CN116904409B - FDH mutant with improved protein soluble expression and encoding gene thereof - Google Patents

FDH mutant with improved protein soluble expression and encoding gene thereof Download PDF

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CN116904409B
CN116904409B CN202310571222.8A CN202310571222A CN116904409B CN 116904409 B CN116904409 B CN 116904409B CN 202310571222 A CN202310571222 A CN 202310571222A CN 116904409 B CN116904409 B CN 116904409B
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amino acid
mutant
fdh
formate dehydrogenase
soluble expression
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CN116904409A (en
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刘薇
王浩博
陈波
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Hangzhou Liwen Institute Biotechnology Co ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
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    • 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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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/01002—Formate dehydrogenase (1.2.1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Abstract

The invention discloses an FDH mutant with improved protein soluble expression and a coding gene thereof, and the invention carries out sequence modification on wild formate dehydrogenase through a Lesign platform. The protein soluble expression quantity and the thermal stability of the mutant RP 31-37 obtained by calculation are improved greatly. In particular to RP31 and RP33, the soluble expression quantity reaches more than 300% of the wild type, and the residual enzyme activity after incubation for 90min at 60 ℃ reaches about 200% of the wild type. Therefore, the mutant formate dehydrogenase of the invention can obtain more enzyme amount for catalysis under the same fermentation condition, and the NAD involved by the mutant + The NADH reaction system can react at a higher temperature, the catalytic efficiency of the reaction system can be obviously improved, and the method is suitable for industrial popularization.

Description

FDH mutant with improved protein soluble expression and encoding gene thereof
Technical Field
The invention relates to the field of bioengineering, in particular to an FDH mutant with improved protein soluble expression and a coding gene thereof.
Background
Formate dehydrogenase (Formate dehydrogenase, FDH, EC 1.2.1.2) is widely present in microorganisms and higher plants and is distributed among all methylotrophic microorganisms, of which methylotrophic Pseudomonas sp.101 is the most stable wild-type formate dehydrogenase to date. The enzyme is commonly used for recycling and regenerating reduced coenzyme NADH in the production of biologically converted amino acid, and can convert formate and oxidized coenzyme NAD + Reducing power required for simultaneous production reaction of hydrogen for substrate transfer, and CO as by-product 2 Is easily discharged, has a small influence on the pH of the conversion system, and is therefore considered as one of the best enzymes for NADH regeneration in the synthesis of optically active compounds, and is widely used in the food, pharmaceutical and chemical industries.
However, wild-type formate dehydrogenase has the common problems of low enzyme activity, poor catalytic efficiency and poor solubility of proteins expressed in microorganisms, and the high flexibility of the formate dehydrogenase protein structure determines that the wild-type formate dehydrogenase is easy to denature and inactivate under the temperature conditions required for production, so that the product conversion rate is low and the industrial production of the product is affected. Therefore, how to improve the soluble expression and the thermostability of formate dehydrogenase is a technical problem to be solved at present. The molecular modification of formate dehydrogenase refers to directional regulation and control of the structure and function of formate dehydrogenase by genetic engineering or protein engineering and other methods so as to improve the catalytic efficiency and stability of formate dehydrogenase under specific conditions. There are many methods of molecular engineering such as mutation, insertion, deletion, fusion, surface display, etc.
Disclosure of Invention
In view of the shortcomings of the prior art, one of the purposes of the invention is to provide a formate dehydrogenase mutant with better solubility expression and thermostability than wild type.
In order to achieve the above purpose, the present invention provides the following technical solutions: FDH mutant with improved protein soluble expression, and its wild type sequence is shown in SEQ ID NO. 1. The wild-type formate dehydrogenase is derived from the gene library Uniport: p33160. Substitution mutation is carried out on at least one amino acid site selected from the following wild-type formate dehydrogenase: Y63F, L119I, S132A, R136H, S229E, A240E, R242I, Y246L, R Q, F356W, D364E, A373K, yielding formate dehydrogenase mutants.
