Genetically engineered bacteria that produce D-pantothenic acid, methods of construction and uses

Abstract

The present invention relates to a genetically engineered bacterium capable of producing D-pantothenic acid, a method for constructing it, and the use of this recombinant bacterium in the production of D-pantothenic acid by microbial fermentation. The main components of this invention are: (1) increasing the copy number of key genes in the pantothenic acid synthesis pathway in Escherichia coli to further direct carbon flux toward D-pantothenic acid synthesis; (2) attenuating nitrogen-limiting negative regulatory transcription factors and activating front-end genes of glycolysis to increase carbon flux, conserving phosphoenolpyruvate and reducing central carbon flux into the TCA cycle, thereby obtaining a recombinant bacterial strain for D-pantothenic acid production without the addition of plasmids or antibiotics. Finally, the shake flask titer of D-pantothenic acid was increased by 87.2% compared to the original strain, reaching 5.43 g / L. After 84 hours of fermentation in a 5-L fermentation tank, D-pantothenic acid production reached 94.2 g / L.

Description

[Technical Field]

[0001] The present invention relates to genetically modified bacteria that produce D-pantothenic acid, methods for constructing the bacteria, and the use of the bacteria in the production of D-pantothenic acid by microbial fermentation. [Background technology]

[0002] Pantothenic acid, also known as vitamin B5, is a component of coenzyme A and plays a key role in important biochemical reactions such as energy metabolism and the citric acid cycle. Therefore, D-pantothenic acid is widely used as an important vitamin and precursor in feed, pharmaceuticals, and cosmetics. Among the known methods for synthesizing D-pantothenic acid, biofermentation has attracted attention due to its advantages, including inexpensive substrates, easy isolation, and low toxicity. However, current biological methods for producing D-pantothenic acid still have drawbacks, such as an unstable fermentation process and low production yields. Therefore, constructing strains with higher D-pantothenic acid yields remains a challenge. Summary of the Invention

[0003] The object of the present invention is to provide a method for constructing a genetically modified bacterium that produces D-pantothenic acid at high yields using rational design and CRISPR-Cas9 gene editing technology, and the use of the genetically modified bacterium in the production of D-pantothenic acid by microbial fermentation.

[0004] The technical solutions adopted by the present invention are as follows:

[0005] A genetically modified bacterium that produces D-pantothenic acid is constructed and obtained by the following method.

[0006] (1) Using the genetically modified bacterium ZJUTDPAL5 as the chassis bacterium, the copy number of the EcilvD gene controlled by the promoter pTrc on its genome was increased to obtain a recombinant bacterium DPA PA 6 derivative, yjiV::pTrc-EcilvD, which was recorded as the recombinant bacterium DPAP7.

[0007] (2) The copy number of the Bacillus subtilis BspanBA gene under the control of the promoter pTrc was increased on the recombinant bacterial DPAP7 genome by gene knock-in, and a recombinant bacterial DPAP7 derivative, flik::Ptrc-BspanBA, was obtained and recorded as recombinant bacterial DPAP8.

[0008] (3) The copy number of the glutamate-producing CgpanC gene under the control of the pTrc promoter was increased on the recombinant DPAP8 genome by gene knock-in, to obtain a recombinant DPAP8 derivative, ompT::pTrc-CgpanC, which was recorded as the recombinant DPAP9.

[0009] (4) The copy number of the Bacillus subtilis alsS gene under the control of the promoter pTrc was increased on the recombinant bacterial DPAP9 genome by gene knock-in, and a recombinant bacterial DPAP9 derivative, yjiP::Ptrc-alsS, was obtained and recorded as recombinant bacterial DPAP10.

[0010] (5) The copy number of the B. subtilis BspanBB gene under the control of the pTrc promoter was further increased on the recombinant bacterial DPAP10 genome by gene knock-in, resulting in a recombinant bacterial DPAP10 derivative, ydeU::Ptrc-BspanBB, which was designated as recombinant bacterial DPAP11. BspanBB has the same sequence as BspanB, except that the distance between the promoter and the rbs site is narrower than that of BspanBA.

[0011] (6) The initiation codon of the nac gene in the recombinant bacterial DPAP11 genome was replaced with GTG to create a recombinant bacterial DPAP11 derivative, nac GTG The resulting bacterium is recorded as recombinant bacterium DPAP12.

[0012] (7) We knocked out the in situ promoters P3, P4, and P5 in front of the ptsH, ptsI, and crr gene clusters in the recombinant bacterial DPAP12 genome, and created a recombinant bacterial DPAP12 derivative, ΔP 345ptsH The resulting bacterium is recorded as recombinant DPAP13.

[0013] (8) The initiation codon of the gltA gene in the recombinant bacterial DPAP13 genome was replaced with GTG to create a recombinant bacterial DPAP13 derivative, gltA GTG The resulting bacterium is recorded as recombinant bacterium DPAP14.

[0014] (9) The initiation codon of the gltA gene in the recombinant bacterial DPAP14 genome was replaced with TTG to create a recombinant bacterial DPAP14 derivative, gltA TTG The resulting bacterium is recorded as recombinant bacterium DPAP15.

[0015] (10) The in situ promoter of the pfkB gene in the recombinant bacterial DPAP15 genome is replaced with Ptrc to obtain a recombinant bacterial DPAP15 derivative, PpfkB::Ptrc, which is designated as recombinant bacterial DPAP16, i.e., a genetically modified bacterium capable of producing D-pantothenic acid.

[0016] The present invention is based on the chassis fungus ZJUTDPAL5 (E. coli W3110, Trc-panCpanEpanBilvC / ilvG* / ΔavtA / ilvE* / coaA* / ΔilvA / Trc-lpd / Δglk / ilvA* / Trc-pck / Trc-maeB / Trc-ilvBN / gdhA*T, previously disclosed in CN113637618A), and employs a comprehensive systems metabolic engineering strategy to introduce heterologous genes using CRISPR / Cas9 gene editing technology via gene knock-in, thereby enhancing the expression of key genes in the pantoic acid synthesis pathway in vivo in E. coli and further directing carbon flux toward the synthesis of D-pantothenic acid. To continue enhancing carbon flux to synthesize D-pantothenic acid, we attenuated the negative regulatory transcription factors of nitrogen limitation, activated the front-end genes of glycolysis to pull carbon flow, conserving phosphoenolpyruvate and reducing the influx of central carbon into the TCA cycle. Finally, we obtained a recombinant bacterial strain for producing D-pantothenic acid without the addition of plasmids or antibiotics.

[0017] The nucleotide sequences of the EcilvD, BspanBA, CgpanC, BspanBB, and alsS genes controlled by the promoter pTrc are shown in SEQ ID NOs. 1 to 5, respectively. GTG The nucleotide sequence of the in situ promoters P3, P4, and P5 in front of the ptsH, ptsI, and crr gene cluster is as shown in SEQ. ID. NO. 7. The nucleotide sequence of the gltA GTG The nucleotide sequence of gltA is shown in SEQ ID NO. 8. TTG The nucleotide sequence of the in situ promoter of the pfkB gene is shown in SEQ ID NO. 9, and the nucleotide sequence of the in situ promoter of the pfkB gene is shown in SEQ ID NO. 10.

[0018] The present invention provides a method for constructing the genetically modified bacterium, comprising:

[0019] (1) Using the genetically modified bacterium ZJUTDPAL5 as the chassis bacterium, the original pseudogene yjiV on the original strain genome was replaced with the EcilvD gene controlled by the pTrc promoter derived from pTrc99A using CRISPR-Cas9-mediated gene editing technology, thereby increasing the expression intensity of EcilvD. This resulted in the recombinant bacterial DPA PA 6 derivative, yjiV::Ptrc-EcilvD, which was recorded as the recombinant bacterium DPAP7.

[0020] (2) Using the recombinant bacterium DPAP7 as the original strain, the original pseudogene flik on the genome of the original strain was replaced with the BspanBA gene controlled by the pTrc promoter derived from pTrc99A using CRISPR-Cas9-mediated gene editing technology, thereby increasing the expression intensity of BspanBA and obtaining a recombinant bacterium DPAP7 derivative, flik::Ptrc-BspanBA, which was recorded as recombinant bacterium DPAP8.

