A kind of BFD mutant and its application as carbon chain lengthening enzyme

Abstract

The application provides a BFD mutant and application thereof as a carbon chain lengthening enzyme, and comprises: (a) the BFD mutant contains at least one site of L43, A73, T100 and K165 compared with the amino acid sequence shown in SEQ ID NO. 1, and has an amino acid sequence which is subjected to amino acid mutation and has a carbon chain lengthening enzyme activity function; or (b) the BFD mutant has an amino acid sequence which is subjected to substitution, deletion and / or addition of one or more conservative amino acid residues and has the same function. The enzyme activity of the BFD mutant is obviously improved, and the yield and conversion rate of formaldehyde to ethanol aldehyde and / or 1,3-dihydroxyacetone are improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a BFD mutant and its application as a carbon chain elongation enzyme. Background Technology

[0002] One-carbon compounds are promising green energy materials that have attracted widespread attention. They can be used to synthesize basic organic chemical raw materials, fuels, and other high-value-added chemicals. Due to their low cost and easy availability, one-carbon compounds have become the most promising alternatives to petroleum in the preparation of high-value compounds, and they have significant scientific and developmental value in the fields of medicine, food, and chemicals. At the same time, the utilization and conversion of one-carbon compounds can also significantly reduce the negative impacts on the ecological environment caused by the use of fossil fuels and synthetic materials.

[0003] Formaldehyde can be converted from other one-carbon compounds and further transformed into intermediate substances for bioavailability. It also boasts advantages such as wide availability and low price, making it a promising candidate for industrial biotechnology. The enzymatic conversion of formaldehyde into multi-carbon compounds is of great significance for promoting carbon-neutral economic development.

[0004] Benzoylformate decarboxylases (BFD) are thiamine diphosphate (ThDP)-dependent lyases that catalyze the cleavage of benzoylformate into benzaldehyde and carbon dioxide. CN106916794A discloses site-directed mutant BFD proteins, including W86R, N87T, L109G, L110E, H281V, Q282F, and A460M, and for the first time uses BFD mutant proteins to catalyze the synthesis of hydroxyacetaldehyde or 1,3-dihydroxyacetone from formaldehyde, achieving highly efficient polymerization of formaldehyde. Among them, the yields of hydroxyacetaldehyde and 1,3-dihydroxyacetone produced by the BFD mutant with mutation 5 W86R-N87T-L109G-L110E-A460M were increased, reaching 0.756 g / L and 0.27 g / L, respectively. The yield of hydroxyacetaldehyde produced by the BFD mutant with mutation 7 W86R-N87T-L109G-L110E-H281V-Q282F-A460M was the highest, reaching 1.4357 g / L, while the yield of 1,3-dihydroxyacetone was lower than that of the BFD mutant with mutation 5, at 0.221 g / L.

[0005] CN113832120A discloses a BFD mutant (W86R-N87T-L109G-L110E-A460M) with five mutations, which underwent further rounds of random mutation, gene recombination, and screening. These mutations included at least one amino acid residue mutation at one of the following positions: S26F, L43Q, F66L, R86C, T87A, G109S, A204V, H281Y, A322T, F397L / F397S, M460T, W463R, V467A, V473A, and S525A. This further enhanced enzyme activity, resulting in a 90.24% conversion rate of formaldehyde to 1,3-dihydroxyacetone.

[0006] These mutants, generated through random mutagenesis, exhibited enhanced activity in the synthesis of hydroxyacetaldehyde or 1,3-dihydroxyacetone from formaldehyde. However, further mutations of industrially useful enzymes such as BFD enzymes are still needed to further understand the impact of structural changes on their catalytic mechanisms, further improve their catalytic conversion performance, and expand their applications. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a BFD mutant and its application as a carbon chain elongation enzyme. It solves the problem in existing technologies that necessitates further improvement in the catalytic conversion performance of the BFD mutant.

[0008] In a first aspect, the present invention provides a BFD mutant, comprising: (a) the BFD mutant, compared with the amino acid sequence shown in SEQ ID NO.1, containing at least one of the following sites: L43, A73, T100, K165, an amino acid sequence that has undergone amino acid mutation and possesses carbon chain elongation enzyme activity; or

[0009] (b) The BFD mutant is derived from the amino acid sequence shown in (a) by substitution and / or deletion and / or addition of one or more conserved amino acid residues, resulting in an amino acid sequence with the same function.

[0010] Furthermore, the BFD mutant contains at least one site substitution selected from the group consisting of: L43Q, A73T, T100A, and K165E.

[0011] Furthermore, the BFD mutant includes any of the following amino acid mutations:

[0012] A73T; A73T-T100A; A73T-K165E; A73T-T100A-K165E; L43Q-A73T; L43Q-A73T-K165E;

[0013] In a second aspect, the present invention provides a DNA molecule that encodes the aforementioned BFD mutant.

[0014] In a third aspect, the present invention provides a recombinant vector, expression cassette, or host cell containing the DNA molecule described in the second aspect.

[0015] In a fourth aspect, the present invention provides a method for preparing the above-mentioned BFD mutant, wherein the above-mentioned host cells are cultured and the BFD mutant is obtained from the culture; preferably, the host cells include Escherichia coli.

