Genetically engineered bacterium for producing D-pantothenic acid based on corynebacterium glutamicum CPSPC system as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for producing D-pantothenic acid based on a corynebacterium glutamicum CPSPC system as well as a construction method and application of the genetically engineered bacterium. Wild type C.glutamicum ATCC 13032 is taken as a chassis strain, and a functional combination of a glycine ribosome switch BsGR from bacillus subtilis and a promoter Ptuf / Ph36 is verified through fluorescent protein mCherry; a GCS system of escherichia coli and bacillus subtilis is introduced to the strain DPAg-15, genes related to synthesis of 5, 10-dimethyltetrahydrofolic acid are tested, it is found that serA delta197GTG / ATG significantly increases the yield of D-pantothenic acid, the yield of D-pantothenic acid of the constructed strain DPAj-2-tuf is increased to 23.06 g / L (63 h), the yield is 0.37 g / L / h, and the conversion rate is 0.17 g / g; the engineering strain DPAj-2-tuf is obtained by dynamically regulating and controlling a D-pantothenic acid synthesis path through a CPSPC system and combining plasmid overexpression serA delta197GTG / ATG, the yield of the engineering strain DPAj-2-tuf is increased by 2.1 times compared with that of a traditional method, and an efficient technical scheme is provided for producing D-pantothenic acid through a microbial fermentation method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of metabolic engineering, and relates to a D-pantothenic acid-producing genetically engineered bacterium based on a CPSPC system of Corynebacterium glutamicum, a construction method and application thereof, in particular, application in coupling of a one-carbon unit synthesis pathway and a D-pantothenic acid synthesis pathway. BACKGROUND

[0002] Dynamic control strategies are widely used in metabolic engineering, which can precisely regulate gene expression at the transcription, translation or post-translation level by responding to specific substances changing in cells. Due to its modular design and rapid response characteristics, transcription and translation level control provides greater flexibility and practicality in metabolic regulation, especially the translation level control, which can quickly act within a few minutes, and can achieve faster metabolic adjustment. Riboswitch is a translation level control element, which is located in the structural region of the 5'-untranslated region (5'-UTR) of mRNA and can bind to specific small molecules. Glycine riboswitch has been widely used. For example, it is used to control the production of enzymes and metabolic pathways in the 5-aminovalerate synthesis pathway. In addition, there are many other amino acid-related riboswitches, such as glutamine, lysine and tryptophan riboswitches, which have been effectively applied in various biological systems.

[0003] Corynebacterium glutamicum is a recognized safe (GRAS) strain, which is mainly used in industry to produce organic acids, vitamins and amino acids, among which the production of valine has reached a very high level. The production of valine of C. glutamicum R strain reaches 1940 mM, and the production of valine of C. glutamicum ATCC 13032 strain reaches 103 g / L. The biosynthetic pathways of valine and D-pantothenic acid have a common key intermediate α-hydroxyvalerate KIV. If the carbon flow for synthesizing valine can be introduced into the D-PA synthesis pathway in a suitable manner, it indicates that C. glutamicum has great potential in producing D-pantothenic acid. However, unlike Escherichia coli and Bacillus subtilis, C. glutamicum does not have a glycine cleavage system (GCS), and its CH2-THF synthesis is completely supplied through the conversion of serine to glycine. Therefore, glycine will gradually accumulate in the process of D-pantothenic acid biosynthesis, which will block the metabolic flow, and this feedback inhibition will interfere with the supply of precursors, thereby affecting the synthesis efficiency of D-pantothenic acid.

[0004] Therefore, there is an urgent need to construct a new strategy for improving the production of D-pantothenic acid by utilizing the characteristics of glycine accumulation. SUMMARY

[0005] The purpose of the present application is to provide a D-pantothenic acid-producing genetically engineered bacterium based on a CPSPC system of Corynebacterium glutamicum, a construction method and application thereof.

[0006] To achieve the above object of the present application, the technical solution adopted by the present application is as follows:

[0007] In a first aspect, the present application provides a D-pantothenic acid-producing genetically engineered bacterium based on the CPSPC system of Corynebacterium glutamicum, which is prepared by the following construction method:

[0008] (1) Glycine ribosome switch BsGR from Bacillus subtilis is connected with a Corynebacterium glutamicum promoter, and introduced into a host bacterium through plasmid pECXK99e to screen a glycine concentration-responsive engineered strain;

[0009] (2) Using a sacB gene suicide plasmid-mediated homologous recombination gene editing technology, the ilvE gene, avtA gene, ilvA gene, and pqo gene in Corynebacterium glutamicum ATCC 13032 (C. glutamicum ATCC 13032) are sequentially knocked out, and Ptuf promoter and Pefu promoter-controlled ilvBNC-VWB GTG / ATG are integrated at the ΔilvA and ΔavtA sites, respectively, the promoter of the aceE gene is replaced with PdapA A16, and a strain DPAg-15 is obtained.

[0010] (3) Using plasmid pECXK99e as a vector, a co-expression combination of gene cluster BspanBC from Bacillus subtilis 168 and EcpanE gene from E. coli is connected, wherein the promoter of EcpanE is controlled by PdapB, and plasmid pECXK99e-BspanBC+PdapB-EcpanE is obtained.

[0011] (4) Using strain DPAg-15 as a chassis strain, pECXK99e-BspanBC+PdapB-EcpanE plasmid and pXMJ19 recombinant plasmid are sequentially introduced by electroporation to obtain an engineered strain; the pXMJ19 recombinant plasmid is a recombinant plasmid A carrying genes related to coenzyme synthesis pathway and a recombinant plasmid B carrying GCS system (glycine cleavage system) related genes from E. coli or Bacillus subtilis (B. subsills);

[0012] (5) Using a sacB gene suicide plasmid-mediated homologous recombination gene editing technology, the C-terminal 197 amino acid sequence of the serA gene on the genome of strain DPAg-15 is knocked out to obtain an engineered bacterium DPAi-3;

[0013] (6) Using the homologous recombination gene editing technology mediated by the suicide plasmid of the sacB gene, the start codon GTG of the truncated serA gene on the genome of the engineered strain DPAi-3 was replaced with ATG to obtain the engineered strain DPAj-2.

[0014] (7) Design the Corynebacterium glutamicum CPSPC system so that the glycine ribosome switch BsGR under the control of the high glycine concentration responsive promoter screened in step (1) replaces the PdapB gene in the pECXK99e-BspanBC+PdapB-Ecpan E plasmid to obtain the recombinant expression plasmid.

[0015] (8) The recombinant expression plasmid described in step (5) and the recombinant plasmid A are co-transformed into the engineered strain DPAj-2 to obtain the engineered strain DPAj-2-tuf, which is the genetically engineered bacterium that produces D-pantothenic acid.

[0016] As a preferred embodiment of this application, the host bacterium in step (1) is Corynebacterium glutamicum ATCC13032.

[0017] As a preferred embodiment of this application, the C. glutamicum promoter is selected from one or more of the Pefu promoter, Ptuf promoter, Ph36 promoter, Peno promoter, and Psod promoter.

[0018] As is preferred in this application, step (1) includes:

[0019] (a) The glycine ribosome switch BsGR from Bacillus subtilis was linked to the promoters Pefu, Ptuf, Ph36, Peno and Psod from Corynebacterium glutamicum to construct a variety of gene expression units that control the expression of the fluorescent protein mCherry.

