Germacrene a synthase mutant, pichia pastoris engineering bacteria with high yield of beta-elemene and application thereof

Abstract

The application discloses a germacrene A synthase mutant, a high-yield beta-elemene Pichia pastoris engineering bacterium and application thereof, and belongs to the technical field of genetic engineering. The germacrene A synthase mutant with improved catalytic activity compared with a wild-type germacrene A synthase is obtained by single-point and multi-point mutation of germacrene A synthase derived from a variable fishy algae, and the germacrene A synthase mutant is integrated into a Pichia pastoris strain, thereby constructing the Pichia pastoris engineering bacterium with obviously improved beta-elemene biosynthesis level, and the beta-elemene biosynthesis level of the Pichia pastoris engineering bacterium can be improved to 5.31 times of that of the wild-type germacrene A synthase integrated strain, and the Pichia pastoris engineering bacterium has a wide application prospect in the industrialized biosynthesis of beta-elemene.

Description

[0001] This application is a divisional application of application number 202610909439.9, filed on June 23, 2026, entitled "Gemaene A synthase mutant, Pichia pastoris engineered strain with high β-elemene production and its application". Technical Field

[0002] This invention belongs to the field of genetic engineering technology, specifically relating to a germaxyl A synthase mutant, a Pichia pastoris engineered strain that produces high levels of β-elemene, and their applications. Background Technology

[0003] β-elemene is a sesquiterpene natural product isolated and extracted from Curcuma longa, a traditional Chinese medicinal herb from Zhejiang province. It plays an important role in clinical cancer treatment due to its unique multi-target mechanism of action, high efficacy, low toxicity, and broad-spectrum antitumor properties. Currently, β-elemene is mainly produced through extraction from Curcuma longa, which involves a long cultivation cycle, significant environmental influences, and difficulties in isolation and purification, making it challenging to achieve stable, high-quality production. Therefore, there is an urgent need to develop a stable and efficient method for producing β-elemene.

[0004] The biosynthesis of β-elemene using microbial chassis such as *Escherichia coli* and yeast has become a current research hotspot. The biosynthetic pathway of β-elemene has been elucidated. Microorganisms can utilize a carbon source to generate the sesquiterpene precursor farnesyl pyrophosphate (FPP) via the mevalonate (MVA) pathway. FPP is then cyclized to form farnesyl A by germaxyl A synthase (GAS), and finally converted to β-elemene through an in vitro high-temperature, high-pressure reaction. Currently, significant progress has been made in the biosynthesis of β-elemene using microorganisms such as *Escherichia coli*, *Saccharomyces cerevisiae*, *Yersinia lipolytica*, and *Hansenula polymorpha* as cellular chassis.

[0005] However, different hosts exhibit distinct preferences for the source of GAS and its mutants, and its catalytic efficiency becomes one of the main factors limiting β-elemene accumulation. In the Yersinia lipolytica chassis, through rational design of HaGAS from sunflower (Helianthus annuus L.), a mutant HaGAS was created. V436LThe catalytic activity was increased by 2.07 times [LIU SC, XU LX, SUN YJ, et al. Sustainable high-titer production of β-elemene in Yarrowia lipolytica via modular engineering, pathway refactoring, and a chimeric germacrene A synthase [J]. Chemical Engineering Journal, 2026, 528.]. In the Escherichia coli chassis, the catalytic activity of LTC2 derived from lettuce (Lactuca sativa) was increased by 2.07 times [LIU SC, XU LX, SUN YJ, et al. Sustainable high-titer production of β-elemene in Yarrowia lipolytica via modular engineering, pathway refactoring, and a chimeric germacrene A synthase [J]. Chemical Engineering Journal, 2026, 528.]. T392A-T410S The level of biosynthetic β-elemene was increased by 5.44 times [CHEN R, LIU Y, CHEN S, et al. Protein engineering of a germacrene A synthase from Lactuca sativa and its application in high productivity of germacrene A in Escherichia coli [J]. Frontiers in Plant Science, 2022, Volume 13 - 2022.].

