A transporter protein variant and its application in the preparation of R-pantothenic acid

Abstract

This application relates to a variant of the transporter panS and its encoding gene. Six variants of the transporter panS were constructed in this application, and they were introduced into Saccharomyces cerevisiae to construct R-pantothenic acid-producing strains. Fermentation tests showed that the yield of R-pantothenic acid in the fermentation broth was significantly increased. This variant has application prospects in the construction of R-pantothenic acid-producing strains.

Description

[0001] This application is a divisional application of the application with the application date of January 18, 2023, application number 202310062226.3, and invention title "A transporter variant and its application in the preparation of R-pantothenic acid". Technical Field

[0002] The present invention belongs to the field of biotechnology, and particularly relates to a transporter and its application in the preparation of R-pantothenic acid. Background Art

[0003] Pantothenic acid (VB5) is an important precursor for the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP), and is a vitamin necessary for maintaining the normal physiological functions of organisms. Pantothenic acid participates in the metabolism of carbohydrates, fatty acids, proteins and energy in organisms, and also participates in the synthesis of steroids, melatonin, antibodies and heme in the human body, and plays an important role in aspects such as food, medicine, feed additives, etc.

[0004] Pantothenic acid is an important intermediate for the production of pantothenic acid. By biosynthesizing R-pantothenic acid and then dehydrating and condensing with β-alanine, D-pantothenic acid can be obtained. Therefore, designing and reconstructing model strains such as Escherichia coli and Saccharomyces cerevisiae, and using non-natural life function molecules / devices, including components, modules, circuits, chassis, etc. to construct engineering strains with high-yield R-pantothenic acid will lay a foundation for the industrial biological production of D-pantothenic acid.

[0005] panS encodes a transporter that can transport pantothenic acid in cells to the outside of the cells. Zhang Peng et al. studied the transmembrane transport of substances driven by the energy generated by the decomposition of ATP by ABC transporters. Energy-coupling factor (ECF) transporters are a new class of ABC transporters identified in recent years. They exist in microorganisms and plants and are responsible for the transmembrane transport of vitamins and micronutrients (such as B-group vitamins folic acid, pantothenic acid, riboflavin and metal ions, etc.). The ECF transporter structurally includes a substrate-binding protein S on the cell membrane and an energy-coupling module formed by transmembrane protein T and intracellular ATP-binding protein A / A'. The substrate transport is achieved by the flipping of the substrate-binding protein S in the membrane, which gives people a new understanding of the transmembrane transport mechanism of substances that consume ATP (Xu et al, Nature. 497(7448): 268-71. 2013). Furthermore, through the structural comparison and functional analysis of the pantothenic acid ECF transporter complex from the same species, the molecular basis for the sharing of the energy-coupling module by ECF transporters was revealed (Zhang et al, PNAS. 111(52): 18560-5. 2014)).

[0006] Traditional production methods use chemical methods for synthesis, with high costs for chiral resolving agents, difficult separation, and environmental pollution and toxicity problems. With the pursuit of green environmental protection by people, the production of R-pantothenic acid products by the microbial method has received increasing attention. The microbial fermentation method has mild conditions, is environmentally friendly, and has stable product quality, showing great promise. Saccharomyces cerevisiae, as a eukaryotic model microorganism, has characteristics such as a clear genetic background, easy operation, tolerance to low pH, tolerance to high-concentration substrates, simple nutritional requirements, low cost, and facultative anaerobiosis. However, the current yield of R-pantothenic acid produced by Saccharomyces cerevisiae is relatively low. This may be due to the accumulation of R-pantothenic acid in the organism, which inhibits the activity of related synthase enzymes in its metabolic pathway, thereby reducing the extracellular accumulation of R-pantothenic acid. Therefore, using molecular biology techniques to transform the original yeast strain to obtain a high-yield R-pantothenic acid-producing strain derived from yeast is a problem that needs to be solved in this field. Summary of the Invention

[0007] Through the structural study of the panS gene, a series of site-directed mutations were carried out on panS. The mutated panS gene was transferred into the host bacterium and screened, and 6 panS variants that increased the yield of R-pantothenic acid in yeast were obtained. These panS variants can timely discharge the produced R-pantothenic acid outside the cell, avoiding intracellular accumulation, improving the fermentation yield of R-pantothenic acid, and enhancing the phenotypic activity of the panS gene.

