A 4-hydroxyphenylpyruvate dioxygenase mutant for synthesizing beta-hydroxyisovalerate and a recombinant strain

Abstract

The application discloses a kind of 4-hydroxyphenylpyruvate dioxygenase mutant for synthesizing beta-hydroxyisovaleric acid and recombinant strain, biological engineering and enzyme engineering technical field.The wild-type 4-hydroxyphenylpyruvate dioxygenase of Bacteroides origin is used as parent in the application, and a series of mutants are constructed by site-directed saturation mutagenesis, wherein the mutant Bb HPPD F242Y / L271L / K231K Compared with the wild-type enzyme, the catalytic activity of the mutant is increased by 57.1%. On this basis, a double-enzyme co-expression strain is constructed, and HMB is efficiently synthesized using L-leucine as a substrate. The yield of HMB is 12.7 g / L, and the molar conversion rate is 70.6%. The process is green and environmentally friendly, the reaction period is short, the strain is easy to scale up in industry, and can be widely used in the large-scale production of beta-hydroxyisovaleric acid and its calcium salt in the fields of sports nutrition, livestock breeding and medicine.

Description

Technical Field

[0001] This invention relates to a 4-hydroxyphenylpyruvate dioxygenase mutant and recombinant strain for the synthesis of β-hydroxyisovaleric acid, in the fields of bioengineering and enzyme engineering technology. Background Technology

[0002] β 3-Hydroxyisovaleric acid (HMB) is an important metabolite of L-leucine in mammals, possessing anti-inflammatory properties, promoting muscle protein synthesis, regulating catabolism and apoptosis, reducing sports injuries, and accelerating recovery; it is hailed as a "muscle protector." Due to its reactive chemical properties, commercially available products often exist in the form of its calcium salt. Initially, HMB and its calcium salts were primarily used in agriculture, such as animal husbandry and aquaculture, to improve sow milk quality, promote skeletal muscle protein synthesis in piglets, increase poultry growth rate, and improve muscle quality and immune function in aquatic animals through feed additives. With further research and development in food applications, HMB and its calcium salts have been further applied in the health care field, used in the development of sports nutrition supplements to achieve functions such as muscle building, improved athletic performance, reduced sports injuries, and accelerated recovery. In the medical field, HMB is mainly targeted at the elderly and the treatment of muscle diseases such as skeletal muscle atrophy. Clinical studies have confirmed that HMB can promote wound healing, reduce inflammatory responses, and, when used in combination with glutamine and arginine, can effectively improve the negative nitrogen balance in patients, promoting trauma and postoperative recovery. In addition, HMB has shown potential application value in the treatment of various chronic diseases such as cancer and acquired immunodeficiency syndrome (AIDS).

[0003] Given the market value of HMB, researchers have established multiple process routes, including chemical synthesis and biosynthesis, over the past 30 to 40 years. Chemical synthesis mainly includes oxidation and the Reformatsky reaction, but it suffers from low yields and severe pollution, failing to align with national green development policies. In contrast, biosynthesis offers advantages such as mild reaction conditions, ease of operation in aqueous phases, and low pollution, making it a key focus of research for efficient and green HMB synthesis. HMB biosynthesis primarily involves two pathways: enzymatic conversion and microbial fermentation. Currently, research on HMB synthesis via enzymatic conversion is more extensive, with substrates including... β The enzymes used include methylbutyric acid, 3-hydroxy-3-methylbutyronitrile, acetic acid, acetone, pyruvate, 3-methylcrotonyl-CoA, and L-leucine. In 2025, the inventors' team initially constructed a whole-cell catalytic system of L-AAD and 4-HPPD dual-enzyme cascades using L-leucine as a substrate. This system achieved a HMB yield of 11.6 g / L after 18 h of reaction, with a molar conversion rate of 65% and a production intensity of 0.64 g / L / h. In this cascade reaction, 4-HPPD is the rate-limiting enzyme, and its catalytic efficiency still needs improvement.

[0004] In summary, the insufficient catalytic activity of existing natural 4-HPPD enzymes is a core bottleneck restricting the improvement of HMB biosynthetic capacity. Therefore, molecular modification of 4-HPPD to obtain mutants with high catalytic activity, and the development of matching recombinant strains and efficient catalytic processes, are urgent technical problems to be solved in this field. Summary of the Invention

[0005] The present invention provides a 4-hydroxyphenylpyruvate dioxygenase mutant, which, relative to the parent shown in SEQ ID NO.1, has one or more of the following mutations: phenylalanine at position 242 is mutated to tyrosine, a synonymous mutation at position 271 is mutated to lysine, and lysine at position 231 is mutated to lysine.

