CN117511893A - Carbonyl reductase mutant and application thereof - Google Patents

Abstract

The invention discloses a carbonyl reductase mutant and application thereof in the production of 4-chloro-3-hydroxybutyrate. The carbonyl reductase mutant has a mutation at least one of amino acids 60, 171, 182 or 220 relative to the wild-type carbonyl reductase. The carbonyl reductase mutant body provided by the invention can catalyze the ethyl 4-chloroacetoacetate to prepare the ethyl 4-chloro-3-hydroxybutyrate under an acidic condition, so that spontaneous hydrolysis of the ethyl 4-chloroacetoacetate is avoided, and the conversion efficiency is improved.

Description

Carbonyl reductase mutant and application thereof

Technical Field

The invention belongs to the fields of enzyme engineering and industrial bioconversion, and particularly relates to a carbonyl reductase mutant and application thereof in the synthesis of 4-chloro-3-hydroxybutyrate ethyl esterase.

Background

Ethyl 4-chloro-3-hydroxybutyrate was prepared from ethyl 4-chloroacetoacetate catalyzed by carbonyl reductase, ethyl 4-chloro-3-hydroxybutyrate was mainly used for l-carnitine synthesis. At present, the literature reports that carbonyl reductase catalyzing 4-chloroacetoacetic acid ethyl ester is optimal in pH 7.0+/-0.2, but when the reaction pH value is more than or equal to 7.0, the 4-chloroacetoacetic acid ethyl ester can be spontaneously hydrolyzed, and when the reaction pH value is 5.0-5.5, the spontaneous hydrolysis can not be generated, so that the carbonyl reductase capable of efficiently catalyzing the 4-chloroacetoacetic acid ethyl ester under the pH value of 5.0-5.5 needs to be constructed.

Disclosure of Invention

The purpose of the present disclosure is to provide a carbonyl reductase mutant with high expression activity, and to utilize the carbonyl reductase mutant to catalyze 4-chloroacetoacetic acid ethyl ester to prepare 4-chloro-3-hydroxybutyric acid ethyl ester under acidic (pH5.0-5.5) conditions, so as to avoid spontaneous hydrolysis of the 4-chloroacetoacetic acid ethyl ester, and improve conversion efficiency.

An aspect of the present disclosure provides a carbonyl reductase mutant having a mutation at least one of amino acids 60, 171, 182 or 220 relative to a wild-type carbonyl reductase; wherein the amino acid sequence of the wild-type carbonyl reductase is shown as SEQ ID NO. 1.

In some embodiments, the carbonyl reductase mutant has at least one amino acid mutation of L60I (leucine 60 to isoleucine), G171E (glycine 171 to glutamate), G182S (glycine 182 to serine), and V220E (valine 220 to glutamate).

In some embodiments, the carbonyl reductase mutant has the amino acid sequence shown in SEQ ID NO. 5, or an amino acid sequence with equivalent functions, which is formed by adding, deleting, replacing or modifying one or more amino acids to the amino acid sequence shown in SEQ ID NO. 5.

In some embodiments, the wild-type carbonyl reductase is a Rhodococcus (Rhodococcus) ST-10 carbonyl reductase having the amino acid sequence and nucleotide sequence shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

Another aspect of the present disclosure provides nucleic acid molecules encoding the above carbonyl reductase mutants.

In some embodiments, the nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO. 6, or a sequence complementary to the nucleotide sequence set forth in SEQ ID NO. 6, or a sequence having 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5, 99.8% or more than 99.9% homology to the nucleotide sequence set forth in SEQ ID NO. 6.

Yet another aspect of the present disclosure provides a recombinant expression vector comprising the above nucleic acid molecule.

Yet another aspect of the present disclosure provides a host cell containing a nucleic acid molecule or recombinant expression vector as described above.

In some embodiments, the host cell is E.coli (Escherichia Coli). Preferably, the host cell is an E.coli (Escherichia Coli) BL21 (DE 3) strain.