[0021] (3) Using the recombinant bacterial DPAP8 as the original strain, the original pseudogene ompT on the original strain genome was replaced with the CgpanC gene controlled by the pTrc promoter derived from pTrc99A using CRISPR-Cas9-mediated gene editing technology to enhance the expression intensity of CgpanC, and a recombinant bacterial DPAP8 derivative, ompT::Ptrc-CgpanC, was obtained and recorded as recombinant bacterial DPAP9.

[0022] (4) Using the recombinant bacterium DPAP9 as the original strain, the original pseudogene yjiP on the genome of the original strain was replaced with the alsS gene controlled by the promoter pTrc derived from pTrc99A using CRISPR-Cas9-mediated gene editing technology, thereby increasing the expression intensity of alsS. This resulted in the production of a recombinant bacterium DPAP9 derivative, yjiP::Ptrc-alsS, which was recorded as the recombinant bacterium DPAP10.

[0023] (5) Using the recombinant bacterium DPAP10 as the original strain, the original pseudogene ydeU in the original strain genome was replaced with the BspanBB gene controlled by the pTrc promoter derived from pTrc99A using CRISPR-Cas9-mediated gene editing technology, further enhancing the expression intensity of BspanBB. This resulted in the recombinant bacterium DPAP10 derivative, ydeU::Ptrc-BspanBB, which was designated as the recombinant bacterium DPAP11.

[0024] (6) Using the recombinant bacterium DPAP11 as the original strain, the initiation codon ATG of the nac gene in its genome was replaced with GTG using CRISPR-Cas9-mediated gene editing technology to generate a recombinant bacterium DPAP11 derivative, nac GTG The resulting bacterium is recorded as recombinant bacterium DPAP12.

[0025] (7) Using the recombinant bacterial DPAP12 as the original strain, we used CRISPR-Cas9-mediated gene editing technology to knock out the in situ promoters P3, P4, and P5 in front of the ptsH, ptsI, and crr gene clusters in its genome, and created a recombinant bacterial DPAP12 derivative, ΔP 345ptsH The resulting bacterium is recorded as recombinant DPAP13.

[0026] (8) Using the recombinant bacterium DPAP13 as the original strain, the initiation codon ATG of the gltA gene in its genome was replaced with GTG using CRISPR-Cas9-mediated gene editing technology to generate the recombinant bacterium DPAP13 derivative, gltA GTG The resulting bacterium is recorded as recombinant bacterium DPAP14.

[0027] (9) Using the recombinant bacterium DPAP14 as the original strain, the initiation codon of the gltA gene in its genome was replaced with TTG by CRISPR-Cas9-mediated gene editing technology to generate the recombinant bacterium DPAP14 derivative, gltA TTG The resulting bacterium is recorded as recombinant bacterium DPAP15.

[0028] (10) Using the recombinant bacterium DPAP15 as the original strain, the in situ promoter of the pfkB gene in its genome was replaced with Ptrc using CRISPR-Cas9-mediated gene editing technology to obtain a recombinant bacterium DPAP15 derivative, PpfkB::Ptrc, which was designated as the recombinant bacterium DPAP16, i.e., a genetically modified bacterium capable of producing D-pantothenic acid.

[0029] Specifically, the nucleotide sequences of the EcilvD, BspanBA, CgpanC, BspanBB, and alsS genes controlled by the promoter pTrc are shown in SEQ ID NOs. 1 to 5, respectively. GTG The nucleotide sequence of the in situ promoters P3, P4, and P5 in front of the ptsH, ptsI, and crr gene cluster is as shown in SEQ ID NO. 7. The gltA GTG The nucleotide sequence of gltA is shown in SEQ ID NO. 8. TTG The nucleotide sequence of the in situ promoter of the pfkB gene is shown in SEQ ID NO. 9, and the nucleotide sequence of the in situ promoter of the pfkB gene is shown in SEQ ID NO. 10.

[0030] The present invention further relates to the use of the genetically modified bacterium in the production of D-pantothenic acid by microbial fermentation.

[0031] The use is as follows: The genetically modified bacterium capable of producing D-pantothenic acid is inoculated into a fermentation medium, and fermentation culture is carried out under conditions of 28 to 37°C and 300 to 450 rpm for 72 to 96 hours. After the fermentation is completed, the supernatant of the fermentation liquid is separated and purified to obtain the D-pantothenic acid.

[0032] Specifically, the fermentation medium contains 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 β-alanine, and 1-5 ml / L trace element solution, with deionized water as the solvent and unadjusted pH. The trace element solution contains 10 g / L CuCl, 10 g / L FeSO·7H2O, 10 g / L ZnSO·7H2O, 0.2 g / L CuSO, and 0.02 g / L NiCl·7H2O, with deionized water as the solvent.

[0033] Specifically, the method is as follows: A 1-3 L volume of fermentation medium is placed in a 5 L fermentation tank and sterilized at 115°C for 30 minutes. The recombinant bacteria is inoculated into 1-3 L of the fermentation medium, and fermentation culture is carried out under conditions of 28-37°C, an initial aeration rate of 3-6 L / min, and an initial stirring speed of 300-450 rpm. The pH is adjusted with aqueous ammonia, and IPTG at a final concentration of 0-0.4 mM, VB1 at a final concentration of 5 mg / L, and VB2 at a final concentration of 2 mg / L is added. 12 and 5-10 mL of 10-40 g / L isoleucine are simultaneously added. During the fermentation process, the dissolved oxygen is maintained at 10-30% by controlling the serial rotation speed of the dissolved oxygen, the pH is maintained at 6.7-6.9 using aqueous ammonia as a neutralizer, a feed medium is added to the tank by pH-linked feeding, the glucose concentration is controlled to 5 g / L or less, and the culture is carried out at 28-37°C for 72-96 hours to obtain a fermentation broth, and the supernatant of the fermentation broth is separated and purified to obtain the D-pantothenic acid.

[0034] The feed medium is composed of 500 g / L of glucose, 5 to 25 g / L of ammonium sulfate, 2 to 8 g / L of anhydrous betaine, 1 to 5 g / L of yeast powder, 10 to 20 g / L of potassium dihydrogen phosphate, 5 to 15 g / L of anhydrous magnesium sulfate, 40 to 100 g / L of β-alanine, and 1 to 5 ml / L of trace element solution, the solvent is deionized water, and the pH value is not adjusted.

[0035] In this study, we performed heterologous screening of pantoic acid branched genes and further increased the copy numbers of panC (encoding pantothenate synthase), panB (encoding hydroxymethyltransferase), alsS (encoding acetolactate synthase), and ilvD (encoding dihydroxyacid dehydratase) on the genome using the promoter pTrc and RBS sequence derived from pTrc99A, thereby enhancing the pantoic acid synthesis pathway. DPAP11 (DPAP10 derivative, ydeU::pTrc-BspanBB) was constructed.

[0036] This invention uses CRISPR / Cas9 gene editing technology to weaken the nitrogen limitation negative regulatory transcription factor nac, weaken the promoters of the glucose phosphotransferase system genes ptsH, ptsI, and Crr to conserve phosphoenolpyruvate, knock down the citrate synthase gltA to reduce the influx of central carbon into the TCA cycle, and finally activate the front-end gene pfkB of the glycolysis pathway to pull carbon flow, thereby obtaining the recombinant bacterium DPAP16 without the addition of plasmids or antibiotics during the fermentation process.

