Conjugated polyketoreductase mutant, co-expression engineering bacterium and application

Abstract

The invention discloses a conjugated polyketoreductase mutant which is obtained by performing single-point mutation or multi-point combined mutation on the 64th or 206th site of an amino acid sequence shown as SEQ ID NO.1. The invention further discloses a preparation method of the conjugated polyketoreductase mutant. The specific enzyme activity of the mutant of the conjugated polyketoreductase can be improved by more than 25% compared with that of a wild type. The invention also provides a coding gene, a recombinant vector containing the coding gene, a co-expression engineering bacterium and an application of the co-expression engineering bacterium. The mutant of the conjugated polyketide reductase, glucose dehydrogenase and L-pantoic acid lactone dehydrogenase are used for constructing a three-enzyme co-expression engineering bacterium as a biocatalyst to catalyze racemic pantoic acid lactone to synthesize optically pure D-pantoic acid lactone, so that the catalytic efficiency of a multi-enzyme cascade reaction is improved, the hydrolysis loss of an intermediate product KPL is reduced, and the yield of D-pantoic acid lactone is increased. The yield of the DPL is remarkably improved, the yield of the obtained DPL can reach 98%, and the optical purity can reach 99.9%.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a conjugated polyketone reductase mutant, an encoding gene, a recombinant vector, a triple-enzyme co-expression engineering bacterium, and applications thereof. Background Art

[0002] D-Pantothenic acid (D-PA), with the molecular formula C 9 H 17 NO 5 , is a water-soluble vitamin, also known as vitamin B5, and is an essential vitamin for humans and animals. It can be further converted into coenzyme A (CoA) in animals, plants, and microorganisms, and further participates in the energy metabolism in living organisms. Therefore, it is widely used in the fields of food, feed, and medicine. The synthesis methods of D-PA include chemical methods, chemoenzymatic methods, microbial fermentation methods, and bioenzymatic methods. D-Pantolactone (DPL) is a key chiral intermediate for the synthesis of D-PA by chemical or enzymatic methods. Therefore, constructing a synthesis pathway for DPL is of great significance for the synthesis of D-PA.

[0003] The synthesis methods of DPL mainly include chemical methods and bioenzymatic methods. The chemical synthesis of DPL uses isobutyraldehyde and formaldehyde as substrates and cyanide as a nucleophilic addition reagent. First, it is acidified and then cyclized to synthesize D,L-pantolactone (DL-PL), and then DL-PL is resolved into DPL and L-pantolactone (LPL) with a chemical resolving agent. Although this method has a mature process, it has disadvantages such as high energy consumption, excessive use of acids and bases, and complex processes. The bioenzymatic synthesis of DPL uses hydrolase or reductase to replace the chemical resolving agent to resolve DL-PL into DPL and LPL. The hydrolase resolution method includes two pathways. One is to specifically hydrolyze the L-type substrate in DL-PL with L-pantolactone hydrolase to obtain DPL and L-pantothenic acid, and L-pantothenic acid is regenerated into DL-PL through a recovery and esterification process. The other is to specifically hydrolyze the D-type substrate in DL-PL with D-pantolactone hydrolase to generate D-pantothenic acid and LPL. The separated D-pantothenic acid forms DPL through acidification and cyclization, while LPL is re-formed into DL-PL through racemization. Compared with L-pantolactone hydrolase, the catalytic resolution of D-pantolactone hydrolase can obtain DPL with high optical purity, so it is widely used in industry. However, the hydrolase resolution method also has disadvantages such as complex steps and excessive use of acids and bases.

[0004] The redox enzyme resolution method uses L-pantolactone dehydrogenase (LPLDH) to convert LPL in DL-PL into pantolactone (KPL), and then uses conjugated polyketide reductase (CPR) to asymmetrically reduce it to DPL. Meanwhile, glucose dehydrogenase (GDH) drives coenzyme regeneration in the presence of glucose. Compared with the hydrolase resolution route, the oxidation-reduction enzyme method has no racemization step, does not go through the separation steps of lactone and acid, has a high theoretical yield, and has greater potential for industrial production.