[0008] The present invention provides a mutated panS gene, an expression vector containing the mutated gene, an engineered bacterium containing the gene, and their applications in increasing the yield of R-pantothenic acid and fermentation production.

[0009] In the first aspect of the present invention, it relates to a nucleic acid molecule, including substantially a transport protein encoded by the nucleic acid molecule that transfers R-pantothenic acid from the inside of the cell to the outside of the cell, and the transport protein has a mutation at the amino acid level relative to the wild type.

[0010] In one embodiment, the gene of the transport protein with substantially increased enzyme activity is the mutated panS gene. The panS gene of the present invention has mutations compared to the wild-type panS gene (the nucleotide sequence is as shown in sequence 7 in the sequence listing; the amino acid sequence is as shown in sequence 8 in the sequence listing). After the mutated panS is transferred into the R-pantothenic acid-producing strain, the enzyme activity of the transport protein panS is increased. For example, panS*T106I, panS*S283G, panS*S260A, panS*T106I-S283G, panS*T106I-S260A, panS*S260A-S283G, panS*T106I-S260A-S283G, as shown in sequence 9, 10, 11, 12, 13, 14, 15 in the sequence listing of this application respectively.

[0011] The present invention also relates to a genetically engineered strain, which contains the gene of a mutant transporter panS.

[0012] In one embodiment, the gene with increased enzymatic activity of the transporter panS can be the mutant panS gene.

[0013] For the genetically engineered bacterium as described above, the panS gene of the present invention can be inserted into the vector DNA and then introduced into the host. The panS gene of the present invention can be retained in the host as an extrachromosomal DNA such as a plasmid, or the above gene can be incorporated into the chromosome of the host microorganism by using methods such as transduction, transposon, Mu phage or homologous recombination. In order to effectively express the above gene, the panS of the present invention can be placed under the control of promoters such as lac, trp, PL and tac that function in microorganisms.

[0014] In one embodiment, the R-pantothenic acid fermentation engineering strain containing the mutant panS gene has an R-pantothenic acid production rate 5.72 times higher than that of the engineering strain containing the wild-type panS gene. The strain XAB-10 was deposited for patent preservation on December 5, 2022, with the deposit number CCTCC NO: M 20221859, the classification name Saccharomyces cerevisiae XAB-10, and the deposit unit is the China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, Hubei, China.

[0015] The genetically engineered strain of the present invention can be used for the fermentation production of R-pantothenic acid.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects:

[0017] 1. The present invention provides a panS mutant and its coding nucleic acid sequence

[0018] 2. The present invention provides a method for increasing the yield of R-pantothenic acid. By expressing the mutant panS gene in the recombinant strain S. cerevisiae BY 4741 (MATa, his3△1, leu2△0, met15△0, ura3△0), the fermentation yield of R-pantothenic acid is increased, and it is used for the construction of R-pantothenic acid engineering strains and the high-efficiency fermentation of R-pantothenic acid. The final R-pantothenic acid yield of the genetically engineered strain of the present invention is 806 μg / L, the R-pantothenic acid yield of the original strain is 120 μg / L, and the R-pantothenic acid production capacity of the genetically engineered strain containing panS*S260A-S283G is increased by 5.72 times compared with the strain containing the wild-type panS gene, which has great industrial application value. Description of the Drawings

[0019] Figure 1 Molecular docking diagram of the SBF domain of panS and R-pantothenic acid Detailed implementation manners

[0020] The present invention is further illustrated by the following embodiments, but any embodiment or combination thereof should not be construed as limiting the scope or implementation manner of the present invention. The scope of the present invention is defined by the appended claims. Combining this specification and the general common knowledge in the art, those of ordinary skill in the art can clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art can make any modifications or changes to the technical solutions of the present invention, and such modifications and changes are also included within the scope of the present invention.

