Preparation and use of ketopantoate hydroxymethyltransferase mutant for d-pantothenic acid production

Abstract

The present invention belongs to the technical fields of genetic engineering and enzyme engineering. Provided are the preparation and use of a ketopantoate hydroxymethyltransferase mutant for D-pantothenic acid production. By means of genetic engineering and enzyme engineering, a ketopantoate hydroxymethyltransferase from Escherichia coli is mutated, and the obtained single and double mutants are expressed in improved chassis cells, so that the yield of D-pantothenic acid produced by Escherichia coli can be significantly increased, which provides conditions for the expansion of industrial application thereof.

Description

Preparation and application of ketopantolytic acid hydroxymethyltransferase mutants for D-pantothenic acid production Technical Field

[0001] This invention relates to the preparation and application of ketopantolytic acid hydroxymethyltransferase mutants for D-pantothenic acid production, and belongs to the fields of genetic engineering and enzyme engineering technology. Background Technology

[0002] D-pantothenic acid (D-PA), also known as vitamin B5, is an important water-soluble B vitamin. D-PA participates in the metabolism of carbohydrates, proteins, and fatty acids in organisms and is widely used in the food, animal feed, cosmetics, and pharmaceutical industries. Pantothenic acid is most widely used in animal feed and the pharmaceutical industry. Plants and microorganisms can produce pantothenic acid, but humans and animals cannot. Humans can normally obtain D-pantothenic acid through food, so deficiency is generally not a problem. However, taking anti-D-pantothenic acid drugs such as salicylic acid and consuming foods low in D-pantothenic acid over a long period can lead to D-pantothenic acid deficiency. D-pantothenic acid deficiency can cause symptoms such as memory loss, adrenal insufficiency, and arthritis. Clinically, pantothenic acid is often used to treat vitamin B deficiency, chronic inflammation, and loss of appetite. Severe pantothenic acid deficiency in livestock and poultry can cause diarrhea, hair loss, and anemia in pigs, while chickens may experience stunted growth, poor feather growth, and even death in severe cases. Therefore, when raising livestock and poultry, it is generally necessary to add a certain amount of D-pantothenic acid to the feed.

[0003] D-Pantothenic acid (DPA) is widely used in the food, feed, and pharmaceutical industries, with a large market demand. With the continuous growth of domestic and international market demand for DPA, researching efficient methods for its production is undoubtedly crucial. Currently, there are three main methods for DPA production: chemical synthesis, enzyme-catalyzed synthesis, and microbial fermentation. The chemical synthesis method first requires the preparation of two key intermediates: β-alanine and D,L-pantothenic acid lactone, which are then used to synthesize DPA. β-alanine is mainly synthesized via the acrylonitrile method, acrylic acid method, and succinimide method, while D,L-pantothenic acid lactone is mainly obtained via the isobutyraldehyde-formaldehyde method, the isobutyraldehyde-aldehydeacetic acid method, and the isobutyraldehyde-chloroformane method. After obtaining the two intermediates, β-alanine and D,L-pantothenic acid lactone, there are two main methods for producing DPA. The first method involves directly treating β-alanine with calcium hydroxide to form calcium β-aminopropionate. Calcium β-aminopropionate then undergoes an amidation reaction with D,L-pantolactone in methanol solution at high temperature to obtain calcium D,L-pantolactone. High-purity calcium D-pantolactone is obtained by physically resolving it after inducing crystallization by adding D-calcium pantolactone seed crystals to a methanol solution. Excess calcium L-pantolactone is racemized and then further resolved. The second method involves resolving D,L-pantolactone first, using both chemical and enzymatic methods. Chemical resolution primarily employs resolving agents such as quinine, ephedrine, L-leucine, strychnine, and L-1-p-nitrotoluene-2-amino-1,3-propanediol to resolve D,L-pantolactone. Enzymatic resolution utilizes L-pantolactone hydrolase, D-pantolactone hydrolase, L-pantolactone dehydrogenase, ketopantolactone reductase, and alcohol dehydrogenase to resolve D,L-pantolactone. After obtaining D-pantolactone, it undergoes an amidation reaction with calcium β-aminopropionate in methanol solution under high temperature conditions to yield calcium D-pantothenate. Enzymatic synthesis primarily involves the synthesis of D-pantothenic acid from pantothenic acid and β-alanine via D-pantothenic acid synthase, which is the final step in the microbial fermentation production of D-pantothenic acid. While enzymatic synthesis of D-PA is more environmentally friendly than chemical synthesis, it still faces challenges such as high production costs and stringent reaction conditions. The rapid development of synthetic biology has driven the advancement of microbial fermentation technology, thus the production of D-PA through microbial fermentation is attracting increasing attention.

[0004] The conversion of α-ketoisovalerate to ketopantoate is a key step in the D-pantoate synthesis pathway. However, the natural ketopantoate hydroxymethyltransferase that catalyzes this step has poor enzyme activity and stability. Obtaining ketopantoate hydroxymethyltransferase mutants with improved enzyme activity and stability through enzyme engineering is of great significance for the microbial fermentation production of D-pantoate. Summary of the Invention

[0005] This invention provides a ketopantoacid hydroxymethyltransferase mutant, which uses the ketopantoacid hydroxymethyltransferase derived from Escherichia coli as the starting sequence and mutates multiple sites such as position 44, position 123, and position 124 of the ketopantoacid hydroxymethyltransferase.

