Genetically engineered bacterium with high yield of d-pantothenic acid based on one-carbon unit supply, construction method therefor and use thereof

Abstract

Provided are a genetically engineered bacterium with a high yield of D-pantothenic acid based on one-carbon unit supply, a construction method therefor, and the use of said genetically engineered bacterium in the preparation of D-pantothenic acid by microbial fermentation. By enhancing a glycine cleavage system, the supply of a one-carbon cofactor in Escherichia coli organisms is enhanced, so that the carbon flux flows to the synthesis of D-pantothenic acid. In order to continuously enhance the carbon flux, genes related to a THF synthesis pathway are enhanced to regulate a serine synthesis pathway; and by combining the enhancement of the glycine cleavage system and the expression of key genes of a pantoic acid synthesis pathway, a plasmid-free and antibiotic-free engineered bacterial strain used for high-yield production of D-pantothenic acid is finally obtained. Finally, the titer of D-pantothenic acid in shake flask culture is increased by 31.7% relative to that of the original strain and reaches 6.85 g / L, and after fermentation in a 5 L fermentation tank for 92 hours, the D-pantothenic acid yield can reach 112.62 g / L.

Description

High-yield D-pantothenic acid genetic engineering bacteria based on one-carbon unit supply, construction method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to high-yield D-pantothenic acid genetic engineering bacteria based on one-carbon unit supply, a construction method thereof, and application of the high-yield D-pantothenic acid genetic engineering bacteria in microbial fermentation for preparing D-pantothenic acid. BACKGROUND

[0002] Pantothenic acid, also known as vitamin B5, is one of the components of coenzyme A and plays a key role in important biochemical reactions such as energy metabolism and citric acid cycle. Therefore, as an important vitamin and precursor, D-pantothenic acid is widely used in feed, medicine, cosmetics and other aspects. Among the known D-pantothenic acid synthesis methods, the biological fermentation method for producing D-pantothenic acid has the advantages of cheap substrate, easy separation and low toxicity, and thus attracts attention. However, there are still defects in the production of D-pantothenic acid by biological method at present, such as unstable fermentation process and low yield. Therefore, it is still a great challenge to construct a strain with higher yield of D-pantothenic acid.

[0003] SUMMARY

[0004] In order to solve the technical problem of low yield of D-pantothenic acid produced by microbial fermentation in the prior art, the present application uses CRISPR-Cas9 mediated gene editing technology to strengthen the glycine cleavage system and the folic acid synthesis pathway in the genome of the chassis bacteria, regulate the serine pathway and strengthen the expression of key genes in the pantothenic acid synthesis pathway, to obtain a high-yield D-pantothenic acid genetic engineering bacteria, and apply the genetic engineering bacteria to the microbial fermentation for preparing D-pantothenic acid.

[0005] The technical scheme adopted by the present application is a construction method of high-yield D-pantothenic acid genetic engineering bacteria based on one-carbon unit supply, which comprises: using CRISPR-Cas9 mediated gene editing technology to overexpress gcvTHP gene, strengthen folP gene, integrate serA and glyA gene clusters and increase one copy of panB gene after serAglyA gene cluster in the genome of the chassis bacteria, wherein the serAglyA gene cluster is regulated by a dynamic regulatory element, and increase heterologous codon-optimized alsS gene, to obtain the high-yield D-pantothenic acid genetic engineering bacteria.

[0006] The present application is based on one-carbon unit supply, and the chassis bacteria ZJUTDPAP2 (E. coli W3110, Trc-EcilvD* / BspanBA* / CgpanC* / alsS* / BspanB* / nac GTG / ΔP 345ptsH / gltA GTG / gltA TTGOn the basis of / Trc-PpfkB, which has been disclosed in CN116590209A, the gcvTHP gene in-situ promoter is replaced with Ptrc promoter by using CRISPR / Cas9 gene editing technology, so as to eliminate the transcriptional inhibition of gcvA and purR genes on the gcvTHP gene, strengthen the glycine cleavage system, and enhance the supply of one-carbon cofactor in the E. coli organism, so as to further make the carbon flux flow to the synthesis of D-pantothenic acid. On this basis, in order to further enhance the carbon flux to the synthesis of D-pantothenic acid, the present application also strengthens the genes related to the THF synthesis pathway. On the one hand, a molecule of folP gene copy is added at the lafU pseudogene site to enhance the synthesis of folate and provide methyl carriers for one-carbon units. On the other hand, the Apt2#82 glycine ribosome switch is used to control the expression of the serAglyA gene cluster in real time according to the intracellular glycine concentration, so as to realize the moderate strengthening of the key genes of the serine pathway, so as to reasonably regulate the synthesis rate of serine. At the same time, in response to the supply of methyl, a P panB The promoter-regulated panB gene copy number. Finally, the expression efficiency of the genetically engineered bacteria is improved by introducing a heterologous codon-optimized alsS, and the yield of D-pantothenic acid is increased, and finally a plasmid-free and antibiotic-free engineering strain for high-yield D-pantothenic acid is obtained.