[0037] Compared with the prior art, the advantages of the present invention are mainly as follows:

[0038] The present invention utilizes CRISPR / Cas9 gene editing technology to further enhance the expression levels of key enzymes in the D-pantothenic acid biosynthetic pathway based on existing recombinant bacteria, and then up- or down-regulates the expression of one or more key genes in the glycolysis pathway, the TCA cycle, and global cellular regulators, resulting in a high-yielding strain that is plasmid-free and does not require the addition of antibiotics during fermentation. The shake flask titer of the present strain reached 5.43 g / L, an 87.2% increase compared to the original strain. After 84 hours of fermentation in a 5-L fermentation tank, the production of D-pantothenic acid reached 94.2 g / L, significantly improving D-pantothenic acid synthesis and shortening the fermentation cycle. [Brief explanation of the drawings]

[0039] [Figure 1] FIG. 1 shows the D-pantothenic acid metabolic pathway and modification sites. [Figure 2] FIG. 1 shows changes in OD600 and D-pantothenic acid titer of DPAP7. [Figure 3] FIG. 1 shows changes in OD600 and D-pantothenic acid titer of DPAP8. [Figure 4] FIG. 1 shows changes in OD600 and D-pantothenic acid titer of DPAP9. [Figure 5] FIG. 1 shows changes in OD600 and D-pantothenic acid titer of DPAP10. [Figure 6] FIG. 1 shows changes in OD600 and D-pantothenic acid titer of DPAP11. [Figure 7] FIG. 1 shows changes in OD600 and D-pantothenic acid titer of DPAP12. [Figure 8] FIG. 1 shows changes in OD600 and D-pantothenic acid titer of DPAP13. [Figure 9] FIG. 1 shows changes in OD600 and D-pantothenic acid titer for DPAP14 and DPAP15. [Figure 10] FIG. 1 shows changes in OD600 and D-pantothenic acid titer of DPAP16. [Figure 11] This is a diagram showing the fermentation results of DPAP11 and DPAP16 in a 5 L bioreactor. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will be further described below with reference to specific examples, but the scope of protection of the present invention is not limited thereto.

[0041] In the following examples, the final concentration of spectinomycin in the medium is 0.05 mg / L and the final concentration of kanamycin in the medium is 0.05 mg / L.

[0042] The parent strain E. coli W3110 of the present invention was from the Coli Genetic Stock Center at Yale University, deposited on August 5, 1975, with deposit number CGSC#4474, and has been previously disclosed in U.S. Patent Application Publication No. 2009 / 0298135 A1 and U.S. Patent Application Publication No. 2010 / 0248311 A1.

[0043] The primer sequence information used in Examples 2 to 11 is shown in Table 2.

[0044] Table 1. Genes involved in gene editing and corresponding pathways [Table 1]

[0045] Table 2 Primer sequences [Table 2-1] [Table 2-2] [Table 2-3]

[0046] Example 1: HPLC determination of D-pantothenic acid content The detection method is as follows.

[0047] Chromatography conditions: C 18 Column (250 × 4.6 mm, particle size 5 μm, Agilent Technologies Co., Santa Clara, CA, USA), detection wavelength: 200 nm, column temperature: 30°C.

[0048] Sample treatment: Dilute the sample with ultrapure water to maintain the content of D-pantothenic acid between 0.05 g / L and 0.40 g / L. Mobile phase: acetonitrile / water / phosphoric acid: (50 / 949 / 1), data collection time: 25 min.

[0049] Example 2: Construction of DPAP7 (DPA PA 6 derivative, yjiV::Ptrc-EcilvD) and shake flask fermentation ZJUTDPAL5(E.coli W3110 ,Trc-panCpanEpanBilvC / ilvG* / ΔavtA / ilvE* / coaA* / ΔilvA / Trc-lpd / Δglk / ilvA* / Trc-pck / Trc-maeB / Trc-ilvBN / gdhA* T ) was used as the original strain, and CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene yjiV in the genome of the original strain with the EcilvD gene (the nucleotide sequence of which is shown in SEQ ID No. 1) controlled by the pTrc promoter derived from pTrc99A using a gene knock-in method, thereby increasing the expression intensity of EcilvD.

[0050] (1) Construction of pTarget-yjiV plasmid: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-yjiV-F / R as primers. The PCR product was verified by nucleic acid gel electrophoresis and then digested with Dpn I enzyme at 37°C for 3 hours. It was then transformed into E. coli DH5α, screened on a spectinomycin plate, and verified by sequencing to obtain the correct pTarget-yjiV plasmid, which was then used for subsequent ligation with donor DNA.

[0051] (2) Construction of pTD-EcilvD plasmid: First, the upstream portion of donor DNA (F1) was amplified using the E. coli W3110 genome as a template and yjiV-S6F / R as primers, and the downstream portion of donor DNA (F2) was amplified using yjiV-X6F / R as primers. Next, the EcilvD gene fragment (F3) containing the promoter pTrc was amplified using the E. coli W3110 genome as a template and pTrc-EcilvD-18F / R as primers. The PCR fragments were purified by gel collection to obtain F1, F2, and F3. The plasmid pTarget-yjiV was incubated with XbaI and PstI at 37°C for 8 hours, and the DNA fragments were recovered using a cleanup kit. The pTarget-yjiV vector and fragments F1, F2 and F3 were ligated together according to the instructions of ClonExpress® (One step clone kit, Vazyme Biotech, Nanjing, China) and verified by sequencing to obtain the pTD-EcilvD plasmid.

[0052] (3) Transfer pCas plasmid (Addgene Plasmid#62225) to ZJUTDPAL5 (E.coli W3110, Trc-panCpanEpanBilvC / ilvG* / ΔavtA / ilvE* / coaA* / ΔilvA / Trc-lpd / Δglk / ilvA* / Trc-pck / Trc-maeB / Trc-ilvBN / gdhA* T ), and a monoclonal colony was transferred to an LB test tube containing 0.05 mg / L kanamycin and cultured overnight at 30°C. A 1% inoculum was then inoculated into a 250 mL shake flask containing 50 mL of LB medium, and 500 μL of 1 mol / L L-arabinose was added. The OD was measured at 150 rpm and 30°C. 600 The cells were cultured until the chromatin density reached 0.4-0.6. The cells were collected by centrifugation at 4000 rpm and 4°C for 10 minutes, and electrocompetent cells were prepared. The detailed process is described in (Molecular Cloning: A Laboratory Manual, 3rd Edition, pp. 99-102).

[0053] (4) Using a pipette, an appropriate amount of pTD-EcilvD (approximately 200 ng) plasmid was aspirated and mixed with 100 μl of electroporation competent cells prepared in advance. Both were then transferred to a pre-cooled 2 mm electroporation cuvette, and after about 1-2 minutes on ice, the cuvette was placed in an electroporator (MicroPlus TM Electroporation was performed using a 100-kDa PCR kit (BIO-RAD). Immediately after electroporation, 800 μl of LB medium was added and gently aspirated. The resulting solution was transferred to a 2 mL Eppendorf tube and incubated at 30°C for 3-4 hours. The resulting solution was then plated onto an LB solid plate containing 0.05 mg / L kanamycin and 0.05 mg / L spectinomycin and cultured inverted at 30°C for 12-16 hours. Colony PCR verification was performed using ilvD-VF / R as primers. Successful cloning of a fragment of approximately 3500 bp confirmed the identity of the DPAP7 (DPAP7 derivative, yjiV::Ptrc-EcilvD)-positive colony.

[0054] (5) Plasmid removal: Positive single colonies were selected using an inoculating loop and inoculated into LB test tubes containing 1 mM IPTG and 0.05 mg / L spectinomycin. The cells were then cultured overnight at 30°C. The next day, the bacterial solution was streaked onto solid LB plates containing 0.05 mg / L spectinomycin and cultured at 30°C for 24 hours. When the cells reached a certain size, some single colonies were selected and streaked onto LB plates containing 0.05 mg / L kanamycin. Single colonies that failed to grow on LB plates containing 0.05 mg / L kanamycin indicated successful removal of the pTarget-yjiV plasmid. Subsequently, single colonies that had successfully removed the pTarget-yjiV plasmid were selected, placed in LB tubes, and cultured overnight at 37°C to remove the pCas plasmid. The next day, the bacterial solution was streaked onto LB plates and cultured at 37°C for 12 hours. Some single colonies were then selected and streaked onto LB plates containing 0.05 mg / L kanamycin. For single colonies that could not grow on LB plates containing 0.05 mg / L kanamycin, the pCas plasmid was successfully removed, and finally, the plasmid-free strain DPAP7 (DPA PA 6 derivative, yjiV::Ptrc-EcilvD) was obtained.

