CN117757766B - Aldehyde reductase mutant and application thereof in synthesis of D-1,2, 4-butanetriol - Google Patents
Aldehyde reductase mutant and application thereof in synthesis of D-1,2, 4-butanetriol Download PDFInfo
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Abstract
The invention provides an aldehyde reductase mutant and application thereof in synthesis of D-1,2, 4-butanetriol. The mutant protein is non-natural protein, and has the activity of obviously improving the catalysis of D-3, 4-dihydroxybutanal to generate D-1,2, 4-butanetriol. The most suitable aldehyde reductase mutant strain, xylose dehydrogenase, xylitol dehydratase and alpha-keto acid decarboxylase are jointly applied to the production process of D-1,2, 4-butanetriol, so that the capability of synthesizing D-1,2, 4-butanetriol by D-xylose can be improved, and finally the yield of D-1,2, 4-butanetriol can reach 80 g/L.
Description
Technical Field
The invention belongs to the fields of molecular biology and enzyme engineering, and relates to an aldehyde reductase mutant and application thereof in synthesis of D-1,2, 4-butanetriol.
Background
D-1, 2, 4-butanetriol (D-1, 2, 4-Butanetriol, BT for short) is an important four-carbon polyol and is widely applied to the fields of military, medicines, tobacco, high polymer materials, cosmetics and the like. Can be used for synthesizing the butanetriol trinitrate (BTTN) in military, and is a high-efficiency propellant and plasticizer. Can be used for synthesizing cholesterol-lowering drugs Movinolin, anticancer drugs, and drugs AGENERASE (an HIV protease inhibitor) for treating AIDS. In the tobacco industry, the tar can be used as a cigarette additive, and the harm of tar to human bodies can be reduced.
In the production of D-1, 2, 4-butanetriol using D-xylose by a biological pathway, the synthetic pathway comprises: (1) Converting D-xylose to D-xylonic acid under the catalysis of D-xylose dehydrogenase; (2) D-xylonic acid is catalyzed by D-xylonic acid dehydratase to generate D-3-deoxyglycerolpentonic acid; (3) D-3-deoxyglycerolpentonic acid is catalyzed by aldehyde reductase to generate D-3, 4-dihydroxybutanal; (4) D-3, 4-dihydroxybutanal is catalyzed by aldehyde reductase to produce D-1, 2, 4-butanetriol.
Disclosure of Invention
In the earlier study, the inventors performed a dig-screen for four enzymes catalyzing the synthesis of D-1,2, 4-butanetriol from D-xylose, and in a preferred set of combinations, the first three steps of reaction can be performed rapidly, but aldehyde reductase has the problem of slow reaction rate, resulting in the accumulation of a large amount of D-3, 4-dihydroxybutanal in the reaction. Therefore, we mutate the better aldehyde reductase selected at present, and continuously increase the activity of the aldehyde reductase to increase the reaction rate, namely, directionally evolve the aldehyde reductase (927, NCBI: WP_201093303.1) from Entomomonasasaccharolytica to obtain the mutant with improved enzyme activity and substrate tolerance, and catalyze D-3, 4-dihydroxybutanal to generate D-1,2, 4-butanetriol. Thus, the present invention has been completed.
The invention provides an aldehyde reductase mutant, a coding nucleic acid and application thereof, which can improve enzyme activity, substrate tolerance and the like, so that the aldehyde reductase mutant meets the industrial use requirements.
The invention provides an aldehyde reductase mutant, which is characterized in that the mutant protein sequence is identical with SEQ ID NO:1 and the mutant has activity in reducing D-3, 4-dihydroxybutanal, the mutant corresponding to seq id no:1, and a mutant obtained by mutating one or two or three positions in the F50, W86 and L277 of the amino acids 1 to 340.
Specifically, corresponding to seq id no: mutation at position 50 on 1 to G or P; mutation at position 86 to S, A or G; mutation at position 277 to V, N or Q; mutation at position 286 is A or G.
