CN116555214A - Acyl-coa synthetase mutant and efficient synthesis of malonyl-coa thereof - Google Patents
Acyl-coa synthetase mutant and efficient synthesis of malonyl-coa thereof Download PDFInfo
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- CN116555214A CN116555214A CN202310499077.7A CN202310499077A CN116555214A CN 116555214 A CN116555214 A CN 116555214A CN 202310499077 A CN202310499077 A CN 202310499077A CN 116555214 A CN116555214 A CN 116555214A
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- acyl
- coa synthetase
- coa
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/32—Nucleotides having a condensed ring system containing a six-membered ring having two N-atoms in the same ring, e.g. purine nucleotides, nicotineamide-adenine dinucleotide
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses an acyl-CoA synthetase mutant and an efficient synthesis method of malonyl-CoA thereof, belonging to the technical field of enzyme engineering. The recombinant acyl-CoA synthetase mutant of the invention is ACS when the reaction condition is optimal P158R/V197T/W372F The specific enzyme activity of the enzyme is up to 51.3U/mg, which is improved by 1.6 times compared with the wild type, and malonyl-CoA can be efficiently synthesized by using crude enzyme liquid, which is equivalent to the catalytic effect of pure enzyme. Recombinant acylApplication of coenzyme A synthetase in malonyl-coenzyme A production, mutation of enzyme ACS by substrate addition P158R/V197T/W372F The conversion rate of the catalytic synthesis of malonyl-CoA is 98.5%, and the final yield is 26.4g/L. The invention provides a high-quality acyl-CoA synthetase mutant and a high-efficiency preparation method thereof for biocatalytic synthesis of malonyl-CoA.
Description
Technical Field
The invention relates to an acyl-CoA synthetase mutant and an efficient synthesis method of malonyl-CoA, belonging to the technical field of enzyme engineering.
Background
Malonyl-coa (Malonyl-coa), also known as Malonyl-coa, is an important intermediate metabolite, which is mainly used for the synthesis of fatty acids, polyketides, bio-based platform chemicals, etc., and has important significance in the fields of medicine, antibiotics, disease treatment, etc., but its expensive price brings a certain impediment to research and production, so that Malonyl-coa is catalytically synthesized using relatively inexpensive substrate coa and malonic acid, acyl-coa synthase.
Acyl-coa synthetases are one of the adenylate forming enzymes and are widely found in animals, plants and microorganisms in which malonyl-coa synthetases are involved primarily in malonic acid metabolism, and have been found in a variety of microorganisms including rhizobium sojae (Rhizobium japonicum), rhizobium trilobatum (Rhizobium trifolii), rhodopseudomonas palustris (Rhodopseudomonas palustris), streptomyces coelicolor (Streptomyces coelicolor), sinorhizobium meliloti (sinorhizobium meliloti) and pseudomonas fluorescens (Pseudominas fluorescens). At ATP and Mg 2+ Under the participation condition, the malonyl-CoA synthetase can convert coenzyme A and malonic acid into malonyl-CoA, the specific reaction mechanism is divided into two steps, firstly, the malonyl-CoA synthetase reacts with ATP to activate malonic acid to form an adenylate intermediate and PPi, the adenylate intermediate has higher energy and provides activation energy for the second step of reaction, and in the second step of reaction for forming thioester, the thiol group of pantetheine from coenzyme A attacks carboxylic acid carbon to replace an AMP leaving group to form malonyl-CoA.
As the escherichia coli is used as a host for expressing exogenous genes, the genetic background is clear, the technical operation is simple, the protein expression quantity is high, and the escherichia coli is the most widely used expression vector at present, so that the realization of the efficient synthesis of malonyl-CoA by the recombinant acyl-CoA synthetase has great significance for the industrialized production of malonyl-CoA.
Disclosure of Invention
The invention provides acyl-CoA synthetase (ACS) from Streptomyces sp, which has the sequence shown in SEQ ID NO.1 or SEQ ID NO. 2. The acyl-CoA synthetase is applied to the production of malonyl-CoA.
The invention provides an acyl-CoA synthetase mutant, which is obtained by mutating one or more of 158 th, 197 th and 372 th amino acids of acyl-CoA synthetase with an amino acid sequence shown in SEQ ID NO.1 from Streptomyces sp.
The acyl-CoA synthetase is an acyl-CoA synthetase with an amino acid sequence shown as SEQ ID NO. 1; or an acyl-coa synthetase having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the amino acid sequence shown in SEQ ID No. 1; or an acyl-coa synthetase gene having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence with the coding sequence set forth in SEQ ID No. 3.
In one embodiment of the invention, the at least one mutation comprises a substitution, deletion or addition.
In one embodiment of the present invention, the mutant is obtained by substituting at least one of the 158 th, 197 th and 372 th amino acids of the acyl-CoA synthetase having an amino acid sequence shown in SEQ ID NO. 1.