[0005] Conjugated polyketide reductase is the key enzyme for the asymmetric synthesis of DPL by the redox enzyme method. If the activity of conjugated polyketide reductase cannot meet the requirements of the multi-enzyme cascade catalytic reaction, it will lead to the loss of the intermediate KPL due to hydrolysis, reduce the catalytic efficiency of the multi-enzyme cascade reaction, and increase the difficulty of subsequent product separation and purification. Therefore, it is of great significance to improve the catalytic efficiency of conjugated polyketide reductase through molecular modification. For example, Zhang et al. mutated the 126th and 215th amino acids of the conjugated polyketide reductase CviCPR from Candida viswanathii, and the mutant could catalyze the preparation of DPL from KPL with high activity and high stereoselectivity (CN202210519820.6); Liu et al. mutated the 29th, 65th, and 215th amino acids of the conjugated polyketide reductase KbCPR from Kazachstania barnettii, and the enzyme activity increased by 7.43 times. The co-expression engineering bacteria constructed with this mutant and GDH showed good catalytic activity (CN202311035098.X). Summary of the Invention

[0006] In order to solve the problem that in the three-enzyme cascade catalytic DL-PL resolution reaction of L-pantolactone dehydrogenase-conjugated polyketide reductase-glucose dehydrogenase, due to the insufficient catalytic efficiency of conjugated polyketide reductase, the easily hydrolyzable intermediate cannot be quickly converted into the product, the present invention provides a conjugated polyketide reductase modification strategy, and thus obtains a variety of conjugated polyketide reductase mutants with enhanced activity. Among them, the single mutant CorCPR M1 (A64M) has an enzyme activity increased by 25%, and the double mutant CorCPR M2 (A64M / S206C) has an enzyme activity increased by 30%.

[0007] The technical solution adopted by the present invention is:

[0008] The conjugated polyketide reductase mutant is obtained by performing single-site mutation or multi-site combined mutation on the 64th or 206th site 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 of the following or a combination of two:

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

[0011] (2) The serine at position 206 is mutated to cysteine (S206C);

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

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

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

[0015] Due to the particularity of the amino acid sequence, any fragment or variant of the peptide protein containing the amino acid sequence shown in the present invention, such as its conservative variant, bioactive fragment or derivative, as long as the homology between the fragment of the peptide protein or the peptide protein variant and the foregoing amino acid sequence is more than 90%, belongs to the scope of protection of the present invention. Specifically, the changes include deletion, insertion or substitution of amino acids in the amino acid sequence; among them, for conservative changes of the variant, the substituted amino acid has a structure or chemical property similar to the original amino acid, such as replacing isoleucine with leucine, and asparagine with glutamine. Enzyme variants can also have non-conservative changes.

[0016] The present invention also provides the coding gene of the conjugated polyketone reductase mutant described above.

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

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

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

[0020] The full length of the base sequences of the conjugated polyketone reductase and the mutant described in the present invention is 948bp, from the first base to the 948th base, the start codon is ATG, and the stop codon is TAA.

[0021] Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in the present invention, as long as it has a homology of more than 90% with the aforementioned polynucleotide, falls within the scope of protection of the present invention. The variant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide changes. Such variants of the polynucleotide can be natural mutants or non-natural mutants, including substitution mutants, deletion mutants, and insertion mutants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion, or insertion of a polynucleotide, but does not substantially change the function of the peptide protein encoded thereby.

[0022] The conjugate polyketone reductase mutant of the present invention is obtained by mutating the CorCPR gene using site-directed mutagenesis and combinatorial mutagenesis techniques. The obtained mutant plasmid is transformed into competent E. coli BL21(DE3) cells by heat shock. The obtained strain is inoculated, transferred, induced, and the cells are recovered, and the resuspended cell suspension is used to catalyze KPL. The specific method is as follows: First, the amino acid sequence of CorCPR is aligned with the mutants KmAKR M13 , KbCPR M3 and SceCPR M2 from the same protein family with improved enzyme activity. According to the sequence alignment and sequence identity analysis, seven amino acid mutations (F301W / F302H, Q33H, A64M, N114K, S206C, and E272D) that may affect the enzyme activity of CorCPR are obtained. Using pET28b-CorCPR as the template plasmid, site-directed mutagenesis is performed on the above sites, and the single mutants with improved enzyme activity are selected by gas-phase detection of enzyme activity. Further, the dominant single mutants are combined for double mutagenesis, and the enzyme activity is measured. Finally, the single mutant CorCPR M1 (A64M) with an enzyme activity increased by 25% relative to CorCPR and the double mutant CorCPR M2 (A64M / S206C) with an enzyme activity increased by 30% are obtained.

[0023] The present invention also provides a recombinant vector and an expression host containing the coding gene of the conjugate polyketone reductase mutant described above.