[0016] In a fifth aspect, the present invention provides a method for synthesizing glycolaldehyde, using formaldehyde as a substrate and catalyzing a reaction using the BFD mutant described in the first aspect, the DNA molecule described in the second aspect, the recombinant vector described in the third aspect, an expression cassette, or a host cell.

[0017] In a sixth aspect, the present invention provides a method for synthesizing 1,3-dihydroxyacetone, using formaldehyde as a substrate and catalyzing a reaction using the BFD mutant described in the first aspect, the DNA molecule described in the second aspect, the recombinant vector described in the third aspect, an expression cassette, or a host cell.

[0018] In a seventh aspect, the present invention provides the use of the BFD mutant described in the first aspect, the DNA molecule described in the second aspect, the recombinant vector, expression cassette, or host cell described in the third aspect in the preparation of glycolaldehyde and / or 1,3-dihydroxyacetone.

[0019] In an eighth aspect, the present invention provides a composition for preparing glycolaldehyde and / or 1,3-dihydroxyacetone, said composition comprising the BFD mutant described in the first aspect or biological material expressing the BFD mutant;

[0020] Preferably, the biomaterial is a recombinant vector, expression cassette, or host cell.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention obtains a new BFD mutant protein by randomly mutating the BFD mutant protein (starting enzyme), and the enzyme activity of the new BFD mutant protein is significantly improved compared with that of the starting enzyme.

[0023] (2) The new BFD mutant protein obtained by this invention can directly catalyze the formation of formaldehyde into ethanolaldehyde and / or 1,3-dihydroxyacetone. The production method is simple and efficient, which is conducive to large-scale industrial production. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0025] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The benzoylformate decarboxylase mutant W86R-N87T-L109G-L110E-H281V-Q282F-A460M is the BFD mutant protein disclosed in patent CN106916794A.

[0026] Example 1: Construction of Mutant Library

[0027] The starting enzyme was a mutant of benzoylformate decarboxylases (BFD) from *Pseudomonas putida*, W86R-N87T-L109G-L110E-H281V-Q282F-A460M. Its amino acid sequence is shown in SEQ ID NO.1. Random mutations were introduced into the gene sequence of the starting enzyme using error-prone PCR to construct a mutant library. This mutant library was transformed into *Escherichia coli* BL21 Gold(DE3) and cultured at 37°C until single colonies grew.

[0028] Example 2: Plate expression and screening of mutants

[0029] Select the single clones obtained in Example 1 and inoculate them into a 96-well plate containing LB medium. Incubate overnight at 37°C and 800 rpm in a shaker. Transfer the culture to another 96-well plate, add 0.1 mM IPTG, and incubate at 37°C and 800 rpm in a shaker for a certain time. Then adjust the temperature to 20°C for further incubation.

[0030] After culturing, collect the bacterial cells by centrifugation at 3400 rpm for 15 min at 4℃. Resuspend the bacterial cells in potassium phosphate buffer (formulation: 50 mM K2HPO4 and KH2PO4, 5 mM MgSO4, pH 7.4), and then collect the bacterial cells again by centrifugation at 3400 rpm for 15 min at 4℃.

[0031] The bacterial cells were resuspended in 75 μL of potassium phosphate buffer, and then an equal volume of formaldehyde solution (potassium phosphate buffer containing 360 mM formaldehyde and 1 mM thiamine pyrophosphate (TPP)) was added. The reaction was carried out at 30 °C with shaking. To determine the yield of hydroxyacetaldehyde after the reaction was complete, 30 μL of the reaction solution was placed in a new well plate, and 150 μL of diphenylamine solution (6 g diphenylamine + 400 mL glacial acetic acid + 6 mL concentrated sulfuric acid) was added. The plate was heated at 90 °C for 30 min, and the absorbance was measured at 650 nm. A higher absorbance indicates that the mutant catalyzes the conversion of formaldehyde to hydroxyacetaldehyde in the same amount of time, i.e., it has higher catalytic activity.

[0032] The mutants with high absorbance and high catalytic activity obtained through screening were subjected to gene sequencing to determine the mutation sites and amino acids. After three rounds of mutant library screening and combination of beneficial mutation sites, eight mutants with significantly increased enzyme activity were finally obtained. The names of the mutants and their corresponding mutated amino acids are shown in Table 1.

[0033] Table 1. Mutant names and corresponding mutant amino acids

[0034]

[0035] Example 3: Shake-flask expression of the starting enzyme and mutant

[0036] Positive bacteria of the enzyme and mutant were selected and inoculated into 5 mL of LB medium. They were cultured at 37 °C and 200 rpm for 12 h. Then, they were inoculated into 100 mL of LB medium at a 1% (V / V) inoculation rate. After a certain period of culture, 0.1 mM IPTG was added to induce expression.

[0037] Collect the above-mentioned bacterial culture into centrifuge tubes and incubate at 4°C, 4000 rpm for 15 minutes.