In one embodiment of the invention, the mutant is:
the amino acid sequence is shown as SEQ ID NO.1, namely, proline at 158 th position of acyl-CoA synthetase is mutated into arginine; named ACS P158R ；
Or mutated valine at position 197 of acyl-CoA synthetase with amino acid sequence shown in SEQ ID NO.1 into threonine; named ACS V197T ；
Or the tryptophan at 372 nd position of acyl-CoA synthetase with amino acid sequence shown as SEQ ID NO.1 is mutated into phenylalanine; named ACS W372F ；
Or mutation of proline at 158 th position of acyl-CoA synthetase with amino acid sequence shown in SEQ ID NO.1 into arginine, and mutation of valine at 197 th position into threonine; named ACS P158R/V197T ；
Or the proline at 158 th position of acyl-CoA synthetase with the amino acid sequence shown as SEQ ID NO.1 is mutated into arginine, and the tryptophan at 372 th position is mutated into phenylalanine; named ACS P158R/W372F ；
Or mutation of valine at 197 of acyl-CoA synthetase with amino acid sequence shown in SEQ ID NO.1 into threonine, and mutation of tryptophan at 372 into phenylalanine; named ACS V197T/W372F ；
Or mutation of proline at 158 th position of acyl-CoA synthetase with amino acid sequence shown in SEQ ID NO.1 to arginine, mutation of valine at 197 th position to threonine, mutation of tryptophan at 372 nd position to phenylalanine, named ACS P158R/V197T/W372F The method comprises the steps of carrying out a first treatment on the surface of the The amino acid sequence is shown as SEQ ID NO. 2; the nucleotide sequence is shown as SEQ ID NO. 4.
In one embodiment of the invention, the nucleotide sequence of the acyl-coa synthetase parent derived from Streptomyces sp.
The invention also provides a gene for encoding the mutant.
The invention also provides a recombinant vector carrying the gene.
In one embodiment of the present invention, the recombinant vector is a pET series vector or pRSF series vector or pGEX series vector as an expression vector.
In one embodiment of the present invention, the recombinant vector is an expression vector of pET-28 (a), pET-21 (a), pRSF-Duet-1 or pGEX-6P-1.
The invention also provides a recombinant cell for expressing the mutant, carrying the gene or carrying the recombinant vector.
In one embodiment of the invention, the recombinant cell is a bacterial or fungal expression host.
In one embodiment of the invention, the recombinant cell is a host cell of escherichia coli, bacillus subtilis, bacillus licheniformis and pichia pastoris.
The invention also provides recombinant escherichia coli which expresses the mutant.
In one embodiment of the present invention, the recombinant E.coli is a recombinant E.coli having pET-28a (+) vector and expressing the acyl-CoA synthetase shown as SEQ ID NO.1 or ACS shown as SEQ ID NO.2 in E.coli BL21 (DE 3) P158R /V197T/W372F Acyl-coa synthetase mutants or acyl-coa synthetase mutants as described above.
The invention also provides a method for constructing the recombinant bacterium, which comprises the following steps: the acyl-CoA synthetase gene is connected with a vector pET-28a (+) and the obtained recombinant expression vector is transformed into escherichia coli BL21 (DE 3) to obtain recombinant bacteria.
The invention also provides a method for improving the enzyme activity of acyl-CoA synthetase, which comprises the step of mutating one or more of amino acids 158, 197 and 372 of acyl-CoA synthetase with an amino acid sequence shown in SEQ ID NO.1 from Streptomyces sp.
In one embodiment of the invention, the method comprises mutating proline at position 158 of an acyl-coa synthetase having an amino acid sequence as shown in SEQ ID No.1 to arginine; or mutated valine at position 197 of acyl-CoA synthetase with amino acid sequence shown as SEQ ID NO.1 into threonine; or the 372 nd tryptophan of acyl coenzyme A synthetase with the amino acid sequence shown as SEQ ID NO.1 is mutated into phenylalanine;
or the proline at 158 th position of acyl-CoA synthetase with the amino acid sequence shown as SEQ ID NO.1 is mutated into arginine, and the valine at 197 th position is mutated into threonine; or the 158 th proline of acyl-CoA synthetase with the amino acid sequence shown as SEQ ID NO.1 is mutated into arginine, and the 372 nd tryptophan is mutated into phenylalanine; or the valine at 197 of acyl-CoA synthetase with the amino acid sequence shown in SEQ ID NO.1 is mutated into threonine, and the tryptophan at 372 is mutated into phenylalanine;
or the proline at 158 th position of acyl-CoA synthetase with the amino acid sequence shown in SEQ ID NO.1 is mutated into arginine, simultaneously the valine at 197 th position is mutated into threonine, and the tryptophan at 372 nd position is mutated into phenylalanine.
The invention also provides a method for soluble expression of acyl-CoA synthetase, which is prepared by adopting the recombinant cell fermentation.
In one embodiment of the present invention, the recombinant cell is an E.coli cell as an expression host.
In one embodiment of the invention, the method inoculates the recombinant E.coli into the medium, and induces culture at 17℃for 16h with 0.5mM IPTG.
In one embodiment of the invention, the recombinant E.coli is inoculated into a medium at a culture temperature of 17℃for 16 hours under induction conditions of 0.5mM IPTG.
In one embodiment of the invention, the culture medium formula is 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride.
The invention also provides an enzyme preparation for catalyzing and synthesizing malonyl-CoA, and the enzyme preparation contains the acyl-CoA synthetase mutant.
In one embodiment of the invention, the enzyme preparation is a liquid preparation, and the liquid preparation contains the acyl-CoA synthetase mutant and auxiliary materials.
In one embodiment of the present invention, the enzyme preparation is a lyophilized powder of the acyl-coa synthetase, and contains the acyl-coa synthetase and a protecting agent thereof.
The invention also provides a method for preparing malonyl-CoA, which comprises the step of preparing malonyl-CoA by catalyzing and using the acyl-CoA synthetase mutant or the recombinant cell with CoA, malonic acid and ATP as substrates.
