Multi-site mutated phenylpyruvate decarboxylase mutant and application thereof in production of tyrosol

Abstract

The invention provides a phenylpyruvate decarboxylase mutant with multi-site mutation and application of the phenylpyruvate decarboxylase mutant in production of tyrosol, and belongs to the technical field of bioengineering. The problem of low yield of tyrosol is solved. The phenylpyruvate decarboxylase mutant comprises a phenylpyruvate decarboxylase mutant, and the phenylpyruvate decarboxylase mutant is obtained by mutating any two or three sites of the 93rd site, the 106th site, the 153rd site, the 220th site, the 242nd site, the 273rd site and the 285th site by taking amino acid as shown in SEQ ID NO.2 as a starting sequence. The method is mainly used for increasing the yield of tyrosol.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a multi-site mutated phenylpyruvate decarboxylase mutant and its application in the production of tyrosol. Background Technology

[0002] Tyrosol, chemically known as 4-hydroxyphenylethanol, is a phenylpropanoid compound naturally found in various plants and foods, including olive oil and red wine. In recent years, scientific research has revealed that tyrosol possesses excellent antioxidant, anti-inflammatory, neuroprotective, and cardiovascular protective activities, demonstrating significant application potential in the food, health supplement, cosmetic, and pharmaceutical industries. With the increasing market demand for natural and functional ingredients, developing efficient and sustainable tyrosol production methods has become a focus of attention for related industries.

[0003] Traditional methods of obtaining tyrosol primarily rely on extraction from plant-based raw materials such as olive leaves and olive oil processing byproducts. However, this method is limited by factors such as long plant growth cycles, complex extraction processes, low product content, and significant susceptibility to seasonal and geographical climate influences, resulting in high production costs and unstable yields, making it difficult to meet the demands of large-scale industrial production. Therefore, finding a stable, efficient, and environmentally friendly alternative production route is urgently needed.

[0004] With the rapid development of synthetic biology and metabolic engineering technologies, utilizing genetically engineered microbial cell factories to produce high-value-added natural products has become a promising solution. This method offers significant advantages such as short production cycles, lack of reliance on natural resources, process controllability, and environmental friendliness.

[0005] Currently, research has been conducted to construct a tyrosol synthesis pathway in Escherichia coli, develop recombinant E. coli engineered strains capable of high-yield and stable synthesis of tyrosol, and pair them with efficient fermentation processes. This is of vital significance for overcoming the production bottleneck of tyrosol and promoting its industrial application. Summary of the Invention

[0006] In view of this, the present invention aims to provide a phenylpyruvate decarboxylase mutant composition and its application in the production of tyrosol, and to provide a highly efficient phenylpyruvate decarboxylase and its application in the de novo synthesis of tyrosol, so as to solve the problem of low tyrosol yield in the prior art.

