Engineering strain for efficiently producing D-pantothenic acid as well as construction method and application of engineering strain

Abstract

The invention discloses an engineering strain for efficiently producing D-pantothenic acid as well as a construction method and application of the engineering strain, and belongs to the technical field of gene engineering. In the engineering strain, an acetolactate synthase gene alsS *, a keto acid reductoisomerase gene ilvCV412A, a dihydroxy acid dehydratase gene ilvD and other genes are over-expressed; a pantothenic acid kinase gene coAR106A and a phosphoribose enzyme gene prk are expressed; according to the engineering strain, a repressor protein gene lacI, a threonine dehydratase gene ilvA, a pantothenate kinase gene coA and a formate acetyl transferase gene pflD are deleted on a genome. The engineering strain can be used for efficiently synthesizing D-pantothenic acid by taking glucose as a carbon source.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to an engineering strain for efficiently producing D-pantothenic acid, and a construction method and application thereof. Background Art

[0002] D-pantothenic acid (D-PA), also known as vitamin B5, is an important component of coenzyme A (CoA) and acyl carrier protein (ACP). It participates in cell anabolism and catabolism to maintain normal physiological functions. Therefore, D-pantothenic acid is widely used in food, medicine, feed, cosmetics and other fields.

[0003] At present, the main methods for producing D-pantothenic acid are chemical synthesis, enzymatic method and microbial fermentation. The chemical synthesis method is to first generate calcium β-alanine under the action of calcium hydroxide, and then generate calcium DL-pantothenate by amidation with DL-pantoic acid lactone. This reaction needs to be carried out at high temperature and in methanol solution. D-pantothenate calcium is induced to crystallize by adding D-pantothenate calcium to DL-pantothenate calcium-methanol solution and repeated operation to obtain D-pantothenate calcium by physical splitting. The enzymatic method is to first use L-pantothenic acid lactone dehydrogenase and D-pantothenic acid lactone dehydrogenase to split pantothenic acid lactone to obtain D-pantoic acid, and then condense with β-alanine to obtain D-pantothenic acid. The toxic reagents used in the chemical synthesis of D-pantothenic acid are not only harmful to the environment, but also pose a threat to human health. The enzymatic synthesis of D-pantothenic acid has the disadvantages of substrate dependence on chemical synthesis, high cost and harsh reaction conditions.

[0004] With the continuous development of metabolic engineering and synthetic biology, the production of bio-based chemicals by microbial fermentation using cheap carbohydrates has attracted widespread attention from scholars. However, the synthesis of D-pantothenic acid by microbial fermentation involves the balance of metabolic fluxes between multiple modules. In the process of synthesizing D-pantothenic acid by fermentation of Escherichia coli, the existing technology has problems such as carbon metabolism loss, unstable fermentation process, and low yield. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an engineered strain for efficiently producing D-pantothenic acid and a construction method and application thereof, so as to overcome the problem of insufficient D-pantothenic acid production when synthesizing D-pantothenic acid using microbial fermentation in the prior art.

[0006] In a first aspect, the present invention provides an engineered strain for efficiently producing D-pantothenic acid, wherein the engineered strain overexpresses the following genes: Acetolactate synthase gene alsS*, ketoacid reductoisomerase gene ilvC V412A , dihydroxyacid dehydratase gene ilvD, overexpression of methyltransferase gene panB Y25A, anhydropantothenate reductase gene panE, aspartate aminotransferase gene aspC, aspartate decarboxylase gene panD, pantothenate synthase gene panC, pantothenate transporter gene panF, ribulose-1,5-bisphosphate carboxylase / oxidase gene RuBisCO, and molecular chaperone protein genes GroEL-GroES.

[0007] The engineered strain expresses the pantothenate kinase gene coaA R106A , phosphoribosylation enzyme gene prk.

[0008] The engineered strain lacks the following genes in its genome: Repressor protein gene lacI, threonine dehydratase gene ilvA, pantothenate kinase gene coaA, formate acetyltransferase gene pflD.

[0009] Compared with the prior art, the present invention regulates or optimizes the following metabolic pathways by overexpressing, expressing or not expressing specific genes in the engineered strain, so that the engineered strain can efficiently synthesize D-pantothenic acid using glucose as a carbon source, as follows: Enhanced R-pantoic acid (R-PA) anabolism: The engineered strain overexpresses the acetolactate synthase gene alsS* in the genome. The glycine at position 49 of the small subunit of the acetolactate synthase mutates to alanine, the cysteine ​​at position 60 mutates to threonine, the threonine at position 62 mutates to alanine, the alanine at position 63 mutates to cysteine, and the alanine at position 64 mutates to threonine, making the acetolactate synthase gene resistant to feedback inhibition by L-valine, and promoting more carbon flux to enter the R-PA biosynthesis pathway. The engineered strain overexpresses the ketoacid reductoisomerase gene ilvC in the genome. V412A , where the valine at position 412 mutated to alanine, improving the selectivity and catalytic activity of ketoacid reductoisomerase for substrates. The engineered strain overexpressed the dihydroxyacid dehydratase gene ilvD in the genome, strengthening the metabolism of dihydroxyisovalerate to ketoisoglutaric acid. The methyltransferase gene panB was overexpressed in the genome of the strain Y25A , enhancing the activity of ketopantothenate hydroxymethyltransferase and strengthening the metabolism of ketoisoglutaric acid to dehydrated pantothenate. The engineered strain overexpresses the dehydrated pantothenate reductase gene panE in the genome, strengthening the metabolism of dehydrated pantothenate to generate R-pantothenate.

[0010] Enhanced β-Ala anabolism: The engineered strain overexpresses the aspartate aminotransferase gene aspC in the genome, which strengthens the metabolism of oxaloacetyl conversion to aspartate. The engineered strain overexpresses the aspartate decarboxylase gene panD in the genome, which strengthens the metabolism of aspartate decarboxylation to β-alanine.

[0011] Enhanced D-PA (D-pantothenic acid) anabolism: The engineered strain overexpresses the panC gene of pantothenate synthase in the genome, which strengthens the metabolism of β-alanine and R-pantothenic acid to synthesize D-pantothenic acid. The engineered strain lacks the ilvA gene in the genome, which reduces the degradation of threonine and increases the metabolic flow of β-alanine, thereby enhancing the supply of β-alanine, the precursor of D-pantothenic acid.

[0012] Enhanced D-PA accumulation: The engineered strain expresses the pantothenate kinase gene coaA in the genome R106A , where the arginine at position 106 mutated to alanine, weakening the conversion of D-PA to coenzyme A. The engineered strain overexpressed the pantothenate transporter gene panF in the genome, promoting the transport of intracellular pantothenate to the extracellular space. The engineered strain lacked the coaA gene in the genome, preventing the pantothenate kinase encoded by it from converting D-pantothenate to coenzyme A.

[0013] Enhanced pyruvate enrichment: The engineered strain lacks the formate acetyltransferase gene pflD in the genome, weakening the metabolism of pyruvate to formate.

[0014] Establishment of a heterotrophic RuBisCO-based bypass, in situ CO 2 Fix and improve the production of D-PA: The engineered strain overexpresses the phosphoribosylase gene prK in the genome, which strengthens the metabolism of ribulose-5-phosphate to ribulose-1,5-bisphosphate. The engineered strain overexpresses the ribulose-1,5-bisphosphate carboxylase / oxidase gene RuBisCO in the genome, which strengthens the ribulose-1,5-bisphosphate and CO 2 The engineered strain overexpresses the molecular chaperone protein gene groEL-groES in the genome, promoting the functional expression of RuBisCO.

[0015] Furthermore, the engineering strain of the present invention lacks the repressor protein gene lacI, thereby preventing the LacI repressor protein encoded by the lacI gene from inhibiting the transcription of the target gene by the Ptrc promoter.

[0016] In addition, as further preferred, the engineered strain of the present invention also overexpresses the lpd gene, aspA gene, spoT* gene, pykA gene, zwf gene, pck gene and csgD gene, and deletes the poxB gene, avtA gene, ldhA gene and purR gene. The above preferred technical scheme further optimizes the metabolic pathway from glucose to D-pantothenic acid in the engineered strain, thereby further improving the synthesis efficiency of D-pantothenic acid.

[0017] Furthermore, the nucleotide sequence of the acetolactate synthase gene alsS* is shown in SEQ ID NO.1.

[0018] The ketoacid reductoisomerase gene ilvC V412A The nucleotide sequence is shown in SEQ ID NO.2.

[0019] The nucleotide sequence of the dihydroxyacid dehydratase gene ilvD is shown in SEQ ID NO.3.

[0020] The overexpressed methyltransferase gene panB Y25A The nucleotide sequence is shown in SEQ ID NO.4.

[0021] The nucleotide sequence of the dehydropantothenate reductase gene panE is shown in SEQ ID NO.5.

[0022] The nucleotide sequence of the aspartate aminotransferase gene aspC is shown in SEQ ID NO.6.

[0023] The nucleotide sequence of the aspartate decarboxylase gene panD is shown in SEQ ID NO.7.

[0024] The nucleotide sequence of the pantothenate synthase gene panC is shown in SEQ ID NO.8.

