High-yield D-pantothenic acid genetically engineered bacterium based on ilvY activation system as well as construction method and application of high-yield D-pantothenic acid genetically engineered bacterium

Abstract

The invention provides a high-yield D-pantothenic acid gene engineering bacterium based on an ilvY activation system as well as a construction method and application thereof, and belongs to the technical field of gene engineering. According to the invention, high-activity mutants ilvYR257C and S264C are obtained mainly by constructing a fluorescence activated expression system, constructing an ilvY mutant library by using error-prone PCR (Polymerase Chain Reaction) and performing high-throughput screening; the structural stability and binding capacity of the mutant are verified through molecular dynamics simulation; the mutant is integrated into an original strain, and a high-yield genetically engineered bacterium is constructed by combining enhanced expression of a pantoic acid path key gene alsS and inhibition of a D-pantothenic acid degradation path. The genetically engineered bacterium is subjected to fed-batch fermentation in a 5L fermentation tank for 86h, the yield of D-pantothenic acid reaches 137.8 g / L and is increased by 22.5% compared with an original strain, and the yield is 1.64 g / L / h. By optimizing an ilvY activation system, precise regulation and control of a pantoic acid synthesis path are realized, and technical support is provided for industrial efficient production of D-pantothenic acid.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a method based on ilvY High-yield D-pantothenic acid genetically engineered bacteria for activating the system, construction method and application. Background Technology

[0002] Pantothenic acid, also known as vitamin B5, is a component of coenzyme A and plays a crucial role in important biochemical reactions such as energy metabolism and the citric acid cycle. Therefore, as an important vitamin and precursor, D-pantothenic acid (DPA) is widely used in feed, medicine, and cosmetics. Among known methods for synthesizing D-pantothenic acid, bio-fermentation has attracted attention due to its advantages such as inexpensive substrates, easy separation, and low toxicity. For example, Chinese patent CN113637618A discloses a D-pantothenic acid producing bacterium, its construction method, and its application. In the fermentation process of the DPA-producing bacterium constructed in this application, the reaction substrate β-alanine is added exogenously. This method can effectively accumulate DPA in the fermentation broth during the fermentation process and improve the DPA yield and sugar conversion rate. The DPA yield is 66.39 g / L, and the sugar-acid conversion rate reaches 30%, showing potential for large-scale production. However, the production of D-pantothenic acid by biological methods still faces problems such as low pyruvate utilization, insufficient regulation of the pantothenic acid synthesis pathway, and easy degradation of the product, resulting in low yield and production efficiency of D-pantothenic acid, which limits industrial production.

[0003] Therefore, there is an urgent need to construct a D-pantothenic acid strain that can weaken glucose-6-phosphate diversion, reduce PPP pathway flux, and enhance EMP pathway carbon flux, resulting in high yield and short production cycle. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention provides a method based on... ilvY This invention relates to a high-yield D-pantothenic acid-producing genetically engineered bacterium that activates a system, its construction method, and its applications. The invention utilizes CRISPR-Cas9-mediated gene editing technology and a method based on… ilvY A method for constructing high-yield D-pantothenic acid-producing genetically engineered bacteria for activating the system, through screening for high-activity bacteria. ilvY By modifying mutants, optimizing the pantothenic acid synthesis pathway, and inhibiting degradation pathways, a genetically engineered bacterium that produces high levels of D-pantothenic acid was obtained, and this genetically engineered bacterium was applied to the microbial fermentation preparation of D-pantothenic acid.

[0005] To solve the above problems, the technical solution adopted in this application is: The first objective of this invention is to provide a method based on ilvYA method for constructing a high-yield D-pantothenic acid-producing genetically engineered bacterium that activates the system, the method comprising: using CRISPR-Cas9-mediated gene editing technology, combined with... ilvy Mutant activation systems modify one or more of the chassis bacteria genome: (1) To zwf Gene implementation of codon optimization and / or promoter substitution; (2) Introducing heterogeneous sources zmglf Genes and glk Gene; (3) Constructing a high-throughput screening system for fluorescence-activated expression and ilvY Mutant library, and on ilvY The mutant library was screened to obtain ilvY R257C,S264C Mutants; (4) ilvY R257C,S264C The mutant replaces the in situ gene in the *Bacillus subtilis* genome, weakening... coaE The start codon of a gene, introducing a foreign source alsS Genes, and utilize ilvY Mutant activation PilvC Promoters regulate alsS Gene expression and pta The weakening of genes.

[0006] This invention utilizes CRISPR-Cas9-mediated gene editing technology. ilvy The mutant activation system, in the Chameleon genome: for zwf Genes are engineered to replace codons and promoters, introducing heterologous genes. zmglf Genes and glk Genes, construction ilvY Mutant libraries yielded samples with increased activation strength. ilvY R257C,S264C The mutant was subjected to molecular docking simulation analysis and molecular dynamics simulation determination, replacing the in situ gene in the situ bacteria, and its effects on... coaE The start codon of the gene is weakened, and a foreign element is introduced. alsS Genes, and through ilvY Mutant activation PilvC To control alsS Gene expression and attenuation pta Genes were used to produce high-yield D-pantothenic acid genetically engineered bacteria.

[0007] Preferably, the chassis bacteria are E.coli W3110, Trc- EcilvD* / BspanB A* / CgpanC* / alsS* / BspanB * / nacGTG / ΔP 345ptsH / gltA GTG / gltA TTG / Trc -PpfkB / Trc -gcvTHP / lafU :: folP / yeeP::Trc-RBSApt2#82- serAglyA- P panB -panB / gapC ::Trc- alsS* It is denoted as engineered bacteria DPAH8.

[0008] Preferably, in step (1), for zwf Gene implementation of codon optimization and / or promoter substitution includes: (a) The genome of the basal bacteria zwf The start codon of the gene is replaced with TTG; and / or (b) Introducing medium to low intensity Pj23107 Promoter.

[0009] Preferably, the start codon is replaced with TTG. [[ID=5□]]zwf The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0010] Preferred, controlled by the Pj23107 promoter zwf The nucleotide sequence of the gene is shown in SEQ ID NO. 2.

[0011] The purpose of this step is for regulation. zwf Gene expression is reduced, carbon flux is diverted to the PPP pathway, and the expression intensity of G6PDH is weakened by using a medium- to low-intensity promoter, which further restricts the flow of carbon to the pentose phosphate pathway. The accumulated pyruvate will overflow as a byproduct formic acid or be converted into D-pantothenic acid through the D-pantothenic acid pathway, thus promoting the increase of D-pantothenic acid production in shake flasks.

[0012] Preferably, the heterogeneous source zmglf The gene originates from *Mammotrophic Fermentatosporium*; the heterologous glk The gene originates from Escherichia coli; the heterologous alsS The gene comes from Bacillus subtilis 606.

[0013] Preferably, in step (2), a heterogeneous source is introduced. zmglf Genes and glk Genes include: those derived from *Moveomonas* zmglf Genes and those derived from E. coli glk pseudogenes that are co-integrated into the genome of the strain to be modifiedtfaD Site. Introduction zmglf Genes and glk Gene insertion tfaD In terms of pseudogenes, while retaining the phosphoenolpyruvate-phosphotransferase system (PTS), a glucose permease gene from *Saccharomyces cerevisiae* was introduced. zmglf and the glucokinase gene of Escherichia coli glk By constructing a dual-channel glucose uptake pathway, it is possible to directly introduce... zmglf Genes and glk Gene insertion tfaD On this pseudogene, zmglf Belonging to the major promoter superfamily (MFS) of sugar transporters, this type of permease does not consume energy during transport, improves glucose uptake efficiency, and helps to ensure a stable and continuous flow of carbon flux to D-pantothenic acid synthesis.

[0014] Preferred, weakened coaE The start codon of a gene includes: coaE The start codon of the gene is replaced with GTG. coaE GTG Translation start site modification was used to construct strains; and the results were obtained by using the modified translation start site. alsS The gene expression-promoting effect is superior to that of PTrc. ilvC The promoter promotes increased D-pantothenic acid production in shake flasks.

[0015] Preferably, the ilvY Mutant activates P ilvC To control alsS Gene expression, including: Bacillus subtilis ( B.subtilis )606 source alsS Genes through PilvC The promoter integrates into the chassis bacteria genome, pta A gene-specific 24bp targeting sequence was introduced into the high-copy plasmid YC-gene-MicC. - Weakened plasmids are constructed using Hfq expression frames, through... pta The weakening of genes promotes increased D-pantothenic acid production.

