonine-producing ability of the bacterial strain by inactivating 2-formyl-citrate synthase 1, thereby providing a construction method of a threonine-producing (L-threonine) engineered bacterium.
  • the present invention provides a modified microorganism of the genus Corynebacterium, the microorganism has reduced or lost activity of 2-formyl-citrate synthase 1 compared with an unmodified microorganism, And the microorganism has enhanced threonine production ability compared to the unmodified microorganism.
  • the reference sequence number of 2-formyl-citrate synthase 1 on NCBI is WP_011013823.1, or an amino acid sequence with 90% similarity thereto.
  • the reduction or loss of the activity of 2-formyl-citrate synthase 1 in the microorganism is by reducing the expression of the gene encoding 2-formyl-citrate synthase 1 or knocking out the endogenous encoding 2-formyl -Achieved by the citrate synthase 1 gene.
  • Methods such as mutagenesis, site-directed mutation or homologous recombination can be used to reduce the expression of the gene encoding 2-formyl-citrate synthase 1 or to knock out the endogenous gene encoding 2-formyl-citrate synthase 1.
  • the enzyme related to the threonine synthesis pathway is selected from aspartate aminotransferase, aspartate kinase, homoserine dehydrogenase, threonine synthase, NAD kinase, fructose-1,6- At least one of the diphosphatases 2; preferably, their reference sequence numbers on NCBI are respectively WP_011013497.1, WP_003855724.1, WP_003854900.1, WP_011014964.1, NP_600631.1, WP_003856830.1, or the above-mentioned Amino acid sequences with a reference sequence similarity of 90%.
  • the microorganism is any one of the following 1 ⁇ 5:
  • the enhancement of the activity of enzymes related to the threonine synthesis pathway in the microorganism is achieved by being selected from the following 1) to 6), or an optional combination:
  • the microorganism of the genus Corynebacterium before the modification used in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum comprises ATCC13032, ATCC13870, ATCC13869, ATCC21799, ATCC21831, ATCC14067, ATCC13287 etc. (see NCBI Corunebacterium glutamicum Phylogenetic 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 engineered bacterium, the method comprising:
  • the enhanced approach is selected from the following 1) to 5), 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 knockout or reduced expression of the gene encoding 2-formyl-citrate synthase 1 in threonine fermentation production or improvement of threonine fermentation yield.
  • fermentative yield of threonine was improved by inactivating 2-formyl-citrate synthase 1 in Corynebacterium with amino acid production ability.
  • the corynebacterium before the modification used in the present invention is Corynebacterium glutamicum (Corynebacterium glutamicum), and Corynebacterium glutamicum comprises 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 application of the modified Corynebacterium genus microorganism or the threonine-producing engineered bacterium 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 present invention has at least the following advantages and beneficial effects:
  • the invention enhances the supply of oxaloacetate, a precursor of threonine synthesis, by inactivating 2-formyl-citrate synthase 1, and improves the ability of bacterial strains to produce threonine.
  • the 2-formyl-citrate synthase 1 inactivated strain corynebacterium, such as Corynebacterium glutamicum
  • the threonine yield can be increased by about 60% compared with the unmodified strain.
  • the invention improves the yield of threonine produced by bacterial strains (such as Corynebacterium glutamicum) by inactivating 2-formyl-citrate synthase 1 (prpC1).
  • 2-formyl-citrate synthase 1 is not directly related to the threonine synthesis pathway, and there is no report that the inactivation of 2-formyl-citrate synthase 1 can increase the downstream products of threonine.
  • the present invention first uses Corynebacterium glutamicum ATCC 13032 as the starting bacterium to construct the 2-formyl-citrate synthase 1 inactivated bacterial strain, and the threonine yield of the transformed bacteria obtained is lower as 0.2g/L, which is comparable to The expectation is inconsistent, and it is speculated that the threonine synthesis is strictly regulated by the intracellular threonine concentration by aspartokinase and homoserine dehydrogenase in the threonine synthesis pathway.
  • the first step is to open up its synthesis pathway, which mainly includes the deregulation and expression enhancement of aspartokinase and homoserine dehydrogenase.
  • the modified strain SMCT077 is obtained so that the strain has a preliminary threonine synthesis ability.
  • the threonine yield was 2.4g/L, and the threonine yield reached 3.3g/L after further inactivation of prpC1.
  • the SMCT077 strain was further enhanced to express aspartate aminotransferase, threonine synthase, NAD kinase and A series of strains obtained by inactivating prpC1 in at least one enzyme strain of fructose-1,6-bisphosphatase have increased threonine production, and the conversion rate is 42% higher than that of the unmodified strain.
  • the expression enhancement during the transformation process includes the replacement of the promoter, the change of the ribosome binding site, the increase of the copy number, the change of the amino acid sequence to increase the activity and the overexpression of the plasmid, etc.
  • the inactivation includes the reduction of expression activity and inactivity,
  • the above means are well-known means by researchers in the field. The above means cannot be exhausted by examples, and the specific examples are only illustrated by promoter strengthening as a representative; in addition, the present invention only lists partially modified combinations, and all combinations of the above-mentioned sites can increase the production of threonine. They are presented by way of example only, and are not exhaustive.
