Klebsiella pneumoniae for efficiently producing D-pantothenic acid as well as construction method and application of klebsiella pneumoniae

Abstract

The invention discloses klebsiella pneumoniae for efficiently producing D-pantothenic acid as well as a construction method and application of the klebsiella pneumoniae, and the specific construction method comprises the following steps: by taking wild klebsiella pneumoniae as an original strain, overexpressing a ketopantoic acid hydroxymethyltransferase gene panB, a pantothenic acid synthetase gene panC and a ketopantoic acid reductase gene panE from escherichia coli; the invention relates to related genes for weakening the expression of a valine-pyruvate transaminase gene ilvE and inactivating byproduct synthesis in a host, and the related genes comprise an alpha-acetolactate decarboxylase gene budA, an acetoin reductase gene budC, glycerol dehydrogenase gldA, a pyruvate oxidase gene poxB, a pyruvate formate lyase activating enzyme gene pflB, an ethanol dehydrogenase gene adhE and a lactic dehydrogenase gene ldhA. The constructed klebsiella pneumoniae engineering bacterium can efficiently synthesize D-pantothenic acid by fermenting glucose, and has great application potential and economic value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and microbial fermentation, and particularly relates to Klebsiella pneumoniae capable of efficiently producing D-pantothenic acid, and a construction method and application thereof. Background Art

[0002] Pantothenic acid, also known as vitamin B5, is a water-soluble vitamin found in nature in two primary configurations: D and L. Only the D configuration is biologically active. In vivo, D-pantothenic acid (D-PA) plays a crucial role in the formation of coenzyme A (CoA) and acyl carrier protein (ACP), playing a vital role in protein and lipid metabolism, as well as the synthesis of numerous secondary metabolites. Therefore, D-PA is widely used in pharmaceuticals, food, health supplements, and feed additives.

[0003] Currently, the industrial production of D-PA relies primarily on chemical-enzymatic methods, which pose significant environmental pollution and complex procedures. Microbial fermentation, due to its environmental sustainability, has become a research hotspot. Researchers have engineered microorganisms such as Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus megaterium, and Saccharomyces cerevisiae for D-PA fermentation. Escherichia coli has been the most studied due to its clear genetic background and ease of operation. However, its current production costs are still not competitive with chemical-enzymatic methods, preventing its practical application.

[0004] Klebsiella pneumoniae is a Gram-negative bacterium that is widely present in the environment. It has the advantages of wide substrate adaptability (it can utilize cheap carbon sources such as glucose, glycerol, and xylose), fast growth rate, and short fermentation cycle. It has been used to produce a variety of chemicals such as 2,3-butanediol and 1,3-propylene glycol. Summary of the Invention

[0005] In response to the above problems, the present invention aims to provide a Klebsiella pneumoniae strain that can efficiently produce D-pantothenic acid, as well as a construction method and application thereof. Through systemic metabolic engineering, the present invention constructs for the first time an engineered Klebsiella pneumoniae strain that can efficiently synthesize D-pantothenic acid, providing a new production bacterium for the microbial fermentation production of D-pantothenic acid.

[0006] The specific technical solutions are as follows:

[0007] A method for constructing Klebsiella pneumoniae that efficiently produces D-pantothenic acid comprises the following steps:

[0008] (1) Using wild-type Klebsiella pneumoniae ATCC 10031 as the chassis strain, the ketopantoate hydroxymethyltransferase gene panB from Escherichia coli Hfr3000 YA139, the pantothenate synthase gene panC from Escherichia coli BL21 (DE3), and the ketopantoate reductase gene panE were introduced and expressed in tandem through the tac promoter of the pDK6 plasmid;

[0009] (2) Gene knockout technology was used to inactivate the genes involved in the 2,3-butanediol synthesis pathway, namely α-acetolactate decarboxylase budA, acetoin reductase budC, and glycerol dehydrogenase gldA, thereby blocking the competitive carbon flow;

[0010] (3) Gene knockout technology was used to inactivate the pyruvate oxidase gene poxB, the pyruvate formate lyase activating enzyme gene pflB, the alcohol dehydrogenase gene adhE, and the lactate dehydrogenase gene ldhA in the chassis strain to reduce mixed acid fermentation byproducts and enhance pyruvate accumulation;

[0011] (4) Weaken the expression of the valine-pyruvate transaminase gene ilvE and mutate the start codon from ATG to TTG to reduce the competition of branched-chain amino acids for α-ketoisovalerate, thereby constructing Klebsiella pneumoniae that efficiently produces D-pantothenic acid.

[0012] Furthermore, in the pDK6 plasmid, the panB, panC, and panE genes are respectively driven by the tac promoter for expression, wherein panB and panC share a tac promoter, and panE independently uses a tac promoter.