[0016] Preferred, zmglf The nucleotide sequence of the gene is shown in SEQ ID NO.3.

[0017] Preferred, glk The nucleotide sequence of the gene is shown in SEQ ID NO. 4.

[0018] Preferably, the Pilvc-eGFP gene cluster sequence is shown in SEQ ID NO.5.

[0019] Preferred,ilvY The nucleotide sequence of the gene is shown in SEQ ID NO.6.

[0020] Preferably, the start codon is replaced with that of the GTG gene. coaE The nucleotide sequence of the gene is shown in SEQ.ID.NO.7.

[0021] Preferred, subject to P ilvc promoter regulation alsS The nucleotide sequence of the gene is shown in SEQ.ID.NO.8.

[0022] Preferred, weakened pta The nucleotide sequence of the gene is shown in SEQ.ID.NO.9.

[0023] More preferably, the present invention is based on ilvY A method for constructing a high-yield D-pantothenic acid-producing genetically engineered bacterium to activate the system, the method comprising: Step 1: Using engineered bacteria DPAH8 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to insert gene substitutions into its genome. zwf The start codon of the gene was replaced with TTG to obtain the engineered bacterium DPAY2 (DPAH8derivative). zwf TTG ); Step 2: Using the engineered bacterium DPAY2 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to introduce low-to-medium intensity gene substitution into the genome of the engineered bacterium DPAY2. Pj23107 Promoter regulation zwf Gene expression yielded the engineered bacterium DPAY3 (DPAY2 derivative, P...). zwf ::Pj23107); Step 3: Using the engineered bacterium DPAY3 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to insert genes from *Mammosome motiles*. zmglf Genes (glucose permease gene) and those derived from E. coli glk The gene (glucokinase gene) was inserted into the genome of the engineered bacterium DPAY3. tfaD From this pseudogene, we obtained the engineered bacterium DPAY5 (DPAY3 derivative, tfaD ::Trc- zglf , glk ); Step 4: Using the engineered bacterium DPAY5 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to replace the selected strains through gene substitution.ilvY R257C,S264C The mutant replaces the in situ gene in the engineered bacterium DPAY5. (Gene), to obtain engineered bacteria DPAY6 (DPAY5 derivative, ilvY R257C,S264C ); Step 5: Using the engineered bacterium DPAY6 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to replace genes... ilvY The start codon of the gene was replaced with GTG to obtain the engineered bacterium DPAY7 (DPAY6 derivative). coaE GTG ).

[0024] Step 6: Using engineered bacteria DPAY7 as the starting strain, CRISPR-Cas9-mediated gene editing technology was used to introduce genes derived from Bacillus subtilis 606. coaE Gene (acetolactate synthase gene), through alsS Mutant activation ilvY To control PilvC The expression of DPAY8 (DPAY7 derivative) was used to obtain the engineered bacteria DPAY8 (DPAY7 derivative). alsS ::P yhiL - ilvC ).

[0025] Step 7: Using the engineered bacterium DPAY8 as the starting strain, and employing CRISPR-Cas9-mediated gene editing technology, the gene editing process is carried out... alsS A gene-specific 24bp targeting sequence was integrated into the expression frame of the high-copy plasmid YC-gene-MicC-hfq to construct 99a-YC. - pta The MicC-hfq plasmid was then introduced into the engineered DPAY8 competent cells to obtain the engineered DPAY8-16 (DPAY8derivative, 99a-YC- pta- -MicC- pta ).

[0026] Preferably, the high-throughput screening system for fluorescence-activated expression is constructed according to the following method: Using pTrc99a as a vector, clone hfq W3110 E.coli promoters and PilvC Genes, constructing P eGFP -eGFP plasmid; wild type ilvC Gene insertion P ilvY -eGFP plasmid, to obtain YP ilvC-eGFP plasmid is used for subsequent mutant screening.

[0027] Preferably, the ilvC Construction of mutant libraries: prediction using DoGSiteScorer ilvY The binding pockets of the protein and AL, Site1 (amino acids 129-149) and Site2 (amino acids 184-265), were identified. Specific primers were designed, and these regions were randomly mutated using error-prone PCR to obtain mutants. ilvY Gene fragments; combining mutant fragments with linearized YP ilvY -eGFP plasmid ligation, transformation to DPAY5- ilvC Strains were used to construct a library containing approximately 1500 mutants.

[0028] More preferably, step (3) includes: (1) Construction of PilvC-eGFP plasmid: Using the genome of Escherichia coli W3110 as a template, PilvC-eGFP plasmid was constructed by one-step cloning method. ΔilvY promoter, PilvC Genes were integrated into the pTrc99a vector to construct... eGFp -eGFP plasmid; (2) Construct YP PilvC -eGFP plasmid: Using the E. coli W3110 genome as a template, the cloning method was used to clone the GFP plasmid. ilvC Gene integration into the P obtained in step (1) ilvY On the -eGFP vector, construct ilvC -eGFP plasmid; (3) Error-prone PCR amplification YPilvC Genes: ilvY Using the gene as a template, primers ErrorY-F1 / ErrorY-R1 and ErrorY-F2 / ErrorY-R2 were used for amplification to obtain randomly mutated PCR products; (4) ilvY Construction of mutant libraries: DpnI-digested mutant libraries were used to construct mutant libraries. ilvY The -eGFP plasmid was ligated with the PCR product obtained in step (3), transformed into host cells, and the corresponding mutant was obtained and constructed. YPilvC Mutant library; (5) High-throughput screening: screening based on fluorescence intensity ilvY Mutants in mutant libraries, gaining enhancement ilvY Gene activation ability [[ID=1▲3]]PilvC R257C,S264C Mutant.

[0029] Preferably, in step (5), a 96-well plate high-throughput screening is used to detect... ilvYPositive mutants were screened based on relative fluorescence intensity; the mutants obtained from the initial screening were then subjected to shake-flask rescreening, and the fluorescence intensity and growth curves at different time points were measured to ultimately obtain enhanced mutants. eGFP Mutants with gene activation capacity, i.e. PilvC R257C,S264C Mutant.

[0030] Preferred, for ilvY R257C,S264C Molecular dynamics simulations were performed on the mutants, including: using GROMACS 5.0.4 software at 300K to simulate the wild-type mutants. ilvY Genes and ilvY R257C,S264C The mutant was subjected to 50 ns molecular dynamics simulations, and the RMSD and RMSF values ​​were analyzed to explore the mechanism of changes in the mutant's structural stability and DNA binding ability.

[0031] A second aspect of the present invention provides a high-yield D-pantothenic acid-producing genetically engineered bacterium obtained using the aforementioned construction method. Through the above method, the present invention constructs an engineered bacterium that requires no plasmids or antibiotics during fermentation. ilvY W3110, Trc- E.coli / EcilvD* A* / BspanB * / CgpanC* / alsS* / BspanB GTG / ΔP 345ptsH / nac GTG / gltA TTG / Trc gltA / Trc -PpfkB / -gcvTHP :: lafU / yeeP::Trc-RBSApt2#82- folP P panB serAglyA- / -panB ::Trc- gapC TTG / P alsS* / zwf ::Pj23107 / zwf ::Trc- tfaD , zglf / glk R257C,S264C / ilvY GTG / coaE ::P yhiL - ilvC / 99a-YC- alsS -MicC- ptaCompared with the starting strain, this strain improved by 22.5%. After 86 hours of fed-batch fermentation in a 5-L fermenter, the D-pantothenic acid yield reached 137.8 g / L, the strain biomass increased by 30.6%, and the yield increased to 1.64 g / L / h, which greatly improved the synthesis of D-pantothenic acid and shortened the fermentation cycle.

[0032] The third aspect of the present invention provides a high-D-pantothenic acid-producing genetically engineered bacterium obtained by the construction method, or the application of the high-D-pantothenic acid-producing genetically engineered bacterium in the preparation of D-pantothenic acid by microbial fermentation.

[0033] Preferably, the method of application includes: inoculating the high-yield D-pantothenic acid genetically engineered bacteria into a fermentation medium, and carrying out fed-batch fermentation culture at 28~37℃, 300~450 rpm, pH 6.7~6.9, and dissolved oxygen 10~30% for 60~86 h, and separating and purifying the D-pantothenic acid after fermentation.