In one embodiment of the present invention, the malonic acid is added in an amount of 15 to 51mM; the addition amount of coenzyme A is as follows: 5-35 mM; the addition amount of ATP is as follows: 10-70 mM.
The invention also provides application of the acyl-CoA synthetase mutant, the gene, the recombinant vector, the recombinant cell or the enzyme preparation in preparing malonyl-CoA or malonyl-CoA-containing.
In one embodiment of the invention, the method is used for efficient synthesis of malonyl-coa and yields are high.
In one embodiment of the invention, the product is a chemical.
Advantageous effects
(1) According to the invention, an acyl-CoA synthetase gene derived from Streptomyces sp is selected through sequence alignment, the expressed protein of the gene is not characterized yet, so that the expressed protein is expressed in Escherichia coli BL (DE 3), and the recombinant enzyme is characterized, so that the recombinant acyl-CoA synthetase with high expression efficiency and high enzyme activity is obtained, and the cost is reduced for preparing the recombinant acyl-CoA synthetase through downstream separation. The recombinant acyl-CoA synthetase is applied to malonyl-CoA production.
(2) The invention successfully constructs the recombinant strain E.coli BL21/pET28a-ACS capable of expressing the target gene with high efficiency and applies the recombinant strain E.coli BL21/pET28a-ACS to malonyl coenzyme A production. Purifying the crude enzyme liquid expressed by the recombinant bacteria by a His-Trap HP chromatographic column to obtain the recombinant pure enzyme. The optimal reaction pH of the pure enzyme is 8.0, the enzyme activity is most stable under the pH condition, and the residual relative enzyme activity is higher than 80% at the pH of 8.0. The optimal reaction temperature for the pure enzyme is 40℃but at this temperature the enzymeThe stability is poor, the enzyme activity is reduced rapidly, and the enzyme activity is slightly lower than 40 ℃ but the stability is the best at 35 ℃. Under the reaction condition of 35 ℃ and pH 8.0, the specific enzyme activity of the acyl-CoA synthetase can reach 32.0U/mg, and the acyl-CoA synthetase mutant ACS P158R/V197T/W372F The specific enzyme activity of (C) can reach 51.3U/mg, V max The value is higher than that of the malonyl-CoA synthetase which has been reported. The recombinant acyl-CoA synthetase and the mutant thereof have the characteristics of high enzyme activity and good stability.
(3) The recombinant acyl-CoA synthetase is applied to malonyl-CoA production, malonyl-CoA can be efficiently synthesized, high yield is obtained, under the current experimental conditions, the malonyl-CoA is obtained by ACS catalysis after 3.5 hours of reaction in a mode of supplementing substrates in batches, and the yield is 24.2g/L, and the conversion rate is 90.3%; ACS (ACS) P158R/V197T/W372F The yield of the malonyl-CoA obtained by catalysis is 26.4g/L, and the conversion rate is 98.5%. The recombinant acyl-coa synthetases and mutants thereof catalyze the production of malonyl-coa more rapidly and at higher concentrations than the metabolic pathway engineering or cell-free systems currently in use.
Drawings
Fig. 1: alignment of the amino acid sequence of Streptomyces sp.ACS with other known malonyl-CoA synthetases.
Fig. 2: purifying SDS-PAGE patterns of the obtained recombinant enzyme; m: marker,1: wild-type ACS,2: mutant ACS P158R /V197T/W372F 。
Fig. 3: HPLC result diagram of the product obtained after ACS catalysis; CK: blank control with no enzyme added, sample: reaction samples after catalytic ACS, standard: malonyl-coa standard.
Detailed Description
Technical terms:
acyl-coa synthase: the term "acyl-coa synthetase" refers to an enzyme in class EC 6.2.1.3 as defined by enzyme nomenclature. For the purposes of the present invention, the "acyl-CoA synthetase activity" is determined according to the procedure described in the examples. In one aspect, the acyl-coa synthetase of the invention is an acyl-coa synthetase having an amino acid sequence as shown in SEQ ID No. 1; or an acyl-coa synthetase having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the amino acid sequence shown in SEQ ID No. 1; or an acyl-coa synthetase gene having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence with the coding sequence set forth in SEQ ID No. 3.
Expression: the term "expression" includes any step involving the production of acyl-coa synthetase mutants, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: the term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding an acyl-coa synthetase mutant of the invention and operably linked to control sequences that provide for its expression.
Fragments: the term "fragment" means a polypeptide that lacks one or more (e.g., several) amino acids at the amino and/or carboxy terminus of the polypeptide; wherein the fragment has acyl-coa synthetase activity. In one aspect, the fragment of the invention comprises an acyl-coa synthetase having the amino acid sequence shown in SEQ ID No. 1; or an acyl-coa synthetase having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the amino acid sequence shown in SEQ ID No. 1; or an acyl-coa synthetase gene having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence with the coding sequence set forth in SEQ ID No. 3.
Host cell: the term "host cell" means any cell type that is readily transformed, transfected, transduced, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any parent cell progeny that are not identical to the parent cell due to mutations that occur during replication.
The host cell may be any cell useful in the recombinant production of acyl-coa synthetase mutants, such as a prokaryotic cell or a eukaryotic cell.
