D-pantothenic acid producing engineering bacteria, construction method and application thereof

Abstract

The application discloses an engineering bacterium for producing D-pantothenic acid as well as a construction method and application thereof. The engineering bacterium DPA11A (pTrc99A-panBpanC) is a starting strain. The panB gene of Corynebacterium glutamicum is mutated to strengthen the heterologous expression of a key limiting enzyme, improve the yield of pantothenic acid, simultaneously co-express a pyruvate uptake protein to accelerate the reuptake of extracellular pyruvate, reduce carbon diversion and improve the yield of D-pantothenic acid. The yield of D-pantothenic acid produced by the recombinant bacterium DPA11A*01 constructed by the method is increased by about 6.64 g / L, which is increased by about 81.3% compared with the starting strain DPA11A, and has an application prospect in industrial production.

Description

(I) Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a recombinant bacterium co-expressing ketopantolytic acid hydroxymethyltransferase-pyruvate uptake protein and its application in producing D-pantothenic acid. (II) Background Technology

[0002] D-pantothenic acid (D-PPAN) is a water-soluble vitamin, also known as vitamin B5, essential for the growth and development of organisms. It is a key precursor in the synthesis of coenzyme A (CoA) and acyl carrier protein (ACP). D-PPAN can be synthesized enzymatically in the cells of bacteria, fungi, and plants. However, animals cannot synthesize D-PPAN themselves, and therefore, through evolution, they have developed the ability to obtain D-PPAN from their environment and the physiological function of actively accumulating it. The most stable form of D-PPAN is calcium D-pantothenate; therefore, calcium salts of D-PPAN are commonly used as commercial products and have wide applications in animal feed additives, food additives, pharmaceuticals, and cosmetics.

[0003] The synthesis methods for D-pantothenic acid include chemical synthesis, enzyme-catalyzed synthesis, and traditional fermentation. (1) Chemical synthesis utilizes toxic substances such as hydrogen cyanide and sodium cyanide, and requires chiral resolving agents. However, the resolving agents are expensive, the reaction conditions are harsh, the chemical reaction conversion rate is low, and there are also problems of toxicity and environmental pollution. (2) Enzyme-catalyzed synthesis can effectively avoid the problems of expensive chemical resolving agents, difficult separation, and environmental pollution compared to chemical methods. Enzyme catalysis is achieved by pantothenate synthase (panC) catalyzing the ATP-dependent condensation reaction between pantothenate acid and β-alanine. However, it requires the chemical synthesis of two precursors, which fundamentally fails to solve the environmental pollution problem caused by chemical synthesis. (3) Traditional fermentation has attracted attention due to its advantages of low consumption, low toxicity, and low pollution. However, current microbial fermentation still has shortcomings, such as unstable fermentation process and low yield. Therefore, it is necessary to construct a stable and high-yielding engineered strain of D-pantothenic acid.

[0004] A key step in the pantothenic acid biosynthesis pathway is catalyzed by ketopantoate hydroxymethyltransferase (KPHMT), encoded by the gene panB. This enzyme catalyzes the reaction of α-ketoisovalerate with the cofactor CH2-THF to generate ketopantoate, and also generates a methyl-linked intermediate. This intermediate can be further hydrolyzed to form α-ketoisovalerate and a dihydroxymethyl intermediate. The dihydroxymethyl intermediate is then naturally cleaved into formaldehyde and tetrahydrohydroxypterin, the latter of which is returned to folic acid metabolism through the gene floc. Because the reaction catalyzed by ketopantoate hydroxymethyltransferase is reversible and is inhibited by the substrate and products ketopantoate, pantothenic acid, pantothenic acid and coenzyme A (Elevated levels of ketopantoate hydroxymethyltransferase (PanB) lead to a physiologically significant coenzyme Aelevation in Salmonella entericam eovar Tyhimurium.[J].Journal ofbacteriology,2002,184(10)). Since the amino acid sequence determines the spatial structure of an enzyme and its role in the catalytic process, enzyme engineering can be used to promote the rate-limiting reaction step. By enhancing the expression of the panB gene or increasing the catalytic activity of ketopantolysin hydroxymethyltransferase, the yield of pantothenic acid can be increased, and strains more suitable for industrial production can be obtained.