[0006] Pichia pastoris (Komagataella phaffii) is a dominant chassis microorganism for sesquiterpene biosynthesis. Existing research has shown that heterologous expression of GAS derived from Anabaena variabilis in Pichia pastoris, combined with metabolic engineering, has achieved a β-elemene synthesis yield of 1.9 g / L [CHENG JT, ZUO YM, LIU GF, et al. Development of a Pichiapastoris cell factory for efficient production of germacrene A: a precursor of β-elemene (J). Bioresources and Bioprocessing, 2023, 10(1): 38.]. This level is far lower than that of other sesquiterpene compounds such as santalene. Therefore, the catalytic performance of natural AvGAS is limited, severely restricting the efficient synthesis of β-elemene in the Pichia pastoris chassis and failing to meet the requirements for industrial applications. Therefore, molecular modification of AvGAS and screening for advantageous mutants are the core keys to achieving efficient adaptation of β-elemene based on the Pichia pastoris chassis. Summary of the Invention

[0007] This invention provides a germaxyl A synthase mutant, a Pichia pastoris engineered strain producing high levels of β-elemene, and their applications. This invention involves genetically modifying AvGAS, screening for mutants with higher catalytic activity than the original AvGAS, and then using a biosafe strain of Pichia pastoris for heterologous expression to obtain a Pichia pastoris engineered strain producing high levels of β-elemene.

[0008] The germaxyl A synthase mutant described in this invention is a single-point mutant, two-point mutant, or three-point mutant of the germaxyl A synthase derived from the variable Anabaena as shown in SEQ ID NO.1.

[0009] Among them, the single-point mutants are GAS2, which is formed by mutating alanine at position 227 to leucine in the genotype A synthase from the variable algae Anabaena as shown in SEQ ID NO.1; GAS6, which is formed by mutating leucine at position 43 to alanine as shown in SEQ ID NO.13; GAS12, which is formed by mutating valine at position 164 to glycine as shown in SEQ ID NO.25; and GAS13, which is formed by mutating tyrosine at position 44 to leucine as shown in SEQ ID NO.27.

[0010] The two-point mutants are: GAS2-6, a mutant from the variable *Anabaena* sourced in SEQ ID NO. 1, with amino acid sequences formed by mutating alanine at position 227 to leucine and leucine at position 43 to alanine, as shown in SEQ ID NO. 29; GAS2-12, a mutant from SEQ ID NO. 31, with amino acid sequences formed by mutating alanine at position 227 to leucine and valine at position 164 to glycine; GAS2-13, a mutant from SEQ ID NO. 33, with amino acid sequences formed by mutating alanine at position 227 to leucine and tyrosine at position 44 to leucine; GAS6-12, a mutant from SEQ ID NO. 35, with amino acid sequences formed by mutating alanine at position 43 to alanine and tyrosine at position 44 to leucine; and GAS2-12, a mutant from SEQ ID NO. 35, with amino acid sequences formed by mutating alanine at position 43 to alanine and tyrosine at position 44 to leucine. The mutant GAS6-13 shown in NO.37, or the mutant GAS12-13 formed by mutating valine at position 164 to glycine and tyrosine at position 44 to leucine, as shown in SEQ ID NO.39;

[0011] The three-point mutants are the mutant GAS2-6-13, which is formed by mutating alanine at position 227 to leucine, leucine at position 43 to alanine, and tyrosine at position 44 to leucine in the gemmaeene A synthase derived from the variable Anabaena as shown in SEQ ID NO.1; or the mutant GAS2-12-13, which is formed by mutating alanine at position 227 to leucine, valine at position 164 to glycine, and tyrosine at position 44 to leucine; or the mutant GAS12-13-14, which is formed by mutating valine at position 164 to glycine, tyrosine at position 44 to leucine, and asparagine at position 163 to alanine.

[0012] This invention provides a gene encoding the above-mentioned gemmaene A synthase mutant.

[0013] Preferably, the coding gene for mutant GAS2 is shown in SEQ ID NO. 6, the coding gene for mutant GAS6 is shown in SEQ ID NO. 14, the coding gene for mutant GAS12 is shown in SEQ ID NO. 26, the coding gene for mutant GAS13 is shown in SEQ ID NO. 28, the coding gene for mutant GAS2-6 is shown in SEQ ID NO. 30, the coding gene for mutant GAS2-12 is shown in SEQ ID NO. 32, the coding gene for mutant GAS2-13 is shown in SEQ ID NO. 34, the coding gene for mutant GAS6-12 is shown in SEQ ID NO. 36, the coding gene for mutant GAS6-13 is shown in SEQ ID NO. 38, the coding gene for mutant GAS12-13 is shown in SEQ ID NO. 40, the coding gene for mutant GAS2-6-13 is shown in SEQ ID NO. 44, and the coding gene for mutant GAS2-12-13 is shown in SEQ ID NO. 6. As shown in NO.46, the gene encoding the mutant GAS12-13-14 is shown in SEQ ID NO.48.