[0020] (b) The gene expression unit was cloned into plasmid pECXK99e by restriction enzyme digestion and then introduced into Corynebacterium glutamicum ATCC 13032 by electroporation to obtain the transformed strain;

[0021] (c) After culturing each transformed strain in BHIS medium containing gradient concentrations of glycine at 30 °C for 10–30 h, the fluorescence intensity at 597 nm / 608 nm and OD were measured. 600 The ratio was used to determine the high glycine concentration responsive promoter, and the gene expression units controlled by the high glycine concentration responsive promoter and the glycine concentration responsive engineered strains were screened.

[0022] As a preferred embodiment of this application, in step (c), the concentration of glycine is 0, 0.5, 1, 2, 5, 10, 20, or 50 mM.

[0023] As a preferred embodiment of this application, the nucleotide sequence of the BsGR gene is shown in SEQ ID No. 1, the nucleotide sequence of the Ptuf promoter is shown in SEQ ID No. 2, the nucleotide sequence of the Ph36 promoter is shown in SEQ ID No. 3, the nucleotide sequence of the Pefu promoter is shown in SEQ ID No. 4, the nucleotide sequence of the Psod promoter is shown in SEQ ID No. 5, and the nucleotide sequence of the Peno promoter is shown in SEQ ID No. 6.

[0024] As a preferred embodiment of this application, the high glycine concentration-responsive promoter is Ptuf or Ph36.

[0025] As a preferred embodiment of this application, the coenzyme synthesis pathway-related gene carried in the recombinant plasmid A is selected from serA. GTG / ATG ,serAΔ197 GTG / ATG ,serAΔ197 GTG / ATG Any one of +serBC, pabABC, BsfolD, EcfolD, and glyA.

[0026] As a preferred embodiment of this application, the recombinant plasmid A carries the coenzyme synthesis pathway-related gene serAΔ197. GTG / ATG Its nucleotide sequence is shown in SEQ ID No. 7.

[0027] As a preferred embodiment of this application, the method for constructing the pXMJ19 recombinant plasmid is as follows:

[0028] Using plasmid pXMJ19 as a vector, genes related to the coenzyme synthesis pathway were overexpressed to construct recombinant plasmid A;

[0029] Using plasmid pXMJ19 as a vector, GCS-related genes from E. coli and B. subsills were overexpressed to construct recombinant plasmid B.

[0030] As a preferred embodiment of this application, the GCS system-related genes from E. coli include the EccgvT gene, EccgvP gene, EccgvH gene, and EccgvL gene; the nucleotide sequence of the EccgvT gene is shown in SEQ ID No. 8; the nucleotide sequence of the EccgvP gene is shown in SEQ ID No. 9; the nucleotide sequence of the EccgvH gene is shown in SEQ ID No. 10; and the nucleotide sequence of the EccgvL gene is shown in SEQ ID No. 11.

[0031] As a preferred embodiment of this application, the GCS system-related genes from B. subsills include the BscgvT gene, BscgvP gene, BscgvH gene, and BscgvL gene; the nucleotide sequence of the BscgvT gene is shown in SEQ ID No. 12; the nucleotide sequence of the BscgvP gene is shown in SEQ ID No. 13; the nucleotide sequence of the BscgvH gene is shown in SEQ ID No. 14; and the nucleotide sequence of the BscgvL gene is shown in SEQ ID No. 15.

[0032] The formation of D-pantothenic acid is accompanied by the accumulation of glycine. 5,10-Dimethyltetrahydrofolate (5,10-CH2-THF) is one of the active forms of tetrahydrofolate (THF), participating as a coenzyme in one-carbon unit transfer reactions in organisms. In *C. glutamicum*, 5,10-dimethyltetrahydrofolate is entirely provided by the conversion of serine to glycine. Because ketopantolytic acid (KPR) has bifunctionality—it can catalyze the entry of KPR into the D-pantothenic acid synthesis pathway, but it can also prematurely catalyze the precursor KIV, resulting in carbon flux loss—the expression intensity of the *panE* gene needs to be well regulated to ensure that as much precursor KIV as possible enters the D-pantothenic acid synthesis pathway. Therefore, a CPSPC system was designed to dynamically regulate *panE* expression and simultaneously couple the 5,10-dimethyltetrahydrofolate synthesis pathway with the D-pantothenic acid synthesis pathway. The glycine-riboswitch BsGR derived from *B. subsills* is used to control *panE* expression. In the initial stage, without glycine accumulation, EcpanE, controlled by the glycine riboswitch, remains silent or at a low expression level, thus minimizing premature KIV shunting. As KPHMT (encoded by panB) converts KIV into ketopantolytic acid and accumulates, glycine gradually accumulates, thereby activating EcpanE expression. This allows for efficient utilization of ketopantolytic acid and redirects carbon flux towards D-pantothenic acid biosynthesis.

[0033] Secondly, the present invention also provides the application of the genetically engineered bacteria in the microbial fermentation preparation of D-pantothenic acid.

[0034] As a preferred embodiment of this application, the application is as follows: the genetically engineered bacteria are inoculated into a fermentation medium and fermented at 30°C and 200 rpm for 48-60 h. After fermentation, the supernatant of the fermentation broth is separated and purified to obtain the D-pantothenic acid. The fermentation medium has the following composition: glucose: 10-30 g / L, corn steep liquor: 10-20 g / L, ammonium sulfate: 10-20 g / L, yeast extract: 1-5 g / L, KH2PO4: 0.2-1 g / L, MgSO4: 0.5-5 g / L, sodium acetate: 5-8 g / L, β-alanine: 0.5-2 g / L, CaCO3: 3-5 g / L, and a 0.5-2 mL / L trace element solution in deionized water with a natural pH. The trace element solution has the following composition: NiCl2·7H2O 0.02 g / L, CuCl2 10 g / L, FeSO4·7H2O 10 g / L. The concentrations of the following components were: g / L ZnSO4·7H2O 10 g / L, CuSO4 0.2 g / L, and the solvent was deionized water.

[0035] As a preferred embodiment of this application, the fermentation is carried out in a fermenter using fed-batch fermentation: the genetically engineered bacteria are inoculated into BHIS test tube medium containing 25 mg / L kanamycin resistance and 12 mg / L chloramphenicol resistance, and cultured at 30°C for 18 h to obtain the seed culture; the seed culture is then inoculated into BHIS medium at a volume concentration of 5% and cultured at 30°C for 12 h to obtain the seed culture for the upper fermenter; the seed culture from the upper fermenter is then inoculated into the fermenter containing fermentation medium at a volume concentration of 10%, and fermented at 30°C, 400 rpm, and an aeration rate of 1.0 V / V·min, with dissolved oxygen controlled at 20%~40%. A constant-rate feeding method is used to maintain the glucose concentration below 5 g / L, and fermentation is continued for 63 h to obtain a fermentation broth containing D-pantothenic acid. The fermentation medium consisted of: glucose 40 g / L, ammonium sulfate 20 g / L, corn steep liquor 20 g / L, yeast extract 2 g / L, KH₂PO₄ 0.8 g / L, MgSO₄ 0.5 g / L, sodium acetate 5 g / L, β-alanine 2 g / L, CaCO₃ 3 g / L, and a 1 mL / L trace element solution in deionized water at a natural pH. The trace element solution composition was: NiCl₂·7H₂O 0.02 g / L, CuCl₂ 10 g / L, FeSO₄·7H₂O 10 g / L, ZnSO₄·7H₂O 10 g / L, CuSO₄ 0.2 g / L in deionized water. The feed medium consists of 500 g / L glucose, 20 g / L (NH4)2SO4, 1 g / L MgSO4, and 30 g / L β-alanine, dissolved in deionized water and adjusted to pH 6.8 with 50% ammonia.