[0007] Table 1. Genes involved in gene editing and corresponding pathways

[0008] As a preferred, the method for overexpressing the gcvTHP gene comprises: replacing the in-situ promoter of the gcvTHP gene with a Ptrc promoter. By activating the glycine cleavage system through a strong promoter, the influence of gcvA and purR on the supply of one-carbon units can be eliminated, and 5,10-methylenetetrahydrofolate can be provided for the D-pantothenic acid pathway, so as to effectively increase the synthesis of D-pantothenic acid.

[0009] As a preferred, the method for strengthening the folP gene comprises: adding a molecule of folP gene copy at the lafU pseudogene site. Overexpression of the folP gene can further promote the combination of pABA and GTP pathway, which is an effective strategy to strengthen the synthesis of folate to provide methyl carriers for one-carbon units.

[0010] As preferred, the dynamic regulatory element is ribosome switch Apt2#82. Ribosome switch Apt2#82 can control serA and glyA overexpression according to intracellular glycine concentration. When glycine accumulates to a certain extent, it will fold and cover the ribosome site, repressing the translation of the gene cluster serAglyA; when the intracellular content of glycine is less, it will expose the ribosome site to strengthen the translation. Using ribosome switch to control the expression of glyA in real time can achieve moderate strengthening of the key genes of the serine pathway. This strategy can reasonably regulate the synthesis rate of serine, maintain the intracellular glycine at a low level, and effectively promote the synthesis of D-pantothenate.

[0011] As preferred, the panB gene is regulated by PpanB promoter. Adding a copy of panB gene after the serAglyA gene cluster can better respond to the supply of methyl and promote the synthesis of D-pantothenate.

[0012] As preferred, the alsS gene is derived from Bacillus subtilis and is codon-optimized. Acetolactate synthase (alsS) catalyzes the formation of α-acetolactate from two molecules of pyruvate, and is involved in the synthesis of valine, leucine and isoleucine. By introducing a heterologous alsS gene into the genome and codon-optimizing it, the expression efficiency of the gene in the host can be improved, and the strain burden is small. Without adding pyruvate, the D-pantothenate yield in the shake flask can be improved.

[0013] As preferred, the chassis strain is: E. coli W3110, Trc-EcilvD* / BspanBA* / CgpanC* / alsS* / BspanB* / nac GTG / ΔP 345ptsH / gltA GTG / gltA TTG / Trc-PpfkB, denoted as DPAH2.

[0014] As preferred, the nucleotide sequences of the gcvTHP gene, folP gene and alsS gene regulated by Ptrc promoter are shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively; the sequence of the serAglyA gene cluster regulated by Apt2#82 ribosome switch is shown in SEQ ID NO. 4; and the nucleotide sequence of the panB gene regulated by PpanB promoter is shown in SEQ ID NO. 5.

[0015] More specifically, the method for constructing the genetically engineered strain for producing D-pantothenate includes the following steps:

[0016] (1) Using engineered bacterium DPAP2 as the starting strain and naming it H2, CRISPR-Cas9-mediated gene editing technology was used to replace the in situ promoter of gcvTHP gene in its genome with the Trc promoter and delete the transcriptional regulatory region to obtain engineered bacterium DPAH3derivative, Trc-gcvTHP, denoted as engineered bacterium DPAH3.

[0017] (2) Using engineered strain DPAH3 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to add a copy of the folP gene at the lafU pseudogene site to obtain engineered strain DPAH6derivative, lafU::folP, which is denoted as engineered strain DPAH6.

[0018] (3) Using engineered strain DPAH6 as the starting strain, CRISPR / Cas9 gene editing technology was used to integrate the serA and glyA gene clusters into the genome, and a copy of the panB gene was added after the gene clusters to obtain engineered strain DPAH7derivative, yeeP::Trc-Apt2#82-serAglyA-P panB -panB, denoted as engineered bacteria DPAH7;

[0019] (4) Using engineered strain DPAH7 as the starting strain, CRISPR / Cas9 gene editing technology was used to increase the copy number of the Bacillus subtilis alsS gene regulated by promoter pTrc in the genome by gene knock-in, and codon optimization was performed to obtain engineered strain DPAH8derivative, gapC::Trc-alsS*, denoted as engineered strain DPAH8, which is the genetically engineered strain that produces high levels of D-pantothenic acid.

