Conjugated polyketide reductase mutant, co-expression engineering bacteria and application

Abstract

The application discloses a conjugated polyketide reductase mutant, which is obtained by single-point mutation or multi-point combined mutation of the 64th or 206th site of the amino acid sequence shown in SEQ ID NO. 1. The mutant of the conjugated polyketide reductase can improve the enzyme activity by more than 25% compared with the wild type. The application also provides a coding gene, a recombinant vector containing the coding gene, a co-expression engineering bacterium and application. The conjugated polyketide reductase mutant, glucose dehydrogenase and L-panthenolide dehydrogenase are used to construct a three-enzyme co-expression engineering bacterium as a biological catalyst to catalyze the synthesis of optically pure D-panthenolide from racemic panthenolide, improve the catalytic efficiency of multi-enzyme cascade reaction, reduce the hydrolysis loss of intermediate product KPL, and significantly improve the yield of DPL. The yield of the obtained DPL can be up to 98%, and the optical purity can be up to 99.9%.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a conjugated polyketide reductase mutant, its encoding gene, recombinant vector, and engineered bacteria for co-expression of the three enzymes, as well as their applications. Background Technology

[0002] D-Pantothenic acid (D-PA), molecular formula C9H 17 NO5, also known as vitamin B5, is a water-soluble vitamin and an essential vitamin for humans and animals. It can be further converted into coenzyme A (CoA) in plants, animals, and microorganisms, further participating in energy metabolism and thus widely used in food, feed, and medicine. D-pantothenic acid can be synthesized through chemical methods, enzymatic methods, microbial fermentation, and biological enzymatic methods. D-pantothenic acid lactone (DPL) is a key chiral intermediate in the chemical or enzymatic synthesis of D-pantothenic acid; therefore, constructing a synthetic pathway for DPL is of great significance for the synthesis of D-pantothenic acid.

[0003] The main methods for synthesizing DPL include chemical and enzymatic methods. The chemical method uses isobutyraldehyde and formaldehyde as substrates, and cyanide as a nucleophilic addition reagent. First, acidification followed by cyclization synthesizes D,L-pantolactone (DL-PL), then a chemical resolving agent separates DL-PL into DPL and L-pantolactone (LPL). While this method is mature, it suffers from drawbacks such as high energy consumption, frequent use of acids and alkalis, and complex processes. The enzymatic method uses hydrolases or reductases instead of chemical resolving agents to separate DL-PL into DPL and LPL. The enzymatic resolution method includes two pathways: one uses L-pantolactone hydrolases to specifically hydrolyze the L-type substrate in DL-PL to obtain DPL and L-pantolactone. The L-pantolactone is then recovered and esterified to regenerate DL-PL. Another method involves the specific hydrolysis of the D-type substrate in DL-PL by D-pantolactone hydrolase, yielding D-pantolactone and LPL. The separated D-pantolactone is then acidified and cyclized to form DPL, while LPL is racemicized back to DL-PL. Compared to L-pantolactone hydrolase, D-pantolactone hydrolase-catalyzed resolution yields DPL with high optical purity, thus making it more widely used in industrial applications. However, the enzymatic resolution method also has drawbacks such as complex steps and the use of multiple acids and bases.

[0004] The oxidoreductase resolution method utilizes L-pantolactone dehydrogenase (LPLDH) to convert LPL in DL-PL to pantolactone (KPL), which is then asymmetricly reduced to DPL using conjugated polyketide reductase (CPR). Simultaneously, glucose dehydrogenase (GDH) drives coenzyme regeneration in the presence of glucose. Compared to the hydrolytic enzyme resolution route, the oxidoreductase method eliminates the racemic step and the separation of lactone and acid, resulting in a higher theoretical yield and greater potential for industrial production.

[0005] Conjugated polyketide reductases are key enzymes in the asymmetric synthesis of diphenyl ketone (DPL) via oxidoreductase processes. If the activity of conjugated polyketide reductases is insufficient for the multi-enzyme cascade catalytic reaction, the intermediate product KPL will be lost due to hydrolysis, reducing the catalytic efficiency of the multi-enzyme cascade reaction and increasing the difficulty of subsequent product separation and purification. Therefore, improving the catalytic efficiency of conjugated polyketide reductases through molecular modification is of great significance. For example, Zhang et al. mutated amino acids 126 and 215 of the conjugated polyketide reductase CviCPR from Candida viswanathii, and the mutant exhibited high activity and high stereoselectivity in catalyzing the preparation of DPL from KPL (CN202210519820.6); Liu et al. mutated amino acids 29, 65, and 215 of the conjugated polyketide reductase KbCPR from Kazachstania barnettii, increasing the enzyme activity by 7.43 times. The engineered bacteria co-expressed with GDH using this mutant showed good catalytic activity (CN202311035098.X). Summary of the Invention

[0006] To address the problem of insufficient catalytic efficiency of conjugated polyketide reductase in the L-pantolactone dehydrogenase-conjugated polyketide reductase-glucose dehydrogenase three-enzyme cascade catalytic DL-PL resolution reaction, which fails to rapidly convert easily hydrolyzed intermediates into products, this invention provides a conjugated polyketide reductase modification strategy. This strategy yields several conjugated polyketide reductase mutants with enhanced activity, including the single mutant CorCPR. M1 (A64M) showed a 25% increase in enzyme activity, and the double mutant CorCPR M2 The enzyme activity of (A64M / S206C) was increased by 30%.