[0014] The present invention provides a recombinant expression vector containing the gene encoding the above-mentioned gemmaene A synthase mutant.

[0015] In a specific embodiment of the present invention, the recombinant expression vector Int1-TEF1p-GAS-AOX1t-donor is taken as a representative example of the recombinant expression vector. It is a plasmid vector Int1-TEF1p-AOX1t-donor containing the gene encoding the above-mentioned gemmaene A synthase mutant, as well as the TEF1 promoter and AOX1 terminator.

[0016] The present invention also provides a method for constructing the above-mentioned recombinant expression vector, the specific steps of which are as follows:

[0017] The gene fragment encoding the gamma-aluminum A synthase mutant and the vector backbone fragment were amplified separately, and then the gene fragment encoding the gamma-aluminum A synthase mutant and the vector backbone fragment were assembled and transformed into Escherichia coli to obtain a recombinant expression vector.

[0018] Furthermore, the specific steps for constructing the recombinant expression vector Int1-TEF1p-GAS-AOX1t-donor are as follows:

[0019] The gene fragment encoding the gamma-aluminum A synthase mutant and the vector backbone fragment Int1-TEF1p-AOX1t were amplified separately. Then, the gene fragment encoding the gamma-aluminum A synthase mutant and the vector backbone fragment Int1-TEF1p-AOX1t were assembled using a seamless cloning method and transformed into Escherichia coli DH5α strain to obtain the recombinant expression vector Int1-TEF1p-GAS-AOX1t-donor.

[0020] This invention provides a Pichia pastoris engineered strain that produces high levels of β-elemene, whose genome integrates the gene encoding the above-mentioned gemmaene A synthase mutant.

[0021] The present invention also provides a method for constructing the above-mentioned Pichia pastoris engineered strain that produces high levels of β-elemene, the specific steps of which are as follows:

[0022] The recombinant expression vector was amplified by PCR to form an expression cassette DNA fragment containing a homologous arm and a germaxyl A synthase mutant. This fragment was then electroporated into Pichia pastoris strain GS115-Cas9 along with the corresponding guide RNA plasmid to obtain a Pichia pastoris engineered strain that produces high levels of β-elemene.

[0023] This invention provides the application of the above-mentioned engineered Pichia pastoris strain in the fermentation production of β-elemene.

[0024] Furthermore, the specific methods for the above applications are as follows:

[0025] The engineered Pichia pastoris seed culture was inoculated into YPD medium and fermented for 24-36 h. Then, 1 / 4 to 1 / 3 of the fermentation liquid volume of n-dodecane was added for in-situ extraction. After fermentation continued for 24-48 h, the organic phase was collected by centrifugation to obtain biosynthesized β-elemene.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) By genetically modifying AvGAS, this invention has obtained a variety of GAS mutants with higher catalytic activity than the original GAS. Among them, the β-elemene production of Pichia pastoris engineered strain expressing the single-point mutant GAS6 is 3.22 times higher than that of strain expressing GAS0, and the β-elemene production of Pichia pastoris engineered strain expressing the two-point mutant GAS6-13 is 5.31 times higher than that of strain expressing GAS0.

[0028] (2) This invention utilizes Pichia pastoris as a host cell to express GAS mutant to synthesize β-elemene. It is a biosafe strain with the advantage of natural sesquiterpene synthesis, and can carry out high-density fermentation, which helps to save application costs. Attached Figure Description

[0029] Figure 1A schematic diagram of the spectrum of the Int1-TEF1p-GAS-AOX1t-donor vector;

[0030] Figure 2 A diagram illustrating the effect of Pichia pastoris recombinant strains expressing the GAS single-point mutant in synthesizing β-elemene;

[0031] Figure 3 A diagram illustrating the effect of Pichia pastoris recombinant strains expressing the GAS multipoint mutant in synthesizing β-elemene;

[0032] Figure 4 The image shows the GC-MS spectrum of β-elemene, where black represents the 5 ppm standard (STD), blue represents the 10 ppm standard, and red represents the β-elemene sample synthesized by the GAS6-13 mutant Pichia pastoris recombinant strain. Detailed Implementation

[0033] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the following embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or in accordance with the product manual.

