Pinene synthase mutant and application thereof in pinene production

Abstract

The invention discloses a pinene synthase mutant and application thereof in pinene production, and belongs to the technical field of bioengineering. Isoleucine at the 409th site of pinene synthase is mutated into valine, phenylalanine at the 443rd site of pinene synthase is mutated into alanine, overexpression is carried out in serratia marcescens (HBQA7), and an engineering strain ST17 is constructed. Under the condition of shake flask fermentation, the pinene yield of the engineering strain ST17 reaches 0.80 g / L; the yield is obviously increased to 43.2 g / L through amplification culture in a 30 L fermentation tank. The invention provides an efficient, economic and environment-friendly novel method for industrial biosynthesis of pinene.

Description

Technical Field

[0001] This invention relates to a pinene synthase mutant and its application in the production of pinene, belonging to the field of bioengineering technology. Background Technology

[0002] Pinene (C 10 H 16 Pinene, also known as pinene, is a naturally occurring cyclic monoterpene. It possesses unique spatial conformation and physicochemical properties, and as research into its properties deepens, it is playing a significant role in energy, medicine, chemical engineering, agriculture, and materials science. In the pharmaceutical field, pinene exhibits antibacterial, antiviral, and anti-inflammatory properties. Pinene has anti-tumor, anti-inflammatory, anti-allergic, and ulcer-improving effects, as seen in products like eucalyptus-pinene enteric-coated soft capsules and pediatric cold granules. In the energy sector, pinene's unique double bond and bicyclic structure allows it to form pinene dimers under specific conditions through cyclization and hydrogenation. These dimers possess high volumetric energy similar to high-energy fuel JP-10 and are considered alternatives to jet and rocket fuels. In food materials, the most researched application is the synthesis of perilla sugar, a high-sweetness, low-calorie sweetener. Pinene is also an important raw material for flavoring synthesis.

[0003] Pinene is widely found in coniferous plants in nature, which secrete large amounts of turpentine, and pinene is the main component of turpentine, accounting for up to 90%. Currently, pinene is mainly obtained through chemical synthesis and extraction from coniferous plants, followed by purification through vacuum distillation and fractional distillation. However, this method suffers from problems such as strong dependence on raw materials, long plant growth cycles, significant influence from climate and soil conditions, and low distillation extraction rates. Large-scale production can also have adverse effects on the ecological environment. While chemical synthesis routes can circumvent raw material limitations, they generally suffer from drawbacks such as cumbersome procedures, insufficient specificity, and high costs.

[0004] With the development of metabolic engineering and synthetic biology, the production of pinene through biosynthesis is expected to become a strategy to replace chemical synthesis in the future. By using genetic engineering and metabolic engineering techniques, it is possible to increase pinene yield, shorten the production cycle, reduce production costs, minimize environmental impact, and enhance the controllability of the production process, thus providing a new, efficient, economical, and environmentally friendly approach for the industrial production of pinene.

[0005] Microorganisms generally possess the natural ability to synthesize terpenes. Most bacteria synthesize terpenoid precursors via the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, while fungi and other organisms synthesize terpenoid precursors such as isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) via the mevalonic acid (MVA) pathway. These precursors can further generate intermediates required for pinene synthesis, which are then converted into pinene under the catalysis of pinene synthase.

[0006] Current research on the microbial synthesis of pinene mainly focuses on *Escherichia coli* and yeast. Existing studies have achieved pinene biosynthesis by introducing pinene synthase genes into these hosts and enhancing endogenous terpene synthesis pathways. For example, Zhang Haibo introduced *Firmiana simplex* (a type of pine) into *E. coli*. Abies grandis Pinene synthase from a specific source was synthesized, and pinene synthesis was achieved through the introduction of exogenous MVA pathway, with a pinene yield of 5.44 mg / L during shake-flask fermentation; StepHen Sarria coli co-expressed pinene synthase genes (PS) from different sources with geraniyl pyrophosphate synthase genes (GPPS) in pairs and through fusion expression, and after optimizing the culture conditions, the pinene yield reached 32 mg / L; Chen Tianhua et al. expressed pinene synthase derived from pine in Saccharomyces cerevisiae (Pinus tarragon). Pinus taeda The pinene synthase gene was modified, and through a series of optimizations to the metabolic pathway, pinene production was increased from an initial 0.329 mg / L to a final 11.7 mg / L.

[0007] However, bottlenecks remain in the production of pinene using model strains such as *Escherichia coli* and *Saccharomyces cerevisiae*. Pinene exhibits certain biotoxicity, inhibiting the growth and metabolism of host bacteria, and these model hosts have limited tolerance to pinene. Furthermore, the low catalytic efficiency of pinene synthase, a key rate-limiting enzyme in pinene biosynthesis, is also a major factor restricting yield.

[0008] Therefore, it is urgent to explore non-model microorganisms as chassis strains, screen for hosts with fast growth rates, long steady-state periods, and high tolerance to pinene, and improve their terpene synthesis capabilities through genetic engineering, thereby achieving efficient and economical pinene production. Summary of the Invention

[0009] To overcome the problems of poor tolerance, low yield, and insufficient catalytic efficiency in the production of pinene using model microorganisms in existing technologies, this invention constructs an engineered strain ST17 by mutating isoleucine at position 409 to valine and phenylalanine at position 443 to alanine, and then overexpressing these mutants in *Serratia marcescens* (HBQA7). Under shake-flask fermentation conditions, the engineered strain ST17 achieved a pinene yield of 0.8 g / L; after scale-up cultivation in a 30 L fermenter, the yield was significantly increased to 43.2 g / L, achieving efficient pinene synthesis and a significant increase in yield.

