Condensing enzyme mutant and use thereof

Abstract

This invention discloses a mutant of a condensation enzyme and its applications. Several mutant condensation enzymes with enhanced activity were obtained through genetic engineering. These mutants can efficiently catalyze the conversion of formaldehyde, especially significantly improving the efficiency of formaldehyde condensation to 1,3-dihydroxyacetone under low formaldehyde concentration conditions. The product, 1,3-dihydroxyacetone, can serve as a key precursor in the biosynthesis of C6 molecules; therefore, this invention has significant application value.

Description

Technical Field

[0001] This invention discloses a mutant of a condensing enzyme and its application, specifically involving several mutants of condensing enzymes with enhanced activity, belonging to the field of biochemical engineering. Background Technology

[0002] A condensing enzyme capable of catalyzing the condensation of formaldehyde to produce DHA was first reported in 2015; however, its affinity for formaldehyde is limited, exhibiting a very high Km value. Formaldehyde is highly toxic to bacteria, and the high Km value of the condensing enzyme hinders the intracellular synthesis of 1,3-dihydroxyacetone (DHA) from low concentrations of formaldehyde, and further synthesis of high-value compounds.

[0003] There is a great need to improve the enzyme activity and conversion efficiency of this condensing enzyme. Therefore, it is necessary to study and screen for condensing enzymes that improve the efficiency of condensation of formaldehyde to produce 1,3-dihydroxyacetone under low formaldehyde conditions. Summary of the Invention

[0004] Previous applications 202211308892.2 and 202211309020.8 involved mutations in the condensate enzyme (amino acid sequence SEQ ID NO: 1) reported in 2015. This invention further mutates the condensate enzyme (i.e., the mutant V92 therein) to obtain a new mutant, which significantly improves the efficiency of formaldehyde condensation to produce 1,3-dihydroxyacetone under low-concentration formaldehyde conditions. Simultaneously, the new amino acid site mutation obtained in this way, when used in mutants based on the amino acid sequence SEQ ID NO: 1, can also significantly improve the efficiency of formaldehyde condensation to produce 1,3-dihydroxyacetone under low-concentration formaldehyde conditions. Therefore, this invention is proposed.

[0005] This invention provides a mutant protein of a condensing enzyme, which, based on the amino acid sequence corresponding to SEQ ID NO: 1, contains the following site mutation: .

[0006] The present invention also provides a nucleic acid encoding the mutant protein described above.

[0007] The present invention further provides an expression vector containing the aforementioned nucleic acid.

[0008] The present invention also provides recombinant host cells containing the said nucleic acid or the said expression vector, such as recombinant host bacteria.

[0009] The present invention also provides the use of the mutant protein in the formaldehyde condensation to produce 1,3-dihydroxyacetone.

[0010] The present invention further provides a method for the condensation of formaldehyde to produce 1,3-dihydroxyacetone, comprising the following steps: The mutant protein was used as a catalyst to react with formaldehyde as a substrate to carry out a catalytic reaction, thereby obtaining 1,3-dihydroxyacetone.

[0011] Specifically, the mutant protein is used as a catalyst in crude enzyme washing of clothes and pure enzyme solution to express the mutant protein in its whole-cell form.

[0012] In a specific embodiment, the system of the catalytic reaction is as follows: The mixture was prepared by mixing the lysed supernatant of recombinant cells expressing the mutant protein with formaldehyde solution and TPP.

[0013] Preferably, the final concentration of formaldehyde in the reaction system is not higher than 100 mM; In a specific implementation, the reaction is carried out at 25°C-35°C; Optionally, the process may also include a step of further separating the resulting 1,3-dihydroxyacetone.

[0014] The condensate enzyme mutant provided by this invention can efficiently catalyze the conversion of formaldehyde, especially significantly improving the efficiency of formaldehyde condensation to 1,3-dihydroxyacetone under low formaldehyde concentration conditions. The product, 1,3-dihydroxyacetone, can serve as a key precursor in the biosynthesis of C6 molecules; therefore, this invention has significant application value. Detailed Implementation

[0015] Example 1: Construction of expression vector for condensing enzyme The condensation enzyme (FLS) is an amino acid mutant of benzaldehyde lyase derived from Pseudomonas fluorescens biovar I, containing the mutation A28I-A394G-G419N-A480W-L90T-W89K-R188H. Its amino acid sequence is shown in SEQ ID No. 1, and the corresponding coding gene sequence is shown in SEQ ID No. 2 disclosed in patent application 202211308892.2.

