A 4-hydroxyphenylpyruvate dioxygenase mutant for synthesizing beta-hydroxyisovaleric acid and application thereof

Abstract

The application discloses a 4-hydroxyphenylpyruvate dioxygenase mutant for synthesizing beta-hydroxyisovaleric acid and an application thereof, and belongs to the technical field of bioengineering. The 4-hydroxyphenylpyruvate dioxygenase mutant is constructed, and the catalytic efficiency of the 4-hydroxyphenylpyruvate dioxygenase can be significantly improved. The 4-hydroxyphenylpyruvate dioxygenase and L-amino acid deaminase constructed are used together for catalyzing preparation of beta-hydroxyisovaleric acid, and the yield of HMB can reach 7 g / L, and the conversion rate is 77%. Compared with the 4-hydroxyphenylpyruvate dioxygenase capable of catalyzing L-leucine to generate HMB at present, the yield of HMB is higher. The HMB yield of BbHPPD A366P / M359V / N382S is 3.7 times that of the wild enzyme, and the industrialization process of enzyme conversion method for producing HMB is accelerated.

Description

TECHNICAL FIELD

[0001] The present application relates to a 4-hydroxyphenylpyruvate dioxygenase mutant for synthesizing beta-hydroxyisovaleric acid and application, and belongs to the field of bioengineering technology. BACKGROUND

[0002] Beta-hydroxyisovaleric acid (3-Hydroxyisovaleric acid, HMB) is an important metabolite of L-leucine in mammals, which can change the balance of protein metabolism during the growth of new muscle tissue and has anti-decomposition effect. HMB has very wide application: (1) in sports fitness, it promotes protein synthesis and helps muscle gain; (2) in weight management, it can inhibit protein decomposition and help fat loss; (3) in the health care of the elderly, HMB can help the elderly with muscle degradation, osteoporosis and malnutrition problems, and can also reduce the level of cholesterol and low-density lipoprotein in the body to reduce the occurrence of coronary heart disease and cardiovascular disease; (4) in clinical treatment, HMB has been proved to have the effect of accelerating wound healing and inhibiting inflammation, and when used in combination with glutamine, arginine, etc., it can improve the negative nitrogen balance of patients and play a positive role in the recovery of trauma, surgery, etc., and it is expected to play an important role in the treatment of cancer, acquired immune deficiency syndrome (AIDS) and other chronic diseases.

[0003] Currently, the synthesis methods of HMB mainly include chemical synthesis and biological synthesis. The chemical synthesis mainly includes oxidation method and Reformatsky reaction. The oxidation method is to use peroxoacetic acid or sodium hypochlorite to oxidize diacetone alcohol to generate HMB, and the yield is 64.1%. The Reformatsky reaction is to prepare an organic metal halide from halogenated acid ester, react with ketone to prepare hydroxy acid ester, and then hydrolyze, and the yield is 53%. Because the price of diacetone alcohol is relatively low, and the danger of preparing peroxoacetic acid is relatively large, the method of using sodium hypochlorite to oxidize diacetone alcohol to synthesize HMB is still the mainstream method in current industrial production, but this method has the problem of low yield. In addition, because the concentration of industrial sodium hypochlorite is only 10-13%, the amount of wastewater generated is large, and there is a problem of not easy to save. The biological synthesis mainly includes microbial fermentation and enzyme conversion method. The microbial fermentation method: in 2024, Sally J. et al. studied the de novo synthesis of HMB by BL21(DE3), using the mevalonate pathway and the myxobacterial iso-fatty acid pathway, and the fermentation time was 128h, the HMB yield was 17.7g / L, the conversion rate was 0.16g / g glucose, and the production intensity was 0.19g / (L·h). However, IPTG induction and antibiotics are needed, which is not conducive to food industry application. The enzyme conversion method: in 2002, Fei Hai-ming et al. studied the fermentation process characteristics of Galactomyces reessii in the conversion of β-methylbutyric acid to produce β-hydroxy-β-methylbutyric acid, and the biochemical process was carried out through the pathway of leucine catabolism, and the fermentation time was 102h, the HMB yield was 29.0g / L, the conversion rate was 57.3%, and the production intensity was 0.098g / (L·h). However, the survival rate of the strain is low, the product separation is difficult, and the three wastes are much. In 2018, Fan Wen-chao et al. used β-methylbutyric acid as the substrate, and produced HMB under the catalysis of P450 enzyme derived from sugar poly-spore red fungus, and the reaction time was 24h, 10000U / g P450 enzyme was used to convert 10g / L β-methylbutyric acid to synthesize HMB, and the conversion rate was 70%. In 2021, Gao Rui-chen et al. used L-leucine as the substrate, and produced HMB through the double-enzyme cascade whole-cell catalytic system composed of L-AAD and 4-HPPD, and the reaction time was 10h, the HMB yield was 1.42g / L, and the conversion rate was 80%. However, the catalytic efficiency of the rate-limiting enzyme 4-HPPD in the path is low, which leads to low HMB yield. Therefore, it is urgent to develop an effective biological method for high-efficiency production of β-hydroxyisovaleric acid. SUMMARY