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

[0025] The present invention also provides the application of the conjugate polyketone reductase mutant in the asymmetric reduction of ketopantolactone by microbial catalysis to synthesize optically pure D-pantolactone.

[0026] The conjugate polyketone reductase mutant catalyst can be obtained by the following method: culturing a recombinant genetic engineering bacterium containing the coding gene of the conjugate polyketone reductase mutant, inducing the expression of the conjugate polyketone reductase mutant, and centrifuging the obtained culture solution to obtain the conjugate polyketone reductase mutant whole-cell catalyst.

[0027] The present invention also provides a recombinant three-enzyme genetic engineering bacterium containing the coding gene of the conjugate polyketone reductase mutant. The expression host of the recombinant three-enzyme genetic engineering bacterium is usually Escherichia coli E. coli BL21(DE3). The expression vector of the recombinant three-enzyme genetic engineering bacterium is pCDFDuet1, pRSFDuet1 or pACYCDuet1.

[0028] Furthermore, the recombinant three-enzyme genetic engineering bacterium containing the coding gene of the conjugate polyketone reductase mutant is a three-enzyme co-expression engineering bacterium that simultaneously expresses the conjugate polyketone reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase.

[0029] The three-enzyme co-expression engineering bacterium can also express molecular chaperones.

[0030] The recombinant three-enzyme genetic engineering bacterium containing the coding gene of the conjugate polyketone reductase mutant can also be a molecular chaperone three-enzyme co-expression engineering bacterium that simultaneously expresses the molecular chaperone pGro7, the conjugate polyketone reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase.

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

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

[0033] The three - enzyme co - expression engineered bacterium contains the coding gene of the conjugate polyketone reductase mutant, the coding gene of glucose dehydrogenase, and the coding gene of L - pantolactone dehydrogenase.

[0034] The three - enzyme co - expression engineered bacterium is induced to simultaneously express the conjugate polyketone reductase mutant, glucose dehydrogenase, and L - pantolactone dehydrogenase.

[0035] Furthermore, the three - enzyme co - expression engineered bacterium can be constructed by the following method:

[0036] Construct a recombinant vector containing the coding gene of the L - pantolactone dehydrogenase, transform it into a host bacterium to obtain a recombinant genetic engineering bacterium containing the coding gene of the L - pantolactone dehydrogenase, construct a recombinant vector containing the coding genes of glucose dehydrogenase and the conjugate polyketone reductase mutant, and transform it into the recombinant genetic engineering bacterium containing the coding gene of the L - pantolactone dehydrogenase to obtain the three - enzyme co - expression engineered bacterium.

[0037] Furthermore, the molecular chaperone and the three - enzyme co - expression engineered bacterium can be constructed by the following method:

[0038] Co - transform the recombinant vector containing the coding gene of the L - pantolactone dehydrogenase and the recombinant vector containing the coding genes of glucose dehydrogenase and the conjugate polyketone reductase mutant into the E.coli BL21(DE3) / pGro7 competent cells containing the molecular chaperone plasmid to construct the molecular chaperone and the three - enzyme co - expression engineered bacterium.

[0039] Furthermore, preferably in the three - enzyme co - expression engineered bacterium or the molecular chaperone and the three - enzyme co - expression engineered bacterium, the amino acid sequence of the conjugate polyketone reductase mutant is as shown in SEQ ID NO.5; the amino acid sequence of the L - pantolactone dehydrogenase is as shown in SEQ ID NO.10; the amino acid sequence of glucose dehydrogenase is as shown in SEQ ID NO.8.

[0040] Furthermore, the three - enzyme co - expression engineered bacterium is constructed by the following method:

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

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

[0043] pRSFDuet-BmGDH M6 -CorCPR M2 and transformed into E.coli BL21(DE3) / pACYCDuet-RhoLPLDH M5 to obtain the triple-enzyme co-expression engineering bacterium E.coli BL21(DE3) /

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

[0045] Furthermore, the molecular chaperone and the triple-enzyme co-expression engineering bacterium were constructed as follows:

[0046] The coding gene of the L-pantolactone dehydrogenase mutant RhoLPLDH with the amino acid sequence shown in SEQ ID NO.10 M5 was ligated into the pCDFDuet plasmid vector to obtain a recombinant vector pCDFDuet-RhoLPLDH containing the coding gene of the said L-pantolactone dehydrogenase M5 ;

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

[0048] pRSFDuet-BmGDH M6 -CorCPR M2, pCDFDuet-RhoLPLDH M5 and

[0049] pRSFDuet-BmGDH M6 -CorCPR M2 were co-transformed into E. coli BL21(DE3) / pGro7 competent cells containing the chaperone plasmid to construct the recombinant engineering bacterium co-expressing the chaperone and three enzymes, E. coli BL21(DE3) / pCDFDuet-RhoLPLDH M5 / pRSFDuet-BmGDH M6 -CorCPR M2 / pGro7, denoted as DPCA4.

