Construction and application of a screening method for aldehyde dehydrogenase biological component

Abstract

The application discloses an aldolase bioelement screening method and related application of a series of aldolases in catalyzing a condensation reaction of formaldehyde and pyruvic acid to synthesize 4-hydroxy-2-ketobutyric acid. The aldolase provided by the application is a protein shown in SEQ ID NO. 1-NO. 10, or a functionally identical protein obtained through substitution and / or deletion / addition of one or more amino acid residues. The aldolase has the following characteristics: 1) belonging to the HpcH functional domain family protein; 2) the sequence length is 240-320 amino acids; 3) the existing database function annotation is 4-hydroxy-2-oxovalerate aldolase (can catalyze the condensation reaction of acetaldehyde and pyruvic acid); the aldolase can take formaldehyde and pyruvic acid as substrates, and can carry out enzyme catalysis reaction in appropriate conditions and medium, and can efficiently biosynthesize 4-hydroxy-2-ketobutyric acid, and has the remarkable characteristics of mild reaction condition, good stereoselectivity and no pollution.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to the use of a biological catalyst, aldolase, and aldolase catalyzing the condensation of formaldehyde and pyruvic acid to generate a 4-hydroxy-2-ketobutyric acid compound. BACKGROUND

[0002] Formaldehyde is an important one-carbon compound metabolic intermediate. Methanol, methane and other one-carbon compounds can be converted into formaldehyde through enzyme-catalyzed reaction, which can further enter the central metabolic pathway, synthesize biomass, and be used for microbial growth and synthesis of high value-added chemicals (Engineering the bioconversion of methane and methanol to fuels and chemicals in native and synthetic methylotrophs, R Kyle Bennett, Lisa M Steinberg, Wilfred Chen, Eleftherios T Papoutsakis. Curr. Opin. Biotechnol. 2018. 50: 81-93; Current advance in bioconversion of methanol to chemicals, Wenming Zhang, Meng Song, Qiao Yang, Zhongxue Dai, Shangjie Zhang, Fengxue Xin, Weiliang Dong, Jiangfeng Ma, Min Jiang. Biotechnol. Biofuels. 2018. 11,: 11.). The main natural formaldehyde utilization pathways include three types: 1) ribulose monophosphate pathway (RuMP); 2) Calvin Benson Bassham pathway (CBB); 3) serine pathway.In the natural utilization pathway of formaldehyde, the most studied and effective formaldehyde utilization pathway is the RuMP pathway. At present, there are documents reported that the RuMP pathway is used to maintain the growth of microbial organisms with methanol as the carbon source in engineered strains (Engineering Escherichia coli for methanol conversion, Jonas E N Müller, Fabian Meyer, Boris Litsanov, Patrick Kiefer, Eva Potthoff, Stéphanie Heux, Wim J Quax, Volker F Wendisch, Trygve Brautaset, Jean-Charles Portais, Julia A Vorholt. Metab. Eng. 2015. 28: 190-201; Improving formaldehyde consumption drives methanol assimilation in engineered E. coli., Benjamin M. Woolston, Jason R. King, Michael Reiter, Bob Van Hove & Gregory Stephanopoulos. Nat. Commun. 2018. 9: 2387) or to produce high-value chemicals (Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli, W. Brian Whitaker, J. Andrew Jones, R. Kyle Bennett, Jacqueline E. Gonzalez, Victoria R. Vernacchio, Shannon M. Collins, Michael A. Palmer, Samuel Schmidt, Maciek R. Antoniewicz, Mattheos A. Koffas, Eleftherios T. Papoutsakis. Metab. Eng. 2017. 39: 49-59). But the RuMP pathway also has some deficiencies, and the most critical point is that the RuMP pathway is greatly limited by the low efficiency of the regeneration of Ru5P acceptor.Thus, the utilization of one-carbon compounds such as methanol and formaldehyde in microorganisms is limited (Biological conversion of methane to chemicals and fuels: technical challenges and issues, In Yeub Hwang, Anh Duc Nguyen, Thu Thi Nguyen, Linh Thanh Nguyen, Ok Kyung Lee, Eun Yeol Lee. Microbiol. Biotechnot. 2018. 102. 3071-3080).

[0003] Due to the high reactivity of formaldehyde, it can react with various intermediate metabolites containing ketone groups to synthesize advanced metabolites. The ketone group is one of the most common functional groups of metabolites in microorganisms, and the aldol reaction initiated by formaldehyde provides a new research idea for the utilization of one-carbon compounds such as formaldehyde and methanol. At present, there are documents reporting the non-natural utilization pathway of formaldehyde, and the inventors have particularly noticed a new utilization pathway of formaldehyde: the non-natural homoserine cycle (Homoserine cycle) pathway (An optimized methanol assimilation pathway relying on promiscuous formaldehyde-condensing aldolases in E. coli, Hai He, et al. Nat. Commun. 2018. 9. 1-9). Philippe Marliere, Arren Bar-Even. Metab Eng, 2020. 60: 1-13). In this pathway, formaldehyde can undergo an unnatural aldol reaction with the glycolysis end product pyruvate to generate 4-hydroxy 2-ketobutyrate. 4-hydroxy 2-ketobutyrate is an important intermediate in biological manufacturing, which can be further synthesized into a variety of high-value chemicals, including homoserine, 3-hydroxypropionic acid, 1,3-propanediol, etc. (Combining Aldolases and Transaminases for the Synthesis of 2-Amino-4-hydroxybutanoic Acid, Karel Hernandez, Jordi Bujons, Jesus Joglar, Simon J. Charnock, Pablo Dominguez de Maria, Wolf Dieter Fessner, and Pere Clapes. ACS Catal, 2017. 7(3): 1707-1711; An Aldolase-Catalyzed New Metabolic Pathway for the Assimilation of Formaldehyde and Methanol To Synthesize 2-Keto-4-hydroxybutyrate and 1,3-Propanediol in Escherichia coli, Chuang Wang, Jie Ren, Libang Zhou, Zhidong Li, Lin Chen, An-Ping Zeng. ACS Synth. Biol. 2019. 8: 2483-2493). Compared with the natural utilization pathway, the formaldehyde unnatural utilization pathway has the advantages of shortness and high efficiency, and thus has become a hot research direction for the microbial utilization of one-carbon such as methanol, methane, formaldehyde, etc.