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

[0008] The conjugated polyketide reductase mutant is obtained by single-point or multi-point combined mutation at position 64 or 206 of the amino acid sequence shown in SEQ ID NO.1. The conjugated polyketide reductase with the amino acid sequence shown in SEQ ID NO.1 is derived from Candida orthopsilosis.

[0009] The mutation is one or a combination of two of the following:

[0010] (1) The alanine at position 64 is mutated to methionine (A64M);

[0011] (2) Serine at position 206 is mutated to cysteine ​​(S206C);

[0012] Furthermore, the mutant is preferably one of the following:

[0013] (1) Mutate alanine at position 64 of the amino acid sequence shown in SEQ ID NO.1 to methionine (A64M), and the amino acid sequence is shown in SEQ ID NO.3;

[0014] (2) Mutate alanine at position 64 of the amino acid sequence shown in SEQ ID NO.1 to methionine (A64M) and serine at position 206 to cysteine ​​(S206C), and the amino acid sequence is shown in SEQ ID NO.5.

[0015] Due to the specificity of amino acid sequences, any fragment or variant of a peptide protein containing the amino acid sequence shown in this invention, such as its conserved variants, bioactive fragments, or derivatives, is within the scope of protection of this invention, provided that the fragment or variant shares more than 90% homology with the aforementioned amino acid sequence. Specifically, the alterations include the deletion, insertion, or substitution of amino acids in the amino acid sequence; wherein, for conserved alterations of variants, the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine, or asparagine with glutamine; enzyme variants may also have non-conserved alterations.

[0016] The present invention also provides the encoding gene of the conjugated polyketide reductase mutant.

[0017] Specifically, the nucleotide sequence of the gene corresponding to the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.2.

[0018] The nucleotide sequence of the gene encoding the mutant in which alanine at position 64 is mutated to methionine (A64M) is shown in SEQ ID NO.4.

[0019] The nucleotide sequence of the gene encoding the mutant with alanine at position 64 mutated to methionine (A64M) and serine at position 206 mutated to cysteine ​​(S206C) is shown in SEQ ID NO.6.

[0020] The conjugated polyketide reductase and its mutant described in this invention both have a full-length base sequence of 948 bp, from the first base to the 948th base, with the start codon being ATG and the stop codon being TAA.

[0021] Due to the specific nature of nucleotide sequences, any variant of the polynucleotides described in this invention, provided that it shares more than 90% homology with the aforementioned polynucleotides, falls within the scope of protection of this invention. A variant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide alterations. This polynucleotide variant can be a live or non-live variant, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, but does not substantially alter the function of the peptide protein it encodes.

[0022] The conjugated polyketide reductase mutant of this invention is produced by site-directed mutagenesis and combinatorial mutagenesis to mutate the CorCPR gene. The resulting mutant plasmid is then transformed into E. coli BL21(DE3) competent cells via heat shock. The obtained strain is then inoculated, transferred, induced, and the cells are recovered. The resuspended bacterial solution is used to catalyze KPL. The specific method is as follows: First, the amino acid sequence of CorCPR is combined with that of the mutant KmAKR, whose enzyme activity has been enhanced from the same protein family. M13 KbCPR M3 and SceCPR M2 Multiple sequence alignment was performed. Based on sequence alignment and sequence consistency analysis, seven amino acid mutations (F301W / F302H, Q33H, A64M, N114K, S206C, and E272D) that may affect CorCPR enzyme activity were identified. Site-directed mutagenesis was performed at these sites using pET28b-CorCPR as a template plasmid. Enzyme activity was detected by gas chromatography, and single mutants with increased activity were selected. Furthermore, the dominant single mutants were combined with double mutants, and enzyme activity was measured. Finally, a single mutant CorCPR with 25% increased activity compared to CorCPR was obtained. M1 (A64M) and the double mutant CorCPR, which increased by 30%. M2 (A64M / S206C)

[0023] The present invention also provides a recombinant vector containing the coding gene of the conjugated polyketide reductase mutant and an expression host.

[0024] Preferably, the plasmid vector of the recombinant vector is pET28b(+), and the expression host is Escherichia coli BL21(DE3).

[0025] The present invention also provides the application of the conjugated polyketide reductase mutant in the asymmetric reduction of ketopanolactone by microorganisms to synthesize optically pure D-opanolactone.

[0026] The conjugated polyketide reductase mutant catalyst can be obtained by the following method: culturing recombinant genetically engineered bacteria containing the encoding gene of the conjugated polyketide reductase mutant, inducing the expression of the conjugated polyketide reductase mutant, and centrifuging the resulting culture medium to obtain the whole-cell catalyst of the conjugated polyketide reductase mutant.

[0027] This invention also provides a recombinant three-enzyme genetically engineered bacterium containing the coding gene of the aforementioned conjugated polyketide reductase mutant. The expression host of the recombinant three-enzyme genetically engineered bacterium is typically *Escherichia coli* BL21(DE3). The expression vector of the recombinant three-enzyme genetically engineered bacterium is pCDFDuet1, pRSFDuet1, or pACYCDuet1.