[0010] A first object of the present application is to provide a pinene synthetase mutant, the amino acid sequence of which is based on SEQ ID NO: 1, and in which one or more amino acid mutations are made to the isoleucine at position 296, the asparagine at position 300, the isoleucine at position 308, the alanine at position 405, the isoleucine at position 409, the phenylalanine at position 443, and the glycine at position 533.

[0011] In one embodiment of the present application, the amino acid sequence of the pinene synthetase mutant comprises any one of the following amino acid sequences: (1) the amino acid sequence obtained by mutating the isoleucine at position 296 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to asparagine; (2) the amino acid sequence obtained by mutating the isoleucine at position 296 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to methionine; (3) the amino acid sequence obtained by mutating the asparagine at position 300 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to serine; (4) the amino acid sequence obtained by mutating the isoleucine at position 308 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to alanine; (5) the amino acid sequence obtained by mutating the alanine at position 405 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to serine; (6) the amino acid sequence obtained by mutating the alanine at position 405 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to threonine; (7) the amino acid sequence obtained by mutating the alanine at position 405 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to valine; (8) the amino acid sequence obtained by mutating the isoleucine at position 409 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to valine; (9) the amino acid sequence obtained by mutating the isoleucine at position 409 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to lysine; (10) the amino acid sequence obtained by mutating the phenylalanine at position 443 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to alanine; (11) the amino acid sequence obtained by mutating the glycine at position 533 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to alanine; (12) the amino acid sequence obtained by mutating the glycine at position 533 of the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1 into serine; (13) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 308 into alanine and the isoleucine at position 409 into valine of the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1; (14) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 308 into alanine and the phenylalanine at position 443 into alanine of the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1; (15) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 308 into alanine and the glycine at position 533 into alanine of the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1; (16) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 409 into valine and the phenylalanine at position 443 into alanine of the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1; (17) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 409 into valine and the glycine at position 533 into alanine of the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1; (18) the amino acid sequence obtained by simultaneously mutating the phenylalanine at position 443 into alanine and the glycine at position 533 into alanine of the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1; (19) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 308 into alanine, the isoleucine at position 409 into valine, and the phenylalanine at position 443 into alanine of the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1; (20) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 409 into valine, the phenylalanine at position 443 into alanine, and the glycine at position 533 into alanine of the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1.

[0012] In one embodiment of the present application, the pinene synthase mutant has one or more amino acid mutations at positions 308, 409, 443, and 533, based on the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1.

[0013] In one embodiment of the present application, the pinene synthase mutant has one or more amino acid mutations at positions 308, 409, 443, and 533, based on the pinene synthase whose amino acid sequence is shown in SEQ ID NO: 1. on the basis of the pinene synthetase with the amino acid sequence shown in SEQ ID NO: 1, the isoleucine at position 409 is mutated into valine (the amino acid sequence is shown in SEQ ID NO: 4); or, on the basis of the pinene synthetase with the amino acid sequence shown in SEQ ID NO: 1, the phenylalanine at position 443 is mutated into alanine (the amino acid sequence is shown in SEQ ID NO: 5); or, on the basis of the pinene synthetase with the amino acid sequence shown in SEQ ID NO: 1, the glycine at position 533 is mutated into alanine (the amino acid sequence is shown in SEQ ID NO: 6); or, on the basis of the pinene synthetase with the amino acid sequence shown in SEQ ID NO: 1, the isoleucine at position 409 is mutated into valine, and the phenylalanine at position 443 is mutated into alanine (the amino acid sequence is shown in SEQ ID NO: 7).

[0014] A second object of the present application is to provide a nucleotide of the above-mentioned pinene synthetase mutant.

[0015] A third object of the present application is to provide a plasmid vector of the above-mentioned nucleotide, which is pBBR1MCS-2-pG09.

[0016] A fourth object of the present application is to provide a cell of the above-mentioned pinene synthetase mutant.

[0017] A fifth object of the present application is to provide a recombinant bacterium expressing the above-mentioned pinene synthetase mutant, wherein the recombinant bacterium takes Serratia marcescens (S. marcescens) HBQA7 as a host. Serratia marcescens

[0018] A sixth object of the present application is to provide an application of the above-mentioned recombinant bacterium in the production of pinene.

[0019] A seventh object of the present application is to provide a method for producing pinene, which uses the above-mentioned recombinant bacterium for fermentation to produce pinene, comprising the steps of: (1) inoculating the recombinant bacterium single colony into a growth medium, and culturing at 30-37℃ and 180-600 rpm for 10-12 hours to obtain a seed liquid; (2) inoculating the seed liquid into a fermentation medium at an inoculation amount of 1-2%, and culturing at 30-37℃ and 180-600 rpm for 48-72 hours to obtain a fermentation liquid; (3) centrifuging the upper organic phase of the fermentation liquid at 8000-10000 rpm for 5-10 minutes to obtain a supernatant containing pinene.

[0020] ​In an embodiment of the present application, the growth medium comprises LB medium, M9 medium or TB medium.