[0006] In one embodiment, the starting sequence contains the S7A / L8S / Y12C / E15D / A63E / Q124K mutation compared to the pure wild-type ketopantoacid hydroxymethyltransferase. Unless otherwise specified, the term "starting sequence" or "parent" as used below refers to ketopantoacid hydroxymethyltransferase containing the above mutation.

[0007] In one embodiment, the amino acid sequence of the starting sequence is shown in SEQ ID NO.1.

[0008] In one embodiment, the mutant is based on the sequence shown in SEQ ID NO.1 and has at least one of the following mutations:

[0009] (1) Mutate valine at position 123 to glycine, aspartic acid, tyrosine, threonine, methionine, histidine, cysteine, proline, leucine, isoleucine, glutamine, or alanine.

[0010] (2) Mutate the lysine at position 124 to glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine, and histidine.

[0011] In one embodiment, the mutant is based on the sequence shown in SEQ ID NO.1, with valine at position 123 mutated to isoleucine and lysine at position 124 mutated to valine.

[0012] In one embodiment, the mutant is based on the sequence shown in SEQ ID NO.1, with valine at position 123 replaced by isoleucine and lysine at position 124 replaced by tryptophan.

[0013] In one embodiment, the mutant is obtained by changing valine (Val) at position 123 to threonine (Thr), and the mutant is named V123T.

[0014] In one embodiment, the mutant is obtained by changing valine (Val) at position 123 to glutamine (Gln), and the mutant is named V123Q.

[0015] In one embodiment, the mutant is obtained by changing valine (Val) at position 123 to alanine (Ala), and the resulting mutant is named V123A.

[0016] In one embodiment, the mutant is obtained by changing the lysine (Lys) at position 124 to valine (Val), and the resulting mutant is named K124V.

[0017] In one embodiment, the mutant is obtained by changing the lysine (Lys) at position 124 to tryptophan (Trp), and the resulting mutant is named K124W.

[0018] In one embodiment, the mutant is obtained by changing the lysine (Lys) at position 124 to aspartic acid (Asp), and the mutant is named K124D.

[0019] In one embodiment, the mutant is obtained by changing valine (Val) at position 123 to isoleucine (Ile) and lysine (Lys) at position 124 to valine (Val), and the resulting mutant is named V123I / K124V.

[0020] In one embodiment, the mutant is obtained by changing valine (Val) at position 123 to isoleucine (Ile) and lysine (Lys) at position 124 to tryptophan (Trp), and the mutant is named V123I / K124W.

[0021] The present invention also provides a gene encoding a high-D-pantothenic acid mutant of the ketopantothenic acid hydroxymethyltransferase.

[0022] In one embodiment, the nucleotide sequence encoding mutant V123T is shown in SEQ ID NO.3; the nucleotide sequence encoding mutant V123L is shown in SEQ ID NO.4; the nucleotide sequence encoding mutant V123I is shown in SEQ ID NO.5; the nucleotide sequence encoding mutant K124D is shown in SEQ ID NO.6; the nucleotide sequence encoding mutant K124V is shown in SEQ ID NO.7; the nucleotide sequence encoding mutant K124W is shown in SEQ ID NO.8; the nucleotide sequence encoding mutant V123I / K124V is shown in SEQ ID NO.9; and the nucleotide sequence encoding mutant V123I / K124W is shown in SEQ ID NO.10.

[0023] The present invention also provides a recombinant plasmid carrying the said gene.

[0024] In one embodiment, the recombinant plasmid uses pACYC as the starting plasmid.

[0025] The present invention also provides mutants of the ketopantolytic acid hydroxymethyltransferase expressed therefrom or microbial cells carrying the gene therefrom.

[0026] In one embodiment, the recombinant microbial cell is Escherichia coli.

[0027] In one embodiment, the Escherichia coli is Escherichia coli PA104 as the starting strain, and the Escherichia coli PA104 and its construction method have been disclosed in the paper "Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering".

[0028] In one embodiment, the *Escherichia coli* is an *Escherichia coli* capable of synthesizing D-pantothenic acid.

[0029] In one embodiment, the *E. coli* strain capable of D-pantothenic acid synthesis is *E. coli* MG1655, which expresses ketopantolysin hydroxymethyltransferase and pantothenic acid synthase, and has the following improvements (a) or (b):

[0030] (a) The alanine aminotransferase gene avtA and the glucokinase gene glk were knocked out;

[0031] (b) The alanine aminotransferase gene avtA, the glucokinase gene glk, and the acetic acid dehydrogenase gene poxB were knocked out.

[0032] In one embodiment, the nucleotide sequence of the gene panB encoding ketopantolate hydroxymethyltransferase is shown in SEQ ID NO.2; and the nucleotide sequence of the gene panC encoding pantothenic acid synthase is shown in SEQ ID NO.11.

[0033] The present invention also provides a method for increasing the production of D-pantothenic acid in Escherichia coli, the method being to mutate one or more sites at positions 123 and 124 of the amino acid sequence of the ketopantothenic acid hydroxymethyltransferase in Escherichia coli.

[0034] In one embodiment, the method involves mutating ketopantolysin hydroxymethyltransferase by at least one of the following methods to increase the content of D-pantothenic acid in Escherichia coli:

[0035] (1) Mutate valine at position 123 to glycine, aspartic acid, tyrosine, threonine, methionine, histidine, cysteine, proline, leucine, isoleucine, glutamine or alanine.