[0036] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0037] (1) This invention creatively constructs a genetically engineered bacterium that produces D-pantothenic acid based on the CPSPC system of Corynebacterium glutamicum, introduces the glycine riboside switch BsGR from Bacillus subtilis for the first time, verifies mCherry using multiple promoters, and establishes a dynamic regulatory coupling mechanism of CPSPC.

[0038] (2) Optimize metabolic pathways: Using strain DPAg-15 as the chassis strain, the 5,10-dimethyltetrahydrofolate synthesis pathway was enhanced. Taking advantage of the characteristics of glycine accumulation, the GCS system of E. coli and B. subtilis was introduced to achieve precise regulation of glycine metabolic flux.

[0039] (3) During the fermentation process, the engineered bacteria adopted the strategy of exogenously adding the reaction substrate β-alanine to achieve a dynamic balance between the synthesis of one-carbon units and D-pantothenic acid. At the fermenter level, 23.06 g / L of D-pantothenic acid was produced in 63 hours, with a yield of 0.37 g / L / h and a conversion rate of 0.17 g / g, which is the highest level of D-pantothenic acid production by Corynebacterium glutamicum to date. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the working principle of the glycine ribosome switch.

[0041] Figure 2 To determine the fluorescence intensity (OD) of BsGR at different glycine concentrations, it was found that linking BsGR to different promoters controls mCherry expression. 600 Changes in the ratio;

[0042] Figure 3 Metabolic diagram of the 5,10-dimethyltetrahydrofolate synthesis pathway;

[0043] Figure 4 To determine the yield of D-pantothenic acid after plasmid overexpression of genes related to the 5,10-dimethyltetrahydrofolate biosynthesis pathway;

[0044] Figure 5 The yield of D-pantothenic acid, the accumulation of glycine, and the molar ratio of D-pantothenic acid to glycine were determined after plasmid overexpression of the GCS system derived from E. coli and B. subsills.

[0045] Figure 6 The yield of D-pantothenic acid after strains DPAg-15, DPAi-3, and DPAj-2 were transformed into plasmid pECXK99e-BspanBC+PdapB-EcpanE.

[0046] Figure 7 This is a schematic diagram illustrating the working principle of the CPSPC system.

[0047] Figure 8 To control mCherry expression using promoters with and without BsGR, fluorescence intensity and OD were compared at different glycine concentrations. 600 Changes in the ratio;

[0048] Figure 9 The fluorescence intensity OD at different concentrations of glycine was measured to control mCherry expression using PtufGR-EcpanE, Ph36GR-EcpanE, Ptuf-EcpanE, Ph36-EcpanE, and PdapB-EcpanE. 600 Changes in the ratio;

[0049] Figure 10 This is a fermenter test diagram of strain DPAj-2-tuf. Detailed Implementation

[0050] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention 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 the present invention.

[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, the experimental methods described in the following examples are conventional methods.

[0054] Unless otherwise specified, the experimental materials used in the following examples are all conventional biochemical reagents.

[0055] In the following examples, the final concentration of kanamycin in the culture medium was 0.025 mg / L, and the final concentration of chloramphenicol in the culture medium was 0.012 mg / L.

[0056] LB medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, solvent is deionized water, solid medium with 2% agar powder, pH is natural.

[0057] BHIS liquid culture medium: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, 10 g / L glucose, 10 g / L brain and heart extract powder, solvent: deionized water, pH: natural.

[0058] BHIS plates: 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, 10 g / L glucose, 10 g / L brain and heart extract powder, solvent: deionized water, 2% agar powder, pH: natural.

[0059] The strain Corynebacterium glutamicum ATCC13032 (C. glutamicum ATCC 13032, purchased from the ATCC Collection).

[0060] Table 1: Genes involved in gene editing and corresponding pathways

[0061] The primer sequence information used in Examples 2-7 is shown in Tables 2(A)-2(E):

[0062] Table 2(A): Primer sequence listing

[0063] Table 2(B): Primer Sequence Continued Table

[0064] Table 2(C): Primer Sequences Continued

[0065] Table 2(D): Primer Sequences Continued

[0066] Table 2(E): Primer sequences

[0067] Example 1: Determination of the content of α-hydroxyvalerate (KIV), D-pantothenic acid (D-PA) and amino acids

[0068] The detection methods for KIV and D-pantothenic acid are as follows: Chromatographic conditions: C18 column (250×4.6 mm, particle size 5 μm, Agilent Technologies Co., Santa Clara, CA, USA); detection wavelength: 200 nm; column temperature: 30℃; flow rate: 1 ml / min; mobile phase: acetonitrile / water / phosphoric acid: (80 / 919 / 1); data acquisition time: 20 min. Sample preparation: Dilute the sample with ultrapure water to maintain the D-pantothenic acid content between 0.2 g / L and 1 g / L.

[0069] Amino acids: C18 column (250×4.6 mm, particle size 5 μm, Agilent Technologies Co., Santa Clara, CA, USA); mobile phase: methanol: 0.05 mol / L acetic acid, sodium acetate solution (55:45); flow rate: 1.0 mL / min; column temperature: room temperature. Sample derivatization: 100 μL of sample was dissolved in 100 μL of 0.5 mol / L NaHCO3 aqueous solution and 0.1 mL of 1% 2,4-dinitrofluorobenzene aqueous solution in acetonitrile. After reacting at 60 ℃ in the dark for 30 min, 700 μL of 0.2 mol / L phosphate buffer (pH 7) was added.

[0070] Example 2: Construction of DPAg-15

[0071] Using strain C. glutamicum ATCC13032 as the starting strain, homologous recombination gene editing technology mediated by the suicide plasmid of the sacB gene was employed to knock out ilvE in the genome, thereby blocking the synthesis pathways of L-valine and L-isoleucine.

[0072] (1) Construction of pK18-ΔilvE plasmid: Using pK18-mobsacb plasmid (Addgene Plasmid #177838) as a template, amplification was performed using PK18-F / PK18-R primers to obtain a linearized plasmid, which was named PK18; using C. glutamicum ATCC 13032 strain as a template, amplification was performed using ilvEup-F / ilvEup-R primers to obtain a fragment named ilvEup; using C. glutamicum ATCC 13032 strain as a template, amplification was performed using ilvEdown-F / ilvEdown-R primers to obtain a fragment named ilvEdown. The three amplified PCR products were purified by gel extraction to obtain the purified fragments PK18, ilvEup, and ilvEdown. Following the instructions of Clon Express® (One step clone kit, Vazyme Biotech, Nanjing, China), PK18, ilvEup, and ilvEdown were ligated together, transformed into E. coli DH5α, screened by kanamycin plates, and sequenced to verify the correct pK18-ΔilvE plasmid.