[0013] Furthermore, the gene knockout technology uses the Red homologous recombination system, the knockout fragment contains upstream and downstream homologous arms and apramycin resistance screening marker, and the resistance marker is eliminated by Flp recombinase.

[0014] A Klebsiella pneumoniae constructed by the construction method can efficiently synthesize D-pantothenic acid using glucose as a substrate.

[0015] The invention discloses an application of Klebsiella pneumoniae in the fermentation production of D-pantothenic acid. During the fermentation process, IPTG with a final concentration of 0.1 mmol / L is added when the OD600 reaches 0.8 to induce gene expression.

[0016] The beneficial effects of the present invention are:

[0017] (1) The strain with the best performance obtained after the modification of the present invention has a significantly improved level of D-pantothenic acid fermentation production compared with the wild strain. The D-pantothenic acid production in the shake flask is increased from 0.01 g / L to 1.0-1.5 g / L, and the production in the 5 L fermentation tank is increased to 10-15 g / L.

[0018] (2) For the first time, Klebsiella pneumoniae achieved efficient synthesis of D-pantothenic acid, laying the foundation for further increasing the yield of D-pantothenic acid and providing a new strategy for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of metabolic transformation of Klebsiella pneumoniae D-PA high-producing strain.

[0020] Figure 2 The fermentation results of D-PA10 in a 5L fermenter. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only a portion of the embodiments of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0022] The term "plasmid overexpression" refers to the technology of expressing the target gene at an abnormally high level in the host cell by constructing a recombinant plasmid vector. Strong promoters or multi-copy plasmids are usually used to drive gene transcription, thereby producing a large amount of target protein. The term "weakening" refers to weakening the activity of the enzyme encoded by the corresponding polynucleotide, which can be achieved by replacing the expression regulatory sequence of the gene on the genome (non-coding region sequence replacement, etc.). The term "gene inactivation" refers to the temporary or permanent inhibition of the expression or functional activity of the target gene by physical, chemical or biological means.

[0023] The materials and methods involved in the following examples are as follows:

[0024] 1) Sequence:

[0025] The nucleotide sequence of the ketopantoate hydroxymethyltransferase panB gene from Escherichia coli Hfr3000 YA139 is shown in SEQ ID NO. 1;

[0026] The nucleotide sequence of the panC gene of pantothenate synthase from Escherichia coli BL21(DE3) is shown in SEQ ID NO. 2; the nucleotide sequence of the panE gene of ketopantoate reductase from Escherichia coli BL21(DE3) is shown in SEQ ID NO. 3;

[0027] The nucleotide sequence encoding the pyruvate oxidase poxB gene is shown in SEQ ID NO.4;

[0028] The nucleotide sequence of the gene encoding pyruvate formate lyase activating enzyme pflB is shown in SEQ ID NO.5;

[0029] The nucleotide sequence encoding the alcohol dehydrogenase adhE gene is shown in SEQ ID NO.6;

[0030] The nucleotide sequence encoding the lactate dehydrogenase ldhA gene is shown in SEQ ID NO.7;

[0031] The nucleotide sequence encoding the α-acetolactate decarboxylase budA gene is shown in SEQ ID NO.8;

[0032] The nucleotide sequence encoding the acetoin reductase budC gene is shown in SEQ ID NO.9;

[0033] The nucleotide sequence encoding the glycerol dehydrogenase gldA gene is shown in SEQ ID NO.10;

[0034] The nucleotide sequence of the gene in which the start codon of ilvE is changed from ATG to TTG is shown in SEQ ID NO. 11;

[0035] The nucleotide sequence of the Tac promoter is shown in SEQ ID NO.12.

[0036] 2) Strain: The Klebsiella strain used in the present invention is Klebsiella pneumoniae ATCC10031.

[0037] 3) Culture medium and solution composition:

[0038] LB liquid culture medium composition: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, prepared with deionized water, natural pH value, LB plate is prepared by adding agar powder to the LB liquid culture medium at a final concentration of 2 g / L.

[0039] The composition of 1L fermentation medium is as follows: 30g / L glucose, 20g / L (NH4)2SO4, 2g / L K2HPO4, 0.25g / L KH2PO4, 0.4g / L MgSO4·7H2O, 1.5g / Lβ-alanine, and 1mL trace element solution.

[0040] 1L of trace element solution contains: CoCl2 10.0g / L, FeSO4·7H2O 10.0g / L, CuSO4 0.2g / L, NiCl2·7H2O 0.02g / L, ZnSO4·7H2O 1.0g / L.

[0041] 10% SDS solution: Weigh 1 g of SDS (sodium dodecyl sulfate) and dissolve it in distilled water to make up to 10 mL.