[0007] This invention provides a phenylpyruvate decarboxylase mutant, wherein the phenylpyruvate decarboxylase mutant is any one of the following: (1) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and S at position 106 with A; (2) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and L at position 220 with A; (3) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and D at position 242 with E; (4) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and F at position 273 with L; (5) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and N at position 285 with Q; (6) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and S at position 153 with T; (7) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and L at position 220 with A; (8) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and D at position 242 with E; (9) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and F at position 273 with L; (10) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and N at position 285 with Q; (11) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T and L at position 220 with A; (12) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T and D at position 242 with E; (13) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T and F at position 273 with L; (14) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A and N at position 285 with Q; (15) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace D at position 242 with E and F at position 273 with L; (16) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace D at position 242 with E and N at position 285 with Q; (17) Using the amino acid shown in SEQ ID NO.1 as the starting sequence, replace F at position 273 with L and N at position 285 with Q; (18) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 106 with A, and S at position 153 with T; (19) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 106 with A, and L at position 220 with A; (20) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 106 with A, and F at position 273 with L; (21) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 106 with A, and N at position 285 with Q; (22) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 153 with T, and L at position 220 with A; (23) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 153 with T, and D at position 242 with E; (24) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 153 with T, and F at position 273 with L; (25) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 153 with T, and N at position 285 with Q; (26) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, L at position 220 with A, and D at position 242 with E; (27) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, L at position 220 with A, and F at position 273 with L; (28) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, L at position 220 with A, and N at position 285 with Q; (29) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, D at position 242 with E, and F at position 273 with L; (30) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, D at position 242 with E, and N at position 285 with Q; (31) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, F at position 273 with L, and N at position 285 with Q; (32) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace S at position 153 with T, and replace L at position 220 with A; (33) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace S at position 153 with T, and replace D at position 242 with E; (34) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace S at position 153 with T, and replace F at position 273 with L; (35) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace S at position 153 with T, and replace N at position 285 with Q; (36) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace L at position 220 with A, and replace D at position 242 with E; (37) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace L at position 220 with A, and replace F at position 273 with L; (38) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace L at position 220 with A, and replace N at position 285 with Q; (39) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, F at position 273 with L, and N at position 285 with Q; (40) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, L at position 220 with A, and F at position 273 with L; (41) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, L at position 220 with A, and N at position 285 with Q; (42) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, D at position 242 with E, and F at position 273 with L; (43) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, D at position 242 with E, and N at position 285 with Q; (44) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, F at position 273 with L, and N at position 285 with Q; (45) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A, D at position 242 with E, and F at position 273 with L; (46) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A, D at position 242 with E, and N at position 285 with Q; (47) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A, replace F at position 273 with L, and replace N at position 285 with Q; (48) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace D at position 242 with E, F at position 273 with L, and N at position 285 with Q.

[0008] The present invention provides a gene encoding the above-mentioned phenylpyruvate decarboxylase mutant.

[0009] The present invention provides a recombinant vector containing the above-mentioned genes.

[0010] To further specify, the launch vehicle is the pRSFDuet series.

[0011] This invention provides a recombinant microbial cell containing the above-mentioned genes.

[0012] The present invention provides a recombinant Escherichia coli expressing the above-mentioned phenylpyruvate decarboxylase mutant.

[0013] Further specifying, the alcohol dehydrogenase gene ADH6 and the mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase gene aroG are included. fbr Overexpression plasmids of the cyclohexadiene dehydrogenase gene tyrC and the L-amino acid ammonia-lyase gene LAAD were transfected into Escherichia coli BL21(DE3) competent cells.

[0014] The present invention provides the application of the above-mentioned phenylpyruvate decarboxylase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cell in the production of tyrosol and products containing tyrosol.

[0015] The present invention provides a method for producing tyrosol, the steps of which are as follows: fermenting the above-mentioned recombinant Escherichia coli at 30°C for 72 hours.

[0016] Further specified, the fermentation medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract, with 10 g / L CaCO3 used to maintain the pH of the fermentation medium. The culture medium was supplemented with 0.1% by volume of trace elements, consisting of: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O, and 15 g / L ZnSO4·7H2O. The pH was adjusted to 7.0 using NaOH.

[0017] Compared with the prior art, the beneficial effects of the present invention are: the present invention is effective for the treatment of yeast derived from brewing yeast ( Saccharomyces cerevisiae Different combinations of site-directed mutagenesis were performed on phenylpyruvate decarboxylase, resulting in the ARO10 mutant phenylpyruvate decarboxylase. I93V / L220A / N285Q Overexpression in genetically engineered bacteria significantly increased tyrosol production, with shake-flask fermentation levels reaching up to 3.6 g / L, providing valuable components for constructing high-yield tyrosol strains. Attached Figure Description

[0018] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 Figure 1 shows the HPLC chromatogram of tyrosol content in the fermentation broth of the strain according to Example 3 of the present invention. Figure 2 shows the HPLC chromatogram of tyrosol standard; Figure 3 shows the HPLC chromatogram of the genetically engineered strain ARO10. I93V / L220A / N285Q HPLC chromatogram of tyrosol production after 72 h of fermentation. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other, and the described embodiments are only some embodiments of the present invention, not all embodiments.