[0028] Furthermore, the recombinant three-enzyme genetically engineered bacteria containing the coding gene of the conjugated polyketide reductase mutant is a three-enzyme co-expression engineered bacteria that simultaneously expresses the conjugated polyketide reductase mutant, glucose dehydrogenase, and L-pantolactone dehydrogenase.

[0029] The engineered bacteria that co-express the three enzymes can also express molecular chaperones.

[0030] The recombinant three-enzyme genetically engineered bacteria containing the coding gene of the conjugated polyketide reductase mutant can also be molecular chaperone three-enzyme co-expression bacteria that simultaneously express molecular chaperone pGro7, the conjugated polyketide reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase.

[0031] The glucose dehydrogenase is a glucose dehydrogenase derived from Bacillus megaterium or a mutant thereof, and the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO.7. The glucose dehydrogenase mutant is obtained by mutating valine at position 72 to isoleucine, serine at position 100 to proline, lysine at position 137 to arginine, lysine at position 166 to arginine, glutamic acid at position 170 to lysine, and glutamine at position 252 to leucine, and the amino acid sequence is shown in SEQ ID NO.8.

[0032] The L-pantolactone dehydrogenase is derived from Rhodococcus hoagii or a mutant thereof, and the amino acid sequence of the L-pantolactone dehydrogenase is shown in SEQ ID NO. 9. The L-pantolactone dehydrogenase mutant is obtained by mutating isoleucine at position 156 to leucine, asparagine at position 164 to lysine, phenylalanine at position 224 to glutamine, valine at position 241 to isoleucine, and leucine at position 254 to isoleucine in the amino acid sequence shown in SEQ ID NO. 9, and the amino acid sequence is shown in SEQ ID NO. 10.

[0033] The engineered bacteria expressing the three enzymes contain the coding genes for the conjugated polyketide reductase mutant, glucose dehydrogenase, and L-pantolactone dehydrogenase.

[0034] The engineered bacteria that co-expressed the three enzymes were induced to simultaneously express a conjugated polyketide reductase mutant, glucose dehydrogenase, and L-pantolytic acid lactone dehydrogenase.

[0035] Furthermore, the engineered bacteria for the co-expression of the three enzymes can be constructed as follows:

[0036] A recombinant vector containing the encoding gene of L-pantolactone dehydrogenase was constructed and transformed into a host bacterium to obtain a recombinant genetically engineered bacterium containing the encoding gene of L-pantolactone dehydrogenase. A recombinant vector containing the encoding genes of glucose dehydrogenase and conjugated polyketide reductase mutants was constructed and transformed into a recombinant genetically engineered bacterium containing the encoding gene of L-pantolactone dehydrogenase to obtain a three-enzyme co-expression engineered bacterium.

[0037] Furthermore, engineered bacteria co-expressing molecular chaperones and three enzymes can be constructed using the following method:

[0038] A recombinant vector containing the encoding gene of L-pantolactone dehydrogenase and a recombinant vector containing the encoding genes of glucose dehydrogenase and conjugated polyketide reductase mutants were co-transformed into E.coli BL21(DE3) / pGro7 competent cells containing a molecular chaperone plasmid to construct an engineered bacterium that co-expresses molecular chaperone and three enzymes.

[0039] Furthermore, among the preferred engineered bacteria that co-express the three enzymes or the engineered bacteria that co-express the three enzymes with molecular chaperones, the amino acid sequence of the conjugated polyketide reductase mutant is shown in SEQ ID NO.5; the amino acid sequence of L-pantolactone dehydrogenase is shown in SEQ ID NO.10; and the amino acid sequence of glucose dehydrogenase is shown in SEQ ID NO.8.

[0040] Furthermore, the engineered bacteria for the co-expression of the three enzymes were constructed using the following method:

[0041] The recombinant vector containing the encoding gene of the aforementioned L-pantolactone dehydrogenase is pACYCDuet-RhoLPLDH M5 The mixture was transformed into the host bacterium E. coli BL21(DE3) to obtain E. coli BL21(DE3) / pACYCDuet-RhoLPLDH. M5 ;pACYCDuet-RhoLPLDH M5 It is the L-pantolactone dehydrogenase mutant RhoLPLDH with the amino acid sequence shown in SEQ ID NO.10.M5 The encoding gene was obtained by ligating it into the pACYCDuet plasmid vector;

[0042] The glucose dehydrogenase mutant BmGDH with the amino acid sequence shown in SEQ ID NO. 8 was used. M6 and the conjugated polyketide reductase mutant CorCPR with the amino acid sequence shown in SEQ ID NO.5 M2 The coding genes were inserted into the multiple cloning sites I and II of the pRSFDuet1 dual expression vector, respectively, to construct a recombinant vector containing the coding genes for glucose dehydrogenase and conjugated polyketide reductase mutants, denoted as .

[0043] pRSFDuet-BmGDH M6 -CorCPR M2 Transformed to E. coli BL21(DE3) / pACYCDuet-RhoLPLDH M5 In this study, the engineered strain E. coli BL21(DE3) / , which co-expresses three enzymes, was obtained.

[0044] pACYCDuet-RhoLPLDH M5 / pRSFDuet-BmGDH M6 -CorCPR M2 This is denoted as DPA2.