[0020] This invention also provides a genetically engineered bacterium that produces high levels of D-pantothenic acid, constructed using any of the methods described above. Using the methods described above, this invention constructed an engineered bacterium, *E. coli* W3110,Trc-EcilvD* / BspanBA* / CgpanC* / alsS* / BspanB* / nac, which was free of plasmids and antibiotics during fermentation. GTG / ΔP 345ptsH / gltA GTG / gltA TTG / Trc-PpfkB / Trc-gcvTHP / lafU::folP / yeeP::Trc-Apt2#82-serAglyA-P panB-panB / gapC::Trc-alsS*, the shake flask titer of which reaches 6.85 g / L, which is increased by 31.7% compared with the starting strain, and the D-pantothenic acid yield can reach 112.6 g / L after 5-L fermenter fed-batch fermentation for 92 hours, which greatly improves the synthesis of D-pantothenic acid and shortens the fermentation cycle.

[0021] The application further provides application of the genetically engineered strain with high D-pantothenic acid yield in microbial fermentation for preparing D-pantothenic acid.

[0022] As preferred, the application method comprises inoculating the genetically engineered strain with high D-pantothenic acid yield into a fermentation medium, and performing fed-batch fermentation culture at 28-37°C, 300-450 rpm, pH 6.7-6.9, and 10%-30% dissolved oxygen for 72-96 hours, and then separating and purifying the D-pantothenic acid after the fermentation.

[0023] As preferred, the fermentation medium comprises 10-30 g / L glucose, 10-25 g / L ammonium sulfate, 1-5 g / L anhydrous betaine, 1-5 g / L yeast powder, 1-5 g / L potassium dihydrogen phosphate, 0.5-2 g / L anhydrous magnesium sulfate, 1-5 g / L beta-alanine, 1-5 ml / L trace element solution, and deionized water as a solvent, and the pH value is natural; the trace element solution comprises 10 g / L CuCl2, 10 g / L FeSO4·7H2O, 10 g / L ZnSO4·7H2O, 0.2 g / L CuSO4, and 0.02 g / L NiCl2·7H2O, and deionized water as a solvent.

[0024] As preferred, the application method comprises loading 1-3 L fermentation medium into a 5-L fermenter, sterilizing at 115°C for 30 min, inoculating the genetically engineered strain into the 1-3 L fermentation medium, and performing fermentation culture at 28-37°C, 3-6 L / min initial aeration, and 300-450 rpm initial stirring speed, and adjusting the pH value with ammonia water, and adding IPTG with a final concentration of 0.1-0.4 mM, VB1 with a final concentration of 5 mg / L, and VB 12and 10-40 g / L isoleucine 5-10 mL; the dissolved oxygen is maintained at 10%-30% by using dissolved oxygen series rotation speed during the fermentation process, ammonia is used as a neutralizing agent to maintain pH 6.7-6.9, the feed medium is added into the tank by pH linkage feeding, the glucose concentration is controlled below 5 g / L, and the fermentation is carried out at 28-37°C for 72-96 hours to obtain the fermentation liquor, and the D-pantothenic acid is obtained by separating and purifying the fermentation liquor supernatant. The composition of the feed medium is as follows: glucose 500 g / L, ammonium sulfate 5-25 g / L, anhydrous betaine 2-8 g / L, yeast powder 1-5 g / L, potassium dihydrogen phosphate 10-20 g / L, anhydrous magnesium sulfate 5-15 g / L, beta-alanine 40-100 g / L, 1-5 ml / L trace element solution, the solvent is deionized water, and the pH value is natural.

[0025] The beneficial effects of the present application are as follows: the CRISPR / Cas9 gene editing technology is used to strengthen the glycine cleavage system and the folic acid synthesis pathway on the basis of the existing engineering bacteria, to provide one-carbon units and rich methyl carriers for the pantothenic acid synthesis path. On the other hand, the Apt2#82 glycine ribosome switch is used to finely control the synthesis rate of serine and glycine, and the expression level of the key genes in the D-pantothenic acid biosynthesis pathway is further strengthened to continuously supply methyl for the whole reaction system. A high-yield strain without plasmid and without adding antibiotics in the fermentation process is obtained; the strain reaches a shaking flask titer of 6.85 g / L, which is increased by 31.7% compared with the starting strain, and the D-pantothenic acid yield can reach 112.6 g / L after 92 hours of fermentation in a 5L fermenter, which greatly improves the synthesis of D-pantothenic acid and shortens the fermentation period. BRIEF DESCRIPTION OF DRAWINGS

[0026] Fig. 1 is a D-pantothenic acid metabolic pathway diagram and modification site.

[0027] Fig. 2 is the OD600 and D-pantothenic acid titer change of DPAH3 in Example 1.

[0028] Fig. 3 is the OD600 and D-pantothenic acid titer change of DPAH6 in Example 2.

[0029] Fig. 4 is the OD600 and D-pantothenic acid titer change of DPAH7 in Example 3.

[0030] Fig. 5 is the OD600 and D-pantothenic acid titer change of DPAH8 in Example 4.