[0004] In terms of one-carbon utilization, the unnatural condensation reaction of formaldehyde and pyruvate has shown great application potential. However, there are very few aldolases reported to effectively catalyze the condensation reaction of formaldehyde and pyruvate, mainly including 2-keto-4-hydroxyglutarate aldolase (KHB, EC 4.1.3.16), 2-dehydro-3-deoxy-L-rhamnose acid aldolase (RhmA, also named YfaU, EC 4.1.2.53) and 5-keto-4-deoxy-D-glutarate aldolase (GarL, EC 4.1.2.20) etc. The reported aldolases generally have low affinity for formaldehyde and exhibit low catalytic activity, thus limiting the application of the formaldehyde unnatural utilization pathway. SUMMARY

[0005] In order to solve the above technical problems, the present application provides a method strategy for efficiently mining and screening aldehyde condensation biological materials, and successfully screens a series of novel aldehyde condensing enzymes capable of effectively catalyzing the condensation of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid. The enzyme element basis for solving formaldehyde utilization is laid.

[0006] One object of the present application provides an application of an aldehyde condensing enzyme biological element, which is to biosynthesize 4-hydroxy-2-ketobutyric acid and 4-hydroxy-2-ketobutyric acid downstream derivatives from pyruvic acid and formaldehyde as substrates, wherein the aldehyde condensing enzyme can biosynthesize 4-hydroxy-2-ketobutyric acid from formaldehyde and pyruvic acid as substrates; the aldehyde condensing enzyme biological element is B1 or B2 or B3 as follows:

[0007] B1, aldehyde condensing enzyme, B2, nucleic acid sequence encoding the aldehyde condensing enzyme, B3, expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid sequence;

[0008] The aldehyde condensing enzyme is any one of C1-C12 as follows:

[0009] C1, the protein shown as sequence 1 in the sequence listing;

[0010] C2, the protein shown as sequence 2 in the sequence listing;

[0011] C3, the protein shown as sequence 3 in the sequence listing;

[0012] C4, the protein shown as sequence 4 in the sequence listing;

[0013] C5, the protein shown as sequence 5 in the sequence listing;

[0014] C6, the protein shown as sequence 6 in the sequence listing;

[0015] C7, the protein shown as sequence 7 in the sequence listing;

[0016] C8, the protein shown as sequence 8 in the sequence listing;

[0017] C9, the protein shown as sequence 9 in the sequence listing;

[0018] C10, the protein shown as sequence 10 in the sequence listing;

[0019] C11, the protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues to any one of C1-C10;

[0020] C12, the fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any one of C1-C10;

[0021] The tag in C12 above is used for expression, detection, tracing and / or purification of the target protein, etc. The protein tag refers to a polypeptide or protein fused and expressed with the target protein by DNA in vitro recombination technology.

[0022] The tag can be any of the tags shown in the following table:

[0023] Table 1 Protein tag for fusion expression

[0024] Tag Residue Sequence Poly-His 2-10 (usually 6) HHHHHH Poly-Arg 5-6 (usually 5) RRRRR Flag 8 DYKDDDDK c-myc 10 EQKLISEEDL Strep-tag II 8 WSHPQFEK MBP 367 Sequence 10

[0025] The above application of using aldehyde condensing biological material to catalyze the condensation of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid also provides a preparation method of 4-hydroxy-2-ketobutyric acid or a downstream derivative of 4-hydroxy-2-ketobutyric acid.

[0026] Further, the expression cassette of the nucleic acid molecule in B3 refers to the expression of the above-mentioned aldolase DNA in a host cell. The DNA can include not only a promoter for initiating transcription of the aldolase-encoding gene, but also a terminator for terminating transcription of the aldolase-encoding gene. Further, the expression cassette can also include an enhancer sequence.

[0027] The recombinant vector of the nucleic acid molecule in B3 can be a bacterial plasmid (such as a T7 promoter-based expression vector for expression in bacteria, specifically pET-16b, pET28a, etc.; a Trc promoter-based expression vector for expression in bacteria, specifically pTrc99a, pTrc33a, etc.), a bacteriophage, a yeast plasmid, or a retrovirus packaging plasmid carrying the aldolase-encoding gene.

[0028] The recombinant microorganism containing the nucleic acid molecule encoding the above-mentioned dehydrogenase in B3 can be a bacterium, yeast, algae, or fungus such as Escherichia coli, Corynebacterium glutamicum, and methylotrophic bacteria carrying the aldolase-encoding gene.