[0050] The present invention also provides the application of the conjugate polyketone reductase mutant or the recombinant three-enzyme genetic engineering bacterium containing the coding gene of the conjugate polyketone reductase mutant in the microbial catalysis of the synthesis of optically pure D-pantolactone from racemic pantolactone.

[0051] The reaction formula is as follows:

[0052]

[0053] Furthermore, the method of the application is as follows: The wet cells obtained by fermentation culture of the recombinant three-enzyme genetic engineering bacterium containing the coding gene of the conjugate polyketone reductase mutant are used as the catalyst, racemic pantolactone is used as the reaction substrate, glucose is used as the co-substrate, and coenzyme NADP + under the action of, PB buffer is used as the reaction medium to form a transformation system, and D-pantolactone is prepared by reaction; the recombinant three-enzyme genetic engineering bacterium containing the coding gene of the conjugate polyketone reductase mutant is a three-enzyme co-expression engineering bacterium that simultaneously expresses the conjugate polyketone reductase mutant, glucose dehydrogenase, and L-pantolactone dehydrogenase, or a molecular chaperone three-enzyme co-expression engineering bacterium that simultaneously expresses the molecular chaperone pGro7, the conjugate polyketone reductase mutant, glucose dehydrogenase, and L-pantolactone dehydrogenase.

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

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

[0056] Further, in the conversion system, the concentration of the substrate racemic pantolactone is 500 - 1000 mM; the glucose concentration is 350 - 2000 mM; the dosage of the catalyst is 100 - 250 g WCW / L (WCW wet cell weight) based on the wet weight of the bacterial cells; the concentration of the coenzyme NADP + is 50 - 100 mM.

[0057] Further, glucose is preferably added to the conversion system in a batch manner. Initially, 40 - 60% (preferably 50%) of the total amount of glucose is added. After 8 h of reaction, 25 - 35% of the total amount of glucose is added. After 16 h of reaction, the remaining glucose is added. Alternatively, 60 - 70% of the remaining glucose is added after 16 h of reaction, and the remaining glucose is added after 24 h of reaction.

[0058] Further, the wet bacterial cells obtained by fermentation and culture of the recombinant genetically engineered bacterium containing the encoding gene of the conjugate polyketone reductase mutant are prepared as follows: The tri-enzyme co-expression engineered bacterium that simultaneously expresses the conjugate polyketone reductase mutant, glucose dehydrogenase, and L-pantolactone dehydrogenase, or the molecular chaperone tri-enzyme co-expression engineered bacterium that simultaneously expresses the molecular chaperone pGro7, conjugate polyketone reductase mutant, glucose dehydrogenase, and L-pantolactone dehydrogenase is inoculated into an 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 solution; The seed solution is inoculated into a fresh LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol at an inoculation amount of 1% (v / v), and cultured at 37°C and 180 rpm until OD600 = 0.6 - 0.8. Isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.1 - 0.3 mM is added to the culture solution, and after culturing at 24°C for 12 h, centrifuged at 4°C and 8000 rpm for 10 - 15 min to obtain the wet bacterial cells expressing L-pantolactone dehydrogenase, conjugate polyketone reductase, and glucose dehydrogenase.

[0059] For the inoculation, transfer, induction, and recovery of the bacterial cells of the genetically engineered bacterium of the present invention, the culture medium can be any culture medium in the art that can enable the growth of the bacterial cells and produce the present invention. Preferably, it is an LB medium, and the formula is: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, dissolved in distilled water, and the pH is adjusted to 7.0. There are no special restrictions on the culture method and culture conditions, and the culture method and conditions can be optimized according to factors such as the host type and culture method.

[0060] In the present invention, through screening, the co-expression engineered bacterium E. coli BL21(DE3) / pACYCDuet-RhoLPLDH M5 / pRSFDuet-BmGDH M6-CorCPR M2 (designated as DPA2) and E. coli BL21(DE3) / pCDFDuet-RhoLPLDH M5 / pRSFDuet-BmGDH M6 -CorCPR M2 / pGro7 (designated as DPCA4) has the highest DPL production when catalyzing, and also has a high catalytic conversion rate for high-concentration substrates (up to 1000 mM), exceeding the existing level.