[0006] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.1, with the phenylalanine at position 242 mutated to tyrosine, and named as follows: Bb HPPD F242Y The amino acid sequence is shown in SEQ ID NO.3.

[0007] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.1, with the phenylalanine at position 242 mutated to tyrosine, and the leucine at position 271 undergoing a synonymous mutation, wherein the synonymous mutation changes the codon encoding position 271 from CTG to TTG; named Bb HPPD F242Y / L271L The amino acid sequence is shown in SEQ ID NO.5.

[0008] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.1, with phenylalanine at position 242 mutated to tyrosine, and leucine at position 271 and lysine at position 231 undergoing synonymous mutations. Specifically, the synonymous mutation changes the codon encoding leucine at position 271 from CTG to TTG and the codon encoding lysine at position 231 from AAA to AAG. The resulting mutant is named... Bb HPPD F242Y / L271L / K231K The amino acid sequence is shown in SEQ ID NO.7.

[0009] The present invention also provides a gene encoding the mutant.

[0010] In one embodiment, the nucleotide sequence of the gene is as shown in any one of SEQ ID NO.4, SEQ ID NO.6, and SEQ ID NO.8.

[0011] The present invention also provides a recombinant vector carrying the above-mentioned genes.

[0012] In one embodiment of the present invention, the recombinant vector is pET-28a, pCDFDuet-1 or pACYCFDuet-1 as the expression vector.

[0013] The present invention also provides recombinant cells expressing the above-mentioned mutants, or containing the above-mentioned genes, or containing the above-mentioned recombinant vectors.

[0014] In one embodiment of the present invention, the recombinant cells express the host in the form of prokaryotic cells.

[0015] The present invention also provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli expresses the 4-hydroxyphenylpyruvate dioxygenase mutant. Bb HPPD F242Y / L271L / K231K .

[0016] In one embodiment, the recombinant Escherichia coli is... E. coli BL21 (DE3) is the expression host.

[0017] In one embodiment, the recombinant Escherichia coli also expresses an L-amino acid deaminase single mutant. Pm L-AAD Q92A .

[0018] In one embodiment, the recombinant Escherichia coli uses pCDFDuet-1 as a vector to express the 4-hydroxyphenylpyruvate dioxygenase mutant and uses pACYCFDuet-1 as a vector to express the L-amino acid deaminase single mutant.

[0019] The present invention also provides a method for preparing HMB, wherein the method comprises: using a strain co-expressing the 4-hydroxyphenylpyruvate dioxygenase mutant and the L-amino acid deaminase monomutant as a catalyst, and reacting in a Tris-Maleate buffer containing L-leucine, FeSO4·7H2O and L-ascorbic acid.

[0020] In one embodiment of the present invention, the final concentration of the recombinant Escherichia coli in the reaction system is 10-60 g / L, preferably 20 g / L. In one embodiment of the present invention, the final concentration of L-leucine in the reaction system is 10~60 g / L, preferably 20 g / L. In one embodiment of the present invention, the final concentration of FeSO4·7H2O in the reaction system is 1~5 mM, preferably 2 mM.

[0021] In one embodiment of the present invention, the final concentration of L-ascorbic acid in the reaction system is 2~20 mM, preferably 10 mM.

[0022] In one embodiment of the present invention, the final concentration of Tris-Maleate in the reaction system is 40-80 mM, preferably 60 mM.

[0023] In one embodiment, the pH of the reaction system was controlled at 5.5 (adjusted by maleic acid), and the reaction was carried out at 37°C and 220 rpm for 18 h.

[0024] The present invention also provides the application of the 4-hydroxyphenylpyruvate dioxygenase mutant and the recombinant Escherichia coli in the large-scale preparation of HMB or its calcium salt.

[0025] In one embodiment, the applications include sports nutrition, animal husbandry, medicine and health, food additives and other fields.

[0026] The beneficial effects of this invention are: (1) This invention uses Bacteroides ( Bacteroidia bacterium Based on 4-hydroxyphenylpyruvate dioxygenase derived from [source name], a mutant was obtained through protein engineering. Bb HPPD F242Y / L271L / K231K This significantly improved catalytic efficiency. Compared to the wild-type enzyme, HMB yield increased by 57.1%, providing a superior rate-limiting enzyme for the enzymatic conversion of L-leucine to HMB.