[0005] E. coli contains the pyruvate transporter YhjX from the OFA family. Pyruvate is an important intermediate in the biochemical metabolic network of organisms. When the extracellular pyruvate concentration is greater than 600 μmol / L, the YpdA / YpdB system is activated, leading to the expression of the yhjX gene. Pyruvate is a central node in carbon metabolism in cells and is therefore crucial for most living cells. During the fermentation process of industrial microbial production bacteria, the addition of large amounts of carbon causes pyruvate to overflow during the logarithmic growth phase. When the logarithmic growth phase ends and the cell enters the stationary phase, it experiences nutrient restriction and induces rapid pyruvate reuptake. (III) Summary of the Invention

[0006] The purpose of this invention is to construct an engineered bacterium that produces D-pantothenic acid and its applications, particularly to construct a recombinant bacterium that co-expresses ketopantothenic acid hydroxymethyltransferase and pyruvate uptake protein and its application in the production of D-pantothenic acid. By constructing a co-expression strain, the heterologous expression of ketopantothenic acid hydroxymethyltransferase is enhanced, effectively improving the yield of D-pantothenic acid synthesized by biological methods. Overexpression of pyruvate uptake protein YhjX accelerates pyruvate re-uptake, reduces carbon diversion, and further improves the yield of D-pantothenic acid.

[0007] The technical solution adopted in this invention is:

[0008] This invention provides a D-pantothenic acid-producing engineered bacterium. The engineered bacterium is constructed by co-transferring the coding gene of a ketopantoacid hydroxymethyltransferase mutant and the coding gene of pyruvate uptake protein into an expression vector to construct a co-expression recombinant plasmid. The co-expression recombinant plasmid is then transformed into a host bacterium to obtain a ketopantoacid hydroxymethyltransferase-pyruvate uptake protein co-expressing recombinant bacterium, which is the D-pantothenic acid-producing engineered bacterium. The ketopantoacid hydroxymethyltransferase mutant is obtained by single or double mutation at positions 58 and 59 of the amino acid sequence shown in SEQ ID NO.1.

[0009] Furthermore, the ketopanthelic acid hydroxymethyltransferase mutant is obtained by changing glycine at position 58 of the amino acid sequence shown in SEQ ID NO.1 to alanine and / or changing arginine at position 59 to histidine.

[0010] Furthermore, the pyruvate uptake proteins are derived from Escherichia coli (Genbank: NC_000913.3), Corynebacterium glutamicum (Genbank: CP025533.1), and Bacillus subtilis (Genbank: CP053102.1), respectively, and the nucleotide sequences of the encoding genes yhjX, ysbAB, and mctC are shown in one of SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11.

[0011] Furthermore, the host bacterium is DPA11A, which was constructed according to patent application CN113637618A.

[0012] Furthermore, the co-expressing recombinant bacteria of ketopantolysin-pyruvate uptake protein is constructed as follows: the gene encoding the mutant ketopantolysin-hydroxymethyltransferase is transferred into the host bacterium DPA11A to construct the engineered bacterium DPA11A*; the plasmid is extracted and the gene encoding the pyruvate uptake protein is overexpressed to construct a co-expression plasmid, and the constructed co-expression plasmid is transferred into the host bacterium DPA11A to obtain the co-expressing recombinant bacteria DPA11A*01.

[0013] Furthermore, the co-expressed recombinant bacteria were constructed using the following method:

[0014] (1) Using plasmid pTrc99A-panBpanC as a template, panB-R59H was introduced by PCR amplification to construct a single mutant engineered bacterium DPA11A*(panB-R59HpanC), denoted as DPA11A*(R59H). The amino acid sequence of panB-R59H is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6.

[0015] (2) Using the plasmid of the single mutant DPA11A*(panB-R59HpanC) as a template, the double mutant panB-G58A / R59H was introduced by PCR amplification to construct the engineered strain DPA11A*(panB-G58A / R59HpanC), denoted as DPA11A*(G58A / R59H). The amino acid sequence of panB-G58A / R59H is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7.

[0016] (3) Using plasmid pTrc99A-panB-G58A / R59HpanC as a template, linearized plasmid fragments were obtained by PCR amplification. Similarly, yhjX, ysbAB, and mctC target fragments were obtained by PCR amplification. Then, the plasmid framework and target fragments were seamlessly ligated using Novizan's one-step cloning kit. The ligated products were promptly transformed into E. coli DH5α competent cells and plated on LB solid plates containing Kan resistance. The plates were inverted and incubated overnight at 37°C to obtain plasmids pTrc99A-panB-G58A / R59HpanC-yhjX, pTrc99A-panB-G58A / R59HpanC--ysbAB, and pTrc99A-panB-G58A / R59HpanC--mctC.