[0034] The amino acid sequence of GAS (GAS0) derived from wild-type Anabaena polymorpha used in this invention is shown in SEQ ID NO.1. Reference: [ZHANG W, GUO J, WANG Z, et al. Improved production of germmacrene A, a direct precursor of β-elemene, in engineered Saccharomyces cerevisiae by expressing a cyanobacterial germacrene A synthase [J]. Microbial Cell Factories, 2021, 20(1): 7.].

[0035] In the following examples, the culture medium used has the following composition:

[0036] YPD liquid medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose.

[0037] LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride.

[0038] YPD / Ze solid medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, 16 g / L agar, 100 mg / L bleomycin.

[0039] LB / Amp solid-liquid culture medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride, 16 g / L agar, 100 mg / L ampicillin antibiotic.

[0040] In the following examples, the mutation sites and sequences of wild-type GAS and each GAS mutant are shown in Table 1.

[0041] Table 1

[0042]

[0043] Example 1: Construction of Pichia pastoris engineered strain expressing wild-type GAS

[0044] To achieve GAS expression in Pichia pastoris host cells, the Int1-TEF1p-GAS0-AOX1t-donor recombinant plasmid was first constructed using TEF1p as the promoter (SEQ ID NO. 51) and AOX1t as the terminator (SEQ ID NO. 52). Then, primers were designed to amplify the TEF1p-GAS0-AOX1t expression cassette DNA fragment containing the Int1 homologous arm, and simultaneously electroporated into the Pichia pastoris GS115-Cas9 strain along with the sgRNA-Int1-Ze plasmid to obtain an engineered Pichia pastoris strain expressing wild-type GAS. The specific steps are as follows:

[0045] (1) Construction of recombinant expression vector Int1-TEF1p-GAS0-AOX1t-donor

[0046] Using the GAS0 gene (SEQ ID NO.2) as a template, primers Tef-AvGAS-F and Tef-AvGAS-R were designed to amplify the GAS0 target gene fragment. Using the Int1-TEF1p-AOX1t-donor plasmid (SEQ ID NO.53) as a template, primers AOX1t-F and pTEF-R were designed to amplify the plasmid backbone. The two fragments were assembled using a seamless cloning method and transformed into *E. coli* DH5α to obtain the recombinant vector Int1-TEF1p-GAS0-AOX1t-donor. Figure 1 As shown.

[0047] The primer sequences used are shown in Table 2.

[0048] Table 2

[0049]

[0050] The PCR system is shown in Table 3.

[0051] Table 3

[0052]

[0053] The PCR amplification procedure is shown in Table 4 below.

[0054] Table 4

[0055]

[0056] (2) Construction of Pichia pastoris engineered strains expressing GAS0

[0057] Using the verified transformants as templates, primers Int1-F and Int1-R were designed to amplify the TEF1p-GAS0-AOX1t expression cassette DNA fragment with the Int1 homologous arm. This fragment, along with the corresponding guide RNA plasmid sgRNA-Int1-Ze (SEQ ID NO. 54), was simultaneously transformed into Pichia pastoris GS115-Cas9 cells to obtain Pichia pastoris engineered strains with the TEF1p-GAS0-AOX1t expression cassette integrated into the Int1 site.

[0058] The primer sequences used are shown in Table 5.

[0059] Table 5

[0060]

[0061] Example 2: Construction of Pichia pastoris engineered strain expressing GAS mutant

[0062] (1) Construction of recombinant expression vector

[0063] Through rational design of GAS0, 13 candidate single-point mutants and 11 multi-point mutants were obtained. The construction methods of recombinant expression vectors containing each mutant are as follows.

[0064] Construction of the Int1-TEF1p-GAS1-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS1-F and GAS1-R were designed to amplify the plasmid, introducing the A28S mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS1-AOX1t-donor recombinant vector.

