CN116200353A

CN116200353A - Carbonyl reductase mutant, recombinant bacterium and application thereof

Info

Publication number: CN116200353A
Application number: CN202310127729.4A
Authority: CN
Inventors: 黄旺生; 胡建良; 范宇鹏; 陈诗浪; 林传明; 李志江; 朱超; 周定干; 范亚新; 厉金锋
Original assignee: Hangzhou Grascent Co ltd
Current assignee: Hangzhou Grascent Co ltd
Priority date: 2023-02-01
Filing date: 2023-02-01
Publication date: 2023-06-02

Abstract

The invention discloses a carbonyl reductase mutant, recombinant bacteria and application thereof, and relates to the technical field of biology. The invention provides a novel carbonyl reductase mutant, which effectively solves the problems of low diketone conversion rate and poor ketone selectivity in the prior art, and has the advantages of short catalytic time, high substrate conversion rate and good ketone selectivity. In addition, when the carbonyl reductase mutant provided by the invention participates in the catalytic reaction, the reaction can be normally carried out without adding the reduced coenzyme additionally, so that zero addition of the coenzyme is realized in a true sense, and the cost of industrial application is greatly reduced.

Description

Carbonyl reductase mutant, recombinant bacterium and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a carbonyl reductase mutant, recombinant bacteria and application thereof.

Background

Carbonyl reductases belong to one of the enzymes catalyzing the activity of asymmetric prochiral ketones, which have the activity of catalyzing diketone compounds. At present, patent publication No. CN111575258A provides a short-chain dehydrogenase which has low activity in catalyzing aliphatic potential chiral diketones (such as 2,3 butanedione), patent publication No. CN109468291B discloses a carbonyl reductase EbSDR8 mutant, however, the mutant has the problems of excessively long reaction time of 20-24h, low substrate conversion rate and poor ketone selectivity, and the reaction requires the addition of reduced coenzyme, so that the industrialized application cost is high.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a carbonyl reductase mutant, recombinant bacterium and application thereof, which are used for solving the problems of long catalytic reaction time and low substrate conversion rate of the existing carbonyl reductase.

The invention is realized in the following way:

in a first aspect, the invention provides a carbonyl reductase mutant, the amino acid sequence of which has any one of the following mutations compared to a reference sequence:

(1) Phe at position 153 was mutated to Leu (F153L);

(2) Met 207 to Phe (M207F);

(3) Simultaneously, the 153 th Phe is mutated into Leu and the 207 th Met is mutated into Phe; the reference sequence is shown as SEQ ID NO. 1.

The reference sequence (wild type) is derived from Caenibius tardaugens NBRC 16725, NCBI sequence WP_244925490.1, and the DNA sequence is shown in SEQ ID NO.4 and is codon optimized. The inventor constructs a carbonyl reductase mutant library based on a reference sequence, screens and obtains carbonyl reductase mutants with high catalytic activity, and sequences the mutants to find that Phe at 153 th position is mutated into Leu and Met at 207 th position is mutated into Phe compared with a wild type; double mutation increases the enzyme activity of the mutant by 10 times. The final concentration of the thalli is 20g (dcw)/L, the substrate conversion rate of 200g/L can be more than 95% within 12h, the problems of low diketone conversion rate and poor ketone selectivity in the prior art are effectively solved, and the method has the advantages of short catalytic time, high substrate conversion rate and good ketone selectivity.

In addition, when the carbonyl reductase mutant provided by the invention participates in the catalytic reaction, the reaction can be normally carried out without adding the reduced coenzyme additionally, so that zero addition of the coenzyme is realized in a true sense, and the cost of industrial application is greatly reduced.

Based on the double mutants, the inventors also constructed corresponding carbonyl reductase single mutants. Compared with the wild type, the constructed single mutant F153L or M207F can also improve the enzyme activity of carbonyl reductase to a certain extent by 2 times and 3 times respectively, and has stronger catalytic capability than the parent. Therefore, the invention can prepare the monoketone by using the diketone as a substrate through the catalytic reaction of the biological enzyme.

Definition of the unit enzyme activity: under standard reaction conditions, the amount of enzyme required to produce 1umol of monoketone per minute is one enzyme activity unit U.

In a second aspect, the invention also provides a nucleic acid molecule encoding a carbonyl reductase mutant as described above.