The host cells of the present disclosure are capable of highly expressing active carbonyl reductase enzymes.

Yet another aspect of the present disclosure provides a primer pair for PCR amplification of a gene encoding the carbonyl reductase mutant, the primer pair comprising:

forward primer: 5'-CCATATGAAGGCAATCCAGTACACGAG-3' (SEQ ID NO: 3),

reverse primer: 5'-CAAGCTT CTACAGACCAGGGACCACAA-3' (SEQ ID NO: 4).

Yet another aspect of the present disclosure provides the use of the carbonyl reductase mutant described above, the nucleic acid molecule described above, the recombinant expression vector described above, or the host cell described above, for the production or catalytic production of ethyl 4-chloro-3-hydroxybutyrate.

The carbonyl reductase mutant disclosed by the invention can catalyze the ethyl 4-chloroacetoacetate to prepare the ethyl 4-chloro-3-hydroxybutyrate under the acidic (pH 5.0-5.5) condition, so that spontaneous hydrolysis of the ethyl 4-chloroacetoacetate is avoided, and the conversion efficiency is improved.

In yet another aspect of the present disclosure, there is provided a method for producing ethyl 4-chloro-3-hydroxybutyrate, comprising contacting a substrate with the above-described carbonyl reductase mutant, or with a host cell expressing the carbonyl reductase mutant, and performing a fermentation reaction to produce ethyl 4-chloro-3-hydroxybutyrate.

In some embodiments, the substrate is ethyl 4-chloroacetoacetate.

In some embodiments, the host cell is a rhodococcus.

In some embodiments, the pH of the fermentation reaction is from 4.5 to 7.5, preferably from 5.0 to 6.0.

The disclosure further provides a method for constructing the carbonyl reductase mutant, comprising the following steps:

(1) Amplifying a coding gene CBR of carbonyl reductase (CBR) from genomic DNA of Rhodococcus (Rhodococcus) ST-10 by a PCR method;

(2) Respectively carrying out double enzyme digestion on the amplified cbr gene and pET-42a plasmid, connecting the cbr gene and pET-42a plasmid after enzyme digestion, and converting pET42a-cbr into an escherichia coli BL21 (DE 3) strain to obtain a cbr expression vector;

(3) Adopting error-prone PCR technology, taking the coding gene CBR of the CBR as a template, and amplifying to obtain a CBR mutant gene library;

(4) Connecting the error-prone PCR product obtained in the step (2) and an expression plasmid pET28a (+) after double digestion, transferring the connecting product into competent cells of escherichia coli BL21 (DE 3) for amplification, and obtaining and constructing a CBR mutant gene expression vector library;

(5) Randomly picking mutant strains for expression, measuring the CBR enzyme activities of the wild type and mutant type of rhodococcus, selecting one strain with the highest enzyme activity as a carbonyl reductase mutant, and measuring the gene sequence of the carbonyl reductase mutant.

In some embodiments, the construction method comprises the steps of:

(1) Amplifying a carbonyl reductase coding gene cbr in rhodococcus ST-10 by adopting a PCR method to obtain cbr genes;

(2) Respectively carrying out double enzyme digestion on the amplified cbr gene and pET-42a plasmid, connecting the cbr gene and pET-42a plasmid after enzyme digestion, and converting pET42a-cbr into an escherichia coli BL21 (DE 3) strain to obtain a cbr expression vector;

(3) Amplifying carbonyl reductase coding gene CBR in rhodococcus ST-10 by adopting an error-prone PCR method to obtain a CBR mutant gene library;

(4) And (3) respectively carrying out double enzyme digestion on the CBR mutant gene and the pET-42a plasmid, connecting the CBR mutant gene and the pET-42a plasmid after enzyme digestion, and converting pET42a-CBR into an escherichia coli BL21 (DE 3) strain to obtain a CBR mutant gene expression vector library.