[0038] Discard the supernatant, resuspend and wash the cells with 25 mL of potassium phosphate buffer (formulation: 50 mM K2HPO4 and KH2PO4, 5 mM MgSO4, pH 7.4), centrifuge at 4000 rpm for 15 min at 4 °C. Discard the supernatant and store at -80 °C to obtain the cells expressing the starting enzyme and mutant.

[0039] Example 4 Synthesis of Glycoaldehyde

[0040] The starting enzyme and mutant cells from Example 3 were resuspended in potassium phosphate buffer, sonicated on ice, and the supernatant was collected by centrifugation.

[0041] Take 75 μL of bacterial cell lysis supernatant (crude enzyme solution), add an equal volume of formaldehyde solution (potassium phosphate buffer containing 360 mM formaldehyde and 1 mM thiamine pyrophosphate (TPP)), and incubate at 30°C with shaking for 1 h. After the reaction is complete, transfer the reaction solution to a new well plate, add 150 μL of diphenylamine solution (6 g diphenylamine + 400 mL glacial acetic acid + 6 mL concentrated sulfuric acid), heat at 90°C, and measure the absorbance at 650 nm. Based on the linear relationship between absorbance and hydroxyacetaldehyde concentration, calculate the relative enzyme activities of the starting enzyme and the mutant crude enzyme solution in catalyzing the production of glycolaldehyde from formaldehyde. The percentage of mutant enzyme activity relative to the starting enzyme is shown in Table 2 (with the enzyme activity of the cell lysis supernatant expressing the starting enzyme as 100%).

[0042] Table 2. Relative activities of starting enzyme and mutant crude enzyme solutions at 180 mM formaldehyde concentration.

[0043]

[0044]

[0045] As shown in the table above, compared with the starting enzyme, the activities of all eight mutants in catalyzing the production of glycolaldehyde from formaldehyde were improved, with mutant V12 exhibiting the highest enzyme activity, reaching 3.15 times that of the starting enzyme.

[0046] Example 5: Synthesis of DHA (1,3-dihydroxyacetone)

[0047] Similar to Example 4, 75 μL of the bacterial cell lysis supernatant from Example 3 was taken and an equal volume of formaldehyde solution (potassium phosphate buffer containing 360 mM formaldehyde and 1 mM thiamine pyrophosphate (TPP)) was added. The mixture was reacted at 30°C for 1 h. After the reaction was completed, the reaction solution was transferred to a new well plate, and 60 μL of enzyme Buffer 1 (formulation: 0.6 mg / mL galactose oxidase, 32 U / mL horseradish peroxidase) was added. Then, 50 μL of Buffer 2 (formulation: 8 mM ABTS) was added, and the absorbance at 410 nm was measured. The slope of the absorbance change over time was determined. This slope was linearly related to the DHA concentration. Based on this, the relative enzyme activities of the starting enzyme and the mutant crude enzyme solution catalyzing the production of DHA from formaldehyde were calculated, as shown in Table 3 (with the enzyme activity of the lysis supernatant of the cells expressing the starting enzyme as 100%).

[0048] Table 3. Relative activities of starting enzyme and mutant crude enzyme solutions at 180 mM formaldehyde concentration.

[0049]

[0050] As shown in Table 3, compared with the starting enzyme, the enzyme activity of all eight mutants in catalyzing the production of DHA from formaldehyde was improved. Among them, mutant V11 had the highest relative enzyme activity, reaching 1.28 times that of the starting enzyme.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A BFD mutant, characterized in that: Perform any of the following mutations based on the amino acid sequence shown in SEQ ID NO.1: (1) Single mutation: A73T; (2) Double mutation: A73T-T100A or A73T-K165E or L43Q-A73T; (3) Triple mutation: A73T-T100A-K165E or L43Q-A73T-K165E or A73T-T100A-L43Q; (4) Four mutations: L43Q-A73T-T100A-K165E.

2. A DNA molecule, characterized in that, The DNA molecule encodes the BFD mutant of claim 1.

3. A recombinant vector, expression cassette, or host cell containing the DNA molecule of claim 2.

4. A method for preparing BFD mutants, characterized in that, The host cells of claim 3 are cultured, and the BFD mutant is obtained from the culture.

5. The method for preparing a BFD mutant as described in claim 4, characterized in that, The host cells include Escherichia coli.

6. A method for synthesizing glycolaldehyde, characterized in that, It is obtained by using formaldehyde as a substrate and catalyzing the reaction with the BFD mutant described in claim 1 and the host cell described in claim 3.

7. A method for synthesizing 1,3-dihydroxyacetone, characterized in that, It is obtained by using formaldehyde as a substrate and catalyzing the reaction with the BFD mutant described in claim 1 and the host cell described in claim 3.

8. The use of the BFD mutant of claim 1, the DNA molecule of claim 2, the recombinant vector of claim 3, the expression cassette or the host cell in the preparation of glycolaldehyde and / or 1,3-dihydroxyacetone.

9. A composition for preparing glycolaldehyde and / or 1,3-dihydroxyacetone, said composition comprising the BFD mutant of claim 1 or biological material expressing the BFD mutant; said biological material being the recombinant vector, expression cassette, or host cell of claim 3.

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