[0031] Fig. 6 is the fermentation result diagram of DPAH8 in a 5L bioreactor in Example 5. DETAILED DESCRIPTION

[0032] The present application is illustrated by way of example and not limitation in the following detailed description of the application in which advantages and benefits provided by the present application can be understood. Other advantages and benefits will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description of the application with reference made to the accompanying drawings and the appended claims. The application can be implemented in its embodiments in any manner, or with any details, which are not specifically disclosed in this specification, without departing from the spirit or essential characteristics thereof. It is to be expressly understood that the following examples and features thereof are included herein for illustration and description purposes only and are not intended to limit the scope of the application, which is set forth in the appended claims. In the following examples, the spectinomycin final concentration in the culture medium is 0.05 mg / L and the kanamycin final concentration in the culture medium is 0.05 mg / L.

[0033] In the following examples, the spectinomycin final concentration in the culture medium is 0.05 mg / L and the kanamycin final concentration in the culture medium is 0.05 mg / L.

[0034] The parent strain E. coli W3110 of the present application is from Coli Genetic Stock Center of Yale University, which was deposited on August 5, 1975, and has the accession number CGSC#4474. It has been disclosed in US2009 / 0298135A1 and US2010 / 0248311A1.

[0035] The HPLC method for determining the D-pantothenic acid content is as follows: Chromatographic conditions: C18 column (250 x 4.6 mm, particle size 5 μm, Agilent Technologies Co., Santa Clara, CA, USA), detection wavelength: 200 nm, column temperature: 30 °C; sample treatment: dilute the sample with ultrapure water to maintain the D-pantothenic acid content between 0.05 g / L and 0.40 g / L; mobile phase: acetonitrile / water / phosphoric acid: (50 / 949 / 1); data collection time: 25 min.

[0036] Example 1: Construction of DPAH3 and shake flask fermentation

[0037] DPAP2 (E. coli W3110, Trc-EcilvD* / BspanBA* / CgpanC* / alsS* / BspanB* / nac GTG / ΔP 345ptsH / gltA GTG / gltA TTG / Trc-PpfkB) as the starting strain, using CRISPR-Cas9 mediated gene editing technology to overexpress gcvTHP in the genome, and replace the glycine in situ promoter with the Trc promoter (nucleotide sequence as shown in SEQ ID NO. 1), so that the transcriptional regulator gcvA and purR cannot inhibit the expression of the glycine cleavage system, and obtain the strain DPAH3 (DPAH2 derivative, Trc-gcvTHP).

[0038] (1) Construct pTarget-PAM-gcvTHP plasmid: use pTarget plasmid (Addgene Plasmid #62226) as a template, PCR amplification with pT-gcvTHP-PAM-F / R primers, the PCR product is verified by nucleic acid gel electrophoresis, then use Dpn I digestion enzyme to digest at 37°C for 3h, then transform into E. coli DH5a, select by spectinomycin plate, and obtain the correct pTarget-PAM-gcvTHP plasmid by sequencing verification, which is used for subsequent connection of Donor DNA.

[0039] (2) Construct pTD-donor-EcgcvTHP plasmid: first, use E. coli W3110 genome as a template, and gcvTHP-up-F / R primers to amplify the upper part of the donor DNA (F1), and gcvTHP-down-F / R primers to amplify the lower part of the donor DNA (F2). Then, use E. coli W3110 genome as a template, and pTrc-EcgcvTHP-18F / R primers to amplify the EcgcvTHP gene fragment with the pTrc promoter (F3), and purify the PCR fragments F1, F2 and F3 by gel recovery; digest the plasmid pTarget-gcvTHP with Xba I and Pst I at 37°C for 8h, and recover the DNA fragments with Clean up kit; according to the instructions of One step clone kit (Vazyme Biotech, Nanjing, China), connect pTarget-gcvTHP vector, fragments F1, F2 and F3 together, and obtain pTD-donor-EcgcvTHP plasmid by sequencing verification. (One step clone kit, Vazyme Biotech, Nanjing, China) instructions to connect pTarget-gcvTHP vector, fragments F1, F2 and F3 together, and obtain pTD-donor-EcgcvTHP plasmid by sequencing verification.

[0040] (3) Introduce pCas plasmid (Addgene Plasmid #62225) into ZJUTDPAH2 (E. coli W3110, Trc-EcilvD* / BspanBA* / gpanC* / alsS* / BspanB* / nac GTG / ΔP 345ptsH / gltA GTG / gltA TTG / Trc-PpfkB), transfer the monoclonal colony to a test tube containing 0.05 mg / L kanamycin, and incubate at 30°C overnight; then inoculate into a 250 mL flask containing 50 mL LB medium with a 1% inoculation volume, and add 500 μl of 1 mol / L L-arabinose, and incubate at 150 rpm and 30°C until the OD 600 0.4-0.6; centrifuge at 4000 rpm and 4°C for 10 min to collect the cells, prepare the electrotransformation competent cells, and the detailed process is described in (Molecular Cloning: A Laboratory Manual, 3ed Edition, 99-102).