The nucleotide coding sequence of the carbonyl reductase mutant with Phe at 153 th position of the reference sequence mutated into Leu is shown as SEQ ID NO. 2; the nucleotide coding sequence of the double mutant with the 153 th Phe mutated to Leu and the 207 th Met mutated to Phe is shown in SEQ ID NO. 3.

Based on the known amino acid sequence, the nucleotide sequence encoding the carbonyl reductase mutant can be deduced by a person skilled in the art based on the degeneracy of codons, and is not limited to the above nucleotide sequence, and the person skilled in the art can optimize codons according to the need and is also within the scope of the invention.

The term "nucleic acid molecule" as used herein refers to a sequence of nucleoside (nucleoside) or nucleotide (nucleotidide) monomers consisting of natural bases, sugars and inter-sugar (backbone) linkages. The term also includes modified or substituted sequences that contain non-naturally occurring monomers or portions thereof. The nucleic acid molecules of the invention may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may comprise natural bases including adenine, guanine, cytosine, thymine, and uracil. Modified bases may also be included. Examples of such modified bases include nitrogen-containing and deazaadenine, guanine, cytosine, thymine, and uracil; xanthine and hypoxanthine.

In a third aspect, the invention also provides an expression cassette or recombinant vector comprising a nucleic acid molecule as described above.

The term "vector" is used herein in its most general sense and includes any intermediate vector for a nucleic acid that enables the nucleic acid to be introduced, for example, into a prokaryotic and/or eukaryotic cell, and where appropriate, integrated into the genome. Vectors of this type are preferably replicated and/or expressed in cells. The vector comprises a plasmid, phagemid, phage or viral genome. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, typically circular DNA duplex, that can replicate independently of chromosomal DNA.

For example, the vector is selected from the commercial plasmid pET-3a.

The expression cassette comprises: promoters, screening genes, terminators, and the like.

In a fourth aspect, the present invention also provides a recombinant bacterium or recombinant cell comprising: the nucleic acid molecules described above or the expression cassettes or recombinant vectors described above.

In a preferred embodiment of the invention, the recombinant bacterium is selected from E.coli, for example E.coli BL21 (DE 3).

The term "recombinant cell" refers to any cell that can be transformed or transfected with an exogenous nucleic acid. The term "recombinant cell" according to the invention comprises prokaryotic (e.g. E.coli) or eukaryotic cells (e.g. mammalian cells, in particular human cells, yeast cells and insect cells). Mammalian cells, such as cells from humans, mice, hamsters, pigs, goats or primates, are particularly preferred. Cells may be derived from a plurality of tissue types and comprise primary cells and cell lines. The nucleic acid may be present in the host cell in a single copy or in two or more copies, and in one embodiment is expressed in a recombinant cell.

In a fifth aspect, the present invention also provides a method for producing the above carbonyl reductase mutant, comprising the steps of: culturing the recombinant bacterium or recombinant cell under conditions suitable for expression, thereby expressing a carbonyl reductase mutant; and/or isolating carbonyl reductase mutants.

For example, the seed solution is induced to express for 12 hours at 10-45 ℃, and the supernatant is discarded after centrifugation, and wet cells are collected. The culture condition of the seed solution is that the seed solution is cultured for 8-10 hours at 37 ℃.

In a sixth aspect, the invention also provides an application of a carbonyl reductase mutant or the recombinant bacterium or recombinant cell in asymmetric catalysis of diketone compounds to synthesis of damascone series compounds.

The carbonyl reductase mutant can be catalyzed in a whole cell form, and can also be catalyzed by crude enzyme liquid of cell disruption or pure enzyme of complete disruption. In addition, the above enzymes may be prepared as immobilized enzymes or as immobilized cell-form enzymes using specific immobilization techniques.

In a preferred embodiment of the invention, the reaction medium in the reaction system for selectively reducing and synthesizing the damascone series compound is isopropanol or other hydrogen donors.

Isopropanol acts as both a solvent and a hydrogen donor in the reaction. The invention can normally carry out the reaction without adding the reduced coenzyme additionally, realizes zero addition of the coenzyme in the true sense, and greatly reduces the cost of industrialized application. In addition, the separation of the byproduct acetone and the residual isopropanol generated in the reaction is simpler, so that the residual isopropanol can be combined with the production of the acetone, and the purified isopropanol can be recycled. In a word, the utilization of the input reaction raw materials is close to 100% in theory, which accords with the concept of green chemical industry.