[0027] (2) This invention constructs a dual-enzyme co-expression strain, and by optimizing the expression of the dual enzymes, expresses the rate-limiting enzyme using a high-copy vector. Bb HPPD F242Y / L271L / K231K Low-copy vector expression Pm L-AAD Q92A The production of HMB using L-leucine as a raw material was achieved, with a yield of 10.2 g / L and a molar conversion rate of 56.6%.

[0028] (3) To reduce the accumulation of intermediate α-KIC and optimize the RBS sequence and gene copy number, under the catalysis of 20 g / L HMB 3.0 wet cells, 20 g / L L-leucine, 2 mM FeSO4·7H2O and 10 mM L-ascorbic acid were added to 10 mL Tris-Maleic buffer (60 mM, pH 5.5) and incubated at 37℃ for 18 h. The HMB yield was 12.7 g / L, the molar conversion rate was 70.6% and the production intensity was 0.71 g / L / h. Attached Figure Description

[0029] Figure 1 HMB production performance of the mutant strain; Figure 2 mutant Bb HPPD F242Y / L271L / K231KThe expression indicates the situation; where M: Marker; 1: Bb HPPD F242Y / L271L / K231K 2: Supernatant of the breaking fluid; Bb HPPD F242Y / L271L / K231K Sedimentation of the breakup fluid; Figure 3 For different substrate concentrations on Bb HPPD F242Y / L271L / K231K Verification of production capacity; Figure 4 To verify the production performance of the dual-enzyme co-expression strain; Figure 5 To verify the production performance of the repetitive expression strain. Detailed Implementation

[0030] In the following examples, pET-28a(+) was purchased from Novagen (Madison, WI, USA), and restriction endonucleases, T4 DNA ligases, primeSTAR Mix (2x), etc., were purchased from TaKaRa (Dalian, China). Standard α-KIC and HMB were purchased from Shanghai Maclean Biochemical Technology Co., Ltd., and all other reagents were purchased from the market. Bb All HPPD mutants were obtained through molecular modification.

[0031] The culture media involved in the following examples are as follows: LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, sterilized at 121℃ for 20 min.

[0032] LB solid medium: LB liquid medium with 2% agar added.

[0033] TB liquid medium: KH2PO4 2.31 g / L, K2HPO4·3H2O 16.42 g / L, yeast extract 24 g / L, peptone 12 g / L, glycerol 4 g / L.

[0034] Sample preparation: Take 500 μL of the converted solution, add an equal volume of 2 M HCl, centrifuge at 12000 rpm for 3 min 30 s, take the supernatant and filter it through a 0.22 μm aqueous filter membrane, and use the filtrate for liquid chromatography analysis.

[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: Heterologous Expression Bb Construction and expression of HPPD engineered bacteria Bacteroides ( Bacteroidia bacterium ) target protein sequence in Bb HPPD is codon-optimized and synthesized by Genewiz (as shown in SEQ ID NO.2) before being... BamH I and Hind III was ligated into the pET-28a vector. The recombinant expression plasmid pET-28a- was obtained. Bb HPPD will recombinant plasmid pET-28a- Bb HPPD conversion E. coli BL21(DE3), the engineered bacteria that obtained positive results was named E. coli A01.

[0037] strain E. coli A01 was transferred to 3 mL of LB liquid medium and incubated overnight at 37°C. Subsequently, it was inoculated at a 1:100 ratio into 200 mL of TB liquid medium and incubated at 37°C and 200 rpm. When OD... 600 When the pH value is between 0.4 and 0.8, add IPTG to a final concentration of 0.4 mM, induce culture at 25℃ for 16 h, collect the bacterial cells after centrifugation, and store the bacterial cells at -40℃.

[0038] Example 2: Construction of mutants Construction of mutants: design Bb The saturation mutation primers for the three sites F242, L271 and K231 of HPPD are shown in Table 1. Mutants were constructed by whole plasmid PCR.

[0039] Table 1. Mutant Primer Sequences

[0040] The PrimeSTAR® Max DNA Polymerase system was used to construct the PCR amplification system, with different concentrations of pET-28a- Bb HPPD, pET-28a- BbHPPD F242Y pET-28a- Bb HPPD F242Y / L271L Using plasmids as templates, PCR was performed using the primers shown in Table 1. The PrimeSTAR® Max DNA Polymerase system is shown in Table 2.