[0025] The nucleotide sequence of the pantothenate transporter gene panF is shown in SEQ ID NO.9.

[0026] The pantothenate kinase gene coaA R106A The nucleotide sequence is shown in SEQ ID NO.10.

[0027] The nucleotide sequence of the ribulose-1,5-bisphosphate carboxylase / oxidase gene RuBisCO is shown in SEQ ID NO.11.

[0028] The nucleotide sequence of the phosphoribosylase gene prk is shown in SEQ ID NO.12.

[0029] The nucleotide sequence of the molecular chaperone protein gene GroEL-GroES is shown in SEQ ID NO.13.

[0030] The nucleotide sequence of the repressor protein gene lacI is shown in SEQ ID NO.14.

[0031] The nucleotide sequence of the threonine dehydratase gene ilvA is shown in SEQ ID NO.15.

[0032] The nucleotide sequence of the pantothenate kinase gene coaA is shown in SEQ ID NO.16.

[0033] The nucleotide sequence of the formate acetyltransferase gene pflD is shown in SEQ ID NO.17.

[0034] In addition, the nucleotide sequence of the lpd gene is shown in SEQ ID NO.18.

[0035] The nucleotide sequence of the aspA gene is shown in SEQ ID NO.19.

[0036] The nucleotide sequence of the spoT* gene is shown in SEQ ID NO. 20.

[0037] The nucleotide sequence of the pykA gene is shown in SEQ ID NO.21.

[0038] The nucleotide sequence of the zwf gene is shown in SEQ ID NO.22.

[0039] The nucleotide sequence of the pck gene is shown in SEQ ID NO.23.

[0040] The nucleotide sequence of the csgD gene is shown in SEQ ID NO.24.

[0041] The nucleotide sequence of the poxB gene is shown in SEQ ID NO.25.

[0042] The nucleotide sequence of the avtA gene is shown in SEQ ID NO.26.

[0043] The nucleotide sequence of the ldhA gene is shown in SEQ ID NO.27.

[0044] The nucleotide sequence of the purR gene is shown in SEQ ID NO.28.

[0045] Furthermore, the acetolactate synthase gene alsS* and the ketoacid reductoisomerase gene ilvC V412A , dihydroxyacid dehydratase gene ilvD, overexpression of methyltransferase gene panB Y25A , anhydropantothenate reductase gene panE, aspartate aminotransferase gene aspC, aspartate decarboxylase gene panD, pantothenate synthase gene panC, pantothenate transporter gene panF, ribulose-1,5-bisphosphate carboxylase / oxidase gene RuBisCO, and molecular chaperone protein genes GroEL-GroES are all controlled by the Ptrc promoter.

[0046] The phosphoribosylase gene prk is controlled by the Plac promoter or has no promoter.

[0047] In addition, the lpd gene, aspA gene, spoT* gene, pykA gene, zwf gene, pck gene and csgD gene are also controlled by the Ptrc promoter.

[0048] In the above technical solution, the strong promoter Ptrc is used to control the overexpression of the RuBisCO gene, and the weak promoter Plac is used to control the expression of the prk gene or no promoter is used, thereby controlling the expression of the RuBisCO gene to be stronger than the expression of the prk gene, thereby avoiding toxic effects on the bacteria.

[0049] Furthermore, the alsS* gene is located at the lacI gene site and the yqaC pseudogene site.

[0050] The ilvC V412A The gene is located at the yciQ pseudogene locus.

[0051] The ilvD gene is located at the mbhA pseudogene locus.

[0052] The panB Y25A The gene is located at the ygaY pseudogene locus.

[0053] The panE gene is located at the ycdN pseudogene locus.

[0054] The aspC gene is located at the yncI pseudogene site.

[0055] The panD gene is located at the yjiT pseudogene site.

[0056] The panC gene is located at the ilvA gene site.

[0057] The panF gene is located at the coaA gene locus.

[0058] The coaA R106A The gene is located at the yghE pseudogene locus.

[0059] The RuBisCO gene is located at the gapC pseudogene locus.

[0060] The prk gene is located at the ylbE pseudogene locus.

[0061] The GroEL-GroES gene is located at the yjiP pseudogene site.

[0062] In addition, the lpd gene is located at the avtA gene locus.

[0063] The aspA gene is located at the yghX pseudogene locus.

[0064] The spoT* gene is located at the yeel pseudogene locus.

[0065] The pykA gene is located at the ldhA gene locus.

[0066] The zwf gene is located at the yjiV pseudogene locus.

[0067] The pck gene is located at the yeeP pseudogene locus.

[0068] The csgD gene is located at the purR gene locus.

[0069] In a second aspect, the present invention provides a method for constructing an engineered strain for efficiently producing D-pantothenic acid, which is used to construct the above-mentioned engineered strain for efficiently producing D-pantothenic acid, comprising the following steps: The alsS* gene was overexpressed using the Ptrc promoter at the lacI gene locus.

[0070] The Ptrc promoter was used to control the overexpression of the alsS* gene at the yqaC pseudogene locus.

[0071] Control of ilvC by the Ptrc promoter at the yciQ pseudogene locus V412A Gene overexpression.

[0072] The ilvD gene was overexpressed using the Ptrc promoter at the mbhA pseudogene locus.

[0073] Control of panB by the Ptrc promoter at the ygaY pseudogene locus Y25A Heterologous overexpression of genes.

[0074] The panE gene was overexpressed under the control of the Ptrc promoter at the ycdN pseudogene locus.

[0075] The aspC gene was overexpressed using the Ptrc promoter at the yncI pseudogene locus.

[0076] The panD gene was overexpressed using the Ptrc promoter at the yjiT pseudogene locus.

[0077] The panC gene was overexpressed using the Ptrc promoter at the ilvA gene locus.

[0078] The panF gene was overexpressed using the Ptrc promoter at the coaA gene locus.

[0079] Expression of coaA at the yghE pseudogene locus R106A Gene.

[0080] Knockout of the pflD gene.

[0081] The RuBisCO gene was overexpressed using the Ptrc promoter at the gapC pseudogene locus.

[0082] The prk gene is expressed at the ylbE pseudogene locus.

[0083] GroEL-GroES gene overexpression was controlled by Ptrc promoter at the yjiP pseudogene locus.

[0084] Compared with the prior art, the engineered strain constructed by the present invention generates no toxic metabolic byproducts during fermentation, has high strain stability, and has very good industrial application value.

[0085] In addition, as a further preference, the present invention also uses the Ptrc promoter at the avtA gene site of the above-mentioned engineered strain to control overexpression of the lpd gene; uses the Ptrc promoter at the yghX pseudogene site to control overexpression of the aspA gene; uses the Ptrc promoter at the yeel pseudogene site to control overexpression of the spoT* gene; uses the Ptrc promoter at the ldhA gene site to control overexpression of the pykA gene; deletes the poxB gene; uses the Ptrc promoter at the yjiV pseudogene site to control overexpression of the zwf gene; uses the Ptrc promoter at the yeeP pseudogene site to control overexpression of the pck gene; and uses the Ptrc promoter at the purR gene site to control overexpression of the csgD gene.

[0086] Furthermore, the nucleotide sequence of the Ptrc promoter is shown in SEQ ID NO. 29; the nucleotide sequence of the Plac promoter is shown in SEQ ID NO. 30.

[0087] In addition, the nucleotide sequence of the Ptrc terminator is shown in SEQ ID NO. 31; the nucleotide sequence of the Plac terminator is shown in SEQ ID NO. 32.

[0088] Furthermore, the starting strain used in the construction of the above-mentioned engineering strain is Escherichia coli; the Escherichia coli is E. coli W3110.

[0089] In a third aspect, the present invention provides the use of the above-mentioned engineered strain in the production of D-pantothenic acid.

[0090] Compared with the prior art, the engineered strain of the present invention can utilize glucose as a substrate to efficiently synthesize D-pantothenic acid from scratch. After 68 to 72 hours of fermentation, the yield of D-pantothenic acid in the fermentation broth is as high as 77.9 g / L, and the sugar-acid conversion rate is as high as 32%.

[0091] Furthermore, the production of D-pantothenic acid comprises the following steps: The engineered strain is activated and then seed culture is performed to obtain seed liquid.

[0092] The obtained seed liquid is inoculated into the fermentation medium at an inoculation rate of 15% to 20% for fermentation culture. The culture temperature is 30 to 37°C, the pH is 6.8 to 7.1, the dissolved oxygen value is 25 to 35%, and a glucose solution with a mass fraction of 70% to 80% is added. The concentration of glucose in the fermentation liquid is controlled at ≤ 0.5 g / L. After the fermentation culture is completed, D-pantothenic acid is obtained. The fermentation culture is completed when the OD value no longer increases. The fermentation time can generally be referenced to 68 to 72 hours.

[0093] Furthermore, the activation of the engineered strain includes: taking the above-mentioned engineered strain and inoculating it on an activation slant, culturing it at 30-37° C. for 10-14 hours, and subculturing it 1-2 times to obtain an activated engineered strain.