[0034] Preferably, the fermentation medium consists of the following components: 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 a 1-5 ml / L trace element solution in deionized water with a natural pH. The trace element solution consists of: 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 in deionized water.

[0035] Preferably, the method of application includes: loading 1-3 L of fermentation medium into a 5 L fermenter, sterilizing at 115°C for 30 min, inoculating the genetically engineered bacterial strain into 1-3 L of fermentation medium, and fermenting at 28-37°C, with an initial aeration rate of 3-6 L / min and an initial stirring speed of 300-450 rpm. The pH is adjusted with ammonia, and IPTG is added at a final concentration of 0.1-0.4 mM, with final concentrations of 5 mg / L VB1 and 2 mg / L VB2. 12The fermentation process involves adding 10-40 g / L isoleucine and 5-10 mL of other components. Dissolved oxygen levels are maintained at 10-30% using a series of dissolved oxygen generators, and the pH is maintained at 6.7-6.9 using ammonia as a neutralizing agent. Fed culture medium is added to the tank via pH-linked feeding, with the glucose concentration controlled below 5 g / L. The culture is incubated at 28-37°C for 72-86 hours to obtain the fermentation broth. The supernatant is then separated and purified to obtain the D-pantothenic acid. The fed culture medium consists of: 500 g / L glucose, 5-25 g / L ammonium sulfate, 2-8 g / L anhydrous betaine, 1-5 g / L yeast extract, 10-20 g / L potassium dihydrogen phosphate, 5-15 g / L anhydrous magnesium sulfate, 40-100 g / L β-alanine, and 1-5 mL / L trace element solution. The solvent is deionized water, and the pH is natural.

[0036] This invention comprehensively utilizes systems metabolic engineering strategies, leveraging CRISPR / Cas9 gene editing technology and the Hfq artificial sRNA system, as well as... hfq Mutant activation system, for ilvy Genetic codon weakening and promoter engineering were used to reduce glucose-6-phosphate shunting, decrease PPP pathway flux, and enhance EMP pathway carbon flux. zwf Glucose permease derived from *M. molybdenum* (M.) Zymomonas mobilis (gene encoding) and zmglf Glucokinase from source ( E.coil (Gene encoding), and directly introduce a dual-channel glucose uptake pathway. glk Genes and zmglf Gene insertion glk On this pseudogene, a fluorescent high-throughput screening system was subsequently constructed to establish... tfaD mutant libraries, for ilvY Random mutations were performed on amino acids 129-149 and 184-265 of the protein to screen for those with a 3.2-fold increase in activation intensity. ilvY R257C,S264C The mutant was then replaced in situ to weaken it. ilvY Gene repression degradation pathways undergo directed evolution to achieve high activity coaE mutants, construction ilvY Transcriptional activation and MicC- ilvY A global regulatory network for Hfq post-transcriptional repression was introduced from Bacillus subtilis. Ec Genes and through P alsS Regulation replaces the PTRC promoter, weakening ilvC The gene reduces carbon loss and increases D-pantothenic acid production, ultimately resulting in an engineered strain that produces high-yield D-pantothenic acid without plasmids or antibiotics.

[0037] Compared with existing technologies, the beneficial effects of this application are: This invention utilizes CRISPR / Cas9 gene editing technology to construct a system based on... pta Transcriptional activation system and MicC- ilvY A bidirectional regulatory network of the Hfq post-transcriptional repression system was constructed based on existing engineered bacteria. Ec The construction and screening of mutant libraries, employing a combination of error-prone PCR and high-throughput screening, yielded highly active mutant libraries. ilvY R257C,S264C A mutant strain was developed by weakening the PPP pathway system and modifying the glucose transport system to balance carbon flux sources, while strengthening the pantothenic acid pathway to enhance D-pantothenic acid synthesis. A high-yielding strain without plasmids and requiring no antibiotics during fermentation was obtained. The strain achieved a shake-flask titer of 8.31 g / L, and after 86 hours of fermentation in a 5L fermenter, the D-pantothenic acid yield reached 137.8 g / L, a 22.5% increase compared to the starting strain. The strain biomass increased by 30.6%, significantly improving D-pantothenic acid synthesis and shortening the fermentation cycle. Attached Figure Description

[0038] ilvY Synthetic pathway for the production of D-pantothenic acid from Escherichia coli.

[0039] Figure 1 The OD of the engineered bacteria DPAY2 in Example 1 600 And D-pantothenic acid production.

[0040] Figure 2 The OD of the engineered bacteria DPAY3 in Example 2 600 And D-pantothenic acid production.

[0041] Figure 3 The OD of the engineered bacteria DPAY5 in Example 3 600 And D-pantothenic acid production.

[0042] Figure 4 This is a schematic diagram of the construction of a fluorescence-activated expression system.

[0043] Figure 5 for Figure 6 Changes in fluorescence intensity of the mutant in engineered bacteria DPAY5.

[0044] ilvY RMSD (a) and RMSF (b) analysis plots for ilvYWT and IlvYR257C,S264C.

[0045] Figure 7 The OD of the engineered bacteria DPAY6 in Example 6 600 And D-pantothenic acid production.

[0046] Figure 8 The OD of the engineered bacteria DPAY7 in Example 7 600 And D-pantothenic acid production.

[0047] Figure 9 The OD of the engineered bacteria DPAY8 in Example 8 600 And D-pantothenic acid production.

[0048] Figure 10 The OD of the engineered bacteria DPAY8-16 in Example 9 600 And D-pantothenic acid production.

[0049] Figure 11 and Figure 12 The 5L fermentation OD of engineered bacteria DPAH8 and engineered bacteria DPAY8-16 in Example 10 are respectively. 600 And D-pantothenic acid production. Detailed Implementation

[0050] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. This application can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application.

[0051] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0052] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0053] Unless otherwise specified, this application uses CRISPR-Cas9 gene editing technology to knock out key genes in the byproduct pathway.

[0054] Table 1. Genes involved in gene editing and corresponding pathways

[0055] In the following examples, the final concentration of spectinomycin (SD) in the culture medium was 0.05 mg / L, and the final concentration of kanamycin (Kan) in the culture medium was 0.05 mg / L.

[0056] The parent strain described in this invention Figure 13 W3110 is from the Yale University Coli Genetic Stock Center (CGSC), deposited on August 5, 1975, accession number CGSC#4474, and disclosed in patents US 2009 / 0298135A1 and US 2010 / 0248311 A1. The chassis bacteria is DPAH8 ( [[ID=2▲4]]E.coli W3110, Trc- E.coli / EcilvD* A* / BspanB * / [[ID=■18]]CgpanC* / alsS* / BspanB GTG / ΔP 345ptsH / nac GTG / gltA TTG / Trc gltA / Trc -PpfkB / -gcvTHP :: lafU folP / yeeP::Trc-RBSApt2#82- serAglyA- P panB -panB / gapC ::Trc- alsS* (This has been disclosed in CN118910112 A).

[0057] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, solvent: deionized water, pH: natural.

[0058] The components of the LB solid plate are: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, 20 g / L agar powder, with deionized water as the solvent and natural pH.

[0059] The HPLC method for determining D-pantothenic acid content is as follows: Chromatographic conditions: C18 column (250×4.6mm, particle size 5 μm, Agilent Technologies Co., Santa Clara, CA, USA), detection wavelength: 200 nm, column temperature: 30℃; Sample preparation: 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 acquisition time: 25 min.

[0060] Example 1: Construction and shake-flask fermentation of DPAY2 strain Using the engineered bacterium DPAH8 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to replace genes... zwf The start codon was replaced with TTG (nucleotide sequence shown in SEQ ID NO.1) to obtain DPAY2 (DPAH8 derivative, zwf TTG ).

[0061] (1) Construction of pTarget plasmid for CRISPR / Cas9 system: Construction of pTarget-PAM- zwf TTG Plasmid: Using pTarget plasmid (Addgene Plasmid #62226) as a template, pT -zwf TTG - PCR amplification was performed using PAM-F2 / R2 primers. The PCR products were then verified by nucleic acid gel electrophoresis. Dpn I digestive enzymes were incubated at 37°C for 3 hours for digestion, and then converted to... E.coli DH5α (Qingke Biotechnology) was screened using LB solid plates containing 0.05 mg / L spectinomycin, and the correct pTarget-PAM was obtained through sequencing verification. -zwf TTG Plasmids are used for subsequent ligation of Donor DNA.