[0017] (4) The constructed plasmids pTrc99A-panB-G58A / R59HpanC-yhjX, pTrc99A-panB-G58A / R59HpanC--ysbAB, and pTrc99A-panB-G58A / R59HpanC--mctC were introduced into DPA11A competent cells, spread on LB solid plates containing Kan resistance, and incubated overnight in a 37°C constant temperature incubator to obtain the co-expressing recombinant bacteria DPA11A*01, DPA11A*02, and DPA11A*03.

[0018] This invention also relates to the application of the aforementioned D-pantothenic acid-producing engineered bacteria in the microbial fermentation preparation of D-pantothenic acid. The method of application is as follows: the D-pantothenic acid-producing engineered bacteria are inoculated into a fermentation medium and fermented at 30°C and 200 rpm for 49 hours to obtain a fermentation broth containing D-pantothenic acid. The fermentation medium consists of: glucose 20 g / L, ammonium sulfate 16 g / L, yeast extract 2 g / L, β-alanine 2 g / L, KH₂PO₄ 0.8 g / L, MgSO₄ 0.5 g / L, CaCO₃ 15 g / L, trace element solution 1 mL / L, L-isoleucine 20 mg / L, VB1 5 mg / L, and VB2 5 mg / L. 12 Resistance to 2 mg / L IPTG, 0.1 mM kanamycin (Kan), and 50 mg / L was assessed using water as the solvent, pH 6.8. The trace element solution consisted of: CuCl2 10 g / L, FeSO4·7H2O 10 g / L, ZnSO4·H2O 1 g / L, CuSO4 0.2 g / L, NiCl2·7H2O 0.02 g / L, in deionized water. The fermentation medium was prepared by filling 250 ml shake flasks with 20 ml of medium and autoclaving at 115°C for 30 minutes before use. The medium contained CaCO3, L-isoleucine, VB1, and VB2. 12 IPTG and Kan resistance should be added to the culture medium before fermentation. Note: DPA11A is an L-isoleucine auxotype, so L-isoleucine needs to be added during shake flask culture.

[0019] Furthermore, before fermentation, the D-pantothenic acid engineered bacteria are first inoculated into LB medium containing 50 mg / L kanamycin (KAN) resistance and cultured overnight on a shaker at 37°C and 180 rpm. Then, they are inoculated into the fermentation medium at a volume concentration of 5% for further culture.

[0020] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following aspects: The DPA11A (pTrc99A-panBpanC) engineered strain of this invention serves as the starting strain. By mutating the panB gene of Corynebacterium glutamicum, the yield of pantothenic acid is increased. Simultaneously, co-expression of a pyruvate uptake protein accelerates the uptake of the key precursor pyruvate, and enhanced heterologous expression of the key rate-limiting enzyme further improves D-pantothenic acid production. The recombinant strain DPA11A*01 constructed by the method of this invention shows a significant increase in D-pantothenic acid production through shake-flask fermentation, reaching approximately 6.64 g / L, which is about 81.3% higher than the starting strain DPA11A, demonstrating promising application prospects for industrial production. (iv) Description of the attached drawings

[0021] Figure 1 It has the structural formula of D-pantothenic acid.

[0022] Figure 2 This is the D-pantothenic acid biosynthesis pathway.

[0023] Figure 3 This is a standard curve of D-calcium pantothenate concentration in Example 2.

[0024] Figure 4 The image shows the HPLC liquid chromatogram of the D-calcium pantothenate standard (concentration: 1 g / L) in Example 2.

[0025] Figure 5 The D-pantothenic acid production and OD of the mutant strain constructed in Example 1 and the recombinant strain co-expressed in Example 3 are shown. 600 Bar chart.

[0026] Figure 6 Agarose gel electrophoresis was performed on PCR products of pyruvate transporter genes from different sources. Lane M: 250bp DNA Ladder Marker; Lane 1: Linearized PCR results of pTrc99A-panB*panC plasmid; Lane 2: PCR results of yhjX gene from Escherichia coli; Lane 3: PCR results of ysbAB gene from Corynebacterium glutamicum; Lane 4: PCR results of mctC gene from Bacillus subtilis.

[0027] Figure 7 The growth curves of the mutant strain DPA11*(G58A / R59H) and the co-expressed recombinant strain DPA11A*01 in Example 4 are shown.