[0016] Using the recombinant plasmid pET21a-FLS disclosed in patent application 202211308892.2 as a template, the following primers were designed and synthesized: Upstream primer: 5'-GTTTAACTTTAAGAAGGAGATATACATATGGC-3' (SEQ ID No: 2); Downstream primer: 5'-GCTTTGTTAGCAGCCGGATC-3' (SEQ ID No: 3).

[0017] Error-prone PCR was used to randomly mutate the target gene.

[0018] After PCR, the target gene was recovered using a gel, and the mutated FLS gene was ligated into the vector pET21a.

[0019] The above vector was transformed into Escherichia coli BL21 Gold(DE3) and cultured at 37°C until single colonies grew.

[0020] Example 2: Screening of mutants of condensing enzymes For the mutant clones obtained in Example 1, single clones were picked and placed into 96-well plates containing LB medium. They were cultured overnight at 37°C and 800 rpm in a shaker. The clones were then transferred to 96-well plates and cultured at 37°C and 800 rpm for 3 hours. 0.5 mM IPTG was added, and the temperature was adjusted to 20°C to induce expression for 24 hours.

[0021] After culturing, the bacterial cells were collected by centrifugation. The cells were resuspended in Hepes buffer (formulation: 100 mM Hepes-NaCl, pH 7.4), and formaldehyde solution and TPP were added to the resuspended solution, bringing the final formaldehyde concentration in the reaction system to 15 mM. The reaction was carried out at 30°C. After the reaction was complete, Buffer 1 (formulation: galactose oxidase, horseradish peroxidase, pH 7.4) was added, followed by 50 μL of Buffer 2 (formulation: ABTS). The cells were then detected at 410 nm absorbance.

[0022] The strains obtained were compared with the original condensing enzyme; those with higher catalytic efficiency were identified as beneficial mutant strains. Gene sequencing was then used to identify the corresponding mutated amino acids. After screening a mutant library, mutants with significantly increased and stable enzyme activity were finally obtained.

[0023] The names of the mutants and their corresponding mutated amino acids were obtained through gene sequencing, as shown in Table 1.

[0024] Table 1. Condensation enzyme mutants obtained by screening in this invention

[0025] Note: Mutated amino acids are those compared to SEQ ID No. 1, and the mutation site is represented as "original amino acid (wild type), position (i.e., the position in SEQ ID No. 1), substituted amino acid". For example, replacing histidine with lysine at position 21 in SEQ ID No. 1 is named "H21R". Multiple mutations are separated by a plus sign (+), for example, "H21R+E367G" represents the substitution of histidine (H) and glutamic acid (E) at positions 21 and 367 with arginine (R) and glycine (G), respectively. Mutated nucleotides are those compared to SEQ ID No. 2, and the mutation site is represented as "original codon (wild type), position (i.e., the starting position of the codon in SEQ ID No. 2), substituted codon". For example, replacing CAC with CGC at positions 1447-1449 in SEQ ID No. 2 is named "CAC1447CGC". Multiple codon mutations are separated by a plus sign (+). For example, "CAT61CGT+GAA1099GGA" means that CAT and GAA at positions 61-63 and 1099-1101 of SEQ ID No.2 are replaced by CGT and GGA, respectively.

[0026] Example 3: Expression of condensase and its mutant in Escherichia coli The original condensate and mutant positive bacteria (see Example 2) were picked up with an inoculation needle and inoculated into LB medium. The culture was incubated at 37°C for 12 h, then inoculated into 100 mL of LB medium at a 1% (V / V) inoculation rate. After incubation at 37°C for 2.5 hours, IPTG was added to a final concentration of 0.5 mM and expression was induced at 20°C for 24 h. The culture was collected into centrifuge tubes, centrifuged, and the supernatant was discarded. The cells were resuspended in Hepes buffer (formulation: 100 mM Hepes-NaCl, pH 7.4) and washed. The cells were then stored at low temperature.