[0021] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods; the reagents and materials used, etc., unless otherwise specified, can all be obtained from commercial channels.

[0022] Table 1 Strains and plasmids used in the present invention

[0023]

[0024]

[0025] Table 2 Primers used in the present invention

[0026]

[0027]

[0028] Example 1: Construction of R-pantothenic acid-producing strain

[0029] (1) Overexpression of the ECM31 gene in Saccharomyces cerevisiae S.cerevisiae BY 4741

[0030] The ECM31 gene (nucleotide sequence as shown in Sequence 1 of the sequence listing) was overexpressed by the CRISPR Cas9 method. The specific steps are as follows:

[0031] In the first step, using the p426-SNR52p-gRNA plasmid DNA as a template, a 6263 bp DNA fragment I was amplified using the primers sg-ECM31-F / sg-ECM31-R (Primers 13 and 14), transformed into TOP10 competent cells, and monoclonal colonies were selected for sequencing. The sequencing primers were M13 Reverse / T7 (Primers 19 and 20). After correct sequencing, the plasmid was extracted and named sg-ECM31 for standby.

[0032] Step 2: Using the pSC-1 plasmid DNA as a template, amplify a 675-bp DNA fragment II with primers GAL1-F / GAL1-R (Primers 1 and 2) for later use.

[0033] The amplification system is as follows: 10 μl of Phusion 5X buffer (New England Biolabs), 1 μl of dNTP (dNTP concentration 10 mM), 20 ng of DNA template, 2 μl of each primer (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), and 33.5 μl of distilled water, with a total volume of 50 μl.

[0034] The amplification conditions are: pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 1 minute (30 cycles); extension at 72°C for 10 minutes.

[0035] Use the above DNA fragment II for gene editing: First, transform the pUDP004 plasmid - (purchased from ADDGENE, #101165) into Saccharomyces cerevisiae BY 4741 by electroporation, and then electroporate the DNA fragment II into Saccharomyces cerevisiae BY 4741 carrying pUDP004.

[0036] The electrotransformation conditions are as follows: First, prepare electrocompetent cells of Saccharomyces cerevisiae BY 4741 carrying the pUDP004 plasmid. The preparation method is as follows: Cultivate fresh yeast liquid to make competent cells: According to an inoculation amount of 1%, inoculate 100 μl of the seed liquid into 10 ml of the medium. Collect the cells: Take 1.5 ml of the culture and dispense it into a 1.5 ml EP tube. Centrifuge at 13,000 rpm for 1 min, discard the supernatant, and aspirate it clean with a pipette. Wash the pellet with 1 ml of sterile water, pipette and centrifuge, discard the supernatant, and wash twice. Cell treatment: Add 1 ml of SOB liquid medium (from the 4°C refrigerator) + 10 μl of dithiothreitol solution, heat at 25°C for 20 min. Centrifuge, discard the supernatant, aspirate it completely with a pipette, add 1 ml of pre-cooled 1 M SOB liquid medium (from the 4°C refrigerator), pipette, centrifuge, and discard the supernatant. Wash twice with 1 M SOB liquid medium, aspirate and discard the supernatant, and then add 50 μl of SOB liquid medium to suspend. Add 3 μl of the sg-ECM31 plasmid and 1.1 μl of DNA fragment II, mix well and transfer to a pre-cooled electroporation cuvette, and incubate on ice for 5 min. Wipe the electroporation cuvette dry, and perform electroporation at 2.7 kV. Put 1 ml of the pre-aspirated SOB liquid medium into the electroporation cuvette, mix well and aspirate into a new 1.5 ml EP tube. Incubate on a shaker at 30°C and 250 rpm for 40 - 60 min. Centrifuge the cell suspension, remove part of the supernatant, mix well and spread on the corresponding plate, and incubate in an incubator at 30°C for 36 h. Select single colonies for PCR verification. The primers used are yz-GAL1-F / yz-GAL1-R (primers 7 and 8). The correct colony amplification product is a 1142 bp fragment. Select one correct single colony and name it XAB-1.