The prokaryotic host cell may be any gram-positive or gram-negative bacterium. Gram positive bacteria include, but are not limited to: bacillus, clostridium, enterococcus, geobacillus (Geobacillus), lactobacillus, lactococcus, bacillus, staphylococcus, streptococcus and streptomyces. Gram-negative bacteria include, but are not limited to, campylobacter, escherichia, flavobacterium, fusobacterium, helicobacter, mirobacter, neisseria, pseudomonas, salmonella, and ureaplasma.
The host cell may also be a eukaryotic organism, such as a mammalian, insect, plant or fungal cell.
Malonyl-coa: an organic matter with chemical formula of C 24 H 38 N 7 O 19 P 3 S is a derivative of coenzyme A.
Enzyme preparation: the enzyme-purified and processed biological product with the catalytic function is mainly used for catalyzing various chemical reactions in the production process, has the characteristics of high catalytic efficiency, high specificity, mild acting condition, energy consumption reduction, chemical pollution reduction and the like, and has the application fields of food (bread baking industry, flour deep processing, fruit processing industry and the like), textile, feed, detergent, papermaking, leather, medicine, energy development, environmental protection and the like. The enzyme preparation is of biological origin, is generally safer, and can be used in proper amount according to production requirements.
The following examples relate to the following media:
Luria-Bertani (LB) medium: 10g/L of tryptone, 10g/L of sodium chloride and 5g/L of yeast extract.
The detection method involved in the following examples is as follows:
enzyme activity assay for acyl-coa synthetase
Because malonyl-CoA has a better response value at the ultraviolet 254nm, the enzyme activity of acyl-CoA synthetase is measured by using high performance liquid chromatography, and the reaction conditions are as follows: 9mM MgCl 2 100mM 4-hydroxyethyl piperazine ethane sulfonic acid (HEPES), 5mM coenzyme A, 10mM ATP, 10mM malonic acid, 10% (w/v) glycerol, 10nM acyl-CoA synthetase, pH 8.0, 200. Mu.L of the reaction system, placing in a 1.5mL centrifuge tube, reacting at 35℃in a constant temperature metal bath for 30 minutes, adding 200. Mu.L of methanol to terminate the reaction, standing at room temperature for 30 minutes, centrifuging at 12,000Xg for 10 minutes to remove denatured protein precipitate, and taking the supernatant as a sample. Malonyl-coa was detected using an agilent 1260 liquid chromatograph and Waters C18 column, mobile phase a was ultrapure water containing 0.1% trifluoroacetic acid, and liquid B was methanol containing 0.1% trifluoroacetic acid. The detection conditions are as follows: and (3) carrying out gradient elution on 10 mu L of 10% -67% B liquid at a flow rate of 1mL/min for 20min, and detecting at the ultraviolet 254nm at a column temperature of 30 ℃. And drawing a standard curve by using a malonyl-CoA standard substance, and calculating the content of malonyl-CoA through the peak area and standard curve, and further calculating the enzyme activity and specific enzyme activity of the acyl-CoA synthetase.
Definition of acyl-coa synthetase the enzyme activity units are: the amount of enzyme required to catalyze the conversion of 1. Mu. Mol of substrate to malonyl-CoA per minute is one enzyme activity unit, i.e., 1U=1. Mu. Mol/min.
The calculation formula is enzyme activity (U) = (mAU x s x 0.4 x 1000)/(8766.7 x 853 x T), wherein mAU x s is the peak area of malonyl-CoA detected by liquid phase, 8766.7 is the slope of malonyl-CoA standard curve, 853 is the molecular weight of malonyl-CoA, 0.4 is the total volume of the reaction system after adding equal volume of methanol to terminate the reaction, and T is time (min).
Specific enzyme activity = activity (U)/protein mass (mg)
Example 1: construction of recombinant E.coli BL21/pET28a-ACS
By sequence alignment in the Non-Redundant Protein Sequence database, an acyl-coa synthetase from Streptomyces sp was selected, the amino acid sequence of which was compared to malonyl-coa synthetases from Bradyrhizobium japonicum, rhizobium trifolii and Rhodopseudomonas palustris (fig. 1), and the enzyme was found to have a sequence and active site conserved for malonyl-coa synthetase, presumably for its function in malonyl-coa synthesis.
(1) Construction of recombinant plasmid pET28a-ACS
The amino acid sequence (shown as SEQ ID NO. 1) from Streptomyces sp. Encoding acyl-CoA synthetase was sent to the division of biological engineering (Shanghai) and subjected to codon optimization (shown as SEQ ID NO. 3), and cloned into vector pET-28a (+), the cloning site was BamH I/Xho I, the vector resistance was kanamycin resistance, and recombinant plasmid pET28a-ACS was obtained and stored at-20 ℃.
(2) Construction of recombinant bacteria
1 mu L of plasmid is added into 100 mu L of E.coli BL21 (DE 3) competent cell suspension, after ice bath for 30min, the mixture is placed in a metal bath at 42 ℃ for heat shock for 90s, after heat shock, the mixture is rapidly placed on ice for 3-5 min, 700 mu L of LB liquid medium is added into a centrifuge tube, and the mixture is subjected to shaking culture for 1h at 37 ℃ and 200 rpm. mu.L of the bacterial liquid was plated on LB solid medium plates containing 50. Mu.g/mL kanamycin sulfate. The cells were placed in a constant temperature incubator at 37℃overnight (about 10 hours).
The recombinant strain E.coli BL21 (DE 3)/pET 28a-ACS is prepared.