[0004] The present application uses L-amino acid deaminase single mutant (PmL-AAD Q92Acatalyze the synthesis of alpha-ketoisocaproate (α-KIC) from L-leucine; and a new 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) was mined from Bacteroidia bacterium and protein engineering was performed to make it able to produce HMB from α-KIC as substrate, the synthetic route is shown in Figure 1 .

[0005] The present application provides a 4-hydroxyphenylpyruvate dioxygenase BbHPPD mutant, which has one or more mutations at the following positions based on the parent:

[0006] The alanine at position 366 is changed to proline, or the methionine at position 359 is changed to valine, or the asparagine at position 382 is changed to serine.

[0007] In an embodiment, the alanine at position 366 is changed to proline based on the sequence shown in SEQ ID NO. 1, and the mutant BbHPPD A366P has an amino acid sequence shown in SEQ ID NO. 3.

[0008] In an embodiment, the alanine at position 366 is changed to proline and the methionine at position 359 is changed to valine based on the sequence shown in SEQ ID NO. 1, and the mutant BbHPPD A366P / M359V has an amino acid sequence shown in SEQ ID NO. 5.

[0009] In an embodiment, the alanine at position 366 is changed to proline, the methionine at position 359 is changed to valine, and the asparagine at position 382 is changed to serine, and the mutant BbHPPD A366P / M359V / N382S has an amino acid sequence shown in SEQ ID NO. 7.

[0010] The present application also provides a gene encoding the mutant.

[0011] In an embodiment, the nucleotide sequence of the mutant BbHPPD A366P is shown in SEQ ID NO. 4; the nucleotide sequence of the mutant BbHPPD A366P / M359V is shown in SEQ ID NO. 6, and the nucleotide sequence of the mutant BbHPPD A366P / M359V / N382S is shown in SEQ ID NO. 8.

[0012] The present application also provides a recombinant vector carrying the above-mentioned gene.

[0013] In an embodiment, the recombinant vector is pET-28a as an expression vector.

[0014] The present application also provides a recombinant cell expressing the mutant, or containing the gene, or containing the recombinant vector.

[0015] In one embodiment, the recombinant cell is a prokaryotic cell expression host.

[0016] The present application also provides a recombinant Escherichia coli expressing the 4-hydroxyphenylpyruvate dioxygenase mutant BbHPPD A366P / M359V / N382S (BbHPPD M3 ).

[0017] In one embodiment of the present application, the recombinant Escherichia coli is an expression host of E. coli BL21(DE3).

[0018] The present application also provides a cell catalyst containing the recombinant Escherichia coli.

[0019] The present application also provides a method for preparing β-hydroxyisovaleric acid, which uses microbial cells expressing the mutant as catalyst, and L-amino acid deaminase to catalyze the synthesis of β-hydroxyisovaleric acid from L-leucine.

[0020] In one embodiment, the catalysis is carried out at 37-40℃ for at least 12h, or at least 16h, or at least 18h.

[0021] In one embodiment, the amino acid sequence of the L-amino acid deaminase is shown in SEQ ID NO. 9, and the gene sequence encoding the L-amino acid deaminase is shown in SEQ ID NO. 10.

[0022] In one embodiment, the method uses microbial cells expressing the mutant and microbial cells expressing the L-amino acid deaminase as catalysts to catalyze the synthesis of β-hydroxyisovaleric acid from L-leucine.