[0020] The seed culture system consisted of 10 mL of LB medium in a 100 mL Erlenmeyer flask containing 10 mL of culture medium, which was composed of 5 g / L yeast extract, 10 g / L NaCl, and 10 g / L peptone. The fermentation system for shake-flask fermentation consisted of 250 mL Erlenmeyer flasks containing 50 mL of culture medium. The culture medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract. The pH of the fermentation medium was maintained using 10 g / L CaCO3. The culture medium was supplemented with 0.1% by volume of trace elements, the composition of which was: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O, and 15 g / L ZnSO4·7H2O. The pH was adjusted to 7.0 using NaOH.

[0021] Example 1. Obtaining a phenylpyruvate decarboxylase mutant This invention provides a phenylpyruvate decarboxylase mutant for efficient synthesis of tyrosol. Through co-evolutionary analysis and substrate binding analysis of the catalytic cavity modification, site-directed mutagenesis was performed on the following positions of the phenylpyruvate decarboxylase ARO10: Ile at position 93, Ser at position 106, Ser at position 153, Leu at position 220, Asp at position 242, Phe at position 273, and Asn at position 285, yielding seven mutant phenylpyruvate decarboxylases, namely ARO10... I93V ARO10 S106A ARO10 S153T ARO10 L220A ARO10 D242E ARO10 F273L ARO10 N285Q .

[0022] The above-mentioned single-point phenylpyruvate decarboxylase mutants were subjected to multi-site combination mutations, including any one of the following combinations of mutation sites: I93V+S106A, I93V+S153T, I93V+L220A, I93V+D242E, I93V+F273L, I93V+N285Q, S106A+S153T, S106A+L220A, S106A+D242E, S106A+F273L, S106A+N285Q, S153T+L220A, S153T+D242E, S153T+F273L, S153T+N285Q, L220A+D24 2E, L220A+F273L, L220A+N285Q, D242E+F273L, D242E+N285Q, F273L+N285Q, I93V+S106A+S153T, I93V+S106A+L220A, I93V+S106A+D242E , I93V+S106A+F273L, I93V+S106A+N285Q, I93V+S153T+L220A, I93V+S153T+D242E, I93V+S153T+F273L, I93V+S153T+N285Q, I93V+L220A +D242E, I93V+L220A+F273L, I93V+L220A+N285Q, I93V+D242E+F273L, I93V+D242E+N285Q, I93V+F273L+N285Q, S106A+S153T+L220A, S10 6A+S153T+D242E, S106A+S153T+F273L, S106A+S153T+N285Q, S106A+L220A+D242E, S106A+L220A+F273L, S106A+L220A+N285Q, S106A+D2 42E+F273L, S106A+D242E+N285Q, S106A+F273L+N285Q, S153T+L220A+D242E, S153T+L220A+F273L, S153T+L220A+N285Q, S153T+D242E+F 273L, S153T+D242E+N285Q, S153T+F273L+N285Q, L220A+D242E+F273L, L220A+D242E+N285Q, L220A+F273L+N285Q, D242E+F273L+N285Q.

[0023] The wild-type phenylpyruvate decarboxylase gene enzyme ARO10 is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiaeIts nucleotide sequence is shown in SEQ ID NO.1; its amino acid sequence is shown in SEQ ID NO.2.

[0024] SEQ ID NO.1 (nucleotide sequence of wild-type phenylpyruvate decarboxylase ARO10): SEQ ID NO.2 (Amino acid sequence of wild-type phenylpyruvate decarboxylase ARO10): *

[0025] Example 2: Construction of genetically engineered bacteria containing different phenylpyruvate decarboxylase ARO10 mutants Overexpression derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiae ) phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6, Derived from Escherichia coli ( Escherichia coli The mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase gene aroG fbr Derived from motile fermentation monosporus ( Zymomonas mobilis ) cyclohexadiene dehydrogenase gene tyrC, Derived from Proteus mirabilis (Proteus mirabilis L-amino acid ammonia-lyase gene LAAD recombinant plasmid pRSFDuet -ARO10-ADH6 - aroG fbr -tyrC-LAAD Synthesized by Genewiz Sequencing Company.