[0041] Table 2 PrimeSTAR® Max DNA Polymerase System

[0042] When performing PCR amplification, steps 2-4 need to be repeated 30 times. The specific program settings are shown in Table 3.

[0043] Table 3 PCR Program Settings

[0044] The above PCR reaction system was incubated in a metal bath at 37°C for 30 min to digest the plasmid template (the digestion system consisted of: 0.3 μL of DpnI quick, 8.7 μL of the above PCR product, and 1 μL of 10×T Buffer). The digestion product was obtained after digestion.

[0045] Transformation: The digestion products described above are introduced into the body via heat shock. E. coli BL21(DE3) competent cells, transformation steps: (1) Prepare competent cells: Take them out of the -80℃ freezer E. coli BL21(DE3) competent cells were thawed on ice. (2) Add PCR product: Add 10 μL of PCR product to 100 μL E. coli BL21(DE3) competent cells, gently mixed and then incubated on ice for 30 min; (3) Heat shock: The cells were placed in a 42°C water bath for 90 s for heat shock; (4) Cooling: After the heat shock is completed, quickly return the device to the ice bath to cool for 3-5 minutes; (5) Recovery: Add 600 μL of antibiotic-free LB medium and mix well. Incubate at 37°C and 220 rpm for 1 h to allow the bacteria to recover. (6) Spreading culture medium: After centrifuging at 4000 rpm for 2 min, discard the supernatant, use the remaining 100-200 μL LB medium to mix the bacterial cells by blowing and spreading them onto a plate containing 0.05 mg / mL kanamycin resistance, and incubate at 37℃ for about 12 h.

[0046] Example 3: Containing pET-28a- Bb HPPDF242Y / L271L / K231K Screening of recombinant bacteria Single clones were picked and placed in 96-well plates containing 0.05 mg / mL kanamycin-resistant LB liquid medium. After incubation at 220 rpm and 37°C for 8 h, TBA liquid medium was added and incubated at 220 rpm and 37°C for 2-3 h. After incubation, the plates were transferred to 25°C for 16 h of induction, and then the cells were harvested by centrifugation at 3800 rpm for 10 min.

[0047] Screening conditions: A solution containing 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) was added to a 96-well plate and incubated at 37°C for 1 h. After the reaction, the remaining keto acid levels were detected using the DNPS high-throughput method, as disclosed in patent application CN112626100A. Five mutants superior to wild-type were selected from each well plate, and these mutants were subsequently sent to Tianlin Biotechnology Co., Ltd. for sequencing.

[0048] The correctly sequenced mutant strains were inoculated into LB seed medium and cultured at 220 rpm and 37°C for 8–12 h. Then, they were inoculated at a 1% inoculum into shake-flask fermentation medium (TB medium) and cultured at 220 rpm and 37°C until OD (Organic Degrees). 600 =0.6~0.8, add IPTG to a final concentration of 0.4 mM for induction, and incubate at 220 rpm and 25℃ for 16 h. The induced bacterial cells are then used to determine HMB production. The first round of 10 mL reaction system contained (at the final concentration): 20 g / L of expressed HMB. Bb The second and third rounds of reaction were performed using wet HPPD cells or their mutants, 15 g / L α-KIC, 1.5 mM FeSO4·7H2O, and 7.5 mM L-ascorbic acid in Tris-Maleic buffer (60 mM, pH 5.5). The mixture was incubated at 37°C for 18 h. The 10 mL reaction mixtures for the second and third rounds contained (at final concentrations): 20 g / L α-KIC, 2 mM FeSO4·7H2O, and 10 mM L-ascorbic acid in Tris-Maleic buffer (60 mM, pH 5.5), and were incubated at 37°C for 18 h. After the reaction, 500 μL of the transformed solution was added, and an equal volume of 2 M HCl was added. The mixture was centrifuged at 12000 rpm for 3 min 30 s. The supernatant was filtered through a 0.22 μm aqueous filter and analyzed by HPLC. After the first round of saturated mutagenesis, mutants were obtained through screening. Bb HPPD F242Y The result is as follows Figure 1The HMB yield shown was 12.2 g / L, with a molar conversion rate of 67%, compared to the control group. Bb HPPD production increased by 24.5%; after a second round of saturation mutagenesis, mutants were obtained through screening. Bb HPPD F242Y / L271L The result is as follows Figure 1 The yield of HMB shown was 14.7 g / L, with a molar conversion rate of 81%, which is higher than... Bb HPPD F242Y The improvement was 20.5%; after the third round of saturation mutagenesis, mutants were obtained through screening. Bb HPPD F242Y / L271L / K231K The result is as follows Figure 1 The yield of HMB shown was 15.4 g / L, with a molar conversion rate of 85.4%, which is higher than... Bb HPPD F242Y / L271L The improvement was 5%. In summary, after three rounds of iterative saturation mutagenesis, the optimal mutant was obtained through screening. Bb HPPD F242Y / L271L / K231K The result is as follows Figure 1 The HMB yield shown was 15.4 g / L, with a molar conversion rate of 85.4%, compared to the initial strain. Bb HPPD production increased by 57.1%.