[0045] Furthermore, the engineered bacteria co-expressing molecular chaperones and three enzymes were constructed using the following method:

[0046] The amino acid sequence of the L-indohydrin dehydrogenase mutant RhoLPLDH, as shown in SEQ ID NO.10, was used. M5 The encoding gene was ligated into the pCDFDuet plasmid vector to obtain the recombinant vector pCDFDuet-RhoLPLDH containing the encoding gene of the L-pantolactone dehydrogenase. M5 ;

[0047] The glucose dehydrogenase mutant BmGDH with the amino acid sequence shown in SEQ ID NO. 8 was used. M6 and the conjugated polyketide reductase mutant CorCPR with the amino acid sequence shown in SEQ ID NO.5 M2 The coding genes were inserted into the multiple cloning sites I and II of the pRSFDuet1 dual expression vector, respectively, to construct a recombinant vector containing the coding genes for glucose dehydrogenase and conjugated polyketide reductase mutants, denoted as .

[0048] pRSFDuet-BmGDH M6 -CorCPR M2pCDFDuet-RhoLPLDH M5 and

[0049] pRSFDuet-BmGDH M6 -CorCPR M2 The cells were co-transformed into E. coli BL21(DE3) / pGro7 competent cells containing a molecular chaperone plasmid to construct the engineered strain E. coli BL21(DE3) / pCDFDuet-RhoLPLDH, which co-expresses the molecular chaperone and three enzymes. M5 / pRSFDuet-BmGDH M6 -CorCPR M2 / pGro7, denoted as DPCA4.

[0050] The present invention also provides the application of conjugated polyketide reductase mutants or recombinant three-enzyme genetically engineered bacteria containing the coding gene of the conjugated polyketide reductase mutants in the microbial catalytic synthesis of optically pure D-indomethacin from racemic pantothenic acid lactones.

[0051] The reaction formula is shown below:

[0052]

[0053] Furthermore, the application method is as follows: wet bacterial cells obtained by fermentation culture of recombinant three-enzyme genetically engineered bacteria containing the encoding gene of the conjugated polyketide reductase mutant are used as catalysts; racemic pantothenic acid lactone is used as the reaction substrate; glucose is used as the co-substrate; and NADP coenzyme is used. + Under the action of [unclear], a transformation system is constructed using PB buffer as the reaction medium to prepare D-pantolactone; the recombinant three-enzyme genetic engineered bacteria containing the encoding gene of the conjugated polyketide reductase mutant is a three-enzyme co-expression engineered bacteria that simultaneously expresses the conjugated polyketide reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase, or a molecular chaperone three-enzyme co-expression engineered bacteria that simultaneously expresses the molecular chaperone pGro7, the conjugated polyketide reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase.

[0054] Furthermore, the pH value of the PB buffer solution is 6.0–8.0, preferably 7.0. The concentration of the PB buffer solution is 50–100 mM, preferably 50 mM.

[0055] Furthermore, the reaction temperature is 30–40°C (preferably 30°C), and the stirring speed is 600–800 rpm (preferably 800 rpm).

[0056] Furthermore, in the transformation system, the concentration of the racemic pantothenic acid lactone substrate is 500–1000 mM; the glucose concentration is 350–2000 mM; the catalyst dosage is 100–250 g WCW / L (wCW cell wet weight); and the coenzyme NADP... + The concentration is 50–100 mM.

[0057] Furthermore, glucose is preferably added to the conversion system in batches, with 40-60% (preferably 50%) of the total glucose initially added, 25-35% of the total glucose added after 8 hours of reaction, and the remaining glucose added after 16 hours of reaction, or 60-70% of the remaining glucose added after 16 hours of reaction, and the remaining glucose added after 24 hours of reaction.

[0058] Further, the wet cells obtained by fermentation culture of the recombinant genetically engineered bacteria containing the coding gene of the conjugated polyketide reductase mutant are prepared as follows: A three-enzyme co-expression engineered bacterium simultaneously expressing the conjugated polyketide reductase mutant, glucose dehydrogenase, and L-pantolactone dehydrogenase, or a three-enzyme co-expression engineered bacterium simultaneously expressing the molecular chaperone pGro7, conjugated polyketide reductase mutant, glucose dehydrogenase, and L-pantolactone dehydrogenase, is inoculated into LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured at 37°C for 10–12 h to obtain a seed culture; The seed culture was inoculated at a volume concentration of 1% into fresh LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, and cultured at 37℃ and 180 rpm until OD600 = 0.6-0.8. Isopropyl β-D-thiogalactoside (IPTG) was added to the culture medium to a final concentration of 0.1-0.3 mM, and cultured at 24℃ for 12 h. After centrifugation at 4℃ and 8000 rpm for 10-15 min, wet cells expressing L-pantolactone dehydrogenase, conjugated polyketide reductase, and glucose dehydrogenase were obtained.

[0059] The present invention relates to the inoculation, transfer, induction, and cell recovery of genetically engineered bacteria. The culture medium can be any medium in the art that can promote bacterial growth and produce the present invention, preferably LB medium, with the following formulation: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in distilled water, and adjusted to pH 7.0. There are no special limitations on the culture method and conditions, which can be optimized according to factors such as host type and culture method.