[0065] Construction of the Int1-TEF1p-GAS2-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS2-F and GAS2-R were designed to amplify the plasmid, introducing the A227L mutation. The fragment was then recombined using seamless cloning to obtain the Int1-TEF1p-GAS2-AOX1t-donor recombinant vector.

[0066] Construction of the Int1-TEF1p-GAS3-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS3-F and GAS3-R were designed to amplify the plasmid, introducing the F23E mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS3-AOX1t-donor recombinant vector.

[0067] Construction of the Int1-TEF1p-GAS4-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS4-F and GAS4-R were designed to amplify the plasmid, introducing the C63A mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS4-AOX1t-donor recombinant vector.

[0068] Construction of the Int1-TEF1p-GAS5-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS5-F and GAS5-R were designed to amplify the plasmid, introducing the I38A mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS5-AOX1t-donor recombinant vector.

[0069] Construction of the Int1-TEF1p-GAS6-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS6-F and GAS6-R were designed to amplify the plasmid, introducing the L43A mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS6-AOX1t-donor recombinant vector.

[0070] Construction of the Int1-TEF1p-GAS7-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS7-F and GAS7-R were designed to amplify the plasmid, introducing the M196E mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS7-AOX1t-donor recombinant vector.

[0071] Construction of the Int1-TEF1p-GAS8-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS8-F and GAS8-R were designed to amplify the plasmid, introducing the M196T mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS8-AOX1t-donor recombinant vector.

[0072] Construction of the Int1-TEF1p-GAS9-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS9-F and GAS9-R were designed to amplify the plasmid, introducing the M196S mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS9-AOX1t-donor recombinant vector.

[0073] Construction of the Int1-TEF1p-GAS10-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS10-F and GAS10-R were designed to amplify the plasmid, introducing the N72T mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS10-AOX1t-donor recombinant vector.

[0074] Construction of the Int1-TEF1p-GAS11-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS11-F and GAS11-R were designed to amplify the plasmid, introducing the R34D mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS11-AOX1t-donor recombinant vector.

[0075] Construction of the Int1-TEF1p-GAS12-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS12-F and GAS12-R were designed to amplify the plasmid, introducing the V164G mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS12-AOX1t-donor recombinant vector.

[0076] Construction of the Int1-TEF1p-GAS13-AOX1t-donor single-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS13-F and GAS13-R were designed to amplify the plasmid, introducing the Y44L mutation. The fragment was then recombinated using seamless cloning to obtain the Int1-TEF1p-GAS13-AOX1t-donor recombinant vector.

[0077] Construction of the Int1-TEF1p-GAS2-6-AOX1t-donor two-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS2-AOX1t-donor vector as a template, primers GAS6-F and GAS6-R were designed to amplify the plasmid, and two-point mutations L43A and A227L were introduced. The fragment was recombined through seamless cloning to obtain the Int1-TEF1p-GAS2-6-AOX1t-donor recombinant vector.

[0078] Construction of the Int1-TEF1p-GAS2-12-AOX1t-donor two-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS2-AOX1t-donor vector as a template, primers GAS12-F and GAS12-R were designed to amplify the plasmid, and two-point mutations A227L and V164G were introduced. The fragment was recombined through seamless cloning to obtain the Int1-TEF1p-GAS2-12-AOX1t-donor recombinant vector.

[0079] Construction of the Int1-TEF1p-GAS2-13-AOX1t-donor two-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS2-AOX1t-donor vector as a template, primers GAS13-F and GAS13-R were designed to amplify the plasmid, and two-point mutations A227L and Y44L were introduced. The fragment was recombined through seamless cloning to obtain the Int1-TEF1p-GAS2-13-AOX1t-donor recombinant vector.

[0080] Construction of the Int1-TEF1p-GAS6-12-AOX1t-donor two-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS6-AOX1t-donor vector as a template, primers GAS12-F and GAS12-R were designed to amplify the plasmid, and two-point mutations of L43A and V164G were introduced. The fragment was recombined through seamless cloning to obtain the Int1-TEF1p-GAS6-12-AOX1t-donor recombinant vector.

[0081] Construction of the Int1-TEF1p-GAS6-13-AOX1t-donor two-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS0-AOX1t-donor vector as a template, primers GAS6-13-F and GAS6-13-R were designed to amplify the plasmid, and two-point mutations L43A and Y44L were introduced. The fragment was recombined through seamless cloning to obtain the Int1-TEF1p-GAS6-13-AOX1t-donor recombinant vector.