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

[0062] (1) The present invention provides a single mutant CorCPR of conjugated polyketone reductase with improved catalytic activity M1 (A64M), double mutant CorCPR M2 (A64M / S206C), and the specific activity of the mutants is increased by 25% and 30% respectively compared with the wild type.

[0063] (2) A three-enzyme co-expression engineering bacterium is constructed with a conjugated polyketone reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase. Using wet cells as a biocatalyst for whole-cell catalysis. The three enzymes can be highly expressed simultaneously in a recombinant bacterium, and the recombinant bacterium can be cultured in large quantities without going through processes such as cell disruption and enzyme solution purification, with low cost and simple operation.

[0064] (3) In the present invention, using wet cells as a biocatalyst, DL-PL can be catalytically split and synthesized into DPL by a "one-pot method". The substrate is racemic pantolactone, and the LPL in it is catalytically reacted, converted into 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 KPL, the yield of DPL is significantly improved, and the co-expression engineering bacterium of the present invention can perform high-conversion catalytic conversion on high-concentration substrates, significantly improving the production efficiency. In the present invention, the co-expression engineering bacterium DPA2 catalyzes the splitting of 500 mM and 1000 mM DL-PL, and the DPL yields are 98.4% (64.0 g / L) and 91.3% (118.7 g / L) respectively. The co-expression engineering bacterium DPCA4 catalyzes the splitting of 500 mM and 1000 mM DL-PL respectively, and the DPL yields are 97.6% and 88.6% respectively. Description of the Drawings

[0065] Figure 1 Alignment analysis diagram of the CorCPR sequence and other mutant sequences.

[0066] Figure 2 Relative enzyme activity comparison diagram of seven CorCPR mutants.

[0067] Figure 3 Process diagram of the catalytic DL-PL resolution reaction by different co-expressing engineered bacteria.

[0068] Figure 4 Process diagram of the catalytic 500 mM DL-PL resolution reaction by co-expressing engineered bacteria.

[0069] Figure 5 Process diagram of the catalytic 500 and 1000 mM DL-PL resolution reactions by co-expressing engineered bacteria with fed-batch glucose addition. Specific implementation mode

[0070] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0071] Example 1: Screening of mutation sites by sequence alignment

[0072] Sequence comparison is a common method for quickly obtaining beneficial mutations. KmAKR derived from Kluyveromyces marxianus is an aldehyde-ketone reductase. Research shows 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, realizes co-evolution, and improves the catalytic efficiency. In addition, the literature reports that KbCPR M3 (Y29K / R65N / K215R) and SceCPR M2 (S157A / Y298H) have improved catalytic ability for the substrate KPL. In order to further improve the catalytic efficiency of CorCPR, CorCPR derived from Candida orthopsilosis was multi-sequence aligned with the mutants KbCPR M3 , SceCPR M2 , KmAKR M13 (see Figure 1 ). According to sequence alignment and sequence identity analysis, seven amino acid mutations (F301W / F302H, Q33H, A64M, N114K, S206C and E272D) that may affect the enzyme activity of CorCPR were selected.

[0073] Example 2: Construction and screening of conjugate polyketone reductase mutants

[0074] 1. Starting strain:

[0075] The coding gene of CorCPR was inserted into the restriction enzyme sites Nde I / Xho I of plasmid pET28b to construct the recombinant plasmid pET28b-CorCPR, which was then transformed into BL21(DE3) competent cells as the original strain. The amino acid sequence of CorCPR 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 preparation of CorCPR mutants was achieved by site-directed mutagenesis. Using CorCPR as a template, mutant primers were designed for polymerase chain reaction (PCR). The PCR products digested with DpnI enzyme were introduced into E. coli BL21(DE3) competent cells by heat shock and spread on LB solid plate medium with Kan resistance, and cultured at 37°C for 12 - 16 h. After single colonies grew, single colonies were picked and cultured overnight in 10 mL LB liquid medium (Kan). The bacterial solution was added with 30% glycerol for storage and verified by sequencing.

[0078] The mutant strains stored with glycerol and the original strain were respectively inoculated into 10 mL LB resistant medium and cultured for 12 - 16 h as seed solutions, and then inoculated into 100 mL LB shake flask medium (with Kan resistance) at an inoculation amount of 1%. After induced culture, wet bacteria were obtained. An appropriate amount of bacteria was weighed and resuspended with PB (pH 7.0) buffer to make a 5 g / L cell suspension, which was sonicated at 20% power and centrifuged at 12,000 rpm for 10 min, and the supernatant was collected as the crude enzyme solution.