[0060] In this invention, the engineered bacterium E. coli BL21(DE3) / pACYCDuet-RhoLPLDH was co-expressed through screening. M5 / pRSFDuet-BmGDH M6-CorCPR M2 (denoted as DPA2) and E. coli BL21(DE3) / pCDFDuet-RhoLPLDH M5 / pRSFDuet-BmGDH M6 -CorCPR M2 / pGro7 (denoted as DPCA4) catalyzes the highest DPL production yield and also exhibits high catalytic conversion rates for high-concentration substrates (up to 1000 mM), exceeding current levels.

[0061] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0062] (1) This invention provides a single mutant of conjugated polyketide reductase CorCPR with enhanced catalytic activity. M1 (A64M), double mutant CorCPR M2 (A64M / S206C), the mutant enzyme activity was increased by 25% and 30% compared to the wild type, respectively.

[0063] (2) A three-enzyme co-expression engineered bacterium was constructed using a conjugated polyketide reductase mutant, glucose dehydrogenase, and L-pantolactone dehydrogenase. The wet bacterial cells were used as a biocatalyst for whole-cell catalysis. The three enzymes can be expressed simultaneously and efficiently in a single recombinant bacterium. The recombinant bacterium can be cultured in large quantities without the need for cell disruption, enzyme purification, or other processing steps, resulting in lower costs and simpler operation.

[0064] (3) This invention uses wet bacterial cells as a biocatalyst to catalyze the resolution of DL-PL to synthesize DPL in a one-pot process. The substrate is a racemic pantothenic acid lactone, in which LPL is catalyzed to convert to KPL, and then DPL is obtained. By improving the catalytic efficiency of the multi-enzyme cascade reaction and reducing the hydrolysis loss of the intermediate product KPL, the yield of DPL is significantly improved. Moreover, the co-expressed engineered bacteria of this invention can catalyze the high-conversion rate of high-concentration substrates, significantly improving production efficiency. In this invention, the co-expressed engineered bacteria DPA2 catalyzes the resolution of 500mM and 1000mM DL-PL, obtaining DPL yields of 98.4% (64.0 g / L) and 91.3% (118.7 g / L), respectively. The co-expressed engineered bacteria DPCA4 catalyzes the resolution of 500mM and 1000mM DL-PL, respectively, with DPL yields of 97.6% and 88.6%, respectively. Attached Figure Description

[0065] Figure 1 Figure showing the alignment analysis of the CorCPR sequence with other mutant sequences.

[0066] Figure 2 Comparison of relative enzyme activities of seven CorCPR mutants.

[0067] Figure 3 Graph showing the DL-PL resolution reaction process catalyzed by different co-expressed engineered bacteria.

[0068] Figure 4 Graph of the resolution reaction catalyzed by co-expressed engineered bacteria with 500mM DL-PL.

[0069] Figure 5 Flowchart of the reaction process of co-expressed engineered bacteria undergoing glucose-catalyzed resolution at 500 and 1000 mM DL-PL. Detailed Implementation

[0070] 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:

[0071] Example 1: Sequence alignment for screening mutation sites

[0072] Sequence comparison is a common method for rapidly obtaining beneficial mutations. KmAKR, derived from Kluyveromyces marxianus, is an aldehyde-ketone reductase. Studies have shown that the KmAKR mutant KmAKR... M13 (W297H / Y296W / K29H / Y28A / T63M / A30P / K164E / T302S / N109K / S196C / S232A / S182H / Q266D) overcomes the trade-off between enzyme activity and stability, achieving co-evolution and improving catalytic efficiency. Additionally, literature reports KbCPR... M3 (Y29K / R65N / K215R) and SceCPR M2 (S157A / Y298H) enhances the catalytic activity of the substrate KPL. To further improve the catalytic efficiency of CorCPR, CorCPR derived from Candida orthopsilosis was combined with the mutant KbCPR. M3 SceCPR M2 KmAKR M13 Perform multiple sequence alignment (see) Figure 1 Based on sequence alignment and sequence identity analysis, seven amino acid mutations that may affect CorCPR enzyme activity were selected (F301W / F302H, Q33H, A64M, N114K, S206C, and E272D).

[0073] Example 2: Construction and screening of conjugated polyketide reductase mutants

[0074] 1. Starting strain:

[0075] The CorCPR coding gene was inserted into the Nde I / Xho I restriction site of plasmid pET28b to construct the recombinant plasmid pET28b-CorCPR, which was then transformed into BL21(DE3) competent cells as the original strain. The CorCPR amino acid sequence is shown in SEQ ID NO.1, and the coding gene sequence is shown in SEQ ID NO.2.

[0076] 2. Single mutation:

[0077] The CorCPR mutant was prepared through site-directed mutagenesis. Using CorCPR as a template, mutant primers were designed, and polymerase chain reaction (PCR) was performed. The PCR product, digested with DpnI enzyme, was introduced into E. coli BL21(DE3) competent cells via heat shock and plated on Kan-resistant LB agar plates, incubated at 37°C for 12-16 h. Once a single colony grew, it was picked and incubated overnight in 10 mL of LB liquid medium (Kan). The bacterial culture was then preserved with 30% glycerol for later use and sequenced for verification.