[0055] (6) Shake flask fermentation: DPAP7 (DPA PA 6 derivative, yjiV::Ptrc-EcilvD) and the original strain ZJUTDPAL5 (control) were inoculated into 10 mL of LB medium and cultured at 37°C and 200 rpm as preculture. After 8-12 hours, 1 mL of the preculture was inoculated into 50 mL of MS medium in a 500 mL shake flask at a 2% inoculum size, and then cultured in a thermostatic shaker at 30°C and 180 rpm for 48 hours to perform strain fermentation. After fermentation was completed, 1 mL of the fermented broth was collected and the OD 600 The OD value was measured, and 1 mL of the fermentation broth was taken with a pipette and centrifuged at 12,000 rpm for 3 minutes at room temperature. The fermentation supernatant was diluted 5-fold. The diluted sample was then filtered through a microporous filtration membrane to remove impurities, and then subjected to HPLC detection according to Example 1. 600The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0056] As can be seen from the figure, adding the ilvD gene copies to the genome had no obvious effect on bacterial growth, nor did it have any effect on improving the D-pantothenic acid shake flask titer, which remained at approximately 2.9 g / L, the same as the original strain. This may be due to a lack of carbon flux in the previous pathway. Considering the need for further modifications, we continued to modify the strain accordingly.

[0057] LB medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, the solvent was deionized water, and the pH value was not adjusted.

[0058] MS medium: 20 g / L glucose, 16 g / L (NH4)2SO4, 2 g / L KH2PO4, 0.5 g / L MgSO4, 2 g / L yeast extract, 10 g / L CaCO3, and 1 ml / L trace element solution (deionized water, pH unadjusted). 10 g / L calcium carbonate (sterilized separately) was used. The trace element solution was 10 g / L CuCl2, 10 g / L FeSO4·7H2O, 1 g / L ZnSO4·7H2O, 0.20 g / L CuSO4, and 0.02 g / L NiCl2·7H2O (deionized water).

[0059] Example 3: Construction of DPAP8 and shake flask fermentation Using DPAP7 as the original strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene flik in the genome of the original strain with the BspanBA gene (nucleotide sequence shown in SEQ ID No. 2) controlled by the pTrc promoter derived from pTrc99A through gene knock-in, thereby increasing the expression intensity of BspanBA.

[0060] (1) Construction of pTarget-flik plasmid: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-flik-F / R as primers. The PCR product was verified by nucleic acid gel electrophoresis and then incubated and digested with Dpn I digestion enzyme at 37°C for 3 hours. It was then transformed into E. coli DH5α, screened on a spectinomycin plate, and verified by sequencing to obtain the correct pTarget-flik plasmid, which was then used for subsequent ligation with donor DNA.

[0061] (2) Construction of pTD-BspanBA plasmid: First, the upstream portion of donor DNA (F1) was amplified using the E. coli W3110 genome as a template and flik-S8F / R as primers, and the downstream portion of donor DNA (F2) was amplified using flik-X8F / R as primers. Next, the BspanBA gene fragment (F3) containing the promoter pTrc was amplified using the B. subtilis 168 genome as a template and pTrc-BspanBA-9F / R as primers. The PCR fragments were purified by gel collection to obtain F1, F2, and F3. The plasmid pTarget-flik was incubated with XbaI and PstI at 37°C for 8 hours, and the DNA fragments were recovered using a cleanup kit. The pTarget-flik vector and fragments F1, F2 and F3 were ligated together according to the instructions of ClonExpress® (One step clone kit, Vazyme Biotech, Nanjing, China) and verified by sequencing to obtain the pTD-BspanBA plasmid.

[0062] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAP7 competent cells obtained in Example 2. The method for preparing DPAP7 electrocompetent cells was the same as in Example 2 (3).

[0063] (4) DPAP8-positive colonies were obtained by construction. The construction method was the same as in Example 2 (4).

[0064] (5) Removal of the plasmid: Plasmid-free DPAP8 was obtained in the same manner as in Example 2(5).

[0065] (6) The constructed DPAP8-producing strain was subjected to shake flask testing and detection according to the method described in (6) of Example 2, using DPAP7 constructed in Example 2 as a control. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0066] As can be seen, after increasing the copy number of the heterologous BspanBA gene in the genome by gene knock-in, the growth of the bacteria was not affected, but the shake flask titer of D-pantothenic acid increased to 3.72 g / L. This may be because the introduction of this gene diverted part of the carbon flux to branched-chain amino acids to the synthesis of pantoic acid, thereby promoting the biosynthesis of D-pantothenic acid in the strain.

[0067] Example 4: Construction and shake flask fermentation of DPAP9 Using DPAP8 as the original strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene ompT in the genome of the original strain with the CgpanC gene (nucleotide sequence shown in SEQ ID No. 3) controlled by the pTrc promoter derived from pTrc99A through gene knock-in, thereby increasing the expression intensity of CgpanC.

[0068] (1) Construction of pTarget-ompT plasmid: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-ompT-F / R as primers. The PCR product was verified by nucleic acid gel electrophoresis and then incubated with Dpn I digestion enzyme at 37°C for 3 hours. It was then transformed into E. coli DH5α, screened on a spectinomycin plate, and verified by sequencing to obtain the correct pTarget-ompT plasmid, which was then used for subsequent ligation with donor DNA.

[0069] (2) Construction of pTD-CgpanC plasmid: First, the upstream portion of donor DNA (F1) was amplified using the E. coli W3110 genome as a template and ompT-S6F / R as primers, and the downstream portion of donor DNA (F2) was amplified using ompT-X6F / R as primers. Next, the CgpanC gene fragment (F3) containing the promoter pTrc was amplified using the C. glutamicum ATCC 13032 genome as a template and pTrc-CgpanC-8F / R as primers. The PCR fragments were purified by gel collection to obtain F1, F2, and F3. The plasmid pTarget-ompT was incubated with XbaI and PstI at 37°C for 8 hours, and the DNA fragments were recovered using a cleanup kit. The pTarget-ompT vector and fragments F1, F2 and F3 were ligated together according to the instructions of ClonExpress® (One step clone kit, Vazyme Biotech, Nanjing, China) and verified by sequencing to obtain the pTD-CgpanC plasmid.

[0070] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAP8 competent cells obtained in Example 3. The method for preparing DPAP8 electrocompetent cells was the same as in Example 2 (3).

[0071] (4) DPAP9-positive colonies were obtained by construction. The construction method was the same as in Example 2 (4).

[0072] (5) Removal of the plasmid: Plasmid-free DPAP9 was obtained in the same manner as in Example 2(5).

[0073] (6) The constructed DPAP8-producing strain was subjected to shake flask testing and detection according to the method of (6) in Example 2, using the DPAP8 constructed in Example 3 as a control. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0074] As can be seen from the figure, after increasing the copy number of the heterologous CgpanC gene in the genome using gene knock-in, the shake flask titer of D-pantothenic acid was slightly improved to 3.76 g / L without significantly affecting the growth of the strain, but the improvement was not as great as that obtained by the heterologous introduction of BspanB. This may be because the enzyme activity encoded by this gene in the genome was sufficient, and further increase in carbon flux to pantothenic acid synthesis was required to significantly increase the D-pantothenic acid content in the fermentation broth.

[0075] Example 5: Construction of DPAP10 and shake flask fermentation Using the DPAP9 strain as the original strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene yjiP in the genome of the original strain with the alsS gene (nucleotide sequence shown in SEQ ID No. 4) controlled by the pTrc promoter derived from pTrc99A through gene knock-in, thereby enhancing the expression intensity of alsS.

[0076] (1) Construction of pTarget-yjiP plasmid: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-yjiP-F / R as primers. The PCR product was verified by nucleic acid gel electrophoresis and then incubated with Dpn I digestion enzyme at 37°C for 3 hours. It was then transformed into E. coli DH5α, screened on a spectinomycin plate, and verified by sequencing to obtain the correct pTarget-yjiP plasmid, which was then used for subsequent ligation with donor DNA.