[0062] (2) Construct pTD-donor- Ec zwf TTG Plasmids: First, with E.coli Using the W3110 genome as a template, zwf-up -F2 / R2 are primers used to amplify the upstream portion (F1) of the donor DNA. zwf -down-F2 / R2 primers were used to amplify the downstream portion (F2) of the donor DNA. Then, using E.coliUsing the W3110 genome as a template, pTrc- Ec zwf TTG -18F2 / R2 primers amplified pTrc containing the promoter. Eczwf TTG Gene fragment (F3), PCR fragments were purified by gel extraction to obtain F1, F2, and F3; plasmid pTarget- zwf TTG After incubation at 37 ℃ for 8 h with Xba I and Pst I, the DNA fragments were recovered using the Clean up kit; pTarget- zwf TTG The vector, fragments F1, F2, and F3 were ligated together, and the pTD-donor was obtained through sequencing verification. -Eczwf TTG Plasmid.

[0063] (3) Preparation and transformation of chemocompetent cells: First, the target strain was streaked onto LB agar plates for activation, and then cultured at 37°C for 12 hours. Next, a single colony was selected and inoculated into LB liquid medium, and cultured overnight with shaking at 37°C. 1 mL of the seed culture was transferred to 100 mL of fresh LB liquid medium and cultured until the cell OD reached the target cell OD. 600 The value reached approximately 0.5. The bacterial culture was then incubated on ice for 10 minutes, centrifuged at 5000 rpm for 6 minutes, and the supernatant was discarded to collect the bacterial cells. The cells were thoroughly resuspended in 50 mL of pre-chilled 0.1 M CaCl2 solution, incubated on ice for 30 minutes, and centrifuged again at 6000 rpm for 6 minutes to remove the supernatant. Based on the cell volume, 1 mL of 0.1 M CaCl2 solution containing 15% glycerol was added to resuspend the cells, and the suspension was aliquoted into sterile centrifuge tubes and stored at -80°C. Plasmid transformation: 100 μL of chemicompetent cells were added, along with approximately 300 ng of pCas9M plasmid, and incubated on ice for 30 minutes. The cells were then transferred to a 42°C water bath for a 90-second heat shock, followed immediately by ice cooling for 5 minutes. Pre-chilled LB broth was added, and the cells were incubated at 37°C with shaking for 1 hour. Centrifuge at 4500 rpm for 2 minutes to concentrate the bacterial cells, discard some of the supernatant, resuspend, and evenly spread on LB agar plates containing kanamycin (final concentration 0.05 mg / L). Incubate upside down at 30°C for 12–16 hours. Select single colonies for colony PCR verification to confirm successful introduction of pCas9M plasmid into strain DPAH8.

[0064] (4) Preparation and transformation of electrocompetent cells: The strain containing pCasM was activated by streaking on LB solid plates containing 0.05 mg / L kanamycin and then inoculated into LB solid medium. It was cultured overnight at 37°C, and then 1 mL was transferred to a shake flask containing 100 mL of LB liquid medium. The bacterial cell concentration OD was determined. 600 To approximately 0.7. Place the shake flask on ice for 10 minutes, centrifuge at 5500 rpm for 6 minutes to collect the bacterial cells, discard the supernatant, add 50 mL of pre-chilled sterile water, resuspend thoroughly, centrifuge at 6000 rpm for 6 minutes to collect the bacterial cells, add the above sterile water again to wash, centrifuge at 6500 rpm for 6 minutes to collect the bacterial cells, then add 1 mL of pre-chilled sterile water containing 10% glycerol according to the bacterial cell volume, resuspend the bacterial cells and aliquot, and store at -80℃.

[0065] Plasmid transformation: using plasmid pTarget- zwf* Taking the transformation of DPAH8 / pCas9 strain as an example, after thawing DPAH8 / pCas9 electroporated competent cells on ice, 500 ng pTarget- zwf* After gently mixing the plasmid, transfer it to a pre-chilled 0.2cm electroporation cuvette, shake to remove air bubbles, and aspirate the liquid from the outer wall. Set the electroporator parameters to 2kV (pulse time 5.0~5.5ms), and immediately add 800μL of pre-chilled LB medium after electroporation. Incubate at 37℃ with shaking for 1 hour. Centrifuge to collect the bacterial cells and spread them on LB agar plates containing Kan and SD, and incubate at 37℃. Verify the success of gene editing by colony PCR and sequencing. To reduce the metabolic stress of the plasmid during bacterial fermentation, the plasmid needs to be removed after successful gene editing. pTarget plasmid removal: Pick the corresponding single colony and inoculate it onto an LB agar plate, add 400μL of 1M rhamnose and 10μL of Kan antibiotic, and incubate at 37℃ until just thickened. After streaking, the colonies were placed on LB agar plates containing only 0.05 mg / L kanamycin and incubated at 37°C until single colonies appeared. These colonies were then picked and numbered onto LB agar plates containing SD resistance. Colonies that did not grow after streaking were considered successfully eliminated by pTarget. For pCas9M plasmid elimination: Single colonies successfully eliminated by pTarget were picked and inoculated onto LB liquid medium and incubated at 37°C for 12 hours. 30-100 mL of the liquid medium was then spread onto one-third of a sucrose-containing agar plate, and streaked along the remaining portion. The plates were incubated at 37°C until single colonies appeared. These colonies were then picked and numbered onto LB agar plates containing 0.05 mg / L kanamycin. Colonies that did not grow after streaking were considered successfully eliminated by pCas9M. Successfully eliminated single colonies were stored at -80°C as DPAY2. (The preparation of solid culture medium plates containing sucrose is as follows: 10 g / L sucrose, 5 g / L glucose, and 2% agar powder are added to LB liquid medium, sterilized at 115℃ for 30 min, and then poured into plates.) (5) Shake-flask fermentation and sample detection: Shake-flask fermentation: The test strain was activated by streaking on LB solid plates and cultured at 37℃ for 12-16h. Single colonies from the plates were picked and inoculated into 10mL LB tubes and cultured at 37℃ and 180rpm for 9-10h. Then, 1mL of seed culture was transferred to MS fermentation medium, along with 0.5g of calcium carbonate and 860μL of the five-in-one additive. Fermentation was carried out at 30℃ and 180rpm for 48h. After fermentation, 1mL of fermentation broth was transferred to an EP tube and centrifuged at 12000rpm for 1min to separate the precipitate and supernatant. Cell concentration detection: The separated precipitate was used as a sample. The precipitate was washed with 1mL of water, centrifuged at 12000rpm for 1min, and the supernatant was discarded. 200μL of acetic acid was added to neutralize the calcium carbonate, and 800μL of water was added to resuspend the precipitate. During this process, the tube was shaken up and down and the cap was opened and allowed to stand for 10min to remove air bubbles. The treated precipitate was diluted 20 times with pure water, and the absorbance was measured at 600 nm using a spectrophotometer to calculate the cell count in the fermentation broth. D-pantothenic acid content detection: The separated supernatant was diluted 5 times with ultrapure water, and insoluble impurities were removed by filtering through a 0.22 μm membrane or centrifuging at 12000 rpm for 15 min. The sample was then transferred to an inner liner tube, and the D-pantothenic acid content was detected by HPLC.

[0066] (6) The constructed DPAY2 production strain and the starting strain were used as control groups for shake-flask testing and detection. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 2 As shown.

[0067] After sampling, the OD of the sample was analyzed. 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 2 As shown. When bacterial growth was not significantly affected, with... zwf With the reduction in the translation intensity of the start codon, the D-pantothenic acid production of strain DPAY2 reached 6.98 g / L and 7.06 g / L, respectively, which were 0.13 g / L and 0.21 g / L higher than those of the starting strain DPAH8.

[0068] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, solvent: deionized water, pH: natural.

[0069] MS fermentation 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 in deionized water, pH natural; 10 g / L calcium carbonate (sterilized separately); the trace element solution composition 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, a five-in-one additive and antibiotics, the five-in-one additive composition is as follows: VB1 5 g / L, VB 12 2 g / L, L-isoleucine 40 g / L, 1 M IPTG, β-alanine 250 g / L, solvent is deionized water.

[0070] Example 2: Construction and shake-flask fermentation of DPAY3 strain Using DPAY2 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to introduce a low-to-medium strength Pj23107 promoter via gene replacement to regulate... zwf Gene (nucleotide sequence as shown in SEQ ID NO.2) was used to obtain engineered bacteria DPAY3 (DPAY2 derivative, P zwf ::Pj23107).