[0036] (2) Mutate the lysine at position 124 to glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine, or histidine.

[0037] In one embodiment, the method involves mutating valine at position 123 of the ketopantolysin hydroxymethyltransferase to isoleucine and lysine at position 124 to valine.

[0038] In one embodiment, the method involves mutating valine at position 123 to isoleucine and lysine at position 124 to tryptophan using ketopantolysin hydroxymethyltransferase.

[0039] In one embodiment, the ketopantolytic acid hydroxymethyltransferase has the amino acid sequence shown in SEQ ID NO.1.

[0040] The present invention also provides the application of the engineered Escherichia coli in the production of D-pantothenic acid.

[0041] In one embodiment, the application involves fermenting the engineered strain of Escherichia coli in a culture medium for a period of time and collecting D-pantothenic acid from the fermentation broth.

[0042] In one embodiment, the fermentation is carried out at 35–40°C.

[0043] In one embodiment, the culture medium contains carbon sources, nitrogen sources, and inorganic salts required for the growth of Escherichia coli.

[0044] In one embodiment, the culture medium is LB medium.

[0045] In one embodiment, the culture medium contains glucose, tryptone, yeast extract, and inorganic salts; the inorganic salts include sodium salts and ammonium salts; optionally, the sodium salt is sodium chloride, and the ammonium salt is ammonium sulfate.

[0046] In one embodiment, the culture medium contains glucose, corn steep liquor, yeast extract, peptone, ammonium sulfate, magnesium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and β-alanine.

[0047] In one implementation, the fermentation process is also supplemented with glucose and β-alanine.

[0048] The present invention also provides the application of the engineered Escherichia coli in the production of products containing D-pantothenic acid.

[0049] In one embodiment, the product includes, but is not limited to, pharmaceuticals, health supplements, or animal feed. Beneficial effects:

[0050] (1) This invention targets ketopantolate hydroxymethyltransferase (KPHMT) in *Escherichia coli* MG1655. The amino acid sequence of KPHMT is modified using site-directed mutagenesis. After initial screening in well plates and subsequent screening in shake flasks, mutants V123T, V123L, V123I, V123Q, K124D, K124V, and K124W were obtained. The D-pantolate yields of the recombinant strains expressing these mutants were 2.7 g / L, 2.62 g / L, 3.19 g / L, 1.7 g / L, 2.71 g / L, 3.25 g / L, and 3.38 g / L, respectively. These yields represent increases of 11.5%, 8.2%, 31.8%, 12%, 34.3%, and 39.7% compared to strains expressing the parental ketopantolate hydroxymethyltransferase.

[0051] (2) The present invention also constructed a recombinant Escherichia coli expressing the double mutant V123I / K124W, with a D-pantothenic acid yield of up to 48.99 g / L; the fermentation intensity of this strain was significantly improved, and the production intensity of the mutant V123I / K124W in the first 24 hours of fermentation was 87% higher than that of the starting sequence. Attached Figure Description

[0052] Figure 1 is a schematic diagram of the metabolism of D-pantothenic acid synthesis in Escherichia coli.

[0053] Figure 2 shows a plasmid containing a fragment of the parent gene for ketopantolytic acid hydroxymethyltransferase.

[0054] Figure 3 shows the OD of the parent strain and the recombinant strain with the 44th amino acid ketone pantothenic acid hydroxymethyltransferase mutant in a 24-well plate. 600 And a graph showing the production of D-pantothenic acid.

[0055] Figure 4 shows the OD values ​​of the parent strain and the recombinant strain with the ketone pantothenic acid hydroxymethyltransferase mutant at position 123 in a 24-well plate. 600 And a graph showing the production of D-pantothenic acid.

[0056] Figure 5 shows the OD of the parent strain and the recombinant strain with the ketone pantothenic acid hydroxymethyltransferase mutant at position 124 in a 24-well plate. 600 And a graph showing the production of D-pantothenic acid.

[0057] Figure 6 shows the shake-flask screening results of the parent strain and the recombinant strain OD, which is a high-yielding D-pantothenic acid pantothenic acid hydroxymethyltransferase mutant. 600 And a graph showing the production of D-pantothenic acid.

[0058] Figure 7 shows the relative yields of the parent strain and the recombinant strain with high D-pantothenic acid hydroxymethyltransferase mutant, as well as the strain screened in shake flasks.

[0059] Figure 8 shows the OD values ​​of the parental line and the double mutant V123I / K124W in a 5L fermenter. 600 Figures showing the production of residual sugar and D-pantothenic acid. Detailed Implementation

[0060] I. Culture medium:

[0061] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L. Adding 15 g / L agar powder prepares a solid medium.

[0062] Shake flask culture medium: glucose 25g / L, yeast extract 5g / L, peptone 10g / L, ammonium sulfate 16g / L, β-alanine 4g / L. Depending on the situation, antibiotics with a final concentration of 0.1% may need to be added. β-alanine and glucose need to be sterilized separately before being added to the culture medium.