[0026] pRSFDuet- ARO10 - ADH6 and pETDuet- aroG fbr - tyrC - AtUGT85A1 Published in the references Engineering Escherichia coli for Efficient De Novo Synthesis of Salidroside), pETDuet- aroG fbr - tyrC - AtUGT85A1 The carrier is connected to pRSFDuet- ARO10 - ADH6 In, and connect to LAAD Gene (SEQ ID NO.4), the obtained recombinant plasmid pRSFDuet -ARO10-ADH6 - aroG fbr -tyrC-LAAD.

[0027] SEQ ID NO.3 (Amino acid sequence of L-amino acid ammonia-lyase LAAD) MNISRRKLLLGVGAAGVLAGGAATLVPMVRRDGKFVESKSRALFVESTEGALPSESDVVIIGGGIQGIMTAINLAERGMSVTILEKGEVAGEQSGRAYSQIISYQTSPEIFPLHHYGKILWRGMNEKIGADTSYRTQGRVEALADEKALDRAQEWIKTAKETAGFDVPLNTRIIKGEELSNRLVGAQTPWTVAAFEEDSGSVDPETGTPTLARYAKQIGVKIYTHCAVRGIETAGGKISDVVTEKGAIRTSNVVLAGGIWSRLFMGNMGVDLPTLNVYLSQQRVSGVPGAPRGNVHLPNGIHFREQADGTYAVAPRIFTSSIVKDSFLLGPKFMHLLGGGELPLEFSIGEDLFNSFKMPTSWKLDEKSPFEQYRIATATQNTEHLDAVFQRMKTEFPVFEKSQIVERWGAVVSPTFDELPIISEVKEYPGLVINTATVWGMTEGPAAGEVTADIVTGKKPVIDPTPFSLDRFKK; SEQ ID NO.4 (Nucleotide sequence of L-amino acid deaminase LAAD) overexpression plasmid pRSFDuet -ARO10-ADH6 - aroG fbr -tyrC-LAAD The wild-type phenylpyruvate decarboxylase ARO10 gene fragment was replaced with the mutant phenylpyruvate decarboxylase gene fragment (mutant phenylpyruvate decarboxylase ARO10). I93V+S106A Mutant phenylpyruvate decarboxylase ARO10 I93V+S153T Mutant phenylpyruvate decarboxylase ARO10 I93V +L220A Mutant phenylpyruvate decarboxylase ARO10 I93V+D242E Mutant phenylpyruvate decarboxylase ARO10 I93V+F273L Mutant phenylpyruvate decarboxylase ARO10 I93V+N285Q Mutant phenylpyruvate decarboxylase ARO10 S106A+S153T Mutant phenylpyruvate decarboxylase ARO10 S106A+L220A Mutant phenylpyruvate decarboxylase ARO10 S106A+D242E Mutant phenylpyruvate decarboxylase ARO10 S106A+F273L Mutant phenylpyruvate decarboxylase ARO10 S106A+N285Q Mutant phenylpyruvate decarboxylase ARO10 S153T +L220A Mutant phenylpyruvate decarboxylase ARO10 S153T+D242E Mutant phenylpyruvate decarboxylase ARO10 S153T+F273L Mutant phenylpyruvate decarboxylase ARO10 264N+N285Q Mutant phenylpyruvate decarboxylase ARO10 L220A+D242E Mutant phenylpyruvate decarboxylase ARO10 L220A+F273L Mutant phenylpyruvate decarboxylase ARO10 L220A+N285Q Mutant phenylpyruvate decarboxylase ARO10 D242E+F273L Mutant phenylpyruvate decarboxylase ARO10 D242E+N285Q Mutant phenylpyruvate decarboxylase ARO10 F273L +N285Q Mutant phenylpyruvate decarboxylase ARO10 I93V+S106A+S153T Mutant phenylpyruvate decarboxylase ARO10 I93V +S106A+L220A Mutant phenylpyruvate decarboxylase ARO10 I93V+S106A+D242E Mutant phenylpyruvate decarboxylase ARO10 I93V +S106A+F273L Mutant phenylpyruvate decarboxylase ARO10 I93V+S106A+N285Q Mutant phenylpyruvate decarboxylase ARO10 I93V +S153T+L220A Mutant phenylpyruvate decarboxylase ARO10I93V+S153T+D242E Mutant phenylpyruvate decarboxylase ARO10 I93V +S153T+F273L Mutant phenylpyruvate