(5) Randomly picking mutant strains for expression, and after expression, ultrasonically crushing thalli and purifying CBR mutant proteins. And (3) performing enzyme activity detection to finally obtain a strain CBR mutant with the highest enzyme activity, and extracting plasmid sequencing to determine the sequence.

The carbonyl reductase mutant constructed by the method can catalyze 4-chloro-3-hydroxybutyric acid ethyl ester to prepare 4-chloro-3-hydroxybutyric acid ethyl ester under the acidic (pH 5.0-5.5) condition, so that spontaneous hydrolysis of the 4-chloro-acetoacetic acid ethyl ester is avoided, and the conversion efficiency is improved.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

Definition of the definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.

The term "substitution" as used herein with respect to amino acids refers to the replacement of at least one amino acid residue in an amino acid sequence with another, different "replacement" amino acid residue. The term "insertion" as used herein with respect to amino acids refers to the incorporation of at least one additional amino acid into an amino acid sequence. Although the inserts typically consist of insertions of 1 or 2 amino acid residues, larger "peptide inserts" may also be made, for example insertions of about 3 to 5 or even up to about 10, 15 or 20 amino acid residues. As disclosed above, the inserted residues may be naturally occurring or non-naturally occurring. The term "deletion" as used herein with respect to amino acids refers to the removal of at least one amino acid residue from an amino acid sequence.

Mutants of the present disclosure or fragments thereof may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A "conservative amino acid substitution" is a substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, in this context, an essential or non-essential amino acid residue in a mutant is preferably replaced with another amino acid residue from the same side chain family.

"percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target sequence and the reference sequence. Since the gaps are not nucleotides or amino acids, the gaps present in the target sequence are not taken into account. Also, since the target sequence nucleotide or amino acid is counted, and the nucleotide or amino acid from the reference sequence is not counted, gaps in the reference sequence are not counted.

Percent sequence identity can be calculated by the following procedure: determining the number of positions in which the same amino acid residue or nucleobase occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software that is readily available for online use and download. Suitable software programs are available from a variety of sources for alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is the bl2seq, which is part of the BLAST suite of programs available from the national center for Biotechnology information, BLAST website (BLAST. Ncbi. Lm. Nih. Gov) of the U.S. government. Bl2seq uses BLASTN or BLASTP algorithms to make a comparison between two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, needle, stretcher, water or a part of the Matcher, bioinformatics program EMBOSS kit, and are also available from European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

The following examples are provided to aid in the understanding of the present invention. It is to be understood that these examples are illustrative of the present invention only and are not to be construed as limiting in any way. The actual scope of the invention is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the invention.

Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art and commercially available usual instruments and reagents, and can be referred to in the molecular cloning test guidelines (3 rd edition), the scientific press, the microbiological experiments (4 th edition), the higher education press, and the manufacturer's instructions for the corresponding instruments and reagents.

EXAMPLE 1 obtaining of Rhodococcus carbonyl reductase Gene (cbr)

The primer sequence is designed according to the sequence of the carbonyl reductase gene sequence (SEQ ID NO:2, the amino acid sequence of which is shown as SEQ ID NO: 2) of rhodococcus ST-10 by taking rhodococcus ST-10 genome DNA (1 mug) as a PCR reaction template. PCR amplification was performed according to the primer sequences shown below; cbr-F: reverse primer sequence cbr-R: /> Wherein, the italic letter parts are respectively enzyme cutting sites Nde I and HindIII; the PCR reaction was performed in a 50. Mu.L system under the following conditions: denaturation at 95℃for 3min, denaturation at 95℃for 50s, annealing at 58℃for 1min, extension at 72℃for 3min for 35 cycles; extending at 72 ℃ for 10min, and taking 3 mu L of PCR amplified products for agarose gel electrophoresis verification; and taking 100 mu L of PCR products for agarose gel electrophoresis, and recovering the target fragment according to the specification of a gel recovery kit.