[0077] (2) Construction of pTD-alsS plasmid: First, the upstream portion of the donor DNA (F1) was amplified using the E. coli W3110 genome as a template and yjiP-S5F / R as primers, and the downstream portion of the donor DNA (F2) was amplified using yjiP-X5F / R as primers. Next, the alsS gene fragment (F3) containing the promoter pTrc was amplified using the B. subtilis 168 genome as a template and pTrc-alsS-17F / R as primers. The PCR fragments were purified by gel collection to obtain F1, F2, and F3. The plasmid pTarget-yjiP was incubated with XbaI and PstI at 37°C for 8 hours, and the DNA fragments were recovered using a cleanup kit. The pTarget-yjiP vector and fragments F1, F2 and F3 were ligated together according to the instructions of ClonExpress® (One step clone kit, Vazyme Biotech, Nanjing, China) and verified by sequencing to obtain the pTD-alsS plasmid.

[0078] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAP9 competent cells obtained in Example 4. The method for preparing DPAP9 electrocompetent cells was the same as in Example 2 (3).

[0079] (4) DPAP10-positive colonies were obtained by construction. The construction method was the same as in Example 2 (4).

[0080] (5) Removal of the plasmid: Plasmid-free DPAP10 was obtained in the same manner as in Example 2(5).

[0081] (6) The constructed DPAP10-producing strain was subjected to shake flask testing and detection according to the method of (6) in Example 2, using the DPAP9 constructed in Example 4 as a control. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0082] As can be seen from the figure, after increasing the copy number of the heterologous alsS gene in the genome using gene knock-in, the shake flask titer of D-pantothenic acid increased to 3.87 g / L, but the effect was not significant. Therefore, the supply of pyruvate precursors or the synthetic capacity of the downstream D-pantothenic acid pathway is still insufficient and needs to be further strengthened.

[0083] Example 6: Construction and shake flask fermentation of DPAP11 Using DPAP10 as the original strain, CRISPR-Cas9-mediated gene editing technology was used to replace the original pseudogene ydeU in the genome of the original strain with the BspanBB gene (nucleotide sequence shown in SEQ ID No. 5) controlled by the pTrc promoter derived from pTrc99A through gene knock-in, thereby further enhancing the expression intensity of BspanBB.

[0084] (1) Construction of pTarget-ydeU plasmid: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-ydeU-F / R as primers. The PCR product was verified by nucleic acid gel electrophoresis and then incubated with Dpn I digestion enzyme at 37°C for 3 hours. It was then transformed into E. coli DH5α, screened on a spectinomycin plate, and verified by sequencing to obtain the correct pTarget-ydeU plasmid, which was then used for subsequent ligation with donor DNA.

[0085] (2) Construction of pTD-BspanBB plasmid: First, the upstream part of the donor DNA (F1) was obtained by amplification using the E. coli W3110 genome as a template and ydeU-S6F / R as primers, and the downstream part of the donor DNA (F2) was obtained by amplification using ydeU-X6F / R as primers.

[0086] Next, the B. subtilis 168 genome was used as a template to amplify the BspanBB gene fragment (F3) carrying the promoter pTrc using pTrc-BspanBB-9F / R as primers. The PCR fragments were purified by gel collection to obtain F1, F2, and F3. The plasmid pTarget-ydeU was incubated with XbaI and PstI at 37°C for 8 hours, and the DNA fragments were recovered using a Clean Up Kit. The pTarget-ydeU vector and fragments F1, F2, and F3 were ligated together according to the instructions of the ClonExpress® (One Step Clone Kit, Vazyme Biotech, Nanjing, China) and verified by sequencing to obtain the pTD-BspanBB plasmid.

[0087] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAP10 competent cells obtained in Example 5. The method for preparing DPAP10 electrocompetent cells was the same as in Example 2 (3).

[0088] (4) DPAP11-positive colonies were obtained by construction. The construction method was the same as in Example 2 (4).

[0089] (5) Removal of the plasmid: Plasmid-free DPAP11 was obtained in the same manner as in Example 2(5).

[0090] (6) The constructed DPAP11-producing strain was subjected to shake flask testing and detection according to the method of (6) in Example 2, using the DPAP10 constructed in Example 5 as a control. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0091] As can be seen from the figure, after the copy number of the heterologous BspanBB gene was increased again in the genome by gene knock-in, the D-pantothenic acid production was still significantly improved to about 4.45 g / L. Therefore, after the previous gene integration, part of the carbon flux in the pathway needed to be pulled further downstream toward the synthesis of D-pantothenic acid, which promoted the increase in D-pantothenic acid production in shake flasks.

[0092] Example 7: Construction of DPAP12 and shake flask fermentation Using DPAP11 as the original strain, the nac initiation codon ATG was replaced with GTG (the nucleotide sequence is shown in SEQ ID NO. 6) in the DPAP11 genome using CRISPR / Cas9 gene editing technology to generate DPAP12 (DPAP11 derivative, nac GTG ) was obtained.

[0093] (1) pTarget-nac GTG Plasmid construction: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-nac-F / pT-nac-R as primers, and the PCR product was digested with Dpn I at 37°C for 3 hours. GTG The PCR product was amplified with linearized primers pTarget-XF and pTarget-XR, and then gel-collected to obtain pTarget-nac GTG The linearized vector was purified and used for subsequent ligation of Donor DNA.

[0094] (2) pTD-nac GTGPlasmid construction: Using the E. coli W3110 genome as a template, the upstream part of the donor DNA (F1) was amplified using primers nac-up-F and nac-up-R, and the downstream part of the donor DNA (F2) was amplified using primers nac-down-F and nac-down-F. The PCR fragments were purified by gel collection to obtain F1 and F2. pTarget-nac was constructed according to the instructions of the ClonExpress® (One step clone kit, Vazyme Biotech, Nanjing, China). GTG The linearized vector and fragments F1 and F2 were ligated together and verified by sequencing to give pTD-nac GTG The plasmid was obtained.

[0095] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAP11 competent cells obtained in Example 6. The method for preparing DPAP11 electrocompetent cells was the same as in Example 2 (3).

[0096] (4) DPAP12-positive colonies were obtained by construction. The construction method was the same as in Example 2 (4).

[0097] (5) Removal of the plasmid: Plasmid-free DPAP12 was obtained in the same manner as in Example 2(5).

[0098] (6) The constructed DPAP12-producing strain was subjected to shake flask testing and detection according to the method of Example 2(6), using the DPAP11 constructed in Example 6 as a control. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0099] As can be seen from the figure, gene editing of the genome to replace the nac start codon (ATG) with GTG resulted in a modest increase in D-pantothenic acid production, at approximately 4.54 g / L. The E. coli nitrogen regulatory protein Nac can suppress the expression of multiple genes, including those related to D-pantothenic acid production, such as cycA, ilvN, ilvH, and ilvM, under nitrogen-limited conditions. The degree of downregulation of nac was insufficient, likely limiting the significant increase in production.

[0100] Example 8: Construction of DPAP13 and shake flask fermentation Using DPAP12 as the original strain, CRISPR / Cas9 gene editing technology was used to knock out the in situ promoters P3, P4, and P5 (the nucleotide sequence of which is shown in SEQ ID NO. 7) in front of the ptsH, ptsI, and Crr gene clusters in the genome of the strain DPAP12, resulting in DPAP13 (DPAP12 derivative, ΔP345ptsH).

[0101] (1) Construction of pTarget-ΔP345ptsH plasmid: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-PptsH-F / pT-PptsH-F as primers, and the PCR product was digested with Dpn I at 37°C for 3 hours. 345ptsH The PCR product was amplified with linearized primers pTarget-XF and pTarget-XR and purified by gel collection to give pTarget-ΔP. 345ptsH The linearized vector was purified and used for subsequent ligation of Donor DNA.