[0042] 4) Sample processing:

[0043] Take an appropriate amount of fermentation broth in a centrifuge tube, centrifuge at 12000 rpm for 2 minutes, take the supernatant, dilute the D-pantothenic acid content to 0.05-0.40 g / L with purified water, and filter with a 0.22 μm filter membrane.

[0044] 5) Detection method of D-pantothenic acid:

[0045] HPLC detection: The chromatographic column was C18-AQ (5 μm, A 4.6×250 mm column (Yuexu, China) was used, the mobile phase was acetonitrile / water / phosphoric acid (volume ratio 50 / 949.5 / 0.5), the detection wavelength was 200 nm, the column temperature was 30°C, and the data acquisition time was 18 min.

[0046] 6) Preparation of Klebsiella competent cells:

[0047] The preserved strain was streaked and activated, and cultured in a 37°C constant temperature incubator overnight; a single colony was selected and inoculated into 5 mL of LB liquid medium, and activated in a constant temperature oscillator at 37°C and 220 rpm for 12 hours; the activated bacterial solution was inoculated into 50 mL of LB liquid medium at a 1% inoculum volume (500 μL) and cultured at 37°C and 220 rpm until the OD600 was about 0.7. When the bacterial solution concentration OD600 was about 0.7, ice bathed for 30 minutes, and pre-cooled sterile water was used; the pre-cooled bacterial suspension was centrifuged at 4°C and 7500 rpm for 10 minutes, and the supernatant was discarded; the bacteria were resuspended in 25 mL of sterile water, and centrifuged at 4°C and 7500 rpm for 10 minutes, and the supernatant was discarded; the above steps were repeated twice; the final precipitate was resuspended in 5 mL of sterile water, and 100 μL per tube was dispensed into sterile 1.5 mL centrifuge tubes.

[0048] 7) Electrotransformation of Klebsiella competent cells:

[0049] Klebsiella competent cells were prepared and used immediately. 100uL of Klebsiella competent cells was taken, 2μL of exogenous plasmid was added, the mixture was mixed gently by pipetting, and the cells were placed on ice for 30min. The electroporator parameters were set to 1.8kV voltage for blank shock, and the return time was 6.0ms, indicating that the electroporator was operating normally and electroporation could be performed. All the bacterial liquid in the EP tube was added to the pre-cooled sterile electroporation cup and electroporated at 1.8kV voltage. After the electroporation, 1mL of pre-cooled LB liquid culture medium was added and the cells were revived at 37℃ and 220rpm for 1h. The revived bacterial liquid was spread on a solid plate containing the corresponding resistance and cultured in a 37℃ constant temperature incubator overnight. Single colonies were selected for PCR amplification verification and sequencing.

[0050] 8) Metabolic pathway modification of D-PA production strains Figure 1 shown.

[0051] Example 1: Screening and expression of panBC gene

[0052] To construct a basic D-pantothenate biosynthesis pathway in wild Klebsiella pneumoniae, the endogenous ketopantoate hydroxymethyltransferase panB and pantothenate synthase panC from Escherichia coli BL21(DE3), Bacillus subtilis 168, Corynebacterium glutamicum ATCC 13032, Escherichia coli Hfr3000YA139, and Klebsiella pneumoniae were cloned into the expression vector pDK6 (laboratory preservation) and transformed into Klebsiella pneumoniae ATCC10031 to obtain an engineered strain and apply it to the fermentation production of D-pantothenate.

[0053] 1. Construction of pDK6-panB / panC Plasmid

[0054] 1) Using vector-F and vector-R as primers and the diluted pDK6 plasmid as a template, amplify the vector backbone fragment. Digest with DpnI enzyme to remove the template DNA to obtain the vector fragment pDK6-vector;

[0055] 2) Amplify the Ec-panBC fragment using primers E-panBC-F and E-panBC-R and the E. coli BL21 genome as a template;

[0056] 3) Amplify the Bs-panBC fragment using primers B-panBC-F and B-panBC-R and the Bacillus subtilis 168 genome as a template;

[0057] 4) Using C-panBC-F and C-panBC-R as primers and the genome of Corynebacterium glutamicum ATCC 13032 as a template, amplify the Cg-panBC fragment;

[0058] 5) Using K-panBC-F and K-panBC-R as primers and the Klebsiella pneumoniae genome as a template, amplify the Kp-panBC fragment;

[0059] 6) Using primers H-panB-F and H-panB-R and the synthetic Hfr3000 YA139 E. coli panB gene as a template, amplify the H-panB fragment to replace the panB gene from E. coli BL21;

[0060] 7) The enzyme-digested plasmid obtained in step (1) and the gene fragments obtained in steps (2), (3), (4), (5), and (6) were purified separately, and then recombined using the ClonExpress II One Step Cloning Kit (Novagen) to obtain a recombinant plasmid.