[0049] Example 4: Verification of soluble expression of the target protein (1) Sample preparation Weigh 0.01 g of induced bacterial cells into a 1.5 mL EP tube, add 1 mL of Tris-HCl buffer to resuspend the cells, and place in an ice bath for sonication (parameters: power 100 W, working time 3.5 s, interval time 2.5 s, total 5 min) to ensure thorough cell disruption. After disruption, centrifuge the EP tube at 10,000 rpm for 10 min in a pre-chilled centrifuge at 4°C to separate the soluble protein supernatant (containing soluble cytoplasmic proteins) and the insoluble protein precipitate (containing inclusion bodies).

[0050] (2) SDS-PAGE gel electrophoresis analysis Prepare 12.5% ​​separating gel (acrylamide concentration 12.5%) and 12.5% ​​stacking gel for separating proteins of different molecular weights. Take 30 μL each of the soluble protein supernatant and the insoluble protein precipitate (the precipitate was resuspended in an equal volume of PBS), and add them separately to 4×SDS loading buffer (containing... βMix β-mercaptoethanol (β-mercaptoethanol) at a 3:1 volume ratio and boil for 10 min to denature the protein. Electrophoresis conditions: Using a low molecular weight protein marker (range 10-180 kDa) as a reference, the sample loading volume is 10 μL / lane, the stacking gel voltage is 80 V, the separating gel voltage is 120 V, and the electrophoresis time is approximately 90 min, until the bromophenol blue indicator migrates to the bottom of the gel. After electrophoresis, stain with Coomassie Brilliant Blue R-250 for 1 h, then destain with destaining solution until the background is clear and the target protein band is visible. Figure 2 ).

[0051] Example 5: Verification at different substrate concentrations Bb HPPD F242Y / L271L / K231K Production performance The recombinant bacteria constructed in Example 3 Bb HPPD F242Y / L271L / K231K Inoculate into LB seed medium and incubate at 220 rpm and 37°C for 8–12 h. Then, inoculate into shake flask fermentation medium (TB medium) at a 1% inoculum and incubate at 220 rpm and 37°C until OD (dose-to-discharge ratio) is reached. 600 The concentration of the substrate was approximately 0.6-0.8. IPTG was added to a final concentration of 0.4 mM for induction at 220 rpm and 25°C for 16 h. The induced bacterial cells were then subjected to a reaction to determine HMB yield, investigating the effect of different substrate concentrations on HMB production. Bb HPPD F242Y / L271L / K231K The effect of the strain on HMB production. A 10 mL reaction system was prepared containing (on a final concentration): Tris-Maleic buffer (60 mM, pH 5.5), 20 g / L. Bb HPPD F242Y / L271L / K231K Wet cells were used, and different substrate concentrations (20, 40, 60, 80, 100 g / L) were set. The concentrations of FeSO4·7H2O and L-ascorbic acid (VC) were adjusted proportionally, as shown in Table 4. The cells were incubated at 37℃ for 18 h, and the effect of substrate concentration on HMB yield was determined.

[0052] Table 4 Validation System

[0053] The results are as follows Figure 3 As shown, at a substrate concentration of 20 g / L, the yield of HMB was 15.4 ± 0.1 g / L. With further increases in substrate concentration, the yield of HMB showed a decreasing trend, suggesting... Bb HPPD F242Y / L271L / K231K Substrate inhibition may exist.