[0079] Determination of the enzyme activity of conjugated polyketone reductase (1 mL reaction system): 5 g / L crude enzyme solution, 10 mM KPL, 15 mM NADPH, 50 mM PB (pH 7.0) buffer. The absorbance value was measured at room temperature and 340 nm for 3 minutes, and the enzyme activity was calculated as the decrease in absorbance value / OD 600 The larger the value, the higher the enzyme activity. Using the original strain as the control group, mutants with higher enzyme activity than the control group were considered dominant mutants. The relative enzyme activities of 7 mutants are as Figure 2 shown.

[0080] 3. Combinatorial mutation

[0081] The mutation sites with higher enzyme activity than the control group in the previous round of mutation were subjected to double-site combinatorial mutation. Using the single-mutation plasmid as a template, new mutation sites were added, and double mutants were obtained through steps such as PCR, transformation, and sequencing. Using the original strain as the control, mutants with improved enzyme activity were screened. The enzyme activities of the mutants are as Figure 2 shown, and mutants CorCPR M1 (A64M), CorCPR M2(A64M / S206C), the enzyme activity of the mutant increased by 25% and 30% respectively compared with the wild type.

[0082] Example 3: Construction and Induced Expression of Engineered Bacteria Co-expressing Three Enzymes

[0083] The coding gene of RhoLPLDH with the amino acid sequence shown in SEQ ID NO.10 M5 was inserted into the restriction enzyme sites Nde I / Xho I of the multiple cloning site 2 (MCS2) of plasmid pACYCDuet to construct plasmid pACYCDuet-RhoLPLDH. M5 , and transformed into E. coli BL21(DE3) competent cells. After overnight culture on plates, single colonies were picked to prepare E. coli BL21(DE3) / pACYCDuet-RhoLPLDH M5 competent cells. Using glucose dehydrogenase (BmGDH, BmGDH M6 ), the coding genes of conjugated polyketone reductase (CorCPR, CorCPR M1 , CorCPR M2 ) were respectively inserted into the restriction enzyme sites (Nco I / Not I) of the multiple cloning site I and the restriction enzyme sites (Nde I / Xho I) of the multiple cloning site II of the pRSFDuet1 dual-expression vector to construct recombinant plasmids 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, and the amino acid sequence of BmGDH M6 is shown in SEQ ID NO.8. The amino acid sequence of the conjugated polyketone reductase CorCPR M1 is shown in SEQ ID NO.3, the coding gene sequence is shown in SEQ ID NO.4, the amino acid sequence of CorCPR M2 is shown in SEQ ID NO.5, and the coding gene sequence is shown in SEQ ID NO.6.

[0084] Then the newly constructed recombinant plasmids were respectively transformed into E. coli BL21(DE3) / pACYCDuet-RhoLPLDH M5 competent cells, and spread on plates with kanamycin and chloramphenicol double resistance. After overnight culture, single colonies were picked for verification. In addition, the newly constructed recombinant plasmids pRSFDuet-BmGDHM6 -CorCPR M2 、pRSFDuet-BmGDH M6 -CorCPR M1、 pRSFDuet-BmGDH-CorCPR and pRSFDuet-BmGDH M6 -CorCPR and plasmid pCDFDuet-RhoLPLDH M5 were co-transformed into competent E. coli BL21(DE3) / pGro7 cells and spread on plates with triple resistance to streptomycin, kanamycin and chloramphenicol. After overnight culture, single colonies were picked for verification. Finally, triple-enzyme co-expression strains DPA1 - DPA4 and triple-enzyme and molecular chaperone co-expression engineering bacteria DPCA1 - DPCA4 were obtained, as shown in Table 1.

[0085] The co-expression engineering bacteria were separately inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol (for molecular chaperone co-expression engineering bacteria: additionally add 50 μg / mL streptomycin), and cultured at 37 °C and 180 rpm for 12 h to obtain seed solutions. The seed solutions were inoculated into fresh 100 mL of LB liquid medium shake flasks containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol (for molecular chaperone co-expression engineering bacteria: additionally add 50 μg / mL streptomycin) at an inoculation amount of 1.0%, and cultured at 37 °C and 180 rpm until the OD 600 was between 0.6 - 0.8. Then, IPTG with a final concentration of 0.3 mM was added to the culture medium, and after culturing at 24 °C for 12 h, centrifuged at 4 °C and 8000 rpm for 10 min to obtain wet cell bodies.