[0061] 2. Construction of recombinant strains

[0062] Each recombinant plasmid pDK6-panBC was transformed into competent Escherichia coli DH5α and cultured on LB plates containing 50 mg / L kanamycin. The correct recombinant plasmid was obtained by PCR and sequencing.

[0063] After extracting the correct recombinant plasmid, it was electrotransformed into Klebsiella pneumoniae ATCC10031. The obtained recombinant strains were named Kp-panBCbs, Kp-panBCec, Kp-panBCcg, Kp-panBCkp, and Kp-panBC Hfr Cec (corresponding to the fragments of steps 2), 3), 4), 5), and 6) respectively).

[0064] 3. Strains Kp-panBCbs, Kp-panBCec, Kp-panBCcg, Kp-panBCkp, Kp-panB Hfr Fermentation of Cec

[0065] Using wild Klebsiella as the control strain, Kp-panBCbs, Kp-panBCec, Kp-panBCcg, Kp-panBCkp, Kp-panBC HfrCec was inoculated into 5 mL of LB medium and cultured at 37°C and 220 rpm to serve as a seed solution. After 12 hours, 500 μL of the seed solution was inoculated into a 250 mL shake flask containing 50 mL of fermentation medium. Fermentation was then continued at 37°C and 150 rpm. When the bacterial concentration reached OD600 = 0.8, IPTG was added to a final concentration of 0.1 mM and culture was continued for 48 hours. After fermentation, 1 mL of the fermentation broth was centrifuged at 12,000 rpm for 2 minutes at room temperature. The supernatant was collected and analyzed by HPLC. The yield is shown in Table 1.

[0066] Table 1 D-pantothenic acid yield of each recombinant strain in shake flask fermentation

[0067] strain Shake flask yield (mg / L) WT 10 Kp-panBCbs 51 Kp-panBCec 83 Kp-panBCcg 122 Kp-panBCkp 70 <![CDATA[Kp-panB Hfr Cec]]> 161

[0068] The experimental results showed that after the introduction of the heterologous panBC gene, the Klebsiella strain's ability to accumulate D-pantothenic acid outside the cell was greatly enhanced, and it was better able to utilize carbon sources such as glucose for the synthesis of D-pantothenic acid than the wild type. Among them, the strain expressing anti-feedback inhibition panBC produced the highest yield of D-pantothenic acid in fermentation, reaching 0.161g / L. The recombinant strain Kp-panB Hfr Cec was named D-PA1 and used as the strain for subsequent further modification.

[0069] Example 2: Tandem expression of panE and panBC genes

[0070] To direct more carbon metabolic flux to the main D-pantothenate synthesis pathway, the ketopantoate reductase encoding gene panE from Escherichia coli BL21 was introduced into the Klebsiella pneumoniae D-PA1 strain.

[0071] pDK6-panB Hfr The construction process of C-panE plasmid is as follows:

[0072] (1) Using panBC-Hfr-F and panBC-Hfr-R as primers, construct pDK6-panB Hfr Plasmid C was used as a template to amplify the vector backbone containing the panBC-Hfr gene.

[0073] (2) Using E-panE-F and E-panE-R as primers and the E. coli BL21 genome as a template, the E-panE fragment was amplified, and the upstream and downstream primers carried the tac promoter respectively.

[0074] The construction and fermentation steps of the recombinant strain were the same as those in Example 1, and the strain yield reached 389 mg / L.

[0075] The experimental results showed that after the introduction of panE gene expression in the D-PA1 strain, the fermentation production level of D-pantothenic acid increased from 0.161g / L to 0.389g / L. The recombinant strain was named D-PA2.

[0076] Example 3: Knockout of genes related to the 2,3-butanediol synthesis pathway

[0077] Wild-type Klebsiella can synthesize high concentrations of 2,3-butanediol, indicating that the metabolic flux of this synthesis pathway is large. In order to increase the synthesis flux of D-pantothenic acid and reduce the byproduct 2,3-butanediol, the present invention knocks out the genes budA, budC and gldA related to the 2,3-butanediol synthesis pathway to block the synthesis of 2,3-butanediol metabolism.