Preferably, the amino acid sequence of the formate dehydrogenase mutant is substituted with at least one mutation selected from the following sets of mutations:
S132A/R136H/S229E/A240E/R241P/Y246L/R302Q/A373K, said amino acid positions being referred to as SEQ ID NO:1, and a wild-type formate dehydrogenase.
Preferably, the amino acid sequence of the formate dehydrogenase mutant is substituted with one or more of the following sets of mutations: S132A/R136H/S229E/A240E/R241P/Y246L/R302Q/A373K, Y63F, L119I, F356W, D E, said amino acid position being referred to as SEQ ID NO:1, and a wild-type formate dehydrogenase.
Preferably, the amino acid sequence of the formate dehydrogenase mutant is selected from the following set of mutations:
S132A/R136H/S229E/A240E/R241P/Y246L/R302Q/A373K、
Y63F/L119I/S132A/R136H/S229E/A240E/R241P/Y246L/R302Q/D364E/A373K, Y F/L119I/S132A/R136H/S229E/A240E/R242I/Y246L/R302Q/F356W/D364E/A373K, said amino acid position being referred to as SEQ ID NO:1, and a wild-type formate dehydrogenase.
The second object of the present invention is to provide a DNA or RNA capable of expressing the formate dehydrogenase mutant.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a formate dehydrogenase recombinant gene capable of expressing the DNA or RNA of the formate dehydrogenase mutant.
It is a further object of the present invention to provide a recombinant plasmid comprising the above DNA capable of expressing a formate dehydrogenase mutant having a better protein-soluble expression level and thermostability.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a formate dehydrogenase recombinant plasmid comprising the formate dehydrogenase recombinant gene described above.
The fourth object of the present invention is to provide a recombinant cell capable of expressing a formate dehydrogenase mutant having a better protein-soluble expression level and thermostability.
In order to achieve the above purpose, the present invention provides the following technical solutions: a formate dehydrogenase recombinant cell comprising a formate dehydrogenase recombinant plasmid as described above.
Preferably, the chassis cell is selected from one of E.coli, pichia pastoris, bacillus, escherichia, salmonella, clostridium, streptomyces, staphylococcus, neisseria, and Shigella.
The fifth object of the present invention is to provide NAD involved in formate dehydrogenase mutant having better protein solubility expression and thermal stability + NADH reaction, providing NADH to the primary enzyme reaction system or only for the production of NADH product.
In order to achieve the above purpose, the present invention provides the following technical solutions: a biological catalytic system with NADH participation comprises the formate dehydrogenase mutant, wherein the formate dehydrogenase mutant participates in NAD + NADH reaction.
Preferably, the formate dehydrogenase recombinant cell is involved in NAD + NADH reaction.
Compared with the prior art, the invention has the advantages that: the mutant formate dehydrogenase RP 31-37 has greatly improved protein soluble expression and thermal stability. In particular to RP31 and RP33, the soluble expression quantity reaches more than 300% of the wild type, and the residual enzyme activity after incubation for 90min at 60 ℃ reaches about 200% of the wild type. Therefore, the mutant formate dehydrogenase of the present invention can obtain more enzyme amount for catalysis under the same fermentation condition, and the mutantParticipating NAD + The NADH reaction system can react at a higher temperature, the catalytic efficiency of the reaction system can be obviously improved, and the method is suitable for industrial popularization.
Drawings
FIG. 1 concentration of NADH solution-A 340 A standard graph of absorbance of (2);
FIG. 2 is a graph showing the relationship between the enzyme activity of wild-type formate dehydrogenase and temperature.
Detailed Description
DNA construct means a sequence capable of expressing formate dehydrogenase and formate dehydrogenase mutants of the present invention. Typically, the DNA construct is synthesized in vitro by PCR or other suitable techniques known in the art. In certain embodiments, the DNA construct further comprises other accessory elements, such as control elements (e.g., promoters, etc.). The DNA construct may also include a labeling substance (e.g., fluorescence, etc.). The DNA construct may also include other sequences that do not affect expression of the gene of interest.