  • the present invention adopts following technical scheme:
  • One of the technical solutions of the present invention is to provide a method for producing threonine using corynebacteria with 2-formyl-citrate synthase 1 inactivated.
  • the second technical solution of the present invention provides a method that utilizes 2-formyl-citrate synthase 1 inactivation and aspartate aminotransferase, aspartate kinase, homoserine dehydrogenase, threonine synthase A method for enhancing the production of threonine by expressing at least one of NAD kinase and fructose-1,6-bisphosphatase 2.
  • the third technical solution of the present invention is to provide a method for producing threonine by using aspartokinase and homoserine dehydrogenase hydrolysis regulation and expression enhancement and 2-formyl-citrate synthase 1 inactivation.
  • the fourth technical solution of the present invention provides a method for inactivating 2-formyl-citrate synthase 1 and enhancing the expression of aspartokinase, homoserine dehydrogenase, and threonine synthase to produce threonine .
  • the fifth technical solution of the present invention provides a method for producing threonine by inactivating 2-formyl-citrate synthase 1 and enhancing the expression of aspartokinase, homoserine dehydrogenase and NAD kinase.
  • the sixth technical solution of the present invention is to provide a combination of 2-formyl-citrate synthase 1 inactivation and aspartokinase, homoserine dehydrogenase, NAD kinase, fructose-1,6-bisphosphatase 2 Methods for expressing enhanced threonine production.
  • the seventh technical solution of the present invention is to provide a method that utilizes 2-formyl-citrate synthase 1 inactivation and aspartate aminotransferase, aspartate kinase, homoserine dehydrogenase, threonine synthase , NAD kinase, and fructose-1,6-bisphosphatase expression to enhance the production of threonine method.
  • Corynebacterium preferred Corynebacterium glutamicum, most preferably Corynebacterium glutamicum ATCC 13032.
  • 2-formyl-citrate synthase 1 encoding gene name prpC1, NCBI number: cg0798, Cgl0696, NCgl0666.
  • Aspartate aminotransferase encoding gene name aspB, NCBI number: cg0294, Cgl0240, NCgl0237.
  • Aspartokinase encoding gene name lysC, NCBI number: cg0306, Cgl0251, NCgl0247.
  • Homoserine dehydrogenase encoding gene name hom, NCBI number: cg1337, Cgl1183, NCgl1136.
  • Threonine synthase encoding gene name thrC, NCBI number: cg2437, Cgl2220, NCgl2139.
  • NAD kinase encoding gene name ppnK, NCBI number: cg1601, Cgl1413, NCgl1358.
  • Fructose-1,6-bisphosphatase 2 encoding gene name fbp/glpX, NCBI number: cg1157, Cgl1019, Ncgl0976.
  • the PCR amplification procedure is as follows:
  • Transformation method refer to the instructions of Trans1-T1 Phage Resistant Chemically Competent Cell.
  • 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 P25/P26 primer lysCg1a-T311I was obtained by PCR amplification
  • the downstream homology arm dn was obtained by PCR amplification with the P27/P28 primer pair. Fusion PCR was carried out with P21/P24 primer pair and up and Psod as templates to obtain the fragment up-Psod.
  • the full-length fragment up-Psod-lysCg1a-T311I-dn was obtained by fusion PCR with P21/P28 primer pair and up-Psod, lysCg1a-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-P sod -lysC g1a-T311I .
  • g1a means that the first base of the start codon of the lysC gene (see SEQ ID NO: 1 for the wild-type gene sequence of lysC) is mutated from g to a
  • T311I means that the 311th base of the aspartokinase encoded by the lysC gene is The amino acid is mutated from T to I.
  • PCR amplification was performed with the P29/P30 primer pair to obtain the upstream homology arm up, and the ATCC14067 genome was used as a template to perform PCR amplification with the P31/P32 primer pair to obtain the promoter fragment PcspB
  • the ATCC13032 genome was used as a template to obtain homG378E by PCR amplification with P33/P34 primer pair, and the downstream homology arm dn was obtained by PCR amplification with P35/P36 primer pair.
  • the fragment up-PcspB was obtained.
  • the full-length fragment up-PcspB-homG378E-dn was obtained by fusion PCR with P29/P36 primer pair and up-PcspB, homG378E, dn as template.
  • 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-P cspB -hom G378E .
  • the upstream homology arm up was obtained by PCR amplification with the P103/P104 primer pair
  • the promoter fragment Psod was obtained by PCR amplification with the P105/P106 primer pair
  • the P107/P108 primer Perform PCR amplification to obtain the downstream homology arm dn.
  • the full-length fragment up-Psod-dn was obtained by fusion PCR with P103/P108 primer pair and up, Psod, 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-P sod -aspB.
  • the upstream homology arm up was obtained by PCR amplification with the P37/P38 primer pair
  • the promoter fragment Psod-thrCg1a was obtained by PCR amplification with the P39/P40 primer pair
  • PCR amplification was performed with the P41/P42 primer pair.
  • Add the downstream homology arm dn The full-length fragment up-Psod-thrCg1a-dn was obtained by fusion PCR with P37/P42 primer pair and up, Psod-thrCV1M, 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-P sod -thrC g1a .
