Formaldehyde lyase as well as mutant and application thereof

Abstract

The invention provides formaldehyde lyase as well as a mutant and application thereof. The amino acid sequence of the formaldehyde lyase mutant is shown as SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and contains at least one of the following sites: S26, W86, N87 or Y87, L109 or M109, L110, H281, Q282 or E282 and A460, and the amino acid sequence is subjected to amino acid mutation and has a formaldehyde lyase activity function. The amino acid sequences of the formaldehyde lyase and the mutants of the formaldehyde lyase have at least 99% of sequence identity with SEQ ID NO.1-7. The formaldehyde lyase and the mutant thereof can catalyze formaldehyde to synthesize 1, 3-dihydroxyacetone, and a new way for converting formaldehyde into 1, 3-dihydroxyacetone is provided.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a formaldehyde lyase, its mutant and application. Background Art

[0002] One-carbon compounds have received extensive attention as green energy substances with great application prospects, and can be used to synthesize basic organic chemical raw materials, fuels and other high-value chemicals. Due to their cheap and easily available characteristics, one-carbon compounds have become the most promising compounds for replacing petroleum to prepare high-value compounds, and have important scientific significance and development value in the fields of medicine, food and chemical industry. At the same time, the utilization and conversion of one-carbon compounds can also significantly reduce the negative impact on the ecological environment caused by people in the process of using fossil fuels and synthetic materials. Therefore, carbon chain elongation enzymes that can catalyze one-carbon compounds to form new carbon-carbon bonds, thereby realizing their conversion and utilization, have high application value.

[0003] Formaldehyde, as the most reactive one-carbon compound, its conversion and utilization has always been a research hotspot. In 2013, the global formaldehyde production capacity was about 64 million tons. Formaldehyde can be converted from other one-carbon compounds, and then converted into intermediate substances for biological utilization. At the same time, it has the characteristics of wide source and low price, and has broad application prospects in industrial biotechnology. Therefore, it is of great significance to explore and excavate carbon chain elongation enzymes using formaldehyde as a substrate.

[0004] Formaldehyde lyase (FLS, Sequence ID: 4QQ8_A) was initially obtained by researchers at the University of Washington (Justin B. Siegel, Amanda Lee Smith, et al. Proc Natl Acad Sci USA (2015) 3704-3709) through the transformation of benzaldehyde lyase at A28I, W89R, L90T, R188H, A394G, G419N and A480W to endow it with formaldehyde catalytic ability, and can catalyze three molecules of formaldehyde to form one molecule of 1,3-dihydroxyacetone (DHA), but its catalytic efficiency is low, and kcat / Km is only 4.7±0.1 M -1 S -1 In the prior art, generally, the above formaldehyde lyase is subjected to site-directed mutagenesis to obtain a formaldehyde lyase mutant protein with strong activity. For example, CN107475281A performs site-directed mutagenesis on the above formaldehyde lyase at M473Y, L482R or L482D respectively, and the enzyme activity of the obtained FLS (M473Y / L482R) reaches 1.166 U / mg, which is 2.5 times higher than that of the original FLS (wild), and the catalytic efficiency reaches 82.44 s -1 mM -1, which is 2.8 times higher than the original. CN113151230A mutated the above-mentioned formaldehyde lyase at positions 28 (Ile to Thr), 480 (Trp to Phe), 481 (Thr to Ser), 482 (Leu to Glu), 483 (His to Val), 484 (Phe to Ala), and 485 (Gln to Phe) from the N-terminus to the C-terminus. One or several of these mutations were used to construct mutants with a 4.67-fold increase in enzyme activity and a 1,3-dihydroxyacetone production reaching 9.59 g / L.

[0005] A series of mutants generated by site-directed mutagenesis showed enhanced activity for the synthesis of 1,3-dihydroxyacetone from formaldehyde. However, it is still necessary to isolate new and useful formaldehyde lyases in industry for mutagenesis to further understand the effect of structural changes on their catalytic mechanism, further improve their catalytic conversion performance, and expand their application scope. Summary of the Invention

[0006] Aiming at the deficiencies in the prior art, the present invention provides a formaldehyde lyase and its mutants, which solve the problems existing in the prior art.