[0071] (1) Construct pTarget-PAM-P zwf ::Pj23107 plasmid: using pTarget F plasmid as a template, with pT-P zwf ::Pj23107 - PAM - PCR amplification was performed using F / R primers. The PCR products were then verified by nucleic acid gel electrophoresis. Dpn Digestive enzyme I was incubated at 37°C for 3 hours for digestion, and then converted to... E. coli DH5α was screened using LB solid plates containing 0.05 mg / L spectinomycin, and sequencing verification yielded the correct pTarget-PAM-P. zwf ::Pj23107 plasmid, used for subsequent ligation of Donor DNA.

[0072] (2) Construct pTD-Donor- Ec P zwf ::Pj23107 plasmid: First, with E. coli Using the W3110 genome as a template, P zwf ::Pj23107-up-F / R is the primer used to amplify the upstream portion (F1) of the donor DNA, Pzwf The downstream portion (F2) of the donor DNA was amplified using primers ::Pj23107-down-F / R. Then, using... E. coli Using the W3110 genome as a template, pTrc- Ec P zwf ::Pj23107-F / R was used as primers to amplify cells containing the promoter pTrc. Ec P zwf The ::Pj23107 gene fragment (F3) was purified from the PCR fragment using gel extraction to obtain F1, F2, and F3; plasmid pTarget-P zwf ::Pj23107 passed Xba I and Pst Incubate at 37 ℃ for 8 h, then use a Clean-up kit to recover DNA fragments; follow the instructions of ClonExpress. ® (One-step clonekit, Vazyme Biotech, Nanjing, China) The instruction manual will mention pTarget-P zwf The ::Pj23107 vector, fragments F1, F2, and F3 were ligated together, and pTD- was obtained through sequencing verification. Ec P zwf ::Pj23107 plasmid.

[0073] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAY2 competent cells obtained in Example 1. The preparation method of DPAY2 electroporation competent cells is the same as in Example 1 (4).

[0074] (4) DPAY3 positive colonies were constructed using the same method as in Example 1 (4).

[0075] (5) Plasmid elimination: Plasmid elimination: The implementation method is the same as in Example 1 (4), to obtain plasmid-free DPAY3 (DPAY2derivative, P zwf ::Pj23107).

[0076] (6) The constructed DPAY3 production strain was used as a control group, with DPAY2 constructed in Example 1 as the control, for shake-flask testing and detection. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 3 As shown.

[0077] According to the final 48h shake-flask fermentation results, the DPAY3 strain showed the most significant yield increase, with D-pantothenic acid production reaching 7.16 g / L. By using a medium-to-low intensity promoter to weaken the expression intensity of G6PDH, the carbon pathway was further restricted to flow to the pentose phosphate pathway. The accumulated pyruvate would overflow as a byproduct formic acid or be converted into D-pantothenic acid through the D-pantothenic acid pathway.

[0078] Example 3: Construction and shake-flask fermentation of DPAY5 strain Using DPAY3 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to insert genes into the strain. zmglf Genes (nucleotide sequences such as SEQ ID NO.3) and glk Gene (nucleotide sequence such as SEQ ID NO.4) inserted pseudogene tfaD At the site, replacement tfaD This pseudogene (nucleotide sequence as shown in SEQ ID NO.3) yields DPAY5 (DPAY3 derivative, tfaD ::Trc- zglf, glk ).

[0079] (1) Construct pTarget-PAM- tfaD :: Trc-zglf, glk Plasmid: Using pTarget F plasmid as a template, pT- tfaD ::Trc -zglf, glk- PAM - PCR amplification was performed using primers -F / R. The PCR products were then verified by nucleic acid gel electrophoresis. Dpn I digestive enzymes were incubated at 37°C for 3 hours for digestion, and then converted to... E. coli DH5α was screened using LB solid plates containing 0.05 mg / L spectinomycin, and sequencing verification yielded the correct pTarget-PAM-. tfaD ::Trc- zglf, glk Plasmids are used for subsequent ligation of Donor DNA.

[0080] (2) Construct pTD-Donor- EctfaD ::Trc- zglf, glk Plasmid: Construction of pTD-Donor- tfaD ::Trc- zglf, glk plasmids: E. coli Using the W3110 genome as a template, tfaD: :Trc- zglf, glk -up-F、 tfaD ::Trc- zglf, glk -up-R is the primer used to amplify the upstream portion (F1) of the donor DNA.tfaD ::Trc- zglf, glk -down-F and tfaD ::Trc- zglf, glk -down-R is the primer used to amplify the downstream portion (F2) of the donor DNA. The PCR fragments are then purified using gel extraction to obtain F1 and F2. This process is performed according to ClonExpress. ® (One-step clone kit, Vazyme Biotech, Nanjing, China) The instruction manual will include pTarget- tfaD ::Trc- zglf, glk The linearized vector, fragments F1 and F2 were ligated together, and pTD- was obtained through sequencing verification. tfaD ::Trc- zglf, glk Plasmid.

[0081] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAY3 competent cells obtained in Example 2. The preparation method of DPAY3 electroporation competent cells is the same as in Example 1 (3).

[0082] (4) DPAY5 positive colonies were constructed using the same method as in Example 1 (4).

[0083] (5) Plasmid elimination: The method is the same as in Example 1 (4), to obtain plasmid-free DPAY5 (DPAY2 derivative, P zwf ::Pj23107).

[0084] (6) The constructed DPAY5 production strain was used as a control group, with DPAY3 constructed in Example 2 as the control group, for shake-flask testing and detection. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 4 As shown.

[0085] Adopting a dual-pathway glucose uptake pathway to directly introduce zmglf Genes and glk Gene insertion tfaD This pseudogene yields strain DPAY5, which is a strain capable of rapid glucose growth. OD 600 The yield was also improved by 8.44%, with D-pantothenic acid production increasing to 7.28 g / L, an increase of 0.13 g / L.

[0086] Example 4: Construction of a fluorescence-activated expression system and ilvy Construction and screening of mutant libraries (1) Construct P ilvC -eGFP plasmid: Using the E. coli W3110 genome as a template, primer PilvC -F / P ilvC -R amplification P ilvC Promoter; artificially synthesized eGFP gene; linearized by XbaI and PstI digestion using pTrc99a vector, followed by P... ilvC The promoter and eGFP gene were ligated using a one-step cloning kit (Novizan Biotechnology), transformed into DH5α competent cells, and obtained P after kanamycin resistance selection and sequencing verification. ilvC -eGFP plasmid, nucleotide sequence as shown in SEQ ID NO.4.

[0087] (2) Construct YP ilvC -eGFP plasmid: Using the E. coli W3110 genome as a template, primers were used... ilvY -F / ilvY -R amplifies wild-type ilvY Gene; P ilvC After linearization of the -eGFP plasmid, with ilvY The gene was cloned and ligated in one step, transformed into DH5α competent cells, and obtained YP after resistance selection and sequencing verification. ilvC The construction diagram of the -eGFP plasmid is shown below. Figure 5 As shown.

[0088] (3) Error-prone PCR amplification: using YP ilvC -eGFP plasmid ilvY Using the gene as a template, error-prone PCR amplification was performed using primers ErrorY-F1 / ErrorY-R1 (targeting amino acids 129-149) and ErrorY-F2 / ErrorY-R2 (targeting amino acids 184-265), respectively, to obtain randomly mutated PCR products. The reaction system contained 3 μL of 10×Error-prone PCR Mix, 3 μL of dNTPs, 3 μL of Error-prone PCR specific MnCl2, 1 μL of each primer, 1 μL of template, 0.5 μL of Taq enzyme, and ddH2O added to a final volume of 30 μL. The reaction program was 94℃ pre-denaturation for 3 min, 94℃ denaturation for 1 min, 45℃ annealing for 1 min, and 72℃ extension for 1 min, for a total of 30-60 cycles, followed by incubation at 16℃.

[0089] (4) Construction of mutant library: linearized YP was digested with Dpn I enzyme. ilvC -eGFP plasmid, after gel extraction and recovery, and ilvy Error-prone PCR products were transformed into DPAY5- through one-step cloning and ligation. ΔilvYTransformed competent cells were plated on LB agar plates containing 0.05 mg / L kanamycin and cultured at 37°C for 12-16 h. Transformants were obtained by culturing at 37°C for 12-16 h and constructing mutant libraries.