  • g1a means that the first base of the start codon of the thrC gene (see SEQ ID NO: 2 for the wild-type gene sequence of thrC) is mutated from g to a.
  • the upstream homology arm up was obtained by PCR amplification with the prpC1-UF/prpC1-UR primer pair, and the downstream homology arm was obtained by PCR amplification with the prpC1-DF/prpC1-DR primer pair. source arm dn. Fusion PCR was performed with prpC1-UF/prpC1-DR primer pair and up and dn 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, transformed into Trans1T1 competent cells, and obtained the recombinant plasmid pK18mobsacB- ⁇ prpC1.
  • the upstream homology arm up was obtained by PCR amplification with the P109/P110 primer pair
  • the promoter fragment Ptuf was obtained by PCR amplification with the P111/P112 primer pair
  • the P113/P114 primer Perform PCR amplification to obtain the downstream homology arm dn.
  • the full-length fragment up-Ptuf-dn was obtained by fusion PCR using the P109/P114 primer pair and up, Ptuf, and 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-P tuf -ppnK.
  • fructose-1,6-bisphosphatase expression enhanced plasmid pK18mobsacB-P tuf -fbp
  • the upstream homology arm up was obtained by PCR amplification with the P61/P62 primer pair
  • the promoter fragment Ptuf was obtained by PCR amplification with the P63/P64 primer pair
  • the P65/P66 primer Perform PCR amplification to obtain the downstream homology arm dn.
  • the full-length fragment up-Ptuf-dn was obtained by fusion PCR with P61/P66 primer pair and up, Ptuf, dn as template.
  • 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-P tuf -fbp.
  • the primers used in the construction process are shown in Table 1:
  • Competent cells of Corynebacterium glutamicum ATCC13032 were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB- ⁇ prpC1 was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, in which the gene of interest 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.
  • 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 sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strain was obtained and named SMCT089.
  • ATCC13032 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -lysC g1a-T311I was used to transform the competent cells by electroporation, and the transformant was screened on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was eliminated due to homology. inserted into the chromosome.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm.
  • the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. 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 SMCT076.
  • SMCT076 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P cspB -hom G378E was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the in the chromosome.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. 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. 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 SMCT077.
  • SMCT077 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -thrC g1a was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the in the chromosome.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. 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. 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 SMCT078.
  • SMCT077 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P tuf -ppnK 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 gene of interest was inserted into the chromosome due to homology middle.
  • 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. 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 SMCT079.
  • Competent cells of SMCT077 and SMCT079 were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P tuf -fbp 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 gene of interest was inserted into the chromosome due to homology middle.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm.
  • the transformants undergo a second recombination, whereby the vector sequence is removed from the genome by gene exchange.
  • the culture was serially diluted (from 10 -2 to 10 -4 ), the diluted solution was spread on common solid brain heart infusion medium containing 10% sucrose, and cultured at 33°C for 48 hours. 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 strains were obtained and named SMCT080 and SMCT081.
  • SMCT081 competent cells were prepared according to the classical glutamicum method (C. glutamicum Handbook, Chapter 23).
  • the recombinant plasmid pK18mobsacB-P sod -aspB was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, in which the gene of interest was inserted into the chromosome due to homology middle.
  • 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. Strains grown on sucrose media do not carry the inserted vector sequence in their genome. Amplify the target sequence by PCR, and perform nucleotide sequencing analysis to obtain the target mutant strain and continue to prepare competent cells.
  • the recombinant plasmid pK18mobsacB-P sod -thrC g1a was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, wherein the gene of interest was inserted into the in the chromosome.
  • the screened transformants were cultured overnight in common liquid brain-heart infusion medium at a temperature of 30° C. on a rotary shaker at 220 rpm. 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. 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 to obtain the target mutant strain SMCT082.
  • SMCT077, SMCT078, SMCT079, SMCT080, SMCT081, SMCT082, and competent cells were prepared according to the classical method of glutamicum (C. glutamicum Handbook, Charpter 23).
  • the recombinant plasmid pK18mobsacB- ⁇ prpC1 was used to transform the competent cells by electroporation, and the transformants were selected on the selection medium containing 15mg/L kanamycin, in which the gene of interest 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.
  • 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 sequence was amplified by PCR and analyzed by nucleotide sequencing, and the target mutant strains were named SMCT083, SMCT084, SMCT085, SMCT086, SMCT087, SMCT088.
  • Embodiment 3 constructs bacterial strain shaking flask verification
  • 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 SMCT076, SMCT077, SMCT078, SMCT079, SMCT080, SMCT081, SMCT082, SMCT083, SMCT084, SMCT085, SMCT086, SMCT088 slant seeds 1 ring and connect them to a 500mL Erlenmeyer flask containing 20mL seed medium, at 30°C , 220r/min shaking culture 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.
  • 2-formyl-citrate synthase 1 was further inactivated on the basis of threonine-producing bacteria, as can be seen from Table 3
  • the threonine production of all 2-formyl-citrate synthase 1-modified strains increased to varying degrees, and the highest increase was 42% compared with the control strain. It can be seen that the inactivation of 2-formyl-citrate synthase 1 is beneficial to the improvement of threonine production of the strain.