[0021] In an embodiment of the present application, the composition of the fermentation medium comprises: 10~14 g / L peptone, 22~26 g / L yeast extract, 1~2 g / L antifoam, 2~2.4 g / L K2HPO4, 9.2~9.4 g / L KH2PO4, 30~35 g / L glycerol, 1.5~1.6 g / L MgSO4, 0.1~0.2% trace metal element stock solution (27~28 g / L FeCl3·6H2O, 2~3 g / L ZnCl2·4H2O, 2~3 g / L CoCl2·6H2O, 2~3 g / L Na2MoO4·2H2O, 1~2 g / L CaCl2·2H2O, 1~1.5 g / L CuCl2·6H2O, 0.5~1 g / L H3BO3), 1~2% vitamin stock solution (30~32 g / L vitamin B5, 50~52 g / L citric acid, 30~32 g / L vitamin B1), kanamycin 50~60 mg / L, spectinomycin 50~60 mg / L, deionized water as solvent, and pH 7.0.

[0022] In an embodiment of the present application, an organic solvent is added during the fermentation process to achieve in-situ extraction.

[0023] In an embodiment of the present application, after the fermentation is completed, the upper organic phase is collected by centrifugation to obtain the pinene product.

[0024] In an embodiment of the present application, the carbon source in the medium is 10~20 g / L glucose or glycerol.

[0025] In an embodiment of the present application, the medium is LB, M9 or TB medium; preferably, the medium is TB medium.

[0026] In an embodiment of the present application, the amount of organic solvent added is 20~30% (v / v) of the volume of the fermentation broth; Optionally, the organic solvent comprises isopropyl myristate and pentane; preferably, the organic solvent is n-dodecane.

[0027] In an embodiment of the present application, the fermentation pH is controlled at 6~8.

[0028] In an embodiment of the present application, the composition of the medium comprises: 12 g / L peptone, 24 g / L yeast extract, 1 g / L defoamer, 2.2 g / L K2HPO4, 9.4 g / L KH2PO4, 30 g / L glycerol, 1.5 g / L MgSO4, 0.1% trace metal element stock solution, 1% vitamin stock solution, kanamycin 50 mg / L, spectinomycin 50 mg / L, solvent: deionized water, pH 7.0.

[0029] In one embodiment of the present invention, the mother liquor containing trace metal elements comprises: 27 g / L FeCl3·6H2O, 2 g / L ZnCl2·4H2O, 2 g / L CoCl2·6H2O, 2 g / L Na2MoO4·2H2O, 1 g / L CaCl2·2H2O, 1.3 g / L CuCl2·6H6O, and 0.5 g / L H3BO3.

[0030] In one embodiment of the present invention, the vitamin stock solution comprises: 30 g / L vitamin B5, 50 g / L citric acid, and 30 g / L vitamin B1.

[0031] In one embodiment of the present invention, a feed medium is provided during the fermentation process, and the feed medium includes K2HPO4, KH2PO4, glycerol, MgSO4, trace metal elements, citric acid and vitamins (vitamin B1 and vitamin B5).

[0032] Beneficial effects This invention constructs an engineered strain ST17 by mutating isoleucine at position 409 of pinene synthase to valine and phenylalanine at position 443 to alanine, followed by overexpression in *Serratia marcescens*. This engineered strain, after fermentation in 50 mL of culture medium for 48 h, achieves a pinene yield of over 0.8 g / L; after fermentation in a 30 L fermenter (20 L of culture medium) for 180 h, a pinene yield of 43.2 g / L can be achieved. Compared with existing technologies, the method of this invention has advantages such as high yield, controllable fermentation cycle, high in-situ extraction efficiency, and good process stability, making it suitable for industrial-scale production.

[0033] Preservation of biological materials Serratia marcescens ( Serratia marcescens HBQA7, taxonomically named Serratia marcescens HBQA7 was deposited on February 23, 2023, at the China Center for Type Culture Collection (CCTCCNO: M 2023184), located at Wuhan University, Wuhan, China. Attached Figure Description

[0034] Figure 1Pinene yield of PlPS mutants. DETAILED DESCRIPTION

[0035] The present application is further described in connection with the following specific examples, which are not intended to limit the application, and by reference to the accompanying drawings in which:

[0036] The culture media used in the following examples are as follows: Fermentation medium: 10 g·L -1 Peptone, 5 g·L -1 Yeast extract, 10 g·L -1 Sodium chloride, 0.5 g·L -1 Magnesium sulfate, 20% (v / v) n-dodecane; LB medium: 10 g·L -1 Peptone, 5 g·L -1 Yeast extract, 10 g·L -1 NaCl, 0.5 g·L -1 Magnesium sulfate and 20% (v / v) n-dodecane; M9 medium: 6.78 g·L -1 Na2HPO4, 3 g·L -1 KH2PO4, 0.5 g·L -1 NaCl, 1 g·L -1 NH4Cl, 0.0111 g·L -1 CaCl2, 0.1 g·L -1 Thiamine, 0.5 g·L -1 Magnesium sulfate and 20% (v / v) n-dodecane; TB medium: 12 g·L -1 Tryptone, 24 g·L -1 Yeast extract, 9.4 g·L -1 K2HPO4, 2.2 g·L -1 KH2PO4, 0.5 g·L -1 Magnesium sulfate and 20% (v / v) n-dodecane; 30 L fermenter fermentation medium: 12 g·L -1 Peptone, 24 g·L -1 Yeast extract, 1 g·L -1 Antifoam, 2.2 g·L -1 K2HPO4, 9.4 g·L -1 KH2PO4, 30 g·L -1 Glycerol, 1.5 g·L -1MgSO4, 20% (v / v) n-dodecane, 0.1% trace metals stock (27 g·L -1 FeCl3·6H2O, 2 g·L -1 ZnCl2·4H2O, 2 g·L -1 CoCl2·6H2O, 2 g·L -1 Na2MoO4·2H2O, 1 g·L -1 CaCl2·2H2O, 1.3 g·L -1 CuCl2·6H2O and 0.5 g·L - 1 HBO3), 1% vitamin stock (30 g·L -1 Vitamin B5, 50 g·L -1 Citric acid, 30 g·L -1 Vitamin B1). -1 , spectinomycin 50 mg·L -1 , with deionized water as the solvent and pH 7.0; Feed medium: 2.2 g·L -1 K2HPO4, 9.4 g·L -1 KH2PO4, 1000 g / L -1 Glycerol, 1.5 g·L - 1 MgSO4, 0.1% trace metals stock (27 g·L -1 FeCl3·6H2O, 2 g·L -1 ZnCl2·4H2O, 2 g·L - 1 CoCl2·6H2O, 2 g·L -1 Na2MoO4·2H2O, 1 g·L -1 CaCl2·2H2O, 1.3 g·L -1 CuCl2·6H2O and 0.5 g·L -1 HBO3), 1% vitamin stock (30 g·L -1 Vitamin B5, 50 g·L -1 Citric acid, 30 g·L -1 Vitamin B1).