[0037] (2) Overexpress the PAN5 gene in Saccharomyces cerevisiae XAB-1

[0038] Use the CRISPR Cas9 method to overexpress the PAN5 gene (the nucleotide sequence is shown in Sequence 2 of the sequence listing). The specific steps are as follows:

[0039] First step, using the p426-SNR52p-gRNA plasmid DNA as a template, amplify a 6263 bp DNA fragment I using the primers sg-pan5-F / sg-pan5-R (primers 15 and 16), transform it into TOP10 competent cells, select monoclonal colonies for sequencing, and the sequencing primers are M13 Reverse / T7 (primers 19 and 20). After correct sequencing, extract the plasmid and name it sg-pan5 for standby.

[0040] Second step, using the pSC-1 plasmid DNA as a template, amplify a 727 bp DNA fragment II using the primers ENO2-F / ENO2-R (primers 3 and 4) for standby.

[0041] The amplification system is as follows: 10 μl of Phusion 5X buffer (New England Biolabs), 1 μl of dNTP (dNTP concentration 10 mM), 20 ng of DNA template, 2 μl of each primer (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), and 33.5 μl of distilled water, with a total volume of 50 μl.

[0042] The amplification conditions are as follows: pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 1 minute (30 cycles); extension at 72°C for 10 minutes.

[0043] The above DNA fragment II was used for gene editing: DNA fragment II was electrotransformed into XAB-1 carrying pUDP004.

[0044] The electrotransformation conditions are as follows: First, prepare electrocompetent cells of XAB-1 carrying the pUDP004 plasmid. The preparation method is as follows: Culture fresh yeast liquid to make competent cells. According to an inoculation amount of 1%, inoculate 100 μl of seed liquid into 10 ml of medium to collect the bacteria. Take 1.5 ml of the culture and dispense it into 1.5 ml EP tubes. Centrifuge at 13,000 rpm for 1 min, discard the supernatant, and aspirate it clean with a pipette. Wash the precipitate with 1 ml of sterile water, pipette and centrifuge, discard the supernatant, and wash twice. Bacterial treatment: Add 1 ml of SOB liquid medium (from a 4°C refrigerator) + 10 μl of dithiothreitol solution, and heat at 25°C for 20 min. Centrifuge, discard the supernatant, aspirate it completely with a pipette, add 1 ml of pre-cooled 1 M SOB liquid medium (from a 4°C refrigerator), pipette, centrifuge, and discard the supernatant. Wash twice with 1 M SOB liquid medium, aspirate and discard the supernatant, and then add 50 μl of SOB liquid medium to suspend. Add 3 μl of sg-pan5 plasmid and mix well, and then add 1.1 μl of DNA fragment II and transfer it into a pre-cooled electroporation cuvette. Incubate on ice for 5 min. Wipe the electroporation cuvette dry and perform electroporation at 2.7 kV. Put 1 ml of the previously aspirated SOB liquid medium into the electroporation cuvette, mix well and aspirate it into a new 1.5 ml EP tube. Incubate on a shaker at 30°C and 250 rpm for 40 - 60 min. Centrifuge the bacterial liquid, remove some supernatant, mix well, spread it on the corresponding plate, and culture it in an incubator at 30°C for 36 h. Select single colonies for PCR verification. The primers used are yz-ENO2-F / yz-ENO2-R (primers 9 and 10). The correct colony amplification product is a 1251 bp fragment. Select one correct single colony and name it XAB-2.

[0045] (3) Overexpress the PAN6 gene in Saccharomyces cerevisiae XAB-2

[0046] The CRISPR Cas9 method was used to overexpress the PAN6 gene (nucleotide sequence as shown in Sequence 3 of the sequence listing). The specific steps are as follows:

[0047] First step: Using the p426-SNR52p-gRNA plasmid DNA as a template, amplify a 6263-bp DNA fragment I with primers sg-pan6-F / sg-pan6-R (primers 17 and 18), transform it into TOP10 competent cells, select monoclonal colonies for sequencing, with the sequencing primers being M13 Reverse / T7 (primers 19 and 20). After correct sequencing, extract the plasmid and name it sg-pan6 for standby.