[0073] (2) Preparation of competent cells of wild-type strain C. glutamicum ATCC 13032: A single colony of C. glutamicum was picked and placed in 10 mL of BHIS liquid medium and cultured at 30 °C and 100 r / min for about 10 h. 3 mL of the above culture solution was then inoculated into 100 mL of BHIS liquid medium and cultured at 30 °C and 200 rpm until OD. 600 ≈0.9 (approximately 5 h of culture); transfer the culture medium to sterile centrifuge tubes and incubate on ice for approximately 30 min, then centrifuge at 4°C, 4000 r / min for 10 min, discard the supernatant, and collect the bacterial cells; then add 30 mL of pre-chilled 10% glycerol solution to the bacterial cells, gently agitate in ice water to resuspend the cells, and centrifuge at 4°C, 4000 r / min for 10 min after complete resuscitation, discard the supernatant, and collect the bacterial cells. Wash the collected bacterial cells once more with pre-chilled 10% glycerol solution. Resuspend the collected bacterial cells in a certain amount of pre-chilled 10% glycerol solution, aliquot into 100 μL tubes, and store at -80°C. These are the electrocompetent cells, ready for use.

[0074] (3) Electroporate plasmid pK18-ΔilvE into competent cells prepared in Example 2 (2): Take 2 μg of plasmid pK18-ΔilvE prepared in step (1) and 100 μL of electroporation competent cells prepared in step (3), mix them, and transfer them into a 1 mm electroporation cuvette pre-cooled at 4 ℃. Incubate on ice for 1 min, and perform electroporation transformation using an electroporator (Micro Pluser, BIO-RAD). After two consecutive electroporations, immediately add the mixture to 1 mL of BHIS medium pre-cooled at 4 ℃ and immediately transfer it to a sterile 1.5 mL centrifuge tube. After recovery at 30 ℃ and 200 rpm for 2 h, spread it on a BHIS plate containing 25 μg / L kanamycin and incubate upside down at 30 ℃ for 48 h until single colonies appear.

[0075] (4) Transformant verification and engineered strain screening verification: Colony PCR was performed using PK18-SEQ-F / ilvE-down-R as verification primers. Five to ten colonies that amplified the correct bands were selected and inoculated into LB liquid medium and cultured overnight at 30 °C and 200 rpm. The bacterial culture was diluted 10 times with antibiotic-free LB liquid medium. 3 10 4 10 5 The samples were spread onto LB agar containing 10% sucrose and incubated at 30 °C for approximately 48 h. Single colonies were simultaneously validated on both LB agar containing 10% sucrose and LB agar containing kanamycin resistance. Several single colonies that could grow on LB agar containing 10% sucrose but not on LB agar containing kanamycin resistance were selected. Validation was performed using primers ilvE-up-F / ilvE-down-R. PCR products with significantly smaller bands compared to the wild-type strain were sequenced. Sequencing results confirmed that the single colonies corresponding to successful gene knockout were the correct engineered strain.

[0076] (5) Knock out the ilvA gene sequentially using the corresponding primers using the same method; insert Pefu-ilvBNC-VWB at the avtA gene site. GTG / ATG ; pqo gene locus insertion Ptuf-ilvBNC-VWB GTG / ATG(Zhang, H., Li, Y., Wang, C., Wang, X., 2018. Understanding the high l-valine production in Corynebacterium glutamicum VWB-1 using transcriptomics and proteomics. Sci. Rep. 8 (1)); The promoter of the aceE gene was replaced with PdapA A16. The engineered strain DPAg-15 was obtained.

[0077] Example 3: Construction of pECXK99e-BspanBC+PdapB-EcpanE

[0078] The gene clusters BspanBC from Bacillus subtilis 168 and EcpanE from E. coli were selected and constructed on plasmid pECXK99e.

[0079] (1) Construction of plasmid pECXK99e-BspanBC: Using Bacillus subtilis 168 strain (purchased from Beyotime) as a template, the fragment was amplified using RBS-BspanB-F / Pec-BspanC-R primers and named BspanBC. Using plasmid pECXK99e (purchased from NovoPro) as a template, the fragment was amplified using PECXK99E-F / PECXK99E-R primers and named pECXK99e. The two amplified PCR products were purified by gel extraction to obtain the purified fragments pECXK99e and BspanBC. Following the instructions of Clon Express® (One step clone kit, Vazyme Biotech, Nanjing, China), pECXK99e and BspanBC were ligated together, transformed into E. coli DH5α, screened by kanamycin plates, and sequenced to verify that the correct pECXK99e-BspanBC plasmid was obtained.

[0080] (2) Construction of plasmid pECXK99e-BspanBC+PdapB-EcpanE: Using T2ter-pEC-F / T2terpEC-R as primers and plasmid pECXK99e-BspanBC as template, the linearized plasmid was amplified and named pECXK99e-BspanBC; using T2terpEC-PdapB-F / EcpanE-PdapB-R as primers and C. glutamicum ATCC 13032 strain as template, the obtained fragment was named PdapB; using EcpanE-F / T2terpEC-EcpanE-R as primers and E. coli strain as template, the obtained fragment was named EcpanE. The two amplified PCR products were purified by gel extraction to obtain the purified fragments pECXK99e and BspanBC. Following the instructions of Clon Express® (One step clone kit, Vazyme Biotech, Nanjing, China), pECXK99e-BspanBC, PdapB, and EcpanE were ligated together, transformed into E. coli DH5α, screened by kanamycin plates, and sequenced to verify that the correct pECXK99e-BspanBC+PdapB-EcpanE plasmid was obtained.

[0081] Example 4 Functionalization of glycine riboswitch BsGR in Corynebacterium glutamicum

[0082] In the absence of glycine, glycinosomes shut down the expression of downstream genes; as the concentration of glycine increases, the expression intensity of downstream genes gradually increases. Figure 1 Using *C. glutamicum* ATCC13032 as the starting strain, the glycine ribosome switch BsGR from *B. subtilis* was introduced and linked to the strong promoters Pefu, Ptuf, Ph36, Peno, and Psod in *C. glutamicum* to control the expression of the fluorescent protein mCherry. The fluorescence intensity and OD values ​​at different glycine concentrations were tested. 600 The ratio change.

[0083] (1) Construction of plasmid pECXK99E-PtufBsGR-mCherry: Using pECXK99E plasmid (purchased from NovoPro) as a template, amplification was performed using p-ECXK99E-F / pEXK99E-nolaq-R primers to obtain a linearized plasmid, which was named pEXK99E; using mCherry (Addgene Plasmid #176016) as a template, amplification was performed using RBS-mCherry-F / pECXK99E-mCherry-R primers. The obtained fragment was named mCherry; using C. glutamicum ATCC13032 as a template and BsGR-Ptuf-R / pECXK-Ptuf-F as primers, the obtained fragment was named BsGR-Ptuf; using B. subtilis 168 (purchased from Beyotime) as a template and mCherry-BsGR-R / pEC-BsGR-F as primers, the obtained fragment was named BsGR. The four amplified PCR products were purified by gel extraction to obtain the purified fragments pEXK99E, mCherry, BsGR-Ptuf, and BsGR. Following the instructions of Clon Express® (One step clone kit, Vazyme Biotech, Nanjing, China), pEXK99E, mCherry, BsGR-Ptuf, and BsGR were ligated together, transformed into E. coli DH5α, screened by kanamycin plates, and sequenced to verify the correct pECXK99E-PtufBsGR-mCherry plasmid.