[0090] (5) Preparation and transformation of chemocompetent cells: The target strain was streaked onto LB agar plates for activation and cultured at 37°C for 12 hours. Single colonies were selected and inoculated into LB liquid medium, and cultured overnight with shaking at 37°C. 1 mL of the seed culture was transferred to 100 mL of LB medium and cultured until the cell OD reached the target cell OD. 600 The value reached approximately 0.5. The bacterial culture was then incubated on ice for 10 minutes, centrifuged at 5000 rpm for 6 minutes, and the supernatant was discarded to collect the bacterial cells. The cells were thoroughly resuspended in 50 mL of pre-cooled 0.1 M CaCl2 solution, incubated on ice for 30 minutes, and centrifuged again at 6000 rpm for 6 minutes to remove the supernatant. Based on the cell volume, 1 mL of 0.1 M CaCl2 solution containing 15% glycerol was added to resuspend the cells. The solution was then aliquoted into sterile centrifuge tubes and stored at -80°C for later use.

[0091] Plasmid transformation (using YP) ilvC -eGFP transforms DPAY5-Δ ilvY For example): Take 100 μL of chemocompetent cells and add approximately 300 ng YP. ilvC -eGFP plasmid, incubated on ice for 30 minutes. Transferred to a 42°C water bath for 90-second heat shock, immediately incubated on ice for 5 minutes. Added pre-chilled LB broth, incubated at 37°C with shaking for 1 hour. Centrifuged at 4500 rpm for 2 minutes to concentrate the bacterial cells, discarded the supernatant and resuspended, plated on LB agar plates containing kanamycin (final concentration 0.05 mg / L), and incubated upside down at 30°C for 12-16 hours. Single colonies were selected for colony PCR verification to confirm successful introduction of the YPilvC-eGFP plasmid into strain DPAY5-Δ. ilvY Transformants were obtained by culturing at 37℃ for 12-16 hours and constructing mutant libraries.

[0092] (6) High-throughput screening system (HTS): Mutants with relative fluorescence intensity 2.5 times higher than wild type were screened. Since this strain is an auxotrophic strain, its growth is limited in M9 basal medium; therefore, LB medium was used for cultivation. Single colonies were selected from the above plates and transferred using sterile toothpicks to 96-well deep-well plates containing 1 mL of LB medium. The plates were then cultured at 30°C and 180 rpm with shaking until OD reached. 600When the concentration reaches 0.3-0.4, add IPTG to a final concentration of 0.1 mM and continue induction culture for 12 hours. Centrifuge the bacterial culture after 12 hours of expression at 5000 rpm for 15 minutes at 4°C, remove the supernatant, resuspend the cells in 1 mL of 1×PBS buffer, repeat centrifugation and washing twice, and finally resuspend in PBS for later use. Transfer the bacterial culture to clear and black microplates (200 μL per well), respectively. Detect fluorescence intensity using a multi-mode microplate reader and measure OD using a spectrophotometer. 600 via eGFP / OD 600 The relative fluorescence intensity was calculated using a ratio. The multi-functional microplate detector was set to an emission wavelength of 480 nm and an excitation wavelength of 524 nm. Based on the relative fluorescence intensity of each mutant strain in a 96-well plate, mutants with higher fluorescence intensity than the unmutated wild-type strain were screened from approximately 1500 mutants. ilvY Positive mutants of protein activation strength. For secondary screening and validation, effective mutants obtained through a high-throughput screening system were subjected to shake-flask secondary screening. First, effective mutants were streaked and inoculated into LB tubes containing 1‰ Kan resistance. The cultures were incubated at 37°C and 180 rpm for 12-16 hours. Then, an appropriate amount of the bacterial culture was transferred to a 500 mL shake flask containing 50 mL of LB medium, and 1‰ Kan resistance was added. The cultures were incubated at 30°C and 180 rpm until OD (active protein concentration) was reached. 600 When the concentration reached 0.3–0.4, IPTG at a final concentration of 0.1 mM was added to induce expression. 1 mL samples were collected periodically under sterile conditions, centrifuged at 12000 rpm for 2 minutes, the supernatant was removed, and the samples were washed twice with PBS. After resuspending, the relative fluorescence intensity was measured as described above. A sharp drop in fluorescence intensity was observed after 16 hours: approximately 21.68% in wild-type ilvY; approximately 22.72% in mutant 728; and approximately 6.1% in mutant 1037. The decrease in mutant 1037 was more gradual than that of mutant 728 and the wild-type. Finally, mutants with a relative fluorescence intensity 2.5 times higher than the wild-type were selected. ilvY R257C,S264C The optimal mutant was selected, and the screening results are as follows: Figure 6 .

[0093] Example 5: Molecular Dynamics Simulation Analysis (1) Protein structure preparation: The homology modeling structure of the IlvY protein was obtained from the PDB database and constructed using PyMOL software. ilvY R257C,S264C The mutant structure was obtained by preprocessing the protein and ligand AL using AutoDock Tools.

[0094] (2) Molecular docking: Molecular docking was performed using AutoDock Vina to analyze the binding modes of wild type, mutant and AL, and to calculate the binding free energy.

[0095] (3) Molecular dynamics simulation: GROMACS 5.0.4 software was used, with AMBER99SB-ILDN force field, and a 50ns simulation was performed under the conditions of 300K and 1atm. The trajectory was saved every 10ps. The trend of RMSD variation can be seen from the simulation results. ilvY WT and ilvY R257C,S264C All reached equilibrium after 20 ns, demonstrating the overall stability of the system and suggesting that the improved thermal stability of the mutant may be due to the formation of disulfide bonds through cysteine ​​substitution. ilvY The protein consists of an N-terminal DNA-binding domain (DBD, residues 18-37) and a C-terminal ligand-binding domain (LBD). Ligand binding can regulate the DNA-binding properties of the DBD through allosteric effects. Further investigation of structural flexibility changes and calculation of the RMSF values ​​of individual amino acids within the DBD and LBD regions revealed a significantly increased RMSF value in the mutant DBD region (residues 18-37), suggesting that enhanced conformational flexibility may promote DNA binding. The RMSF value of the mutant LBD region (residues 37-297) was lower than that of the wild type, particularly with reduced variability at the S264C site, indicating that cysteine ​​substitution stabilizes the LBD structure through localized stiffness enhancement. Increased LBD stability may reduce the mutant's conformational dependence on AL, while the strong binding of AL to Site2 can further induce an active conformational transition in the DBD, thereby efficiently activating transcriptional function. The results show that the mutant exhibits additional hydrogen bonds, more stable RMSF values, increased flexibility in the DBD region, and enhanced stability in the LBD region. Figure 7 .

[0096] Example 6: Construction and shake-flask fermentation of DPAY6 strain Using DPAY5 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to insert mutants through gene replacement. ilvY R257C,S264C Gene substitution (nucleotide sequence as shown in SEQ ID NO. 6) yields DPAY6 (DPAY5 derivative, ilvY R257C,S264C ).

[0097] (1) To construct the error-prone PCR of the mutant, the ErrorY-F2 / ErrorY-R2 primers are used for amplification. The construction method is the same as in Example 1 (1-2).

[0098] (2) Preparation and transformation of chemocompetent cells: The construction method is the same as in Example 4 (5). (3) The ilvy mutant was obtained by high-throughput screening, using the same method as in Example 4 (6). (4) DPAY6 positive colonies were constructed using the same method as in Example 1 (4).

[0099] (5) Plasmid elimination: The method is the same as in Example 1 (4), to obtain plasmid-free DPAY6 (DPAY5 derivative, ilvY R257C,S264C ). (6) The constructed DPAY6 production strain was used as a control group, with DPAY5 constructed in Example 3 as the control, for shake-flask testing and detection. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as Figure 8 As shown.

[0100] As shown in the figure, the D-pantothenic acid production of strain DPAY6 increased from 7.28 g / L to 7.50 g / L compared to the control. These results confirm that rational design... ilvY The allosteric regulation module can effectively optimize the metabolic network efficiency of the host bacteria.

[0101] Example 7: Construction and shake-flask fermentation of DPAY7 strain Using DPAY6 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to replace genes... coaE The start codon of the gene is replaced with GTG (nucleotide sequence as shown in SEQ ID NO.7) to obtain DPAY7 (DPAY6 derivative, coaE GTG ).