[0094] Seed culture is as follows: the activated engineered strain is cultured at a temperature of 30-37°C, a pH of 6.8-7.1, and a dissolved oxygen of 25-35% until the OD of the seed solution reaches 600 Reach 13~18. DETAILED DESCRIPTION

[0095] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0096] It should be understood that the raw materials used in the following examples are all commercially available raw materials unless otherwise specified.

[0097] The present invention uses a CRISPR / Cas9-mediated gene editing method to edit the gene of E. coli W3110 (refer to Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). CRISPR / Cas9 is a precise and efficient new gene targeting modification technology. The two plasmids used in this method are pGRB and pREDCas9. The pREDCas9 plasmid is a temperature-sensitive plasmid that carries the gRNA plasmid elimination system, the Red recombination system of λ phage and the Cas9 protein expression system. It has resistance to spectinomycin (working concentration: 100 mg / L) and the optimal culture temperature is 32°C. The pGRB plasmid uses pUC18 as the backbone, contains the promoter J23100, the gRNA-Cas9 binding region sequence and the terminator sequence, has resistance to ampicillin (working concentration: 100 mg / L) and the optimal culture temperature is 37°C. Example 1

[0098] Use the Ptrc promoter to control the overexpression of the alsS* gene at the lacI gene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the lacI gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers lacI-US (SEQ ID NO. 33), lacI-UA (SEQ ID NO. 34), lacI-DS (SEQ ID NO. 35), and lacI-DA (SEQID NO. 36).

[0099] Using the alsS* gene carrying a mutation point from Bacillus subtilis synthesized by Anshengda Biotechnology Company as a template and alsS*-S (SEQ ID NO. 37) and alsS*-A (SEQ ID NO. 38) as primers, the Ptrc-alsS* gene fragment was amplified by HS enzyme PCR.

[0100] Among them, the Ptrc promoter is designed in the antisense strand primer of the upstream homologous arm of the lacI gene and the positive strand primer of the alsS* gene; the Ptrc terminator is designed in the antisense strand primer of the alsS* gene and the positive strand primer of the downstream homologous arm of the lacI gene.

[0101] Then, using the upstream homology arm, downstream homology arm and Ptrc-alsS* gene fragment of the above-mentioned lacI as templates, HS enzyme was used to obtain the lacI::Ptrc-alsS* gene integration fragment by overlapping PCR. The gene integration fragment consists of the lacI upstream homology arm, the Ptrc-alsS* target gene and the lacI downstream homology arm.

[0102] Using pGRB-lacI-S (SEQ ID NO. 39) and pGRB-lacI-A (SEQ ID NO. 40) as primers, a DNA fragment containing the lacI target sequence was constructed through a PCR annealing program. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-lacI.

[0103] The lacI::Ptrc-alsS* gene integration fragment obtained in the above steps and the pGRB-lacI plasmid were electroporated into the E. coli DPAH0 (i.e., E. coli W3110) strain containing the pREDCas9 plasmid. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-1. Example 2

[0104] The Ptrc promoter was used to control the overexpression of the alsS* gene at the yqaC pseudogene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the yqaC pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yqaC-US (SEQ ID NO.41), yqaC-UA (SEQ ID NO.42), yqaC-DS (SEQ ID NO.43), and yqaC-DA (SEQ ID NO.44) of the yqaC pseudogene, respectively.

[0105] Using the alsS* gene carrying a mutation point from Bacillus subtilis synthesized by Anshengda Biotechnology Company as a template and alsS*-S (SEQ ID NO. 37) and alsS*-A (SEQ ID NO. 38) as primers, the Ptrc-alsS* gene fragment was amplified by HS enzyme PCR.

[0106] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the yqaC pseudogene and the positive chain primer of the alsS* gene; the Ptrc terminator was designed in the antisense primer of the alsS* gene and the positive chain primer of the downstream homologous arm of the yqaC pseudogene.

[0107] Then, using the upstream homology arm, downstream homology arm and Ptrc-alsS* gene fragment of the above-mentioned yqaC as templates, HS enzyme was used to obtain the yqaC::Ptrc-alsS* gene integration fragment by overlapping PCR. The gene integration fragment consists of the yqaC upstream homology arm, the Ptrc-alsS* target gene and the yqaC downstream homology arm.

[0108] Using pGRB-yqaC-S (SEQ ID NO. 45) and pGRB-yqaC-A (SEQ ID NO. 46) as primers, a DNA fragment containing the yqaC target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-yqaC.

[0109] The yqaC::Ptrc-alsS* gene integration fragment obtained in the above steps and the pGRB-yqaC plasmid were electroporated into the E. coli DPAH1-1 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-2. Example 3

[0110] Control of ilvC by the Ptrc promoter at the yciQ pseudogene locus V412A Gene overexpression, the specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the yciQ pseudogene were amplified by HS enzyme PCR using the upstream and downstream homologous arms primers yciQ-US (SEQ ID NO.47), yciQ-UA (SEQ ID NO.48), yciQ-DS (SEQ ID NO.49), and yciQ-DA (SEQ ID NO.50) of the yciQ pseudogene.

[0111] ilvC with mutation point from E. coli synthesized by Anshengda Biotechnology V412A Gene as template, using ilvC V412A -S (SEQ ID NO. 51), ilvC V412A -A (SEQ ID NO. 52) was used as primer, and Ptrc-ilvC was obtained by HS enzyme PCR amplification. V412A Gene fragment.

[0112] Among them, the antisense primer of the upstream homology arm of the yciQ pseudogene and the ilvC V412AThe Ptrc terminator was designed in the positive strand primer of the ilvC V412A The antisense strand primer of the gene and the sense strand primer of the downstream homology arm of the yciQ pseudogene.

[0113] The upstream homology arm, downstream homology arm and Ptrc-ilvC of the above yciQ pseudogene were used. V412A The gene fragment was used as a template and HS enzyme was used to obtain yciQ::Ptrc-ilvC by overlapping PCR. V412A The gene integration fragment consists of the upstream homology arm of yciQ, Ptrc-ilvC V412A The target gene and the downstream homology arm of yciQ.

[0114] Using pGRB-yciQ-S (SEQ ID NO. 53) and pGRB-yciQ-A (SEQ ID NO. 54) as primers, a DNA fragment containing the yciQ target sequence was constructed through a PCR annealing procedure. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-yciQ.

[0115] The yciQ::Ptrc-ilvC V412A The gene integration fragment and the pGRB-yciQ plasmid were electroporated into the E. coli DPAH1-2 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-3. Example 4

[0116] The Ptrc promoter was used to control the overexpression of the ilvD gene at the mbhA pseudogene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the mbhA pseudogene were amplified by HS enzyme PCR using the upstream and downstream homologous arms primers mbhA-US (SEQ ID NO.55), mbhA-UA (SEQ ID NO.56), mbhA-DS (SEQ ID NO.57), and mbhA-DA (SEQ ID NO.58).

[0117] Using the E. coli W3110 genome as a template and ilvD-S (SEQ ID NO.59) and ilvD-A (SEQ ID NO.60) as primers, the Ptrc-ilvD target gene fragment was amplified by HS enzyme PCR.

[0118] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the mbhA pseudogene and the sense primer of the ilvD gene; the Ptrc terminator was designed in the antisense primer of the ilvD gene and the sense primer of the downstream homologous arm of the mbhA pseudogene.

[0119] Using the upstream homology arm, downstream homology arm and Ptrc-ilvD target gene fragment of the above-mentioned mbhA pseudogene as templates, HS enzyme was used to obtain the mbhA::Ptrc-ilvD gene integration fragment by overlapping PCR. The gene integration fragment consisted of the mbhA upstream homology arm, the Ptrc-ilvD target gene and the mbhA downstream homology arm.

[0120] Using pGRB-mbhA-S (SEQ ID NO. 61) and pGRB-mbhA-A (SEQ ID NO. 62) as primers, a DNA fragment containing the mbhA pseudogene target sequence was constructed through a PCR annealing program. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-mbhA from them.

[0121] The mbhA::Ptrc-ilvD gene integration fragment obtained in the above steps and the pGRB-mbhA plasmid were electroporated into the E. coli DPAH1-3 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-4. Example 5

[0122] Control of panB by the Ptrc promoter at the ygaY pseudogene locus Y25A Gene overexpression, the specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the ygaY pseudogene were amplified by HS enzyme PCR using the upstream and downstream homologous arms primers ygaY-US (SEQ ID NO.63), ygaY-UA (SEQ ID NO.64), ygaY-DS (SEQ ID NO.65), and ygaY-DA (SEQ ID NO.66) of the ygaY pseudogene.

[0123] PanB carrying mutations from E. coli synthesized by Anshengda Bio Y25A gene as template, using panB Y25A -S (SEQ ID NO. 67), panB Y25A -A (SEQ ID NO. 68) was used as primer, and Ptrc-panB was obtained by HS enzyme PCR amplification.Y25A Target gene fragment.

[0124] Among them, the antisense primer of the upstream homology arm of the ygaY pseudogene and the panB Y25A The Ptrc terminator was designed in the panB Y25A The antisense primer of the gene and the sense primer of the downstream homology arm of the ygaY pseudogene.

