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

Abstract

The invention discloses a 4-hydroxyphenylpyruvate dioxygenase mutant for synthesizing beta-hydroxyisovaleric acid and application of the 4-hydroxyphenylpyruvate dioxygenase mutant, and belongs to the technical field of biological engineering. According to the invention, the 4-hydroxyphenylpyruvate dioxygenase mutant is constructed, and the catalytic efficiency of the 4-hydroxyphenylpyruvate dioxygenase can be obviously improved. The constructed 4-hydroxyphenylpyruvate dioxygenase and L-amino acid deaminase are jointly used for catalytic preparation of beta-hydroxyisovaleric acid, the yield of HMB can reach 7 g / L, and the conversion rate is 77%. Compared with the existing 4-hydroxyphenylpyruvate dioxygenase capable of catalyzing L-leucine to generate HMB, the 4-hydroxyphenylpyruvate dioxygenase disclosed by the invention has the advantage that the yield of HMB is higher. Wherein the HMB yield of the BbHPPD A366P / M359V / N382S is 3.7 times that of a wild enzyme, and the industrial process of producing the HMB by an enzyme conversion method is accelerated.

Description

Technical Field

[0001] The invention relates to a 4-hydroxyphenylpyruvate dioxygenase mutant for synthesizing beta-hydroxyisovalerate and application thereof, belonging to the technical field of bioengineering. Background Art

[0002] β-Hydroxyisovaleric acid (HMB) is an important metabolite of L-leucine in mammals. It can change the balance of protein metabolism during the growth of new muscle tissue and has an anti-degradation effect. HMB has a wide range of applications: (1) In sports and fitness, it promotes protein synthesis and helps build muscle; (2) In weight management, it can inhibit protein breakdown and help reduce fat; (3) In elderly care, HMB can help with muscle degeneration, osteoporosis and malnutrition in the elderly, and can also reduce cholesterol and low-density lipoprotein levels in the body to reduce the incidence of coronary heart disease and cardiovascular disease; (4) In clinical treatment, HMB has been shown to accelerate wound healing and inhibit inflammation. When used in combination with glutamine, arginine, etc., it can improve the patient's negative nitrogen balance and play a positive role in the recovery of trauma and surgery. In addition, it is expected to play an important role in the treatment of cancer, such as tumors, acquired immunodeficiency syndrome (AIDS) and other chronic diseases.

[0003] At present, the synthesis methods of HMB mainly include chemical synthesis and biosynthesis. Chemical synthesis mainly includes oxidation method and Reformatsky reaction. The oxidation method is to use peracetic acid or sodium hypochlorite to oxidize diacetone alcohol to produce HMB, with a yield of 64.1%. The Reformatsky reaction is a method in which halogenated acid esters are made into organic metal halides, reacted with ketones to prepare hydroxy acid esters, and then hydrolyzed, with a yield of 53%. Because diacetone alcohol is relatively low in price and the preparation of peracetic acid is relatively dangerous, the use of sodium hypochlorite to oxidize diacetone alcohol to synthesize HMB is still the mainstream method of industrial production, but this method has always had the problem of low yield. In addition, since the concentration of industrial sodium hypochlorite is only 10-13%, the amount of wastewater generated is large, and there is a problem of difficult preservation. Biosynthesis is mainly divided into microbial fermentation and enzyme conversion. 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 myxomycete iso-fatty acid pathway. After fermentation for 128 hours, the HMB yield was 17.7 g / L, the conversion rate was 0.16 g / g glucose, and the production intensity was 0.19 g / (L·h). However, IPTG induction and antibiotics are required, which is not conducive to food industry application. Enzyme conversion method: In 2002, Fei Haiming et al. studied the fermentation process characteristics of the microorganism Galactomyces reessii to convert β-methylbutyric acid to produce β-hydroxy-β-methylbutyric acid. The biochemical process was carried out through the leucine catabolism pathway. After fermentation for 102 hours, the HMB yield was 29.0 g / L, the conversion rate was 57.3%, and the production intensity was 0.098 g / (L·h). However, the survival rate of the strain is low, the product separation is difficult, and there are many three wastes. In 2018, Fan Wenchao et al. used β-methylbutyrate as a substrate to produce HMB under the catalysis of P450 enzyme from Saccharopolyspora erythrocytes. After 24 hours of reaction, 10000U / gP450 enzyme converted 10g / L β-methylbutyrate to synthesize HMB, with a conversion rate of 70%. In 2021, Gao Ruichen et al. used L-leucine as a substrate and produced HMB through a dual-enzyme cascade whole-cell catalytic system composed of L-AAD and 4-HPPD. After 10 hours of reaction, 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 this pathway is low, resulting in a low yield of HMB. Therefore, there is an urgent need for an effective biological method to efficiently produce β-hydroxyisovaleric acid. Summary of the invention

[0004] The present invention uses a single mutant of L-amino acid deaminase (PmL-AAD) derived from Proteus viscosus Q92A) catalyzes L-leucine to synthesize α-keto-leucine (α-KIC); and a new 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) was discovered from Bacteroidia bacterium, and its protein engineering was performed to enable it to efficiently produce HMB using α-KIC as a substrate. The synthesis route is shown in Figure 1 .