In an alternative embodiment, the final concentration of recombinant bacteria or recombinant cells in the reaction system is 10-300g/L and the final concentration of diketone compound is 0.3-3M. For example, the final concentration of the recombinant bacterium is 10g/L,12g/L,15g/L,18g/L,20g/L, or 100g/L. The final concentration of the diketone compound is, for example, 0.3 to 1M, or 0.8 to 2M.

In an alternative embodiment, the reaction conditions are 10-45℃for 1-12h. For example, the reaction condition is 37-40 ℃, and the carbonyl reductase mutant has good catalytic activity under the reaction condition of 1-12 hours. For example, the reaction is carried out for 1h, 2h, 4h, 6h and 10h. The carbonyl reductase mutant provided by the invention can greatly shorten the catalytic reaction time.

In an alternative embodiment, the reaction is carried out at 200-700 rpm; for example, the reaction is carried out at 200 to 500rpm, or at 300 to 700 rpm.

In an alternative embodiment, the reaction is carried out in 80% to 85% isopropanol reaction medium.

In a preferred embodiment of the present invention, the carbonyl reductase mutant is provided in the reaction system in the form of wet bacterial cells obtained by induction culture of genetically engineered bacteria containing the carbonyl reductase mutant.

In a preferred embodiment of the use of the invention, the damascone is selected from the group consisting of damascone A, damascone B and damascone B. The carbonyl reductase mutant provided by the invention has good ketone selectivity.

In an alternative embodiment, the damascone is selected from 3-hydroxy-1- (2, 6-trimethyl-3-cyclohexenyl) -1-butanone; the diketone compound is selected from 1- (2, 6-trimethyl-3-cyclohexen-1-yl) -1,3 butanedione.

The invention has the following beneficial effects:

according to the invention, a carbonyl reductase mutant library is constructed based on a reference sequence, carbonyl reductase mutants with high catalytic activity are obtained through screening, and the mutants are sequenced to find that Phe at 153 th position is mutated into Leu and Met at 207 th position is mutated into Phe compared with a wild type; double mutation improves the enzyme activity of the mutant by 10 times. The final concentration of the thalli is 20g (dcw)/L, the substrate conversion rate of 200g/L can reach more than 95% within 12h, the problems of low diketone conversion rate and poor ketone selectivity in the prior art are effectively solved, and the method has the advantages of short catalytic time, high substrate conversion rate and good ketone selectivity.

Based on the double mutants, the inventors also constructed corresponding carbonyl reductase single mutants. Compared with the wild type, the constructed single mutant F153L or M207F can also improve the enzyme activity of carbonyl reductase to a certain extent, and has stronger catalytic capability than the parent. Therefore, the invention can prepare the monoketone by using the diketone as a substrate through the catalytic reaction of the biological enzyme.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing the results of gel electrophoresis verification of carbonyl reductase gene CTSDR, lane M: DNA marker, lane: both 1 and 2 are PCR products.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The embodiment provides a method for amplifying carbonyl reductase gene CTSDR and constructing recombinant genetic engineering bacteria E.coli BL21 (DE 3)/pET-3 a-CTSDR containing carbonyl reductase gene.

1. Amplification of carbonyl reductase Gene

The target gene amplification primers are as follows:

an upstream primer: p1 5'-TGGCTGATATCGGATCCATG-3'

A downstream primer: p2 5'-GCAGCCGGATCTCAGTGTTA-3'

The gene CTSDR for encoding carbonyl reductase is used as a template for PCR amplification, wherein the PCR amplification system is as follows: 50. Mu.L of reaction system: 2 Xphantamax Buffer 25. Mu.L; dNTP Mix (10 mM): 1. Mu.L; upstream primer (50. Mu.M): 1 μl; downstream primer (50. Mu.M): 1 μl; phanta Max super-Fidelity DNA Polymerase, 1 μl; template DNA: 0.5. Mu.L; ddH ₂ O：18.5μL。

The reaction procedure was 95℃for 5min of pre-denaturation followed by a cycle: denaturation at 94℃for 1min, 15s run at 55℃as annealing temperature, extension at 72℃for 30s for 30 cycles, final extension at 72℃for 10min, and storage at 4 ℃. mu.L of the PCR product was mixed with 2. Mu.L of a locking buffer, and the result of gel electrophoresis was shown in FIG. 1.

The result shows that the target PCR product is obtained through successful amplification, the PCR product after successful verification is purified according to the Clean up kit, and the purified product is stored in a refrigerator at 4 ℃ for the next round of use.