[0054] Case Study 6: Pm L-AAD Q92A and BbHPPD F242Y / L271L / K231K Construction of dual-enzyme co-expression strains (1) Gene amplification and vector selection and preparation L-amino acid deaminase single mutant ( Pm L-AAD Q92A Gene amplification: using Proteus mirabilis ( Proteus mirabilis Using genomic DNA as a template, design specific primers. Pm L-AAD Q92A -F / Pm L-AAD Q92A -R, obtained through PCR amplification Pm L-AAD Q92A Gene fragment (nucleotide sequence as shown in SEQ ID NO.9).

[0055] 4-Hydroxyphenylpyruvate dioxygenase mutant ( Bb HPPD F242Y / L271L / K231K Gene amplification: Using the mutant plasmid constructed in Example 2 as a template, primers were designed. Bb HPPD F242Y / L271L / K231K -F / Bb HPPD F242Y / L271L / K231K -R, obtained by PCR amplification Bb HPPD F242Y / L271L / K231K Gene fragment (nucleotide sequence as shown in SEQ ID NO.8). PCR conditions are as described in Example 2.

[0056] Table 5 Amplification Primer Sequences

[0057] The co-expression of the two enzymes was optimized by selecting four vectors with different copy numbers. The selected vectors are shown in Table 6. These four vectors have different resistances and are compatible with each other.

[0058] Table 6 Selection of Expression Vectors

[0059] Vector fragment preparation: Using pACYCDuet-1, pCDFDuet-1, pETDuet-1, and pRSFDuet-1 plasmids as templates, vector-specific primers were designed, and linearized vector fragments were obtained by PCR amplification. Since the sequences inserted into the target gene are identical for the four plasmid vectors, only two pairs of upstream and downstream primers are needed; one pair contains primers specific to the target gene. Pm L-AAD Q92A Primer sequence with homologous arm G-Plasmid-F1 ( Pm L-AAD Q92A) / G-Plasmid-R1( Pm L-AAD Q92A The other pair contains the target gene. Bb HPPD F242Y / L271L / K231K The primer sequence with homologous arms is G-Plasmid-F2 ( Bb HPPD F242Y / L271L / K231K ) / G-Plasmid-R2( Bb HPPD F242Y / L271L / K231K As shown in Table 7, the underlined parts are homologous arms.

[0060] Table 7 Amplification Primer Sequences

[0061] (2) Construction of recombinant plasmids Construction of single enzyme expression vectors: Using Gibson Assembly technology, the single enzyme expression vector was constructed... Pm L-AAD Q92A The gene and pACYCDuet-1 vector fragment were incubated at a molar ratio of 2:1 at 50°C for 30 min, cooled on ice, and then transformed into... E. coli BL21(DE3) competent cells were plated on LB agar plates containing 34 μg / mL chloramphenicol and cultured at 37°C for 12 h. Positive clones were picked and sequenced for verification, yielding the recombinant plasmid pACYCDuet-1- Pm L-AAD Q92A Similarly, constructing connections involves... Bb HPPD F242Y / L271L / K231K The recombinant plasmid pACYCDuet-1- Bb HPPD F242Y / L271L / K231K .

[0062] Similarly, following the above method, Pm L-AAD Q92A Genes were used to construct recombinant plasmids with three other vectors, and... Bb HPPD F242Y / L271L / K231K The gene was used to construct recombinant plasmids with three other vectors. A total of four types of plasmids containing the gene were obtained. Pm L-AAD Q92A The recombinant plasmid pACYCDuet-1- of the gene Pm L-AAD Q92A pCDFDuet-1- Pm L-AAD Q92A pETDuet-1- Pm L-AAD Q92A pRSFDuet-1- Pm L-AAD Q92Aand four kinds of Bb HPPD F242Y / L271L / K231K The recombinant plasmid pACYCDuet-1- of the gene Bb HPPD F242Y / L271L / K231K pCDFDuet-1- Bb HPPD F242Y / L271L / K231K pETDuet-1- Bb HPPD F242Y / L271L / K231K pRSFDuet-1- Bb HPPD F242Y / L271L / K231K .

[0063] (3) Construction of dual-enzyme co-expression strains Verify the correct pACYCDuet-1- Pm L-AAD Q92A Plasmid and pCDFDuet-1- Bb HPPD F242Y / L271L / K231K plasmid concomitant conversion to E. coli BL21(DE3) competent cells were plated on plates containing streptomycin (100 μg / mL) and chloramphenicol (34 μg / mL) to screen for strains co-expressing the two enzymes. Similarly, the strains shown in Table 8 were constructed by combining plasmids of the two different resistance types in pairs.