[0041] (4) Use a pipette to take an appropriate amount of pTD-donor-EcgcvTHP (about 200 ng) plasmid and mix with the previously prepared 100 μl electroshock competent cells, and transfer them into a pre-cooled 2 mm electroshock cup, and then ice bath for 1-2 min, and then use an electroporator (MicroPluser TM , BIO-RAD) to perform electroshock transformation, and immediately add 800 μl of LB medium after the electroshock is completed, and then immediately gently aspirate, transfer to a 2 mL Ep tube, and incubate at 30°C for 3-4 h, then plate on an LB solid plate containing 0.05 mg / L kanamycin and 0.05 mg / L spectinomycin, and incubate at 30°C for 12-16 h, and then perform colony PCR using gcvTHP-VF / R as the primer, and if a fragment of about 3500 bp can be successfully cloned, then it is a DPAH3 (DPAH2 derivative, Trc-gcvTHP) positive colony.

[0042] (5) Plasmid elimination: positive single colonies were inoculated into LB liquid tubes containing 1 mM IPTG and 0.05 mg / L kanamycin, and incubated at 30°C overnight. The next day, the bacterial solution was streaked on LB solid plates containing 0.05 mg / L kanamycin, and incubated at 30°C for 24 h. When the bacterial bodies reached a certain size, some single colonies were streaked on LB plates containing 0.05 mg / L spectinomycin. Single colonies that could not grow on LB plates containing 0.05 mg / L spectinomycin indicated that the pTarget-gcvTHP plasmid had been successfully eliminated. The single colonies in which the pTarget-gcvTHP plasmid had been successfully eliminated were then inoculated into LB tubes and incubated at 37°C overnight to eliminate the pCas plasmid. The next day, the bacterial solution was streaked on LB plates and incubated at 37°C for 12 h. Some single colonies were then streaked on LB plates containing 0.05 mg / L kanamycin. Single colonies that could not grow on LB plates containing 0.05 mg / L kanamycin indicated that the pCas plasmid had been successfully eliminated. Finally, the plasmid-free strain was obtained. The starting strain H2 was used as a control group and inoculated into 10 mL of LB medium at 37°C, 200 rpm for pre-culture. After 8-12 h, 1 mL of the pre-culture was inoculated into a 500 mL shake flask containing 50 mL of MS medium at a 2% inoculation amount, and then incubated at 30°C, 180 rpm on a constant temperature shaker for 48 h for strain fermentation. After fermentation, 1 mL of the fermentation broth was used to measure the OD 600 value, and 1 mL of the fermentation broth was taken using a pipette, centrifuged at 12,000 rpm at room temperature for 3 min. The fermentation supernatant was diluted 5-fold, and the diluted sample was treated with a water-based filter membrane to remove impurities. HPLC detection was then performed. 600 The D-pantoic acid content in the fermentation supernatant is shown in FIG. 2.

[0043] As can be seen from the figure, after increasing the copy number of gcvTHP genes on the genome, the D-pantoic acid yield of DPAH3 increased by 11.7% after 48 h of shake flask fermentation, reaching 5.81 g / L. By activating the glycine cleavage system with a strong promoter and eliminating the influence of gcvA and pur R on one-carbon unit supply, 5,10-methylenetetrahydrofolate was provided for the D-pantoic acid pathway, successfully increasing the D-pantoic acid yield.

[0044] LB medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, solvent is deionized water, pH value is natural.

[0045] MS medium: glucose 20 g / L, (NH4)2SO4 16 g / L, KH2PO4 2 g / L, MgSO4 0.5 g / L, yeast extract 2 g / L, CaCO3 10 g / L, 1 ml / L trace element solution, solvent is deionized water, pH value is natural; 10 g / L calcium carbonate (sterilized separately); the composition of the trace element solution is: 10 g / L CuCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.20 g / L CuSO4, 0.02 g / L NiCl2·7H2O, solvent is deionized water.

[0046] Example 2: DPAH6 construction and shake flask fermentation

[0047] Taking DPAH3 as the starting strain, using CRISPR-Cas9 mediated gene editing technology, a molecule of folP gene copy (nucleotide sequence as shown in SEQ ID NO. 2) was added at the lafU pseudogene site by gene knock-in to obtain DPAH6 (derivative, lafU::folP).

[0048] (1) Constructing pTarget-PAM-folP plasmid: taking pTarget F plasmid (Addgene Plasmid #62226) as a template, PCR amplification was performed with pT-folP-PAM--F / R as primers, and the PCR product was verified by nucleic acid gel electrophoresis. Then, Dpn I digestion enzyme was used for 3h incubation at 37℃, and then transformed into E. coli DH5a. After spectinomycin plate screening and sequencing verification, the correct pTarget-PAM-folP plasmid was obtained for subsequent connection of Donor DNA.