[0125] The upstream homology arm, downstream homology arm and Ptrc-panB of the above ygaY pseudogene were used. Y25A The target gene fragment was used as a template and HS enzyme was used to obtain ygaY::Ptrc-panB by overlapping PCR. Y25A The gene integration fragment consists of the upstream homology arm of ygaY, Ptrc-panB Y25A The target gene and the downstream homology arm of ygaY.

[0126] Using pGRB-ygaY-S (SEQ ID NO. 69) and pGRB-ygaY-A (SEQ ID NO. 70) as primers, a DNA fragment containing the ygaY target sequence was constructed through a PCR annealing program. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-ygaY.

[0127] The ygaY::Ptrc-panB Y25A The gene integration fragment and the pGRB-ygaY plasmid were electroporated into the E. coli DPAH1-4 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-5. Example 6

[0128] The Ptrc promoter was used to control the overexpression of the panE gene at the ycdN pseudogene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of ycdN were amplified by HS enzyme PCR using the upstream and downstream homology arm primers ycdN-US (SEQ ID NO.71), ycdN-UA (SEQ ID NO.72), ycdN-DS (SEQ ID NO.73), and ycdN-DA (SEQ ID NO.74) of the ycdN pseudogene.

[0129] Using the E. coli W3110 genome as a template and panE-S (SEQ ID NO.75) and panE-A (SEQ ID NO.76) as primers, the Ptrc-panE target gene fragment was amplified by HS enzyme PCR.

[0130] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the ycdN pseudogene and the positive chain primer of the panE gene; the Ptrc terminator was designed in the antisense primer of the panE gene and the positive chain primer of the downstream homologous arm of the ycdN pseudogene.

[0131] Then, using the upstream homologous arm, downstream homologous arm and Ptrc-panE target gene fragment of the above-mentioned ycdN as templates, HS enzyme was used to obtain the ycdN::Ptrc-panE gene integration fragment by overlapping PCR. The gene integration fragment consists of the ycdN upstream homologous arm, the Ptrc-panE target gene and the ycdN downstream homologous arm.

[0132] Using pGRB-ycdN-S (SEQ ID NO. 77) and pGRB-ycdN-A (SEQ ID NO. 78) as primers, a DNA fragment containing the ycdN target sequence was constructed through a PCR annealing program. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-ycdN.

[0133] The ycdN::Ptrc-panE gene integration fragment obtained in the above steps and the pGRB-ycdN plasmid were electroporated into the E. coli DPAH1-5 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-6. Example 7

[0134] The Ptrc promoter was used to control the overexpression of the lpd gene at the avtA gene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of avtA were amplified by HS enzyme PCR using the upstream and downstream homology arm primers of the avtA gene, avtA-US (SEQ ID NO.79), avtA-UA (SEQ ID NO.80), avtA-DS (SEQ ID NO.81), and avtA-DA (SEQID NO.82), respectively.

[0135] Using the E. coli W3110 genome as a template and lpd-S (SEQ ID NO.83) and lpd-A (SEQ ID NO.84) as primers, the Ptrc-lpd target gene fragment was amplified by HS enzyme PCR.

[0136] Among them, the Ptrc promoter is designed in the antisense primer of the upstream homologous arm of the avtA gene and the positive primer of the lpd gene; the Ptrc terminator is designed in the antisense primer of the lpd gene and the positive primer of the downstream homologous arm of the avtA gene.

[0137] Then, using the upstream homology arm, downstream homology arm and Ptrc-lpd target gene fragment of avtA as templates, HS enzyme was used to obtain the avtA::Ptrc-lpd gene integration fragment by overlapping PCR. The gene integration fragment consisted of the upstream homology arm of avtA, the Ptrc-lpd target gene and the downstream homology arm of avtA.

[0138] Using pGRB-avtA-S (SEQ ID NO. 85) and pGRB-avtA-A (SEQ ID NO. 86) as primers, a DNA fragment containing the avtA target sequence was constructed through a PCR annealing program. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-avtA from them.

[0139] The avtA::Ptrc-lpd gene integration fragment obtained in the above step and the pGRB-lpd plasmid were electroporated into the E. coli DPAH1-6 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-7. Example 8

[0140] Use the Ptrc promoter to control aspC gene overexpression at the yncI pseudogene site. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the yncI pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yncI-US (SEQ ID NO.87), yncI-UA (SEQ ID NO.88), yncI-DS (SEQ ID NO.89), and yncI-DA (SEQ ID NO.90).

[0141] Using the E. coli W3110 genome as a template and aspC-S (SEQ ID NO.91) and aspC-A (SEQ ID NO.92) as primers, the Ptrc-aspC target gene fragment was amplified by HS enzyme PCR.

[0142] Among them, the Ptrc promoter is designed in the antisense strand primer of the upstream homologous arm of the yncI pseudogene and the positive strand primer of the aspC gene; the Ptrc terminator is designed in the antisense strand primer of the aspC gene and the positive strand primer of the downstream homologous arm of the yncI pseudogene.

[0143] Then, using the upstream homology arm, downstream homology arm and Ptrc-aspC target gene fragment of the above-mentioned yncI as templates, HS enzyme was used to obtain the yncI::Ptrc-aspC gene integration fragment by overlapping PCR. The gene integration fragment consists of the yncI upstream homology arm, the Ptrc-aspC target gene and the yncI downstream homology arm.

[0144] Using pGRB-yncI-S (SEQ ID NO. 93) and pGRB-yncI-A (SEQ ID NO. 94) as primers, a DNA fragment containing the yncI target sequence was constructed through a PCR annealing program. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-yncI.

[0145] The yncI::Ptrc-aspC gene integration fragment obtained in the above steps and the pGRB-yncI plasmid were electroporated into the E. coli DPAH1-7 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-8. Example 9

[0146] Use the Ptrc promoter to control the overexpression of the aspA gene at the yghX pseudogene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of yghX were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yghX-US (SEQ ID NO.95), yghX-UA (SEQ ID NO.96), yghX-DS (SEQ ID NO.97), and yghX-DA (SEQ ID NO.98) of the yghX pseudogene.

[0147] Using the E. coli W3110 genome as a template and aspA-S (SEQ ID NO.99) and aspA-A (SEQ ID NO.100) as primers, the Ptrc-aspA target gene fragment was amplified by HS enzyme PCR.

[0148] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the yghX pseudogene and the sense primer of the aspA gene; the Ptrc terminator was designed in the antisense primer of the aspA gene and the sense primer of the downstream homologous arm of the yghX pseudogene.

[0149] Then, using the upstream homology arm, downstream homology arm and Ptrc-aspA target gene fragment of the above-mentioned yghX as templates, HS enzyme was used to obtain the yghX::Ptrc-aspA gene integration fragment by overlapping PCR. The gene integration fragment consisted of the yghX upstream homology arm, the Ptrc-aspA target gene and the yghX downstream homology arm.

[0150] Using pGRB-yghX-S (SEQ ID NO. 101) and pGRB-yghX-A (SEQ ID NO. 102) as primers, a DNA fragment containing the yghX target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-yghX.

[0151] The yghX::Ptrc-aspA gene integration fragment obtained in the above step and the pGRB-yghX plasmid were electroporated into the E. coli DPAH1-8 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-9. Example 10

[0152] The Ptrc promoter was used to control the overexpression of the panD gene at the yjiT pseudogene site. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of yjiT were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yjiT-US (SEQ ID NO. 103), yjiT-UA (SEQ ID NO. 104), yjiT-DS (SEQ ID NO. 105), and yjiT-DA (SEQ ID NO. 106) of the yjiT pseudogene.

[0153] Using the E. coli W3110 genome as a template and panD-S (SEQ ID NO. 107) and panD-A (SEQ ID NO. 108) as primers, the Ptrc-panD target gene fragment was amplified by HS enzyme PCR.

[0154] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the yjiT pseudogene and the sense primer of the panD gene; the Ptrc terminator was designed in the antisense primer of the panD gene and the sense primer of the downstream homologous arm of the yjiT pseudogene.

[0155] Then, using the upstream homology arm, downstream homology arm and Ptrc-panD target gene fragment of the above-mentioned yjiT as templates, HS enzyme was used to obtain the yjiT::Ptrc-panD gene integration fragment by overlapping PCR. The gene integration fragment consists of the yjiT upstream homology arm, the Ptrc-panD target gene and the yjiT downstream homology arm.

[0156] Using pGRB-yjiT-S (SEQ ID NO. 109) and pGRB-yjiT-A (SEQ ID NO. 110) as primers, a DNA fragment containing the yjiT target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-yjiT.

[0157] The yjiT::Ptrc-panD gene integration fragment obtained in the above steps and the pGRB-yjiT plasmid were electroporated into the E. coli DPAH1-9 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-10. Embodiment 11

[0158] The Ptrc promoter was used to control the overexpression of the panC gene at the ilvA gene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the ilvA gene were amplified by HS enzyme PCR using the upstream and downstream homologous arms primers ilvA-US (SEQ ID NO.111), ilvA-UA (SEQ ID NO.112), ilvA-DS (SEQ ID NO.113), and ilvA-DA (SEQ ID NO.114), respectively.