Further, corresponding to seq id no:1, a mutation at any one of the following sites:
a combined mutation at positions 50 and 86; a combined mutation at positions 86 and 277; a combined mutation at positions 86 and 286; a combined mutation at positions 50, 86 and 277; a combined mutation at positions 50, 86 and 286; combined mutations at positions 86, 277 and 286.
Preferably, corresponding to seq id no: mutation at position 50 to G and mutation at position 86 to S on 1; mutation at position 50 to G and mutation at position 86 to A; mutation at position 86 to a and mutation at position 277 to V; mutation at position 86 to A and mutation at position 286 to A; mutation at position 50 to G and at position 86 to A and at position 286 to A; mutation at position 50 to G and at position 86 to A and at position 277 to V.
The invention provides the coding nucleic acid of the mutant and an expression vector containing the coding nucleic acid.
The invention also provides recombinant host bacteria of the coding nucleic acid or the expression vector of claim 6. Specifically, it is a microorganism of the genus Escherichia (Escherichia), erwinia (Erwinia), serratia (Serratia), providia (Providencia), enterobacter (Enterobacteria), salmonella (Salmonella), streptomyces (Streptomyces), pseudomonas (Pseudomonas), brevibacterium (Brevibacterium), bacillus (Bacillus) or Corynebacterium (Corynebacterium).
The invention also provides the mutant, the coding nucleic acid of the mutant, the application of the expression vector or the recombinant host bacterium in the synthesis of D-1,2, 4-butanetriol.
The invention further provides a method for preparing D-1, 2, 4-butanetriol by reducing D-xylose, which is characterized by comprising the following steps:
(i) The mutant is combined with xylose dehydrogenase, xylitol dehydratase and alpha-keto acid decarboxylase, D-xylose is used as a reaction substrate, and a catalytic reaction is carried out, so that the D-1, 2, 4-butanetriol is obtained.
(Ii) The method comprises the steps of catalyzing D-xylose by xylose dehydrogenase (xylB), xylitol dehydratase (yjhG), alpha-keto acid decarboxylase (SaPDC, derived from Staphylococcus aureus) and aldehyde reductase mutant strains together, carrying out 4-step catalytic reaction on the D-xylose to generate D-xylonic acid under the action of xylose dehydrogenase, then generating 3-deoxy-D-glycerolpentanoic acid under the action of xylitol dehydratase, generating D-3, 4-dihydroxybutanal under the action of alpha-keto acid decarboxylase, and finally generating D-1,2, 4-butanetriol under the action of aldehyde reductase.
Specifically, the pH of the system for the catalytic reaction is 6.0-10.0; (ii) a reaction time of 24-48 hours; (iii) The reaction temperature is 20 ℃ to 40 ℃ (iv) and the concentration of the substrate is 100-200g/L.
Preferably, the pH of the system of catalytic reactions is pH6.5-8.0; (ii) a reaction time of 32 to 40 hours; (iii) a reaction temperature of 25 ℃ to 35 ℃; (iv) the concentration of the substrate is 50-100g/L.
Specifically, the mutant is added in the form of crude enzyme liquid prepared from the whole cells or fermentation liquid thereof.
Optionally, the method further comprises the step of separating and purifying the D-1, 2, 4-butanetriol.
The mutant protein provided by the invention is non-natural protein, and has the effect of remarkably improving the activity of catalyzing D-3, 4-dihydroxybutanal to generate D-1,2, 4-butanetriol. The most suitable aldehyde reductase mutant strain, xylose dehydrogenase, xylitol dehydratase and alpha-keto acid decarboxylase are jointly applied to the production process of D-1,2, 4-butanetriol, so that the capability of synthesizing D-1,2, 4-butanetriol by D-xylose can be improved, and finally the yield of D-1,2, 4-butanetriol can reach 80 g/L.
Drawings
FIG. 1A shows a xylose liquid phase profile.
FIG. 1B shows the xylitol acid liquid phase diagram.
FIG. 1C shows a liquid phase diagram of D-3-deoxy-glycerylvaleric acid.
FIG. 1D shows a liquid phase diagram of D-3, 4-dihydroxybutanal.