[0064] Table 8. Strains and Plasmids

[0065] Example 7: Induction culture of engineered bacteria with dual-enzyme co-expression The engineered bacteria constructed in Example 6 E. coli 1.0~ E. coli 12.0 g of the culture medium was inoculated into LB liquid medium and cultured for 12 h to obtain seed culture. The seed culture was then inoculated into fresh TB liquid medium at an inoculation rate of 1% (v / v) and cultured for 2.5-3 h. OD was then measured. 600 IPTG was added to a final concentration of 0.4 mM at a concentration of 0.6-0.8, and the mixture was incubated at 25°C for 16 h to induce the expression of the recombinant target protein. 150 mL of the induced fermentation broth was centrifuged at 8000 rpm for 8 min to collect the bacterial cells, obtaining bacterial cells that co-expressed the two enzymes, and then stored at -40°C.

[0066] Example 8: Preparation of HMB from Whole Cells Prepare a 60 mM Tris-Maleate buffer solution in a 100 mL Erlenmeyer flask, adjust the pH to 5.5 with maleic acid, add 20 g / L L-Leu, 2 mM Fe2SO4·7H2O, and 10 mM ascorbic acid, then add ultrapure water to a final volume of 10 mL, adjust the pH to 5.5 with NaOH solid, and then add 20 g / L of the 12 recombinant Escherichia coli wet cells prepared in Example 7. E. coli 1.0~ E. coli 12.0), and reacted in a constant temperature shaker at 37℃ and 220 rpm for 18 h. After the reaction, 500 μL of the transformed solution was taken, and an equal volume of 2 M HCl was added. The mixture was centrifuged at 12000 rpm for 3 min 30 s. The supernatant was filtered through a 0.22 μm aqueous filter membrane and analyzed by HPLC. Twelve recombinant Escherichia coli strains were transformed into whole cells to prepare HMB (Table 9), among which the recombinant strains... E. coli The highest yield of HMB was 10.2 g / L with a molar conversion rate of 56.6%, and it was designated as strain HMB 1.0.

[0067] Table 9. Construction and HMB production of 12 recombinant strains

[0068] Example 9: Increased HMB production through repeated expression of rate-limiting enzyme Since the engineered strain HMB 1.0 still accumulated 8.5 g / L α-KIC, it indicates that... Bb HPPD F242Y / L271L / K231K This remains the rate-limiting step in the cascade reaction. In multi-enzyme cascade reactions, increasing gene copy number can improve gene expression levels. Following these steps, the expression is sequentially performed in pCDFDuet-1- Bb HPPD F242Y / L271L / K231K and pACYCFDuet-1- Pm L-AAD Q92A Add to each Bb HPPD F242Y / L271L / K231K The copy number was determined to obtain engineered bacteria HMB 2.0 and HMB 3.0. The specific steps are as follows: (1) Gene amplification 4-Hydroxyphenylpyruvate dioxygenase mutant ( Bb HPPD F242Y / L271L / K231K Gene amplification: Using the mutant plasmid constructed in Example 2 as a template, primers RBS- were designed. Bb HPPD F242Y / L271L / K231K -F / RBS- Bb HPPD F242Y / L271L / K231K-R1 / RBS- ​ HPPD F242Y / L271L / K231K -R2 (Table 10, underlined arms are homologous), PCR amplification yielded RBS- ​ HPPD F242Y / L271L / K231K Gene fragment. PCR conditions are the same as those for gene amplification in Case Study 6.

[0069] Table 10 Amplification Primer Sequences

[0070] Vector fragment amplification: using pCDFDuet-1- ​ HPPD F242Y / L271L / K231K and pACYCDuet-1- ​ L-AAD Q92A Using the plasmid as a template, two pairs of vector-specific primers were designed, and the linearized vector fragment was obtained by PCR amplification. One primer pair, pCDFDuet-1-F / pCDFDuet-1-R, was used to amplify the vector pCDFDuet-1- ​ HPPD F242Y / L271L / K231K Another primer pair, pACYCDuet-1-F / pACYCDuet-1-R, is used to amplify the vector pACYCDuet-1- ​ L-AAD Q92A Table 11, underlined parts are homologous arms.