EXAMPLE 2 construction of carbonyl reductase Gene expression vector

The PCR product of example 1 was digested with restriction enzymes NdeI and HindIII, and subjected to ligation with the plasmid pET-42a digested with NdeI and HindIII (available from Novagen Co.), the constructed vector was designated pET42a-cbr, and then E.coli BL21 (DE 3) strain (available from Promega Co.) was transformed with the ligation product pET42 a-cbr.

EXAMPLE 3 error-prone PCR amplification of the Rhodococcus Gene cbr

By utilizing the property that Taq DNA polymerase does not have 3'-5' proofreading function, under the conditions of high magnesium ion concentration (8 mmol/L) and dNTP concentration with different concentrations (wherein dATP and dGTP concentration are 1.5mmol/L and dTTP and dCTP concentration are 3.0 mmol/L), the frequency of random mutation is controlled, random mutation is introduced into a target gene, a mutation library is constructed, the value of template concentration A260 is 1000ng/mL, the enzyme concentration is 5U/mu L, the primer concentration is 100 mu M, and the optimal mutation rate in an experiment is about 0.6%;

error-prone PCR reaction System (100. Mu.L):

the PCR procedure was: pre-denaturation at 95℃for 3min; denaturation at 94℃for 1min, annealing at 56℃for 1min, extension at 75℃for 3min,45 cycles; finally, the PCR product is recovered by a gel recovery method after the extension at 75 ℃ for 15min, 5 mu L of the product is taken for 1% agarose gel electrophoresis inspection, and the product is preserved at-20 ℃ for standby.

EXAMPLE 4 construction of carbonyl reductase Gene mutant library

The error-prone PCR product was digested with restriction enzymes Nde I and HindIII, and subjected to ligation with pET-42a plasmid digested with Nde I and HindIII to construct a vector library pET42a-cbrM, and then pET42a-cbrM was transformed into E.coli BL21 (DE 3) strain to construct an expression mutant library.

EXAMPLE 5 construction of expression mutant library and screening of mutants

Randomly picking mutant strains into a 6-hole plate containing LB culture medium of 60 mug/mL kanamycin, culturing at 37 ℃ and 150rpm, adding IPTG (final concentration of 0.1 mmol/L) when the OD600 value reaches 0.6-0.8, and continuously culturing at 30 ℃ for 12h; the cells were collected by centrifugation, suspended in 50mmol/L, pH 8.0.0 Tris-HCl (containing 1mmol/L imidazole) buffer (the ratio of wet cells to buffer: 1g wet cells: 5mL buffer), and the cells were broken by ultrasonic waves in an ice bath, and the supernatant was collected after centrifugation, and then subjected to protein purification and enzyme activity measurement.

The specific method is as follows:

ni is taken ²⁺ 5mL of NTAAgarose was packed into a column, washed with 25mL of water, repeatedly washed twice, and then the column was equilibrated with 25mL of NAT-0 buffer (20 mM Tris-HCl pH7.9,0.5M NaCl), the above-mentioned disrupted cells were collected, and the supernatant obtained after centrifugation was added to Ni ²⁺ After repeated addition of 2-3 times to the NTA column, 25mL of NAT-1 buffer (i.e. NAT-0 buffer contains 80mmol/L imidazole) is used to wash the medium to remove the foreign proteins, finally the target proteins are eluted by using the buffer containing 300mmol/L imidazole, SDS-PAGE analysis is carried out to obtain specific protein bands meeting the expected size, and the concentration of the proteins is measured by the Brandford method.

The enzyme activity determination method comprises the following steps: to 5ml of a substrate reaction solution (0.1M potassium phosphate buffer, pH5.5, 100mM ethyl 4-chloroacetoacetate, 200mM glucose, 10mM NADPH), 0.5% (mass/volume ratio) of the purified protein was added and mixed uniformly, and the reaction was carried out at 30℃for 1 hour, the reaction solution was centrifuged at 12000rpm at 4℃for 10 minutes, and the supernatant was examined for the ethyl 4-chloroacetoacetate and ethyl 4-chloro-3-hydroxybutyrate contents.