[0078] Using wild-type Klebsiella pneumoniae ATCC 10031 as the starting strain, knockout was achieved using the red recombinase-assisted homologous recombination system, containing two plasmids, pDK6-red and pDK6-flp. The red homologous recombination technology is derived from the homologous recombinase system of bacteriophage lambda. This system comprises three derived recombination proteins: Gam, Exo, and Beta. Gam inhibits host exonuclease activity, preventing degradation of transfected exogenous DNA fragments; Exo acts on the 5'-3' exonuclease of double-stranded DNA, creating single-stranded nicks at both ends of the linear DNA; and Beta binds to these nicks, mediating recombination between the exogenous DNA and the targeted homologous sequence in the genome. The pDK6-red plasmid contains the structural genes encoding Gam, Exo, and Beta, whose expression is induced by the lactose operator LacO, responsible for the targeted knockout of the target genomic band. Plasmid pDK6-flp encodes Flp recombinase, which recognizes specific FRT sequences and causes the gene sequence between the FRT sites to be deleted or recombined.

[0079] 1. Construction of budA gene knockout strain

[0080] (1) The pDK6-red plasmid was introduced into the wild Klebsiella pneumoniae ATCC 10031 strain to construct the red recombination system;

[0081] (2) Amplify the target gene: Based on the wild Klebsiella pneumoniae ATCC 10031 genome sequence, determine the location of budA in the genome, select the 800bp upstream and downstream fragments of the target gene as homology arms, and use Snapgene to design primers. Using budA-up-F, budA-up-R and budA-down-F, budA-down-R as primers, and wild-type Klebsiella as template, amplify the upstream and downstream DNA fragments. Using apr-F and apr-R as primers, and the pIJ773 plasmid (laboratory collection) with apramycin resistance gene as template, amplify the resistance gene with FRT sequence. DNA fragments are easily degraded, and connecting them to T vectors can better preserve the repair template. Using TF and TR as primers, and pMD18-T As a template, linearized pMD18-T vector was amplified and Dpn1 was eliminated from the pMD18-T plasmid.

[0082] (3) Construction of replacement template plasmid: The four fragments obtained in step (2) (upstream and downstream DNA fragments, resistance gene with FRT sequence, linearized pMD18-T vector) were ligated using the Ultra One Step Cloning Kit (Novozymes). The reaction was allowed to react at 50°C for 15 minutes and then immediately placed on ice. The above reaction system was transformed into Escherichia coli DH5α, and colonies were screened using a solid plate containing apramycin. Single colonies were verified by PCR using T-check-F and T-check-R primers and sequenced. The positive clone was identified as DH5α-T-budA.

[0083] (4) Using budA-up-F and budA-down-R as primers and DH5α-T-budA obtained in step (3) as a template, amplify the resistance cassette fragment containing the FRT sequence and upstream and downstream homology arms. Prepare competent cells of Klebsiella carrying the pDK6-red plasmid, and under the induction of IPTG, electroporate the resistance cassette fragment into the competent cells. After recovery, spread it on LB solid culture medium of the corresponding resistance and place it in a constant temperature incubator at 37°C overnight.

[0084] (5) Select a single colony, verify it with primers budA-up-F and budA-down-R, and sequence it. If the sequencing results are correct, the budA gene knockout strain is successfully constructed.

[0085] The methods for constructing other knockout strains were as described above.

[0086] 2. Elimination of resistance in knockout strains

[0087] (1) Elimination of plasmid pDK6-red: The knockout strain contains the pDK6-red plasmid, which affects the growth rate of the strain and subsequent operations, and the plasmid needs to be eliminated. The knockout strain was inoculated into 5 mL of LB liquid culture medium, and 1% of the inoculum was added with 10% (w / v) SDS solution without adding kanamycin. The culture was subcultured every 12 hours at 37°C and 220 rpm. After four subcultures, a small amount of bacterial solution was diluted and spread on an LB solid culture dish without kanamycin, and cultured at 37°C overnight. After obvious single colonies grew, they were transferred to LB solid culture media containing kanamycin and LB solid culture media without kanamycin, and screened using the stamp method. The engineered strain that eliminated pDK6-red grew on a plate without kanamycin, but did not grow on a plate containing kanamycin.

[0088] (2) Elimination of resistance screening marker: After gene knockout is completed by homologous recombination mediated by the red system, resistance sequences and two FRT sites will be left on the genome. The plasmid pDK6-flp is electroporated into competent cells and single colonies are screened. The single colony is inoculated into 5 mL LB liquid culture medium, and IPTG is added at an inoculum rate of 0.1% to induce FLP expression. The culture is subcultured every 12 hours at 37°C and 220 rpm. After ten subcultures, a small amount of bacterial solution is diluted and spread on an LB solid culture plate without resistance, and cultured at a constant temperature of 37°C overnight. After the growth of a single colony, it is transferred to LB solid culture medium containing relevant resistance and LB solid culture medium without resistance, and screened using the stamp method. The engineered strain with eliminated resistance grows on a plate without resistance and does not grow on a plate containing relevant resistance.