[0071] Table 11 Amplification Primer Sequences

[0072] (2) Construction of recombinant plasmids Construction of repetitive expression vectors: Using Gibson Assembly technology, RBS- ​ HPPD F242Y / L271L / K231K Gene fragments and pCDFDuet-1- ​ HPPD F242Y / L271L / K231K The vector fragment (molar ratio 2:1) was incubated at 50°C for 30 min, cooled on ice, and then transformed into... ​ BL21(DE3) competent cells were plated on LB agar plates containing 100 μg / mL streptomycin and cultured at 37°C for 12 h. Positive clones were picked and sequenced for verification, yielding the recombinant plasmid pCDFDuet-1-. ​ HPPD F242Y / L271L / K231K -RBS- ​ HPPD F242Y / L271L / K231K Similarly, construct the recombinant plasmid pACYCDuet-1- ​ L-AAD Q92A -RBS- ​ HPPDF242Y / L271L / K231K The RBS sequence is TCTAGAGAAAGAGGGGACAAACTAGATG.

[0073] (3) Construction of dual-enzyme co-expression strains Verify the correct pCDFDuet-1- ​ HPPD F242Y / L271L / K231K -RBS- ​ HPPD F242Y / L271L / K231K plasmids and pACYCDuet-1- ​ L-AAD Q92A plasmid concomitant conversion to ​ BL21(DE3) competent cells were plated on plates containing streptomycin (100 μg / mL) and chloramphenicol (34 μg / mL) antibiotics to screen for the dual-enzyme co-expression strain HMB 2.0. Based on this, the validated pCDFDuet-1- ​ HPPD F242Y / L271L / K231K -RBS- ​ HPPD F242Y / L271L / K231K plasmids and pACYCDuet-1- ​ L-AAD Q92A -RBS- ​ HPPD F242Y / L271L / K231K plasmid concomitant conversion to ​ BL21(DE3) competent cells were plated on double-antibody plates containing streptomycin (100 μg / mL) and chloramphenicol (34 μg / mL) to screen for the dual-enzyme co-expression strain HMB 3.0. The plasmid and yield data are shown in Table 12. Whole-cell testing was performed according to Example 8, with shake-flask yields of 11.2 g / L and 12.7 g / L, respectively, representing increases of 9.8% and 24.5% compared to the HMB1.0 strain constructed in Example 8. The molar conversion rates were 62.4% and 70.6%, respectively.

[0074] Table 12 HMB production by the dual-enzyme co-expression strains

[0075] Comparative Example 1: Following the methods of Examples 2-3, the mutants shown in Table 13 were also constructed, and the HMB yields of different mutants were verified following the methods of Example 3.

[0076] Table 13 HMB production of different mutants

[0077] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1,4-Hydroxyphenylpyruvate dioxygenase mutant, characterized in that, Compared to the parent shown in SEQ ID NO.1, phenylalanine at position 242 was mutated to tyrosine.

2. The 4-hydroxyphenylpyruvate dioxygenase mutant according to claim 1, characterized in that, It also has synonymous mutations at position 271 and / or position 231.

3. The 4-hydroxyphenylpyruvate dioxygenase mutant according to claim 2, characterized in that, The synonymous mutation is to change the codon encoding position 271 from CTG to TTG; or to change the codon encoding position 231 (lysine) from AAA to AAG.

4. A gene encoding any of the mutants described in claims 1 to 3.

5. A recombinant vector carrying the gene of claim 4.

6. A recombinant cell expressing any of the mutants described in claims 1 to 3, or containing the gene described in claim 4, or containing the recombinant vector described in claim 5.

7. Recombinant Escherichia coli, characterized in that, by E. coli BL21 (DE3) is used as the host to express the 4-hydroxyphenylpyruvate dioxygenase mutant as described in any one of claims 1 to 3.

8. The recombinant Escherichia coli according to claim 7, characterized in that, It also expressed a single mutant of L-amino acid deaminase. Pm L-AAD Q92A .

9. A method for preparing β The method for preparing 1-hydroxyisovaleric acid is characterized by, The recombinant Escherichia coli of claim 8 was used as a catalyst in a Tris-Maleate buffer containing L-leucine, FeSO4·7H2O and L-ascorbic acid.

10. The 4-hydroxyphenylpyruvate dioxygenase mutant according to any one of claims 1 to 3, or the recombinant Escherichia coli according to claim 8, for the preparation of a product containing... β Application in products containing hydroxyisovalerate or its salts.

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