FIG. 1E shows a liquid phase pattern of D-1,2, 4-butanetriol.
FIG. 2 shows protein electrophoresis patterns of several crude enzymes. Wherein M represents Marker,1 xylose dehydrogenase, 2 xylitol dehydratase, 3 alpha-keto acid decarboxylase, and 4 aldehyde reductase (927).
Detailed Description
The experimental techniques and methods used in this example are conventional techniques unless otherwise specified, such as those not specified in the following examples, and are generally performed under conventional conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Materials, reagents and the like used in the examples are all available from a regular commercial source unless otherwise specified.
Example 1: construction of aldehyde reductase 927 mutant library and screening of mutants
First round vitality reconstruction
The aldehyde reductase 927 (from Entomomonas asaccharolytica aldehyde reductase 927, NCBI: WP_201093303.1, amino acid sequence shown in SEQ ID NO: 1) is used as template to search in PDB database to find a protein 4GKV with 68% similarity and crystal structure. And 4GKV is used as a template, DS software is used for carrying out homologous modeling, and after a 927 simulated configuration is obtained, small molecules of a substrate are butted and then selected.
The substrate is taken as a center, and the points of the 9A range are 32, wherein C39, H40 and C147 are catalytic triplets. Taking C 4 N of NADH as a center, taking 28 points in the 8A range, analyzing the structure, and selecting the following sites from the points to establish a mutation library: the saturation mutations were performed for T41, H44, F50, H60, T86, A148, V150, T151, L175, V261, G262, L263, V276, L277, S285, L286, V287, G288, R332, respectively, and degenerate codon NNK was used to design mutation primers, using pET21a-927 as templates. PCR was performed using a two-step method, using a high fidelity polymerase Pfx.
1) Construction of pET21a-927 plasmid
The wild type amino acid sequence of aldehyde reductase is shown as SEQ ID NO. 1, the corresponding nucleotide sequence is fully synthesized and cloned between restriction enzyme sites NdeI and XhoI of a pET-21a vector to obtain recombinant plasmids pET21a-927, the recombinant plasmids pET21a-927 are further transformed into an expression host E.coli BL21 (DE 3), and positive clones are selected to obtain recombinant expression transformant E.coli BL21 (DE 3)/pET 21a-927.
2) Construction of aldehyde reductase single-point mutant library
Using pET21a-927 as a template, forward primers of T41, H44, P50, H60, and T86 were combined with reverse primer 1R, respectively, and forward primers of a148, V150, T151, and L175 were combined with reverse primer 2R, respectively, and reverse primers of V261, G262, L263, V276, L277, S285, L286, V287, G288, and R332 were used, respectively, and forward primer 3F were used, respectively, and the PCR reaction system and reaction conditions were as follows:
a. first-step PCR reaction system and reaction conditions
Round 1: to a PCR reaction system having a total volume of 25. Mu.L, 15 ng of the template, 12.5. Mu.L of 2 XPfx MIX, 0.5. Mu.L (10. Mu.M) of each of a pair of mutation primers was added, and distilled water was added to 25. Mu.L after sterilization.
First step PCR reaction procedure: ① The steps ②～④ are carried out for 30 cycles in total by pre-denaturation at 98℃of 2 min, denaturation at ② ℃of 30 sec, ③ Tm-5℃annealing at 30 sec, extension at ④ 72 ℃of 30 sec, final extension at ⑤ 72 ℃of 5 min.
B. Second step PCR reaction system and reaction condition
Round 2: to a PCR reaction system with a total volume of 50. Mu.L, 30 ng of template, 25. Mu.L of 2 XPfx MIX, 1. Mu.L of primer as the first round PCR product fragment, and distilled water to 50. Mu.L were added.
Second step PCR reaction procedure: ① Pre-denaturation at 98℃for 2min, denaturation at ② 98 deg.C for 30 sec, annealing at ③ 55 ℃for 30 sec, extension at ④ 72 ℃for 3min, extension at ⑤ 72 ℃for 10min, step ②～④ performed a total of 25 cycles.