[0089] (3) The elimination method of plasmid pDK6-flp is the same as that of pDK6-red, see step (1)

[0090] After obtaining the strain with the budA gene knocked out, the knocked-out strain was prepared into Klebsiella competent state as in step 2 of Example 1, and the pDK6-B constructed in Example 22 was cloned into Hfr The CE plasmid was electroporated into Kp-ΔbudA; the fermentation steps were the same as in Example 1.

[0091] The Klebsiella with the budA gene knocked out was named Kp-ΔbudA, and the high-yield recombinant strain obtained after the plasmid was introduced was named D-PA3. The D-pantothenic acid production of the strain is shown in Table 2.

[0092] Table 2 D-pantothenic acid production by shake flask fermentation of strain D-PA3

[0093] strain Shake flask yield (mg / L) D-PA2 389 D-PA3 639

[0094] After knocking out the budA gene, the strain with the best performance increased its D-pantothenic acid production level from 0.389 g / L to 0.639 g / L compared with the starting strain.

[0095] 3. Knockout of budC and gldA genes

[0096] Using Kp-ΔbudA as the starting strain, two targeting fragments were designed to knock out the budC and gldA genes. The two targeting fragments are as follows:

[0097] (1) budC-up-F, budC-up-R and budC-down-F, budC-down-R were used as primers and wild-type Klebsiella as template to amplify upstream and downstream DNA fragments.

[0098] (2) Using gldA-up-F, gldA-up-R and gldA-down-F, gldA-down-R as primers and wild-type Klebsiella as template, upstream and downstream DNA fragments were amplified.

[0099] After obtaining the strain with knockout budC and gldA genes, the knockout strain was made into Klebsiella competent state as in step 2.(1)(2) of Example 11, and the pDK6-B constructed in Example 2 was cloned into Hfr The CE plasmid was electroporated into Kp-ΔbudA-ΔbudC and Kp-ΔbudA-ΔbudC-ΔgldA; and the fermentation steps were the same as in Example 1.

[0100] The steps of construction of knockout strains and plasmid loss were the same as in Example 3 to obtain Kp-ΔbudA-ΔbudC and Kp-ΔbudA-ΔbudC-ΔgldA. The high-yield recombinant strains obtained after plasmid introduction were named D-PA4 and D-PA5, respectively. Their yields are shown in Table 3.

[0101] Table 3 D-pantothenic acid yields of D-PA4 and D-PA5 strains in shake flask fermentation

[0102] strain Shake flask yield (mg / L) D-PA4 732 D-PA5 801

[0103] Example 5: Knockout of ldhA, pflB, poxB, and adhE genes

[0104] Wild-type Klebsiella pneumoniae produces a large number of mixed acid byproducts, including lactic acid, formic acid, acetic acid, and ethanol. To enrich the pyruvate pool and reduce carbon metabolic loss, attempts were made to inactivate the ldhA, pflB, poxB, and adhE genes that metabolize pyruvate to lactic acid, formic acid, acetic acid, and ethanol.

[0105] Using Kp-ΔbudA-ΔbudC-ΔgldA as the starting strain, four targeting fragments were designed to knock out the ldhA, pflB, poxB, and adhE genes, respectively. The four targeting fragments are as follows:

[0106] (1) ldhA-up-F, ldhA-up-R and ldhA-down-F, ldhA-down-R were used as primers, and wild-type Klebsiella total DNA was used as a template to amplify the upstream and downstream DNA fragments of ldhA.

[0107] (2) pflB-up-F, pflB-up-R and pflB-down-F, pflB-down-R were used as primers and wild-type Klebsiella total DNA was used as a template to amplify the upstream and downstream DNA fragments of pflB.

[0108] (3) Using poxB-up-F, poxB-up-R and poxB-down-F, poxB-down-R as primers and wild-type Klebsiella total DNA as a template, the upstream and downstream DNA fragments of poxB were amplified.

[0109] (4) Using primers adhE-up-F, adhE-up-R and adhE-down-F, adhE-down-R, and wild-type Klebsiella total DNA as a template, adhE upstream and downstream DNA fragments were amplified.

[0110] The steps of construction of knockout strain and plasmid loss were the same as in Example 3, and the obtained

[0111] Kp-ΔbudA-ΔbudC-ΔgldA-ΔldhA, Kp-ΔbudA-ΔbudC-ΔgldA-ΔldhA-ΔpflB, Kp-ΔbudA-ΔbudC-ΔgldA-ΔldhA-ΔpflB-ΔpoxB,

[0112] Kp-ΔbudA-ΔbudC-ΔgldA-ΔldhA-ΔpflB-ΔpoxB-ΔadhE.