[0082] Construction of the Int1-TEF1p-GAS12-13-AOX1t-donor two-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS12-AOX1t-donor vector as a template, primers GAS13-F and GAS13-R were designed to amplify the plasmid, and two-point mutations V164G and Y44L were introduced. The fragment was recombined through seamless cloning to obtain the Int1-TEF1p-GAS12-13-AOX1t-donor recombinant vector.

[0083] Construction of the Int1-TEF1p-GAS2-6-12-AOX1t-donor three-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS2-6-AOX1t-donor vector as a template, primers GAS12-F and GAS12-R were designed to amplify the plasmid, introducing L43A, A227L and V164G three-point mutations. The two fragments were then assembled through seamless cloning to obtain the Int1-TEF1p-GAS2-6-12-AOX1t-donor recombinant vector.

[0084] Construction of the Int1-TEF1p-GAS2-6-13-AOX1t-donor three-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS6-13-AOX1t-donor vector as a template, primers GAS2-F and GAS2-R were designed to amplify the plasmid, introducing L43A, A227L, and Y44L three-point mutations. The two fragments were then assembled through seamless cloning to obtain the Int1-TEF1p-GAS2-6-13-AOX1t-donor recombinant vector.

[0085] Construction of the Int1-TEF1p-GAS2-12-13-AOX1t-donor three-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS2-12-AOX1t-donor vector as a template, primers GAS13-F and GAS13-R were designed to amplify the plasmid, introducing the A227L, V164G, and Y44L three-point mutations. The two fragments were then assembled through seamless cloning to obtain the Int1-TEF1p-GAS2-12-13-AOX1t-donor recombinant vector.

[0086] Construction of the Int1-TEF1p-GAS12-13-14-AOX1t-donor three-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS12-13-AOX1t-donor vector as a template, primers GAS12-14-F and GAS12-14-R were designed to amplify the plasmid, introducing the N163A, V164G, and Y44L three-point mutations. The two fragments were then assembled through seamless cloning to obtain the Int1-TEF1p-GAS12-13-14-AOX1t-donor recombinant vector.

[0087] Construction of the Int1-TEF1p-GAS2-6-12-13-AOX1t-donor four-point mutation recombinant expression vector: Using the Int1-TEF1p-GAS2-6-12-AOX1t-donor vector as a template, primers GAS13-F and GAS13-R were designed to amplify the plasmid, introducing four-point mutations: A227L, L43A, V164G, and Y44L. The two fragments were then assembled through seamless cloning to obtain the Int1-TEF1p-GAS2-6-12-13-AOX1t-donor recombinant vector.

[0088] The primer sequences used are shown in Table 6.

[0089] Table 6

[0090]

[0091] (2) Construction of Pichia pastoris engineered strains expressing GAS mutant