[0029] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; or the nucleic acid molecule can be RNA, such as mRNA or hnRNA, etc.

[0030] In the above application, the aldolase catalyzes in the form of crude enzyme solution, crude enzyme solution freeze-dried powder, whole cell, or pure enzyme. Further, the crude enzyme solution, crude enzyme solution freeze-dried powder, and pure enzyme can be prepared according to the following method: expressing the aldolase in a host cell to obtain recombinant cells; lysing the recombinant cells to obtain the crude enzyme solution, crude enzyme solution freeze-dried powder, or pure enzyme. The whole cell can be prepared according to the following method: expressing the aldolase in a host cell, and the obtained recombinant cells are the whole cells.

[0031] Further, the recombinant cell can be prepared according to the method comprising the following steps: introducing a nucleic acid molecule capable of expressing the aldolase into the host cell, and obtaining the recombinant cell expressing the aldolase after induction culture.

[0032] Further, the "nucleic acid molecule capable of expressing the aldolase" is introduced into the host cell in the form of a recombinant vector. The recombinant vector can be a bacterial plasmid (such as a T7 promoter-based expression vector for expression in bacteria, specifically pET-16b, or a Trc promoter-based expression vector for expression in bacteria, specifically pTrc99a) carrying the coding gene of the aldolase, a bacteriophage, a yeast plasmid, or a retrovirus packaging plasmid.

[0033] Further, the coding gene of the aldolase is introduced into the host cell in the form of a recombinant vector. The recombinant vector can be a bacterial plasmid (such as a T7 promoter-based expression vector for expression in bacteria, specifically pET-16b, or a Trc promoter-based expression vector for expression in bacteria, specifically pTrc99a) carrying the coding gene of the aldolase, a bacteriophage, a yeast plasmid, or a retrovirus packaging plasmid.

[0034] In an embodiment of the present application, the host cell selected is a microbial cell such as Escherichia coli, Corynebacterium glutamnicum, Bacillus subtilis, Lactic acid bacteria, Pseudomonas putida, Methylorubrum extorquens, Saccharomyces cerevisiae, etc. Preferably, the host cell is Escherichia coli, and more preferably, the host cell is Escherichia coli W3110.

[0035] In an embodiment of the present application, the methanol dehydrogenase and the aldolase are co-expressed using the plasmid pTrc99a as an expression vector.

[0036] In an embodiment of the present application, the sarcosine oxidase and the aldolase are co-expressed using the plasmid pTrc99a as an expression vector.

[0037] The present application further provides a method for producing 4-hydroxy-2-ketobutyric acid using the genetically engineered recombinant strain described above.

[0038] In an embodiment of the present application, the method for producing 4-hydroxy-2-ketobutyric acid using the recombinant strain described above using methanol or sarcosine as a substrate is as follows:

[0039] 1) Seed culture:

[0040] Carbenicillin (50 μg / mL) was added to LB medium according to plasmid resistance, and a single colony was inoculated into a test tube containing LB medium, and cultured at 37°C and 220 r / min overnight;

[0041] 2) Fermentation culture:

[0042] The seed liquid was obtained by overnight culture, and the initial OD 600 = 0.1 was transferred into inorganic salt medium with the addition of carbenicillin (50 μg / mL), and the glucose concentration was 10 g / L, and the culture was carried out at 37°C and 220 rpm, and when the OD600 of the bacteria was 0.5, 0.1 mM IPTG was added to induce gene expression, and after 3 hours of induction, 10 g / L of methanol or 10 mM of sarcosine was added, and the culture was continued at 30°C for 24 hours.

[0043] In one experiment, the results showed that the addition of 10 mM sarcosine can generate 5.33 mM of 4-hydroxy-2-ketobutyric acid, and the addition of 10 g / L of methanol can generate 0.95 mM of 4-hydroxy-2-ketobutyric acid.

[0044] The present application thus provides a method for screening an aldolase capable of being applied to effectively catalyze the condensation of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid, comprising the following steps:

[0045] (1) Screening a candidate aldolase satisfying the following characteristics:

[0046] A1) belongs to the HpcH functional domain family protein;

[0047] A2) the database function annotation is 4-hydroxy-2-oxovalerate aldolase capable of catalyzing the condensation of acetaldehyde and pyruvic acid;

[0048] A3) the length of the amino acid sequence ranges from 240 to 320 amino acids;

[0049] A4) capable of performing enzyme catalytic reaction with formaldehyde and pyruvic acid as substrates in appropriate conditions and media to biosynthesize 4-hydroxy-2-ketobutyric acid;

[0050] (2) verifying the function of the candidate aldolase, specifically with pyruvic acid and formaldehyde as substrates, and the candidate aldolase as a catalytic enzyme, verifying whether it can catalyze the generation of 4-hydroxy-2-ketobutyric acid or its catalytic efficiency.

[0051] The application provides an efficient strategy for screening aldehyde lyases capable of catalyzing condensation reaction of formaldehyde and pyruvic acid, and a series of aldehyde lyases capable of efficiently catalyzing condensation reaction of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid are screened, the aldehyde lyases catalyze the reaction for the first time, and lay a good screening strategy and efficient enzyme element foundation for microbial one-carbon utilization and development of methanol, methane, formaldehyde, formic acid, carbon dioxide and the like. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 It is an SDS-PAGE detection graph of the purified aldehyde lyase.