decarboxylase ARO10 I93V+S153T+N285Q Mutant phenylpyruvate decarboxylase ARO10 I93V +L220A+D242E Mutant phenylpyruvate decarboxylase ARO10 I93V+L220A+F273L Mutant phenylpyruvate decarboxylase ARO10 I93V +L220A+N285Q Mutant phenylpyruvate decarboxylase ARO10 I93V+D242E+F273L Mutant phenylpyruvate decarboxylase ARO10 I93V +D242E+N285Q Mutant phenylpyruvate decarboxylase ARO10 I93V+F273L+N285Q Mutant phenylpyruvate decarboxylase ARO10 S106A +S153T+L220A Mutant phenylpyruvate decarboxylase ARO10 S106A+S153T+D242E Mutant phenylpyruvate decarboxylase ARO10 S106A +S153T+F273L Mutant phenylpyruvate decarboxylase ARO10 S106A+S153T+N285Q Mutant phenylpyruvate decarboxylase ARO10 S106A +L220A+D242E Mutant phenylpyruvate decarboxylase ARO10 S106A+L220A+F273L Mutant phenylpyruvate decarboxylase ARO10 S106A +L220A+N285Q Mutant phenylpyruvate decarboxylase ARO10 S106A+D242E+F273L Mutant phenylpyruvate decarboxylase ARO10 S106A +D242E+N285Q Mutant phenylpyruvate decarboxylase ARO10 S106A+F273L+N285Q Mutant phenylpyruvate decarboxylase ARO10 S153T +L220A+D242E Mutant phenylpyruvate decarboxylase ARO10 S153T+L220A+F273L Mutant phenylpyruvate decarboxylase ARO10 S153T +L220A+N285Q Mutant phenylpyruvate decarboxylase ARO10 S153T+D242E+F273L Mutant phenylpyruvate decarboxylase ARO10 S153T +D242E+N285Q Mutant phenylpyruvate decarboxylase ARO10 S153T+F273L+N285Q Mutant phenylpyruvate decarboxylase ARO10 L220A +D242E+F273L Mutant phenylpyruvate decarboxylase ARO10 L220A+D242E+N285Q Mutant phenylpyruvate decarboxylase ARO10 L220A +F273L+N285Q Mutant phenylpyruvate decarboxylase ARO10D242E+F273L+N285Q The gene was obtained, and 56 different combinations of mutant sites were obtained. The gene of wild-type phenylpyruvate decarboxylase ARO10 was also introduced into the chassis strain Escherichia coli BL21(DE3), resulting in 57 engineered strains as shown in Table 1. They are named WT, I93V+S106A, I93V+S153T, I93V+L220A, I93V+D242E, I93V+F273L, I93V+N285Q, S106A+S153T, S106A+L220A, S106A+D242E, S106A+F273L, S106A+N285Q, S153T+L220A, S153T+D242E, S153T+F273L, S153T+N285Q, L220A+D242E, L220A+F273L, and L220A+N285Q respectively. , D242E+F273L, D242E+N285Q, F273L+N285Q, I93V+S106A+S153T, I93V+S106A+L220A, I93V+S106A+D242E, I93V+S106A+F273L, I93 V+S106A+N285Q, I93V+S153T+L220A, I93V+S153T+D242E, I93V+S153T+F273L, I93V+S153T+N285Q, I93V+L220A+D242E, I93V+L220A +F273L、I93V+L220A+N285Q、I93V+D242E+F273L、I93V+D242E+N285Q、I93V+F273L+N285Q、S106A+S153T+L220A、S106A+S153T+D24 2E, S106A+S153T+F273L, S106A+S153T+N285Q, S106A+L220A+D242E, S106A+L220A+F273L, S106A+L220A+N285Q, S106A+D242E+F273 L. S106A+D242E+N285Q, S106A+F273L+N285Q, S153T+L220A+D242E, S153T+L220A+F273L, S153T+L220A+N285Q, S153T+D242E+F273L , S153T+D242E+N285Q, S153T+F273L+N285Q, L220A+D242E+F273L, L220A+D242E+N285Q, L220A+F273L+N285Q, D242E+F273L+N285Q.