[0078] The glycerol-preserved mutant strain and the original strain were inoculated into 10 mL of LB resistant medium and cultured for 12-16 h to obtain seed culture. They were then inoculated into 100 mL of LB shake flask medium (Kan resistant) at a 1% inoculation rate and induced to obtain wet cells. An appropriate amount of cells was weighed and resuspended in PB (pH 7.0) buffer to prepare a 5 g / L cell suspension. The suspension was sonicated at 20% power and centrifuged at 12,000 rpm for 10 min. The supernatant was collected as crude enzyme solution.

[0079] Determination of conjugated polyketide reductase activity (1 mL reaction system): 5 g / L crude enzyme solution, 10 mM KPL, 15 mM NADPH, 50 mM PB (pH 7.0) buffer. Measure absorbance over 3 minutes at room temperature and 340 nm. The absorbance decrease / OD value is recorded. 600 The higher the value, the higher the enzyme activity. Using the original strain as the control group, mutants with values ​​higher than the control group are considered dominant mutants. The relative enzyme activities of the seven mutants are as follows: Figure 2 As shown.

[0080] 3. Combinatorial mutation

[0081] The mutation sites in the previous round of mutations that were higher than the control group were subjected to dual-site combination mutations. Using a single-mutant plasmid as a template, new mutation sites were added, and double mutants were obtained through PCR, transformation, and sequencing. The original strain was used as a control to screen for mutants with enhanced enzyme activity. The mutant enzyme activity was as follows: Figure 2 As shown, a mutant with increased enzyme activity, CorCPR, was obtained. M1 (A64M), CorCPR M2(A64M / S206C), the mutant enzyme activity was increased by 25% and 30% compared with the wild type, respectively.

[0082] Example 3: Construction and Induction of Three-Enzyme Co-expression Engineered Bacteria

[0083] The amino acid sequence is shown in SEQ ID NO.10. RhoLPLDH M5 The coding gene was inserted into the Nde I / Xho I restriction site of the multiple cloning site 2 (MCS2) of plasmid pACYCDuet to construct plasmid pACYCDuet-RhoLPLDH. M5 Transformed into E. coli BL21(DE3) competent cells, plated overnight, and single colonies were selected to prepare E. coli BL21(DE3) / pACYCDuet-RhoLPLDH. M5 Competent cells. Utilizing glucose dehydrogenase (BmGDH, BmGDH) M6 ), conjugated polyketide reductase (CorCPR, CorCPR) M1 CorCPR M2 The coding gene of pRSFDuet1 was inserted into the restriction enzyme sites (Nco I / Not I) of multiple cloning site I and (Nde I / Xho I) of multiple cloning site II of the pRSFDuet1 dual expression vector to construct the recombinant plasmid pRSFDuet-BmGDH. M6 -CorCPR M2 pRSFDuet-BmGDH M6 -CorCPR M1、 pRSFDuet-BmGDH-CorCPR and pRSFDuet-BmGDH M6 -CorCPR. The amino acid sequence of BmGDH is shown in SEQ ID NO.7. M6 The amino acid sequence is shown in SEQ ID NO. 8, and the conjugated polyketide reductase CorCPR M1 The amino acid sequence is shown in SEQ ID NO.3, and the encoding gene sequence is shown in SEQ ID NO.4, CorCPR M2 The amino acid sequence is shown in SEQ ID NO.5, and the encoding gene sequence is shown in SEQ ID NO.6.

[0084] The newly constructed recombinant plasmids were then transformed into E. coli BL21(DE3) / pACYCDuet-RhoLPLDH. M5 Competent cells were plated on kanamycin and chloramphenicol-treated plates, incubated overnight, and single colonies were picked for verification. Additionally, the newly constructed recombinant plasmid pRSFDuet-BmGDH was separately...M6 -CorCPR M2 pRSFDuet-BmGDH M6 -CorCPR M1、 pRSFDuet-BmGDH-CorCPR and pRSFDuet-BmGDH M6 -CorCPR and plasmid pCDFDuet-RhoLPLDH M5 The enzymes were co-transformed into E. coli BL21(DE3) / pGro7 competent cells and plated on triple-resistance plates containing streptomycin, kanamycin, and chloramphenicol. After overnight incubation, single colonies were picked for verification. The resulting strains, DPA1–DPA4, co-expressing the three enzymes, and DPCA1–DPCA4, co-expressing the three enzymes and molecular chaperone, are shown in Table 1.

[0085] The co-expressed engineered bacteria were inoculated into 10 mL LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol (for the molecular chaperone co-expressed engineered bacteria, an additional 50 μg / mL streptomycin was added), and cultured at 37°C and 180 rpm for 12 h to obtain seed culture. The seed culture was then inoculated at a 1.0% inoculation rate into 100 mL LB liquid medium shake flasks containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol (for the molecular chaperone co-expressed engineered bacteria, an additional 50 μg / mL streptomycin was added), and cultured at 37°C and 180 rpm until OD (dose elapsed). 600 The concentration was set between 0.6 and 0.8. Then, IPTG was added to the culture medium to a final concentration of 0.3 mM. After incubation at 24°C for 12 h, the culture was centrifuged at 4°C and 8000 rpm for 10 min to obtain wet bacterial cells.