[0053] Figure 2 It is a schematic diagram of the aldehyde lyase catalyzing condensation of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid

[0054] Figure 3 It is in vitro catalysis of the purified aldehyde lyase to condense formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid.

[0055] Figure 4 It is in vivo participation of the aldehyde lyase in synthesis of 4-hydroxy-2-ketobutyric acid by taking methanol as a substrate.

[0056] Figure 5 It is in vivo participation of the aldehyde lyase in synthesis of 4-hydroxy-2-ketobutyric acid by taking sarcosine as a substrate. DETAILED DESCRIPTION

[0057] The method of the application is described below through specific embodiments. Unless otherwise specified, the technical means used in the application are methods known to those skilled in the art. The examples are implemented on the premise of the technical solutions of the application, and detailed implementation modes and specific operation processes are given. The examples will help to understand the application, but the protection scope of the application is not limited to the following examples, i.e., does not constitute a limitation on the application. For those skilled in the art, various changes or modifications to the material components and amounts in these embodiments without departing from the essence and scope of the application also belong to the protection scope of the application.

[0058] Among them, the determination method used in the application is as follows:

[0059] 1) Formaldehyde analysis and determination: acetylacetone colorimetric method is used for formaldehyde analysis and determination, acetylacetone colorimetric reagent (Nash): 2M ammonium acetate, 50mM glacial acetic acid, 20mM acetylacetone. Nash reagent (100μL) is mixed with sample (100μL) in a 96-well plate, and the reaction is carried out at 60℃ for 10min, after cooling, an enzyme marker is used for detection, and the detection wavelength is 414nm.

[0060] 2) 4-hydroxy-2-ketobutyric acid assay A sample was taken from the reaction mixture and diluted with deionized water to obtain a concentration range of 3 to 50 mM. The diluted solution (25 μL) was mixed with O-benzyl hydroxylamine hydrochloride solution (50 μL, 130 mM stock solution in pyridine:methanol:water = 33:15:2). After 10 min of reaction at 25 °C, the sample was diluted with methanol (500 μL), centrifuged through a membrane (0.22 μm), and analyzed by HPLC with a 120 EC-C18 column (2.7 μm, 3.0 x 150 mm) equipped. The mobile phase A: deionized water (ddH2O) with 0.1% (v / v) trifluoroacetic acid (TFA); mobile phase B: acetonitrile (CH3CN) with 0.095% (v / v) TFA, flow rate 1 mL min -1 at 215 nm, column temperature 30 °C. Elution conditions: gradient elution, mobile phase (B) changed from 8 to 80% in 30 min.

[0061] The culture and reaction system used in the present application is as follows:

[0062] 1) Inorganic salt medium: the basic components include (1 L): 47.8 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 93 mM NH4Cl, 2 mM MgSO4 100 μM CaCl2; trace elements (1 L): 134 μM EDTA, 31 μM FeCl3, 6.2 μM ZnCl2, 0.76 μM CuCl2, 0.42 μM CoCl2, 1.62 μM H3BO3, 0.081 μM MnCl2; glucose: 30 g / L, and other additives such as antibiotics, amino acids, etc. are added according to the situation.

[0063] 2) LB medium (1 L): 10 g of proteose peptone, 10 g of sodium chloride, 5 g of yeast powder, pH 7.0.

[0064] 3) Enzyme reaction system buffer: HEPES buffer (100 mM): 100 mM HEPES, 1500 mM KCl, 100 mM KH2PO4, pH = 7.5.

[0065] Example 1 Screening of aldehyde dehydrogenase biological elements

[0066] 1. Establishment of aldehyde dehydrogenase biological material screening method

[0067] To screen new aldehyde lyases that can effectively catalyze the condensation of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid, based on the presumed active functional domain of aldehyde lyase that can catalyze the condensation of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid, and combined with the substrate similarity strategy, a method for screening new aldehyde lyases that can catalyze the condensation of formaldehyde and pyruvic acid was developed, and a series of new aldehyde lyases that can effectively catalyze the condensation of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid were successfully screened. The specific screening strategy is as follows:

[0068] 1) First, based on the aldehyde lyase protein library in Uniprot and NCBI databases, protein elements with HpcH functional domain were screened;

[0069] 2) Further, combined with the substrate similarity strategy, protein elements of 4-hydroxy-2-oxovalerate aldehyde lyase that can or can catalyze the condensation reaction of acetaldehyde and pyruvic acid were screened;

[0070] 3) Further, the preliminary screening candidate aldehyde lyase library combining the above two characteristics was subjected to phylogenetic tree analysis;

[0071] 4) Further, according to the phylogenetic tree, one protein sequence was selected from each main branch of the phylogenetic tree for synthesis and expression;

[0072] 5) Finally, the screened aldehyde lyase elements were subjected to protein expression and purification, and the efficiency of each candidate aldehyde lyase element in catalyzing the condensation of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid was evaluated in vitro reaction with formaldehyde and pyruvic acid as substrates.

[0073] Based on the screening strategy steps 1 and 2, a library of 960 aldehyde lyase protein sequences was initially screened; further analysis found that the amino acid sequence length of the protein elements in the candidate aldehyde lyase protein sequence library was relatively uniform, and the sequence length ranged from 240 to 320 amino acids; further, phylogenetic analysis was performed on the 960 aldehyde lyase protein elements in the aldehyde lyase protein sequence library, and 20 protein sequences were initially screened from the main branches of the phylogenetic tree for gene synthesis, and 2 protein sequences were further screened from the branches with high activity, and the aldehyde lyase library information composed of a total of 22 aldehyde lyase elements is shown in Table 2. Further, protein expression and function verification were performed.