[0102] (2) pTD-ΔP 345ptsHPlasmid construction: Using the E. coli W3110 genome as a template, the upstream part of donor DNA (F1) was amplified using primers PptsH-up-F and PptsH-up-R to obtain the upstream part of donor DNA, and the downstream part of donor DNA (F2) was amplified using primers PptsH-down-R and PptsH-down-R to obtain the downstream part of donor DNA. The PCR fragments were purified by gel collection to obtain F1 and F2. The linearized pTarget-ΔP345ptsH vector and fragments F1 and F2 were ligated together according to the instructions of ClonExpress® (One step clone kit, Vazyme Biotech, Nanjing, China) and verified by sequencing to obtain pTD-ΔP 345ptsH The plasmid was obtained.

[0103] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAP12 competent cells obtained in Example 7. The method for preparing DPAP12 electrocompetent cells was the same as in Example 2 (3).

[0104] (4) DPAP13-positive colonies were obtained by construction. The construction method was the same as in Example 2 (4).

[0105] (5) Removal of the plasmid: Plasmid-free DPAP13 was obtained in the same manner as in Example 2(5).

[0106] (6) The constructed DPAP13-producing strain was subjected to shake flask testing and detection according to the method of Example 2(6), using the DPAP12 constructed in Example 7 as a control. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0107] As can be seen from the figure, after knocking out the in situ promoters P3, P4, and P5 in front of the ptsH, ptsI, and Crr gene clusters in the genome using gene knockout methods, D-pantothenic acid production increased to 4.70 g / L. The phosphotransferase system consumes PEP to transport glucose, and by slightly weakening the phosphotransferase system to conserve PEP, PEP is instead used to generate pyruvate and ATP through the action of the pykAF gene, thereby promoting increased D-pantothenic acid production in shake flasks.

[0108] Example 9: Construction of DPAP14 and shake flask fermentation Using DPAP13 as the original strain, the CRISPR / Cas9 gene editing technique was used to replace the gltA initiation codon ATG with GTG (the nucleotide sequence is as shown in SEQ ID NO. 8) in the genome of the strain DPAP13, resulting in DPAP14 (DPAP13 derivative, gltAGTG).

[0109] (1) pTarget-gltA GTG Plasmid construction: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-gltA-F / pT-gltA-R as primers, and the PCR product was digested with Dpn I at 37°C for 3 hours. GTG The PCR product was amplified with linearized primers pTarget-XF and pTarget-XR and purified by gel collection to give pTarget-gltA. GTG The linearized vector was purified and used for subsequent ligation of Donor DNA.

[0110] (2) pTD-gltA GTGPlasmid construction: Using the E. coli W3110 genome as a template, the upstream portion of donor DNA (F1) was amplified using primers gltA-up-F and gltA-up-R. The middle portion of donor DNA (F2) was amplified using primers gltA-gRNA-F and gltA-gRNA-R. The downstream portion of donor DNA (F3) was amplified using primers gltA-down-F1 and gltA-down-R. The linearized vector pTarget-gltAGTG and fragments F1, F2, and F3 were ligated together according to the instructions of the ClonExpress® (One Step Clone Kit, Vazyme Biotech, Nanjing, China). The pTD-gltAGTG plasmid was constructed using the same procedures as in Example 2(2).

[0111] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAP13 competent cells obtained in Example 8. The method for preparing DPAP13 electrocompetent cells was the same as in Example 2 (3).

[0112] (4) DPAP14-positive colonies were obtained by construction. The construction method was the same as in Example 2 (4).

[0113] (5) Removal of the plasmid: Plasmid-free DPAP14 was obtained in the same manner as in Example 2(5).

[0114] (6) The constructed DPAP14-producing strain was subjected to shake flask testing and detection according to the method of (6) in Example 2, using the DPAP13 constructed in Example 8 as a control. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0115] As can be seen from the figure, after gene editing in the genome to replace the ATG start codon of gltA with GTG, D-pantothenic acid production increased to 4.85 g / L. During the growth phase of the strain, large amounts of NADH, ATP, and several metabolites were required, resulting in a large carbon flux into the TCA cycle. Therefore, we considered weakening the TCA cycle to enhance pyruvate accumulation and inactivating the gltA gene to slow strain growth. Therefore, by replacing the ATG start codon of the gltA gene with GTG, we weakened the TCA cycle and promoted the improvement of D-pantothenic acid production.

[0116] Example 10: Construction of DPAP15 and shake flask fermentation Using DPAP14 as the original strain, the gltA initiation codon GTG was replaced with TTG (the nucleotide sequence is shown in SEQ ID NO. 9) in the genome of the strain DPAP14 using CRISPR / Cas9 gene editing technology to obtain DPAP15 (DPAP14 derivative, gltATTG).

[0117] (1) pTarget-gltA TTG Plasmid construction: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-gltA-F / pT-gltA-R as primers, and the PCR product was digested with Dpn I at 37°C for 3 hours. TTG The PCR product was amplified with linearized primers pTarget-XF and pTarget-XR, and then gel-collected to obtain pTarget-gltA. TTG The linearized vector was purified and used for subsequent ligation of Donor DNA.

[0118] (2) pTarget-gltA TTGPlasmid construction: Using the E. coli W3110 genome as a template, the upstream part of donor DNA (F1) was amplified using primers gltA-up-F and gltA-up-R. The middle part of donor DNA (F2) was amplified using primers gltA-gRNA-F and gltA-gRNA-R. The downstream part of donor DNA (F3) was amplified using primers gltA-down-F2 and gltA-down-R. pTarget-gltA was constructed according to the instructions of the ClonExpress® (One step clone kit, Vazyme Biotech, Nanjing, China). TTG The linearized vector and fragments F1, F2, and F3 were ligated together and constructed in the same manner as in Example 2(2), pTD-gltA TTG The plasmid was obtained.

[0119] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAP14 competent cells obtained in Example 9. The method for producing DPAP14 competent cells was the same as in Example 2 (3).

[0120] (4) DPAP15-positive colonies were obtained by construction. The construction method was the same as in Example 2 (4).

[0121] (5) Removal of the plasmid: Plasmid-free DPAP15 was obtained in the same manner as in Example 2(5).

[0122] (6) The constructed DPAP15-producing strain was subjected to shake flask testing and detection according to the method of Example 2(6), using DPAP13 constructed in Example 9 as a control. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0123] As can be seen from the figure, after gene editing in the genome to replace the gltA start codon GTG with TTG, D-pantothenic acid production increased to 5.16 g / L. We considered weakening the TCA cycle by replacing the start codon ATG of the gltA gene with GTG, but this had no obvious effect on cell growth. Therefore, we further downregulated the start codon of the gltA gene to TTG, further improving D-pantothenic acid production.

[0124] Example 11: Construction of DPAP16 and shake flask fermentation Using DPAP15 as the original strain, the in situ promoter of the pfkB gene in the genome of strain DPAP15 was replaced with Ptrc (the nucleotide sequence is shown in SEQ ID NO. 10) using CRISPR / Cas9 gene editing technology to obtain DPAP16 (DPAP15 derivative, PpfkB::Ptrc).

[0125] (1) Construction of pTarget-PpfkB::Ptrc plasmid: PCR amplification was performed using pTarget F plasmid (Addgene Plasmid #62226) as a template and pT-PpfkB-F / pT-PpfkB-R as primers. The PCR product was digested with Dpn I at 37°C for 3 hours. The pTarget-PpfkB::Ptrc PCR product was amplified with linearization primers pTarget-XF and pTarget-XR. The linearized pTarget-PpfkB::Ptrc vector was purified by gel collection and used for subsequent ligation with donor DNA.

[0126] (2) Construction of pTD-PpfkB::Ptrc plasmid: Using the E. coli W3110 genome as a template, the upstream part of donor DNA (F1) was amplified using primers PpfkB-up-F and PpfkB-up-R to obtain the upstream part of donor DNA, and the downstream part of donor DNA (F2) was amplified using primers PpfkB-down-F and PpfkB-down-R to obtain the downstream part of donor DNA. The pTarget-PpfkB::Ptrc linearized vector and fragments F1 and F2 were ligated together according to the instructions of ClonExpress® (One step clone kit, Vazyme Biotech, Nanjing, China). The construction was carried out in the same manner as in Example 2(2), and the pTD-PpfkB::Ptrc plasmid was obtained.