[0048] Second step: Using the pSC-1 plasmid DNA as a template, amplify a 263-bp DNA fragment II with primers URA3-F / URA3-R (primers 5 and 6) for standby.

[0049] The amplification system is as follows: 10 μl of Phusion 5X buffer (New England Biolabs), 1 μl of dNTP (dNTP concentration 10 mM), 20 ng of DNA template, 2 μl of each primer (10 μM), 0.5 μl of Phusion High-Fidelity DNA polymerase (2.5 U / μl), and 33.5 μl of distilled water, with a total volume of 50 μl.

[0050] The amplification conditions are as follows: Pre-denaturation at 98°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 56°C for 10 seconds, extension at 72°C for 1 minute (30 cycles); extension at 72°C for 10 minutes.

[0051] Use the above DNA fragment II for gene editing: Electroporate the DNA fragment II into XAB-2 carrying pUDP004.

[0052] The electrotransformation conditions are as follows: First, prepare electrocompetent cells of XAB-2 carrying the pUDP004 plasmid. The preparation method is as follows: Culture fresh yeast liquid to make competent cells. According to an inoculation amount of 1%, inoculate 100 μl of the seed liquid into 10 ml of the culture medium and collect the bacteria: Take 1.5 ml of the culture and dispense it into 1.5 ml EP tubes. Centrifuge at 13,000 rpm for 1 min, discard the supernatant, and aspirate it clean with a pipette. Wash the precipitate with 1 ml of sterile water, pipette and centrifuge, discard the supernatant, and wash twice. Bacterial treatment: Add 1 ml of SOB liquid medium (from the 4°C refrigerator) + 10 μl of dithiothreitol solution, and heat at 25°C for 20 min using a metal heater. Centrifuge, discard the supernatant, aspirate it completely with a pipette, add 1 ml of pre-cooled 1 M SOB liquid medium (from the 4°C refrigerator), pipette, centrifuge, and discard the supernatant. Wash twice with 1 M SOB liquid medium, aspirate and discard the supernatant, and then add 50 μl of SOB liquid medium to suspend. Add 3 μl of sg-pan6 plasmid and mix well, and then transfer 1.1 μl of DNA fragment II into a pre-cooled electroporation cuvette, and incubate on ice for 5 min. Wipe the electroporation cuvette dry, and perform electroporation at 2.7 kV. Put 1 ml of the pre-aspirated SOB liquid medium into the electroporation cuvette, mix well and then aspirate it into a new 1.5 ml EP tube. Incubate on a shaker at 30°C and 250 rpm for 40 - 60 min. Centrifuge the bacterial liquid, remove part of the supernatant, mix well, spread it on the corresponding plate, and culture it in an incubator at 30°C for 36 h. Select single colonies for PCR verification. The primers used are yz-URA3-F / yz-URA3-R (primers 11 and 12). The correct colony amplification product is a 768 bp fragment. Select one correct single colony and name it XAB-3.

[0053] Example 2: Bioinformatics structure simulation of the panS gene

[0054] The transport protein encoded by the panS gene mainly has three domains, namely the N-terminal domain, the C-terminal domain, and the catalytic domain SBF. Among them, using R-pantothenic acid as the substrate, molecular docking with the SBF domain of panS was performed in AutoDock Vina to observe their interactions. As Figure 1 shown, R-pantothenic acid formed hydrogen bonds with the residues at positions G105, T106, A107, S108, S283, and S260 of panS. The above residues are the key residues of the active pocket. The inventor of the present invention conducted a series of structural studies on the panS gene and found that mutations at the three sites of T106, S260, and S283 are closely related to the activity of the panS gene.