[0113] The pDK6-B constructed in Example 2 was Hfr The CE plasmid was electroporated into the four knockout strains described above. The recombinant strains obtained after plasmid introduction were named D-PA6, D-PA7, D-PA8, and D-PA9. The fermentation steps were the same as in Example 1. The D-pantothenic acid production was shown in Table 4, where "+" represents knockout.

[0114] Table 4 D-pantothenic acid yields of D-PA6, D-PA7, D-PA8, and D-PA9 strains in shake flask fermentation

[0115] D-PA6 D-PA7 D-PA8 D-PA9 ΔldhA + + + + ΔpflB + + + ΔpoxB + + ΔadhE + Shake flask yield (mg / L) 950 1003 1121 1207

[0116] Example 6: Attenuating the expression of the ilvE gene

[0117] In the D-pantothenic acid biosynthetic pathway, α-ketoisovalerate serves as a key precursor, participating in both the tricarboxylic acid cycle and branched-chain amino acid anabolism. The transamination reaction catalyzed by the ilvE gene significantly diverts α-ketoisovalerate from the synthesis of valine, resulting in a loss of carbon flux to the target product, D-pantothenic acid. To achieve this metabolic flux redirection, ilvE expression must be attenuated through a translation initiation control strategy, without completely blocking branched-chain amino acid synthesis to avoid nutritional deficiencies, thereby balancing cell growth and product synthesis.

[0118] Using the Kp-ΔbudA-ΔbudC-ΔgldA-ΔldhA-ΔpflB-ΔpoxB-ΔadhE strain as the starting strain, the translation start codon of the ilvE gene was rationally modified. The original sequence, which contained the highly expressed ATG, was replaced with the less efficient start codons GTG or TTG through site-directed mutagenesis.

[0119] 1. Construction of ilvE gene attenuated strain

[0120] (1) The pDK6-red plasmid was introduced into wild Klebsiella pneumoniae ATCC 10031 to construct the red recombination system;

[0121] (2) Amplify the target gene. Based on the genome sequence of wild Klebsiella pneumoniae ATCC 10031, the location of ilvE in the genome was determined. 800 bp upstream and downstream fragments of the target gene were selected as homology arms, and primers were designed using Snapgene. Using ilvE-up-F, ilvE-up-R and ilvE-down-F, ilvE-down-R as primers, and wild-type Klebsiella as template, upstream and downstream DNA fragments were amplified. Using primers GTG-ilvE-F, GTG-ilvE-R, TTG-ilvE-F, TTG-ilvE-R with a mutant start codon, and wild-type Klebsiella as template, the mutated ilvE fragment was amplified. Using apr-F and apr-R as primers, and the pIJ773 plasmid carrying the apramycin resistance gene as a template, the resistance gene with the FRT sequence was amplified. DNA fragments are easily degraded, and connecting them to the T vector can better preserve the repair template. Using TF and TR as primers and pMD18-T as a template, the linearized pMD18-T vector was amplified and Dpn1 was eliminated from the pMD18-T plasmid.

[0122] (3) Construct a replacement template plasmid. The five fragments obtained in step (2) were ligated using the Ultra One Step Cloning Kit (C115). The reaction was allowed to react at 50°C for 15 minutes, followed by immediate ice bath. The reaction system was transformed into Escherichia coli DH5α, and colonies were screened using a solid plate containing apramycin. Single colonies were verified by PCR using T-check-F and T-check-R primers and sequenced. Positive clones were identified as DH5α-T-ilvE-GTG / TTG.

[0123] (4) Using ilvE-up-F and ilvE-down-R as primers and DH5α-T-ilvE-GTG / TTG obtained in step (3) as a template, amplify the resistance cassette fragment containing the FRT sequence, upstream and downstream homology arms, and the mutated ilvE. Prepare competent cells of Klebsiella carrying the pDK6-red plasmid, and electroporate the resistance cassette fragment into the competent cells under the induction of IPTG. After recovery, spread it on LB solid culture medium of the corresponding resistance and place it in a constant temperature incubator at 37°C overnight.

[0124] (5) Single colonies were selected and verified using ilvE-up-F and ilvE-down-R as primers and sequenced.

[0125] If the sequencing results are aligned correctly (especially the start codon), the ilvE gene mutant strain is successfully constructed.

[0126] The steps of plasmid loss are the same as those in Example 3. Hfr The CE plasmid was electroporated into the strain with the mutated ilvE. The recombinant strains obtained after the introduction of the plasmid were named D-PA10-GTG and D-PA10-TTG. The corresponding D-pantothenic acid production is shown in Table 5.