2. Carrier linearization

The vector linearization primer is as follows:

an upstream primer: p3 5'-CACTGAGATCCGGCTGCTAACAAAGCCCGA-3'

A downstream primer: p4 5'-GGATCCGATATCAGCCATGGCCTTGTCGTC-3'

PCR amplification was performed using pET-3a as a template, 2 XPhatamax Buffer 25. Mu.L; dNTP Mix (10 Mm each): 1. Mu.L; upstream primer (50. Mu.M): 1 μl; downstream primer (50. Mu.M): 1 μl; phanta Max super-Fidelity DNA Polymerase, 1 μl; template DNA (plasmid): 0.5. Mu.L; ddH ₂ O：18.5μL。

The PCR reaction conditions were: pre-denaturation at 95℃for 10min, then temperature cycling at 95℃for 30s,55℃for 30s, and 72℃for 6min for 30 cycles, and final extension at 72℃for 10min with a termination temperature of 4 ℃.

mu.L of the PCR product was mixed with 2. Mu.L of a locking buffer, and the band size was verified by gel electrophoresis. And (3) purifying the PCR product after successful verification according to a Clean up kit, and storing the purified product in a refrigerator at 4 ℃ for the next round of use.

3. Recombinant vector construction: and (3) cloning the carbonyl reductase gene and the linearization vector in one step to obtain a recombinant vector pET-3a-CTSDR, and purifying the product by adopting a PCR clean Kit and preserving at 4 ℃ after 1% agarose gel electrophoresis analysis and verification.

4. Recombinant engineering bacteria construction: the recombinant vector is transformed into E.coli BL21 (DE 3) competent cells, an LB plate containing kanamycin (50 mug/mL) is coated, and the culture is carried out at 37 ℃ overnight, at the moment, a plurality of single colonies are presented on the LB plate, and the single colonies are recombinant genetically engineered bacteria E.coli BL21 (DE 3)/pET-3 a-CTSDR containing carbonyl reductase genes.

Under standard reaction conditions, the enzyme activity of the E.coli BL21 (DE 3)/pET-3 a-CTSDR wet thalli for catalyzing diketone to single ketone is 150U/g wet thalli.

Definition of unit enzyme activity: under standard reaction conditions, the amount of enzyme required to produce 1umol of monoketone per minute is one enzyme activity unit U.

The reaction system: the diketone with the final concentration of 1M and the E.coli BL21 (DE 3)/pET-3 a-CTSDR wet bacterial body with the final concentration of 20g/L form a reaction system by taking 10mL of isopropanol as a reaction medium. At 37 ℃,600rpm for 1h, adding 1mL of ethyl acetate into 500 mu L of reaction liquid for extraction, centrifuging at 12000rpm for 1min, taking organic phase, drying by anhydrous sodium sulfate, detecting the peak area of monoketone and residual substrate diketone in gas phase, calculating the product yield and the consumption of substrate by the ratio of the peak area of the product to the peak area of the substrate, and calculating the enzyme activity according to the definition of the enzyme activity.

The method for detecting the monoketone comprises the following steps: using the fuli system, column type: rt-beta DEXsa Column (30m x 0.32mm x 0.25um,Restek,Germany) capillary Column, chromatographic conditions: column temperature 174 ℃, sample injection chamber temperature 230 ℃, FID detector 230 ℃, N ₂ :0.1Mpa；H ₂ :0.1Mpa；Air:0.1Mpa。

Carbonyl reductase bacterial E.coli BL21 (DE 3)/pET 3a-CTSDR 200g/L obtained by fermentation is added into a reaction system of diketone with the final concentration of 0.3M and 80% isopropanol, and the conversion rate of a reaction substrate is only 17% under the stirring of 200rpm at 37 ℃ and the activity is not high.

Example 2

To increase the conversion efficiency of diketones, this example provides 3 carbonyl reductase mutants. The mutant is obtained by single-point single mutation or multi-point combined mutation of 153 th phenylalanine and 207 th methionine of an amino acid sequence shown as SEQ ID NO. 1; the 153 th glycine is mutated into leucine, the 207 th methionine is mutated into phenylalanine, the nucleotide sequence of the single mutant pET3a-CTSDR-F153L is shown as SEQ ID NO.2, and the nucleotide sequence of the double mutant pET3a-CTSDR-F153L-M207F is shown as SEQ ID NO. 3.