The substrate conversion rate of the purified wild strain is calculated to be 0.57%, and the highest substrate conversion rate of the mutant strain M1 (CBRM 1) reaches 93.82%.

The mutant strain CBRM1 is selected for cloning, and plasmid pET42a-cbrM1 is extracted for PCR verification and sequencing identification, and the amino acid sequence corresponding to the determined carbonyl reductase mutant M1 is shown as SEQ ID NO. 5, and the nucleotide sequence is shown as SEQ ID NO. 6.

To 5ml of the substrate reaction solution (0.1M potassium phosphate buffer, pH5.5, 100mM ethyl 4-chloroacetoacetate, 200mM glucose, 10mM NADPH), 0.5% (mass/volume ratio) of the purified protein was added and mixed, and the reaction was carried out at pH 4.5 to pH 7.5 (30 ℃ C.) for 1 hour, to determine the ethyl 4-chloroacetoacetate and ethyl 4-chloro-3-hydroxybutyrate contents, and the conversion was calculated (see Table 1). As can be seen from the results, pH5.0-pH6.0 is the optimal reaction pH for CBRM 1.

TABLE 1 4 determination of ethyl chloroacetoacetate conversion

The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.

Claims (10)

1. A carbonyl reductase mutant, wherein the carbonyl reductase mutant has a mutation at least one of amino acids 60, 171, 182 or 220 relative to a wild-type carbonyl reductase, wherein the amino acid sequence of the wild-type carbonyl reductase is set forth in SEQ ID No. 1.

2. The carbonyl reductase mutant of claim 1, wherein the mutant amino acid position of the carbonyl reductase mutant comprises at least one of L60I, G171E, G S and V220E,

preferably, the carbonyl reductase mutant has an amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence with one or more amino acid additions, deletions, substitutions or modifications of the amino acid sequence shown in SEQ ID NO. 5.

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

4. The nucleic acid molecule of claim 3, wherein the nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO. 6, or a sequence complementary to the nucleotide sequence shown in SEQ ID NO. 6, or a sequence having homology of 95% or more to the nucleotide sequence shown in SEQ ID NO. 6.

5. A recombinant expression vector comprising the nucleic acid molecule of claim 3 or 4.

6. A host cell comprising the nucleic acid molecule of claim 3 or 4 or the recombinant expression vector of claim 5.

7. The host cell according to claim 6, wherein the host cell is an E.coli (Escherichia Coli), preferably an E.coli (Escherichia Coli) BL21 (DE 3) strain.

8. A primer pair for PCR amplification of a gene encoding the carbonyl reductase mutant of claim 1 or 2, the primer pair comprising:

forward primer: 5'-CCATATGAAGGCAATCCAGTACACGAG-3' (SEQ ID NO: 3),

reverse primer: 5'-CAAGCTT CTACAGACCAGGGACCACAA-3' (SEQ ID NO: 4).

9. Use of a carbonyl reductase mutant according to claim 1 or 2, a nucleic acid molecule according to claim 3 or 4, a recombinant expression vector according to claim 5, a host cell according to claim 6 or 7 for the production or catalytic production of ethyl 4-chloro-3-hydroxybutyrate.

10. A process for producing ethyl 4-chloro-3-hydroxybutyrate, which comprises contacting a substrate with the carbonyl reductase mutant of claim 1 or 2 or with a host cell expressing the carbonyl reductase mutant, performing a fermentation reaction to produce ethyl 4-chloro-3-hydroxybutyrate,

preferably, the substrate is ethyl 4-chloroacetoacetate,

preferably, the host cell is a rhodococcus,

preferably, the pH of the fermentation reaction is in the range of 4.5 to 7.5, preferably 5.0 to 6.0.