[0084] (2) The connection method of the remaining promoters is the same as in Example 4(1);

[0085] (3) Preparation of competent cells of wild-type strain C. glutamicum ATCC 13032: A single colony of C. glutamicum was picked and placed in 10 mL of BHIS liquid medium and cultured at 30 °C and 100 r / min for about 10 h. 3 mL of the above culture solution was then inoculated into 100 mL of BHIS liquid medium and cultured at 30 °C and 200 rpm until OD. 600≈0.9 (approximately 5 h of culture); transfer the culture medium to sterile centrifuge tubes and incubate on ice for approximately 30 min, then centrifuge at 4°C, 4000 r / min for 10 min, discard the supernatant, and collect the bacterial cells; then add 30 mL of pre-chilled 10% glycerol solution to the bacterial cells, gently agitate in ice water to resuspend the cells, and centrifuge at 4°C, 4000 r / min for 10 min after complete resuscitation, discard the supernatant, and collect the bacterial cells. Wash the collected bacterial cells once more with pre-chilled 10% glycerol solution. Resuspend the collected bacterial cells in a certain amount of pre-chilled 10% glycerol solution, aliquot into 100 μL tubes, and store at -80°C. These are the electrocompetent cells, ready for use.

[0086] (4) Electroporate the plasmids obtained in Example 4 (1-2) into competent cells of Corynebacterium glutamicum ATCC 13032: Take 2 μg of plasmid pECXK99E-PtufBsGR-mCherry prepared in step (1) and other plasmids and 100 μL of electroporation competent cells prepared in step (3), mix them and transfer them into a 1 mm electroporation cuvette pre-cooled at 4 ℃, incubate on ice for 1 min, and perform electroporation transformation using an electroporator (Micro Pluser, BIO-RAD). After two consecutive electroporations, immediately add the mixture to 1 mL of BHIS liquid medium pre-cooled at 4 ℃ and immediately transfer it to a sterile 1.5 mL centrifuge tube. After recovery at 30 ℃ and 200 rpm for 2 h, spread it on a BHIS plate containing 25 μg / L kanamycin and incubate upside down at 30 ℃ for 48 h until single colonies appear.

[0087] (5) Verification of transformed strains and screening of engineered strains: PK18-SEQ-F / PK18-SEQ-R were used as verification primers for colony PCR. Sequencing results proved that the single colony corresponding to the successful gene knockout was the strain transformed by the correct engineered strain.

[0088] (6) Shake-flask test: The strain obtained in Example 4 (5) was used as a control group by transforming blank pECXK99E with C. glutamicum ATCC 13032. Each strain was inoculated into 5 mL of BHIS liquid medium and cultured at 30 ℃ and 200 rpm as a pre-culture. After 12-24 h, 0.5% inoculum was added to 1 mL of BHIS liquid medium in a 96-well plate, with different concentrations of glycine added, including 0, 0.5, 1, 2, 5, 10, 20, and 50 mM, with three replicates for each concentration. The plates were then cultured in a constant temperature shaker at 30 ℃ and 200 rpm for 20 h. After the culture, 1 mL of fermentation broth was taken to determine the OD. 600Simultaneously, 1 mL of bacterial culture was pipetted, washed twice with PBS, and fluorescence was measured using an EnSpire multimode plate reader (PerkinElmer). The excitation and emission wavelengths used for mCherry were 597 nm and 608 nm, respectively. Fluorescence intensity was calculated relative to OD. 600 The ratio of .

[0089] (7) Fermentation culture: The strain obtained in Example 4 (5) was precultured and then inoculated into the fermentation medium. Fermentation culture was carried out at 30℃ and 200rpm for 48~60 h. After fermentation, the supernatant of the fermentation broth was separated and purified to obtain the D-PA. The fermentation medium composition is as follows: glucose: 30 g / L, ammonium sulfate: 16 g / L, corn steep liquor: 10 g / L, yeast powder: 2 g / L, KH2PO4: 0.8 g / L, MgSO4: 0.5 g / L, sodium acetate: 5 g / L, β-alanine: 2 g / L, CaCO3 3 g / L, 1 mL / L trace element solution, the solvent is deionized water, and the pH value is natural. The trace element solution consists of: NiCl2·7H2O 0.02 g / L, CuCl2 10 g / L, FeSO4·7H2O 10 g / L, ZnSO4·7H2O 10 g / L, CuSO4 0.2 g / L, with deionized water as the solvent.

[0090] Depend on Figure 2 It can be seen that the fluorescence intensity of the control group is related to OD. 600 The ratio is low and remains stable across different concentrations of glycine. Among the five constitutive strong promoters, Ptuf exhibits the best sensitivity to glycine concentration, followed by Ph36, while the other three promoters show lower sensitivity.

[0091] Example 5: Plasmid overexpression of genes related to coenzyme synthesis pathway

[0092] Using plasmid pXMJ19 as a vector, genes related to the coenzyme synthesis pathway were overexpressed. These genes included key genes serA, serB, and serC in the 3-phosphate-glucose to serine synthesis pathway, with the C-terminus of serA being a feedback inhibition site for serine recognition; genes related to the tetrahydrofolate synthesis pathway pabABC; the gene glyA for the conversion of serine to glycine; and the genes EcfolD (from E. coli) and BsfolD (from B. subsills) for the conversion of 5,10-methylenetetrahydrofolate to 5,10-dimethyltetrahydrofolate.

[0093] (1) Construct relevant plasmids using pXMJ19 as a vector. The construction process is the same as in Example 4(1). Multiple plasmids containing the above genes were obtained.

[0094] (2) Prepare competent cells of DPAg-15. The preparation process is the same as in Example 4 (3).

[0095] (3) Electroporate the plasmid pECXK99e-BspanBC+PdapB-EcpanE (Example 3) into the competent cells prepared in Example 5 (2), and the preparation process is the same as in Example 4 (4).

[0096] (4) Validation of transformed strains and screening and validation of engineered strains: The process is the same as in Example 4 (5).

[0097] (5) Prepare competent cells from the correct transformed strain obtained in Example 5 (4), the process is the same as in Example 4 (3), and kanamycin needs to be added to a final concentration of 0.025 mg / ml;

[0098] (6) Electroporate the various plasmids constructed in Example 5 (1) into the competent cells in Example 5 (5);

[0099] (7) Validation of transformed strains and screening and validation of engineered strains: The process is the same as in Example 4 (5).

[0100] (8) Shake-flask fermentation: The engineered strain obtained in Example 5 (7), with the starting strain mCherry as the control group, was inoculated into 5 mL of BHIS liquid medium and cultured at 30 ℃ and 200 rpm as a pre-culture. After 12 h-24 h, 2 mL of the pre-culture was inoculated into a 500 mL shake flask containing 50 mL of fermentation medium at a 4% inoculation rate. Then, the strain was cultured in a constant temperature shaker at 30 ℃ and 200 rpm for 48 h for fermentation. After fermentation, 1 mL of fermentation broth was taken to determine the OD. 600 Simultaneously, 1 mL of fermentation broth was pipetted and centrifuged at 12000 rpm for 3 min at room temperature. After removing impurities using an aqueous filter membrane, the sample was analyzed by HPLC according to Example 1. The D-pantothenic acid content in the fermentation broth supernatant was as follows: Figure 4 As shown.