[0028] Figure 8 This study examines the expression level of the pyruvate transporter gene yhjX in the co-expression strain DPA11A*01 in Example 5 and its relationship with the pyruvate concentration in the fermentation broth. (V) Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0030] The culture media involved in the following examples are as follows:

[0031] LB liquid culture medium composition: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, solvent: water. Autoclave at 121°C for 20 minutes before use.

[0032] LB fixation medium is LB liquid medium with 20 g / L agar powder added.

[0033] Fermentation medium composition: glucose 20 g / L, ammonium sulfate 16 g / L, yeast extract 2 g / L, β-alanine 2 g / L, KH₂PO₄ 0.8 g / L, MgSO₄ 0.5 g / L, CaCO₃ 15 g / L, trace element solution 1 mL / L, L-isoleucine 20 mg / L, VB1 5 mg / L, VB 12 2 mg / L, IPTG 0.1 mM, Kan resistance 50 mg / L, solvent: water, pH 6.8; Trace element solution composition: CuCl2 10 g / L, FeSO4·7H2O 10 g / L, ZnSO4·H2O 1 g / L, CuSO4 0.2 g / L, NiCl2·7H2O 0.02 g / L, solvent: deionized water. The fermentation medium volume is 20 ml of medium in a 250 ml shake flask, sterilized at 115℃ for 30 minutes before use. The medium contains CaCO3, L-isoleucine, VB1, and VB2. 12 IPTG and Kan resistance should be added to the fermentation medium before fermentation. Note: DPA11A is an L-isoleucine auxotype, so L-isoleucine needs to be added during shake flask culture.

[0034] The embodiments of the present invention use DPA11A engineered bacteria as the starting strain and are constructed in accordance with patent application CN113637618A.

[0035] The preparation steps of DPA11A competent cells in this embodiment of the invention are as follows:

[0036] Strawberry strain DPA11A, stored at -80℃, was streaked onto antibiotic-free LB agar plates and incubated at 37℃ for approximately 12 hours, resulting in single colonies. A single colony was picked and inoculated into a sterile 10 mL LB tube, then incubated at 37℃ with shaking at 180 rpm for 12 hours. The culture was then transferred to 50 mL LB liquid medium at a 1% (v / v) inoculum concentration and incubated at 37℃ with shaking at 18 rpm for approximately 2 hours (to allow the bacterial culture to reach OD500). 600 Cells were kept between 0.4 and 0.6 μg / mL and incubated on ice for 20 min to stop growth. They were then transferred to sterile, pre-chilled 50 mL centrifuge tubes and centrifuged at 4000 rpm for 6 min at 4°C to collect the cells, discarding the supernatant. The cells were then resuspended in 15 mL of pre-chilled 0.1 M CaCl2 solution and incubated on ice for 30 min. The centrifugation and resuspension with 0.1 M CaCl2 solution were repeated. Cells were then collected by centrifugation at 4000 rpm for 15 min at 4°C, discarding the supernatant. The cells were resuspended in 1 mL of 0.1 M CaCl2 solution containing 15% glycerol, aliquoted into sterile 1.5 mL EP tubes (100 μL / tube), and stored at -80°C for later use.

[0037] The construction steps of the pTrc99A-panBpanC plasmid used in this embodiment of the invention are as follows:

[0038] Using plasmid pTrc99A (purchased from Addgene, product number VT1294) as a template, linearized plasmid fragments were obtained by PCR amplification. Similarly, panB (nucleotide sequence as shown in SEQ ID NO.5, amino acid sequence as shown in SEQ ID NO.1) and panC (nucleotide sequence as shown in SEQ ID NO.8, amino acid sequence as shown in SEQ ID NO.4) fragments derived from Corynebacterium glutamicum ATCC 14007 were obtained by PCR amplification. The plasmid framework and target fragments were then seamlessly ligated using a one-step cloning kit from Novizan. The ligated product was promptly transformed into commercially competent E. coli DH5α cells, plated on LB agar plates containing Kan resistance, and incubated overnight at 37°C. Plasmids were then extracted to obtain pTrc99A-panBpanC.

[0039] The construction steps of DPA11A (pTrc99A-panBpanC) in this embodiment of the invention are as follows:

[0040] The constructed pTrc99A-panBpanC plasmid was introduced into DPA11A competent cells, plated on LB solid plates containing Kan resistance, and incubated overnight in a 37°C incubator to obtain the engineered Escherichia coli strain DPA11A (pTrc99A-panBpanC).