The term "expression" refers to the process by which DNA is transcribed into messenger RNA (mRNA) and then translated into protein.
An "expression vector" has the ability to incorporate and express heterologous polynucleic acid fragments in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. The choice of an appropriate expression vector is within the knowledge of the skilled person.
The term "host cell" refers to a suitable host vector for expressing a DNA comprising the invention. The host may comprise any organism capable of comprising and expressing the nucleic acids or genes disclosed herein, but is not limited thereto. The host may be a prokaryote or eukaryote, single or multiple cells, including mammalian cells, plant cells, fungi, and the like. Examples of single cell hosts include cells of Escherichia, salmonella, bacillus, clostridium, streptomyces, staphylococcus, neisseria, lactobacillus, shigella and Mycoplasma. Suitable E.coli strains (including many others) include BL21 (DE 3), C600, DH 5. Alpha. F',1113101, JM83, JM101, JM103, JM105, JM107, JM109, JM110, MC1061, MC4100, M294, NM522, NM554, TGI, χ1776, XL1-Blue and Y1089 + All of which areAre commercially available.
The term "identity" means that the residues in the two sequences are identical when aligned for maximum correspondence, as measured using sequence comparison or analysis algorithms such as those described herein. For example, two sequences are said to have 50% identity if, when properly aligned, the corresponding fragments of the two sequences have identical residues at 5 of the 10 positions. Most bioinformatic programs report percent identity of aligned sequence regions, which are typically not the entire molecule. If the alignment is long enough and contains enough identical residues, then the expected value can be calculated, indicating that the same level in alignment is unlikely to occur randomly.
The invention will now be described in further detail with reference to the drawings and examples.
Example 1
Proteins are the material basis of life and are important components of human cells and tissues. All important components in the human body require the participation of proteins, which play a very important role in the vital activities of cells and organisms. It can be said that there is no life without protein. The protein in human body has very various kinds and different functions, and some of the protein can form human tissues, some of the protein can provide energy, some of the protein can participate in metabolism and transportation of substances, some of the protein can promote growth and development, and some of the protein can regulate immune function. Different proteins take on different roles and roles, the function of which is determined by the structure of the protein. The 3D structure of the protein is determined by the amino acid sequence of the protein. Therefore, the design of proteins depends on the correspondence between the structure and the sequence, and it is necessary to design proteins having specific functions, and it is necessary to design sequences conforming to the functional structures. Knowing and designing proteins is of great importance to drive innovative advances in biology and medicine.
It is a very difficult task to design a protein sequence for a specific function, and what structure and function the designed sequence finally assumes is unexpected. And the sample space for a fixed length protein sequence is also quite large. In order to accomplish the above work, the literature developed a protein design platform based on deep learning algorithms, lesign. The platform integrates the most advanced protein design method at present, and realizes the functions of protein structure prediction, sequence design, result evaluation and the like. The functional modules cooperate through interfaces to form a calculation pipeline integrating prediction, design and evaluation. The structure prediction module takes the sequence, the co-evolution information MSA and the template structure of database search as input, predicts the protein structure of the sequence, and outputs the structure credibility, and the representatives of the models are alpha fold2 and Rosettafold. The sequence design module has a plurality of modes, one takes a certain structure as input, then predicts the sequence, and the model can also be used for evaluating the structure, such as ESM-if1 and ProteinMPNN; another way is to predict the structure by initializing the sequence, comparing the structure prediction model with the real structure, calculating the error, and optimizing the initial sequence by error gradient back-propagation until the structure error converges, such as AFDesign. There are also protein language models that use masked sequence sampling to generate complete sequences. Combining these modules can derive a variety of protein design and evaluation methods from sequence to structure, structure to sequence, sequence to sequence, structure to structure.