[0159] Using the genome of Corynebacterium glutamicum as a template and panC-S (SEQ ID NO. 115) and panC-A (SEQ ID NO. 116) as primers, the Ptrc-panC target gene fragment was amplified by HS enzyme PCR.

[0160] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the ilvA gene and the sense primer of the panC gene; the Ptrc terminator was designed in the antisense primer of the panC gene and the sense primer of the downstream homologous arm of the ilvA gene.

[0161] Then, using the upstream homology arm, downstream homology arm and Ptrc-panC target gene fragment of the above-mentioned ilvA as templates, HS enzyme was used to obtain the ilvA::Ptrc-panC gene integration fragment by overlapping PCR. The gene integration fragment consists of the ilvA upstream homology arm, the Ptrc-panC target gene and the ilvA downstream homology arm.

[0162] Using pGRB-ilvA-S (SEQ ID NO. 117) and pGRB-ilvA-A (SEQ ID NO. 118) as primers, a DNA fragment containing the ilvA target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-ilvA from them.

[0163] The ilvA::Ptrc-panC gene integration fragment obtained in the above step and the pGRB-ilvA plasmid were electroporated into the E. coli DPAH1-10 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-11. Example 12

[0164] The Ptrc promoter was used to control the overexpression of the panF gene at the coaA gene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of coaA were amplified by HS enzyme PCR using the upstream and downstream homologous arm primers of the coaA gene, coaA-US (SEQ ID NO. 119), coaA-UA (SEQ ID NO. 120), coaA-DS (SEQ ID NO. 121), and coaA-DA (SEQ ID NO. 122).

[0165] Using the E. coli W3110 genome as a template and panF-S (SEQ ID NO. 123) and panF-A (SEQ ID NO. 124) as primers, the Ptrc-panF target gene fragment was amplified by HS enzyme PCR.

[0166] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the coaA gene and the positive primer of the panF gene; the Ptrc terminator was designed in the antisense primer of the panF gene and the positive primer of the downstream homologous arm of the coaA gene.

[0167] Then, using the upstream homology arm, downstream homology arm and Ptrc-panF target gene fragment of the above-mentioned coaA as templates, HS enzyme was used to obtain the coaA::Ptrc-panF gene integration fragment by overlapping PCR. The gene integration fragment consists of the coaA upstream homology arm, the Ptrc-panF target gene and the coaA downstream homology arm.

[0168] Using pGRB-coaA-S (SEQ ID NO. 125) and pGRB-coaA-A (SEQ ID NO. 126) as primers, a DNA fragment containing the coaA target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-coaA from them.

[0169] The coaA::Ptrc-panF gene integration fragment obtained in the above steps and the pGRB-coaA plasmid were electroporated into the E. coli DPAH1-11 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH1-12. Embodiment 13

[0170] Expression of coaA at the yghE pseudogene locus R106A Gene, the specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of yghE were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yghE-US (SEQ ID NO. 127), yghE-UA (SEQ ID NO. 128), yghE-DS (SEQ ID NO. 129), and yghE-DA (SEQ ID NO. 130) of the yghE pseudogene.

[0171] The coaA with mutation point was synthesized by Anshengda Biotechnology Co., Ltd. R106Agene as template, using coaA R106A -S (SEQ ID NO. 131), coaA R106A -A (SEQ ID NO. 132) was used as primer, and Ptrc-coaA was obtained by HS enzyme PCR amplification. R106A Target gene fragment.

[0172] Then, the upstream homology arm, downstream homology arm and Ptrc-coaA of yghE were used. R106A The target gene fragment was used as a template and yghE::Ptrc-coaA was obtained by overlapping PCR using HS enzyme. R106A The gene integration fragment consists of the upstream homology arm of yghE, Ptrc-coaA R106A The target gene and the downstream homology arm of yghE.

[0173] Using pGRB-yghE-S (SEQ ID NO. 133) and pGRB-yghE-A (SEQ ID NO. 134) as primers, a DNA fragment containing the yghE target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-yghE.

[0174] The yghE::PcoaA obtained in the above step R106A The gene integration fragment and the pGRB-yghE plasmid were electroporated into the E. coli DPAH1-12 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH2. Embodiment 14

[0175] The Ptrc promoter was used to control the overexpression of the spoT* gene at the Yeel pseudogene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of Yeel were amplified by HS enzyme PCR using the upstream and downstream homology arm primers of the Yeel pseudogene, Yeel-US (SEQ ID NO. 135), Yeel-UA (SEQ ID NO. 136), Yeel-DS (SEQ ID NO. 137), and Yeel-DA (SEQ ID NO. 138).

[0176] Using the spoT* gene carrying a mutation point from Escherichia coli synthesized by Anshengda Biotechnology Co., Ltd. as a template and spoT*-S (SEQ ID NO. 139) and spoT*-A (SEQ ID NO. 140) as primers, the Ptrc-spoT* target gene fragment was amplified by HS enzyme PCR.

[0177] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the Yeel pseudogene and the positive chain primer of the spoT* gene; the Ptrc terminator was designed in the antisense primer of the spoT* gene and the positive chain primer of the downstream homologous arm of the Yeel pseudogene.

[0178] Then, using the upstream homology arm, downstream homology arm and Ptrc-spoT* target gene fragment of the above-mentioned yghE as templates, HS enzyme was used to obtain the Yeel::Ptrc-spoT* gene integration fragment by overlapping PCR. The gene integration fragment consists of the Yeel upstream homology arm, the Ptrc-spoT* target gene and the Yeel downstream homology arm.

[0179] Using pGRB-Yeel-S (SEQ ID NO. 141) and pGRB-Yeel-A (SEQ ID NO. 142) as primers, a DNA fragment containing the Yeel target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-Yeel.

[0180] The Yeel::Ptrc-spoT* gene integration fragment obtained in the above steps and the pGRB-Yeel plasmid were electroporated into the E. coli DPAH2 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH3. Embodiment 15

[0181] The steps of using the Ptrc promoter to control the overexpression of the pykA gene at the ldhA gene locus are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the ldhA gene were amplified by HS enzyme PCR using the upstream and downstream homologous arms primers ldhA-US (SEQ ID NO. 143), ldhA-UA (SEQ ID NO. 144), ldhA-DS (SEQ ID NO. 145), and ldhA-DA (SEQ ID NO. 146), respectively.

[0182] Using the E. coli W3110 genome as a template and pykA-S (SEQ ID NO. 147) and pykA-A (SEQ ID NO. 148) as primers, the Ptrc-pykA target gene fragment was amplified by HS enzyme PCR.

[0183] Among them, the Ptrc promoter is designed in the antisense strand primer of the upstream homologous arm of the ldhA gene and the sense strand primer of the pykA gene; the Ptrc terminator is designed in the antisense strand primer of the pykA gene and the sense strand primer of the downstream homologous arm of the ldhA gene.

[0184] Then, using the upstream homology arm, downstream homology arm and Ptrc-pykA target gene fragment of the above-mentioned ldhA as templates, HS enzyme was used to obtain the ldhA::Ptrc-pykA gene integration fragment by overlapping PCR. The gene integration fragment consists of the ldhA upstream homology arm, the Ptrc-pykA target gene and the ldhA downstream homology arm.

[0185] Using pGRB-ldhA-S (SEQ ID NO. 149) and pGRB-ldhA-A (SEQ ID NO. 150) as primers, a DNA fragment containing the ldhA target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-ldhA.

[0186] The ldhA::Ptrc-pykA gene integration fragment obtained in the above steps and the pGRB-ldhA plasmid were electroporated into the E. coli DPAH3 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH3-1. Example 16

[0187] The specific steps for knocking out the pflD gene are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the pflD gene were amplified by HS enzyme PCR using the upstream and downstream homologous arm primers pflD-US (SEQ ID NO. 151), pflD-UA (SEQ ID NO. 152), pflD-DS (SEQ ID NO. 153), and pflD-DA (SEQ ID NO. 154) of the pflD gene, respectively.

[0188] Then, the upstream homology arm and the downstream homology arm of the pflD gene were used as templates and HS enzyme was used to obtain the pflD gene integration fragment by overlapping PCR. The gene integration fragment consisted of the pflD upstream homology arm and the pflD downstream homology arm.

[0189] Using pGRB-pflD-S (SEQ ID NO. 155) and pGRB-pflD-A (SEQ ID NO. 156) as primers, a DNA fragment containing the pflD target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-pflD.

[0190] The pflD gene integration fragment obtained in the above steps and the pGRB-pflD plasmid were electroporated into the E. coli DPAH3-1 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH3-2. Embodiment 17

[0191] The specific steps for knocking out the poxB gene are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the poxB gene were amplified by HS enzyme PCR using the upstream and downstream homologous arm primers poxB-US (SEQ ID NO.157), poxB-UA (SEQ ID NO.158), poxB-DS (SEQ ID NO.159), and poxB-DA (SEQ ID NO.160).

[0192] Then, the upstream homology arm and the downstream homology arm of the poxB gene were used as templates, and the HS enzyme was used to obtain the poxB gene integration fragment by overlapping PCR. The gene integration fragment consisted of the poxB upstream homology arm and the poxB downstream homology arm.