[0055] Using the panS gene as a template, homologous sequences were obtained through BLAST in NCBI, and multiple sequence alignments were performed using Muscle in MEGA11. The Seqlogo was analyzed, and it was determined whether the above residues were non-fully conserved residues in the Seqlogo. If so, site-directed mutagenesis could be performed according to the Seqlogo. Seven variants, namely panS*T106I, panS*S283G, panS*S260A, panS*T106I-S283G, panS*T106I-S260A, panS*S260A-S283G, and panS*T106I-S260A-S283G, were obtained.

[0056] Example 3: Overexpression of the panS mutant gene

[0057] Construct recombinant plasmids containing panS mutations. The panS genes carried on the plasmids respectively contain mutations of panS*T106I (the amino acid sequence is shown in Sequence 9 of the sequence listing), panS*S283G (the amino acid sequence is shown in Sequence 10 of the sequence listing), panS*S260A (the amino acid sequence is shown in Sequence 11 of the sequence listing), panS*T106I-S283G (the amino acid sequence is shown in Sequence 12 of the sequence listing), panS*T106I-S260A (the amino acid sequence is shown in Sequence 13 of the sequence listing), panS*S260A-S283G (the amino acid sequence is shown in Sequence 14 of the sequence listing), and panS*T106I-S260A-S283G (the amino acid sequence is shown in Sequence 15 of the sequence listing). The above plasmids were introduced into Saccharomyces cerevisiae XAB-3 to overexpress the mutant panS gene. The specific steps are as follows:

[0058] The pYes2.0 plasmids each carrying the above panS mutant genes and the wild-type panS gene (the nucleotide and amino acid sequences of the wild-type panS gene are shown in Sequences 7 and 8 of the sequence listing respectively) were electrotransformed into XAB-3.

[0059] The conditions for electrotransformation are as follows: First, prepare electrocompetent cells of XAB-3. The preparation method is as follows: Culture fresh yeast liquid to make competent cells. According to an inoculation amount of 1%, inoculate 100 μl of seed liquid into 10 ml of medium to collect the thalli: Take 1.5 ml of the culture and dispense it into a 1.5 ml EP tube. Centrifuge at 13000 rpm for 1 min, discard the supernatant, and aspirate it clean with a pipette. Wash the precipitate with 1 ml of sterile water, pipette and centrifuge, discard the supernatant, and wash twice. Thallus treatment: Add 1 ml of SOB liquid medium (from the 4°C refrigerator) + 10 μl of dithiothreitol solution, and heat at 25°C for 20 min using a metal heater. Centrifuge, discard the supernatant, aspirate it completely with a pipette, add 1 ml of pre-cooled 1 M SOB liquid medium (from the 4°C refrigerator), pipette, centrifuge, and discard the supernatant. Wash twice with 1 M SOB liquid medium, aspirate and discard the supernatant, and then add 50 μl of SOB liquid medium to suspend. Add 3 μl of the plasmid with pYes2.0, mix well, and transfer it into a pre-cooled electroporation cuvette, and incubate on ice for 5 min. Wipe the electroporation cuvette dry, and perform electroporation at 2.7 kV. Put 1 ml of the pre-aspirated SOB liquid medium into the electroporation cuvette, mix well, and aspirate it into a new 1.5 ml EP tube. Incubate on a shaker at 30°C and 250 rpm for 40 - 60 min. Centrifuge the bacterial liquid, remove part of the supernatant, mix well, spread it on the corresponding plate, and culture it in an incubator at 30°C for 36 h, and pick the transformants. The transformant strains and their corresponding panS genotypes are shown in the following table:

[0060] Table 3: Transformed strains containing the panS gene and their panS genotypes

[0061] Strain panS gene mutation XAB-4 panS*T106I XAB-5 panS*S283G XAB-6 panS*S260A XAB-7 panS*T106I-S283G XAB-8 panS*T106I-S260A XAB-9 panS*T106I-S260A-S283G XAB-10 panS*S260A-S283G XAB-11 panS