[0101] Depend on Figure 4 It is evident that overexpression of genes related to the serine biosynthesis pathway significantly enhances D-pantothenic acid production, while overexpression of other genes reduces D-pantothenic acid production, indicating that increased serine production is a key factor in enhancing 5,10-dimethyltetrahydrofolate production. Among these, serAΔ197... GTG / ATG Overexpression of the drug alone significantly increased D-pantothenic acid production.

[0102] Fermentation medium: glucose: 30 g / L, ammonium sulfate: 16 g / L, corn steep liquor: 10 g / L, yeast extract: 2 g / L, KH₂PO₄: 0.8 g / L, MgSO₄: 0.5 g / L, sodium acetate: 5 g / L, β-alanine: 2 g / L, CaCO₃: 3 g / L, 1 mL / L trace element solution, solvent: deionized water, pH: natural. The trace element solution composition is: NiCl₂·7H₂O 0.02 g / L, CuCl₂ 10 g / L, FeSO₄·7H₂O 10 g / L, ZnSO₄·7H₂O 10 g / L, CuSO₄ 0.2 g / L, solvent: deionized water.

[0103] Example 6: Plasmid overexpression of GCS system-related genes

[0104] The GCS system, present in *E. coli* and *B. subsills*, further converts accumulated glycine into CO2 and NH3, accompanied by the production of 5,10-dimethyltetrahydrofolate. This system is absent in *C. glutamicum*, necessitating the introduction of a heterologous GCS system. Using plasmid pXMJ19 as a vector, GCS system-related genes from *E. coli* and *B. subsills* were overexpressed, including *EccgvT*, *EccgvP*, *EccgvH*, and *EccgvL* from *E. coli* and *BscgvT*, *BscgvP*, *BscgvH*, and *BscgvL* from *B. subsills*. *EccgvT*, *EccgvP*, *EccgvH*, *BscgvT*, *BscgvP*, and *BscgvH* played crucial roles.

[0105] (1) Plasmid construction and strain verification are the same as in Example 3;

[0106] (2) Shake-flask fermentation: The engineered strain obtained in Example 6 (1), with the starting strain mCherry as the control group, was inoculated into 5 mL of BHIS medium and cultured at 30 ℃ and 200 rpm as a pre-culture. After 12 h-24 h, 2 mL of the pre-culture was inoculated into a 500 mL shake flask containing 50 mL of MS medium at a 4% inoculation rate. Then, the strain was cultured in a constant temperature shaker at 30 ℃ and 200 rpm for 48 h for fermentation. After fermentation, 1 mL of fermentation broth was taken to determine the OD. 600 Simultaneously, 1 mL of fermentation broth was pipetted and centrifuged at 12000 rpm for 3 min at room temperature. After removing impurities using an aqueous filter membrane, the sample was analyzed by HPLC according to Example 1. The contents of D-pantothenic acid and glycine in the fermentation broth supernatant were as follows: Figure 4 As shown.

[0107] Depend onFigure 4 It is evident that, regardless of whether the strong promoter Ptac (inherent in pXMJ19) or the weak promoter PdapB is used, the heterologous GCS does not promote the production of D-pantothenic acid; in fact, due to the pressure of protein expression, it has a slight inhibitory effect on production. Glycine accumulation is also not significantly reduced. The molar ratio of glycine to D-pantothenic acid remains unchanged compared to the control group. This indicates that the heterologous introduction of GCS does not function to convert the accumulated glycine for the production of 5,10-dimethyltetrahydrofolate.

[0108] Example 7: Construction of DPAi-3 and DPAj-2

[0109] (1) Construction of pK18-ΔilvE plasmid: Using pK18-mobsacb plasmid (Addgene Plasmid #177838) as a template, amplification was performed using PK18-F / PK18-R primers to obtain a linearized plasmid, which was named PK18; using C. glutamicum ATCC 13032 strain as a template, amplification was performed using M13r-serArup-F / down-serArup-R primers to obtain a fragment named serAup; using C. glutamicum ATCC 13032 strain as a template, amplification was performed using serArdown-F / M13f-serArdown-R primers to obtain a fragment named serArdown. The three amplified PCR products were purified by gel extraction to obtain purified fragments PK18, serAup, and serArdown. Following the instructions of Clon Express® (One step clone kit, Vazyme Biotech, Nanjing, China), PK18, serAup, and serArdown were ligated together, transformed into E. coli DH5α, screened by kanamycin plates, and sequenced to verify that the correct pK18-serAr plasmid was obtained.

[0110] (2) Electroporation of plasmid pK18-ΔserAr into competent cells of DPAg-15: Take 2 μg of plasmid pK18-serAr prepared by method (1) and 100 μL of electroporation competent cells prepared by method (3), mix them, and transfer them into a 1 mm electroporation cuvette pre-cooled at 4 ℃. After ice bath for 1 min, electroporate using an electroporator (Micro Pluser, BIO-RAD). After two consecutive electroporations, immediately add the mixture to 1 mL of BHIS medium pre-cooled at 4 ℃ and immediately transfer it to a sterile 1.5 mL centrifuge tube. After recovery at 30 ℃ and 200 rpm for 2 h, spread it on a BHIS plate containing 25 μg / L kanamycin and incubate upside down at 30 ℃ for 48 h until single colonies appear.

[0111] (3) Transformant verification and engineered strain screening verification: Colony PCR was performed using PK18-SEQ-F / serAr-down-R as verification primers. Five to ten colonies that amplified the correct bands were selected and inoculated into LB medium and cultured overnight at 30 ℃ and 200 rpm. The bacterial culture was diluted 10 times with antibiotic-free LB liquid medium. 3 10 4 10 5 The samples were spread onto LB agar containing 10% sucrose and incubated at 30 °C for approximately 48 h. Single colonies were simultaneously validated on both LB agar containing 10% sucrose and LB agar containing kanamycin resistance. Several single colonies that could grow on LB agar containing 10% sucrose but not on LB agar containing kanamycin resistance were selected. Validation was performed using primers serAr-up-F / serAr-down-R. PCR products with significantly smaller bands compared to the wild-type strain were sequenced. Sequencing results confirmed that the single colonies corresponding to the successful gene knockout were the correct engineered strain DPAi-3.

[0112] (4) Using the same method, DPAj-2 was constructed on the basis of DPAi-3 with the corresponding primers, that is, the start codon GTG of serAΔ197 in DPAi-3 was replaced with the strong start codon ATG.

[0113] (5) Electroporate pECXK99e-BspanBC+PdapB-EcpanE into DPAg-15, DPAi-3 and DPAj-2 to obtain the corresponding strains DPAg-15-P, DPAi-3-P and DPAj-2-P.

[0114] (6) Shake flask fermentation: The method is the same as in Example 3 (8), and the KIV and D-pantothenic acid content in the supernatant of the fermentation broth is tested.

[0115] Depend on Figure 6As can be seen, compared with DPAg-15-P, the production of D-pantothenic acid in DPAi-3-P and DPAj-2-P was significantly increased, while the accumulation of KIV gradually decreased. This indicates that both strains enhanced serine production, and the production of 5,10-dimethyltetrahydrofolate during the conversion of serine to glycine contributes to the formation of D-pantothenic acid.