[0193] Using pGRB-poxB-S (SEQ ID NO. 161) and pGRB-poxB-A (SEQ ID NO. 162) as primers, a DNA fragment containing the poxB target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-poxB from them.

[0194] The poxB gene integration fragment obtained in the above steps and the pGRB-poxB plasmid were electroporated into the E. coli DPAH3-2 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH4. Embodiment 18

[0195] The Ptrc promoter was used to control the overexpression of the zwf gene at the yjiV pseudogene site. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homologous arms of the yjiV pseudogene were amplified by HS enzyme PCR using the upstream and downstream homologous arms primers yjiV-US (SEQ ID NO.163), yjiV-UA (SEQ ID NO.164), yjiV-DS (SEQ ID NO.165), and yjiV-DA (SEQ ID NO.166) of the yjiV pseudogene.

[0196] Using the E. coli W3110 genome as a template and zwf-S (SEQ ID NO. 167) and zwf-A (SEQ ID NO. 168) as primers, the Ptrc-zwf target gene fragment was amplified by HS enzyme PCR.

[0197] Among them, the Ptrc promoter is designed in the antisense chain primer of the upstream homologous arm of the pseudogene yjiV and the positive chain primer of the zwf gene; the Ptrc terminator is designed in the antisense chain primer of the zwf gene and the positive chain primer of the downstream homologous arm of the pseudogene yjiV.

[0198] Then, using the upstream homologous arm, downstream homologous arm and Ptrc-zwf target gene fragment of the above-mentioned yjiV pseudogene as templates, HS enzyme was used to obtain the yjiV::Ptrc-zwf gene integration fragment by overlapping PCR. The gene integration fragment consists of the yjiV upstream homologous arm, the Ptrc-zwf target gene and the yjiV downstream homologous arm.

[0199] Using pGRB-yjiV-S (SEQ ID NO. 169) and pGRB-yjiV-A (SEQ ID NO. 170) as primers, a DNA fragment containing the yjiV target sequence was constructed through a PCR annealing program. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-yjiV.

[0200] The yjiV::Ptrc-zwf gene integration fragment obtained in the above steps and the pGRB-yjiV plasmid were electroporated into the E. coli DPAH4 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH4-1. Embodiment 19

[0201] The Ptrc promoter was used to control the overexpression of RuBisCO gene at the gapC pseudogene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the gapC pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers of the gapC pseudogene gapC-US (SEQ ID NO. 171), gapC-UA (SEQ ID NO. 172), gapC-DS (SEQ ID NO. 173), and gapC-DA (SEQ ID NO. 174), respectively.

[0202] Using the genome of Bacteroides delbrueckii as a template and RuBisCO-S (SEQ ID NO. 175) and RuBisCO-A (SEQID NO. 176) as primers, the Ptrc-RuBisCO target gene fragment was amplified by HS enzyme PCR.

[0203] Among them, the Ptrc promoter was designed in the antisense primer of the upstream homologous arm of the pseudogene gapC and the positive primer of the RuBisCO gene; the Ptrc terminator was designed in the antisense primer of the RuBisCO gene and the positive primer of the downstream homologous arm of the pseudogene gapC.

[0204] Then, using the upstream homology arm, downstream homology arm and Ptrc-RuBisCO target gene fragment of the above-mentioned gapC pseudogene as templates, HS enzyme was used to obtain the gapC::Ptrc-RuBisCO gene integration fragment by overlapping PCR. The gene integration fragment consists of the gapC upstream homology arm, the Ptrc-RuBisCO target gene and the gapC downstream homology arm.

[0205] Using pGRB-gapC-S (SEQ ID NO. 177) and pGRB-gapC-A (SEQ ID NO. 178) as primers, a DNA fragment containing the gapC target sequence was constructed through a PCR annealing program. After recombining with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-gapC from them.

[0206] The gapC::Ptrc-RuBisCO gene integration fragment obtained in the above step and the pGRB-gapC plasmid were electroporated into the E. coli DPAH4-1 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH4-2. Embodiment 20

[0207] The prk gene is expressed at the ylbE pseudogene locus as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the ylbE pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers ylbE-US (SEQ ID NO.179), ylbE-UA (SEQ ID NO.180), ylbE-DS (SEQ ID NO.181), and ylbE-DA (SEQ ID NO.182).

[0208] Using the genome of Synechococcus elongatus as a template and prk-S (SEQ ID NO. 183) and prk-A (SEQ ID NO. 184) as primers, the prk target gene fragment was amplified by HS enzyme PCR.

[0209] Then, using the upstream homology arm, downstream homology arm and prk target gene fragment of the above-mentioned ylbE pseudogene as templates, HS enzyme was used to obtain the ylbE::prk gene integration fragment by overlapping PCR. The gene integration fragment consisted of the ylbE upstream homology arm, the prk target gene and the ylbE downstream homology arm.

[0210] Using pGRB-ylbE-S (SEQ ID NO. 185) and pGRB-ylbE-A (SEQ ID NO. 186) as primers, a DNA fragment containing the ylbE target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-ylbE.

[0211] The ylbE::prk gene integration fragment obtained in the above steps and the pGRB-ylbE plasmid were electroporated into the E. coli DPAH4-2 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH4-3. Embodiment 21

[0212] The Ptrc promoter was used to control the overexpression of the GroEL-GroES gene at the yjiP pseudogene site. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the yjiP pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yjiP-US (SEQ ID NO. 187), yjiP-UA (SEQ ID NO. 188), yjiP-DS (SEQ ID NO. 189), and yjiP-DA (SEQ ID NO. 190) of the yjiP pseudogene.

[0213] Using the E. coli W3110 genome as a template and GroEL-GroES-S (SEQ ID NO. 191) and GroEL-GroES-A (SEQ ID NO. 192) as primers, the Ptrc-GroEL-GroE target gene fragment was amplified by HS enzyme PCR.

[0214] Among them, the Ptrc promoter was designed in the antisense chain primer of the upstream homologous arm of the pseudogene yjiP and the positive chain primer of the GroEL-GroES gene; the Ptrc terminator was designed in the antisense chain primer of the GroEL-GroES gene and the positive chain primer of the downstream homologous arm of the pseudogene yjiP.

[0215] Then, using the upstream homologous arm, downstream homologous arm and Ptrc-GroEL-GroE target gene fragment of the above-mentioned yjiP pseudogene as templates, HS enzyme was used to obtain the yjiP::Ptrc-GroEL-GroE gene integration fragment by overlapping PCR. The gene integration fragment consists of the yjiP upstream homologous arm, the Ptrc-GroEL-GroES target gene and the yjiP downstream homologous arm.

[0216] Using pGRB-yjiP-S (SEQ ID NO. 193) and pGRB-yjiP-A (SEQ ID NO. 194) as primers, a DNA fragment containing the yjiP target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-yjiP.

[0217] The yjiP::Ptrc-GroEL-GroES gene integration fragment obtained in the above steps and the pGRB-yjiP plasmid were electroporated into the E. coli DPAH4-3 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH4-4. Embodiment 22

[0218] The Ptrc promoter was used to control the overexpression of the pck gene at the yeeP pseudogene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the yeeP pseudogene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers yeeP-US (SEQ ID NO. 195), yeeP-UA (SEQ ID NO. 196), yeeP-DS (SEQ ID NO. 197), and yeeP-DA (SEQ ID NO. 198) of the yeeP pseudogene.

[0219] Using the E. coli W3110 genome as a template and pck-S (SEQ ID NO. 199) and pck-A (SEQ ID NO. 200) as primers, the Ptrc-pck target gene fragment was amplified by HS enzyme PCR.

[0220] Among them, the Ptrc promoter was designed in the antisense strand primer of the upstream homologous arm of the pseudogene yeeP and the positive strand primer of the pck gene; the Ptrc terminator was designed in the antisense strand primer of the pck gene and the positive strand primer of the downstream homologous arm of the pseudogene yeeP.

[0221] Then, using the upstream homologous arm, downstream homologous arm and Ptrc-pck target gene fragment of the above-mentioned yeeP pseudogene as templates, HS enzyme was used to obtain the yeeP::Ptrc-pck gene integration fragment by overlapping PCR. The gene integration fragment consists of the yeeP upstream homologous arm, the Ptrc-pck target gene and the yeeP downstream homologous arm.

[0222] Using pGRB-yeeP-S (SEQ ID NO. 201) and pGRB-yeeP-A (SEQ ID NO. 202) as primers, a DNA fragment containing the yeeP target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain the plasmid pGRB-yeeP.

[0223] The yeeP::Ptrc-pck gene integration fragment obtained in the above steps and the pGRB-yeeP plasmid were electroporated into the E. coli DPAH4-4 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH5. Embodiment 23

[0224] The Ptrc promoter was used to control the overexpression of the csgD gene at the purR gene locus. The specific steps are as follows: Using the E. coli W3110 genome as a template, the upstream and downstream homology arms of the purR gene were amplified by HS enzyme PCR using the upstream and downstream homology arm primers purR-US (SEQ ID NO. 203), purR-UA (SEQ ID NO. 204), purR-DS (SEQ ID NO. 205), and purR-DA (SEQ ID NO. 206).