And (3) after the PCR product obtained in the step is verified by agarose gel electrophoresis analysis, adding restriction enzyme DpnI and digesting for 2 hours at 37 ℃. The digested product was transferred to E. coliBL21 (DE 3) competent cells and plated on plates containing 10. Mu.g/mL ampicillin, and allowed to stand in an incubator at 37℃for about 12 hours to give a single colony, thereby obtaining an aldehyde reductase mutant library.
Meanwhile, the pET21a-927 mutant library plasmid is transferred into E.coliBL21 (DE 3) competent cells and is coated on a plate containing 100mg/mL ampicillin antibiotics, and the plate is placed in a 37 ℃ incubator for static culture for about 12 hours, and single colony is grown, thus obtaining the strain for expressing aldehyde reductase mutant.
TABLE 1 first round of modified primer sequences
Note that: n=a/T/C/G, k=g/T, m=a/C.
3) Induction expression of aldehyde reductase mutants
The monoclonal colony obtained after the culture in the step 2 is picked up to an LB liquid medium (peptone 10g/L, yeast powder 5g/L and NaCl 10 g/L) containing 4mL portions of ampicillin (100 mg/L), and cultured overnight at 37 ℃ and 200rpm to obtain a culture solution. The culture broth was inoculated to a fermentation medium (LB liquid medium) in an inoculum size of 1% (v/v), and was subjected to shaking culture at 37℃and 200rpm until OD 600 was 0.6-0.8, and IPTG was added to a final concentration of 0.1mM, and shaking culture was performed at 25℃and 200rpm for 8-12 hours. And collecting thalli by using 6000 g centrifugal culture solution, crushing under high pressure to obtain crude enzyme solution of aldehyde reductase, and carrying out subsequent enzyme activity detection.
4) Screening of aldehyde reductase mutants
The screening method is to detect the decrease in NADH at 340 nm. The specific method of the reaction is as follows: d-3, 4-dihydroxybutanal 10mM,NADH 0.25mg/mL, crude enzyme solution 20. Mu.L, and potassium phosphate buffer were supplemented to 200. Mu.L. NADH has characteristic light absorption at 340nm, the decrease of the light absorption at 340nm is detected by using an enzyme label instrument, if the enzyme activity is relatively high, the consumption of NADH is rapid, and the slope of the decrease curve is large. The useful mutation sites for which the enzyme activity was improved by screening were 50, 86 and 277.
2. Second round of vitality reconstruction
The combined mutant of single mutation site was constructed according to the saturated mutation result, the obtained mutant was picked up in a test tube containing 4mL of LB medium and cultured, and the activity of the expressed protein was detected and 1mL transformation reaction (containing 100 mM D-3, 4-dihydroxybutyraldehyde, 200 mM glucose, 0.5 g/L NAD +, 3U/mL GDH,20 mg/mL whole cell, reaction at 25 ℃ for 16 hours in ph7.5 potassium phosphate buffer) was performed, and the results are shown in table 2, the activities of mutants 14 and 15 were the highest, 10.5 and 14.3 times higher than the wild type.
TABLE 2 relative Activity of aldehyde reductase 927 and mutants thereof on substrates
EXAMPLE 2 Whole-cell catalytic synthesis of D-1,2, 4-butanetriol Using mutant 10
Mutant 15 was obtained by inducing expression in the same manner as in example 1, and the cells were collected by centrifugation (6000 rpm) and used as a biocatalyst. The cells were resuspended in 20 mL potassium phosphate buffer (pH 7.5, 100 mM), 200 mM D-3, 4-dihydroxybutanal, 300 mM glucose, 0.5 g/L NAD +, 3U/mLGDH, 10 mg/mL whole cells, and reacted at 37℃for 24 hours. After the reaction, the pH was adjusted to 2 with sulfuric acid, the conversion was 95% by HPLC analysis, and the isolation yield was 80%. FIG. 1A to FIG. 1E show the liquid chromatograms of xylose-free, intermediate products and D-1,2, 4-butanetriol, respectively.