[0007] In the first aspect of the present invention, a formaldehyde lyase mutant is provided, and its amino acid sequence is 1) or 2):

[0008] 1) Relative to SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6, it contains at least one of the following sites: S26, W86, N87 or Y87, L109 or M109, L110, H281, Q282 or E282, A460, and has an amino acid sequence with formaldehyde lyase activity function after amino acid mutation;

[0009] 2) The amino acid sequence as shown in 1) has an amino acid sequence with the same function after substitution and / or deletion and / or addition of one or several conservative amino acid residues.

[0010] In some embodiments of the present invention, the formaldehyde lyase mutant has a mutation at at least one of the sites S26, W86, N87, L109, L110, H281, Q282, A460 in the amino acid sequence shown in SEQ ID NO.1 from the N-terminus to the C-terminus.

[0011] In some embodiments of the present invention, the formaldehyde lyase mutant has a mutation at at least one of the sites S26, W86, N87, L109, L110, H281, E282, A460 in the amino acid sequence shown in SEQ ID NO.4 from the N-terminus to the C-terminus.

[0012] In some embodiments of the present invention, the formaldehyde lyase mutant is a mutant at at least one site among S26, W86, Y87, M109, L110, H281, and A460 from the N-terminus to the C-terminus of the amino acid sequence shown in SEQ ID NO.6.

[0013] In some embodiments of the present invention, its amino acid sequence comprises at least one site substitution selected from the group consisting of: S26F, W86R, Y87T or N87T, L109G or L109S or M109S, L110E, H281V or H281Y, Q282F or E282Q, A460M.

[0014] Furthermore, the amino acid sequence has at least 99% sequence identity with SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7.

[0015] Even further, its amino acid sequence is as shown in any one of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7.

[0016] In a second aspect of the present invention, there is provided a DNA molecule encoding the formaldehyde lyase mutant described in the first aspect.

[0017] In a third aspect of the present invention, there is provided a recombinant vector, expression cassette or host cell containing the above DNA molecule.

[0018] In a fourth aspect of the present invention, there is provided a formaldehyde lyase having an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6, the mutant described in the first aspect, a vector, expression cassette, host cell expressing the formaldehyde lyase having an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and its mutant, and the use of a DNA molecule encoding the formaldehyde lyase having an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and the mutant described in the first aspect in catalyzing a carbon chain elongation reaction of a one-carbon compound.

[0019] Furthermore, the one-carbon compound is formaldehyde.

[0020] Furthermore, the catalyzing of the carbon chain elongation reaction of the one-carbon compound refers to catalyzing formaldehyde to generate 1,3-dihydroxyacetone.

[0021] In the fifth aspect of the present invention, a method for synthesizing 1,3-dihydroxyacetone is provided. Using formaldehyde as a substrate, a formaldehyde lyase with an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and the mutant described in the first aspect, a vector, an expression cassette, and a host cell expressing the formaldehyde lyase with an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and the mutant described in the first aspect are used for catalytic reaction to obtain the product.

[0022] In the sixth aspect of the present invention, a composition for synthesizing 1,3-dihydroxyacetone is provided, including a formaldehyde lyase with an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and the mutant described in the first aspect and / or a vector, an expression cassette, and a host cell expressing the formaldehyde lyase with an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and the mutant described in the first aspect.

[0023] The technical principle of the present invention is as follows: In the prior art, generally, mutations or mutagenesis are carried out on known formaldehyde lyases. The inventor unexpectedly found in repeated experiments and screening processes that some wild-type carbon chain elongation enzymes have formaldehyde lyase activity. By performing site-directed mutagenesis on these wild-type carbon chain elongation enzymes, it was further found that after mutation of these enzymes, their activity increased, significantly higher than that of the wild type.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention for the first time discovers that some wild-type carbon chain elongation enzymes have formaldehyde lyase activity. These enzymes include wild-type carbon chain elongation enzymes from Paraburkholderia lacunae, wild-type carbon chain elongation enzymes from Methylocystis parvus, and wild-type carbon chain elongation enzymes from Halomonas gudaonensis, which can catalyze the synthesis of 1,3-dihydroxyacetone from formaldehyde, providing a new way to convert formaldehyde into 1,3-dihydroxyacetone.