[0225] Using the E. coli W3110 genome as a template and csgD-S (SEQ ID NO. 207) and csgD-A (SEQ ID NO. 208) as primers, the Ptrc-csgD target gene fragment was amplified by HS enzyme PCR.

[0226] Among them, the Ptrc promoter was designed in the antisense strand primer of the upstream homology arm of purR and the positive strand primer of the csgD gene; the Ptrc terminator was designed in the antisense strand primer of the csgD gene and the positive strand primer of the downstream homology arm of purR.

[0227] Then, using the upstream homology arm, downstream homology arm and Ptrc-csgD target gene fragment of the above-mentioned purR gene as templates, HS enzyme was used to obtain the purR::Ptrc-csgD gene integration fragment by overlapping PCR. The gene integration fragment consists of the purR upstream homology arm, the Ptrc-csgD target gene and the purR downstream homology arm.

[0228] Using pGRB-purR-S (SEQ ID NO. 209) and pGRB-purR-A (SEQ ID NO. 210) as primers, a DNA fragment containing the purR target sequence was constructed through a PCR annealing program. After recombination with the linearized pGRB vector, it was transformed into E. coli DH5α competent cells, and positive transformants were screened to obtain plasmid pGRB-purR.

[0229] The purR::Ptrc-csgD gene integration fragment obtained in the above steps and the pGRB-purR plasmid were electroporated into the E. coli DPAH5 strain. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH6. Embodiment 24

[0230] The strain E. coli DPAH6 obtained in Example 23 above was used to produce D-pantothenic acid. The specific production method is as follows: S1. Seed activation: The E. coli DPAH6 strain was streaked and inoculated on a non-resistant activation slant, cultured at 37°C for 10 hours, and subcultured twice to obtain the activated strain. The activated bacteria on the slant were washed with sterilized distilled water and transferred to a 5L mechanically stirred fermenter for seed culture.

[0231] S2. Seed culture: The culture temperature was 37°C, and the culture pH was maintained at 6.8 by automatically adding 25% ammonia solution. The culture dissolved oxygen value was maintained at 25% by adjusting the stirring speed or ventilation volume. When the OD of the seed solution 600 The vaccination requirements are met at 15:00.

[0232] S3. Fermentation culture: leave 20% of the seed liquid in the fermenter, and then use a peristaltic pump to pump the fermentation medium into the tank. The culture temperature is 37°C, and the pH is controlled at 6.8 by automatically adding 25% ammonia solution. The dissolved oxygen value is maintained at 25% by adjusting the stirring speed or ventilation volume. The glucose concentration in the fermentation liquid in the tank is controlled to ≤0.5g / L by adding 80% glucose solution by mass. Ferment for 68h to obtain a fermentation liquid containing D-pantothenic acid. Embodiment 25

[0233] The strain E. coli DPAH6 obtained in Example 23 above was used to produce D-pantothenic acid. The specific production method is as follows: S1. Seed activation: The E. coli DPAH6 strain was streaked and inoculated on a non-resistant activation slant, cultured at 30°C for 14 hours, and subcultured once to obtain the activated strain. The activated bacteria on the slant were washed with sterilized distilled water and transferred to a 5L mechanically stirred fermenter for seed culture.

[0234] S2. Seed culture: The culture temperature was 30°C, and the culture pH was maintained at 7.1 by automatically adding 25% ammonia solution. The culture dissolved oxygen value was maintained at 35% by adjusting the stirring speed or ventilation volume. When the OD of the seed solution 600 The vaccination requirements were met at 13:00.

[0235] S3. Fermentation culture: leave 15% of seed liquid in the fermentation tank, and then use a peristaltic pump to pump the fermentation medium into the tank. The culture temperature is 30°C, and the pH is controlled at 7.1 by automatically adding 25% ammonia solution. The dissolved oxygen value is maintained at 35% by adjusting the stirring speed or ventilation volume. The glucose concentration in the fermentation liquid in the tank is controlled to ≤0.5g / L by adding 70% glucose solution by mass. Ferment for 72 hours to obtain a fermentation liquid containing D-pantothenic acid. Embodiment 26

[0236] The strain E. coli DPAH6 obtained in Example 23 above was used to produce D-pantothenic acid. The specific production method is as follows: S1. Seed activation: The E. coli DPAH6 strain was streaked and inoculated on a non-resistant activation slant, cultured at 35°C for 12 hours, and subcultured twice to obtain the activated strain. The activated bacteria on the slant were washed with sterilized distilled water and transferred to a 5L mechanically stirred fermenter for seed culture.

[0237] S2. Seed culture: The culture temperature is 35°C, and the culture pH is maintained at 7 by automatically adding 25% ammonia solution. The culture dissolved oxygen value is maintained at 30% by adjusting the stirring speed or ventilation volume. When the OD of the seed solution is 600 The vaccination requirements must be met by 18:00.

[0238] S3. Fermentation culture: leave 20% of the seed liquid in the fermenter, then pump the fermentation medium into the tank through a peristaltic pump, the culture temperature is 35°C, the pH is maintained at 7 by automatically adding 25% ammonia solution, the dissolved oxygen value is maintained at 30% by adjusting the stirring speed or ventilation volume, and the glucose concentration in the fermentation liquid in the tank is controlled to ≤0.5g / L by adding 80% glucose solution by mass. Ferment for 70 hours to obtain a fermentation liquid containing D-pantothenic acid.

[0239] In the above Examples 24 to 26, the seed culture medium used in the seed culture was: 30 g / L glucose, 5 g / L yeast, 3 g / L peptone, (NH 4 ) 2 SO 4 1g / L, KH 2 PO 4 2g / L, MgSO 4 7H 2 O1g / L, the rest is water.

[0240] The fermentation medium used in the fermentation culture was: glucose 20 g / L, yeast powder 2 g / L, NaHCO 3 1.68 g / L, (NH 4 ) 2 SO 4 16g / L, KH 2 PO 4 4g / L, MgSO 4 0.5g / L, anhydrous betaine 3g / L, MnSO 4 ·H 2 O 10mg / L, FeSO 4 7H 2 O 20mg / L, CoCl 2 10g / L, ZnSO 4 7H 2 O 1.25g / L, CuSO 40.25 g / L, NiCl 2 7H 2 O0.02g / L, β-alanine 3g / L, trace elements VB1, VB3, VB5, VB12, VH are all 2mg / L, and the rest is water. Comparative Example 1

[0241] Using E. coli W3110 as the starting strain, the engineered strain was prepared according to the methods of Examples 1 to 11 and 13 to 23, i.e., the step of Example 12 was not performed to overexpress the panF gene at the coaA gene locus, and in Example 13, yghE::PcoaA R106A The gene integration fragment and the pGRB-yghE plasmid were electroporated into the strain E. coli DPAH1-11 obtained in Example 11. After screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH2′. The strain E. coli DPAH2′ was constructed into an engineered strain according to the methods of Examples 14 to 23, and the engineered strain was used to produce D-pantothenic acid according to the method of Example 24. Comparative Example 2

[0242] Using E. coli W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 18 and 20 to 23, i.e., the step of Example 19 to overexpress the RuBisCO gene at the gapC pseudogene site was not performed. In Example 20, the ylbE::prk gene integration fragment and the pGRB-ylbE plasmid were electroporated into the strain E. coli DPAH4-1 obtained in Example 18, and after screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH4-3′. The strain E. coli DPAH4-3′ was further constructed into an engineered strain according to the methods of Examples 21 to 23, and the engineered strain was used to produce D-pantothenic acid according to the method of Example 24. Comparative Example 3

[0243] Using E. coli W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 19 and 21 to 23, i.e., the step of Example 20 was not performed to express the prk gene at the ylbE pseudogene site. In Example 21, the yjiP::Ptrc-GroEL-GroES gene integration fragment and the pGRB-yjiP plasmid were electroporated into the strain E. coli DPAH4-2 obtained in Example 19, and after screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH4-4′. The strain E. coli DPAH4-4′ was further constructed into an engineered strain according to the methods of Examples 22 to 23, and the engineered strain was used to produce D-pantothenic acid according to the method of Example 24. Comparative Example 4

[0244] Using E. coli W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 20, 22 to 23, that is, the step of Example 21 to overexpress the GroEL-GroES gene at the yjiP pseudogene site was not performed, and in Example 22, the yeeP::Ptrc-pck gene integration fragment and the pGRB-yeeP plasmid were electroporated into the E. coli DPAH4-3 obtained in Example 20, and after screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH5′, and the strain E. coli DPAH5′ was further constructed into an engineered strain according to the method of Example 23, and the engineered strain was used to produce D-pantothenic acid according to the method of Example 24. Comparative Example 5

[0245] Using E. coli W3110 as the starting strain, an engineered strain was prepared according to the methods of Examples 1 to 15 and 17 to 23, i.e., the step of Example 16 to knock out the pflD gene was not performed. In Example 17, the poxB gene integration fragment and the pGRB-poxB plasmid were electroporated into the E. coli DPAH3-1 strain obtained in Example 15, and after screening to obtain positive transformants, the two plasmids used for gene editing were eliminated to obtain the strain E. coli DPAH4′. The strain E. coli DPAH4′ was further constructed into an engineered strain according to the methods of Examples 18 to 23, and the engineered strain was used to produce D-pantothenic acid according to the method of Example 24. Test Case

[0246] The yield of D-pantothenic acid in the fermentation broth after fermentation in Examples 24 to 26 and Comparative Examples 1 to 5 was detected, and the sugar-acid conversion rate (i.e., the conversion rate of glucose to D-pantothenic acid) was calculated. The results are shown in Table 1.