EXAMPLE 3 catalytic Synthesis of D-1,2, 4-butanetriol Using crude enzyme solution of mutant 10
Mutant 15 was obtained by inducing expression in the same manner as in example 1, and the cells were collected by centrifugation (6000 rpm) and the crude enzyme solution after the sterilization was used as a biocatalyst. The other conditions were the same as in example 2, and the conversion was 97% and the isolation yield was 82%.
EXAMPLE 4 Whole-cell catalytic synthesis of D-1,2, 4-butanetriol Using mutant 10
Plasmids of the tetrase coexpression vector were constructed, and xylose dehydrogenase (WP_ 010918706.1), xylitol dehydratase (Q9A9Z2.1), α -keto acid decarboxylase (WP_ 016501746.1) and aldehyde reductase 927 mutant 15 were co-constructed on vector pRSFDuet and designated as G4.
BL21 (DE 3) expression strain carrying G4 plasmid was induced and collected by centrifugation (6000 rpm) as biocatalyst according to the method of example 1. The reaction system contained 160g/L D-xylose, 50g/L G, 1g/L NAD + and NADH,1.5mM TPP,10mM Mg 2+, the other conditions being the same as in example 2. The conversion rate is 99%, and the separation yield is 70%.
Claims (11)
1. A mutant of an aldehyde reductase, wherein the mutant has a protein sequence corresponding to SEQ ID NO: mutation at position 50 on 1to G; mutation at position 50 to P; mutation at position 86 to S; mutation at position 86 to a; mutation of 86 to G, mutation of 277 to N, mutation of 277 to V, mutation of 277 to Q, mutation of 286 to A, mutation of 286 to G, mutation of 50 to G and mutation of 86 to S; mutation at position 86 to a and mutation at position 277 to V; mutation at position 86 to A and mutation at position 286 to A; mutation at position 50 to G and at position 86 to A and at position 286 to A; or a mutation at position 50 to G and a mutation at position 86 to A and a mutation at position 277 to V.
2. The mutant nucleic acid of claim 1 which encodes.
3. An expression vector comprising the coding nucleic acid of claim 2.
4. A recombinant host bacterium comprising the nucleic acid encoding claim 2 or the expression vector of claim 3.
5. The recombinant host bacterium according to claim 4, which is a microorganism belonging to the genus Escherichia (Escherichia), erwinia (Erwinia), serratia (Serratia), providia (Providia), enterobacter (Enterobacteria), salmonella (Salmonella), streptomyces (Streptomyces), pseudomonas (Pseudomonas), brevibacterium (Brevibacterium), bacillus (Bacillus) or Corynebacterium (Corynebacterium).
6. Use of a mutant according to claim 1, a nucleic acid encoding a mutant according to claim 2, an expression vector according to claim 3 or a recombinant host bacterium according to claim 4 or 5 for the synthesis of D-1,2, 4-butanetriol.
7. A method for preparing D-1, 2, 4-butanetriol by reducing D-xylose, which is characterized in that the mutant of claim 1 is combined with xylose dehydrogenase, xylitol dehydratase and alpha-keto acid decarboxylase, and the D-xylose is used as a reaction substrate for catalytic reaction, so that the D-1, 2, 4-butanetriol is obtained.
8. The method of claim 7, wherein the pH of the system of catalytic reactions is from 6.0 to 10.0; the reaction time is 24-48 hours; the reaction temperature is 20 ℃ to 40 ℃; the concentration of the substrate is 100-200g/L.
9. The method of claim 8, wherein the pH of the system of catalytic reactions is between pH6.5 and 8.0; the reaction time is 32-40 hours; the reaction temperature is 25 ℃ to 35 ℃; the concentration of the substrate is 50-100g/L.
10. The method according to claim 9, wherein the mutant is added in the form of a crude enzyme solution prepared from whole cells of the recombinant host bacterium according to claim 4 or 5 or a fermentation broth thereof.
11. The method of claim 10, further comprising the step of isolating and purifying the D-1, 2, 4-butanetriol.
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