[0127] Table 5 D-pantothenic acid yields of strains with attenuated ilvE gene in shake flask fermentation

[0128] strain Shake flask yield (mg / L) D-PA10-GTG 1322 D-PA10-TTG 1468

[0129] Based on the experimental results, it was concluded that mutating the start codon of ilvE, ATG, to TTG had a better effect. D-PA10-TTG was finally named D-PA10.

[0130] Example 7: 5 L fermentation tank fermentation of D-PA10 strain

[0131] D-PA10 was selected for D-pantothenic acid production testing in a 5-L fermentor. The strain was streaked onto LB plates and cultured overnight in a 37°C incubator. A single colony was selected and inoculated into 10 mL of LB liquid medium and incubated at 37°C for 12 hours to obtain a primary seed. 5 mL of the primary seed was then inoculated into a 1000-mL Erlenmeyer flask containing 250 mL of seed medium (LB liquid) and incubated at 37°C at 150 rpm for 12 hours to obtain a secondary seed. This secondary seed solution was inoculated at 10% inoculum into a 5-L fermentor containing 2.5 L of fermentation medium. Feed medium was added via a fed-batch method for a 62-hour fermentation cycle. During fermentation, the glucose concentration was maintained at 1 g / L and the DO level was approximately 30%. The stirring speed was controlled between 400 and 600 rpm using a stirring-coupled dissolved oxygen (DO) mode, and the aeration rate was maintained at 1 vvm. The culture temperature was maintained at 37°C, and the pH was adjusted to 6.8 ± 0.2 using 50% aqueous ammonia. The fermentation medium consists of 30 g / L glucose, 20 g / L (NH4)2SO4, 2 g / L K2HPO4, 0.25 g / L KH2PO4, 0.4 g / L MgSO4·7H2O, 1.5 g / L β-alanine, and 1 mL of trace element solution. 1 L of trace element solution contains 10.0 g / L CoCl2, 10.0 g / L FeSO4·7H2O, 0.2 g / L CuSO4, 0.02 g / L NiCl2·7H2O, and 1.0 g / L ZnSO4·7H2O. The pH is adjusted to 7.0 and the medium is sterilized at 121°C for 25 minutes. Glucose is separately sterilized at 115°C for 20 minutes. The fermentation curve is shown in Figure 2. Figure 2 ,according to Figure 2 It can be seen that the production of D-pantothenic acid was 14.6 g / L after 62 h.

[0132] The primers used in the above examples are shown in Table 6.

[0133] Table 6 Primer list

[0134]

[0135]

[0136]

Claims

1. A method for constructing Klebsiella pneumoniae that efficiently produces D-pantothenic acid, characterized in that: The following steps are involved: (1) Using wild-type Klebsiella pneumoniae ATCC 10031 as the starting strain, the ketopantoate hydroxymethyltransferase gene panB from Escherichia coli Hfr3000 YA139, the pantothenate synthase gene panC from Escherichia coli BL21 (DE3), and the ketopantoate reductase gene panE were introduced and expressed in tandem via the tac promoter of the pDK6 plasmid; (2) Gene knockout technology was used to inactivate genes related to the 2,3-butanediol synthesis pathway of the starting strain, namely α-acetolactate decarboxylase budA, acetoin reductase budC, and glycerol dehydrogenase gldA, thereby blocking the competitive carbon flow; (3) Gene knockout technology was used to inactivate the pyruvate oxidase gene poxB, the pyruvate formate lyase activating enzyme gene pflB, the alcohol dehydrogenase gene adhE, and the lactate dehydrogenase gene ldhA to reduce mixed acid fermentation byproducts and enhance pyruvate accumulation; (4) Weaken the expression of the valine-pyruvate transaminase gene ilvE and mutate the start codon from ATG to TTG to reduce the competition of branched-chain amino acids for α-ketoisovalerate, thereby constructing Klebsiella pneumoniae that efficiently produces D-pantothenic acid.

2. The construction method according to claim 1, characterized in that In the pDK6 plasmid, the panB, panC, and panE genes are expressed by the tac promoter, respectively. PanB and panC share a tac promoter, while panE uses an independent tac promoter.

3. The construction method according to claim 1, characterized in that The gene knockout technology uses the Red homologous recombination system. The knockout fragment contains upstream and downstream homologous arms and apramycin resistance selection markers, and the resistance marker is eliminated by Flp recombinase.

4. A Klebsiella pneumoniae constructed by the construction method according to any one of claims 1 to 3, characterized in that: The strain can efficiently synthesize D-pantothenic acid using glucose as a substrate.

5. Use of the Klebsiella pneumoniae according to claim 4 in fermentative production of D-pantothenic acid.

6. The use according to claim 5, characterized in that During the fermentation process, IPTG with a final concentration of 0.1 mmol / L should be added when OD600 reaches 0.8 to induce gene expression.

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