The construction method of the carbonyl reductase mutant specifically comprises the following steps:

1: constructing a recombinant engineering bacterium mutation library:

error-prone PCR is carried out by taking the gene of an E.coli BL21 (DE 3)/pET-3 a-CTSDR expression vector as a template to obtain an error-prone PCR product; and then taking the gene of E.coli BL21 (DE 3)/pET-3 a-CTSDR expression vector as a template, taking error-prone PCR products as primers to carry out full plasmid amplification, then converting the amplified products into host cells, then coating an LB plate containing kanamycin, and culturing to obtain recombinant genetically engineered bacteria containing carbonyl reductase mutant genes.

The error-prone PCR primers were:

an upstream primer: p1 5'-TGGCTGATATCGGATCCATG-3'

A downstream primer: p2 5'-GCAGCCGGATCTCAGTGTTA-3'

The vector linearization primer is as follows:

an upstream primer: p3 5'-CACTGAGATCCGGCTGCTAACAAAGCCCGA-3'

A downstream primer: p4 5'-GGATCCGATATCAGCCATGGCCTTGTCGTC-3'

The method comprises the following steps:

firstly, using pET-3a-CTSDR gene as a template, and performing error-prone PCR according to the primer.

Wherein the PCR amplification system is as follows: 50. Mu.L of reaction system: 2×t5 Taq DNA Polymerase:1 μl; mnCl ₂ (1 mM): 2.5. Mu.L; upstream primer (50. Mu.M): 1 μl; downstream primer (50. Mu.M): 1 μl; template DNA (plasmid): 1 μl; ddH ₂ O：13.5μL。

The error-prone PCR reaction procedure was 95℃pre-denatured for 5min, followed by cycling: denaturation at 94℃for 1min, 15s run at 55℃as annealing temperature, extension at 72℃for 30s for 30 cycles, final extension at 72℃for 10min, and storage at 4 ℃.

After the error-prone PCR was completed, 3. Mu.L of the error-prone PCR product was mixed with 2. Mu.L of Loading buffer, and the size of the band was checked by gel electrophoresis to verify that the error-prone PCR was successful. And (3) purifying the error-prone PCR product after successful verification according to a Clean up kit, and storing the purified product in a refrigerator at 4 ℃ for the next round of use.

Then, the pET-3a-CTSDR gene is used as a template, and error-prone PCR products are used as primers to carry out full plasmid amplification.

Wherein the whole plasmid amplification system is as follows: 2 Xphantamax Buffer 25. Mu.L; dNTP Mix (10 mM each): 1. Mu.L; upstream primer (50. Mu.M): 1 μl; downstream primer (50. Mu.M): 1 μl; phanta Max super-Fidelity DNA Polymerase, 1 μl; template DNA (plasmid): 0.5. Mu.L;ddH ₂ O：18.5μL。

After the PCR product is verified by 1% agarose gel electrophoresis analysis, 1 mu L of DpnI and 5 mu L of buffer are added into the PCR product, template plasmid DNA is removed by digestion for 2 hours at 37 ℃, after the PCR product is inactivated for 10 minutes at 65 ℃, the PCR product is purified by adopting a PCR clean up Kit and then is transformed into E.coli BL21 (DE 3) competent cells, an LB plate containing kanamycin (50 mu g/mL) is coated, and the LB plate is cultured overnight at 37 ℃ to obtain a mutation library of carbonyl reductase, at the moment, a plurality of single colonies with different mutations are presented on the LB plate, and the single colonies are recombinant genetic engineering bacteria containing carbonyl reductase mutant genes.

In the embodiment, the LB culture medium is peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, and the solvent is water with pH of 7.0.LB plates were prepared by adding 20g/L agar to LB liquid medium.

The specific preparation method of competent cells of escherichia coli BL21 (DE 3) (Invitrogen) comprises the following steps:

step one, obtaining E.coli BL21 (DE 3) strain preserved by glycerol tubes from a refrigerator at the temperature of minus 80 ℃, streaking on an LB plate without antibiotics, and culturing for 10 hours at the temperature of 37 ℃ to obtain single colony;

step two, picking single colony of the LB plate, inoculating the single colony into a test tube containing 5mL of LB culture medium, and culturing for 9h at 37 ℃ and 180 rpm;

step three, 200 mu L of bacterial liquid is taken from a test tube and inoculated into 50mL of LB culture medium, and OD is cultivated at 37 ℃ and 180rpm ₆₀₀ To 0.4-0.6;