[0037] The strains and vectors involved in the following examples are as follows: The plasmid pBBR1MCS-2 used in this example was purchased from Shanghai Zeye Biotechnology Co., Ltd.; The vector backbone pBBR1MCS-2-pG09 used in this example is obtained by connecting pG09 (P5- atoB Ec - mvaS Lc - mvaE Lc , P1- mvk Mc - pmd Ha - ipk Mt - idi Hp , P2- AgGPPS - AaFPPase -2 F38L / Y98E ; pBBRl oriV; Km R ), which is disclosed in the article "Engineering an Extremely Monoterpene-Tolerant Serratia marcescens for High-Yield Geraniol Production via a Rationally Modified Insect Phosphatase" and can be constructed according to the disclosure in the article.

[0038] The chassis strain used in this example is the Serratia marcescens engineering strain HBQA7, which is disclosed in the patent CN118853436A "A Serratia marcescens Resistant to Organic Solvents", and has high MVA pathway metabolic capacity, which can significantly improve the synthesis efficiency of terpenoids, and is suitable as an engineering chassis strain for pinene production.

[0039] Example 1: Construction of plasmid with pinene synthase from peony The pinene synthase gene used in this example is derived from peony (Paeonia suffruticosa) Paeonia lactiflora ), and its amino acid sequence is shown in SEQ ID NO. 1. The gene sequence is optimized for codon bias of Serratia marcescens, and the optimized nucleotide sequence is shown in SEQ ID NO: 2 (encoding pinene synthase), and is synthesized by Beijing Genomics Institute (BGI).

[0040] The PlPS gene fragment with homologous arms is obtained by PCR amplification using the primer PlPS-F and PlPS-R with SEQ ID NO: 2 as the template. The vector backbone pBBR1MCS-2-pG09 is also obtained by PCR amplification, and the primers used are pBBR1MCS-2-pG09-F and pBBR1MCS-2-pG09-R.

[0041] The amplified vector skeleton and the PlPS gene fragment carrying the homologous arm were connected by homologous recombination, and the connection product was transformed into Escherichia coli JM109 competent cells, and coated on LB solid medium plates containing kanamycin selection pressure and cultured at 37°C overnight. Single colonies were picked and identified by colony PCR using primers yzhP-vF and PlPS-vR. The PCR system used 2x Rapid Taq Master Mix (low fidelity, Nanjing Novogene Bioinformatics Technology Co., Ltd.). The positive clones that showed the target band were subjected to small-scale plasmid extraction (kit purchased from Nanjing Novogene Bioinformatics Technology Co., Ltd.), and were sent to Suzhou Kingfisher Biological Technology Co., Ltd. for first-generation sequencing verification. The plasmid with correct sequencing results was named pBBR1MCS-2-pG09-PlPS.

[0042] The pBBR1MCS-2-pG09-PlPS plasmid was introduced into the chassis strain HBQA7 by electroporation to obtain the ST1 recombinant strain.

[0043] Table 1 Plasmid construction primers

[0044] Example 2 Pinene yield detection of each recombinant strain The recombinant genetically engineered strain ST1 constructed in Example 1 was used for pinene yield detection.

[0045] The engineered strain ST1 was streaked on LB solid medium and cultured at 37°C overnight. Then a single colony was inoculated into a culture tube containing 3 mL of LB liquid medium and cultured at 37°C, 200 rpm for 12 hours. The above seed liquid was inoculated into a fermentation medium (250 mL flask) containing 20 g / L glycerol at a volume of 1% of the seed liquid, and cultured at 30°C, 200 rpm for 48 hours.

[0046] The upper organic phase of the fermentation liquid was centrifuged at 12000 rpm for 10 minutes to obtain the supernatant, and the pinene content was determined by gas chromatography (GC). The detection results showed that the pinene yield of the recombinant strain ST1 containing peony-derived pinene synthase was 0.61 g / L.