[0041] Example 1: Construction of DPA11A*(pTrc99A-panBpanC) mutant strain by site-directed mutagenesis

[0042] 1. Mutation site screening

[0043] Homology modeling was performed on the panB gene (nucleotide sequence shown in SEQ ID NO. 5, amino acid sequence shown in SEQ ID NO. 1) encoding ketopantolysin hydroxymethyltransferase from Corynebacterium glutamicum (Genbank: CP022614.1) using Swiss-Model. Mutation sites were predicted using the HotSpot Wizard online server, and sites where the target enzyme differed from most similar enzymes were screened. Site analysis was performed using Discovery Studio docking. The results showed that amino acids at positions 58 and 59 are located at the cleft where the two subunits overlap, which can easily interact with nearby sites to maintain the stability of the two subunits and thus maintain the activity of the overall protein.

[0044] 2. Unit-point fixed-site mutation

[0045] The plasmid pTrc99A-panBpanC (the amino acid sequence of panB is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.5; the amino acid sequence of panC is shown in SEQ ID NO.4, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.8) of the engineered strain DPA11A (pTrc99A-panBpanC) was used as a template for PCR amplification using primers R59H-F / R in Table 1. The PCR product was digested with DpnI at 37°C for 15 min and then transformed into competent cells of the DPA11A engineered strain. The cells were plated on LB agar plates containing a final concentration of 50 mg / L kanamycin and cultured at 37°C for 12 h. Single colony clones were selected for sequencing. D-pantothenic acid fermentation production and detection were performed using the method in Example 2. A single mutant engineered strain, DPA11A*(panB-R59HpanC), was obtained and designated as DPA11A*(R59H). The amino acid sequence of panB-R59H is shown in SEQ ID NO.1. As shown in NO.2, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6.

[0046] 3. Two-site site-directed mutagenesis

[0047] Using the plasmid of the single mutant DPA11A*(panB-R59HpanC) as a template, PCR amplification was performed using primers G58A / R59H-F / R in Table 1, following the same method as in step 2. The double mutant engineered bacteria DPA11A*(panB-G58A / R59HpanC) was obtained by screening and designated as DPA11A*(G58A / R59H). The amino acid sequence of panB-G58A / R59H is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.7.

[0048] Table 1 Primers for the ketopanol acid hydroxymethyltransferase mutation site

[0049]

[0050]

[0051] The PCR reaction procedure is as follows:

[0052] 95℃ for 5 min, 95℃ for 30 s, 58℃ for 30 s, 72℃ for 0.5 min (2 kbp / min), repeat for 30 cycles; extend the treatment at 72℃ for another 10 min.

[0053] Example 2: Detection of D-pantothenic acid production by fermentation of *Bacillus subtilis* and mutant strains

[0054] 1. Fermentation culture of engineered bacteria

[0055] The following strains were inoculated onto LB plates: the bottom strain DPA11A, the engineered strains DPA11A*(R59H), DPA11A*(G58A / R59H) screened in Example 1, and the strains DPA11A*01, DPA11A*02, and DPA11A*03 constructed in Example 3. They were cultured at 37°C for 12 hours. Single clones were selected and inoculated into 10 ml LB tubes and cultured overnight at 37°C. The culture was then inoculated at a volume concentration of 5% into 250 ml shake flasks containing 20 ml of fermentation medium and cultured at 30°C and 200 rpm for 49 hours. Take 1 mL of fermentation broth and transfer it to a 1.5 mL EP tube. Centrifuge at 12,000 rpm for 1 min. Transfer the supernatant to a clean EP tube. Retain the precipitate for subsequent determination of cell biomass. Dilute the supernatant by a certain factor and filter it through a 0.22 μm aqueous filter membrane. Detect the peak area of ​​D-pantothenic acid by high-performance liquid chromatography (HPLC). Obtain the D-pantothenic acid content according to the D-pantothenic acid calcium standard curve. See the bar chart for D-pantothenic acid yield. Figure 5 .from Figure 5 It can be seen that, compared with the starting strain DPA11A, the D-pantothenic acid production of the engineered strains DPA11A*(R59H), DPA11A*(G58A / R59H) screened in Example 1 and the DPA11A*01, DPA11A*02, and DPA11A*03 constructed in Example 3 increased by 26.9%, 54.8%, 81.3%, 62.7%, and 61.2%, respectively. This indicates that the constructed DPA11A*01 can effectively accumulate D-pantothenic acid in the fermentation broth by enhancing the expression of the panB gene and the transport protein YhjX during the fermentation process.