step four, precooling the bacterial liquid on ice, taking the bacterial liquid into a sterilized centrifuge tube, placing the bacterial liquid on the ice for 10min, and centrifuging the bacterial liquid at 4 ℃ for 10min at 5000 rpm;

pouring out the supernatant, taking care to prevent bacterial contamination, and using pre-cooled CaCl of 0.1mol/L ₂ The precipitated cells were resuspended in aqueous solution and placed on ice for 30min;

step six, centrifuging at 4 ℃ for 10min at 5000rpm, discarding the supernatant, and using pre-cooled CaCl containing 15% glycerol and 0.1mol/L ₂ The precipitated cells were resuspended in aqueous solution, 100. Mu.L of the resuspended cells were dispensed into sterilized 1.5mL centrifuge tubes and stored in a-80℃freezer and removed as needed.

E.coli BL21 (DE 3) (Invitrogen) competent cells stored at-80℃were ice-bathed at 0℃for 10min, 5. Mu.L of the error-prone PCR-derived recombinant plasmid was added to each of the above-described superclean benches, ice-bathed at 0℃for 30min, hot-shocked in a water bath at 42℃for 90s, ice-bathed at 0℃for 2min, 600. Mu.L of LB medium was added, and shaking culture was performed at 37℃and 200rpm for 1h;

finally, the recombinant engineering bacteria mutant library is obtained by coating the recombinant engineering bacteria mutant library on an LB plate containing 50 mug/ml kanamycin resistance and culturing for 8-12 hours at 37 ℃, randomly picking up clone extraction plasmids for sequencing and identification, and obtaining recombinant escherichia coli containing expression recombinant plasmids through screening.

2. High-flux screening of recombinant escherichia coli;

the method for high throughput screening of recombinant genetically engineered bacteria employed in this example was to integrate the mutant sequence to replace the original wild-type sequence.

The method comprises the following steps: first, using wild CTSDR as reference, picking Shan Junla clones from carbonyl reductase mutation library to 2mL deep 96-well plate for culturing, adding 600 μl LB culture solution containing 50 μg/mL kanamycin as final concentration in advance, and picking 2 parent strains in the last 2 wells of 96-well plate as control.

2mL of 96-well plate was cultured at 37℃for 8 hours as a seed solution, then 200. Mu.L of the seed solution was added to a new sterile 600. Mu.L of LB medium containing 50. Mu.g/mL kanamycin and 0.1mM IPTG, the seed solution was subjected to induction expression at 26℃for 12 hours, and then centrifuged at 4000rpm for 20 minutes, the supernatant was discarded, and wet cells were collected for the next step of high-throughput screening.

In the embodiment, an effective high-throughput screening method is adopted, so that the time cost for obtaining the mutant with high catalytic property from a library containing tens of thousands of mutants is greatly saved: since conversion of ketone to alcohol is a hydrogenation reduction step, carbonyl reductase catalyzes the formation of alcohol from carbonyl and releases a molecule of H ⁺ Thus, as the reaction proceeds, the concentration of hydrogen ions in the reaction systemA change occurs. By selecting bromothymol blue which can generate color change in the effective range as an indicator, the activity and chiral selectivity of each mutant on ketone substances can be rapidly and directly detected under an enzyme-labeled instrument.

Wherein wet cells collected by centrifugation in 96-well plates were added with 200 μl of PB buffer (ph=8.0, 200 mM) per well to prepare cell suspensions. The colorimetric reaction was carried out in a 96-well quartz plate with a reaction system of 200. Mu.L: 80. Mu.L of the cell suspension, 20mM diketone and 0.09mg/ml bromothymol blue were added to 200. Mu.L of ethyl acetate and sodium phosphate buffer (200 mM, pH=8.0) in a volume ratio of 6:4, and after mixing, the mixture was incubated at 37℃for 20 minutes, and the reaction was carried out for the same period of time, whereby the enzyme activity of carbonyl reductase was determined according to the rate of change in the color of the reaction solution (from blue to yellow).

In the bacterial screening process, about 400 single colonies are screened in each round of mutation library, and in the same time, according to the color change speed and the color change depth of a pH indicator, the color of a reaction liquid with higher enzyme activity than that of a parent CTSDR control group is found to be more yellow than that of the parent reaction liquid, so that a mutant of recombinant bacteria containing carbonyl reductase mutant genes with higher activity is obtained through preliminary screening, and double mutants E.coli BL21 (DE 3)/pET 3a-CTSDR-F153L-M207F are obtained through sequencing, wherein the nucleotide sequence is shown as SEQ ID NO. 3.