[0049] (2) Constructing pTD-Donor-EcfolP plasmid: first, taking E. coli W3110 genome as a template, folP-up-F / R primers were used to amplify the upper part of donor DNA (F1), and folP-down-F / R primers were used to amplify the lower part of donor DNA (F2). Then, taking E. coli W3110 genome as a template, pTrc-EcfolP-F / R primers were used to amplify the EcfolP gene fragment with the promoter pTrc (F3), and F1, F2 and F3 were obtained by gel recovery and purification of PCR fragments; the plasmid pTarget-folP was incubated at 37℃ for 8h, and the DNA fragment was recovered by Clean up kit; according to the manufacturer's instructions, the F1, F2 and F3 were ligated into the pTarget-PAM-folP plasmid, and the ligation product was transformed into E. coli DH5a. After spectinomycin plate screening and sequencing verification, the correct pTD-Donor-EcfolP plasmid was obtained for subsequent transformation. (One step clone kit, Vazyme Biotech, Nanjing, China) according to the instruction manual. The pTarget-ydeU vector, fragments F1, F2 and F3 were ligated together, and the pTD-EcfolP plasmid was obtained by sequencing verification.

[0050] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAH3 competent cells obtained in Example 1, and the DPAH3 electrocompetent cell preparation method was the same as that in Example 1 (3).

[0051] (4) The DPAH6 positive colonies were constructed, and the construction method was the same as that in Example 1 (4).

[0052] (5) Plasmid elimination: the implementation method was the same as that in Example 1 (5), and the plasmid-free DPAH6 (derivative, lafU::folP) was obtained.

[0053] (6) The constructed DPAH6 production strain was compared with the DPAH3 constructed in Example 1 as a control group, and shake flask test and detection were performed. OD 600 The D-pantothenic acid content in the fermentation supernatant is shown in Figure 3.

[0054] As can be seen from the figure, we increased one molecule of folP gene copy in DPAH3 using the lafU pseudogene site, and the D-pantothenic acid yield of DPAH6 was increased to 6.01 g / L. Therefore, overexpression of folP promotes the combination of pABA and GTP pathway, which is an effective strategy to strengthen the provision of one-carbon units for folate synthesis.

[0055] Example 3: Construction of DPAH7 and shake flask fermentation

[0056] Using DPAH6 as the starting strain, CRISPR / Cas9 gene editing technology was used to integrate the serA and glyA gene clusters in the genome of the strain DPAH6 using the pseudogene site, and a dynamic regulation element was designed to adjust the expression state of the gene cluster in real time. In response to the supply of methyl, we increased one molecule of panB gene copy after the gene cluster, which is controlled by the in situ promoter PpanB (nucleotide sequences are shown in SEQ ID NO. 4, SEQ ID NO. 5), and obtained DPAH7 (DPAH6 derivative, yeeP::Trc-RBSApt2#82-serAglyApanB).

[0057] (1) Construction of pTarget-PAM-serAglyApanB plasmid: pTarget F plasmid (Addgene Plasmid#62226) as a template, pT-PAM-serAglyApanB-F / R as primers for PCR amplification, and the PCR product was digested with Dpn I at 37°C for 3h; the PCR product of pTarget-PAM-serAglyApanB was amplified by linear primers pTarget-XF and pTarget-XR, and the purified pTarget-PAM-serAglyA linearized vector was used for subsequent ligation of Donor DNA.

[0058] (2) Construction of pTD-Donor-serAglyApanB plasmid: E. coli W3110 genome as a template, serAglyApanB-up-F and serAglyApanB-up-R as primers to amplify the upstream part of the donor DNA (F1), and serAglyApanB-down-F and serAglyApanB-down-R as primers to amplify the downstream part of the donor DNA (F2), and F1 and F2 were obtained by gel recovery and purification; according to the instructions of One step clone kit (Vazyme Biotech, Nanjing, China), the linearized vector of pTarget-serAglyApanB and fragments F1 and F2 were ligated together, and the pTD-serAglyApanB plasmid was obtained by sequencing verification. (One step clone kit, Vazyme Biotech, Nanjing, China) instructions to ligate the linearized vector of pTarget-serAglyApanB and fragments F1 and F2 together, and the pTD-serAglyApanB plasmid was obtained by sequencing verification.

[0059] (3) Introduce pCas plasmid (Addgene Plasmid#62225) into the competent cells obtained in Example 4, and the preparation method of DPAH6 electrocompetent cells is the same as that in Example 1(3).

[0060] (4) Construct DPAH7 positive colonies, and the construction method is the same as that in Example 1(4).

[0061] (5) Plasmid elimination: the implementation method is the same as that in Example 1(5), and the plasmid-free DPAH7 (DPAH6 derivative, yeeP::Trc-RBSApt2#82-serAglyApanB) is obtained.

[0062] (6) Shake flask test and detection were performed on the constructed DPAH7 production strain with DPAH6 constructed in Example 2 as the control group. OD 600 The D-pantothenic acid content in the fermentation supernatant is shown in Figure 4.