[0102] (1) Construct pTarget-PAM -coaE GTG Plasmid: Using pTarget F plasmid (Addgene Plasmid #62226) as a template, pT-PAM- coaE GTG PCR amplification was performed using primers -F / R. The PCR products were then verified by nucleic acid gel electrophoresis. Dpn I digestive enzymes were incubated at 37°C for 3 hours for digestion, and then converted to... E. coli DH5α, after screening on LB solid plates containing 0.05 mg / L spectinomycin, and sequencing verification, yielded the correct pTarget-PAM-. coaE GTG Plasmids are used for subsequent ligation of Donor DNA.

[0103] (2) Construct pTD-Donor- s GTG plasmids: coaE Using the W3110 genome as a template, E. coli GTG -up-F、 coaE GTG-up-R is the primer used to amplify the upstream portion (F1) of the donor DNA. coaE GTG -down-F and coaE GTG -down-R is the primer used to amplify the downstream portion (F2) of the donor DNA. The PCR fragments are then purified using gel extraction to obtain F1 and F2. This process is performed according to ClonExpress. ® (One-step clone kit, Vazyme Biotech, Nanjing, China) The instruction manual will include pTarget- coaE GTG The linearized vector, fragments F1 and F2 were ligated together, and pTD- was obtained through sequencing verification. coaE GTG Plasmid.

[0104] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAY6 competent cells obtained in Example 6. The preparation method of DPAY6 electrotransfer competent cells is the same as in Example 3 (3).

[0105] (4) DPAY7 positive colonies were constructed using the same method as in Example 1 (4).

[0106] (5) Plasmid elimination: The method is the same as in Example 1 (4), to obtain plasmid-free DPAY7 (DPAY6 derivative, coaE GTG ).

[0107] (6) The constructed DPAY7 production strain was used as a control group, with DPAY6 constructed in Example 6 as the control group, for shake-flask testing and detection. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as coaE As shown.

[0108] Compared to strain DPAY6, DPAY7 showed an increase in D-pantothenic acid production from 7.50 g / L to 7.91 g / L, and OD... 600 The value decreased from 9.12 to 8.82, and bacterial growth was slightly inhibited. The inhibition of key genes in the degradation pathway and the balance between cell growth and production successfully promoted the synthesis of D-pantothenic acid products.

[0109] Example 8: Construction and shake-flask fermentation of DPAY8 strain Using DPAY7 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to introduce P through gene substitution. Figure 9 Promoter controls heterogeneous sources ilvc ( alaS) regulation (nucleotide sequence as shown in SEQ ID NO.8) yields DPAY8 (DPAY7 derivative, B.subtilis606 ::P yhiL ).

[0110] (1) Construct pTarget-PAM ilvC-alsS ::P -yhiL Plasmid: Using pTarget F plasmid (AddgenePlasmid #62226) as a template, pT-PAM- ilvC-alsS ::P yhiL PCR amplification was performed using primers -F / R. The PCR products were then verified by nucleic acid gel electrophoresis. ilvC-alsS I digestive enzymes were incubated at 37°C for 3 hours for digestion, and then converted to... Dpn DH5α was screened using LB solid plates containing 0.05 mg / L spectinomycin, and sequencing verification yielded the correct pTarget-PAM-. E. coli :: yhiL PilvC- Plasmids are used for subsequent ligation of Donor DNA.

[0111] (2) Construct pTD-Donor- alsS ::P yhiL Plasmids: First, with ilvC-alsS Using the W3110 genome as a template, E. coli ::P yhiL -up-F / R refers to primers used to amplify the upstream portion (F1) of the donor DNA. ilvC-alsS ::P yhiL - ilvC -down-F / R primers were used to amplify the downstream portion (F2) of the donor DNA. Then, using... alsS Using the 606 genome as a template, pTrc- B. subtilis ::P yhiL - s (2) F / R primers amplify the product with the promoter p ilvC of alsS Gene fragment (F3), PCR fragments were purified by gel extraction to obtain F1, F2, and F3; plasmid pTarget- ilvC ::P BsalsS - yhiL go through ilvC I and alsS Incubate at 37 ℃ for 8 h, then use a Clean-up kit to recover DNA fragments; follow the instructions of ClonExpress. ®(Onestep clone kit, Vazyme Biotech, Nanjing, China) The instruction manual will include pTarget- Xba ::P Pst The vector, fragments F1, F2, and F3 were ligated together, and pTD- was obtained through sequencing verification. yhiL ::P ilvC-alsS Plasmid.

[0112] (3) The pCas plasmid (Addgene Plasmid #62225) was introduced into the DPAY7 competent cells obtained in Example 7. The preparation method of DPAY7 electrotransfer competent cells is the same as in Example 1 (3).

[0113] (4) DPAY8 positive colonies were constructed using the same method as in Example 1 (4).

[0114] (5) Plasmid elimination: The method is the same as in Example 1 (4), to obtain plasmid-free DPAY8 (DPAY6 derivative, yhiL GTG ).

[0115] (6) The constructed DPAY8 production strain was used as a control group, with DPAY7 constructed in Example 7 as the control group, for shake-flask testing and detection. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as ilvC-alsS As shown.

[0116] As shown in the figure, P coaE Controlled Figure 10 Editing the genome of strain DPAY7 yielded strain DPAY8, with a shake-flask concentration of 8.12 g / L. ilvC promoter pairs alsS Gene expression promoting effects are superior to PTrc promoters, making them more suitable for... ilvC Dynamic regulation of genes can, in turn, help increase the production of D-pantothenic acid.

[0117] Example 9: Construction and shake-flask fermentation of DPAY8-16 strain Using DPAY8 as the starting strain, CRISPR-Cas9-mediated gene editing technology was employed to integrate transcriptional activation. alsS Systemic and post-transcriptional repression, by alsS A 24bp specific target sequence for the gene (nucleotide sequence as shown in SEQ ID NO.9) was introduced into the expression frame of the high-copy plasmid YC-gene-MicC-Hfq to construct a weakened plasmid mechanism. This plasmid was then introduced into the DPAY8 strain to obtain DPAY8-16 (DPAY8 derivative, 99a-YC-). ilvY-MicC-hfq).

[0118] (1) Construct 99a-YC pta MicC- pta Plasmid: Using the existing YC-gene-MicC-Hfq plasmid as a template, -pta- PCR amplification was performed using primers -F / R. After verification by nucleic acid gel electrophoresis, the PCR products were digested with Dpn I digestive enzyme at 37 °C for 3 h, and then transformed into... hfq DH5α was screened using solid plates containing 0.05 mg / L spectinomycin, and sequencing verification yielded the correct 99a-YC-. pta -MicC-hfq plasmid.

[0119] (2) 99a-YC- E.coli The MicC-hfq plasmid was introduced into the DPAY8 competent cells obtained in Example 8, and the preparation method was the same as in Example 1 (3) to obtain DPAY8-16.

[0120] (3) The constructed DPAY8-16 production strain was used as a control group, with DPAY8 constructed in Example 8 as the control group, for shake-flask testing and detection. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as pta As shown.

[0121] Among them, the attenuation of the pta gene, OD 600 The concentration of D-pantothenic acid decreased from 8.82 to 7.26, while the yield increased by 0.19 g / L to 8.31 g / L. pta The gene is responsible for encoding phosphotransacetase, which is related to acetate kinase (Pt-transacetase). Figure 11 (Encoding) Co-catalyzes the production of acetic acid. By weakening it, the overflow of pyruvate into acetic acid can be reduced, the accumulation of pyruvate pool can be increased, and more carbon flow can enter the D-pantothenic acid pathway, thereby increasing the yield of D-pantothenic acid.

[0122] Example 10: Application of strain DPAY8-16 in the preparation of D-pantothenic acid by microbial fermentation Fermentation was carried out in a 5 L fermenter (Shanghai Baoxing, BIOTECH-5BG) and included the following steps: (1) Seed culture: DPAH8-16 was inoculated into 10 mL of LB medium on a plate and cultured overnight on a shaker at 37℃ and 180 rpm. Then, the seed culture was inoculated into two bottles containing 100 mL of LB medium at a volume concentration of 1% for 7-12 h.