The comparative results of the activity of the carbonyl reductase in asymmetrically catalytic reduction of diketones to monoketones are given in this example, and are specifically:

The reaction system: the diketone with the final concentration of 1M and the recombinant escherichia coli mutant with the final concentration of 20g/L form a reaction system by taking 10mL of isopropanol as a reaction medium. At 37 ℃,600rpm for 1h, adding 1mL of ethyl acetate into 500 mu L of reaction liquid for extraction, centrifuging at 12000rpm for 1min, taking organic phase, drying by anhydrous sodium sulfate, detecting the peak area of monoketone and residual substrate diketone in gas phase, calculating the product yield and the consumption of substrate by the ratio of the peak area of the product to the peak area of the substrate, and calculating the enzyme activity according to the definition of the enzyme activity.

The final experimental results are shown in the following table:

the results of the table show that the activity of each single-point mutant is improved, but the enzyme activity of the double mutant is improved to the maximum extent from 150U/g wet thalli to 1500U/g wet thalli, 10 times is improved, 3.2 times is improved after F153L single-point mutation, and 1.6 times is improved after M207F single-point mutation.

As shown by the experimental results, the recombinant escherichia coli containing the carbonyl reductase gene has stronger catalytic capability compared with a parent, and can utilize diketone as a substrate to carry out bioconversion reaction to prepare the diketone.

The recombinant vector constructed by the carbonyl reductase mutant coding gene and the recombinant genetic engineering bacteria prepared by the conversion of the recombinant vector realize the heterologous expression in the carbonyl reductase escherichia coli engineering bacteria. Compared with the wild parent, the enzyme activity of the mutant is improved by 10 times through the superposition mutation of the two mutation sites of the 153-position Phe mutation to Leu and the 207-position Met mutation to Phe. And the final concentration of the thalli is 20g (dcw)/L, so that the substrate with the concentration of 200g/L can be converted to more than 95% within 12h, and the problems of low conversion rate and poor ketone selectivity in the prior art are solved.

In addition, isopropanol can be used as a solvent and also as a hydrogen donor in the reaction. The invention can normally carry out the reaction without adding the reduced coenzyme additionally, realizes zero addition of the coenzyme in the true sense, and greatly reduces the cost of industrialized application. In addition, the acetone which is a byproduct generated in the reaction is separated from the residual isopropanol simply, so that the acetone can be combined with the production of the acetone, and the purified isopropanol can be recycled. In a word, the utilization of the input reaction raw materials is close to 100% in theory, which accords with the concept of green chemical industry.

Example 3

The embodiment provides an application of carbonyl reductase mutant in preparing monoketone by asymmetrically catalyzing diketones, in particular to a method for synthesizing monoketone by catalyzing diketone selective reduction by recombinant genetic engineering bacteria containing carbonyl reductase mutant genes.

1. Preparation of recombinant bacterial cells of carbonyl reductase and mutants thereof.

The wet bacterial cells obtained by fermenting E.coli BL21 (DE 3)/pET 3a-CTSDR-F153L-M207F in example 2 are used as catalysts, 1- (2, 6-trimethyl-3-cyclohexene-1-yl) -1,3 butanedione is used as a substrate, the reaction is carried out in a reaction system formed by taking an isopropanol-water biphasic system as a reaction medium, the reaction is carried out at 300-700rpm and 37 ℃, after the reaction is finished, a reaction solution containing monoketone is obtained, and the reaction solution is separated and purified to obtain monoketone.

Wherein the content of the isopropanol is 10% -100%. The dosage of the catalyst is 100-300g/L based on the weight of wet thalli, and the initial adding concentration of diketone is 0.5-3M.

The specific preparation method of the wet thalli in the embodiment comprises the following steps:

step S1, inoculating the recombinant engineering bacteria into LB culture solution containing kanamycin with the final concentration of 50mg/L, and culturing for 8 hours at 37 ℃ to obtain seed solution;

step S2, inoculating the seed solution obtained in the step S1 into a sterile LB liquid medium containing kanamycin with a final concentration of 50mg/L at an inoculum size of 2% by volume, and culturing at 37 ℃ for about 1.5-2.5h to obtain a thallus concentration OD ₆₀₀ 0.4-0.8; the LB liquid medium comprises: 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride, wherein the solvent is deionized water, and the pH value is 7.0.