[0023] The present application also provides the use of the 4-hydroxyphenylpyruvate dioxygenase mutant, or the recombinant cell, or the cell catalyst, or the method in the preparation of β-hydroxyisovaleric acid or a product containing β-hydroxyisovaleric acid.

[0024] Advantages:

[0025] (1) The present application constructs a 4-hydroxyphenylpyruvate dioxygenase mutant, which can significantly improve the catalytic efficiency of 4-hydroxyphenylpyruvate dioxygenase.

[0026] (2) The present application provides a method for preparing β-hydroxyisovaleric acid, which uses L-leucine as raw material and utilizes L-amino acid deaminase PmL-AAD Q92Aand constructed 4-hydroxyphenylpyruvate dioxygenase mutants BbHPPD A366P / M359V / N382S Biosynthesis of HMB was carried out. In 10 g / L PmL-AAD Q92A wet cells and 20 g / L wet cells expressing BbHPPD A366P / M359V / N382S catalyzed by 10 g / L L-leucine, 1 mM FeS04-7H20 and 5 mM L-ascorbic acid in 10 mL Tris-Maleic buffer (60 mM, pH 5.5) at 37 °C for 18 h, the yield of HMB was 7 g / L with a conversion rate of 77%. Compared with the 4-hydroxyphenylpyruvate dioxygenase capable of catalyzing L-leucine to generate HMB, the yield of HMB of the present application is higher. Among them, the HMB yield of BbHPPD A366P / M359V / N382S is 3.7 times that of the wild enzyme, which accelerates the industrialization process of enzyme conversion method for producing HMB. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The present application provides a method for catalytically synthesizing HMB from L-leucine.

[0028] Figure 2 PmL-AAD Q92A and BbHPPD parent enzyme expression analysis results; M: Marker, 1: BbHPPD supernatant after crushing, 2: BbHPPD precipitate after crushing. 3: PmL-AAD Q92A supernatant after crushing; 4: PmL-AAD Q92A precipitate after crushing.

[0029] Figure 3 PmL-AAD Q92A and BbHPPD to synthesize HMB from L-leucine, HPLC (A) and MS (B) product identification of HMB generated by catalysis. DETAILED DESCRIPTION

[0030] The pET-28a(+) involved in the following examples was purchased from Novagen (Madison, WI, U.S.A.), restriction endonuclease, T4 DNA ligase, primeSTAR, etc. were purchased from TaKaRa (Dalian, China). BbHPPD mutants were obtained by molecular modification.

[0031] The medium involved in the following examples is as follows:

[0032] LB liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, sterilized at 121 °C for 20 min.

[0033] LB solid medium: LB liquid medium with 2% agar added.

[0034] TB liquid culture medium: KH2PO4 2.31g / L, K2HPO4·3H2O 16.42g / L, yeast extract 24g / L, peptone 12g / L, glycerol 4g / L.

[0035] HMB was determined by HPLC: An Aminex HPX-87H column (7.8 mm × 300 mm, 5 μm) was used as the chromatographic column, with 5 mM dilute sulfuric acid (filtered and degassed by sonication) as the mobile phase. The injection volume was 10 μL, the column temperature was 60 °C, a Waters differential detector was used, the flow rate was 0.6 mL / min, and the sample processing time was 20 min. Under these detection conditions, the retention times of α-KIC and HMB were 12.6 min and 14.1 min, respectively.

[0036] Example 1: Construction and expression of BbHPPD-pET28a-BL21(DE3) engineered bacteria

[0037] The target protein sequence BbHPPD from Bacteroidia bacterium was synthesized by Genewiz (SEQ ID NO.1) and codon-optimized (nucleotide sequence shown in SEQ ID NO.2). The resulting plasmid was then ligated into the pET-28a vector using BamHI and HindIII. The recombinant expression plasmid BbHPPD-pET-28a was obtained and transformed into E. coli BL21(DE3). The resulting positive engineered bacterium was named BbHPPD-pET-28a-BL21(DE3).

[0038] The BbHPPD-pET28a-BL21(DE3) strain was transferred to 3 mL of LB liquid medium and cultured overnight at 37°C. Subsequently, it was inoculated at a 1:100 ratio into 200 mL of TB liquid medium and cultured at 37°C and 200 rpm. When OD... 600 When the value is between 0.4 and 0.8, add IPTG to a final concentration of 0.4 mM, and induce culture at 25℃ for 16 h. After centrifugation, collect the bacterial cells and store them at -40℃. For the bacterial expression after sonication, please refer to [link to relevant documentation]. Figure 2 .