[0063] As can be seen from the figure, according to the intracellular glycine concentration control serA and glyA overexpression, when glycine accumulates to a certain extent, it will fold and cover the ribosome site, and repress the translation of the gene cluster serA-glyA, when the intracellular content of glycine is less, it will expose the ribosome site to strengthen the translation. In response to the supply of methyl, we increase a copy of the panB gene molecule after the gene cluster to obtain strain H7, and the D-pantothenate yield of DPAH7 is successfully improved to 6.52 g / L. The ribosome switch is used to control the expression of glyA in real time, and the key genes of the serine pathway are moderately strengthened. This strategy can reasonably regulate the serine synthesis rate and maintain the intracellular glycine at a low level, and successfully promote the synthesis of D-pantothenate.

[0064] Example 4: Construction of DPAH8 and shake flask fermentation

[0065] Taking DPAH7 as the starting strain, using CRISPR / Cas9 gene editing technology, introducing a heterologous alsS gene into the genome of strain DPAH7, and performing codon optimization (nucleotide sequence as shown in SEQ ID NO. 3), DPAH8 (DPAH7 derivative, gapC::Trc-alsS*) is obtained.

[0066] (1) Construct pTarget-PAM-alsS plasmid: use pTarget F plasmid (Addgene Plasmid#62226) as a template, and use pT-PAM-alsS-F / R as primers for PCR amplification. After the PCR product is verified by nucleic acid gel electrophoresis, it is digested with Dp n I at 37°C for 3h, then transformed into E. coli DH5α, selected by spectinomycin plate, and the correct pTarget-PAM-alsS plasmid was obtained by sequencing verification, which was used for subsequent connection of Donor DNA.

[0067] (2) Construct pTD-Donor-BsalsS plasmid: first, use E. coli W3110 genome as a template, and use alsS-up-F / R as primers to amplify the upper part of donor DNA (F1), and use alsS-down-F / R as primers to amplify the lower part of donor DNA (F2). Then, use B. subtilis 168 genome as a template, and use pTrc-BsalsS(2) F / R as primers to amplify the BsalsS gene fragment with the promoter pTrc (F3), and purify the PCR fragments F1, F2 and F3 by gel recovery; the plasmid pTarget-alsS is digested with Xba I and Pst I at 37°C for 8h, and the DNA fragment is recovered by Clean up kit; according to the manufacturer's instructions, the purified F1, F2 and F3 are ligated with pTarget-PAM-alsS, and the ligation product is transformed into E. coli DH5α, and the positive clones are selected by spectinomycin plate, and the correct pTD-Donor-BsalsS plasmid is obtained by sequencing verification. (One step clone kit, Vazyme Biotech, Nanjing, China) according to the instruction manual. The pTarget-alsS vector, fragments F1, F2 and F3 were ligated together, and the pTD-BsalsS plasmid was obtained by sequencing verification.

[0068] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAH7 competent cells obtained in Example 5, and the preparation method of the DPAH7 electrocompetent cells was the same as that in Example 1 (3).

[0069] (4) The DPAH8 positive colonies were constructed, and the construction method was the same as that in Example 1 (4).

[0070] (5) Plasmid elimination: the implementation method was the same as that in Example 1 (5), and the plasmid-free DPAH8 (DPAH7 derivative, gapC::Trc-alsS*) was obtained.

[0071] (6) The constructed DPAH8 production strain was compared with the DPAH7 constructed in Example 3 as a control group, and shake flask tests and detections were performed. OD 600 The D-pantothenic acid content in the fermentation supernatant is shown in Figure 5.

[0072] Acetolactate synthase (alsS) catalyzes the formation of alpha-acetolactate from two molecules of pyruvate, and acetolactate synthase is involved in the synthesis of valine, leucine and isoleucine. As can be seen from the figure, by introducing a heterologous alsS gene on the genome and performing codon optimization, the expression efficiency of the gene in the host is improved, and the strain burden is smaller. Under the premise of not adding pyruvate, the shake flask yield is promoted, and the D-pantothenic acid yield of the DPAH8 strain increases to 6.85 g / L.

[0073] Example 5: Application of D-pantothenic acid production strain DPAH8 in microbial fermentation for preparing D-pantothenic acid

[0074] The fermentation was carried out in a 5L fermenter (Shanghai Baohong, BIOTECH-5BG), including the following steps:

[0075] (1) Seed culture: plate inoculate DPAH8 into 10 mL LB medium, and incubate overnight at 37°C, 180 rpm on a shaker, then inoculate into two bottles each containing 100 mL LB medium with a 1% volume concentration of inoculum for secondary seed culture for 7-12 h.