[0116] Example 8: Design and Construction of a CPSPC System

[0117] As mentioned above, the formation of D-pantothenic acid is accompanied by the accumulation of glycine. 5,10-Dimethyltetrahydrofolate (5,10-CH2-THF) is one of the active forms of tetrahydrofolate (THF), and it participates in one-carbon unit transfer reactions in organisms as a coenzyme. In *C. glutamicum*, 5,10-dimethyltetrahydrofolate is entirely provided by the conversion of serine to glycine. Because ketopantolytic acid (KPR) has bifunctionality—it can catalyze the entry of KPR into the D-pantothenic acid synthesis pathway, but it can also prematurely catalyze the precursor KIV, causing carbon flow loss—the expression intensity of the *panE* gene needs to be well regulated to ensure that as much precursor KIV as possible enters the D-pantothenic acid synthesis pathway. Therefore, a CPSPC system was designed to dynamically regulate *panE* expression and simultaneously couple the 5,10-dimethyltetrahydrofolate synthesis pathway with the D-pantothenic acid synthesis pathway. The glycine riboswitch BsGR, derived from *B. subsills*, was used to control *panE* expression. In the early stages, without glycine accumulation, EcpanE, controlled by the glycine riboswitch, is either silent or at a low expression level, thus minimizing premature KIV shunting. As KPHMT (encoded by panB) converts KIV into ketopantolytic acid and accumulates, glycine gradually accumulates, thereby activating EcpanE expression. This allows for efficient utilization of ketopantolytic acid and redirects carbon flux towards D-pantothenic acid biosynthesis. Figure 7 ).

[0118] First, the expression of mCherry was controlled by Ptuf and Ph36 linked to BsGR. Fluorescence intensity at different glycine concentrations was measured relative to OD. 600 The change in the ratio was compared with the control of mCherry expression by the strong promoter Ptrc (pECXK99E itself) and the weak promoter PdapB. Figure 8 It can be seen that Ptuf and Ph36 linking to BsGR controls mCherry expression, and its fluorescence intensity is related to OD. 600 The ratio increases with increasing glycine concentration, with intensity varying between that of the strong promoter Ptrc and the weak promoter PdapB. Ptrc and PdapB control mCherry, and their fluorescence intensity varies with OD. 600The ratio does not change with increasing glycine concentration.

[0119] The PdapB plasmid on pECXK99e-BspanBC+PdapB-EcpanE was replaced with Ptuf and Ph36 ligated to BsGR, while PdapB was replaced with unligated Ptuf and Ph36 as controls. The constructed plasmids were named PtufGR-EcpanE, Ph36GR-EcpanE, Ptuf-EcpanE, and Ph36-EcpanE, respectively. Using pECXK99e-BspanBC+PdapB-EcpanE as a control, these plasmids were transformed into strain DPAj-2, followed by shake-flask fermentation to detect the KIV and D-pantothenic acid content in the supernatant. The construction, preparation, culture, and testing procedures were the same as in Example 7.

[0120] Depend on Figure 9 It is evident that the Ptuf-EcpanE and Ph36-EcpanE plasmids can completely convert accumulated KIV, but the yield of D-pantothenic acid is actually lower compared to the control group. This indicates that excessive panE expression prematurely converts KIV, leading to carbon flux loss. Compared to the control group, PtufGR-EcpanE and Ph36GR-EcpanE, as well as Ptuf-EcpanE and Ph36-EcpanE, showed higher D-pantothenic acid yields with some KIV remaining. PtufGR-EcpanE was particularly effective, achieving a D-pantothenic acid yield of 4.23 g / L. This demonstrates that the CPSPC system dynamically regulates panE expression, directing more carbon flux towards the D-pantothenic acid synthesis pathway.

[0121] Example 9: Construction of DPAj-2-tuf and fermentation test in a fermenter

[0122] (1) PtufGR-EcpanE constructed in Example 7 was transformed in DPAj-2, and competent cells were prepared from the obtained strain. The preparation process was the same as in Example 3 (2);

[0123] (2) The plasmid pXMJ19-serAΔ197 constructed in Example 5 GTG / ATG Transformed into competent cells constructed in Example 7(1). Preparation and verification processes were the same as in Example 5. The correct transformant obtained was DPAj-2-tuf;

[0124] (3) Fermentation tank test: Fresh single colonies of DPAj-2-tuf from the plate were inoculated into BHIS test tube medium containing 25 mg / L kanamycin resistance and 12 mg / L chloramphenicol resistance, and cultured at 30℃ for 18 h as seed culture; 5% by volume was inoculated into BHIS medium and cultured at 30℃ for 12 h as seed culture for the upper tank; the seed culture from the upper tank was inoculated into the fermentation tank containing fermentation medium at 10% by volume, and fermented at 30℃, 400 rpm, and an aeration rate of 1.0 V / V·min, with dissolved oxygen controlled at 20%~40%. Constant feed rate was used to keep the glucose concentration below 5 g / L, and fermentation was continued for 63 h to obtain fermentation broth containing D-pantothenic acid.

[0125] The fermentation medium consisted of: glucose 40 g / L, ammonium sulfate 20 g / L, corn steep liquor 20 g / L, yeast extract 2 g / L, KH₂PO₄ 0.8 g / L, MgSO₄ 0.5 g / L, sodium acetate 5 g / L, β-alanine 2 g / L, CaCO₃ 3 g / L, and a 1 mL / L trace element solution in deionized water at a natural pH. The trace element solution composition was: NiCl₂·7H₂O 0.02 g / L, CuCl₂ 10 g / L, FeSO₄·7H₂O 10 g / L, ZnSO₄·7H₂O 10 g / L, CuSO₄ 0.2 g / L in deionized water. The feed medium consists of 500 g / L glucose, 20 g / L (NH4)2SO4, 1 g / L MgSO4, and 30 g / L β-alanine, dissolved in deionized water and adjusted to pH 6.8 with 50% ammonia.

[0126] Depend on Figure 10 It can be seen that the production of D-pantothenic acid at 23.06 g / L in 63 hours was achieved with a yield of 0.37 g / L / h and a conversion rate of 0.17 g / g, with the highest OD value being... 600 It reached 81.90.

[0127] In summary, this invention first introduces the glycine riboswitch BsGR from Bacillus subtilis and links it to different strong constitutive promoters, verifying its effectiveness in *Corynebacterium glutamicum* by controlling the expression of mCherry. Then, the engineered strain DPAg-15, preserved in the laboratory, was selected as the starting strain to test the effect of overexpression of genes related to coenzyme synthesis pathways on D-pantothenic acid production. A GCS system from *E. coli* and *B. subsills* was introduced to catalyze the accumulation of glycine. Genes related to the 5,10-dimethyltetrahydrofolate synthesis pathway were enhanced in the genome. Utilizing the characteristics of glycine accumulation, a CPSPC system was designed to dynamically regulate the coupling of one-carbon units and D-pantothenic acid synthesis, thereby increasing D-pantothenic acid production. The mutant strain obtained in this invention exhibits dispersed mycelial growth, inhibiting agglomeration during *Streptomyces* fermentation, facilitating the reduction of fermentation broth viscosity in industrial production. The strain maintains a good growth state during growth, and the engineered strain shows an improvement of more than 8.1% compared to the control strain.