[0039] Example 2: Determining the rate-limiting enzyme for the pathway

[0040] PmL-AAD Q92APreparation of wet bacteria: PmL-AAD was prepared according to the method of the paper "Production of a-ketoisocaproate and a-keto-β-methylvalerate by engineered L-amino acid deaminase" Q92A Wet bacteria; wherein, the amino acid sequence of AAD Q92A is shown in SEQ ID NO. 9, and the nucleotide sequence encoding AAD Q92A is shown in SEQ ID NO. 10.

[0041] The reaction system contains 10 g / L PmL-AAD Q92A wet bacteria and 20 g / L BbHPPD wet bacteria, 10 g / L L-leucine, 1 mM FeSO4·7H2O and 5 mM L-ascorbic acid in 10 mL Tris-maleic buffer (60 mM, pH 5.5), incubated at 37°C for 18 h, and the synthesis of HMB was verified by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). The results show that the yield of product HMB is 1.9 g / L, the conversion rate is 21.2%, and the accumulation of intermediate a-KIC is 5.9 g / L, indicating that the rate-limiting enzyme is BbHPPD.

[0042] Example 3: Construction of BbHPPD mutants

[0043] Construction of mutants: Saturation mutation primers for three sites A366P / M359V / N382S of BbHPPD were designed, as shown in Table 1, and the mutants were constructed by whole plasmid PCR.

[0044] Table 1 Mutant primer sequences

[0045]

[0046] KOD system was used when constructing the PCR amplification system, and BbHPPD-pET-28a, BbHPPD A366P -pET-28a and BbHPPD A366P / M359V -pET-28a plasmids were used as templates, and the primers shown in Table 1 were used for PCR. The KOD system is shown in Table 2. The PCR reaction conditions are as follows: 1: 98°C for 5 min; 2: 94°C for 30 s; 3: 55°C for 30 s; 4: 72°C for 3 min 20 s; cycle 2-4 for 30 times; 5: 72°C for 10 min; 6: 12°C for incubation.

[0047] Table 2 KOD system table

[0048]

[0049] The above PCR reaction system was incubated at 37°C in a metal bath for 30 min to digest the plasmid template (digestion system: DpnI quick 0.3 μL, the above reaction PCR product 8.7 μL, 10 × T Buffer 1 μL), and the digestion product was obtained after digestion.

[0050] Transformation: the above digestion product was introduced into E. coli BL21 (DE3) competent cells by heat shock method, and the transformation specific steps were as follows:

[0051] (1) 10 μL of PCR product was introduced into 100 μL of E. coli BL21 (DE3) competent cells;

[0052] (2) ice bath for 30 min;

[0053] (3) 42°C water bath heat shock for 60 s, and then quickly placed in ice for 3-5 min;

[0054] (4) add 600 μL of non-resistant LB medium and mix, incubate at 37°C, 220 rpm for 1 h;

[0055] (5) centrifuge at 4000 rpm for 2 min;

[0056] (6) discard the supernatant, use the remaining 100-200 μL of LB medium to blow and mix the bacterial cells and spread on a 0.05 mg / mL kanamycin-resistant plate, and incubate at 37°C for about 12 h.

[0057] Example 4: Recombinant BbHPPD A366P / M359V / N382S Construction and screening of pET-28a-BL21 (DE3)

[0058] Single colonies were picked and inoculated in 0.05 mg / mL kanamycin-resistant LB liquid medium, and incubated at 220 rpm and 37°C for 12 h, then TBA liquid medium was added and incubated at 220 rpm and 37°C; after 2-3 h, the temperature was shifted to 25°C for induction for 16 h, and then centrifuged at 10,000 rpm for 30 min.

[0059] Screening conditions: 0.6 g / L α-KIC, 1 mM FeSO4·7H2O, 5 mM L-ascorbic acid, and 60 mM Tris-maleic buffer (60 mM, pH 5.5) were added to the 96-well plate, and the reaction was carried out at 37°C for 2 h. After the reaction was completed, the residual amount of keto acid was detected by Dnps high-throughput method (for specific steps, see patent application document CN112626100A). Three mutants better than the wild type were selected from each well plate, and then these mutants were sent to Tianlin Biotechnology Company for sequencing.