[0062] Example 3: Fermentative production of R-pantothenic acid using recombinant strains

[0063] Pick monoclonal colonies of XAB-4, XAB-5, XAB-6, XAB-7, XAB-8, XAB-9, XAB-10, and XAB-11 on the plate and inoculate them into 5 mL of SD-Ura liquid medium (purchased from Beijing PanGenotech Co., Ltd., used as the fermentation medium), and culture overnight on a shaker at 30°C. Wait until the OD of the seed liquid 600 grows to 2.0, inoculate 150 μl of the seed liquid into a 100 mL Erlenmeyer flask containing 15 mL of SD-Ura liquid medium, and culture with shaking at 30°C and 250 rpm for 6 days. Centrifuge the fermentation broths of XAB-4, XAB-5, XAB-6, XAB-7, XAB-8, XAB-9, XAB-10, and XAB-11 at 12000 r / min for 5 min, take 1 mL of the supernatant, dilute it according to a certain ratio so that the detected value of the content of R-pantothenic acid is within the detection range of the instrument; after filtering through a sterile filter membrane, the concentration of R-pantothenic acid is detected using HPLC.

[0064] HPLC detection conditions: The instrument is Primaide from Hitachi, Japan. The chromatographic column model is Eclipse XD8-C18 (5μm, 4.6mm×250mm). The column temperature is 30°C. The detector is an ultraviolet detector with a detection wavelength of 210nm. The volume ratio of the mobile phase is A (phosphoric acid): B (acetonitrile): C (water) = 1:50:950, the flow rate is 1mL / min, and the injection volume is 10μL. The detection results are shown in the following table.

[0065] Table 4: Comparison of the production capacity of R-pantothenic acid strains

[0066]

[0067]

[0068] As presented in Table 4, compared with the control group XAB-11, XAB-8, XAB-9, and XAB-10 have increased R-pantothenic acid yields. In particular, the production capacity of the XAB-10 (panS*S260A-S283G) strain is 5.72 times higher than that of the XAB-11 (wild-type panS) strain for R-pantothenic acid. Experimental data show that the R-pantothenic acid yield of XAB-10 reaches 806μg / L, and the panS activity is significantly improved, which can transport R-pantothenic acid extracellularly, consistent with the results of molecular docking analysis.

[0069] The strain XAB-10 was deposited with the patent deposit on December 5, 2022, with the deposit number CCTCC NO: M 20221859, and the taxonomic name is Saccharomyces cerevisiae XAB-10. The depository is the China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, Hubei, China.

[0070] The above are only the preferred embodiments of the present invention. The protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the concept of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and refinements made without departing from the principle of the present invention should be regarded as within the protection scope of the present invention.

Claims

1. A panS variant, characterized in that: Based on the amino acid sequence shown in Sequence Listing Sequence 8, the following mutations are made: T106I and / or S283G.

2. A nucleic acid molecule encoding the panS variant according to claim 1.

3. A recombinant vector containing the nucleic acid according to claim 2.

4. Use of the panS variant according to claim 1, the nucleic acid molecule according to claim 2, or the recombinant vector according to claim 3 in the preparation of Saccharomyces cerevisiae producing R-pantothenic acid.

5. A method for increasing the yield of R-pantothenic acid, characterized in that, Comprising the following steps: Expressing the encoding gene of the panS mutant according to claim 1 in Saccharomyces cerevisiae XAB-3 to obtain recombinant Saccharomyces cerevisiae; Using the recombinant Saccharomyces cerevisiae to ferment and produce R-pantothenic acid; Wherein, the Saccharomyces cerevisiae XAB-3 is obtained by overexpressing the ECM31 gene, PAN5 gene and PAN6 gene in Saccharomyces cerevisiae BY4741; the nucleotide sequence of the ECM31 gene is shown in Sequence Listing Sequence 1, the nucleotide sequence of the PAN5 gene is shown in Sequence Listing Sequence 2, and the nucleotide sequence of the PAN6 gene is shown in Sequence Listing Sequence 3.

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