[0247] Table 1

[0248] From the above results, it can be seen that compared with the engineered strains in Examples 24 to 26, after the panF gene was not overexpressed at the coaA gene site in Comparative Example 1, the yield of D-pantothenic acid and the sugar-acid conversion rate both decreased, indicating that not expressing the coaA gene and overexpressing the panF gene in the engineered strain can increase the yield of D-pantothenic acid and the sugar-acid conversion rate.

[0249] After the comparative example 2 did not overexpress the RuBisCO gene, the comparative example 3 did not express the prk gene, and the comparative example 4 did not overexpress the GroEL-GroES gene, the yield of D-pantothenic acid and the sugar-acid conversion rate in the fermentation broth decreased, indicating that overexpressing the RuBisCO gene, expressing the prk gene or overexpressing the GroEL-GroES gene in the engineered strain can increase the yield of D-pantothenic acid and the sugar-acid conversion rate in the fermentation broth.

[0250] In comparative example 5, after the pflD gene was not knocked out, the yield of D-pantothenic acid and the sugar-acid conversion rate in the fermentation broth both decreased, indicating that knocking out the pflD gene in the engineered strain to prevent the gene from being expressed can further increase the yield of D-pantothenic acid and the sugar-acid conversion rate in the fermentation broth.

[0251] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An engineered strain for efficiently producing D-pantothenic acid, characterized in that: The engineered strain overexpresses the following genes: Acetolactate synthase gene alsS*, ketoacid reductoisomerase gene ilvC V412A , dihydroxyacid dehydratase gene ilvD, overexpression of methyltransferase gene panB Y25A , anhydropantothenate reductase gene panE, aspartate aminotransferase gene aspC, aspartate decarboxylase gene panD, pantothenate synthase gene panC, pantothenate transporter gene panF, ribulose-1,5-bisphosphate carboxylase / oxidase gene RuBisCO, molecular chaperone protein genes GroEL-GroES; The engineered strain expresses the pantothenate kinase gene coaA R106A , phosphoribosylase gene prk; The engineered strain has the following genes missing from its genome: Repressor protein gene lacI, threonine dehydratase gene ilvA, pantothenate kinase gene coaA, formate acetyltransferase gene pflD.

2. The engineered strain for efficiently producing D-pantothenic acid according to claim 1, characterized in that: The nucleotide sequence of the acetolactate synthase gene alsS* is shown in SEQ ID NO.1; The ketoacid reductoisomerase gene ilvC V412A The nucleotide sequence is shown in SEQ ID NO. 2; The nucleotide sequence of the dihydroxyacid dehydratase gene ilvD is shown in SEQ ID NO. 3; The overexpressed methyltransferase gene panB Y25A The nucleotide sequence is shown in SEQ ID NO. 4; The nucleotide sequence of the dehydropantothenate reductase gene panE is shown in SEQ ID NO.5; The nucleotide sequence of the aspartate aminotransferase gene aspC is shown in SEQ ID NO.6; The nucleotide sequence of the aspartate decarboxylase gene panD is shown in SEQ ID NO.7; The nucleotide sequence of the pantothenate synthase gene panC is shown in SEQ ID NO.8; The nucleotide sequence of the pantothenate transporter gene panF is shown in SEQ ID NO.9; The pantothenate kinase gene coaA R106A The nucleotide sequence is shown in SEQ ID NO. 10; The nucleotide sequence of the ribulose-1,5-bisphosphate carboxylase / oxidase gene RuBisCO is shown in SEQ ID NO. 11; The nucleotide sequence of the phosphoribosylase gene prk is shown in SEQ ID NO. 12; The nucleotide sequence of the molecular chaperone protein gene GroEL-GroES is shown in SEQ ID NO. 13; The nucleotide sequence of the repressor protein gene lacI is shown in SEQ ID NO. 14; The nucleotide sequence of the threonine dehydratase gene ilvA is shown in SEQ ID NO. 15; The nucleotide sequence of the pantothenate kinase gene coaA is shown in SEQ ID NO. 16; The nucleotide sequence of the formate acetyltransferase gene pflD is shown in SEQ ID NO.

17.

3. The engineered strain for efficiently producing D-pantothenic acid according to claim 1, characterized in that: The acetolactate synthase gene alsS*, the ketoacid reductoisomerase gene ilvC V412A , dihydroxyacid dehydratase gene ilvD, overexpression of methyltransferase gene panB Y25A , anhydropantothenate reductase gene panE, aspartate aminotransferase gene aspC, aspartate decarboxylase gene panD, pantothenate synthase gene panC, pantothenate transporter gene panF, ribulose-1,5-bisphosphate carboxylase / oxidase gene RuBisCO, and molecular chaperone protein genes GroEL-GroES are all controlled by the Ptrc promoter; The phosphoribosylase gene prk is controlled by a Plac promoter or has no promoter control.

4. The engineered strain for efficiently producing D-pantothenic acid according to claim 1, characterized in that: The alsS* gene is located at the lacI gene site and the yqaC pseudogene site; The ilvC V412A The gene is located at the yciQ pseudogene locus; The ilvD gene is located at the mbhA pseudogene site; The panB Y25A The gene is located at the ygaY pseudogene locus; The panE gene is located at the ycdN pseudogene site; The aspC gene is located at the yncI pseudogene site; The panD gene is located at the yjiT pseudogene site; The panC gene is located at the ilvA gene site; The panF gene is located at the coaA gene locus; The coaA R106A The gene is located at the yghE pseudogene locus; The RuBisCO gene is located at the gapC pseudogene site; The prk gene is located at the ylbE pseudogene locus; The GroEL-GroES gene is located at the yjiP pseudogene site.

5. A method for constructing an engineered strain for efficiently producing D-pantothenic acid, for constructing an engineered strain for efficiently producing D-pantothenic acid according to any one of claims 1 to 4, characterized in that: The following steps are involved: The Ptrc promoter was used to control the overexpression of the alsS* gene at the lacI gene locus; The Ptrc promoter was used to control the overexpression of the alsS* gene at the yqaC pseudogene locus; Control of ilvC by the Ptrc promoter at the yciQ pseudogene locus V412A Gene overexpression; The Ptrc promoter was used to control the overexpression of the ilvD gene at the mbhA pseudogene locus; Control of panB by the Ptrc promoter at the ygaY pseudogene locus Y25A Heterologous overexpression of genes; The Ptrc promoter was used to control the overexpression of the panE gene at the ycdN pseudogene locus; The Ptrc promoter was used to control the overexpression of the aspC gene at the yncI pseudogene site; The Ptrc promoter was used to control the overexpression of the panD gene at the yjiT pseudogene locus; The Ptrc promoter was used to control the overexpression of the panC gene at the ilvA gene locus; The Ptrc promoter was used to control the overexpression of the panF gene at the coaA gene locus; Expression of coaA at the yghE pseudogene locus R106A Gene; Knockout of the pflD gene; The Ptrc promoter was used to control the overexpression of the RuBisCO gene at the gapC pseudogene locus; prk gene is expressed at the ylbE pseudogene locus; GroEL-GroES gene overexpression was controlled by Ptrc promoter at the yjiP pseudogene locus.

6. The construction method according to claim 5, characterized in that: The nucleotide sequence of the Ptrc promoter is shown in SEQ ID NO. 29; the nucleotide sequence of the Plac promoter is shown in SEQ ID NO.

30.

7. The construction method according to claim 5, characterized in that: The starting strain used in the construction of the engineering strain is Escherichia coli; the Escherichia coli is E. coli W3110.

8. Use of the engineered strain according to any one of claims 1 to 4 in producing D-pantothenic acid.

9. The use according to claim 8, characterized in that: The production of D-pantothenic acid comprises the following steps: activating the engineered strain according to any one of claims 1 to 4 and then performing seed culture to obtain a seed solution; The obtained seed liquid is inoculated into the fermentation medium at an inoculation rate of 15% to 20% for fermentation culture. The culture temperature is 30 to 37°C, the pH is 6.8 to 7.1, the dissolved oxygen value is 25 to 35%, and a glucose solution with a mass fraction of 70% to 80% is added. The concentration of glucose in the fermentation liquid is controlled at ≤0.5g / L. After the fermentation culture is completed, D-pantothenic acid is obtained.

10. The use according to claim 9, characterized in that: The engineering strain activation comprises: taking the engineering strain according to any one of claims 1 to 4 and inoculating it on an activation slant, culturing it at 30 to 37° C. for 10 to 14 hours, and subculturing it 1 to 2 times to obtain an activated engineering strain; The seed culture is as follows: the activated engineering strain is cultured at a temperature of 30-37°C, a pH of 6.8-7.1, and a dissolved oxygen value of 25-35% until the OD of the seed solution reaches 0. 600 Reach 13~18.

Images