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

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

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

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

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

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

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

[0012] The invention also provides a recombinant vector carrying the gene.

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

[0014] The present invention 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 invention also provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli expresses the 4-hydroxyphenylpyruvate dioxygenase mutant BbHPPD A366P / M359V / N382S (BbHPPD M3 ).

[0017] In one embodiment of the present invention, the recombinant Escherichia coli uses E. coli BL21 (DE3) as the expression host.

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

[0019] The present invention also provides a method for preparing β-hydroxyisovaleric acid, which uses microbial cells expressing the mutant as catalysts, L-leucine as a substrate, and the like to catalyze the synthesis of β-hydroxyisovaleric acid in the presence of L-amino acid deaminase.

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

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

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

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

[0024] Beneficial effects:

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

[0026] (2) The present invention provides a method for preparing β-hydroxyisovaleric acid, using L-leucine as a raw material and utilizing L-amino acid deaminase PmL-AAD Q92Aand the constructed 4-hydroxyphenylpyruvate dioxygenase mutant BbHPPD A366P / M359V / N382S Biosynthesis of HMB. Q92A Wet cells and 20 g / L expressed BbHPPD A366P / M359V / N382S Under the catalysis of wet bacteria, 10g / L L-leucine, 1mM FeSO4·7H2O and 5mM L-ascorbic acid were incubated in 10mL Tris-Maleic buffer (60mM, pH 5.5) at 37°C for 18h, and the yield of HMB was 7g / L, with a conversion rate of 77%. Compared with the current 4-hydroxyphenylpyruvate dioxygenase that can catalyze L-leucine to produce HMB, the yield of HMB in the present invention is higher. Among them, BbHPPD A366P / M359V / N382S The HMB yield of the enzyme is 3.7 times that of the wild enzyme, which accelerates the industrialization process of HMB production by enzymatic conversion. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The invention relates to a method for synthesizing HMB catalyzed by L-leucine.

[0028] Figure 2 PmL-AAD Q92A Analysis results of the expression of the parent enzyme of BbHPPD; M: Marker, 1: supernatant of BbHPPD fragmentation liquid, 2: precipitate of BbHPPD fragmentation liquid. 3: PmL-AAD Q92A Supernatant after disruption; 4: PmL-AAD Q92A After crushing, precipitation.

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

[0030] The pET-28a(+) used in the following examples was purchased from Novagen (Madison, WI, USA), and restriction enzymes, T4 DNA ligase, primeSTAR, etc. were purchased from TaKaRa (Dalian, China). All BbHPPD mutants were obtained by molecular modification.

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

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

[0033] LB solid medium: Add 2% agar to LB liquid medium.

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

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

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

[0037] The target protein sequence BbHPPD in Bacteroidia bacterium was synthesized by Jinweizhi Company (shown in SEQ ID NO.1) and after codon optimization (nucleotide sequence shown in SEQ ID NO.2), it was connected to the pET-28a vector through BamHI and HindIII. The recombinant expression plasmid BbHPPD-pET-28a was obtained, and the recombinant plasmid BbHPPD-pET-28a was transformed into E.coli BL21 (DE3), and the positive engineering bacteria were named BbHPPD-pET-28a-BL21 (DE3).

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

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

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

[0041] The reaction system contains 10g / L PmL-AAD according to the final concentration. Q92A Wet bacteria and 20g / L BbHPPD wet bacteria, 10g / L L-leucine, 1mM FeSO4·7H2O and 5mM L-ascorbic acid were incubated in 10mL Tris-Maleic buffer (60mM, pH5.5) at 37°C for 18h, and the synthesis of HMB was confirmed by high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). The results showed that the yield of product HMB was 1.9g / L, the conversion rate was 21.2%, and the accumulation of intermediate α-KIC was 5.9g / L, indicating that the rate-limiting enzyme was BbHPPD.

[0042] Example 3: Construction of BbHPPD mutants

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

[0044] Table 1 Mutation primer sequences

[0045]

[0046] The KOD system was used to construct the PCR amplification system, with BbHPPD-pET-28a, BbHPPD A366P -pET-28a, BbHPPD A366P / M359V -pET-28a plasmid was used as template, and PCR was performed using the primers shown in Table 1. The KOD system is shown in Table 2. The PCR reaction conditions were: 1: 98°C for 5 min; 2: 94°C for 30 s; 3: 55°C for 30 s; 4: 72°C for 3 min and 20 s; the three steps 2-4 were cycled 30 times; 5: 72°C for 10 min; 6: 12°C insulation.