And (3) carrying out sequence design on the wild formate dehydrogenase (the amino acid sequence is shown as SEQ ID NO. 1) by using a Lesign platform, and finally obtaining the optimal enzyme variant on the calculation level.
Construction of the DNA construct:
the nucleotide sequences of the genes of interest were all synthesized by Beijing qing Biotechnology Co., ltd, and these nucleotide sequences were inserted into expression vectors. Specifically, it was inserted into plasmid pET28a (+) to obtain the corresponding plasmid. The synthesized plasmids were then transferred into host cells (e.coli BL21 (DE 3)), whereby e.coli strains containing different plasmids were constructed.
Expression and purification of formate dehydrogenase:
3. Mu.L of recombinant bacteria were streaked on LB solid medium and placed in an incubator at 37℃overnight for cultivation. Picking single colony cultured overnight, inoculating into fresh LB culture medium, shaking to OD at 37deg.C with 200rpm shaker 600 0.6-0.8, isopropyl thiogalactoside (IPTG) was added at a final concentration of 0.5mM, and expression was induced at 16℃and 200rpm for 16h.
Wherein, LB culture medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride.
LB solid medium: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 2% of agar powder.
Cells were collected by centrifugation and cells were resuspended in lysis buffer. Ultrasonic disruption, separation of supernatant and precipitate. The mixture was filtered through a 0.45 μm filter, and the filtrate was placed on ice. The NTA-20, NTA-80 and NTA-100 are used for eluting the hetero protein in sequence, and then NTA-200 is used for eluting the target protein. Then, the target protein eluted from NTA-200 is added into a clean ultrafiltration tube, and is centrifuged for 10min at 3000g, and 1mL of the target protein is used for gently blowing and mixing each time. Repeating for 2-3 times, adding precooled 0.1MPB15mL when NTA-200 eluent remains 1-1.5mL, and centrifuging at 4deg.C for 10min at 3000 g. When 1-2mL of liquid remains, use A 280 The protein concentration was roughly measured, and then the protein concentration was measured by using a BCA kit and stored in a refrigerator at-80℃in a split manner, and specific data of the soluble expression level of formate dehydrogenase are shown in Table 1.
TABLE 1
As is clear from Table 1, the mutant formate dehydrogenase RP 31-37 of the present invention showed a significantly improved soluble expression level. In particular, the expression level of soluble protein of RP31 reaches 1.61mg/mL, which is 303.8% of the wild type; the expression level of soluble protein of RP33 reaches 1.75mg/mL, which is 330.2% of the wild type.
The enzyme activity determination method comprises the following steps: 100. Mu.L of formate dehydrogenase solution at a concentration of 25. Mu.g/ml was added to 100. Mu.L of substrate solution (4 mM NAD + In 200mM HOONa), after incubation for 10min at 30deg.C, immediately measuring A with an ELISA reader 340 Is a solid phase, and is a liquid phase. Wherein, the enzyme activity unit U is recorded as follows: the amount of enzyme required to produce 1. Mu.M NADH per minute at 30℃and pH7.0 is one enzyme activity unit U. Specific enzyme activity U/mg: enzyme activity per mg of enzyme proteinUnits of (3).
Determination of optimum temperature: four portions of 150. Mu.L of formate dehydrogenase solution at a concentration of 25. Mu.g/ml were each taken and added to four portions of 150. Mu.L of substrate solution (4 mM NAD + 200mM HOONa), the four reaction solutions are reacted at 45 ℃, 50 ℃, 55 ℃ and 60 ℃ for 10min respectively, and then the enzyme-labeled instrument is used for measuring A immediately 340 Is a solid phase, and is a liquid phase.
Thermal stability determination: the initial enzyme activity was measured according to the above-described enzyme activity measurement method. Then, the formate dehydrogenase solution was incubated at 60℃for 90 minutes, and then rapidly taken out, and after 5 minutes of standing on ice, the remaining enzyme activity was measured according to the above-mentioned enzyme activity measurement method.