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Abstract

本发明提供一种产苏氨酸工程菌的构建方法。本发明将2-甲酰-柠檬酸合酶1失活菌株(棒杆菌)应用于苏氨酸生产，其苏氨酸的产量较未改造菌株可提高42％左右。进一步将其与苏氨酸合成路径中的天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、NAD激酶、果糖-1,6-二磷酸酶2等中的至少一个表达强化相组合时，苏氨酸的产量均有所提升。为大规模生产苏氨酸提供了新途径，具有较高的应用价值。

Description

产苏氨酸工程菌的构建方法 技术领域
本发明属于微生物工程技术领域，具体地说，涉及一种产苏氨酸工程菌的构建方法。
背景技术
L-苏氨酸(L-Threonin)，化学名称β-羟基-α-氨基丁酸，分子式C 4H 9NO 3，相对分子质量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)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)。
目前利用谷氨酸棒状杆菌生产苏氨酸的报道主要集中在其合成路径中，关于前体供应等方面的报道较少。且现有报道仅对苏氨酸合成路径做了初步研究，尚未形成系统。
发明内容
本发明的目的是通过失活2-甲酰-柠檬酸合酶1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)工程菌的构建方法。
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2-甲酰-柠檬酸合酶1的活性降低或丧失，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，2-甲酰-柠檬酸合酶1在NCBI上的参考序列编号为WP_011013823.1，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2-甲酰-柠檬酸合酶1的活性降低或丧失是通过降低编码2-甲酰-柠檬酸合酶1基因的表达或敲除内源的编码2-甲酰-柠檬酸合酶1的基因来实现的。
可以采用诱变、定点突变或同源重组等方法来降低编码2-甲酰-柠檬酸合酶1基因的表达或敲除内源的编码2-甲酰-柠檬酸合酶1的基因。
进一步地，所述微生物与未修饰的微生物相比，其体内苏氨酸合成途径相关的酶的活性增强；
其中，所述与苏氨酸合成途径相关的酶选自天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、NAD激酶、果糖-1,6-二磷酸酶2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_003855724.1、WP_003854900.1、WP_011014964.1、NP_600631.1、WP_003856830.1，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①～⑤中的任一种：
①2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸激酶和/或高丝氨酸脱氢酶活性增强的微生物；
②2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶和/或苏氨酸合酶活性增强的微生物；
③2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶和/或NAD激酶活性增强的微生物；
④2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、NAD激酶和/或果糖-1,6-二磷酸酶2活性增强的微生物；
⑤2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、NAD激酶和果糖-1,6-二磷酸酶2活性增强的微生物。
所述微生物体内苏氨酸合成途径相关的酶的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。
优选地，本发明所用的修饰前的棒杆菌属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工程菌的构建方法，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2-甲酰-柠檬酸合酶1的基因，获得基因弱化菌株；所述弱化包括敲除或降低2-甲酰-柠檬酸合酶1编码基因的表达；和/或
B、增强步骤A基因弱化菌株中与苏氨酸合成途径相关的酶，获得酶活增强菌株；
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。
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)培养所述修饰的棒状杆菌属微生物，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2-甲酰-柠檬酸合酶1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2-甲酰-柠檬酸合酶1来提高苏氨酸的发酵产量。
优选地，本发明所用的修饰前的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工程菌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.
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明通过失活2-甲酰-柠檬酸合酶1，增强苏氨酸合成前体草酰乙酸的供应，提高菌株生产苏氨酸的能力。本发明将2-甲酰-柠檬酸合酶1失活菌株(棒杆菌，如谷氨酸棒状杆菌)应用于苏氨酸生产，其苏氨酸的产量较未改造菌株可提高60％左右。进一步将其与苏氨酸合成路径中的天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、NAD激酶、果糖-1,6-二磷酸酶2等中的至少一个表达强化相组合时，苏氨酸的产量均有所提升。为大规模生产苏氨酸提供了新途径，具有较高的应用价值。
具体实施方式
本发明通过失活2-甲酰-柠檬酸合酶1(prpC1)提高了菌株(如谷氨酸棒状杆菌)生产苏氨酸的产量。
2-甲酰-柠檬酸合酶1与苏氨酸合成路径不直接相关，且目前尚未由2-甲酰-柠檬酸合酶1失活可提高苏氨酸下游产物的相关报道。为此，本发明先以谷氨酸棒状杆菌ATCC 13032为出发菌，构建2-甲酰-柠檬酸合酶1失活菌株，获得的改造菌苏氨酸产量较低为0.2g/L，与预期是不符，推测其苏氨酸合成受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，改造菌株生产苏氨酸，首先打通其合成路径，主要包括天冬氨酸激酶、高丝氨酸脱氢酶的解调控及表达强化，获得改造菌SMCT077使得菌株具备初步的苏氨酸合成能力，其苏氨酸产量为2.4g/L，进一步的失活prpC1，苏氨酸的产量达到3.3g/L。由此可知虽然2-甲酰-柠檬酸合酶1的失活有利于苏氨酸的生产，当其与苏氨酸生产菌株中的其他与苏氨酸合成相关的位点相结合时，其苏氨酸的生产能力会进一步提升。