[0047] Example 3 Comparison of the extraction effects of different extractants on pinene The yield of pinene was detected based on the recombinant genetically engineered strain ST1 obtained in Example 1 and according to the method of Example 2. In order to investigate the influence of different extractants on the extraction effect of the product, dodecane, isopropyl myristate, pentane, dioctyl phthalate, diisooctyl sebacate and silicone oil were selected as extractants for parallel experiments in the fermentation medium. The results showed that the above different extractants had no significant difference in the extraction effect of pinene, and effective product extraction could be achieved.

[0048] Example 4 Construction and yield detection of pinene synthase mutants In this example, the overlap extension PCR method was used to construct the pinene synthase mutants. The primers containing the target mutation sites were designed, and the vector plasmid pBBR1MCS-2-pG09-PlPS was used as the template for PCR amplification. After the PCR product was digested with Dpn I enzyme to remove the methylated template plasmid, it was transformed into E. coli JM109 competent cells. The bacterial solution was spread on LB solid medium containing kanamycin and incubated at 37°C overnight. Single colonies were picked for plasmid extraction and sent to Suzhou Jinweizhi Biological Technology Co., Ltd. for first-generation sequencing verification. After confirming the successful mutation, the mutant plasmid was introduced into the chassis strain HBQA7 using electroporation to obtain the corresponding recombinant strains. The specific single-mutation recombinant strains are as follows: The isoleucine at position 296 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to asparagine to obtain mutant plasmid pBBR1MCS-2-pG09-PlPS-I296N, which was transformed into HBQA7 to obtain recombinant strain HBQA7-I296A, which was named ST2 (I296N); The isoleucine at position 296 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to methionine to obtain mutant plasmid pBBR1MCS-2-pG09-PlPS-I296M, which was transformed into HBQA7 to obtain recombinant strain HBQA7-I296M, which was named ST3 (I296M); The asparagine at position 300 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to serine to obtain mutant plasmid pBBR1MCS-2-pG09-PlPS-N300S, which was transformed into HBQA7 to obtain recombinant strain HBQA7-N300S, which was named ST4 (N300S); The isoleucine at position 308 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to alanine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-I308A, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-I308A, which was named ST5(I308A); The alanine at position 405 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to serine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-A405S, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-A405S, which was named ST6(A405S); The alanine at position 405 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to threonine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-A405T, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-A405T, which was named ST7(A405T); The alanine at position 405 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to valine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-A405V, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-A405V, which was named ST8(A405V); The isoleucine at position 409 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to valine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-I409V, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-I409V, which was named ST9(I409V); The isoleucine at position 409 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to lysine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-I409K, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-I409K, which was named ST10(I409K); The phenylalanine at position 443 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to alanine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-F443A, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-F443A, which was named ST11(F443A); The glycine at position 533 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to alanine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-G533A, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-G533A, which was named ST12 (G533A); The glycine at position 533 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to serine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-G533S, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-G533S, which was named ST13 (G533S). The primer design of the above mutants is shown in Table 2.

[0049] The pinene production of the above single mutant recombinant strains was determined using the same fermentation conditions and detection methods as in Example 2. The results are shown in Table 3, which show that the recombinant strains carrying the I308A, I409V, F443A and G533A mutations all showed different degrees of improvement in pinene production.

[0050] Further, a combination type recombinant strain of I308A, I409V, F443A and G533A four kinds of mutations was constructed: The isoleucine at position 308 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to alanine, and the isoleucine at position 409 was mutated to valine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-I308A / I409V, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-I308A / I409V, which was named ST14 (I308A / I409V); The isoleucine at position 308 of the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS was mutated to alanine, and the phenylalanine at position 443 was mutated to alanine to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-I308A / F443A, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-I308A / F443A, which was named ST15 (I308A / F443A); Meanwhile, the isoleucine at position 308 and the glycine at position 533 in the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS were mutated into alanine to obtain a mutant plasmid pBBR1MCS-2-pG09-PlPS-I308A / G533A; and the mutant plasmid was transformed into HBQA7 to obtain a recombinant bacterium HBQA7-I308A / G533A, which was named ST16 (I308A / G533A); Meanwhile, the isoleucine at position 409 and the phenylalanine at position 443 in the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS were mutated into valine and alanine, respectively, to obtain a mutant plasmid pBBR1MCS-2-pG09-PlPS-I409V / F443A; and the mutant plasmid was transformed into HBQA7 to obtain a recombinant bacterium HBQA7-I409V / F443A, which was named ST17 (I409V / F443A); Meanwhile, the isoleucine at position 409 and the glycine at position 533 in the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS were mutated into valine and alanine, respectively, to obtain a mutant plasmid pBBR1MCS-2-pG09-PlPS-I409V / G533A; and the mutant plasmid was transformed into HBQA7 to obtain a recombinant bacterium HBQA7-I409V / G533A, which was named ST18 (I409V / G533A); Meanwhile, the phenylalanine at position 443 and the glycine at position 533 in the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS were mutated into alanine to obtain a mutant plasmid pBBR1MCS-2-pG09-PlPS-F443A / G533A; and the mutant plasmid was transformed into HBQA7 to obtain a recombinant bacterium HBQA7-F443A / G533A, which was named ST19 (F443A / G533A); Meanwhile, the isoleucine at position 308, the isoleucine at position 409 and the phenylalanine at position 443 in the pinene synthase amino acid sequence in pBBR1MCS-2-pG09-PlPS were mutated into alanine, valine and alanine, respectively, to obtain a mutant plasmid pBBR1MCS-2-pG09-PlPS-I308A / I409V / F443A; and the mutant plasmid was transformed into HBQA7 to obtain a recombinant bacterium HBQA7-I308A / I409V / F443A, which was named ST20 (I308A / I409V / F443A); Meanwhile, the isoleucine at position 409, the phenylalanine at position 443 and the glycine at position 533 in the amino acid sequence of the pinene synthase in pBBR1MCS-2-pG09-PlPS were mutated into valine, alanine and alanine respectively to obtain the mutant plasmid pBBR1MCS-2-pG09-PlPS-I409V / F443A / G533A, which was transformed into HBQA7 to obtain the recombinant bacteria HBQA7-I409V / F443A / G533A, which was named ST21 (I409V / F443A / G533A). The pinene yield was determined by the method of Example 2, and the results are shown in Table 3. Figure 1 The results show that the pinene yield of the recombinant strain ST17 (I409V / F443A) is the highest, reaching 0.80 g / L.