[0026] (2) The catalytic activity of the formaldehyde lyase mutant of the present invention is higher than that of the wild-type formaldehyde lyase. Mutants pLM1, pLM2, MpM1, and HgM1 not only still have the function of formaldehyde lyase, but also have increased activity compared to their respective wild-type enzymes.

[0027] (3) The method of the present invention is simple and efficient, realizing the efficient synthesis of 1,3-dihydroxyacetone, which is beneficial to large-scale industrial production. Specific embodiments

[0028] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative explanation of the present invention and should not be construed as limiting the protection scope 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.

[0029] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0030] Technical terms

[0031] The term "enzyme activity" refers to the ability of an enzyme to catalyze a certain chemical reaction; in the present invention, the enzyme activity is represented by the amount of 1,3-dihydroxyacetone produced by catalyzing formaldehyde.

[0032] The term "wild type" refers to an individual obtained from nature, whose genes have not been artificially mutated, that is, there are no mutations in the amino acid sequence; in the present invention, it refers to wild-type carbon chain elongation enzymes, including the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:6.

[0033] The term "mutant" refers to a change in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA, which results in a variation in the expressed amino acid sequence; in the present invention, it refers to carbon chain elongation enzyme mutants, including the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7 or amino acid sequences having at least 99% sequence identity thereto.

[0034] Example 1 Obtaining of a carbon chain elongation enzyme gene with the catalytic function of formaldehyde lyase and construction of a vector plasmid

[0035] First, by constructing a phylogenetic tree, potential new carbon chain elongation enzymes capable of catalyzing formaldehyde to produce dihydroxyacetone were mined. The following three carbon chain elongation enzymes were synthesized by a company and found to have the catalytic function of formaldehyde lyase.

[0036] SEQ ID NO.1: Amino acid sequence of a wild-type carbon chain elongation enzyme (also known as Pl) from Paraburkholderia lacunae;

[0037] SEQ ID NO.4: Amino acid sequence of a wild-type carbon chain elongation enzyme (also known as Mp) from Methylocystis parvus;

[0038] SEQ ID NO.6: Amino acid sequence of the wild-type carbon chain elongase (also known as Hg) from Halomonas gudaonensis.

[0039] After artificially synthesizing the gene sequences encoding the above three carbon chain elongases, they were inserted between the restriction enzyme sites NdeI and XhoI of the pET-28a vector to obtain vector plasmids expressing Pl, Mp, and Hg, respectively.

[0040] Example 2 Construction of vector plasmids for carbon chain elongase mutants

[0041] By analyzing and comparing the three amino acid sequences and the three-dimensional protein structures in Example 1, 8 sites were selected for site-directed mutagenesis in SEQ ID NO.1, namely S26F, W86R, N87T, L109S or L109G, L110E, H281V or H281Y, Q282F, and A460M; 7 sites were selected for site-directed mutagenesis in SEQ ID NO.4, namely S26F, W86R, N87T, L109S, L110E, L281Y, A460M; 7 sites were selected for site-directed mutagenesis in SEQ ID NO.6, namely S26F, W86R, Y87T, M109S, L110E, H281Y, and A460M. The mutant genes were inserted between the original restriction enzyme sites by mutant PCR technology to obtain vector plasmids expressing mutants PlM1, PlM2, MpM1, and HgM1, respectively. The names of the mutants and their corresponding amino acid sequences are shown in Tables 1-3.

[0042] Table 1 Names of Pl mutants and corresponding mutant amino acids

[0043]

[0044] Example 3 Expression of carbon chain elongase and its mutants in Escherichia coli

[0045] For in vitro detection of enzyme activity, the enzyme was heterologously expressed in Escherichia coli.

[0046] The plasmids constructed in Examples 1 and 2 were transferred into E. coli BL21(DE3) to obtain recombinant bacteria. After induced expression, the cells were centrifuged and collected in centrifuge tubes.