[0127] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAP15 competent cells obtained in Example 10. The method for producing DPAP15 competent cells was the same as in Example 2 (3).

[0128] (4) DPAP16-positive colonies were obtained by construction. The construction method was the same as in Example 2 (4).

[0129] (5) Removal of the plasmid: Plasmid-free DPAP16 was obtained in the same manner as in Example 2(5).

[0130] (6) The constructed DPAP16-producing strain was subjected to shake flask testing and detection according to the method of Example 2(6), using the DPAP15 constructed in Example 10 as a control. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0131] As can be seen from the figure, after gene editing was used to replace the in situ promoter of the pfkB gene with Ptrc, D-pantothenic acid production increased to 5.43 g / L. Because the original pfkB gene expression level was insufficient, a strong promoter was used to increase expression, strengthening the front-end pathway of glycolysis and promoting the improvement of D-pantothenic acid production.

[0132] Example 12: Fermentation of D-pantothenic acid producing strains DPAP11 and DPAP16 in 5 L bioreactors Fermentation was carried out in a 5L fermentation tank (Shanghai Baoxing, BIOTECH-5BG) and included the following steps:

[0133] (1) Seed culture: The plate was inoculated into 10 mL of LB medium and cultured overnight in a shaking incubator at 37°C and 180 rpm. Then, the plate was inoculated into two flasks containing 100 mL of LB medium at a volumetric concentration of 1% and cultured for 7 to 12 hours as the secondary seed solution.

[0134] (2) Inoculation and fermentation: The volume of fermentation medium in a 5-L fermentation tank was 2 L, and the tank was sterilized at 115°C for 30 minutes. The temperature was 25-30°C, the initial aeration rate was 3-6 L / min, and the initial stirring speed was 300-450 rpm. The pH was adjusted with aqueous ammonia. A total of 200 mL of the secondary seed solution was transferred to a 5-L fermentation tank containing 2 L of intolerant fermentation medium. At the same time, IPTG was added to a final concentration of 0.2 mM, and VB1 and VB2 were added to final concentrations of 5 mg / L and 2 mg / L, respectively. 12 Fermentation was initiated by adding 5-10 mL of 10-40 g / L isoleucine and the dissolved oxygen concentration was maintained at 10-30% by controlling the serial oxygen rotation rate. The pH was maintained at 6.7-6.9 using aqueous ammonia as a neutralizer. The glucose concentration in the tank was controlled to 5 g / L or less by pH-linked feed. The culture was continued at 28-37°C for 3-4 days, and the fermentation broth was obtained. The fermentation broth contained all of the material in the fermentation tank.

[0135] (3) The fermentation supernatant was diluted 80 times, and the diluted sample was filtered through a microporous filtration membrane to remove impurities. HPLC detection was then performed according to Example 1. 600 The D-pantothenic acid content in the fermentation supernatant is shown in FIG.

[0136] As can be seen from the figure, after strengthening the pantothenic acid pathway, the D-pantothenic acid production of the D-pantothenic acid-producing strain DPAP11 was increased to 63.3 g / L after 84 hours of fermentation in a 5 L fermentation tank. Based on this, the D-pantothenic acid-producing strain DPAP16 was obtained by optimizing the supply of pyruvate precursor, weakening the TCA cycle, and introducing more carbon flow into the pantothenic acid pathway. After 84 hours of fermentation in a 5 L fermentation tank, the D-pantothenic acid production was increased to 94.2 g / L, significantly increasing the synthesis of D-pantothenic acid and shortening the fermentation cycle.

[0137] (4) The fermentation medium consisted of 20 g / L glucose, 16 g / L ammonium sulfate, 2 g / L anhydrous betaine, 2 g / L yeast powder, 2 g / L potassium dihydrogen phosphate, 0.5 g / L anhydrous magnesium sulfate, 1.5 g / L β-alanine, and 1 ml / L trace element solution (deionized water, pH unadjusted). The trace element solution consisted of 10 g / L CuCl, 10 g / L FeSO 7H O, 10 g / L ZnSO 7H O, 0.2 g / L CuSO, and 0.02 g / L NiCl 7H O (deionized water).

[0138] (5) The composition of the feed medium was: glucose 500 g / L, ammonium sulfate 10 g / L, anhydrous betaine 4 g / L, yeast powder 2 g / L, potassium dihydrogen phosphate 14 g / L, anhydrous magnesium sulfate 8 g / L, β-alanine 60 g / L, and trace element solution 2 ml / L. The solvent was deionized water, and the pH was not adjusted.

Claims

1. A genetically modified bacterium that produces D-pantothenic acid, the method of construction comprising: (1) using the genetically modified bacterium ZJUTDPAL5 as a chassis bacterium, increasing the copy number of the EcilvD gene controlled by the promoter pTrc on its genome, to obtain a recombinant bacterium DPA PA 6 derivative, yjiV::Ptrc-EcilvD, which is recorded as a recombinant bacterium DPAP7; (2) increasing the copy number of the Bacillus subtilis BspanBA gene under the control of the promoter pTrc on the recombinant bacterial DPAP7 genome by gene knock-in to obtain a recombinant bacterial DPAP7 derivative, flik::Ptrc-BspanBA, which is designated as recombinant bacterial DPAP8; (3) increasing the copy number of the glutamic acid-producing bacterium CgpanC gene under the control of the promoter pTrc on the recombinant bacterium DPAP8 genome by gene knock-in to obtain a recombinant bacterium DPAP8 derivative, ompT::pTrc-CgpanC, which is recorded as recombinant bacterium DPAP9; (4) increasing the copy number of the Bacillus subtilis alsS gene under the control of the promoter pTrc on the recombinant bacterial DPAP9 genome by gene knock-in to obtain a recombinant bacterial DPAP9 derivative, yjiP::Ptrc-alsS, which is designated as recombinant bacterial DPAP10; (5) further increasing the copy number of the Bacillus subtilis BspanBB gene under the control of the promoter pTrc on the recombinant bacterial DPAP10 genome by gene knock-in to obtain a recombinant bacterial DPAP10 derivative, ydeU::Ptrc-BspanBB, which is designated as recombinant bacterial DPAP11; (6) The initiation codon ATG of the nac gene in the recombinant bacterial DPAP11 genome was replaced with GTG to generate a recombinant bacterial DPAP11 derivative, nac GTG obtaining and recording as recombinant bacterial DPAP12; (7) The in situ promoters P3, P4, and P5 in front of the ptsH, ptsI, and crr gene clusters in the recombinant bacterial DPAP12 genome were knocked out, and the recombinant bacterial DPAP12 derivative, ΔP 345ptsH obtaining and recording as recombinant bacterial DPAP13; (8) The initiation codon ATG of the gltA gene in the recombinant bacterial DPAP13 genome was replaced with GTG to generate a recombinant bacterial DPAP13 derivative, gltA GTG obtaining and recording as recombinant bacterial DPAP14; (9) The start codon of the gltA gene in the recombinant bacterial DPAP14 genome was replaced with TTG to generate a recombinant bacterial DPAP14 derivative, gltA TTG obtaining and recording as recombinant bacterial DPAP15; (10) replacing the in situ promoter of the pfkB gene in the recombinant bacterial DPAP15 genome with Ptrc to obtain a recombinant bacterial DPAP15 derivative, PpfkB::Ptrc, which is recorded as recombinant bacterial DPAP16; The recombinant bacterium obtained in step (10) is a genetically modified bacterium that produces D-pantothenic acid.

2. The nucleotide sequences of the EcilvD, BspanBA, CgpanC, BspanBB, and alsS genes controlled by the promoter pTrc are shown in SEQ ID NOs. 1 to 5, respectively. GTG The nucleotide sequence of the in situ promoters P3, P4, and P5 in front of the ptsH, ptsI, and crr gene cluster is as shown in SEQ ID NO.