[0063] Fermentation medium for a 5L fermenter: glucose 25g / L, corn steep liquor 5g / L, yeast extract 5g / L, peptone 5g / L, ammonium sulfate 15g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 1g / L, β-alanine 10g / L, vitamin B1 0.0075g / L, manganese sulfate 0.02g / L, zinc sulfate 0.01g / L, ferrous sulfate 0.02g / L; among which, vitamin B1, manganese sulfate, zinc sulfate, and ferrous sulfate are prepared into a 1000-fold diluted mother liquor, filtered and sterilized, and then added to the fermentation medium at the corresponding concentrations. Depending on the situation, appropriate antibiotics may need to be added. β-alanine and glucose are sterilized separately. Sterilization program: 115℃, 20min.

[0064] Feeding medium 1 (1L): 250g / L β-alanine.

[0065] Feeding medium 2 (1L): 500g / L glucose.

[0066] II. Production of D-pantothenic acid via perforated plate fermentation:

[0067] Select a single plump colony and inoculate it into a 24-well plate. Add 4.8 mL of shake flask culture medium to each well of the plate and incubate at 30°C and 220 rpm for 48 h.

[0068] III. Production of D-pantothenic acid by shake-flask fermentation:

[0069] The bacterial culture was collected from the glycerol tube using an inoculation loop and streaked onto a plate. It was then incubated overnight at 37°C. Single colonies were then picked and inoculated into shake tubes containing 2-3 mL of LB medium, and incubated at 37°C and 220 rpm for 12 h. 500 μL of the seed culture was inoculated into a 250 mL Erlenmeyer flask containing 24.5 mL of shake flask medium. Three replicates were set up for each strain, and the cultures were incubated at 30°C and 220 rpm for 48 h. After fermentation, 1 mL of the fermentation broth was used to determine the OD value. 600 Take another 1 mL of fermentation broth, centrifuge at 12000 rpm for 2 min, dilute the supernatant with water 5 times, and detect it according to the HPLC detection method in the specific implementation method.

[0070] IV. Fermentation production of D-pantothenic acid in a 5L fermenter:

[0071] Using an inoculation loop, collect bacterial culture from a glycerol tube and streak it onto a plate. Incubate overnight at 37°C. Then, pick colonies and inoculate them onto a slant agar, adding appropriate antibiotics if necessary. Incubate at 37°C for 12 hours. Next, streak colonies from the slant agar into 2L Erlenmeyer flasks, adding 300mL of LB liquid medium. Incubate at 37°C and 220rpm for 10 hours. During incubation, maintain a gas flow rate of 2L / min and an initial rotation speed of 200rpm / min. Adjust the rotation speed according to dissolved oxygen and stirring requirements, but do not exceed 900rpm / min. When the initial sugar is almost depleted, use a continuous feeding method to maintain the sugar concentration below 5g / L. Maintain the pH at approximately 6.80 using pure ammonia. Maintain the fermentation temperature at 37°C. Slowly add 100mL, 100mL, and 50mL of β-alanine at 15h, 25h, and 35h of fermentation, respectively.

[0072] V. D-Pantothenic Acid Analysis Method:

[0073] The biomass of *Escherichia coli* was determined by ultraviolet spectrophotometry. An appropriate amount of fermentation broth was diluted with ultrapure water and mixed thoroughly. Using water as a control, the absorbance (OD) at a wavelength of 600 nm was measured. 600 Take 1 mL of fermentation broth and aspirate it into an EP tube. Centrifuge at 12000 rpm for 2 minutes at room temperature. Transfer the supernatant to a new EP tube. Dilute the supernatant fermentation broth with ddH2O to a certain factor, and then filter it through a 0.22 μm inorganic filter membrane to obtain the sample to be tested. Determine the residual sugar content in the sample using a residual sugar analyzer.

[0074] 1 mL of fermentation broth was aspirated into an EP tube and centrifuged at 12,000 rpm for 2 minutes at room temperature. The supernatant was transferred to a new EP tube, and the D-pantothenic acid content in the sample was determined by high-performance liquid chromatography (HPLC). The supernatant fermentation broth was diluted with ddH2O to a certain factor and filtered through a 0.22 μm inorganic filter membrane to obtain the test sample. The D-pantothenic acid content in the test sample was determined by HPLC. Chromatographic conditions: The column was an Aminex HPX-87H column (Bio-Rad, Richmond, CA, USA); the column oven temperature was 30℃; the mobile phase was 95% 0.02 mol / L potassium dihydrogen phosphate and 5% acetonitrile, with the pH of the mobile phase adjusted to 4 using phosphoric acid; the mobile phase flow rate was 1 mL / min; the injection volume was 10 μL; and the detection wavelength was 200 nm. The retention time of pantothenic acid varied slightly depending on the instrument used.

[0075] VI. Strain Information

[0076] The strain information is shown in Table 1. Escherichia coli PA104 is a chassis cell obtained by genetic modification of Escherichia coli MG1655 through genes such as Δglk and ΔavtA. The strain and its construction method are disclosed in the paper "Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering".

[0077] Table 1. Strains involved in this invention

[0078] Table 2 Primer sequences

[0079] Example 1: Construction of chassis cells DPA04

[0080] Using Escherichia coli PA104 (published in the paper "Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering") as the starting strain, the pyruvate dehydrogenase gene poxB was knocked out using CRISPR-Cas9-mediated gene editing technology, which reduced the accumulation of the byproduct acetic acid and increased the accumulation of pyruvate, the precursor of pantothenic acid synthesis.