为了进一步验证苏氨酸产量的提升是由于2-甲酰-柠檬酸合酶1失活造成的，在对SMCT077菌株进一步强化表达天冬氨酸氨基转移酶、苏氨酸合酶、NAD激酶以及果糖-1,6-二磷酸酶中至少一个酶的菌株进行prpC1失活获得的一系列菌株，其苏氨酸的产量均有所提高，转化率较未改造菌株提高42％。
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、氨基酸序列的改变造成活性增加及质粒过表达等手段，失活包括表达活性的降低及无活性，且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，具体实施例中仅以启动子强化作为代表进行说明；另外本发明仅列出部分改造的组合，有关上述位点的所有组合均可提高苏氨酸的产量，本文仅举例说明，并未以穷尽的方式全部说明。
本发明采用如下技术方案：
本发明的技术方案之一，提供一种利用2-甲酰-柠檬酸合酶1失活的棒杆菌生产苏氨酸的方法。
本发明的技术方案之二，提供一种利用2-甲酰-柠檬酸合酶1失活及天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、NAD激酶、果糖-1,6-二磷酸酶2至少一个表达强化生产苏氨酸的方法。
本发明的技术方案之三，提供一种利用天冬氨酸激酶和高丝氨酸脱氢酶解调控及表达强化和2-甲酰-柠檬酸合酶1失活生产苏氨酸的方法。
本发明的技术方案之四，提供一种利用2-甲酰-柠檬酸合酶1失活和天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶表达强化生产苏氨酸的方法。
本发明的技术方案之五，提供一种利用2-甲酰-柠檬酸合酶1失活和天冬氨酸激酶、高丝氨酸脱氢酶、NAD激酶表达强化生产苏氨酸的方法。
本发明的技术方案之六，提供一种利用2-甲酰-柠檬酸合酶1失活和天冬氨酸激酶、高丝氨酸脱氢酶、NAD激酶、果糖-1,6-二磷酸酶2表达强化生产苏氨酸的方法。
本发明的技术方案之七，提供一种利用2-甲酰-柠檬酸合酶1失活及天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、NAD激酶、果糖-1,6-二磷酸酶表达强化生产苏氨酸的方法。
上述菌株为棒杆菌，优选谷氨酸棒状杆菌，最优选谷氨酸棒状杆菌ATCC 13032。
本发明涉及的蛋白及其编码基因如下：
2-甲酰-柠檬酸合酶1，编码基因名称prpC1，NCBI编号：cg0798、Cgl0696、NCgl0666。
天冬氨酸氨基转移酶，编码基因名称aspB，NCBI编号：cg0294、Cgl0240、NCgl0237。
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。
苏氨酸合酶，编码基因名称thrC，NCBI编号：cg2437、Cgl2220、NCgl2139。
NAD激酶，编码基因名称ppnK，NCBI编号：cg1601、Cgl1413、NCgl1358。
果糖-1,6-二磷酸酶2，编码基因名称fbp/glpX，NCBI编号：cg1157、Cgl1019、Ncgl0976。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
以下实施例中使用的实验材料如下：
Figure PCTCN2022143105-appb-000001
Figure PCTCN2022143105-appb-000002
以下实施例中涉及的实验方法如下：PCR扩增体系如下：
成分 体积(微升)
灭菌的去离子水 29
5×pfu buffer 10
2.5mM dNTP 5
10μM上游引物 2
10μM下游引物 2
Pfu 1
模板 1(融合PCR模板最大加到2微升)
共计 50
PCR扩增程序如下：
Figure PCTCN2022143105-appb-000003
菌株改造方法：
1、无缝组装反应程序：参照ClonExpress MultiS One Step Cloning Kit说明书。
2、转化方法：参照Trans1-T1 Phage Resistant Chemically Competent Cell说明书。
3、感受态细胞的制备：参照C.glutamicum Handbook,Charpter 23。
实施例1菌株基因组改造质粒的构建
1、天冬氨酸激酶表达强化质粒pK18mobsacB-P sod-lysC g1a-T311I的构建
以谷氨酸棒状杆菌ATCC 13032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lysCg1a-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为模板进行融合PCR，获得片段up-Psod。以P21/P28引物对以up-Psod、lysCg1a-T311I、dn为模板进行融合PCR获得全长片段up-Psod-lysCg1a-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-P sod-lysC g1a-T311I。
其中，g1a表示lysC基因(lysC野生型基因序列见SEQ ID NO:1)起始密码子的第1位碱基由g突变为a，T311I表示lysC基因编码的天冬氨酸激酶的第311为氨基酸由T突变为I。
2、高丝氨酸脱氢酶表达强化质粒pK18mobsacB-P cspB-hom G378E的构建
以谷氨酸棒状杆菌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ATCC14067基因组为模板以P31/P32引物对进行PCR扩增得到启动子片段PcspB，以ATCC13032基因组为模板以P33/P34引物对进行PCR扩增得到homG378E，以P35/P36引物对进行PCR扩增得到下游同源臂dn。以P29/P32引物对以up、PcspB为模板进行融合PCR，获得片段up-PcspB。以P29/P36引物对以up-PcspB、homG378E、dn为模板进行融合PCR获得全长片段up-PcspB-homG378E-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-P cspB-hom G378E。
3、天冬氨酸氨基转移酶表达强化质粒pK18mobsacB-P sod-aspB的构建
以谷氨酸棒状杆菌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P105/P106引物对进行PCR扩增得到启动子片段Psod，以P107/P108引物对进行PCR扩增得到下游同源臂dn。以P103/P108引物对以up、Psod、dn为模板进行融合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-P sod-aspB。
4、苏氨酸合酶表达强化质粒pK18mobsacB-P sod-thrC g1a的构建