[0051] Table 2 Primers for plasmid construction

[0052] Table 3 Yield and OD 600

[0053] Example 5 Scale-up culture and yield determination of pinene engineering strain ST17 The engineering strain ST17 obtained in Example 4 was taken and scale-up culture was carried out in a 30 L fermenter to prepare pinene, and the steps were as follows: The strain ST17 was streaked on LB solid medium and cultured at 37°C overnight. A single colony was inoculated into 3 mL TB liquid medium and cultured at 30°C, 200 rpm for 12 hours to obtain a primary seed solution.

[0054] The primary seed solution was inoculated into 100 mL TB medium (500 mL flask) at an inoculation amount of 1% (v / v) and cultured under the same conditions for 12 hours to obtain a secondary seed solution.

[0055] The secondary seed solution was inoculated into a 5 L fermenter containing 1 L TB medium at an inoculation amount of 5% (v / v) to obtain a tertiary seed solution.

[0056] Production culture was carried out in a 30 L fermenter with an initial medium volume of 20 L containing glycerol 60 g / L, and the pH was adjusted to 7.0. The fermentation conditions were 30°C, initial stirring speed 200 rpm, and aeration amount 1 vvm. The dissolved oxygen (DO) in the tank was calibrated at full scale (100%). The tertiary seed solution was inoculated into the 30 L fermenter at an inoculation amount of 5% (v / v) (OD 600 about 28.6). In order to keep the dissolved oxygen not less than 30%, the dissolved oxygen and stirring speed were controlled in linkage during the fermentation.

[0057] After about 8 hours of fermentation, a dissolved oxygen rebound appeared, indicating that the initial carbon source was exhausted, and then a dissolved oxygen linkage flow feeding strategy was used: when the dissolved oxygen was greater than 80%, the feeding was started, and when the dissolved oxygen was reduced to below 60%, the feeding was stopped.

[0058] During the fermentation process, 5 M ammonia water and 1 M hydrochloric acid were used to adjust the pH, and the pH was maintained at about 7.0. The total fermentation time was 180 hours.

[0059] After the fermentation was completed, the organic phase of the dodecane layer was taken, and the pinene content was detected by gas chromatography (GC). The results showed that the OD of the bacteria at the end of the fermentation was 95.2, and the final yield of pinene was 43.2 g / L. 600