[0123] (2) Inoculation and fermentation: The fermentation medium volume in the 5L fermenter was 2L, sterilized at 115℃ for 30min. The initial aeration rate was 3-6 L / min, the initial stirring speed was 300-450 rpm, and the pH was adjusted with ammonia. 200mL of the two secondary seed cultures were transferred to the 5L fermenter containing 2L of antibiotic-free fermentation medium, and IPTG was added at a final concentration of 0.2 mM, with final concentrations of 5 mg / L VB1 and 2 mg / L VB2. 12 Add 5-10 mL of isoleucine (10-40 g / L) to the fermentation tank. During fermentation, maintain dissolved oxygen at 10%-30% using a series of dissolved oxygen generators, maintain pH at 6.7-6.9 using ammonia as a neutralizing agent, and control the glucose concentration in the tank below 5 g / L through pH-linked feeding. Incubate at 28-37℃ for 3-4 days to obtain the fermentation broth. The fermentation broth contains all substances from the fermenter.

[0124] (3) After diluting the fermentation supernatant 80 times, the diluted sample was filtered using an aqueous filter membrane to remove impurities. Then, HPLC analysis was performed according to Example 3. OD 600 and the D-pantothenic acid content in the fermentation broth supernatant, such as pta and ackA As shown.

[0125] By weakening the PPP pathway in glucose metabolism, modifying the glucose transport system, and mitigating the CCR effect. Figure 12 Activation system and enhancement of the pantothenic acid pathway and based on Figure 13 A global regulatory network was constructed. After fermentation for 86 hours, the fermentation broth was centrifuged, and the supernatant was diluted and filtered through a filter membrane. The results showed that the D-pantothenic acid yield reached 137.8 g / L, which was 22.5% higher than that of the starting strain. The strain biomass increased by 30.6%, and the yield increased to 1.64 g / L / h, significantly improving the synthesis of D-pantothenic acid.

[0126] The fermentation medium consists of the following components: 20 g / L glucose, 16 g / L ammonium sulfate, 2 g / L anhydrous betaine, 2 g / L yeast extract, 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 in deionized water at a natural pH. The trace element solution consists of: 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 in deionized water.

[0127] The feed culture medium is composed of the following: 500 g / L glucose, 10 g / L ammonium sulfate, 4 g / L anhydrous betaine, 2 g / L yeast powder, 14 g / L potassium dihydrogen phosphate, 8 g / L anhydrous magnesium sulfate, 60 g / L β-alanine, and 2 ml / L trace element solution. The solvent is deionized water, and the pH value is natural.

[0128] The primer sequence information used in Examples 1-9 is shown in Tables 2A-2D.

[0129] Table 2A: Primer Sequences

[0130] Table 2B: Primer Sequences (Continued)

[0131] Table 2C: Primer Sequences (Continued)

[0132] Table 2D: Primer Sequences (Continued)

[0133] This invention utilizes CRISPR / Cas9 gene editing technology to construct a system based on... ilvy Transcriptional activation system and MicC- ilvY A bidirectional regulatory network of the Hfq post-transcriptional repression system was constructed based on existing engineered bacteria. ilvY The construction and screening of mutant libraries, employing a combination of error-prone PCR and high-throughput screening, yielded highly active mutant libraries. Ec ilvY ilvY R257C,S264C A mutant strain was developed by weakening the PPP pathway system and modifying the glucose transport system to balance carbon flux sources, while strengthening the pantothenic acid pathway to enhance D-pantothenic acid synthesis. A high-yielding strain without plasmids and requiring no antibiotics during fermentation was obtained. The strain achieved a shake-flask titer of 8.31 g / L, and after 86 hours of fermentation in a 5L fermenter, the D-pantothenic acid yield reached 137.8 g / L, a 22.5% increase compared to the starting strain. The strain biomass increased by 30.6%, significantly improving D-pantothenic acid synthesis and shortening the fermentation cycle.

[0134] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for constructing a high-yield D-pantothenic acid-producing genetically engineered bacterium based on the ilvY activation system, characterized in that, The method includes: using CRISPR-Cas9-mediated gene editing technology, combined with the ilvy mutant activation system, to modify one or more aspects of the Chameleon genome: (1) Perform codon optimization and / or promoter substitution on the zwf gene; (2) Introduce heterologous zmglf and glk genes; (3) Construct a high-throughput screening system for fluorescence-activated expression and an ilvY mutant library, and screen the ilvY mutant library to obtain ilvY. R257C,S264C Mutants; (4) ilvY R257C,S264C The mutant replaced the in situ gene in the Chagas gene genome, weakened the start codon of the coaE gene, introduced the heterologous alsS gene, and used the ilvY mutant to activate the PilvC promoter to regulate the expression of the alsS gene and weaken the pta gene.

2. The construction method according to claim 1, characterized in that, The basal bacteria is E. coli W3110, Trc-EcilvD* / BspanBA* / CgpanC* / alsS* / BspanB* / nac GTG / ΔP 345ptsH / gltA GTG / gltA TTG / Trc-PpfkB / Trc-gcvTHP / lafU::folP / yeeP::Trc-RBSApt2#82-serAglyA-P panB -panB / gapC::Trc-alsS*.

3. The construction method according to claim 2, characterized in that, In step (1), codon optimization and / or promoter replacement of the zwf gene includes: (a) replacing the start codon of the zwf gene in the Chameleon genome with TTG; and / or, (b) introducing the Pj23107 promoter.

4. The construction method according to claim 1, characterized in that, The heterologous zmglf gene is derived from *Mammotrophic motility*; the heterologous glk gene is derived from *Escherichia coli*; and the heterologous alsS gene is derived from *Bacillus subtilis* 606.

5. The construction method according to claim 4, characterized in that, In step (2), the introduction of heterologous zmglf and glk genes includes: integrating the zmglf gene from Cytosoma motilityis and the glk gene from Escherichia coli into the pseudogene tfaD site of the genome of the strain to be modified.

6. The construction method according to claim 1, characterized in that, Step (3) includes: (1) Construction of PilvC-eGFP plasmid: Using the genome of Escherichia coli W3110 as a template, the PilvC promoter and eGFp gene were integrated into the pTrc99a vector by one-step cloning method to construct the PilvC-eGFP plasmid; (2) Construction of YPilvC-eGFP plasmid: Using the genome of Escherichia coli W3110 as a template, the ilvY gene was integrated into the PilvC-eGFP vector obtained in step (1) by one-step cloning method to construct the YPilvC-eGFP plasmid; (3) Error-prone PCR amplification of the ilvY gene: Using the ilvY gene as a template, primers ErrorY-F1 / ErrorY-R1 and ErrorY-F2 / ErrorY-R2 were used for amplification to obtain PCR products with random mutations; (4) Construction of ilvY mutant library: The YPilvC-eGFP plasmid digested with Dpn I was ligated with the PCR product obtained in step (3), transformed into host cells, and the corresponding transformants were obtained. The ilvY mutant library was then constructed. (5) High-throughput screening: Transformants in the ilvy mutant library were screened based on fluorescence intensity to obtain ilvY that enhances the activation ability of the PilvC gene. R257C,S264C Mutant.

7. The construction method according to claim 6, characterized in that, The nucleotide sequence of the zwf gene with its start codon replaced by TTG is shown in SEQ ID NO. 1; the nucleotide sequence of the zwf gene regulated by the Pj23107 promoter is shown in SEQ ID NO. 2; the gene sequence of zmglf is shown in SEQ ID NO. 3; the gene sequence of glk is shown in SEQ ID NO. 4; the nucleotide sequence of the PilvC-eGFP plasmid is shown in SEQ ID NO. 5; the nucleotide sequence of the ilvY gene is shown in SEQ ID NO. 6; the nucleotide sequence of the coaE gene with its start codon replaced by the GTG gene is shown in SEQ ID NO. 7; the nucleotide sequence of the alsS gene regulated by the Pilvc promoter is shown in SEQ ID NO. 8; and the nucleotide sequence of the weakened pta gene is shown in SEQ ID NO.

9.

8. A high-yield D-pantothenic acid genetically engineered bacterium obtained by any of the construction methods described in claims 1 to 7.

9. The application of the high-D-pantothenic acid-producing genetically engineered bacteria obtained by any of the construction methods described in claims 1 to 7, or the high-D-pantothenic acid-producing genetically engineered bacteria described in claim 8, in the microbial fermentation preparation of D-pantothenic acid.

10. The application as described in claim 9, characterized in that, The application method includes: inoculating the high-yield D-pantothenic acid genetically engineered bacteria into a fermentation medium, and carrying out fed-batch fermentation culture at 28~37℃, 300~450 rpm, pH 6.7~6.9, and dissolved oxygen 10~30% for 60~86 h, and separating and purifying the D-pantothenic acid after fermentation.

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