以ATCC13032基因组为模板，以P37/P38引物对进行PCR扩增得到上游同源臂up，以 P39/P40引物对进行PCR扩增得到启动子片段Psod-thrCg1a，以P41/P42引物对进行PCR扩增得到下游同源臂dn。以P37/P42引物对以up、Psod-thrCV1M、dn为模板进行融合PCR获得全长片段up-Psod-thrCg1a-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-P sod-thrC g1a。
其中，g1a表示thrC基因(thrC野生型基因序列见SEQ ID NO:2)起始密码子的第1位碱基由g突变为a。
5、2-甲酰-柠檬酸合酶1失活质粒pK18mobsacB-△prpC1的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以prpC1-UF/prpC1-UR引物对进行PCR扩增得到上游同源臂up，以prpC1-DF/prpC1-DR引物对进行PCR扩增得到下游同源臂dn。以prpC1-UF/prpC1-DR引物对以up、dn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-△prpC1。
6、NAD激酶表达强化质粒pK18mobsacB-P tuf-ppnK的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P109/P110引物对进行PCR扩增得到上游同源臂up，以P111/P112引物对进行PCR扩增得到启动子片段Ptuf，以P113/P114引物对进行PCR扩增得到下游同源臂dn。以P109/P114引物对以up、Ptuf、dn为模板进行融合PCR获得全长片段up-Ptuf-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-P tuf-ppnK。
7、果糖-1,6-二磷酸酶表达强化质粒pK18mobsacB-P tuf-fbp的构建
以谷氨酸棒状杆菌ATCC 13032基因组为模板，以P61/P62引物对进行PCR扩增得到上游同源臂up，以P63/P64引物对进行PCR扩增得到启动子片段Ptuf，以P65/P66引物对进行PCR扩增得到下游同源臂dn。以P61/P66引物对以up、Ptuf、dn为模板进行融合PCR获得全长片段up-Ptuf-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-P tuf-fbp。
构建过程中所用引物如表1所示：
表1
名称 序列(5′-3′)(依次为SEQ ID NO:3-46)
P21 AATTCGAGCTCGGTACCCGGGGATCCAGCGACAGGACAAGCACTGG
P22 CCCGGAATAATTGGCAGCTATGTGCACCTTTCGATCTACG
P23 CGTAGATCGAAAGGTGCACATAGCTGCCAATTATTCCGGG
P24 TTTCTGTACGACCAGGGCCA TGGGTAAAAAATCCTTTCGTA
P25 TACGAAAGGATTTTTTACCC ATGGCCCTGGTCGTACAGAAA
P26 TCGGAACGAGGGCAGGTGAAGGTGATGTCGGTGGTGCCGTCT
P27 AGACGGCACCACCGACATCACCTTCACCTGCCCTCGTTCCGA
P28 GTAAAACGACGGCCAGTGCCAAGCTTAGCCTGGTAAGAGGAAACGT
P29 AATTCGAGCTCGGTACCCGGGGATCCCTGCGGGCAGATCCTTTTGA
P30 ATTTCTTTATAAACGCAGGTCATATCTACCAAAACTACGC
P31 GCGTAGTTTTGGTAGATATGACCTGCGTTTATAAAGAAAT
P32 GTATATCTCCTTCTGCAGGAATAGGTATCGAAAGACGAAA
P33 TTTCGTCTTTCGATACCTATTCCTGCAGAAGGAGATATAC
P34 TAGCCAATTCAGCCAAAACC CCCACGCGATCTTCCACATCC
P35 GGATGTGGAAGATCGCGTGG GGGTTTTGGCTGAATTGGCTA
P36 GTAAAACGACGGCCAGTGCCAAGCTTGCTGGCTCTTGCCGTCGATA
P37 ATTCGAGCTCGGTACCCGGGGATCCGCCGTTGATCATTGTTCTTCA
P38 CCCGGAATAATTGGCAGCTAGGATATAACCCTATCCCAAG
P39 CTTGGGATAGGGTTATATCCTAGCTGCCAATTATTCCGGG
P40 ACGCGTCGAAATGTAGTCCA TGGGTAAAAAATCCTTTCGTA
P41 TACGAAAGGATTTTTTACCC ATGGACTACATTTCGACGCGT
P42 GTAAAACGACGGCCAGTGCCAAGCTTGAATACGCGGATTCCCTCGC
P61 AATTCGAGCTCGGTACCCGGGGATCCTCATCTGCGGTGACATATCC
P62 CATTCGCAGGGTAACGGCCACTGAAGGGCCTCCTGGGGCA
P63 TGCCCCAGGAGGCCCTTCAGTGGCCGTTACCCTGCGAATG
P64 TCGGGGTTCTTTAGGTTCATTGTATGTCCTCCTGGACTTC
P65 GAAGTCCAGGAGGACATACAATGAACCTAAAGAACCCCGA
P66 GTAAAACGACGGCCAGTGCCAAGCTTGTGACGTCGGAAGGGTTGAT
P103 GAGCTCGGTACCCGGGGATCCGCAGGGTATTGCAGGGACTCA
P104 CAAGCCCGGAATAATTGGCAGCTAAACTGCGTACCTCCGCATGTGGTGG
P105 TAGCTGCCAATTATTCCGGGCTTGT
P106 GGGTAAAAAATCCTTTCGTAGGTTT
P107 GGAAACCTACGAAAGGATTTTTTACCCATGAGTTCAGTTTCGCTGCAGGATTT
P108 ACGACGGCCAGTGCCAAGCTTACACCGGAACAACCCACATG
P109 GAGCTCGGTACCCGGGGATCCGAAGCGTCTGAAGTAGTGGCAGT
P110 ACATTCGCAGGGTAACGGCCATTATTGCGGACCTTCCTTTACAGC
P111 TGGCCGTTACCCTGCGAATGTCCAC
P112 TGTATGTCCTCCTGGACTTCGTGG
P113 CACCACGAAGTCCAGGAGGACATACAATGACTGCACCCACGAACGCTGGGGA
P114 ACGACGGCCAGTGCCAAGCTTGCATCGAGCACTCCCCTGC
prpC1-UF AATTCGAGCTCGGTACCCGGGGATCCACGTGATGGTTCGACGCATC
prpC1-UR AAATCAGCCTCACTGATTAGTCACTCATTGTTTTCTCCTT