[0086] Table 1 Triple-enzyme co-expression strains and plasmids

[0087]

[0088] Example 4: Catalytic verification of triple-enzyme co-expression engineering bacteria

[0089] The conversion efficiency of the whole cells of strains DPA1 - DPA4 and DPCA1 - DPCA4 obtained in Example 3 for the conversion of DL-PL was measured. The reaction system was as follows: 5 mL reaction system, the concentration of the substrate DL-pantolactone (DL-PL) was 100 mM, NADP + concentration was 10 mM, CaCO 3The concentration is 100 mM (to slow down the pH decrease), the wet cell concentration is 50 g / L, and the reaction pH is adjusted with 2 M NaOH to maintain at 7.0 - 8.0. The reaction temperature is 35 °C, and the magnetic stirring speed is 800 rpm. Samples are taken regularly, the reaction is terminated with 6 M HCL solution, and then extracted three times repeatedly with ethyl acetate, and the concentrations of the substrate, intermediate product, and product are detected by gas phase.

[0090] Gas phase detection conditions: Gas chromatography thermoscienfic TRACE1610, chromatographic column Agilent CYCLOSIL - B (30 m × 0.25 mm, 0.25 μm), carrier gas: helium, flow rate: 1 mL / min, inlet and detector temperatures: 250 °C, injection volume: 1 μL, split mode, program: initial temperature 100 °C, heated at a rate of 10 °C / min to 140 °C, held for 5 min, cooled at a rate of 20 °C / min to 100 °C, detected for 15 min.

[0091] The catalytic process of the DL - PL resolution reaction is as Figure 3 shown. It can be seen from the figure that the yields of DPL catalyzed by DPCA4 and DPA2 are the highest, 93.5% and 90.1% respectively, and the remaining LPL are 3.6% and 7.6% respectively. The common point of the DPCA4 and DPA2 engineered bacteria is that both contain the recombinant plasmid pRSFDuet - BmGDH M6 -CorCPR M2 , and the difference is that the recombinant plasmids expressing L - pantolactone are pACYCDuet - RhoLPLDH M5 and pCDFDuet - RhoLPLDH M5 / pGro7 respectively. Thus, DPCA4 and DPA2 are the dominant 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 - pantolactone catalyzed by a three - enzyme co - expression engineered bacterium

[0093] Using the whole cells of the co - expression engineered bacteria DPA2 and DPCA4 as catalysts, the cascade reaction of the one - pot catalytic resolution of DL - PL is carried out to synthesize the product DPL. The resolution of DL - PL is catalyzed by 100 g / L, 180 g / L, 250 g / L DPA2 and 100 g / L, 250 g / L DPCA4 respectively. Among them, 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 is as follows: 5 mL reaction system, the substrate DL - PL concentration is 500 mM, the glucose concentration is 375 mM, NADP +The concentration is 100 mM, and the reaction pH is 6.0 - 8.0 (adjusted with 2 M NaOH). The reaction temperature is 30 °C, and the stirring speed is 800 rpm. Samples are taken regularly, and the reaction is terminated with 6 M HCl solution, followed by three repeated extractions with ethyl acetate. The concentrations of the substrate, intermediate product, and product are detected by gas chromatography.

[0094] The reaction process is as Figure 4 shown. The yields of DPL catalyzed by 100 g / L, 180 g / L, and 250 g / L DPA2 are 88.8%, 93.9%, and 92.5% respectively, and the yields of DPL catalyzed by 100 g / L and 250 g / L DPCA4 are 92.1% and 90.1% respectively. The higher the cell concentration, the faster the reaction rate of LPL, and the substrate LPL is completely converted after 30 h of reaction. The concentration of KPL continuously increases with the progress of the reaction time, and the final accumulation reaches 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 initial stage of the reaction, the intermediate product KPL is rapidly converted to DPL. As time prolongs, glucose is depleted, and the reaction cannot provide sufficient NADPH for the multi-enzyme combination to synthesize DPL, resulting in a large accumulation of KPL.