[0060] The mutant strains with correct sequencing were inoculated into LB seed culture medium, and cultured at 220 rpm and 37°C for 8-12 h, and then inoculated into shake flask fermentation medium at 2%, and cultured at 220 rpm and 37°C until OD 600 =0.6-0.8, and 0.2 mM IPTG was added to induce, and the induction conditions were 220 rpm, 25°C for 16 h. The mutant bacterial cells after expression induction were collected and put into the reaction system. The reaction system contained (in terms of final concentration): 10 g / L PmL-AAD Q92A wet bacterial cells and 20 g / L bacterial cells expressing BbHPPD or its mutants, 10 g / L L-leucine, 1 mM FeSO4·7H2O and 5 mM L-ascorbic acid in 10 mL Tris-maleic buffer (60 mM, pH 5.5), 37°C for 18 h, and the HMB yield was determined. After the reaction, part of the conversion solution was centrifuged at 10,000 rpm for 30 min, the supernatant was filtered with a 0.22 μm microfiltration membrane, and then determined by HPLC method, such as Figure 3 A and Figure 3 B, and the product formation was further determined. The results showed that the yield of HMB was 7 g / L after the reaction of the wet bacterial cells expressing BbHPPD A366P / M359V / N382S , and the conversion rate was 77%, which was 3.7 times of the wild enzyme.

[0061] Comparative Example:

[0062] The specific implementation is the same as that in Examples 3-4, except that the mutants BbHPPD S375Q , BbHPPD A366P / A381N , BbHPPD A366P / T385N , BbHPPD A366P / M359V / T385N were constructed, and the reaction was carried out according to the procedure of Example 4, and the results are shown in Table 3.

[0063] Table 3 Conversion rate of different mutants in HMB catalytic reaction

[0064] Mutant Conversion rate BbHPPD A366P ]]> 45.6％ BbHPPD S375Q ]]> 25.6％ BbHPPD A366P / M359V ]]> 65.6％ BbHPPD A366P / A381N ]]> 44.2％ BbHPPD A366P / T385N ]]> 50.5％ BbHPPD A366P / M359V / T385N ]]> 60.7％ BbHPPD A366P / M359V / N382S ]]> 77.1％

[0065] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1,4-Hydroxyphenylpyruvate dioxygenase mutant, characterized in that, Based on the parent, alanine at position 366 was replaced with proline, methionine at position 359 was mutated to valine, and asparagine at position 382 was mutated to serine; the amino acid sequence of the parent is shown in SEQ ID NO.

1.

2. The gene encoding the 4-hydroxyphenylpyruvate dioxygenase mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. Recombinant cells expressing the 4-hydroxyphenylpyruvate dioxygenase mutant of claim 1, or containing the gene of claim 2, or containing the recombinant vector of claim 3.

5. A recombinant Escherichia coli, characterized in that, by E. coli BL21 (DE3) was used as the host to express the 4-hydroxyphenylpyruvate dioxygenase mutant of claim 1.

6. A cell catalyst containing the recombinant Escherichia coli of claim 5.

7. A method for preparing β-hydroxyisovaleric acid, characterized in that, Using microbial cells expressing the 4-hydroxyphenylpyruvate dioxygenase mutant of claim 1 as a catalyst, β-hydroxyisovaleric acid is synthesized with L-leucine as a substrate in the presence of L-amino acid deaminase.

8. The method according to claim 7, characterized in that, The method uses microbial cells expressing the 4-hydroxyphenylpyruvate dioxygenase mutant of claim 1 and microbial cells expressing L-amino acid deaminase as catalysts, and L-leucine as substrate, to catalyze the synthesis of β-hydroxyisovaleric acid; the amino acid sequence of the L-amino acid deaminase is shown in SEQ ID NO.

9.

9. The use of the 4-hydroxyphenylpyruvate dioxygenase mutant of claim 1, or the recombinant cell of claim 4, or the recombinant Escherichia coli of claim 5, or the cell catalyst of claim 6, or the method of any one of claims 7-8 in the preparation of β-hydroxyisovaleric acid or products containing β-hydroxyisovaleric acid.

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