Example 2: mutant design and construction of acyl-coa synthetase
Amino acid residues of the acyl-CoA synthetase that may have interactions with the substrate CoA or malonate or intermediates individually were found to be different from other acyl-CoA synthetases during sequence alignment, wherein P158 interacted with the intermediates and V197 interacted with carboxylic acid; w372, a362, R374 interact with CoA.
The specific process is as follows:
(1) Primers shown in Table 1 were designed, mutants were obtained by single point mutation of whole plasmid PCR, and then the obtained positive mutants were subjected to combinatorial mutation.
Table 1: primer for acyl-CoA synthetase mutation design
The PCR amplification reaction system is as follows: 2 XPimeSTAR 25. Mu. L, pET28a-ACS plasmid 1. Mu. L, ddH 2 O10. Mu.L, 2. Mu.L each of the primers. PCR procedure: 98 ℃ for 5min; 30 cycles were performed at 98℃for 30s,55℃for 30s, and 72℃for 30 s; 72℃for 10min and 16℃for 10min.
(2) mu.L of 10X Quickcut Green Buffer was added to each PCR tube after completion of PCR, and the mixture was homogenized. The correct band size was verified by 1% agarose gel electrophoresis and product purification was performed.
(3) The DNA fragment and the linearization vector pET28a are subjected to homologous recombination according to the specification by using a Vazyme single fragment homologous recombination kit, a homologous recombination system is added into 100 mu L E.coli BL21 (DE 3) competent cells, the competent cells are subjected to ice bath for 30min, then the mixture is subjected to metal bath heat shock at 42 ℃ for 90s, the mixture is rapidly subjected to ice for 3-5 min after heat shock, 700 mu L LB liquid culture medium is added into a centrifuge tube, the temperature is 37 ℃, the speed is 200rpm, and the mixture is subjected to shaking culture for 1h.
mu.L of the bacterial liquid was plated on LB solid medium plates containing 50. Mu.g/mL kanamycin sulfate. The cells were placed in a constant temperature incubator at 37℃overnight (about 10 hours). Single colonies were picked up and cultured in 5mL LB liquid medium tubes containing 50. Mu.g/mL kanamycin sulfate at 37℃and 200rpm with shaking for about 12 hours, and plasmids were extracted for sequencing to verify correctness.
Recombinant bacteria containing different mutants are respectively prepared:
E.coli BL21(DE3)/pET28a-ACS P158R 、E.coli BL21(DE3)/pET28a-ACS V197T 、E.coli BL21(DE3)/pET28a-ACS A362K 、E.coli BL21(DE3)/pET28a-ACS W372F 、E.coli BL21(DE3)/pET28a-ACS R374I 、E.coli BL21(DE3)/pET28a-ACS P158R/V197T 、E.coli BL21(DE3)/pET28a-ACS P158R/W372F 、E.coli BL21(DE3)/pET28a-ACS V197T/W372F 、E.coli BL21(DE3)/pET28a-ACS P158R/V197T/W372F 。
example 3: expression purification of recombinant acyl-coa synthetases and mutants thereof
The method comprises the following specific steps:
(1) The positive clone single colony prepared in the example 2 is respectively picked up and cultured in a 5mL LB liquid medium test tube containing 50 mug/mL kanamycin sulfate at 37 ℃ and 200rpm under shaking for about 8 hours; seed solutions are prepared respectively.
(2) Inoculating the seed solution into 250mL LB liquid medium shake flask containing 50 μg/mL kanamycin sulfate according to 1% (v/v) inoculum size, respectively, culturing at 37deg.C under shaking at 200rpm to OD 600 After reaching 0.6 to 0.8 (about 2 hours), the culture was induced by cooling the flask in an ice-water mixture at 10min,0.5mM IPTG,17 ℃for about 16 hours, and then the cells were collected by centrifugation at 6,000Xg for 10 minutes and washed twice with physiological saline.
The cells were resuspended in protein purification buffer A (100mM HEPSE,500mM NaCl,10% (w/v) glycerol, pH 7.5), disrupted with an ultrasonic disrupter under ice bath conditions, centrifuged at 10,000Xg at 4℃for 30min to remove cell debris, and the supernatants were filtered through a 0.22 μm aqueous filter to give crude enzyme solutions, respectively.
(3) The crude enzyme solution was purified using a HisTrap HP column (GE Healthcare, chicago, USA). First, 5 column volumes were equilibrated with protein purification buffer a. After loading, the hybrid protein was washed with 3% protein purification buffer B (100mM HEPSE,500mM NaCl,10% (w/v) glycerol, 1M imidazole, pH 7.5), then the target protein was eluted with 30% protein purification buffer B and collected.
And (3) desalting the enzyme solution purified by the nickel column by using a Sephadex G-25 desalting column to remove imidazole in the enzyme solution. Washing and balancing 3 column volumes with ultrapure water and protein purification buffer solution A respectively, loading, eluting protein with the protein purification buffer solution A, performing SDS-PAGE electrophoresis on desalted pure enzyme solution, observing the band result to determine whether a single target purified protein is obtained, and purifying to obtain pure enzyme as shown in figure 2: wild-type ACS, ACS P158R 、ACS V197T 、ACS A362K 、ACS W372F 、ACS R374I 、ACS P158R/V197T 、ACS P158R/W372F 、ACS V197T/W372F 、ACS P158R/V197T/W372F 。
Example 4: verification of acyl-coa synthetase function
The method for determining the enzyme activity is utilized to verify and identify the product obtained after ACS catalysis, and comprises the following specific steps:
the reaction system is as follows: 200. Mu.L, 9mM MgCl 2 100mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), 5mM coenzyme A, 10mM ATP, 10mM malonic acid, 10% (w/v) glycerol, 10nM wild-type acyl-CoA synthetase ACS;
reaction conditions: at pH 8.0, placing in a 1.5mL centrifuge tube, reacting at 35deg.C in a constant temperature metal bath for 30min, adding 200 μl methanol to terminate the reaction, standing at room temperature for 30min, centrifuging at 12,000Xg for 10min to remove denatured protein precipitate, and collecting supernatant as sample. Malonyl-coa was detected using an agilent 1260 liquid chromatograph and a Waters C18 column, the results are shown in figure 3.