[0056] 2. D-Calcium Pantothenate Standard Curve

[0057] Weigh 0.05 g of D-calcium pantothenate reference standard (accurate to 0.001 g), place it in a 10 mL volumetric flask, dissolve and dilute with water to different concentrations (0.5, 1, 1.5, 2, 2.5 g / L), filter through a 0.22 μm organic filter membrane, and detect the peak area using the high-performance liquid chromatography (HPLC) conditions described in step 3. Plot a D-calcium pantothenate standard curve with D-calcium pantothenate concentration on the x-axis and peak area on the y-axis. See [reference needed]. Figure 3 The HPLC chromatogram of D-calcium pantothenate standard (concentration: 1 g / L) is shown below. Figure 4 .

[0058] 3. HPLC detection of D-pantothenic acid yield in mutant strains

[0059] HPLC determination conditions for product D-pantothenic acid: Thermo Fisher Scientific Ultimate HPLC system was used for detection, ACQUITYUPLC BEH C18 column (100mm×2.1mm, 1.7μm, waters, UK).

[0060] The operating conditions for high performance liquid chromatography are as follows:

[0061] Mobile phase composition: 95% ultrapure water, 4.9% acetonitrile, 0.1% phosphoric acid, filtered through a 0.20μm organic microporous membrane and degassed by ultrasonication;

[0062] B. Column type: ACQUITY UPLC BEH C18 column (100mm×2.1mm, 1.7um, Waters, UK); C. Parameter settings: Injection volume: 15μL, column temperature: 30℃, flow rate: 0.9mL / min, detection wavelength: 200nm.

[0063] D retention time: 15 min.

[0064] 4. Spectrophotometer detection of bacterial OD 600

[0065] Pretreatment and determination of bacterial biomass: After retaining the precipitate, wash the precipitate with 1 ml of ultrapure water, centrifuge at 12000 rpm for 1 min, discard the supernatant, add 200 μL of 0.6 mol / L hydrochloric acid and 800 ml of ultrapure water to the precipitate, mix well, open the cap and let stand until the precipitate is completely dissolved, take 100 μL of the mixture, dilute it 20-30 times, and measure the OD using a spectrophotometer. 600 Finally, the actual OD of the fermentation broth was obtained after conversion based on the dilution factor. 600 Values. OD values ​​for each strain. 600 See bar chart Figure 5 .

[0066] Example 3: Cloning of the pyruvate uptake protein gene and construction of engineered bacteria co-expressing ketopanolate hydroxymethyltransferase-pyruvate uptake protein

[0067] Using pTrc99A-panB-G58A / R59HpanC from the engineered strain DPA11A*(G58A / R59H) in Example 1 as a template, the linearized plasmid fragment pTrc99A-panB-G58A / R59HpanC was obtained by PCR amplification using primers Pbc-line-F / R (Table 2).

[0068] Using the genomes of *Escherichia coli* (Genbank: NC_000913.3), *Corynebacterium glutamicum* (Genbank: CP025533.1), and *Bacillus subtilis* (Genbank: CP053102.1) as templates, the target fragments yhjX, ysbAB, and mctC were obtained by PCR amplification using the primers listed in Table 2. The nucleotide sequences are shown in SEQ ID NO. 9, SEQ ID NO. 10, and SEQ ID NO. 11, respectively. See the agarose gel electrophoresis image of the PCR products. Figure 6 Lane M: 250bp DNA Ladder Marker; Lane 1: PCR results of plasmid linearization of pTrc99A-panB-G58A / R59HpanC; Lane 2: PCR results of yhjX gene from Escherichia coli; Lane 3: PCR results of ysbAB gene from Corynebacterium glutamicum; Lane 4: PCR results of mctC gene from Bacillus subtilis.

[0069] Homologous arms were designed based on the upstream and downstream sequences of the linearized plasmid fragment. The plasmid framework and target fragment were seamlessly ligated using a one-step cloning kit from Novizan. The ligated product was promptly transformed into DPA11A competent cells and plated on LB agar plates containing 50 mg / L Kan resistance. The plates were incubated overnight at 37°C. Single colonies were randomly selected, and colony PCR was performed using Pbc-yz-F and Pbc-yz-R primers for verification. The gene fragments yz-yhjX (1200 bp), yz-ysbAB (1600 bp), and yz-mctC (1100 bp) were successfully cloned, confirming positive colonies. Further sequencing confirmed the results, ultimately yielding co-expression strains DPA11A*01, DPA11A*02, and DPA11A*03.