[0028] Table 1 Genetically engineered bacteria containing different phenylpyruvate decarboxylase mutants

[0029] Example 3: Tyrosol fermentation experiment of genetically engineered bacteria containing different phenylpyruvate decarboxylase mutant compositions These 57 genetically engineered bacteria were inoculated into LB broth containing antibiotic resistance. The culture system consisted of 10 mL aliquots of the medium in 100 mL Erlenmeyer flasks, supplemented with 50 μg / mL kanamycin and 100 μg / mL ampicillin. The culture conditions were constant temperature shaking incubator at 37°C and 220 rpm overnight to obtain seed culture. This seed culture was then inoculated into fresh fermentation medium at a volume of 2%. The culture system consisted of 50 mL aliquots of the medium in 250 mL Erlenmeyer flasks, supplemented with 50 μg / mL kanamycin and 100 μg / mL ampicillin. The culture conditions were constant temperature shaking incubator at 37°C and 220 rpm. 600 When the concentration reaches 0.6-0.8, add 0.1 mM IPTG to induce fermentation. Fermentate at 30℃ and 220 rpm for 72 h to prepare samples.

[0030] Example 4: Extraction and liquid chromatography of tyrosol Transfer 1 mL of fermentation broth to a 1.5 mL centrifuge tube and centrifuge at 12000 rpm for 5 min. Mix 200 μL of the supernatant with 800 μL of mobile phase A. Dilute the sample 5-fold and analyze the tyrosol yield using a Shimadzu high-performance liquid chromatograph. Quantification was performed using a reversed-phase C18 column. Mobile phase A was water containing 1% trifluoroacetic acid (TFA); mobile phase B was acetonitrile containing 1% TFA. The flow rate was 1 mL / min. -1 The absorbance of the sample was measured at 274 nm. The HPLC program was as follows: 10% B: 0-5 min, 60% B: 5-10 min, 90% B: 10-12 min, 2% B: 12-16 min, 0% B: 16-20 min. The results after 72 hours of shake-flask fermentation are shown in Table 2. The tyrosol yield of the control strain overexpressing wild-type phenylpyruvate decarboxylase ARO10 was 2.02 g / L. L220A The strain produced tyrosol at a yield of 3.04 g / L and overexpressed phenylpyruvate decarboxylase ARO10. I93V / L220A / N285QThe strain produced tyrosol at 3.60 g / L, representing a 78.42% increase compared to the wild-type control strain, and was comparable to the mutant phenylpyruvate decarboxylase ARO10. L220A The strain produced 18.42% more tyrosol than the strain.

[0031] Table 2. Statistics on tyrosol yield from shake-flask fermentation of different phenylpyruvate decarboxylase ARO10 mutant compositions.

[0032] The formula for the increase in tyrosol production is as follows: Increase (%) = (Tyrosol production of mutant strain (g / L) - Tyrosol production of control strain (g / L)) / Tyrosol production of control strain (g / L) × 100%.