[0092] Using the verified clones as templates, primers Int1-F and Int1-R were designed to amplify the following expression cassettes containing the Int1 homologous arm: TEF1p-GAS1-AOX1t, TEF1p-GAS2-AOX1t, TEF1p-GAS3-AOX1t, TEF1p-GAS4-AOX1t, TEF1p-GAS5-AOX1t, TEF1p-GAS6-AOX1t, TEF1p-GAS7-AOX1t, TEF1p-GAS8-AOX1t, TEF1p-GAS9-AOX1t, TEF1p-GAS10-AOX1t, TEF1p-GAS11-AOX1t, TEF1p-GAS12-AOX1t, and TEF1p-GAS13-AOX1t. TEF1p-GAS2-6-AOX1t expression box, TEF1p-GAS2-12-AOX1t expression box, TEF1p-GAS2-13-AOX1t expression box, TEF1p-GAS6-12-AOX1t expression box, TEF1p-GAS6-13-AOX1t expression box, TEF1p-GAS12-13-AOX1t expression box, TEF1p-GAS2-6-12A DNA fragments of the following expression cassettes were simultaneously transformed into Pichia pastoris cells along with their corresponding guide RNA plasmids: OX1t, TEF1p-GAS2-6-13AOX1t, TEF1p-GAS2-12-13AOX1t, TEF1p-GAS12-13-14-AOX1t, and TEF1p-GAS2-6-12-13-AOX1t.The following expression cassettes were obtained: TEF1p-GAS1-AOX1t, TEF1p-GAS2-AOX1t, TEF1p-GAS3-AOX1t, TEF1p-GAS4-AOX1t, TEF1p-GAS5-AOX1t, TEF1p-GAS6-AOX1t, TEF1p-GAS7-AOX1t, TEF1p-GAS8-AOX1t, TEF1p-GAS9-AOX1t, TEF1p-GAS10-AOX1t, TEF1p-GAS11-AOX1t, TEF1p-GAS12-AOX1t, and TEF1p-GAS13-AOX1t. Pichia pastoris engineered strains integrated into the Int1 site using the AS2-6-AOX1t expression cassette, TEF1p-GAS2-12-AOX1t expression cassette, TEF1p-GAS2-13-AOX1t expression cassette, TEF1p-GAS6-12-AOX1t expression cassette, TEF1p-GAS6-13-AOX1t expression cassette, TEF1p-GAS12-13-AOX1t expression cassette, TEF1p-GAS2-6-12-AOX1t expression cassette, TEF1p-GAS2-6-13-AOX1t expression cassette, TEF1p-GAS2-12-13-AOX1t expression cassette, TEF1p-GAS12-13-14-AOX1t expression cassette, and TEF1p-GAS2-6-12-13-AOX1t expression cassette.

[0093] Example 3: Fermentation test of Pichia pastoris β-elemene synthesizing strain

[0094] Pichia pastoris engineered strains expressing wild-type GAS and mutants GAS1~GAS13, GAS2-6, GAS2-12, GAS2-13, GAS6-12, GAS6-13, GAS12-13, GAS2-6-12, GAS2-6-13, GAS2-12-13, GAS12-13-14, and GAS2-6-12-13 were selected and cultured in 24-well plates containing 3 mL of YPD medium at 30 °C and 300 rpm for 18 h. Then, they were transferred at a 10% inoculum to new 24-well plates containing 2 mL of YPD medium and cultured for 24 h. 500 μL of n-dodecane was added, and two-phase fermentation was continued for 48 h. After fermentation, the upper n-dodecane layer was diluted to an appropriate concentration, and the titer of β-elemene was quantified by GC-MS. GC-MS Quantitative Method for β-elemene: The processed sample was detected by GC-MS, and the products were separated using an HP-5MS capillary column. GC settings were as follows: column temperature 500℃, injection port temperature 280℃, splitless operation, and column flow rate 1.20 mL / min. The temperature program was set as follows: initial temperature 50℃, hold for 1 min, ramp to 200℃ at a rate of 10℃ / min, hold for 10 min, ramp to 280℃ at a rate of 20℃ / min, and hold for 3 min. Mass spectrometry settings were as follows: electron impact (EI) ion source was used, with an ion source temperature of 280℃. For quantitative analysis, MRM acquisition mode was selected, and the detection channels were set to m / z 93.00>77.10, 79.00>77.1, and 93.00>91.1. The β-elemene elution time was 12.700 min. GC-MS image of β-elemene as shown Figure 4 As shown.