[0096] Example 6: Fed-batch addition of glucose by the multi-enzyme co-expressing engineering bacteria to catalyze the synthesis of DPL from DL-PL

[0097] Investigate the effect of fed-batch addition of glucose on the "one-pot" catalytic resolution cascade reaction of DL-PL by the co-expressing engineering bacteria. The reaction system is as follows: In a 5 mL reaction system, the concentrations of the substrate DL-PL are 500 mM and 1000 mM respectively, and the concentrations of NADP + are 50 mM and 100 mM respectively. The cell concentrations of DPA2 or DPCA4 are 150 g / L and 250 g / L respectively. When the concentration of the substrate DL-PL is 500 mM, 375 mM, 250 mM, and 125 mM glucose are added at 0, 8, and 16 h respectively. When the concentration of the substrate DL-PL is 1000 mM, 750 mM, 500 mM, 250 mM, and 125 mM glucose are added at 0, 8, 16, and 24 h respectively. The reaction pH is 6.0 - 8.0 (adjusted with 2 M NaOH). The reaction temperature is 30 °C, and the stirring speed is 800 rpm. Samples are taken at regular intervals, and the concentrations of the substrate, intermediate product, and product are detected by gas chromatography.

[0098] Specific reaction process:

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

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

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

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

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

[0104] The reaction results are as Figure 5 shown. When 150 g / L DPA2 catalyzed 500 mM DL-PL for 24 h, the DPL concentration reached 98.4%, the LPL completely reacted, and only less than 2% of KPL accumulated. When DPCA4 catalyzed 500 mM DL-PL, the DPL yield could still reach 97.5% after 28 h. The results indicate that the strategy of feeding glucose in batches effectively controls the coenzyme cycle during the reaction of 500 mM DL-PL. When DPA2 and DPCA4 catalyzed the resolution of 1000 mM DL-PL for 48 h, the accumulated amounts of DPL were 91.3% and 88.6%, respectively, and the remaining amounts of LPL were both about 2.2%.

[0105] The method of adding glucose in batches can improve the DPL yield and reduce the accumulation of KPL.

Claims

1. A conjugated polyketide reductase mutant, characterized in that The conjugated polyketide reductase mutant is obtained by performing single-point mutation or multi-point combined mutation on the 64th or 206th position of the amino acid sequence shown in SEQ ID NO.

1.

2. The conjugated polyketide reductase mutant according to claim 1, characterized in that The mutation is one or a combination of two of the following: (1) Alanine at position 64 was mutated to methionine; (2) The serine at position 206 was mutated to cysteine.

3. The conjugated polyketide reductase mutant according to claim 1, 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. The amino acid sequence is shown in SEQ ID NO.

5.

4. A gene encoding the conjugated polyketide reductase mutant according to any one of claims 1 to 3.

5. A recombinant vector containing the gene encoding the conjugated polyketide reductase mutant according to claim 4.

6. A recombinant three-enzyme genetic engineering bacterium containing the coding gene of the conjugated polyketide reductase mutant according to claim 4.

7. The recombinant three-enzyme genetic engineering bacteria according to claim 6, characterized in that The recombinant three-enzyme genetic engineering bacteria is a three-enzyme co-expressing engineering bacteria that simultaneously expresses a conjugated polyketide reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase; Alternatively, the recombinant genetically engineered bacteria is a molecular chaperone triple enzyme co-expressing engineering bacteria that simultaneously expresses a molecular chaperone pGro7, a conjugated polyketide reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase.

8. Use of the conjugated polyketide reductase mutant according to claim 1 or the recombinant three-enzyme genetic engineering bacteria according to claim 6 or 7 in the synthesis of optically pure D-pantolactone from racemic pantolactone catalyzed by microorganisms.

9. The use according to claim 8, characterized in that The application method is: the wet bacteria obtained by fermentation culture of the recombinant three-enzyme genetic engineering bacteria containing the coding gene of the conjugated polyketide reductase mutant is used as a catalyst, racemic pantolactone is used as a reaction substrate, glucose is used as a cosubstrate, and the coenzyme NADP is used as a catalyst. + Under the action of , a conversion system is formed with PB buffer as a reaction medium, and a reaction is carried out to obtain D-pantolactone; the recombinant three-enzyme genetic engineering bacteria containing the coding gene of the conjugated polyketide reductase mutant is a three-enzyme co-expressing engineering bacteria that simultaneously expresses the conjugated polyketide reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase, or a molecular chaperone three-enzyme co-expressing engineering bacteria that simultaneously expresses the molecular chaperone pGro7, the conjugated polyketide reductase mutant, glucose dehydrogenase and L-pantolactone dehydrogenase.

10. The use according to claim 9, characterized in that In the three-enzyme co-expressing engineering bacteria or the molecular chaperone and three-enzyme co-expressing engineering bacteria, 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.

Images