prpC1-DF AAGGAGAAAACAATGAGTGACTAATCAGTGAGGCTGATTT
prpC1-DR GTAAAACGACGGCCAGTGCCAAGCTTTGGTTGTCGGATCT
注：加粗字体及下划线为引入相应点突变的引物。
实施例2基因组改造菌株的构建
1、2-甲酰-柠檬酸合酶1失活菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备谷氨酸棒状杆菌ATCC13032感受态细胞。重组质粒pK18mobsacB-△prpC1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，其中感兴趣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SMCT089。
2、天冬氨酸激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ATCC13032感受态细胞。重组质粒pK18mobsacB-P sod-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，其中感兴趣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SMCT076。
3、高丝氨酸脱氢酶表达强化菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT076感受态细胞。重组质粒pK18mobsacB-P cspB-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，其中感兴趣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SMCT077。
4、苏氨酸合酶表达强化菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT077感受态细胞。重组质粒pK18mobsacB-P sod-thrC g1a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，其中感兴趣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SMCT078。
5、NAD激酶表达强化菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT077感受态细胞。重组质粒pK18mobsacB-P tuf-ppnK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，其中感兴趣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SMCT079。
6、果糖-1,6-二磷酸酶表达强化菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT077、SMCT079感受态细胞。重组质粒pK18mobsacB-P tuf-fbp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，其中感兴趣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SMCT080、SMCT081。
7、在SMCT081上进行天冬氨酸氨基转移酶和苏氨酸合酶的表达强化的改造
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT081感受态细胞。重组质粒pK18mobsacB-P sod-aspB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，其中感兴趣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，获得目的突变菌株继续制备感受态细胞。重组质粒pK18mobsacB-P sod-thrC g1a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，其中感兴趣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SMCT082。
8、2-甲酰-柠檬酸合酶1失活菌株的构建
按照谷棒经典方法(C.glutamicum Handbook,Charpter 23)制备SMCT077、SMCT078、SMCT079、SMCT080、SMCT081、SMCT082、感受态细胞。重组质粒pK18mobsacB-△prpC1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，其中感兴趣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SMCT083、SMCT084、SMCT085、SMCT086、SMCT087、SMCT088。
获得的菌株如表2所示：
表2
菌株 基因型
SMCT089 ATCC13032,△prpC1
SMCT076 ATCC13032,P sod-lysC g1a-T311I
SMCT077 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E
SMCT078 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P sod-thrC g1a
SMCT079 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P tuf-ppnK
SMCT080 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P tuf-fbp
SMCT081 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P tuf-ppnK,P tuf-fbp
SMCT082 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P tuf-ppnK,P tuf-fbp,P sod-aspB,P sod-thrC g1a
SMCT083 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,△prpC1
SMCT084 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P sod-thrC g1a,△prpC1
SMCT085 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P tuf-ppnK,△prpC1
SMCT086 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P tuf-fbp,△prpC1
SMCT087 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P tuf-ppnK,P tuf-fbp,△prpC1