[0074] Table 2 Information Table of Aldehyde Lyase Sub-library

[0075]

[0076]

[0077] 2. Preparation of recombinant bacteria expressing aldehyde lyase

[0078] 1) Preparation of recombinant plasmids and strains expressing aldehyde lyase

[0079] The recombinant plasmid expressing aldolase is obtained by replacing the DNA molecule between the Ndel and BamHI sites of the expression vector pET16b with the coding gene of each aldolase in the aldolase library of Table 2 (column 4 of Table 2), to obtain a series of vectors: pET16b-CsAld, pET16b-AmAld, pET16b-AaAld, pET16b-PwAld, pET16b-TtAld, pET16b-PbAld, pET16b-CbAld, pET16b-RiAld, pET16b-ViAld, pET16b-LmAld, pET16b-AsAld, pET16b-RsAld, pET16b-BtAld, pET16b-AfAld, pET16b-AnAld, pET16b-BlAld, pET16b-SfAld, pET16b-PsAld, pET16b-XcAldl, pET16b-XcAld2, pET16b-ToAld, pET16b-BsAld. The series of vectors express recombinant proteins fused with His tag at the N-terminus of each aldolase. Among them, the vector pET16b is resistant to ampicillin and has a His tag. Further, each of the prepared recombinant plasmids is transformed into E. coli BL21 (DE3) to obtain recombinant bacteria capable of expressing aldolase.

[0080] 2) Induced expression and purification of aldolase

[0081] The above expression vectors are transformed into the E. coli expression host E. coli BL21 (DE3) respectively, and cultured at 37°C to OD 600 When OD reaches 0.5, 0.1 mM IPTG is added to induce gene expression, and the bacteria are collected after overnight induction at 16°C. After ultrasonic disruption, the protein is purified by nickel ion affinity chromatography. The purified protein is detected by SDS-PAGE. Part of the detection results are shown in Figure 1 As shown in the figure, M: protein marker; lane 1 is protein CsAld, and the protein size is 28.1 kDa; lane 2 is protein AmAld, and the protein size is 28.7 kDa; lane 3 is protein TtAld, and the protein size is 28.2 kDa; lane 4 is protein LmAld, and the protein size is 27.2 kDa; lane 5 is protein RsAld, and the protein size is 27.1 kDa; lane 6 is protein BtAld, and the protein size is 26.8 kDa; lane 7 is protein AnAld, and the protein size is 28.2 kDa; lane 8 is protein BlAld, and the protein size is 27.6 kDa; lane 9 is protein ToAld, and the protein size is 29.5 kDa; lane 10 is protein BsALd, and the protein size is 28.8 kDa.

[0082] Example 2 Aldolase catalyzing synthesis of 4-hydroxy-2-ketobutyric acid from formaldehyde and pyruvate in vitro

[0083] Figure 2 Schematic diagram of aldolase catalyzing condensation of formaldehyde and pyruvate to generate 4-hydroxy-2-ketobutyric acid.

[0084] Each of the purified aldolases CsAld, AmAld, AaAld, PwAld, TtAld, PbAld, CbAld, RiAld, ViAld, LmAld, AsAld, RsAld, BtAld, AfAld, AnAld, BlAld, SfAld, PsAld, XcAld1, XcAld2, ToAld, BsAld obtained in the above Example 1 was respectively used to conduct a pure enzyme catalysis reaction in vitro with formaldehyde and pyruvate as substrates, so as to realize screening of the aldolase element library.

[0085] The above-mentioned in vitro catalysis reaction was conducted by using the following reaction system: the reaction system contained 10 mM HEPES (pH = 7.5), 5 mM MgCl2, added substrates sodium pyruvate (50 mM) and formaldehyde (20 mM), 5 μM aldolase as catalyst, and the catalysis reaction was conducted at 30°C, the reaction time was 60 min, after the reaction, 10% TCA was added to terminate the reaction, the supernatant was obtained by centrifugation, and 0.22 uM filter membrane was used for filtration, and the sample was prepared for liquid phase detection, and the detection results are shown in Table 2. Figure 3 The results show that the aldolases CsAld, AmAld, TtAld, LmAld, RsAld, BtAld, AnAld, BlAld, ToAld, BsAld can efficiently catalyze the condensation of pyruvate and formaldehyde to generate 4-hydroxy-2-ketobutyric acid, and the conversion rate is 9-99%, and the specific conversion rate is shown in Table 3. The remaining aldolases in Table 2 have lower catalysis efficiency or no product is detected in the condensation reaction of formaldehyde and pyruvate.