The results show that: compared with blank control, the reaction sample has one more chromatographic peak at 7.410min, and compared with the retention time 7.328min of the malonyl-CoA standard, the time shift of 7.410min is in a reasonable range, and is determined as the chromatographic peak of malonyl-CoA, and the product is malonyl-CoA.
The acyl-CoA synthetase has the function of synthesizing malonyl-CoA.
Example 5: acyl-coa synthetase and mutant enzyme activity assay thereof
As malonyl-CoA has a better response value at the ultraviolet 254nm, the enzyme activity of the acyl-CoA synthetase is measured by using high performance liquid chromatography to detect malonyl-CoA, and the specific enzyme activity of the acyl-CoA synthetase mutant pure enzyme prepared in the example 3 is detected. The results are shown in Table 2, and Table 2 shows specific enzyme activities of various acyl-CoA synthetases and mutants thereof.
Table 2: specific enzyme activity of different mutants of acyl-coa synthetase
The results show that the positive mutantACS P158R 、ACS V197T 、ACS W372F And ACS R374I The specific enzyme activities of the mutant are 39.0U/mg, 41.2U/mg, 44.5U/mg and 34.7U/mg respectively, and the ACS is finally obtained after the combined mutation of the positive mutant P158R /V197T/W372F The specific enzyme activity reaches 51.3U/mg.
It can be seen that the forward mutant ACS P158R/V197T/W372F The specific enzyme activity is highest, thus, subsequent studies use ACS P158R /V197T/W372F Is the study object.
Example 6: acyl-coa synthetase and mutant enzyme activity optimum pH
The optimal reaction pH of the recombinant acyl-CoA synthetase mutant pure enzyme prepared in example 3 was measured under different pH conditions, and the specific reaction system was the same as that of example 4, except that the pH was adjusted to 6.0, 7.0, 7.5, 8.0, 8.5, 9.0 and 10.0, respectively, and the reaction was terminated by adding 200. Mu.L of methanol after 10min reaction at 35 ℃; recombinant acyl-CoA synthetase wild-type enzyme and mutant ACS measured at different pH values P158R/V197T/W372F The specific enzyme activities of (2) are shown in Table 3.
Table 3: specific enzyme activities of recombinant acyl-coa synthetases and mutants at different pH
The results show that the enzyme has higher enzyme activity under alkaline condition, the optimal pH value is 8.0, and the enzyme activity is greatly reduced under acidic condition.
Example 7: acyl-coa synthetase and mutant enzyme activity optimum temperature
The optimum reaction temperature of the recombinant acyl-CoA synthetase mutant pure enzyme prepared in example 3 was measured under different temperature conditions, and the specific reaction system was the same as that of example 4, except that the reaction was carried out at 20℃at 30℃at 35℃at 40℃at 50℃with pH of 8.0, and after 10min of reaction, 200. Mu.L of methanol was added to terminate the reaction. The specific enzyme activities of the recombinant acyl-CoA synthetase and mutant measured at different temperatures are shown in Table 4.
Table 4: specific enzyme activity of recombinant acyl-coa synthetase and mutants at different temperatures
The results show that the enzyme activity is highest at 40 ℃.
Example 8: acyl-coa synthetase and mutant enzyme activity pH stability
The recombinant acyl-CoA synthetase mutant pure enzyme prepared in the example 3 is treated in protein purification buffer solution A with different pH values at 35 ℃ for 1h and then is used for reaction in reaction solutions with different pH values to determine specific enzyme activity. Residual relative enzyme activities at different pH are shown in Table 5.
Table 5: residual relative enzyme activities of recombinant acyl-CoA synthetase and mutant after heat preservation for 1h at different pH values
The results show that the stability is best at pH 8.0, the relative specific enzyme activity is higher than 80%, and the stability of the mutant is better than that of the wild type.
Example 9: acyl-coa synthetase and mutant enzyme activity temperature stability
The recombinant acyl-CoA synthetase mutant pure enzyme prepared in the example 3 is treated in protein purification buffer solution A with different temperatures for 1h and then is used for reaction in reaction solutions with different temperatures to determine specific enzyme activity. Residual relative enzyme activities at different temperatures are shown in table 6.
Table 6: residual enzyme activity of recombinant acyl-CoA synthetase and mutant after heat preservation for 1h at different temperatures
The results show that the stability of the enzyme is best at 35 ℃, the relative specific enzyme activity is higher than 80%, and the stability of the mutant is better than that of the wild type.