[0060] The sequence involved in the application is as follows: The pinene synthase amino acid sequence SEQ ID NO: 1 is: MVATSTTEVIERRSGNYQPCIWDIEFLQTVDSDYSKEIYKKRSENLKRQVKTMLVSEVEPLVQLELIDVLQRLCLGYEFETEIKSILQSIYNNNSSSSGKYKSSQDLHAAALEFRLLRQHHYIVPQEIFESFRDENGFKKSIYDDVKGMLSLYEASFFAFEDESIMEEAWEFTSEKLKSLKTEDMDPTLAMLVPHALEIPLSTRLPRFDARWFIEVYEKSRDMNPILLELGKLDFNIVQSMYQEDLRVMSSWWKEIALREKLDFARDRLVPSYLWGVGVSSEPQYAYSRRIIGMDIAIINMIDDIYDIYGTLEEVELFTDAVKKWDINAMKQLPDYMKIAYLAFYNTVNEMIYVILKEHGVDVTDHLTKAWLGLLNGYLTEARWYHTKHKPSLAEYMKNACMSIAGPLIATLAYLTTHKSITEEEMKYLETIPDVMSTTSYVFRISDDYGTSSDELKRGDVPKAIQCYMHDSGVSEEEARLHMRKLVRNGWKKVNKYRFMESQESPLTPTLVEMMQNLTRVSQNLYEHGDGHGIEDGETKDRVLRLLFQPIPM The pinene synthase nucleotide sequence SEQ ID NO: 2 is: ​ Mutant I308A amino acid sequence SEQ ID NO: 3 MVATSTTEVIERRSGNYQPCIWDIEFLQTVDSDYSKEIYKKRSENLKRQVKTMLVSEVEPLVQLELIDVLQRLCLGYEFETEIKSILQSIYNNNSSSSGKYKSSQDLHAAALEFRLLRQHHYIVPQEIFESFRDENGFKKSIYDDVKGMLSLYEASFFAFEDESIMEEAWEFTSEKLKSLKTEDMDPTLAMLVPHALEIPLSTRLPRFDARWFIEVYEKSRDMNPILLELGKLDFNIVQSMYQEDLRVMSSWWKEIALREKLDFARDRLVPSYLWGVGVSSEPQYAYSRRIIGMDIAIINMIDDIYDAYGTLEEVELFTDAVKKWDINAMKQLPDYMKIAYLAFYNTVNEMIYVILKEHGVDVTDHLTKAWLGLLNGYLTEARWYHTKHKPSLAEYMKNACMSIAGPLIATLAYLTTHKSITEEEMKYLETIPDVMSTTSYVFRISDDYGTSSDELKRGDVPKAIQCYMHDSGVSEEEARLHMRKLVRNGWKKVNKYRFMESQESPLTPTLVEMMQNLTRVSQNLYEHGDGHGIEDGETKDRVLRLLFQPIPM Mutant I409V amino acid sequence SEQ ID NO: 4 MVATSTTEVIERRSGNYQPCIWDIEFLQTVDSDYSKEIYKKRSENLKRQVKTMLVSEVEPLVQLELIDVLQRLCLGYEFETEIKSILQSIYNNNSSSSGKYKSSQDLHAAALEFRLLRQHHYIVPQEIFESFRDENGFKKSIYDDVKGMLSLYEASFFAFEDESIMEEAWEFTSEKLKSLKTEDMDPTLAMLVPHALEIPLSTRLPRFDARWFIEVYEKSRDMNPILLELGKLDFNIVQSMYQEDLRVMSSWWKEIALREKLDFARDRLVPSYLWGVGVSSEPQYAYSRRIIGMDIAIINMIDDIYDIYGTLEEVELFTDAVKKWDINAMKQLPDYMKIAYLAFYNTVNEMIYVILKEHGVDVTDHLTKAWLGLLNGYLTEARWYHTKHKPSLAEYMKNACMSIAGPLVATLAYLTTHKSITEEEMKYLETIPDVMSTTSYVFRISDDYGTSSDELKRGDVPKAIQCYMHDSGVSEEEARLHMRKLVRNGWKKVNKYRFMESQESPLTPTLVEMMQNLTRVSQNLYEHGDGHGIEDGETKDRVLRLLFQPIPM Mutant F443A amino acid sequence SEQ ID NO: 5 MVATSTTEVIERRSGNYQPCIWDIEFLQTVDSDYSKEIYKKRSENLKRQVKTMLVSEVEPLVQLELIDVLQRLCLGYEFETEIKSILQSIYNNNSSSSGKYKSSQDLHAAALEFRLLRQHHYIVPQEIFESFRDENGFKKSIYDDVKGMLSLYEASFFAFEDESIMEEAWEFTSEKLKSLKTEDMDPTLAMLVPHALEIPLSTRLPRFDARWFIEVYEKSRDMNPILLELGKLDFNIVQSMYQEDLRVMSSWWKEIALREKLDFARDRLVPSYLWGVGVSSEPQYAYSRRIIGMDIAIINMIDDIYDIYGTLEEVELFTDAVKKWDINAMKQLPDYMKIAYLAFYNTVNEMIYVILKEHGVDVTDHLTKAWLGLLNGYLTEARWYHTKHKPSLAEYMKNACMSIAGPLIATLAYLTTHKSITEEEMKYLETIPDVMSTTSYVARISDDYGTSSDELKRGDVPKAIQCYMHDSGVSEEEARLHMRKLVRNGWKKVNKYRFMESQESPLTPTLVEMMQNLTRVSQNLYEHGDGHGIEDGETKDRVLRLLFQPIPM Mutant G533A amino acid sequence SEQ ID NO: 6 MVATSTTEVIERRSGNYQPCIWDIEFLQTVDSDYSKEIYKKRSENLKRQVKTMLVSEVEPLVQLELIDVLQRLCLGYEFETEIKSILQSIYNNNSSSSGKYKSSQDLHAAALEFRLLRQHHYIVPQEIFESFRDENGFKKSIYDDVKGMLSLYEASFFAFEDESIMEEAWEFTSEKLKSLKTEDMDPTLAMLVPHALEIPLSTRLPRFDARWFIEVYEKSRDMNPILLELGKLDFNIVQSMYQEDLRVMSSWWKEIALREKLDFARDRLVPSYLWGVGVSSEPQYAYSRRIIGMDIAIINMIDDIYDIYGTLEEVELFTDAVKKWDINAMKQLPDYMKIAYLAFYNTVNEMIYVILKEHGVDVTDHLTKAWLGLLNGYLTEARWYHTKHKPSLAEYMKNACMSIAGPLIATLAYLTTHKSITEEEMKYLETIPDVMSTTSYVFRISDDYGTSSDELKRGDVPKAIQCYMHDSGVSEEEARLHMRKLVRNGWKKVNKYRFMESQESPLTPTLVEMMQNLTRVSQNLYEHGDGHAIEDGETKDRVLRLLFQPIPM Mutant I409V / F443A amino acid sequence SEQ ID NO: 7 MVATSTTEVIERRSGNYQPCIWDIEFLQTVDSDYSKEIYKKRSENLKRQVKTMLVSEVEPLVQLELIDVLQRLCLGYEFETEIKSILQSIYNNNSSSSGKYKSSQDLHAAALEFRLLRQHHYIVPQEIFESFRDENGFKKSIYDDVKGMLSLYEASFFAFEDESIMEEAWEFTSEKLKSLKTEDMDPTLAMLVPHALEIPLSTRLPRFDARWFIEVYEKSRDMNPILLELGKLDFNIVQSMYQEDLRVMSSWWKEIALREKLDFARDRLVPSYLWGVGVSSEPQYAYSRRIIGMDIAIINMIDDIYDIYGTLEEVELFTDAVKKWDINAMKQLPDYMKIAYLAFYNTVNEMIYVILKEHGVDVTDHLTKAWLGLLNGYLTEARWYHTKHKPSLAEYMKNACMSIAGPLVATLAYLTTHKSITEEEMKYLETIPDVMSTTSYVARISDDYGTSSDELKRGDVPKAIQCYMHDSGVSEEEARLHMRKLVRNGWKKVNKYRFMESQESPLTPTLVEMMQNLTRVSQNLYEHGDGHGIEDGETKDRVLRLLFQPIPM Although the present application has been disclosed in its preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the claims.