[0081] The specific steps are as follows:

[0082] (1) Constructing pTarget-poxB plasmid: Using pTarget plasmid as template, pT-poxB-F / pT-poxB-R primers were used to introduce the first 20bp of the PAM site into plasmid pTarget as a guide signal for sgRNA. The constructed pTarget-poxB was sent for sequencing verification. Plasmids with correct sequencing results were reserved for subsequent operations.

[0083] (2) Construction of poxB knockout fusion homologous arms: Using *E. coli* MG1655 as a template, upstream and downstream homologous arms were obtained by PCR amplification using two pairs of primers (poxB-up-F / poxB-up-R, poxB-down-F / poxB-down-R). Using the upstream and downstream homologous arms as templates, the two homologous arms were connected by overlap extension PCR using poxB-up-F / poxB-down-R primers. Agarose gel electrophoresis was then performed for verification, and PCR products with correct band sizes were purified and recovered.

[0084] (3) The pCas plasmid was introduced into Escherichia coli PA104, and a single colony was transferred into an LB tube containing 0.05 mg / L kanamycin and cultured overnight at 37°C. Then, a 1% (v / v) inoculum was added to a 250 mL Erlenmeyer flask containing 50 mL of LB medium, and 500 μL of 1 mol / L arabinose was added. The flask was incubated at 220 rpm at 37°C until OD reached the target value. 600 Add 0.4-0.6 μL of 1 mol / L arabinose. Then centrifuge at 5000 rpm and 4°C to collect the strain, wash three times with 10% glycerol, and aliquot to prepare electrotransfer competent cells.

[0085] (4) Take 500 ng of the pTarget-poxB plasmid constructed in step (1) and the poxB knockout fusion homologous arm constructed in step (2) and add it to 100 μL of electroporation competent cells. Transfer the cells to a pre-cooled 1 mm electroporation cuvette and perform electroporation transformation. Immediately after electroporation, add 1 mL of LB medium, gently pipette and mix, then transfer to a 1.5 mL centrifuge tube and incubate at 220 rpm and 37°C for 1.5-2 h. Incubate on an LB plate containing 0.05 mg / L kanamycin and streptomycin at 37°C for 16 h. Perform colony PCR using poxB-up-F and pxoB-down-R primers. If the band size is 1000 bp, it indicates that the poxB gene knockout was successful.

[0086] (5) Plasmid removal: Verified colonies were transferred to LB medium containing 1 mM rhamnose and 0.05 mg / L kanamycin and cultured overnight to remove plasmid pTarget-poxB. After overnight culture, the colonies were streaked onto kanamycin-resistant plates and cultured overnight. Once positive colonies had grown, they were streaked onto kanamycin-resistant and streptomycin-resistant plates and marked. Bacteria that could grow on kanamycin-resistant plates but not on streptomycin-resistant plates indicated that plasmid pTarget-poxB had been removed. The colonies with pTarget-poxB removed were then transferred to LB medium containing 1% (1 M) sucrose to remove pCas9 plasmid. After overnight incubation, the bacteria were streaked onto antibiotic-free plates and incubated overnight. Once positive colonies had grown, they were streaked onto antibiotic-free plates, kanamycin-resistant plates, and streptomycin-resistant plates, and marked accordingly. Bacteria that could grow on antibiotic-free plates but not on kanamycin-resistant or streptomycin-resistant plates indicated that plasmids pCas9 and pTarget had been removed. The strain obtained was named DPA04.

[0087] Example 2: Preparation of a single mutant containing the ketopantolate hydroxymethyltransferase gene

[0088] The panB shown in SEQ ID NO.2 and the panC shown in SEQ ID NO.11 were ligated into plasmid pACYC (SEQ ID NO.12) to construct plasmid pACYC-panBC. Using plasmid pACYC as a template, the linearized pACYC-panBC fragment was amplified by reverse PCR using the primers in Table 2. After the PCR product was verified to be correct, it was digested with Dpn I at 37℃ for 1 h and then transformed into E. coli JM109 competent cells. Clones were selected for sequencing to confirm whether the coding gene of the ketopantolytic acid hydroxymethyltransferase single mutant was correct. The correctly sequenced mutant plasmid was transformed into *E. coli* DPA04 competent cells to construct and express single mutants G44L, G44V, G44T, G44C, G44A, G44K, G44D, G44S, V123K, V123G, V123D, V123Y, V123T, V123M, V123H, V123C, V123P, V123L, V123A, and V1 Strains of type 23R, V123E, V123N, V123F, V123I, V123Q, K124D, K124G, K124M, K124T, K124A, K124N, K124E, K124L, K124S, K124V, V124R, K124Q, K124I, K124P, K124Y, K124C, K124H, K124F, and K124W.

[0089] The specific steps for preparing and transforming competent cells are as follows:

[0090] (1) Take the bacterial culture out of the -80℃ freezer, dip a small amount of bacterial culture into a sterile inoculation needle on a sterile laminar flow hood, and streak it on LB solid medium without any antibiotics. Place the streaked plate in a constant temperature incubator at 37℃ and incubate for about 12 hours.

[0091] (2) After 12 hours, select a single colony with good growth and inoculate it into a 50 mL shake flask containing 5 mL of LB liquid medium. Incubate at 37°C and 220 rpm for 8-10 hours.