SMCT088 ATCC13032,P sod-lysC g1a-T311I,P cspB-hom G378E,P tuf-ppnK,P tuf-fbp,P sod-aspB,P sod-thrC g1a,△prpC1
实施例3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SMCT076、SMCT077、SMCT078、SMCT079、SMCT080、SMCT081、SMCT082、SMCT083、SMCT084、SMCT085、SMCT086、SMCT088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。
谷氨酸棒状杆菌生产苏氨酸能力的比较见表3：
表3
菌株编号 OD 562 苏氨酸(g/L) 菌株编号 OD 562 苏氨酸(g/L)
ATCC13032 25 — SMCT089 25 0.2
SMCT076 23 1.2 — — —
SMCT077 23 2.4 SMCT083 23 3.3
SMCT078 24 3.0 SMCT084 24 4.1
SMCT079 24 3.3 SMCT085 24 4.5
SMCT080 23 3.5 SMCT086 23 5.0
SMCT081 22 8.0 SMCT087 22 9.2
SMCT082 22 10.2 SMCT088 22 12.5
由表3可以看出，在野生菌株ATCC13032的基础上进行天冬氨酸激酶的改造后菌株苏氨酸的产量初步的积累，随着苏氨酸合成路径中的酶(高丝氨酸脱氢酶、天冬氨酸氨基转移酶、苏氨酸合酶)表达的强化，苏氨酸的产量有了进一步的提升，随后对NAD激酶和果糖1,6-二磷酸酶表达的强化，菌株还原力的供应进一步加强，这有利于苏氨酸产量的提升。
为了探究2-甲酰-柠檬酸合酶1对苏氨酸产量的影响，在苏氨酸生产菌的基础上，进一步失活2-甲酰-柠檬酸合酶1，由表3可以看出所有2-甲酰-柠檬酸合酶1的改造菌均的苏氨酸产量均有不同程度的提升，其中最高较对照菌株提高42％。由此可见2-甲酰-柠檬酸合酶1失活有利于菌株苏氨酸产量的提升。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对之做一些改进和修饰，比如表达强化可以采用强启动子、RBS序列的改变、起始密码子的改变、氨基酸序列的改变造成活性增强等，表达失活包括蛋白活性的失活及弱化等，这些改进和修饰均属于本发明要求保护的范围。
序列说明
SEQ ID No:1谷氨酸棒状杆菌(Corynebacterium glutamicum)lysC野生型基因
Figure PCTCN2022143105-appb-000004
SEQ ID No:2谷氨酸棒状杆菌(Corynebacterium glutamicum)thrC野生型基因
Figure PCTCN2022143105-appb-000005

Claims (10)

  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2-甲酰-柠檬酸合酶1的活性降低或丧失，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，所述微生物体内2-甲酰-柠檬酸合酶1的活性降低或丧失是通过降低编码2-甲酰-柠檬酸合酶1基因的表达或敲除内源的编码2-甲酰-柠檬酸合酶1的基因来实现的。
  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降低编码2-甲酰-柠檬酸合酶1基因的表达或敲除内源的编码2-甲酰-柠檬酸合酶1的基因。
  4. 根据权利要求1所述的微生物，其特征在于，所述微生物与未修饰的微生物相比，其体内苏氨酸合成途径相关的酶的活性增强；
    其中，所述与苏氨酸合成途径相关的酶选自天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、NAD激酶、果糖-1,6-二磷酸酶2中的至少一种。
  5. 根据权利要求4所述的微生物，其特征在于，所述微生物为如下①～⑤中的任一种：
    ①2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸激酶和/或高丝氨酸脱氢酶活性增强的微生物；
    ②2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶和/或苏氨酸合酶活性增强的微生物；
    ③2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶和/或NAD激酶活性增强的微生物；
    ④2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸激酶、高丝氨酸脱氢酶、NAD激酶和/或果糖-1,6-二磷酸酶2活性增强的微生物；
    ⑤2-甲酰-柠檬酸合酶1活性降低或丧失且天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、NAD激酶和果糖-1,6-二磷酸酶2活性增强的微生物。
  6. 根据权利要求4所述的微生物，其特征在于，所述微生物体内苏氨酸合成途径相关的酶的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1-6任一项所述的微生物，其特征在于，所述微生物为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
  8. 产苏氨酸工程菌的构建方法，其特征在于，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2-甲酰-柠檬酸合酶1的基因，获得基因弱化菌株；所述弱化包括敲除或降低2-甲酰-柠檬酸合酶1编码基因的表达；和可选的
    B、增强步骤A基因弱化菌株中与苏氨酸合成途径相关的酶，获得酶活增强菌株；
    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；
    其中，所述与苏氨酸合成途径相关的酶选自天冬氨酸氨基转移酶、天冬氨酸激酶、高丝氨酸脱氢酶、苏氨酸合酶、NAD激酶、果糖-1,6-二磷酸酶2中的至少一种。
  9. 根据权利要求8所述的方法，其特征在于，所述棒杆菌为谷氨酸棒状杆菌(Corynebacterium glutamicum)。
  10. 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1-7任一项所述的微生物或用权利要求8或9所述的方法构建的工程菌，以获得所述微生物或工程菌的培养物；
    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/CN2022/143105 2022-01-26 2022-12-29 产苏氨酸工程菌的构建方法 WO2023142861A1 (zh)

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