[0086] Table 3 is the detection results of aldolase catalyzing synthesis of 4-hydroxy-2-ketobutyric acid from formaldehyde and pyruvate

[0087] Serial number Aldehyde-lyase name Source Substrate formaldehyde conversion rate (%) 1 CsAld Curvibacter sp. AEP1-3 95.1 2 AmAld Advenellamimigardefordensis 96.2 3 AaAld Acidovorax antarcticus 4.8 4 PwAld Pseudoxanthomonas winnipegensis 1.9 5 TtAld Thermoanaerobacterium thermosaccharolyticum 9.7 6 PbAld Planctomycetaceae bacterium 0 7 CbAld Chloroflexi bacterium 0.1 8 RjAld Rhodococcus jostii (strain RHA1) 0 9 ViAld Vibrio ishigakensis 0 10 LmAld Leucobacter massiliensis 10.4 11 AsAld Aureimonas sp. Leaf460 0 12 RsAld Rhizobium sp. P28RR-XV 54.8 13 BtAld bacterium M00.F.Ca.ET.156.01.1.1 51.2 14 AfAld Aspergillus flavus 2.8 15 AnAld Aspergillus niger 30.7 16 BlAld Brevibacterium linens 9.7 17 SfAld Sinorhizobium fredii 0.1 18 PsAld Pseudopuniceibacterium sediminis 0.5 19 XcAld1 Xanthomonas campestris 1.4 20 XcAld2 Xanthomonas citri pv. punicae 1.9 21 ToAld Thelonectria olida 92.7 22 BsAld Byssochlamys spectabilis 96.6

[0088] The above results can be seen that the 10 proteins of Table 3 (CsAld, AmAld, TtAld, LmAld, RsAld, BtAld, AnAld, BlAld, ToAld, BsAld, and the corresponding amino acid sequences are shown in SEQ ID NO: 1-10) can effectively prepare 4-hydroxy-2-ketobutyric acid compound, wherein CsAld, AmAld, ToAld, BsAld catalyze the effect of formaldehyde and pyruvic acid to generate 4-hydroxy-2-ketobutyric acid is the best, the substrate formaldehyde conversion rate is 95.1%, 96.2%, 92.7%, 96.6% respectively. In addition, the aldolase RsAld, BtAld, AnAld also showed high catalytic activity for catalyzing the condensation reaction of formaldehyde and pyruvic acid.

[0089] Example Three Construction of Recombinant Strains Containing Aldolase

[0090] 1. Construction of frmA-deficient strain

[0091] Based on the screening of novel aldolases with formaldehyde and pyruvic acid as substrates, the screened novel aldolases were further introduced into the cell. We knocked out the gene frmA in the model Escherichia coli W3110 to construct the functional strain W3110△frmA. The purpose of knocking out the gene frmA is to weaken the effect of the natural glutathione-dependent formaldehyde detoxification system (frmRAB) existing in the cell. The formaldehyde detoxification pathway will convert intracellular formaldehyde into formic acid and CO2, so that the intracellular formaldehyde accumulation is little, and knocking out the gene frmA will be beneficial to the utilization of formaldehyde in the non-natural synthesis pathway.

[0092] 2. Construction of functional plasmid containing aldolase

[0093] Since formaldehyde has high toxicity to cells, formaldehyde at a level greater than 2 mM can have a significant inhibitory effect on cells. Therefore, we introduced formaldehyde precursors and related pathway genes, including methanol and methanol dehydrogenase pathway catalyzing the generation of formaldehyde from methanol, or sarcosine and sarcosine oxidase pathway catalyzing the generation of formaldehyde from sarcosine; the above-mentioned methanol or sarcosine as substrate to realize the generation of intracellular formaldehyde, and further catalyze the formaldehyde and pyruvic acid to generate the target product 4-hydroxy-2-ketobutyric acid by the screened aldolase.

[0094] 1) Construction of methanol utilization pathway plasmid

[0095] First, the plasmid pTra99a-BsMdh was constructed:

[0096] The methanol dehydrogenase BsMdh (EC: 1.1.1.6, SEQ ID NO. 11) gene fragment containing the homologous sequence of plasmid pTrc99a was obtained by PCR amplification using corresponding primers; then Gibson assembly was performed with the double enzyme digested vector pTra99a (NcoI / SacI) to obtain the vector plasmid pTra99a-BsMdh.

[0097] Then a series of plasmids containing aldehyde dehydrogenase were constructed:

[0098] The aldehyde dehydrogenase (BsAld, ToAld, AnAld, CsAld, AmAld) gene fragment containing the homologous sequence of plasmid pTrc99a-BsMdh was obtained by PCR amplification using corresponding primers; then Gibson assembly was performed with the double enzyme digested vector pTra99a-BsMdh (BamHI / SalI) to obtain the vector plasmid pTra99a-BsMdh-ToAld, pTra99a-BsMdh-BsAld, pTra99a-BsMdh-AnAld, pTra99a-BsMdh-CsAld, pTra99a-BsMdh-AmAld.

[0099] 2) Construction of sarcosine utilization pathway plasmid

[0100] First, the plasmid pTra99a-Sox was constructed:

[0101] The sarcosine oxidase Sox (EC: 1.5.3.1, SEQ ID NO. 12) gene fragment containing the homologous sequence of plasmid pTrc99a was obtained by PCR amplification using corresponding primers; then Gibson assembly was performed with the double enzyme digested vector pTra99a (NcoI / SacI) to obtain the vector plasmid pTra99a-Sox.

[0102] Then a series of plasmids containing aldehyde dehydrogenase were constructed:

[0103] The aldehyde dehydrogenase (BsAld, ToAld, AnAld, CsAld, AmAld) gene fragment containing the homologous sequence of plasmid pTrc99a-Sox was obtained by PCR amplification using corresponding primers; then Gibson assembly was performed with the double enzyme digested vector pTra99a-Sox (BamHI / SalI) to obtain the vector plasmid pTra99a-Sox-ToAld, pTra99a-Sox-BsAld, pTra99a-Sox-AnAld, pTra99a-BsMdh-CsAld, pTra99a-BsMdh-AmAld.