[0092] (3) Use a pipette to take 500 μL (1% inoculum) of culture medium and inoculate it into a 250 mL shake flask containing 50 mL LB liquid medium. Place the 50 mL centrifuge tube in a -20 °C freezer to pre-cool.

[0093] (4) Place the 250mL shake flask in a shaker at 37℃ and 220rpm for about 2 hours until OD 600 It is in the range of 0.4 to 0.6.

[0094] (5) Transfer the bacterial culture in the shake flask to a 50mL centrifuge tube, place it in an ice box for 10-15 minutes, and pre-cool the 50mL centrifuge to 4℃.

[0095] (6) Place the 50mL centrifuge tube in a 50mL centrifuge and centrifuge at 4000rpm for 5min. After centrifugation, pour out the supernatant.

[0096] (7) In a sterile laminar flow hood, use a pipette to draw 5 mL of solution A and add it to a 50 mL centrifuge tube, then resuspend the bacterial cells.

[0097] (8) Repeat step (6).

[0098] (9) In a sterile laminar flow hood, use a pipette to draw 5 mL of solution B and add it to a 50 mL centrifuge tube, then resuspend the bacterial cells. Use a pipette to draw 100 μL of the bacterial suspension and add it to a sterile EP tube placed on ice for aliquoting. After aliquoting, store the competent E. coli cells at -80°C.

[0099] (10) Remove competent cells from the -80℃ freezer and place them in an ice box for 5-10 minutes to thaw. Use a pipette to add 1 μL of the correctly sequenced mutant plasmid to the EP tube containing competent cells, slowly rinse 3-4 times to mix thoroughly, and then place on ice for 30 minutes.

[0100] (11) Remove the EP tube from the ice box, heat shock it in a 42℃ constant temperature water bath for 1-2 minutes, and then quickly place it on ice for 2 minutes.

[0101] (12) On a sterile laminar flow hood, use a pipette to add 1 mL of antibiotic-free LB liquid culture medium to the EP tube, mix gently, and then transfer to a constant temperature shaker at 37°C and 220 rpm for 40-60 min.

[0102] (13) Remove the EP tube from the shaker and transfer it to a centrifuge. Centrifuge at 5000 rpm for 3 min. Then, in a sterile laminar flow hood, discard part of the supernatant, keeping about 70-100 μL. After mixing the bacterial cells with a pipette, add them to LB solid medium containing the corresponding antibiotic. Spread the medium evenly with a sterile spreader, label it, and incubate it upside down in a 37°C incubator overnight.

[0103] Example 3: Fermentation of a single mutant of ketopantolysin hydroxymethyltransferase in well plates for the production of D-pantothenic acid.

[0104] The mutant strains constructed in Example 2 were inoculated into 24-well round-bottom plates containing shake-flask culture medium. 100 μg / L ampicillin was added as needed, and fermentation was carried out at 30°C and 220 rpm for 48 h. The fermentation broth was collected to detect the OD of the strains. 600 And D-pantothenic acid production. Using the DPA04 strain expressing the starter sequence (panBC) as a control (WT), fermentation was performed according to the same method described above.

[0105] The results showed that the mutant strain at position 44 did not show any improvement compared to the parent strain. The strains with single-point mutations at amino acid position 123 (V123G, V123D, V123Y, V123T, V123H, V123C, V123P, V123L, V123A, V123I, V123Q, V123N, V123F) all had higher D-pantothenic acid production than the strain expressing the starting sequence. Among them, the mutants V123L, V123I, or V123Q, which showed better results, had D-pantothenic acid production of 1.77 g / L, 1.33 g / L, and 1.48 g / L, respectively, which were 197%, 122%, and 149% higher than the control. The mutants with single-point mutations at amino acid position 124 (K124D, K124G, K124M, K124T, K124E, K124L, K124V, K124Q, K124I, K124P, K124R, K124Y, K124C, K124H, K124W) all produced higher D-pantothenic acid yields than the strain expressing the starting sequence. Among them, the mutants K124D and K124V showed better results, with D-pantothenic acid yields of 1.48 g / L and 1.56 g / L, respectively, representing increases of 148% and 164% compared to the control.

[0106] Example 4: Fermentation of a single mutant of ketopantolysin hydroxymethyltransferase in shake flasks for the production of D-pantothenic acid.

[0107] The expression mutants screened in Example 3 were G44L, G44V, G44T, G44C, G44A, G44K, G44D, G44S, V123K, V123G, V123D, V123Y, V123T, V123M, V123H, V123C, V123P, V123L, V123A, V123R, V123E, V123N, V123F, V123I, V123Q, K124D, K124G, and K124G. From strains 124M, K124T, K124A, K124N, K124E, K124L, K124S, K124V, V124R, K124Q, K124I, K124P, K124Y, K124C, K124H, K124F, or K124W, mutants V123T, V123L, V123I, V123Q, K124D, K124V, and K124W, which showed high yields in plate testing, were selected for shake-flask re-screening verification. The specific steps are as follows:

[0108] Plump recombinant *E. coli* bacteria were inoculated into shake tubes containing 2-3 mL of LB medium and cultured at 37°C and 220 rpm for 12 h to obtain seed culture. 500 μL of the seed culture was inoculated into 250 mL Erlenmeyer flasks containing 24.5 mL of shake culture medium. Each strain was fermented in triplicate, and cultured at 30°C and 220 rpm for 48 h. The fermentation broth was collected to detect the OD of the strains. 600 And D-pantothenic acid production.