[0070] Table 2 Primers

[0071]

[0072] Example 4: Growth curve analysis of D-pantothenic acid production by recombinant bacteria DPA11A*(G58A / R59H) and DPA11A*01 using glucose as a substrate.

[0073] The strain DPA11A*(G58A / R59H) constructed in Example 1 and the strain DPA11A*01 constructed in Example 3 were respectively inoculated onto LB agar plates and cultured at 37°C for 12 h. Single clones were then selected and inoculated into 10 ml LB tubes and cultured overnight at 37°C. A 5% (v / v) inoculum was then inoculated into 250 ml shake flasks containing 20 ml of fermentation medium and fermented at 30°C and 200 rpm. Samples were taken every 2 hours from 0 to 24 h, and the concentrations of D-pantothenic acid and OD in the fermentation samples were determined using the method described in Example 2. 600 After 24 hours, the concentration of D-pantothenic acid was measured every 4 hours. Fermentation was stopped when the D-pantothenic acid concentration stabilized. The differences in D-pantothenic acid yield and strain growth characteristics during fermentation were investigated.

[0074] Fermentation curves of mutant strain DPA11A*(G58A / R59H) and co-expressed recombinant strain DPA11A*01 are shown below. Figure 7 During fermentation from 0 to 10 hours, the D-pantothenic acid production curves of DPA11A*(G58A / R59H)-DPA and DPA11A*01-DPA largely overlapped. After 10 hours, the D-pantothenic acid production of both strains gradually increased, suggesting that pyruvate accumulated intracellularly within 10 hours. Since DPA11A*(G58A / R59H) enhanced the expression of key enzymes, it enhanced the synthesis of ketopantothenic acid from α-ketoisovaleric acid. Based on this, DPA11A*01 further accelerated the rapid uptake of pyruvate, promoting the D-pantothenic acid pathway. At 48 hours, the co-expressed strain DPA11A*01-DPA produced as much as 6.64 g / L. The growth curves show that the growth trends of DPA11A*(G58A / R59H)-OD and DPA11A*01-OD are similar. Both end their lag phase before 2 hours, then enter the logarithmic growth phase, ending around 28 hours, followed by a stationary phase. The highest biomass of DPA11A*01 is 12.96, while that of DPA11A*(G58A / R59H) is 13.44. Overall, the cell biomass of the two strains is not significantly different, indicating that the construction of the co-expressed recombinant strain DPA11A*01 has no significant impact on bacterial growth.

[0075] Example 5: RT-qPCR was used to determine the changes in the transcriptional level of the pyruvate transporter gene yhjX during the fermentation of co-expressed strain DPA11A*01 and its relationship with the pyruvate concentration in the fermentation broth.

[0076] YhjX is known to be a pyruvate uptake protein. To further characterize the transcriptional level of yhjX during the fermentation process of the strain and its relationship with the pyruvate concentration in the fermentation broth, based on the experimental results in Example 4, the transcriptional level of yhjX in DPA11A*01 was measured at 24h, 28h, 32h, 36h, 40h, 44h, and 48h. The rpoB gene in E. coli was used as an internal reference gene. The fermentation broth at the seven time points selected in Example 4 was used as samples. Total RNA was extracted from the samples using FastPure Cell / Tissue Total RNA Isolation Kil V2. Reverse transcription was performed using HiScript IIQ RT SuperMix for qPCR. Using the cDNA generated by reverse transcription as a template, specific upstream and downstream primers for the pyruvate transporter gene yhjX and the internal reference gene rpoB were designed. Real-time quantitative PCR amplification was performed under the following PCR amplification systems and reaction conditions. The changes in the transcriptional level of the yhjX gene were quantitatively analyzed based on the changes in fluorescence in the reaction system.