[0033] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The present invention performs site-directed mutagenesis on phenylpyruvate decarboxylase ARO10 derived from Saccharomyces cerevisiae in different combinations. Compared with wild-type phenylpyruvate decarboxylase, the combined mutant phenylpyruvate decarboxylase ARO10 exhibits superior performance. I93V / L220A / N285Q Tyrosol production levels increased by 78.42% in shake-flask experiments. This was achieved with the dominant phenylpyruvate decarboxylase mutant ARO10. L220A In comparison, the combined mutant phenylpyruvate decarboxylase mutant ARO10 I93V / L220A / N285Q Tyrosol production in shake flasks increased by 18.42%, and other mutant strains also showed increased tyrosol production compared to the control strain. See the table for details.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A phenylpyruvate decarboxylase mutant, characterized in that, The phenylpyruvate decarboxylase mutant is any one of the following: (1) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and S at position 106 with A; (2) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and L at position 220 with A; (3) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and D at position 242 with E; (4) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and F at position 273 with L; (5) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V and N at position 285 with Q; (6) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and S at position 153 with T; (7) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and L at position 220 with A; (8) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and D at position 242 with E; (9) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and F at position 273 with L; (10) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A and N at position 285 with Q; (11) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T and L at position 220 with A; (12) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T and D at position 242 with E; (13) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T and F at position 273 with L; (14) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A and N at position 285 with Q; (15) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace D at position 242 with E and F at position 273 with L; (16) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace D at position 242 with E and N at position 285 with Q; (17) Using the amino acid shown in SEQ ID NO.1 as the starting sequence, replace F at position 273 with L and N at position 285 with Q; (18) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 106 with A, and S at position 153 with T; (19) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 106 with A, and L at position 220 with A; (20) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 106 with A, and F at position 273 with L; (21) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 106 with A, and N at position 285 with Q; (22) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 153 with T, and L at position 220 with A; (23) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 153 with T, and D at position 242 with E; (24) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 153 with T, and F at position 273 with L; (25) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, S at position 153 with T, and N at position 285 with Q; (26) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, L at position 220 with A, and D at position 242 with E; (27) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, L at position 220 with A, and F at position 273 with L; (28) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, L at position 220 with A, and N at position 285 with Q; (29) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, D at position 242 with E, and F at position 273 with L; (30) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, D at position 242 with E, and N at position 285 with Q; (31) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace I at position 93 with V, F at position 273 with L, and N at position 285 with Q; (32) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace S at position 153 with T, and replace L at position 220 with A; (33) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace S at position 153 with T, and replace D at position 242 with E; (34) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace S at position 153 with T, and replace F at position 273 with L; (35) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace S at position 153 with T, and replace N at position 285 with Q; (36) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace L at position 220 with A, and replace D at position 242 with E; (37) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace L at position 220 with A, and replace F at position 273 with L; (38) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, replace L at position 220 with A, and replace N at position 285 with Q; (39) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A, F at position 273 with L, and N at position 285 with Q; (40) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, L at position 220 with A, and F at position 273 with L; (41) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, L at position 220 with A, and N at position 285 with Q; (42) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, D at position 242 with E, and F at position 273 with L; (43) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, D at position 242 with E, and N at position 285 with Q; (44) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T, F at position 273 with L, and N at position 285 with Q; (45) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A, D at position 242 with E, and F at position 273 with L; (46) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A, D at position 242 with E, and N at position 285 with Q; (47) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A, replace F at position 273 with L, and replace N at position 285 with Q; (48) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace D at position 242 with E, F at position 273 with L, and N at position 285 with Q.

2. The gene encoding the phenylpyruvate decarboxylase mutant of claim 1.

3. A recombinant vector containing the gene described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The launch carrier is the pRSFDuet series.

5. A recombinant microbial cell containing the gene of claim 2.

6. A recombinant Escherichia coli, characterized in that, The phenylpyruvate decarboxylase mutant of claim 1 is expressed.

7. The recombinant Escherichia coli according to claim 6, characterized in that, alcohol dehydrogenase gene ADH6 Gene containing a mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase aroG fbr Cyclohexadiene dehydrogenase gene tyrC L-amino acid ammonia-lyase gene LAAD The overexpression plasmid was transferred into Escherichia coli BL21(DE3) competent cells.

8. The use of the phenylpyruvate decarboxylase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, or the recombinant microbial cell of any one of claims 5-7 in the production of tyrosol and products containing tyrosol.

9. A method for producing tyrosol, characterized in that, The method involves fermenting the recombinant Escherichia coli as described in claim 6 or 7 at 30°C for 72 hours.

10. The method according to claim 9, characterized in that, The fermentation medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract. The pH of the fermentation medium was maintained using 10 g / L CaCO3. The culture medium was supplemented with 0.1% by volume of trace elements, the composition of which was: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O and 15 g / L ZnSO4·7H2O. The pH was adjusted to 7.0 using NaOH.

Images