[0027] Example 4: DHA Standard Curve A reaction system (200 μL) was prepared with the following composition: 1,3-dihydroxyacetone (DHA) solutions of different concentrations: 0.125 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and 8 mM; enzyme buffer 1 (Hepes buffer containing galactose oxidase and horseradish peroxidase); and buffer 2 (Hepes buffer containing ABTS). The 200 μL reaction system was then detected under 410 nm absorption light. A standard curve of DHA concentration was obtained by plotting DHA concentration on the x-axis (x) and ΔA410 / t value on the y-axis. R0 2 Greater than 0.99.

[0028] Example 5: Activity Detection of Condensing Enzymes and Their Mutants The bacterial cells from Example 3 were collected by centrifugation at 4°C, resuspended in Hepes buffer (formulation: 100 mM Hepes-NaCl, pH 7.4) and sonicated to break down the bacteria. The supernatant was collected by centrifugation.

[0029] Cell lysis supernatant was mixed with formaldehyde solution and TPP (formaldehyde concentration 5 mM). After reaction at 30℃, enzyme buffer 1 (Hepes buffer containing galactose oxidase and horseradish peroxidase) was added, followed by buffer 2 (Hepes buffer containing ABTS). The reaction was detected at 410 nm. The rate of increase of A410 over time was calculated to represent the activities of wild-type and mutant enzymes. The percentage of mutant enzyme activity relative to wild-type is shown in Table 2 (wild-type enzyme activity is 100%). It can be seen that V1-V10 increased by 1.15-2.31 times.

[0030] Table 2. Relative activities of condensate enzyme variants

[0031] Example 6. Detection of the activity of condensing enzyme I28L-T90L-N283H-F484W-F425M-M167T-H188R (V92 in patent applications 202211308892.2 and 202211309020.8) and its mutants. The bacterial cells from Example 3 were collected by centrifugation at 4°C, resuspended in Hepes buffer (formulation: 100 mM Hepes-NaCl, pH 7.4) and sonicated to break down the bacteria. The supernatant was collected by centrifugation.

[0032] The cell lysis supernatant was mixed with formaldehyde solution and TPP. The formaldehyde concentration was 20 mM. The mixture was reacted at 30°C for 1 h. Then, enzyme buffer 1 (Hepes buffer containing galactose oxidase and horseradish peroxidase) was added, followed by buffer 2 (Hepes buffer containing ABTS). The reaction was detected under 410 nm light absorption. The whole-cell enzyme activities of I28L-T90L-N283H-F484W-F425M-M167T-H188R (V92 in patent applications 202211308892.2 and 202211309020.8) and mutants were calculated. The percentage of mutant enzyme activity relative to I28L is shown in Table 3 (with the enzyme activity of I28L-T90L-N283H-F484W-F425M-M167T-H188R as 100%). It can be seen that the enzyme activity of V11-V31 is 1.16-1.78 times that of I28L-T90L-N283H-F484W-F425M-M167T-H188R.

[0033] Table 3. Relative activities of condensate enzyme variants

[0034] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A mutant protein of a condensation enzyme, characterized in that, The mutant protein has the following site mutations based on the amino acid sequence corresponding to SEQ ID NO: 1: 。 2. A nucleic acid encoding the mutant protein as described in claim 1.

3. An expression vector containing the nucleic acid as described in claim 2.

4. A recombinant host cell containing the nucleic acid as described in claim 2 or the expression vector as described in claim 3.

5. The use of the mutant protein as described in claim 1 in the formaldehyde condensation to produce 1,3-dihydroxyacetone.

6. A method for the condensation of formaldehyde to produce 1,3-dihydroxyacetone, characterized in that, Including the following steps: The mutant protein described in claim 1 is used as a catalyst to react with formaldehyde as a substrate to carry out a catalytic reaction, thereby obtaining 1,3-dihydroxyacetone.

7. The method as described in claim 6, characterized in that, The mutant protein catalyst is a crude enzyme, a pure enzyme, or a whole-cell form expressing the mutant protein.

8. The method as described in claim 6 or 7, characterized in that, The system for the catalytic reaction contains TPP.

9. The method according to any one of claims 6-8, characterized in that, The final formaldehyde concentration in the reaction system is less than 100 mM.

10. The method as described in claim 9, characterized in that, It also includes a step of further separating the produced 1,3-dihydroxyacetone.

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