Example 10: optimization of conditions for catalytic synthesis of malonyl-coenzyme by recombinant acyl-coa synthetase
(1) Influence of different substrate ratios on conversion
The reaction system is as follows: 200. Mu.L, 9mM MgCl 2 100mM HEPES, 15mM ATP, 10% (w/v) glycerol, 10. Mu.M acyl-CoA synthetase ACS P158R/V197T/W372F The ratio of the mutant, coenzyme A and malonic acid is 1: 15. 1: 5. 1: 3. 1: 2. 1:1.5, the addition amount of coenzyme A and malonic acid is as follows: 15mM malonic acid, 1-10 mM coenzyme A;
the reaction conditions are as follows: the conversion of the product was determined after 2h reaction at 35℃at pH 8.0. The conversion of malonyl-coa at different substrate ratios is shown in table 7.
Wild ACS is found in coa: malonic acid = 1: the conversion rate at 3 is up to 91.8%, ACS P158R/V197T/W372F Mutants were found to be at coa: malonic acid = 1: the conversion rate at 2 is up to 94.9%. It is indicated that the ratio of coenzyme A to malonic acid should not differ too much in the catalytic reaction.
Table 7: influence of different substrate concentration ratios on malonyl-CoA conversion
(2) Effect of different ATP concentrations on conversion
In the same manner as in step (1), the conversion of the product was measured after the reaction was performed for 0.5 hours by adding 5mM CoA, 15mM malonic acid and adjusting the ATP concentration to 5mM, 10mM, 15mM, 20mM and 25mM, respectively, without changing the other reaction conditions (Table 8).
Table 8: effect of ATP concentration on malonyl-coa conversion
The conversion of malonyl-CoA at various ATP concentrations is shown in Table 8 whenAt an ATP concentration of 10mM, the conversion was highest, with wild-type ACS and ACS P158R/V197T/W372F 93.8% and 95.3%, respectively. Indicating that the ATP is needed in excess but the concentration is not easily too high in the catalytic reaction, which reduces the conversion rate of the reaction products.
(3) Different Mg 2+ Effect of concentration on conversion
The embodiment is the same as in step (1), except that the other reaction conditions are unchanged, and 5mM CoA, 15mM malonic acid, 10mM ATP, mgCl is added 2 The concentrations were adjusted to 3mM, 6mM, 9mM, 10mM and 15mM, respectively, and the conversion of the products was measured after 0.5 hour of the reaction (Table 9).
Table 9: mg of 2+ Effect of concentration on malonyl-CoA conversion
The results show that different Mg 2+ The conversion of malonyl-CoA at concentration is shown in Table 9 when Mg 2+ At a concentration of 3mM, the conversion was highest, wild type and ACS P158R/V197T/W372F 97.6% and 99.7%, respectively; along with Mg 2+ The concentration is increased, the conversion rate is reduced continuously, which indicates that Mg in the catalytic reaction 2+ The concentration is not easily too high, and the conversion rate of the reaction is reduced.
Example 11: application of recombinant acyl-coa synthetase crude enzyme in high malonyl-coa production
In order to reduce the industrial production cost, the crude enzyme liquid obtained by ultrasonic disruption of recombinant strain cells and removal of cell fragments is used for catalytic reaction, and the result shows that the crude enzyme liquid can obtain the same catalytic effect as that of pure enzyme, and the conversion rate is higher than 98%. Under the optimal reaction condition, expanding the reaction system to 100mL and increasing the substrate concentration, and finding that the reaction rate is reduced when the substrate concentration is too high, possibly including substrate inhibition, optimizing the condition of biocatalysis synthesis by adding substrates in batches in order to reduce the influence of the substrate inhibition on the enzyme; the specific process is as follows:
initial conditions were 3mM MgCl 2 、100mM HEPES、5mM CoA, 15mM malonic acid, 10mM ATP, 10% (w/v) glycerol, pH 8.0, crude enzyme solution (addition amount corresponds to 5g/L fresh cell), 100rpm,35℃were reacted, and after every 30 minutes of reaction, 5mM CoA, 6mM malonic acid and 10mM ATP were added; the total reaction time was 3.5 hours and the results are shown in Table 10.
Table 10: recombinant acyl-CoA synthetase and mutant catalyzed high concentration substrate conversion rate and yield under different reaction time conditions
The result shows that after six times of substrate addition, the yield of malonyl-CoA obtained by ACS catalysis is 24.2g/L, and the conversion rate is 90.3%; the malonyl-CoA yield obtained by the catalysis of the P158R/V197T/W372F mutant enzyme is 26.4g/L, and the conversion rate is 98.5%.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (24)
1. An acyl-coa synthetase mutant, characterized in that the mutant is obtained by mutating at least one of amino acids 158, 197, 372 of an acyl-coa synthetase;
the acyl-CoA synthetase is an acyl-CoA synthetase with an amino acid sequence shown as SEQ ID NO. 1; or an acyl-coa synthetase having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the amino acid sequence shown in SEQ ID No. 1; or an acyl-coa synthetase gene having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence with the coding sequence set forth in SEQ ID No. 3.
2. The acyl-coa synthetase mutant according to claim 1, wherein the at least one mutation comprises a substitution, a deletion or an addition.
3. The mutant acyl-coa synthetase according to claim 2, wherein the mutant is obtained by substituting at least one of amino acids 158, 197 and 372 of the acyl-coa synthetase having an amino acid sequence shown in SEQ ID No. 1.