[0104] 3. Construction of pathway recombinant strains

[0105] 1) Construction of recombinant strain for methanol pathway

[0106] The plasmids pTra99a-BsMdh-ToAld, pTra99a-BsMdh-BsAld, pTra99a-BsMdh-AnAld, pTra99a-BsMdh-CsAld, pTra99a-BsMdh-AmAld containing methanol dehydrogenase gene and aldehyde dehydrogenase gene constructed in the above 2 were respectively transformed into the functional strain W3110△frmA, to obtain the recombinant strains W3110△frmA(pTra99a-BsMdh-ToAld), W3110△frmA(pTra99a-BsMdh-BsAld), W3110△frmA(pTra99a-BsMdh-AnAld), W3110△frmA(pTra99a-BsMdh-CsAld), W3110△frmA(pTra99a-BsMdh-AmAld), with the resistance of carbenicillin. Meanwhile, the empty plasmid pTra99a was transformed into the functional strain W3110△frmA, to obtain the recombinant strain W3110△frmA(pTra99a) as a control strain.

[0107] 2) Construction of recombinant strain for sarcosine pathway

[0108] The plasmids pTra99a-Sox-ToAld, pTra99a-Sox-BsAld, pTra99a-Sox-AnAld, pTra99a-Sox-CsAld, pTra99a-Sox-AmAld containing sarcosine oxidase gene and aldehyde dehydrogenase gene constructed in the above 2 were respectively transformed into the functional strain W3110△frmA, to obtain the recombinant strains W3110△frmA(pTra99a-Sox-ToAld), W3110△frmA(pTra99a-Sox-BsAld), W3110△frmA(pTra99a-Sox-AnAld), W3110△frmA(pTra99a-Sox-CsAld), W3110△frmA(pTra99a-Sox-AmAld), with the resistance of carbenicillin. Meanwhile, the empty plasmid pTra99a was transformed into the functional strain W3110△frmA, to obtain the recombinant strain W3110△frmA(pTra99a) as a control strain.

[0109] Example Four Fermentation of recombinant strain with methanol as substrate to synthesize 4-hydroxy-2-ketobutyric acid

[0110] First, single clones of the series of strains W3110△frmA(pTra99a-BsMdh-ToAld), W3110△frmA(pTra99a-BsMdh-BsAld), W3110△frmA(pTra99a-BsMdh-AnAld), W3110△frmA(pTra99a-BsMdh-CsAld), and W3110△frmA(pTra99a-BsMdh-AmAld) from the methanol pathway were picked from the plate and added to a test tube containing 4 mL of LB medium. Carbobenzylmycin (50 μg / mL) was added, and the culture was carried out overnight at 37°C and 220 r / min to obtain the seed culture.

[0111] Then with the initial OD 600 =0.1 Transfer to inorganic salt medium, add initial concentration of 10 g / L methanol and 20 g / L glucose, and culture at 37℃ and 220 rpm, while adding carbenicillin (50 μg / mL) and the OD of the bacterial cells. 600 When the concentration of the sample was 0.5, 0.1 mM IPTG was added to induce gene expression. The sample was then cultured at 30°C for 24 h. 1 mL of the sample was taken, centrifuged to collect the supernatant, and filtered through a 0.22 μM filter membrane. The resulting sample was then subjected to liquid chromatography analysis. The results are as follows: Figure 4 This indicates that aldolases BsAld, ToAld, AnAld, CsAld, and AmAld can all bind to methanol dehydrogenase intracellularly, using methanol and pyruvate as substrates to synthesize 4-hydroxy-2-keto-butyric acid in two steps. Among them, strain W3110△frmA(pTra99a-BsMdh-BsAld) can produce 0.95mM of 4-hydroxy-2-keto-butyric acid, strain W3110△frmA(pTra99a-BsMdh-ToAld) can produce 0.85mM of 4-hydroxy-2-keto-butyric acid, strain W3110△frmA(pTra99a-BsMdh-AnAld) can produce 0.79mM of 4-hydroxy-2-keto-butyric acid, strain W3110△frmA(pTra99a-BsMdh-CsAld) can produce 0.85mM of 4-hydroxy-2-keto-butyric acid, and strain W3110△frmA(pTra99a-BsMdh-AmAld) can produce 0.8mM of 4-hydroxy-2-keto-butyric acid.

[0112] Example 5: 4-Hydroxy-2-ketobutyric acid synthesized by fermentation of recombinant strains using sarcosine as a substrate.

[0113] First, single clones of the series of strains W3110△frmA(pTra99a-Sox-ToAld), W3110△frmA(pTra99a-Sox-BsAld), W3110△frmA(pTra99a-Sox-AnAld), W3110△frmA(pTra99a-Sox-CsAld), and W3110△frmA(pTra99a-Sox-AmAld) from the methanol pathway were picked from the plate and added to a test tube containing 4 mL of LB medium. Carbobenzylmycin (50 μg / mL) was added, and the culture was carried out overnight at 37°C and 220 r / min to obtain the seed culture.