Standard curve: NADH solutions of different concentrations were prepared and measured at A respectively 340 A corresponding standard curve was prepared, and the obtained standard curve is shown in fig. 1.
Specifically, the present invention measured the optimum temperature of wild-type formate dehydrogenase (WT, amino acid sequence shown in SEQ ID NO. 1) according to the above-mentioned optimum temperature measurement method, and the results obtained are shown in FIG. 2.
The following point mutations were performed using wild-type formate dehydrogenase (WT, amino acid sequence shown in SEQ ID NO. 1) as a parent. The initial enzyme activities and the residual enzyme activities of the wild type and the mutant were measured according to the above-mentioned thermostability measurement method, and specific values are shown in Table 2.
TABLE 2
As is clear from Table 2, the thermal stability of the mutant formate dehydrogenase RP 31-37 was greatly improved. In particular, the residual enzyme activity of RP31 after incubation at 60 ℃ for 90min reaches 23.15U, which is 195.4% of that of the wild type; the residual enzyme activity of RP32 after incubation at 60 ℃ for 90min reaches 22.27U, which is 187.9% of that of the wild type; the residual enzyme activity of RP33 after incubation at 60℃for 90min reached 25.23U, which was 212.9% of the wild-type.
In conclusion, the protein soluble expression quantity and the thermal stability of the mutant RP 31-37 of the formate dehydrogenase are greatly improved. In particular to RP31 and RP33, the soluble expression quantity reaches more than 300% of the wild type, and the residual enzyme activity after incubation for 90min at 60 ℃ reaches about 200% of the wild type. Therefore, the mutant formate dehydrogenase of the invention can obtain more enzyme amount for catalysis under the same fermentation condition, and the NAD involved by the mutant + The NADH reaction system can react at a higher temperature, and the catalytic efficiency of the reaction system can be obviously improved.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention can be made by one of ordinary skill in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.

Claims (10)

1. An FDH mutant having increased soluble expression of a protein, characterized in that said FDH mutant has the amino acid sequence set forth in SEQ ID NO:1 as female parent, the following mutations were made in the wild-type FDH:
S132A/R136H/S229E/A240E/R241 P/Y246L/R302Q/A373K。
2. FDH mutant with increased protein soluble expression according to claim 1, characterized in that the amino acid mutation site of said FDH mutant further comprises Y63F, said amino acid site being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type FDH.
3. FDH mutant with increased protein soluble expression according to claim 1, characterized in that the amino acid mutation site of said FDH mutant further comprises L119I, said amino acid site being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type FDH.
4. FDH mutant with increased protein soluble expression according to claim 1, characterized in that the amino acid mutation site of said FDH mutant further comprises F356W, said amino acid site being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type FDH.
5. FDH mutant with increased protein soluble expression according to claim 1, characterized in that the amino acid mutation site of said FDH mutant further comprises D364E, said amino acid site being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type FDH.
6. FDH mutant with increased protein soluble expression according to claim 1, characterized in that the amino acid mutation site of said FDH mutant further comprises Y63F/L119I/D364E, said amino acid site being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type FDH.
7. FDH mutant with increased protein soluble expression according to claim 1, characterized in that the amino acid mutation site of said FDH mutant further comprises Y63F/L119I/F356W/D364E, while at the same time mutating proline at position 242 to isoleucine, said amino acid site being referred to as SEQ ID NO:1, and the amino acid sequence of the wild-type FDH.
8. A formate dehydrogenase recombinant gene capable of expressing a DNA or RNA of the FDH mutant according to any one of claims 1 to 7.
9. A formate dehydrogenase recombinant plasmid comprising the formate dehydrogenase recombinant gene according to claim 8.
10. A formate dehydrogenase recombinant cell comprising the formate dehydrogenase recombinant plasmid of claim 9.
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