And S3, adding isopropyl thio-beta-D-galactoside with the final concentration of 0.1-1.0mM into the culture solution, carrying out induced expression at 26 ℃ for 12 hours, and centrifuging at 4 ℃ and the rotating speed of 4000rpm for 10-20 minutes, and collecting wet thalli.

The carbonyl reductase mutant can be catalyzed in a whole cell form, or can be catalyzed by crude enzyme liquid of cell disruption or pure enzyme of complete disruption. In addition, the two enzymes may be prepared as immobilized enzymes or as immobilized cell-type enzymes using specific immobilization techniques.

2. Selective catalytic synthesis of monoketone by carbonyl reductase

Carbonyl reductase mutant bacterial E.coli BL21 (DE 3)/pET 3a-CTSDR-F153L-M207F 200g/L obtained by fermentation is added with a diketone with the final concentration of 0.3M and a reaction system of 80 percent isopropanol, and the conversion rate of a reaction substrate reaches up to 95 percent under the stirring of 200rpm at 37 ℃.

Example 4

The embodiment uses F153L single mutant strain to carry out selective catalytic synthesis of monoketone, and specifically comprises the following steps:

carbonyl reductase mutant bacterial E.coli BL21 (DE 3)/pET 3a-CTSDR-F153L 200g/L obtained by fermentation is added into a reaction system of diketone with the final concentration of 0.3M and 80% isopropanol, and the conversion rate of a reaction substrate is 32% under the stirring of 200rpm at 37 ℃.

Example 5

The embodiment uses M207F single mutant strain to carry out selective catalytic synthesis of monoketone, and specifically comprises the following steps:

carbonyl reductase mutant bacterial E.coli BL21 (DE 3)/pET 3a-CTSDR-M207F 200g/L obtained by fermentation is added into a reaction system of diketone with the final concentration of 0.3M and 80 percent isopropanol, and the conversion rate of a reaction substrate reaches up to 15 percent under the stirring of 200rpm at 37 ℃.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that the amino acid sequence of the carbonyl reductase mutant has any one of the following mutations compared to a reference sequence:

(1) Phe at position 153 is mutated to Leu;

(2) Met 207 to Phe;

2. A nucleic acid molecule encoding the carbonyl reductase mutant of claim 1.

3. An expression cassette or recombinant vector comprising the nucleic acid molecule of claim 2.

4. A recombinant bacterium or recombinant cell comprising: the nucleic acid molecule of claim 2 or the expression cassette or recombinant vector of claim 3.

5. The recombinant bacterium or recombinant cell according to claim 4, wherein the recombinant bacterium is selected from the group consisting of e.

6. A method of producing the carbonyl reductase mutant of claim 1, comprising the steps of: culturing the recombinant bacterium or recombinant cell of any one of claims 4-5 under conditions suitable for expression, thereby expressing a carbonyl reductase mutant; and/or isolating the carbonyl reductase mutant.

7. Use of a carbonyl reductase mutant according to claim 1 or a recombinant bacterium or recombinant cell according to any one of claims 4-5 for asymmetrically catalyzing the synthesis of a compound of the damascone series from a diketone compound.

8. The use according to claim 7, wherein in the reaction system for the synthesis of the damascone series compound, the reaction medium is isopropanol or other hydrogen donor;

preferably, the final concentration of the recombinant bacteria or recombinant cells in the reaction system is 10-300g/L, and the final concentration of the diketone compound is 0.3-3M;

preferably, the reaction condition is 10-45 ℃ and the reaction is carried out for 1-12 hours;

preferably, the reaction is carried out at 200-700 rpm;

preferably, the reaction is carried out in 80% -85% isopropanol reaction medium.

9. The use according to claim 8, wherein the carbonyl reductase mutant is provided in the reaction system in the form of wet cell obtained by induction culture of genetically engineered bacteria containing the carbonyl reductase mutant.

10. The use according to claim 7, wherein the damascone is selected from damascone a, damascone b or damascone b;

preferably, the damascone is selected from 3-hydroxy-1- (2, 6-trimethyl-3-cyclohexenyl) -1-butanone; the diketone compound is selected from 1- (2, 6-trimethyl-3-cyclohexen-1-yl) -1,3 butanedione.