[0128] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in methods and compositions, will be apparent to those skilled in the art without departing from the scope and spirit of this invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A genetically engineered bacterium producing D-pantothenic acid based on the Corynebacterium glutamicum CPSPC system, characterized in that, Prepared by the following construction method: (1) The glycine ribosome switch BsGR from Bacillus subtilis was linked to the promoter of Corynebacterium glutamicum and introduced into the host bacteria through the plasmid pECXK99e. Engineered strains carrying high glycine concentration responsive promoters were screened. (2) Corynebacterium glutamicum ATCC 13032 was used as the chassis strain. The ilvE gene, avtA gene, ilvA gene and pqo gene were knocked out in sequence. The ilvBNC-VWBGTG / ATG controlled by the Ptuf promoter and the Pefu promoter were integrated at the ΔilvA and ΔavtA sites, respectively. The promoter of the aceE gene was replaced by PdapA A16, and the strain DPAg-15 was obtained. (3) Using plasmid pECXK99e as a vector, a co-expression combination of the gene cluster BspanBC from Bacillus subtilis 168 and the EcpanE gene from E.coli was formed, wherein the promoter of EcpanE was controlled by PdapB, resulting in plasmid pECXK99e-BspanBC+PdapB-EcpanE; (4) Using strain DPAg-15 as the chassis strain, the pECXK99e-BspanBC+PdapB-EcpanE plasmid and pXMJ19 recombinant plasmid were sequentially introduced by electroporation to obtain the engineered strain; the pXMJ19 recombinant plasmid is including but not limited to recombinant plasmid A carrying coenzyme synthesis pathway related genes and recombinant plasmid B carrying GCS system related genes from E. coli or Bacillus subtilis; (5) Using the homologous recombination gene editing technology mediated by the suicide plasmid of the sacB gene, the C-terminal 197 amino acid sequence of serA on the genome of strain DPAg-15 was knocked out to obtain engineered strain DPAi-3. (6) Using the homologous recombination gene editing technology mediated by the suicide plasmid of the sacB gene, the start codon GTG of the truncated serA in the genome of the engineered strain DPAi-3 was replaced with ATG to obtain the engineered strain DPAj-2. (7) Design the Corynebacterium glutamicum CPSPC system so that the glycine ribosome switch BsGR under the control of the high glycine concentration responsive promoter screened in step (1) replaces the PdapB gene in the pECXK99e-BspanBC+PdapB-EcpanE plasmid to obtain the recombinant expression plasmid. (8) The recombinant expression plasmid obtained in step (5) and the recombinant plasmid A obtained in step (2) are co-transformed into the engineered strain DPAj-2 to obtain the engineered strain DPAj-2-tuf, which is the genetically engineered bacterium.

2. The genetically engineered bacteria according to claim 1, characterized in that, Step (1) includes: (a) By linking the glycine ribosome switch BsGR from Bacillus subtilis with the promoters Pefu, Ptuf, Ph36, Peno and Psod from Corynebacterium glutamicum, a variety of gene expression units controlling the fluorescent protein mCherry were constructed. (b) The gene expression unit was cloned into plasmid pECXK99e by restriction enzyme digestion and then introduced into Corynebacterium glutamicum ATCC 13032 by electroporation to obtain the transformed strain; (c) After culturing each transformed strain in BHIS medium containing gradient concentrations of glycine at 30 °C for 10–30 h, the fluorescence intensity at 597 nm / 608 nm and OD were measured. 600 The ratio of glycine concentration to γ- ...

3. The genetically engineered bacteria according to claim 3, characterized in that: The nucleotide sequence of the BsGR gene is shown in SEQ ID No. 1, the nucleotide sequence of the Ptuf promoter is shown in SEQ ID No. 2, the nucleotide sequence of the Ph36 promoter is shown in SEQ ID No. 3, the nucleotide sequence of the Pefu promoter is shown in SEQ ID No. 4, the nucleotide sequence of the Psod promoter is shown in SEQ ID No. 5, and the nucleotide sequence of the Peno promoter is shown in SEQ ID No.

6.

4. The genetically engineered bacteria according to claim 2, characterized in that, The high glycine concentration-responsive promoter is Ptuf or Ph36.

5. The genetically engineered bacteria according to claim 4, characterized in that, The coenzyme synthesis pathway-related genes in the recombinant plasmid A were selected from serA. GTG / ATG serAΔ197 GTG / ATG serAΔ197 GTG / ATG Any one of +serBC, pabABC, BsfolD, EcfolD, and glyA.

6. The genetically engineered bacteria according to claim 4, characterized in that, The recombinant plasmid A carries the coenzyme synthesis pathway-related gene serAΔ197. GTG / ATG Its nucleotide sequence is shown in SEQ ID No.

7.

7. The genetically engineered bacteria according to claim 1, characterized in that, The method for constructing the pXMJ19 recombinant plasmid is as follows: Using plasmid pXMJ19 as a vector, genes related to the coenzyme synthesis pathway were overexpressed to construct recombinant plasmid A; Using plasmid pXMJ19 as a vector, GCS-related genes from E. coli and B. subsills were overexpressed to construct recombinant plasmid B.

8. The genetically engineered bacteria according to claim 4 or 7, characterized in that, Genes related to the GCS system from *E. coli* include the EccgvT, EccgvP, EccgvH, and EccgvL genes; the nucleotide sequence of the EccgvT gene is shown in SEQ ID No. 8; the nucleotide sequence of the EccgvP gene is shown in SEQ ID No. 9; the nucleotide sequence of the EccgvH gene is shown in SEQ ID No. 10; and the nucleotide sequence of the EccgvL gene is shown in SEQ ID No.

11. Genes related to the GCS system from *B. subsills* include the BscgvT, BscgvP, BscgvH, and BscgvL genes; the nucleotide sequence of the BscgvT gene is shown in SEQ ID No. 12; the nucleotide sequence of the BscgvP gene is shown in SEQ ID No. 13; the nucleotide sequence of the BscgvH gene is shown in SEQ ID No. 14; and the nucleotide sequence of the BscgvL gene is shown in SEQ ID No.

15.

9. The use of the genetically engineered bacteria according to any one of claims 1 to 8 in the microbial fermentation preparation of D-pantothenic acid.

10. The application according to claim 9, characterized in that, The application involves inoculating the genetically engineered bacteria into a fermentation medium and fermenting it at 30°C and 200 rpm for 48-60 h. After fermentation, the supernatant of the fermentation broth is collected and purified to obtain the D-pantothenic acid. The fermentation medium consists of the following components: glucose: 10-30 g / L, corn steep liquor: 10-20 g / L, ammonium sulfate: 10-20 g / L, yeast extract: 1-5 g / L, KH₂PO₄: 0.2-1 g / L, MgSO₄: 0.5-5 g / L, sodium acetate: 5-8 g / L, β-alanine: 0.5-2 g / L, CaCO₃: 3-5 g / L, and a 0.5-2 mL / L trace element solution in deionized water at a natural pH. The trace element solution consists of: NiCl₂·7H₂O 0.02 g / L, CuCl₂ 10 g / L, FeSO₄·7H₂O 10 g / L. The concentrations of the following ingredients were: g / L ZnSO4·7H2O 10 g / L, CuSO4 0.2 g / L, and the solvent was deionized water.

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