[0109] The results showed that strain WT achieved a D-pantothenic acid yield of 2.42 g / L after 48 h of shake-flask fermentation. The D-pantothenic acid yields of mutants V123T, V123L, V123I, V123Q, K124D, K124V, and K124W were 2.7 g / L, 2.62 g / L, 3.19 g / L, 1.7 g / L, 2.71 g / L, 3.25 g / L, and 3.38 g / L, respectively. Except for V123Q, all other mutants were superior to the starting sequence, consistent with the plate results. V123T, V123L, V123I, K124D, K124V, and K124W showed increases of 11.5%, 8.2%, 31.8%, 12%, 34.3%, and 39.7% compared to the strain expressing the parental ketopantolysin hydroxymethyltransferase, respectively.

[0110] Example 5: Preparation of a double mutant of ketopantolytic acid hydroxymethyltransferase

[0111] Using the plasmid containing the V123I mutant constructed in Example 2 as a template, primers for introducing the V123I / K124V and V123I / K124W mutants were designed and synthesized. Site-directed mutagenesis was performed using PCR linearization of the template. After verification that the PCR product was correct, it was digested with Dpn I at 37°C for 1 hour and then transformed into *E. coli* JM109 competent cells. Clones were selected and sequenced to confirm the correctness of the coding gene for the ketopantolytic acid hydroxymethyltransferase double mutant. The correctly sequenced mutant plasmid was transformed into *E. coli* DPA04 competent cells to construct strains expressing the double mutants V123I / K124V and V123I / K124W. Specific steps for competent cell preparation and transformation are detailed in Example 2.

[0112] Example 6: Shake-flask fermentation of a double mutant of ketopantolytic acid hydroxymethylase for the production of D-pantothenic acid

[0113] The strains expressing V123I / K124V and V123I / K124W constructed in Example 5 were screened and validated using shake flasks. Plump *E. coli* bacteria were picked and inoculated into shake tubes containing 2-3 mL of LB medium, and cultured at 37°C and 220 rpm for 12 h to obtain seed culture. 500 μL of the seed culture was inoculated into 250 mL Erlenmeyer flasks containing 24.5 mL of shake flask medium. Each strain was fermented in triplicate, and cultured at 30°C and 220 rpm for 48 h. The fermentation broth was collected to detect the OD of the strains. 600 And D-pantothenic acid production. The strain that expressed the parental ketopantothenic acid hydroxymethyltransferase (pACYC-panBC) in strain DPA04 using the pACYC plasmid was used as the control strain.

[0114] The results showed that the control strain WT produced 2.52 g / L of D-pantothenic acid, while the strains expressing single mutants V123I, K124V, and K124W produced 3.16 g / L, 3.31 g / L, and 3.52 g / L, respectively. The strain expressing the double mutant V123I / K124V showed poor results, with a D-pantothenic acid production of only 3.29 g / L. However, the double mutant strain V123I / K124W showed a significant increase in D-pantothenic acid production, reaching 4.16 g / L. This represents a 65.1% increase compared to the starting sequence, and a 31.6% and 18.2% increase compared to the single mutants V123I and K124W, respectively.

[0115] Example 7: Ketopantolysin hydroxymethyltransferase and its mutants were fermented in a 5L fermenter for the production of D-pantothenic acid.

[0116] The mutant strains constructed in Examples 1-5 were used to collect bacterial culture from glycerol tubes using an inoculation loop and streaked onto agar plates. The plates were then incubated overnight at 37°C. Colonies were then picked and inoculated onto slant agar, with appropriate antibiotics added if necessary. The plates were incubated at 37°C for 12 hours. Colonies from the slant agar were then inoculated into 2L Erlenmeyer flasks, with 300 mL of LB liquid medium added. The flasks were incubated at 37°C and 220 rpm for 10 hours to obtain the seed culture.

[0117] The seed culture was inoculated into the fermentation medium at a 10% inoculum (initial OD = 3). During cultivation, the gas flow rate was maintained at 2 L / min, with an initial rotation speed of 200 rpm / min. The rotation speed was adjusted according to dissolved oxygen and stirring requirements, but the maximum rotation speed should not exceed 900 rpm / min. When the initial sugar was almost depleted, fed-batch medium 2 was continuously added to maintain a sugar concentration of ≤5 g / L. The pH was maintained at 6.80 ± 0.1 using pure ammonia. The fermentation temperature was maintained at 37℃. 100 mL, 100 mL, and 50 mL of fed-batch medium 1 were slowly added at 15 h, 25 h, and 35 h of fermentation, respectively. A strain expressing the parent ketopantolytic acid hydroxymethyltransferase using the pACYC plasmid in strain DPA04 was used as a control strain.

[0118] Fermentation lasted 72 hours, with periodic sampling and testing. As shown in Figure 8, the strain expressing the parental ketopantolate hydroxymethyltransferase produced 42.5 g / L of D-pantothenic acid in a 5L fermenter after 72 hours of fermentation, while the strain expressing the double mutant V123I / K124W produced 48.99 g / L of D-pantothenic acid, an increase of 7.49 g / L compared to the control strain. Furthermore, the production intensity of the double mutant strain was significantly higher than that of the control strain; the control strain produced only 22.08 g / L after 24 hours of fermentation, while the double mutant strain produced 41.28 g / L after 24 hours. In the first 24 hours, the production intensity of the control strain was 0.92 g / L / h, while the production intensity of the double mutant strain was 1.72 g / L / h, with the V123I / K124W mutant showing an 87% increase in production intensity compared to the control.