[0047] Example 4 Catalysis of formaldehyde to 1,3-dihydroxyacetone by carbon chain elongase and its mutants

[0048] The cells collected in Example 3 were resuspended with protein buffer (50 mM potassium phosphate buffer, 5 mM MgSO4, pH 7.5), lysed by ultrasonic treatment on ice bath, and the supernatant was collected by centrifugation.

[0049] The cell resuspension was mixed with formaldehyde solution (200 mM) and TPP (1 mM). After the reaction was completed, the reaction solution was taken into a new well plate, and the yield of 1,3-dihydroxyacetone (DHA) was detected by liquid phase. The results are shown in Table 4.

[0050] The detection of DHA was carried out by high performance liquid chromatography using an Agilent 1200 high performance liquid chromatograph equipped with an ultraviolet detector and a differential refractive index detector. Analytical column: 87H, detector: ultraviolet and differential refractive index detectors; mobile phase: 10 mM H2SO4;. Flow rate: 0.5 mL / min, column temperature: 65 °C, sample volume: 20 μL.

[0051] Table 4 Amount of DHA produced by formaldehyde lyase (mM)

[0052] Name DHA production (mM) pL 0.4 pLM1 1.9 pLM2 2.9 Mp 0.8 MpM1 2.5 Hg 0.3 HgM1 0.5

[0053] As can be seen from Table 4, pL, Mp, and Hg have the function of formaldehyde lyase that can catalyze the conversion of formaldehyde to DHA. The mutants pLM1, pLM2, MpM1, and HgM1 still have the function of formaldehyde lyase, and their enzyme activities are all higher than those of their respective wild-type enzymes.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A formaldehyde lyase mutant, characterized in that, Its amino acid sequence is 1) or 2): 1) An amino acid sequence that, as compared with that shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6, contains at least one of the following sites: S26, W86, N87 or Y87, L109 or M109, L110, H281, Q282 or E282, A460, and has been subjected to amino acid mutation and has the activity function of formaldehyde lyase; 2) An amino acid sequence that has the same function as that shown in 1) after substitution and / or deletion and / or addition of one or several conservative amino acid residues.

2. The formaldehyde lyase mutant according to claim 1, wherein Its amino acid sequence contains at least one site substitution selected from the group consisting of: S26F, W86R, Y87T or N87T, L109G or L109S or M109S, L110E, H281V or H281Y, Q282F or E282Q, A460M.

3. The formaldehyde lyase mutant according to claim 2, wherein: Its amino acid sequence has at least 99% sequence identity with SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7; Preferably, its amino acid sequence is as shown in any one of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.

7.

4. A DNA molecule, characterized in that, The DNA molecule encodes the formaldehyde lyase mutant according to any one of claims 1-3.

5. A recombinant vector, expression cassette or host cell containing the DNA molecule according to claim 4.

6. The application of the formaldehyde lyase with the amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6, the formaldehyde lyase mutant according to claim 1, the vector, expression cassette, host cell expressing the formaldehyde lyase with the amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and the formaldehyde lyase mutant according to claim 1, and the DNA molecule encoding the formaldehyde lyase with the amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and the formaldehyde lyase mutant according to claim 1 in catalyzing the carbon chain elongation reaction of one-carbon compounds.

7. The application according to claim 6, characterized in that, The one-carbon compound is formaldehyde.

8. The application according to claim 7, wherein The catalyzing the carbon chain elongation reaction of one-carbon compounds means catalyzing formaldehyde to generate 1,3-dihydroxyacetone.

9. A method for synthesizing 1,3-dihydroxyacetone, characterized in that, Using formaldehyde as a substrate, the catalysis reaction is obtained by using the formaldehyde lyase with the amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6, the formaldehyde lyase mutant according to claim 1, the vector, expression cassette, host cell expressing the formaldehyde lyase with the amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and the formaldehyde lyase mutant according to claim 1.

10. A composition for synthesizing 1,3-dihydroxyacetone, characterized in that, A formaldehyde lyase comprising an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6, a mutant of the formaldehyde lyase as claimed in claim 1, and / or a vector, expression cassette or host cell expressing the formaldehyde lyase comprising an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.4 or SEQ ID NO.6 and the mutant of the formaldehyde lyase as claimed in claim 1.

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