[0077] Table 3. Specific primers for pyruvate transporter gene yhjX and internal reference gene ropB:

[0078]

[0079] The PCR amplification system, i.e., the 25 μL system, is as follows:

[0080] SYBR Premix Ex Taq II enzyme 12.5uL, cDNA template 1uL, yhjX-F and yhjX-R upstream and downstream primers 1uL each, deionized water 9.5uL;

[0081] SYBR Premix Ex Taq II enzyme 12.5uL, cDNA template 1uL, upstream and downstream primers of ropB-F and ropB-R 1uL each, deionized water 9.5uL;

[0082] The reaction conditions are as follows: 95℃ pre-denaturation for 30s, 95℃ denaturation for 15s, 55℃ annealing for 45s, 72℃ extension for 30s, 40 cycles;

[0083] The transcriptional level of yhjX and the method for analyzing the relative transcriptional level of the gene at each time point are as follows:

[0084] T1: ΔCt1=(yhjX Ct1-rpoB Ct1);

[0085] T0: ΔCt0=(yhjX Ct0-rpoB Ct0);

[0086] ΔCt=ΔCt1-ΔCt0;

[0087] yhjX fold change=2^ -ΔΔCt ;

[0088] The relative expression level of the pyruvate uptake gene yhjX in the co-expression strain DPA11A*01 is shown in [reference needed]. Figure 8 Between 24 and 40 hours, the transcription level of yhjX gradually increased, and the pyruvate concentration in the fermentation broth of both strains first increased and then decreased. The pyruvate concentration of the mutant strain DPA11A*(G58A / R59H) in the fermentation broth was greater than that of the co-expressed strain DPA11A*01. At 40 hours, the transcription level of yhjX reached its highest level, while the pyruvate concentration in the co-expressed strain DPA11A*01 reached its lowest level, differing from that of the mutant strain DPA11A*(G58A / R59H) by about 49%. This indicates that the co-expressed strain DPA11A*01 can accelerate the reuptake of pyruvate and reduce carbon diversion. After 40 hours, yhjX basically stopped transcribing, the ability of the co-expressed strain DPA11A*01 to take up pyruvate weakened, and pyruvate gradually accumulated in the fermentation broth. In addition, the pyruvate concentration in the fermentation broth of the co-expression strain DPA11A*01 was greater than 600 μmol / L, which activated the YpdA / YpdB system and induced the expression of yhjX. The ability of DPA11A*01 to take up pyruvate was enhanced, and after taking up pyruvate for a period of time, yhjX stopped transcription.

[0089] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A D-pantothenic acid-producing engineered bacterium, characterized in that, The engineered bacteria were constructed by co-transferring the coding gene of a ketopantolate hydroxymethyltransferase mutant with the introduced panC gene and the coding gene of a pyruvate uptake protein into an expression vector to construct a co-expression recombinant plasmid. The co-expression recombinant plasmid was then transformed into a host bacterium to obtain a recombinant bacterium co-expressing the ketopantolate hydroxymethyltransferase mutant with the introduced panC gene and the pyruvate uptake protein, which is a D-pantothenic acid-producing engineered bacterium. The ketopantolate hydroxymethyltransferase mutant was obtained by changing glycine at position 58 of the amino acid sequence shown in SEQ ID NO. 1 to alanine and arginine at position 59 to histidine. The nucleotide sequence of the pyruvate uptake protein coding gene is shown in one of SEQ ID NO. 9, SEQ ID NO. 10, or SEQ ID NO.

11. The host bacterium is the DPA11A engineered bacterium.

2. The application of the D-pantothenic acid-producing engineered bacteria as described in claim 1 in the microbial fermentation preparation of D-pantothenic acid.

3. The application as described in claim 2, characterized in that, The application method is as follows: the D-pantothenic acid-producing engineered bacteria are inoculated into a fermentation medium and fermented at 30℃ and 200 rpm to obtain a fermentation broth containing D-pantothenic acid; the fermentation medium consists of: glucose 20 g / L, ammonium sulfate 16 g / L, yeast extract 2 g / L, β-alanine 2 g / L, KH2PO4 0.8 g / L, MgSO4 0.5 g / L, CaCO3 15 g / L, trace element solution 1 mL / L, L-isoleucine 20 mg / L, VB1 5 mg / L, VB 12 2 mg / L, IPTG 0.1 mM, kanamycin 50 mg / L, solvent: water, pH 6.8; Trace element solution composition: CuCl2 10 g / L, FeSO4·7H2O 10 g / L, ZnSO4·H2O 1 g / L, CuSO4 0.2 g / L, NiCl2·7H2O 0.02 g / L, solvent: deionized water.

4. The application as described in claim 3, characterized in that, Before fermentation, the D-pantothenic acid engineered bacteria were first inoculated into LB medium containing 50 mg / L kanamycin and cultured overnight on a shaker at 37°C and 180 rpm. Then, they were inoculated into the fermentation medium at a volume concentration of 5% and cultured.

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