[0086] Table 1. Strains and plasmids co-expressing the three enzymes

[0087]

[0088] Example 4: Catalytic verification by engineered bacteria co-expressing three enzymes

[0089] The conversion efficiency of whole-cell DL-PL catalyzed by strains DPA1-DPA4 and DPCA1-DPCA4 obtained in Example 3 was determined. The reaction system was as follows: 5 mL reaction system, substrate racemic pantothenic acid lactone (DL-PL) concentration of 100 mM, NADP + The concentration of the substrate was 10 mM, the concentration of CaCO3 was 100 mM (to slow down the pH decrease), the wet cell concentration was 50 g / L, and the reaction pH was adjusted to maintain 7.0–8.0 using 2 M NaOH. The reaction temperature was 35 °C, and the magnetic stirring speed was 800 rpm. Samples were taken periodically, and the reaction was terminated with 6 M HCl solution. The samples were then extracted three times with ethyl acetate, and the concentrations of the substrate, intermediate, and product were determined by gas chromatography.

[0090] Gas chromatography detection conditions: Thermoscienfic TRACE1610 gas chromatograph, Agilent CYCLOSIL-B column (30m×0.25mm, 0.25μm), carrier gas: helium, flow rate: 1mL / min, injector and detector temperature: 250℃, injection volume: 1μL, split mode, program: initial temperature 100℃, ramp to 140℃ at a rate of 10℃ / min, hold for 5min, cool to 100℃ at a rate of 20℃ / min, detect for 15min.

[0091] The catalytic DL-PL resolution reaction process is as follows Figure 3 As shown in the figure, DPCA4 and DPA2 catalyzed the highest DPL production, at 93.5% and 90.1% respectively, with the remaining LPL production at 3.6% and 7.6% respectively. A common feature of the engineered bacteria DPCA4 and DPA2 is that they both contain the recombinant plasmid pRSFDuet-BmGDH. M6 -CorCPR M2 The difference lies in the recombinant plasmids expressing L-pantolactone, which are pACYCDuet-RhoLPLDH. M5 and pCDFDuet-RhoLPLDH M5 / pGro7. This demonstrates that DPCA4 and DPA2 are the superior engineered bacteria for the three-enzyme cascade catalytic resolution of DL-PL to synthesize DPL.

[0092] Example 5: One-pot synthesis of DPL from DL-pantolytic lactone catalyzed by engineered bacteria with three co-expression enzymes

[0093] Using whole-cell protozoa co-expressing DPA2 and DPCA4 as catalysts, a one-pot catalytic cascade reaction of DL-PL resolution was employed to synthesize the product DPL. DL-PL resolution was catalyzed with 100 g / L, 180 g / L, and 250 g / L DPA2 and 100 g / L and 250 g / L DPCA4, respectively. The reaction ratios were as follows: Reaction 1: 100 g / L DPA2; Reaction 2: 180 g / L DPA2; Reaction 3: 250 g / L DPA2; Reaction 4: 100 g / L DPCA4; Reaction 5: 250 g / L DPCA4. The reaction system consisted of 5 mL of reaction mixture, DL-PL at 500 mM concentration, glucose at 375 mM concentration, and NADP4 at a concentration of 250 g / L. + The concentration was 100 mM, and the reaction pH was 6.0–8.0 (adjusted with 2 M NaOH). The reaction temperature was 30 °C, and the stirring speed was 800 rpm. Samples were taken periodically, and the reaction was terminated with 6 M HCl solution. The mixture was then extracted three times with ethyl acetate, and the concentrations of substrate, intermediate, and product were determined by gas chromatography.

[0094] The reaction process is as follows Figure 4 As shown, the DPL yields catalyzed by 100 g / L, 180 g / L, and 250 g / L DPA2 were 88.8%, 93.9%, and 92.5%, respectively, while the DPL yields catalyzed by 100 g / L and 250 g / L DPCA4 were 92.1% and 90.1%, respectively. Higher cell concentrations resulted in a faster LPL reaction rate, with complete substrate LPL conversion occurring after 30 hours of reaction. The KPL concentration continuously increased with reaction time, eventually accumulating to 6.1%–11.2%.

[0095] The above results indicate that the co-expression system can catalyze the complete reaction of LPL, but the reaction time is long. In the early stage of the reaction, the intermediate product KPL is rapidly converted into DPL. As time goes on, glucose is depleted, and the reaction cannot provide enough NADPH for the synthesis of DPL by the multi-enzyme combination, resulting in a large accumulation of KPL.

[0096] Example 6: Synthesis of DPL from DL-PL catalyzed by glucose addition from engineered bacteria co-expressing multiple enzymes

[0097] The effect of fed-batch glucose on the one-pot catalytic resolution of the DL-PL cascade by co-expressed engineered bacteria was investigated. The reaction system was as follows: 5 mL of reaction system contained DL-PL at concentrations of 500 mM and 1000 mM, and NADP... + The concentrations were 50 mM and 100 mM, respectively. The cell concentrations of DPA2 or DPCA4 were 150 g / L and 250 g / L, respectively. When the substrate DL-PL concentration was 500 mM, 375 mM, 250 mM, and 125 mM glucose were added at 0, 8, and 16 h, respectively. When the substrate DL-PL concentration was 1000 mM, 750 mM, 500 mM, 250 mM, and 125 mM glucose were added at 0, 8, 16, and 24 h, respectively. The reaction pH was maintained at 6.0–8.0 (adjusted with 2 M NaOH). The reaction temperature was 30 °C, and the stirring speed was 800 rpm. Samples were taken periodically, and the concentrations of substrate, intermediate product, and product were determined by gas chromatography.