Claims

1. A mutant pinene synthase enzyme, characterized in that, The amino acid sequence of the pinene synthetase mutant is based on the amino acid sequence shown in SEQ ID NO: 1, and one or more amino acid mutations are made to the isoleucine at position 296, the asparagine at position 300, the isoleucine at position 308, the alanine at position 405, the isoleucine at position 409, the phenylalanine at position 443, and the glycine at position 533.

2. The pinene synthase mutant of claim 1, wherein, The amino acid sequence of the pinene synthetase mutant includes any one of the following amino acid sequences: (1) the amino acid sequence obtained by mutating the isoleucine at position 296 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to asparagine; (2) the amino acid sequence obtained by mutating the isoleucine at position 296 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to methionine; (3) the amino acid sequence obtained by mutating the asparagine at position 300 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to serine; (4) the amino acid sequence obtained by mutating the isoleucine at position 308 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to alanine; (5) the amino acid sequence obtained by mutating the alanine at position 405 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to serine; (6) the amino acid sequence obtained by mutating the alanine at position 405 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to threonine; (7) the amino acid sequence obtained by mutating the alanine at position 405 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to valine; (8) the amino acid sequence obtained by mutating the isoleucine at position 409 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to valine; (9) the amino acid sequence obtained by mutating the isoleucine at position 409 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to lysine; (10) the amino acid sequence obtained by mutating the phenylalanine at position 443 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to alanine; (11) the amino acid sequence obtained by mutating the glycine at position 533 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to alanine; (12) the amino acid sequence obtained by mutating the glycine at position 533 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to serine; (13) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 308 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to alanine and the isoleucine at position 409 to valine; (14) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 308 of the pinene synthetase having the amino acid sequence shown in SEQ ID NO: 1 to alanine and the phenylalanine at position 443 to alanine; (15) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 308 to alanine and the glycine at position 533 to alanine in the pinene synthase whose amino acid sequence is represented by SEQ ID NO: 1; (16) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 409 to valine and the phenylalanine at position 443 to alanine in the pinene synthase whose amino acid sequence is represented by SEQ ID NO: 1; (17) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 409 to valine and the glycine at position 533 to alanine in the pinene synthase whose amino acid sequence is represented by SEQ ID NO: 1; (18) the amino acid sequence obtained by simultaneously mutating the phenylalanine at position 443 to alanine and the glycine at position 533 to alanine in the pinene synthase whose amino acid sequence is represented by SEQ ID NO: 1; (19) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 308 to alanine, the isoleucine at position 409 to valine and the phenylalanine at position 443 to alanine in the pinene synthase whose amino acid sequence is represented by SEQ ID NO: 1; (20) the amino acid sequence obtained by simultaneously mutating the isoleucine at position 409 to valine, the phenylalanine at position 443 to alanine and the glycine at position 533 to alanine in the pinene synthase whose amino acid sequence is represented by SEQ ID NO:

1.

3. A nucleotide encoding the pinene synthase mutant according to any one of claims 1-2.

4. A plasmid vector carrying the nucleotide according to claim 3.

5. A cell expressing the pinene synthase mutant according to any one of claims 1-2.

6. A recombinant bacterium, characterized in that, The recombinant bacteria express the pinene synthase mutant of any one of claims 1-2; wherein the recombinant bacteria use Serratia marcescens (HBQA7) as a host. Serratia marcescens ) HBQA7 as a host.

7. Use of the recombinant bacteria according to claim 6 in the production of pinene.

8. A method of producing pinene, characterized by, The use of the recombinant bacteria according to claim 6 in the fermentation production of pinene comprises the steps of: (1) inoculating the recombinant bacteria single colony into a growth medium, culturing at 30-37°C and 180-600 rpm for 10-12 hours to obtain a seed liquid; (2) inoculating the seed liquid into a fermentation medium at an inoculation amount of 1-2%, culturing at 30-37°C and 180-600 rpm for 48-72 hours to obtain a fermentation liquid; (3) taking the upper organic phase of the fermentation liquid, centrifuging at 8000-10000 rpm for 5-10 minutes to obtain a supernatant containing pinene.

9. The method of claim 8, wherein, The growth medium includes LB medium, M9 medium or TB medium.

10. The method of claim 8, wherein, The composition of the fermentation medium includes: 10-14 g / L proteose peptone, 22-26 g / L yeast extract, 1-2 g / L antifoaming agent, 2-2.4 g / L K2HPO4, 9.2-9.4 g / L KH2PO4, 30-35 g / L glycerol, 1.5-1.6 g / L MgSO4, 0.1-0.2% trace metal element stock solution, 1-2% vitamin stock solution, kanamycin 50-60 mg / L, spectinomycin 50-60 mg / L, deionized water as solvent, and pH 7.0.

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