4. The acyl-coa synthetase mutant according to claim 3, wherein the mutant is:
the amino acid sequence is shown as SEQ ID NO.1, and the 158 th proline of the acyl-CoA synthetase is replaced by arginine; or substitution of valine at position 197 of acyl-CoA synthetase with amino acid sequence shown in SEQ ID NO.1 for threonine; or substituting tryptophan at 372 nd position of acyl-CoA synthetase with amino acid sequence shown in SEQ ID NO.1 into phenylalanine;
or the amino acid sequence is shown in SEQ ID NO.1, the 158 th proline of the acyl-CoA synthetase is replaced by arginine, and the 197 th valine is replaced by threonine; or the proline at 158 th position of acyl-CoA synthetase with the amino acid sequence shown as SEQ ID NO.1 is replaced by arginine, and the tryptophan at 372 th position is replaced by phenylalanine; or substitution of valine at 197 of acyl-CoA synthetase with amino acid sequence shown in SEQ ID NO.1 for threonine, and substitution of tryptophan at 372 for phenylalanine;
or the amino acid sequence is shown in SEQ ID NO.1, the 158 th proline of the acyl-CoA synthetase is replaced by arginine, the 197 th valine is replaced by threonine, and the 372 nd tryptophan is replaced by phenylalanine.
5. The acyl-coa synthetase mutant according to claim 4, wherein the nucleotide sequence encoding the acyl-coa synthetase is set forth in SEQ ID No. 3.
6. A gene encoding the acyl-CoA synthetase mutant as claimed in any of claims 1 to 5.
7. A recombinant vector carrying the gene according to claim 6.
8. The recombinant vector according to claim 7, wherein the recombinant vector is an expression vector comprising a pET-series vector, a pRSF-series vector or a pGEX-series vector.
9. The recombinant vector according to claim 8, wherein the recombinant vector is an expression vector of pET-28 (a), pET-21 (a), pRSF-Duet-1 or pGEX-6P-1.
10. A recombinant cell expressing the mutant according to any one of claims 1 to 5, or carrying the gene according to claim 6, or carrying the recombinant vector according to any one of claims 7 to 9.
11. The recombinant cell of claim 10, wherein the recombinant cell is bacterial or fungal as an expression host.
12. The recombinant cell of claim 11, wherein the recombinant cell is a host cell of escherichia coli, bacillus subtilis, bacillus licheniformis, or pichia pastoris.
13. A method for improving the enzymatic activity of acyl-coa synthetase, which is characterized in that one or more of 158 th, 197 th and 372 th amino acids of the acyl-coa synthetase with an amino acid sequence shown in SEQ ID No.1 are mutated.
14. The method of claim 13, wherein the proline at position 158 of the acyl-coa synthetase having the amino acid sequence shown in SEQ ID No.1 is mutated to arginine; or mutated valine at position 197 of acyl-CoA synthetase with amino acid sequence shown as SEQ ID NO.1 into threonine; or the 372 nd tryptophan of acyl coenzyme A synthetase with the amino acid sequence shown as SEQ ID NO.1 is mutated into phenylalanine;
or the proline at 158 th position of acyl-CoA synthetase with the amino acid sequence shown as SEQ ID NO.1 is mutated into arginine, and the valine at 197 th position is mutated into threonine; or the 158 th proline of acyl-CoA synthetase with the amino acid sequence shown as SEQ ID NO.1 is mutated into arginine, and the 372 nd tryptophan is mutated into phenylalanine; or the valine at 197 of acyl-CoA synthetase with the amino acid sequence shown in SEQ ID NO.1 is mutated into threonine, and the tryptophan at 372 is mutated into phenylalanine;
or the proline at 158 th position of acyl-CoA synthetase with the amino acid sequence shown in SEQ ID NO.1 is mutated into arginine, simultaneously the valine at 197 th position is mutated into threonine, and the tryptophan at 372 nd position is mutated into phenylalanine.
15. A method for the soluble expression of acyl-coa synthetase, characterized in that it is prepared by fermentation of recombinant cells according to any one of claims 10 to 12.
16. The method of claim 15, wherein the method comprises inoculating recombinant escherichia coli into a culture medium for fermentation to obtain the soluble expression acyl-coa synthetase.
17. The method of claim 16, wherein the recombinant E.coli is pET-28a (+) and E.coli BL21 (DE 3) is an expression host.
18. An enzyme preparation for catalytic synthesis of malonyl-coa, characterized in that the enzyme preparation comprises the acyl-coa synthetase mutant according to any one of claims 1 to 5.
19. The enzyme preparation according to claim 18, characterized in that the enzyme preparation is a liquid preparation.
20. The enzyme preparation according to claim 19, wherein the enzyme preparation is a lyophilized powder comprising the acyl-coa synthetase mutant and a protecting agent.
21. A method for preparing malonyl-coa, characterized in that malonyl-coa is prepared by catalytic synthesis using the acyl-coa synthetase mutant according to any one of claims 1 to 5, or the recombinant cell according to any one of claims 10 to 12, with coa and malonic acid as substrates.
22. The method for producing malonyl-coa according to claim 21, wherein the malonic acid is added in an amount of 15 to 51mM; the addition amount of coenzyme A is as follows: 5-35 mM; the addition amount of ATP is as follows: 10-70 mM.
23. Use of an acyl-coa synthetase mutant according to any one of claims 1 to 5, or a gene according to claim 3, or a recombinant vector according to any one of claims 7 to 9, or a recombinant cell according to any one of claims 10 to 12, or an enzyme preparation according to any one of claims 18 to 20, for the preparation of malonyl-coa or a malonyl-coa containing product.
24. The use according to claim 23, wherein the product is a chemical.
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