[0119] Comparative example:

[0120] The specific implementation method is the same as in Examples 2-6, except that mutant strains G44L, G44V, G44T, G44C, G44A, G44K, G44D, G44S, V123K, V123M, V123R, V123E, K124A, and K124S were also constructed. The results showed that the D-pantothenic acid yields of these mutant strains were 0.259 g / L, 0.272 g / L, 0.252 g / L, 0.235 g / L, 0.303 g / L, 0.268 g / L, 0.306 g / L, 0.311 g / L, 0.508 g / L, 0.08 g / L, 0.368 g / L, 0.517 g / L, 0.477 g / L, and 0.539 g / L, respectively. Compared with the strain expressing wild-type ketopantolysinic hydroxymethyltransferase, the levels were reduced by 56.4%, 54.2%, 57.6%, 60.4%, 49%, 54.9%, 48.6%, 47.6%, 14.5%, 86.5%, 38%, 12.9%, 19.7%, and 9.3%, respectively.

[0121] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A ketopantolytic acid hydroxymethyltransferase mutant, characterized in that, Based on the starting sequence shown in SEQ ID NO.1, it has at least one of the following mutations: (1) Mutate valine at position 123 to glycine, aspartic acid, tyrosine, threonine, methionine, histidine, cysteine, proline, leucine, isoleucine, glutamine, or alanine. (2) Mutate the lysine at position 124 to glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine, and histidine.

2. The ketopantolytic acid hydroxymethyltransferase mutant according to claim 1, characterized in that, Based on the sequence shown in SEQ ID NO.1, the valine at position 123 is mutated to threonine, or the valine at position 123 is mutated to glutamine, or the valine at position 123 is mutated to alanine, or the lysine at position 124 is mutated to valine, or the lysine at position 124 is mutated to tryptophan, or the lysine at position 124 is mutated to aspartic acid.

3. The ketopantolytic acid hydroxymethyltransferase mutant according to claim 1, characterized in that, For (a), (b), or (c): (a) Based on the starting sequence shown in SEQ ID NO.1, valine at position 123 is mutated to threonine or isoleucine, or lysine at position 124 is mutated to aspartic acid, valine or tryptophan. (b) Based on the sequence shown in SEQ ID NO.1, valine at position 123 is mutated to isoleucine and lysine at position 124 is mutated to valine. (c) Based on the sequence shown in SEQ ID NO.1, valine at position 123 is mutated to isoleucine and lysine at position 124 is mutated to tryptophan.

4. A gene encoding the ketopantolytic acid hydroxymethyltransferase mutant of any one of claims 1 to 3.

5. A recombinant plasmid carrying the gene of claim 4.

6. A microbial cell expressing any of the ketopantolytic acid hydroxymethyltransferase mutants of claims 1 to 3 or carrying the gene of claim 4.

7. A recombinant Escherichia coli, characterized in that, The mutant of any one of claims 1 to 3 is expressed in *Escherichia coli* capable of D-pantothenic acid synthesis; wherein the *Escherichia coli* capable of D-pantothenic acid synthesis is based on *Escherichia coli* MG1655, having the following improvements (a) or (b): (a) The alanine aminotransferase gene avtA and the glucokinase gene glk were knocked out; (b) The alanine aminotransferase gene avtA, the glucokinase gene glk, and the acetic acid dehydrogenase gene poxB were knocked out.

8. A method for increasing the D-pantothenic acid yield of Escherichia coli, characterized in that, Mutation of any one of (a) to (d) in ketopantolysin in Escherichia coli: (a) Mutate valine at position 123 to glycine, aspartic acid, tyrosine, threonine, methionine, histidine, cysteine, proline, leucine, isoleucine, glutamine, or alanine. (b) Mutate lysine at position 124 to glycine, valine, leucine, isoleucine, methionine, proline, tryptophan, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, arginine, or histidine. (c) Mutate valine at position 123 of ketopantolysin to isoleucine and lysine at position 124 to valine; (d) The ketopantolysin hydroxymethyltransferase was modified to mutate valine at position 123 to isoleucine and lysine at position 124 to tryptophan.

9. The method according to claim 8, characterized in that, Mutation (a) to (d) was performed on any of the ketopantolytic acid hydroxymethyltransferases represented by SEQ ID NO.1 in Escherichia coli: (a) Mutate valine at position 123 to either threonine or isoleucine; (b) Mutate the lysine at position 124 to valine, tryptophan, or aspartic acid; (c) Mutate valine at position 123 to isoleucine and lysine at position 124 to valine; (d) Mutate valine at position 123 to isoleucine and lysine at position 124 to tryptophan.

10. A method for preparing D-pantothenic acid, characterized in that, The recombinant Escherichia coli of claim 7 is fermented in a culture medium.

11. The method according to claim 10, wherein the fermentation is carried out at 35–40°C; optionally, glucose and β-alanine are fed into the fermentation process.

12. The use of any of the mutants described in claims 1 to 3, or the microbial cells described in claim 6, or the recombinant Escherichia coli described in claim 7, or the method described in any of claims 8 to 11 in the production of D-pantothenic acid or products containing D-pantothenic acid.

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