[0098] Specific reaction process:

[0099] Reaction 1: 500mM DL-PL, 150g / L DPA2, 100mM NADP + 375mM, 250mM, and 125mM glucose were added at 0, 8, and 16 hours, respectively.

[0100] Reaction 2: 500mM DL-PL, 150g / L DPA2, 50mM NADP + 375mM, 250mM, and 125mM glucose were added at 0, 8, and 16 hours, respectively.

[0101] Reaction 3: 1000mM DL-PL, 250g / L DPA2, 100mM NADP + 750mM, 500mM, 250mM, and 125mM glucose were added at 0, 8, 16, and 24 hours, respectively.

[0102] Reaction 4: 500mM DL-PL, 150g / L DPCA4, 100mM NADP + 375mM, 250mM, and 125mM glucose were added at 0, 8, and 16 hours, respectively.

[0103] Reaction 5: 1000mM DL-PL, 250g / L DPCA4, 100mM NADP + 750mM, 500mM, 250mM, and 125mM glucose were added at 0, 8, 16, and 24 hours, respectively.

[0104] The reaction results are as follows Figure 5 As shown, after 24 hours of reaction, 150 g / L DPA2 catalyzed 500 mM DL-PL, the DPL concentration reached 98.4%, LPL was completely reacted, and only less than 2% KPL accumulated. After 28 hours of reaction, DPCA4 catalyzed 500 mM DL-PL, the DPL yield still reached 97.5%. This result indicates that the fed-batch glucose addition strategy effectively controls the coenzyme cycle in the 500 mM DL-PL reaction process. After 48 hours of reaction, DPA2 and DPCA4 catalyzed the resolution of 1000 mM DL-PL, and the DPL accumulation was 91.3% and 88.6%, respectively, with approximately 2.2% of LPL remaining in both cases.

[0105] Adding glucose in batches can improve DPL yield and reduce KPL accumulation.

Claims

1. A mutant of a conjugated polyketide reductase, characterized in that The conjugated polyketide reductase mutant is one of the following: (1) the alanine at position 64 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to methionine, and the amino acid sequence is shown in SEQ ID NO. 3; (2) the alanine at position 64 of the amino acid sequence shown in SEQ ID NO. 1 is mutated to methionine, and the serine at position 206 is mutated to cysteine, and the amino acid sequence is shown in SEQ ID NO.

5.

2. The gene encoding the conjugated polyketide reductase mutant of claim 1.

3. A recombinant vector containing the gene encoding the conjugated polyketide reductase mutant of claim 2.

4. A recombinant genetically engineered bacterium containing the gene encoding the conjugated polyketide reductase mutant of claim 2.

5. The recombinant genetically engineered bacteria according to claim 4, wherein The recombinant genetically engineered bacterium is a three-enzyme co-expression engineering bacterium that simultaneously expresses the conjugated polyketide reductase mutant, glucose dehydrogenase and L-pantoate lactone dehydrogenase.

6. The recombinant genetically engineered bacteria according to claim 5, wherein The recombinant genetically engineered bacterium is a molecular chaperone three-enzyme co-expression engineering bacterium that simultaneously expresses the molecular chaperone pGro7, the conjugated polyketide reductase mutant, glucose dehydrogenase and L-pantoate lactone dehydrogenase.

7. Use of the conjugated polyketide reductase mutant of claim 1 or the recombinant genetically engineered bacterium of claim 4 in catalyzing the synthesis of optically pure D-pantoate lactone from racemic pantoate lactone.

8. Use according to claim 7, wherein The application method is: using wet bacteria obtained by fermenting and culturing a recombinant genetically engineered bacterium containing a coding gene of the conjugated polyketide reductase mutant as a catalyst, using racemic ubiquinol acid lactone as a reaction substrate, using glucose as a co-substrate, and using coenzyme NADP + under the action of PB buffer as a reaction medium to form a conversion system, to produce D-ubiquinol acid lactone; the recombinant genetically engineered bacterium containing a coding gene of the conjugated polyketide reductase mutant is a three-enzyme co-expression engineering bacterium simultaneously expressing conjugated polyketide reductase mutant, glucose dehydrogenase and L-ubiquinol acid lactone dehydrogenase, or a molecular chaperone three-enzyme co-expression engineering bacterium simultaneously expressing molecular chaperone pGro7, conjugated polyketide reductase mutant, glucose dehydrogenase and L-ubiquinol acid lactone dehydrogenase.

9. Use according to claim 8, wherein In the three-enzyme co-expression engineering bacterium or the molecular chaperone three-enzyme co-expression engineering bacterium, the amino acid sequence of the conjugated polyketide reductase mutant is shown in SEQ ID NO. 5; the amino acid sequence of L-pantoate lactone dehydrogenase is shown in SEQ ID NO. 10; and the amino acid sequence of glucose dehydrogenase is shown in SEQ ID NO. 8.

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