[0047] Table 2 KOD system table

[0048]

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

[0050] Transformation: The above digestion products were introduced into E.coli BL21 (DE3) competent cells by heat shock method. The specific transformation steps are as follows:

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

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

[0053] (3) Heat shock at 42°C in a water bath for 60 seconds, then quickly place in ice for 3-5 minutes;

[0054] (4) Add 600 μL of resistance-free LB medium, mix well, and culture at 37°C, 220 rpm for 1 h;

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

[0056] (6) Discard the supernatant, use the remaining 100-200 μL LB medium to mix the bacteria by pipetting and spread onto a plate containing 0.05 mg / mL kanamycin resistance, and culture at 37°C for about 12 h.

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

[0058] A single clone was picked and placed in a 96-deep-well plate containing 0.05 mg / mL kanamycin-resistant LB liquid culture medium. After constant temperature culture at 220 rpm and 37°C for 12 h, TBA liquid culture medium was added and cultured at 220 rpm and 37°C. After culture for 2 to 3 h, the culture was transferred to 25°C for induction for 16 h, and then centrifuged at 10,000 rpm for 30 min.

[0059] Screening conditions: 0.6g / L α-KIC, 1mM FeSO4·7H2O, 5mM L-ascorbic acid, 60mM Tris-Maleic buffer (60mM, pH 5.5) were added to a 96-well plate and reacted at 37°C for 2h. After the reaction, the Dnps high-throughput method was used to detect the remaining keto acid (for specific steps, see the patent application document with publication number CN112626100A). Three mutants that were superior to the wild type were selected for each well plate, and then these mutants were sent to Tianlin Biotechnology for sequencing.

[0060] The mutant strain with correct sequencing was inoculated into LB seed medium and cultured at 220 rpm and 37 °C for 8-12 h. A 2% inoculum was inoculated into shake flask fermentation medium and cultured at 220 rpm and 37 °C until OD 600 = 0.6 to 0.8, add IPTG with a final concentration of 0.2mM for induction, and the induction conditions are 220rpm, 25℃ for 16h. Collect the bacterial cells induced to express the mutant and put them into the reaction system. The reaction system contains (by final concentration): 10g / L PmL-AAD Q92A Wet bacteria 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 were incubated in 10 mL Tris-Maleic buffer (60 mM, pH 5.5) at 37 ° C for 18 h, and the HMB production was determined. After the reaction was completed, part of the conversion solution was centrifuged at 10,000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm microfiltration membrane and then determined by HPLC, as shown in FIG. Figure 3 A and Figure 3 As shown in B, the product generation was further confirmed. The results showed that the expression of BbHPPD A366P / M359V / N382S After the wet bacteria participated in the reaction, the HMB yield was 7g / L and the conversion rate was 77%, which was 3.7 times that of the wild enzyme.

[0061] Comparative Example:

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

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

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

[0065] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A 4-hydroxyphenylpyruvate dioxygenase mutant, characterized in that: Based on the parents, there are mutations at one or more of the following sites: The alanine at position 366 was changed to proline, or the methionine at position 359 was mutated to valine, or the asparagine at position 382 was mutated to serine.

2. The mutant according to claim 1, characterized in that The amino acid sequence of the parent is shown in SEQ ID NO.

1.

3. A gene encoding the 4-hydroxyphenylpyruvate dioxygenase mutant according to claim 1 or 2.

4. A recombinant vector carrying the gene according to claim 3.

5. A recombinant cell expressing the 4-hydroxyphenylpyruvate dioxygenase mutant according to claim 1 or 2, or containing the gene according to claim 3, or containing the recombinant vector according to claim 4.

6. A recombinant Escherichia coli, characterized in that Using E. coli BL21 (DE3) as a host, the 4-hydroxyphenylpyruvate dioxygenase mutant according to claim 1 or 2 is expressed.

7. A cell catalyst comprising the recombinant Escherichia coli according to claim 6.

8. A method for preparing β-hydroxyisovaleric acid, characterized in that: The method uses microbial cells expressing the 4-hydroxyphenylpyruvate dioxygenase mutant according to claim 1 or 2 as catalysts, L-leucine as a substrate, and the like, with the participation of L-amino acid deaminase, to catalyze the synthesis of β-hydroxyisovalerate.

9. The method according to claim 8, 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 a 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.

10. Use of the 4-hydroxyphenylpyruvate dioxygenase mutant according to claim 1 or 2, or the recombinant cell according to claim 5, or the recombinant Escherichia coli according to claim 6, or the cell catalyst according to claim 7, or the method according to any one of claims 8 to 9 in the preparation of β-hydroxyisovaleric acid or a product containing β-hydroxyisovaleric acid.

Images