7. The gltA GTG The nucleotide sequence is as shown in SEQ ID NO. 8, and the gltA TTG 2. The genetically modified bacterium according to claim 1, wherein the nucleotide sequence of the pfkB gene in situ promoter is as set forth in SEQ ID NO. 9, and the nucleotide sequence of the pfkB gene in situ promoter is as set forth in SEQ ID NO.

10.

3. 1. A method for constructing a genetically modified bacterium, said method comprising: (1) Using the genetically modified bacterium ZJUTDPAL5 as the chassis bacterium, the original pseudogene yjiV in the original strain genome is replaced with the EcilvD gene controlled by the pTrc promoter pTrc derived from pTrc99A using CRISPR-Cas9 mediated gene editing technology, thereby increasing the expression intensity of EcilvD, and obtaining a recombinant bacterium DPA PA 6 derivative, yjiV::Ptrc-EcilvD, which is recorded as the recombinant bacterium DPAP7; (2) Using the recombinant bacterium DPAP7 as the original strain, use CRISPR-Cas9-mediated gene editing technology to replace the original pseudogene flik in the genome of the original strain with the BspanBA gene controlled by the pTrc promoter derived from pTrc99A, thereby increasing the expression intensity of BspanBA, and obtain a recombinant bacterium DPAP7 derivative, flik::Ptrc-BspanBA, which is recorded as recombinant bacterium DPAP8; (3) Using the recombinant bacterium DPAP8 as the original strain, use CRISPR-Cas9-mediated gene editing technology to replace the original pseudogene ompT in the genome of the original strain with the CgpanC gene controlled by the pTrc promoter derived from pTrc99A, thereby increasing the expression intensity of CgpanC, and obtain a recombinant bacterium DPAP8 derivative, ompT::Ptrc-CgpanC, which is recorded as the recombinant bacterium DPAP9; (4) Using the recombinant bacterium DPAP9 as the original strain, the original pseudogene yjiP in the genome of the original strain is replaced with the alsS gene controlled by the pTrc promoter pTrc from pTrc99A using CRISPR-Cas9-mediated gene editing technology, thereby increasing the expression intensity of alsS, and obtaining a recombinant bacterium DPAP9 derivative, yjiP::Ptrc-alsS, which is recorded as the recombinant bacterium DPAP10; (5) Using the recombinant bacterium DPAP10 as the original strain, use CRISPR-Cas9-mediated gene editing technology to replace the original pseudogene ydeU in the genome of the original strain with the BspanBB gene controlled by the pTrc promoter derived from pTrc99A, thereby further enhancing the expression intensity of BspanBB, and obtain a recombinant bacterium DPAP10 derivative, ydeU::Ptrc-BspanBB, which is recorded as recombinant bacterium DPAP11; (6) Using the recombinant bacterium DPAP11 as the original strain, the initiation codon ATG of the nac gene in its genome was replaced with GTG using CRISPR-Cas9-mediated gene editing technology to generate a recombinant bacterium DPAP11 derivative, nac GTG obtaining and recording as recombinant bacterial DPAP12; (7) Using the recombinant bacterial DPAP12 as the original strain, the in situ promoters P3, P4, and P5 in front of the ptsH, ptsI, and crr gene clusters in the genome were knocked out using CRISPR-Cas9-mediated gene editing technology, and the recombinant bacterial DPAP12 derivative, ΔP 345ptsH obtaining and recording as recombinant bacterial DPAP13; (8) Using the recombinant bacterium DPAP13 as the original strain, the initiation codon ATG of the gltA gene in its genome was replaced with GTG using CRISPR-Cas9-mediated gene editing technology to generate a recombinant bacterium DPAP13 derivative, gltA GTG obtaining and recording as recombinant bacterial DPAP14; (9) Using the recombinant bacterium DPAP14 as the original strain, the start codon of the gltA gene in its genome was replaced with TTG by CRISPR-Cas9-mediated gene editing technology to generate a recombinant bacterium DPAP14 derivative, gltA TTG obtaining and recording as recombinant bacterial DPAP15; (10) using the recombinant bacterium DPAP15 as the original strain, and using CRISPR-Cas9-mediated gene editing technology to replace the in situ promoter of the pfkB gene in its genome with Ptrc to obtain a recombinant bacterium DPAP15 derivative, PpfkB::Ptrc, which is recorded as recombinant bacterium DPAP16; 3. The method for constructing a genetically modified bacterium according to claim 1 or 2, wherein the recombinant bacterium obtained in step (10) is a genetically modified bacterium that produces D-pantothenic acid.

4. The nucleotide sequences of the EcilvD, BspanBA, CgpanC, BspanBB, and alsS genes controlled by the promoter pTrc are shown in SEQ ID NOs. 1 to 5, respectively. GTG The nucleotide sequence of the in situ promoters P3, P4, and P5 in front of the ptsH, ptsI, and crr gene cluster is as shown in SEQ ID NO.

7. The gltA GTG The nucleotide sequence of gltA is shown in SEQ ID NO.

8. TTG The method for constructing a genetically modified bacterium according to claim 3, characterized in that the nucleotide sequence of the pfkB gene is as shown in SEQ ID NO. 9, and the nucleotide sequence of the in situ promoter of the pfkB gene is as shown in SEQ ID NO.

10.

5. Use of the genetically modified bacterium according to claim 1 or 2 in the production of D-pantothenic acid by microbial fermentation.

6. The use according to claim 5, wherein the D-pantothenic acid-producing genetically modified bacterium is inoculated into a fermentation medium, followed by fermentation culture at 28 to 37°C and 300 to 450 rpm for 72 to 96 hours, and after completion of fermentation, the supernatant of the fermentation liquid is separated and purified to obtain the D-pantothenic acid.

7. The fermentation medium is composed of glucose 10-30 g / L, ammonium sulfate 10-25 g / L, anhydrous betaine 1-5 g / L, yeast powder 1-5 g / L, potassium dihydrogen phosphate 1-5 g / L, anhydrous magnesium sulfate 0.5-2 g / L, β-alanine 1-5 g / L, and trace element solution 1-5 ml / L. The solvent is deionized water, the pH is not adjusted, and the trace element solution contains 10 g / L of CuCl. 2 , 10 g / L FeSO 4 ・7H 2 O, 10 g / L ZnSO 4 ・7H 2 0.2 g / L CuSO 4 , 0.02 g / L NiCl 2 ・7H 2 7. The method of claim 6, wherein the solvent is deionized water.

8. The method of use is as follows: a 5-L fermentation tank is charged with 1 to 3 L of the fermentation medium, sterilized at 115°C for 30 minutes, the recombinant bacterium is inoculated into 1 to 3 L of the fermentation medium, and fermentation culture is carried out under conditions of 28 to 37°C, an initial aeration rate of 3 to 6 L / min, and an initial stirring rate of 300 to 450 rpm; pH is adjusted with aqueous ammonia; IPTG at a final concentration of 0 to 0.4 mM, VB at a final concentration of 5 mg / L, and the like. 1 and 2 mg / L VB 12 and 5-10 mL of 10-40 g / L isoleucine are simultaneously added; in the fermentation process, the dissolved oxygen concentration is maintained at 10-30% by controlling the serial rotation speed of the dissolved oxygen, the pH is maintained at 6.7-6.9 using aqueous ammonia as a neutralizer, a feed medium is added to the tank by pH-linked feeding, the glucose concentration is controlled to 5 g / L or less, and the culture is performed at 28-37°C for 72-96 hours to obtain a fermentation broth, and the supernatant of the fermentation broth is separated and purified to obtain the D-pantothenic acid.

9. 9. The use according to claim 8, wherein the feed medium comprises 500 g / L of glucose, 5-25 g / L of ammonium sulfate, 2-8 g / L of anhydrous betaine, 1-5 g / L of yeast powder, 10-20 g / L of potassium dihydrogen phosphate, 5-15 g / L of anhydrous magnesium sulfate, 40-100 g / L of β-alanine, and 1-5 ml / L of trace element solution, the solvent being deionized water, and the pH value is not adjusted.

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