[0114] Then, the culture was transferred to an inorganic salt medium with an initial OD600 of 0.1 and a glucose concentration of 10 g / L. The medium was incubated at 37°C and 220 rpm, with the addition of carbenicillin (50 μg / mL) and the culture medium containing the desired OD600 of the bacterial cells. 600 When the creatine concentration was 0.5, 0.1 mM IPTG was added to induce gene expression. After culturing at 30°C for 3 hours, 5 mM creatine was added, followed by 5 mM creatine every 12 hours for another 24 hours. After induction, 1 mL of sample was collected, the supernatant was centrifuged, and filtered through a 0.22 μM filter membrane. The sample was then prepared for liquid chromatography analysis. The results are as follows: Figure 5 BsAld, ToAld, AnAld, CsAld, and AmAld can all bind to sarcosine oxidase intracellularly. Using sarcosine and pyruvate as substrates, the reaction proceeds in two steps: sarcosine is converted to formaldehyde by sarcosine oxidase Sox, and formaldehyde is then converted to 4-hydroxy-2-keto-butyric acid by aldolase reaction with pyruvate. Using 10 mM sarcosine as a substrate, strain W3110△frmA (pTra99a-Sox-ToAld) can generate 3.56 mM of 4-hydroxy-2-keto-butyric acid, strain W3110△frmA (pTra99a-Sox-BsAld) can generate 5.33 mM of 4-hydroxy-2-keto-butyric acid, strain W3110△frmA (pTra99a-Sox-AnAld) can generate 2.48 mM of 4-hydroxy-2-keto-butyric acid, strain W3110△frmA (pTra99a-Sox-CsAld) can generate 3.75 mM of 4-hydroxy-2-keto-butyric acid, and strain W3110△frmA (pTra99a-Sox-AmAld) can generate 2.87 mM of 4-hydroxy-2-keto-butyric acid.

Claims

1. An application of an aldolase bioelement, wherein the application uses pyruvate and formaldehyde as substrates to biosynthesize 4-hydroxy-2-ketobutyric acid, and the aldolase bioelement is B1, B2, or B3 as follows: B1, aldolase; B2, nucleic acid sequence encoding the aldolase; B3, expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid sequence. The aldolase is the protein shown in Sequence 1 or the protein shown in Sequence 2, or a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein; the tag is selected from: Poly-His, Poly-Arg, Flag, c-myc, Strep-tagII, or MBP. The procedure is carried out by recombinant microorganisms or in vitro enzymatic reactions, wherein the recombinant microorganisms are obtained by overexpressing the aldolase in a frmA-deficient Escherichia coli strain.

2. The application according to claim 1, characterized in that: The recombinant microorganism is a recombinant bacterium obtained by introducing a recombinant vector into a host bacterium, which overexpresses the aldolase and participates in the condensation reaction of formaldehyde and pyruvate to synthesize 4-hydroxy-2-ketobutyric acid; the recombinant vector is a vector obtained by inserting the coding nucleic acid sequence of the aldolase into an expression vector.

3. The application according to claim 1, characterized in that: The in vitro enzymatic reaction system includes the aldolase, which participates in the condensation reaction of formaldehyde and pyruvate to synthesize 4-hydroxy-2-ketobutyric acid.

4. The application according to claim 1, characterized in that: The formaldehyde is used directly as a substrate.

5. The application according to claim 1, characterized in that: The formaldehyde is generated using methanol, sarcosine, and carbon dioxide as substrates in the recombinant microorganism or in vitro enzyme reaction system.

6. The application according to claim 1, characterized in that: The recombinant microorganism is obtained by knocking out the frmA gene in Escherichia coli W3110 to obtain an frmA-deficient strain, and then introducing the nucleic acid sequence encoding methanol dehydrogenase, which catalyzes the production of formaldehyde from methanol, and the aldolase described in claim 1, or introducing the nucleic acid sequence encoding sarcosine oxidase, which catalyzes the production of formaldehyde from sarcosine, and the aldolase described in claim 1.

7. The application according to claim 1, characterized in that: The pyruvate is used directly as a substrate; or the pyruvate is generated in the recombinant microorganism or in vitro enzyme reaction system using glucose, glycerol, or lactic acid as substrates.

8. The in vitro enzyme reaction system according to claim 7, characterized in that, The reaction system contains 8-12 mM HEPES, pH=7.5, 2-2000 mM formaldehyde, 20-2000 mM pyruvate, 1-10 mM MgCl2, and 1-20 μM aldolase. The reaction conditions are: catalytic reaction at 25-37℃ for 0.1-24 hours.

9. The in vitro enzyme reaction system according to claim 8, characterized in that, The reaction system contained 10 mM HEPES, 50 mM pyruvate, 20 mM formaldehyde, 2 mM MgCl2, and 5 μM aldolase. The reaction conditions were: catalytic reaction at 30 °C for 1 hour.

10. The in vitro enzyme reaction system according to claim 9, characterized in that, The aldolase exists in the form of crude enzyme solution, crude enzyme solution lyophilized powder, whole cells, or pure enzyme.

11. The in vitro enzyme reaction system according to claim 10, characterized in that, The crude enzyme solution, crude enzyme solution freeze-dried powder, and pure enzyme can be prepared by a method including the following steps: the recombinant microorganism ferments and expresses the aldolase to obtain recombinant cells; the recombinant cells are lysed to obtain the crude enzyme solution, crude enzyme solution freeze-dried powder, or pure enzyme. The whole cells are prepared by a method including the following steps: the recombinant microorganism ferments and expresses the aldolase, and the resulting recombinant cells are the whole cells.

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