[0076] (2) Fermentation: the volume of fermentation medium in 5L fermenter is 2L, sterilized at 115℃ for 30min. 25-30℃, initial aeration 3-6L / min, initial stirring speed 300-450rpm, ammonia water to adjust pH. Two bottles of secondary seed liquid, 200mL in total, were transferred to 2L fermentation medium without resistance in 5L fermenter, and 0.2mM IPTG, 5mg / L VB1, 2mg / L VB 12 and 10-40g / L isoleucine 5-10mL, fermentation started. During fermentation, dissolved oxygen was maintained at 10%-30% using dissolved oxygen cascade stirring speed, pH was maintained at 6.7-6.9 using ammonia water as neutralizer, glucose concentration in the tank was controlled below 5g / L by pH linkage feeding, and the culture was maintained at 28-37℃ for 3-4 days to obtain fermentation broth. Fermentation broth is all the substances in the fermenter.

[0077] [Corrected according to Rule 91 on 11.12.2024](3) After diluting the fermentation supernatant 80 times, the sample after dilution was treated by using water-based filter membrane to remove impurities, and HPLC detection was carried out according to Example 1, OD 600 and the content of D-pantothenic acid in the fermentation broth supernatant is shown in Figure 6.

[0078] By regulating the glycine metabolic pathway, methyl cofactor supply, folate production and serine synthesis pathway, the supply of one-carbon units is strengthened, and more metabolic flow is involved in the synthesis of D-pantothenic acid. The D-pantothenic acid production strain DPAH8 is obtained. As can be seen from the figure, the yield of D-pantothenic acid of the engineering strain DPAH8 is increased to 112.62g / L in a 5L fermenter for 92 hours, which greatly improves the synthesis of D-pantothenic acid.

[0079] The fermentation medium is composed of the following: glucose 20g / L, ammonium sulfate 16g / L, betaine 2g / L, yeast powder 2g / L, potassium dihydrogen phosphate 2g / L, anhydrous magnesium sulfate 0.5g / L, beta-alanine 1.5g / L, 1ml / L trace element solution, solvent is deionized water, pH value is natural. The composition of the trace element solution is: 10g / L CuCl2, 10g / L FeSO4·7H2O, 10g / L ZnSO4·7H2O, 0.2g / L CuSO4, 0.02g / L NiCl2·7H2O, solvent is deionized water.

[0080] The composition of the feed medium is as follows: glucose 500g / L, ammonium sulfate 10g / L, betaine 4g / L, yeast powder 2g / L, potassium dihydrogen phosphate 14g / L, anhydrous magnesium sulfate 8g / L, beta-alanine 60g / L, 2ml / L trace element solution, solvent is deionized water, pH value is natural.

[0081] The primer sequence information used in Examples 1 to 4 is shown in Table 2.

[0082] Table 2: Primer sequence

[0083] The above-described examples are merely preferred embodiments of the present application, and are not intended to limit the scope of the present application. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A method for constructing a genetically engineered bacterium with high D-pantothenic acid production, characterized by, The method comprises: using CRISPR-Cas9 mediated gene editing technology to overexpress gcvTHP gene, strengthen folP gene, integrate serA and glyA gene cluster and add a copy of panB gene after the serAglyA gene cluster regulated by a dynamic regulatory element in the genome of the chassis strain, and increase the heterologous codon-optimized alsS gene to obtain the genetically engineered strain for high-yield D-pantothenate.

2. The method of claim 1, wherein, The method for strengthening the folP gene comprises: adding a copy of the folP gene at the lafU pseudogene site.

3. The method of claim 1, wherein, The dynamic regulatory element is ribosome switch Apt2#82.

4. The method of claim 1, wherein, The panB gene is regulated by the PpanB promoter.

5. The method of claim 1, wherein, The alsS gene is derived from Bacillus subtilis and is codon-optimized.

6. The method of claim 1, wherein, The chassis is: E. coli W3110, Trc-EcilvD* / BspanBA* / CgpanC* / alsS* / BspanB* / nac GTG / ΔP 345ptsH / gltA GTG / gltA TTG / Trc-PpfkB.

7. The method of claim 1, wherein, The nucleotide sequences of the gcvTHP gene, the folP gene and the alsS gene regulated by the Ptrc promoter are respectively shown in SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3; the sequence of the serAglyA gene cluster regulated by the ribosome switch Apt2#82 is shown in SEQ ID NO. 4; and the nucleotide sequence of the panB gene regulated by the PpanB promoter is shown in SEQ ID NO.

5.

8. The genetically engineered strain for high-yield D-pantothenate obtained by the method of any one of claims 1-7.

9. Use of the genetically engineered strain for high-yield D-pantothenate of claim 8 in the microbial fermentation for preparing D-pantothenate.

10. Use according to claim 9, wherein The method for the use comprises: inoculating the genetically engineered strain for high-yield D-pantothenate into a fermentation medium, and performing fed-batch fermentation culture at 28-37°C, 300-450 rpm, pH 6.7-6.9 and 10-30% dissolved oxygen for 72-96 h, and then isolating and purifying the D-pantothenate after the fermentation.

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