[0095] like Figure 2As shown, in Pichia pastoris chassis cells expressing wild-type GAS0, the titer of β-elemene was 76.00 mg / L; in Pichia pastoris chassis cells expressing the GAS1 single-point mutant, the titer of β-elemene was 80.41 mg / L, increasing to 1.06 times that of the GAS0-expressing strain; in Pichia pastoris chassis cells expressing the GAS2 single-point mutant, the titer of β-elemene was 134.29 mg / L, increasing to 1.77 times that of the GAS0-expressing strain; in Pichia pastoris chassis cells expressing the GAS3 single-point mutant, the titer of β-elemene was 81.61 mg / L, increasing to 1.07 times that of the GAS0-expressing strain; and in Pichia pastoris chassis cells expressing the GAS4 single-point mutant, the titer of β-elemene was 47.90 mg / L. The titer of β-elemene was 57.39 mg / L, only 63.03% of that expressed in the GAS0 strain; the titer of β-elemene was 57.39 mg / L, only 75.51% of that expressed in the GAS0 strain, when expressed in the GAS6 single-point mutant in the Pichia pastoris chassis cells; the titer of β-elemene was 244.85 mg / L, 3.22 times that of the GAS0 strain, when expressed in the GAS7 single-point mutant in the Pichia pastoris chassis cells; the titer of β-elemene was 54.23 mg / L, only 71.35% of that expressed in the GAS0 strain, when expressed in the GAS8 single-point mutant in the Pichia pastoris chassis cells; the titer of β-elemene was 68.36 mg / L, only 89.94% of that expressed in the GAS0 strain, when expressed in the GAS9 single-point mutant in the Pichia pastoris chassis cells; and the titer of β-elemene was 66.07 mg / L. The titer of β-elemene was 12.31 mg / L, only 86.93% of that of the GAS0 strain, when expressed in Pichia pastoris chassis cells; when expressed in Pichia pastoris chassis cells, the titer of β-elemene was 48.60 mg / L, only 63.95% of that of the GAS0 strain; when expressed in Pichia pastoris chassis cells, the titer of β-elemene was 222.01 mg / L, 2.92 times that of the GAS0 strain; and when expressed in Pichia pastoris chassis cells, the titer of β-elemene was 164.23 mg / L, 2.16 times that of the GAS0 strain. As can be seen from the above, among all Pichia pastoris engineered strains expressing single-point mutants, the biosynthetic yield of β-elemene was significantly increased in Pichia pastoris engineered strains expressing GAS2, GAS6, GAS12, and GAS13 mutants.

[0096] like Figure 3As shown, in *Pichia pastoris* chassis cells, expression of the GAS6-12 two-point mutant resulted in a β-elemene titer of 120.02 mg / L, 1.58 times higher than that of the GAS0-expressing strain; expression of the GAS2-13 two-point mutant resulted in a β-elemene titer of 315.54 mg / L, 4.15 times higher than that of the GAS0-expressing strain; expression of the GAS2-12 two-point mutant resulted in a β-elemene titer of 125.78 mg / L, 1.65 times higher than that of the GAS0-expressing strain; expression of the GAS6-13 two-point mutant resulted in a β-elemene titer of 403.27 mg / L, 5.31 times higher than that of the GAS0-expressing strain; and expression of the GAS2-6 two-point mutant resulted in a β-elemene titer of 174.20 mg / L. The β-elemene titer was increased to 2.29 times that of the GAS0-expressing strain when expressed in Pichia pastoris chassis cells; and increased to 285.48 mg / L, 3.76 times that of the GAS0-expressing strain when expressing the GAS12-13 two-point mutant. These results indicate that the biosynthetic yield of β-elemene was significantly enhanced in all Pichia pastoris engineered strains expressing the two-point mutant.

[0097] Similarly, as Figure 3 As shown, in Pichia pastoris chassis cells, the expression of the GAS2-6-13 three-point mutant resulted in a β-elemene titer of 214.97 mg / L, which was 2.83 times higher than that of the GAS0 strain; in Pichia pastoris chassis cells, the expression of the GAS2-6-12 three-point mutant resulted in a β-elemene titer of 49.804 mg / L, which was only 65.53% of that of the GAS0 strain; in Pichia pastoris chassis cells, the expression of the GAS2-12-13 three-point mutant resulted in a β-elemene titer of 157.35 mg / L, which was 2.07 times higher than that of the GAS0 strain; and in Pichia pastoris chassis cells, the expression of the GAS12-13-14 three-point mutant resulted in a β-elemene titer of 472.93 mg / L, which was 6.22 times higher than that of the GAS0 strain. As shown above, except for the Pichia pastoris strain expressing the GAS2-6-12 three-point mutant, the biosynthetic yield of β-elemene in the other three Pichia pastoris strains expressing the three-point mutant was increased. Furthermore, in the Pichia pastoris chassis cells expressing the GAS2-6-12-13 four-point mutant, the β-elemene titer was 44.14 mg / L, showing a significant decrease, only 58.08% of that of the GAS0 strain.

Claims

1. A gemmaene A synthase mutant, characterized in that, It is a single-point mutant or a two-point mutant of the gemmaeene A synthase from the alginate *Anabaenavariabilis* shown in SEQ ID NO.1; Among them, the single-point mutant is the mutant GAS6, which is formed by mutating leucine at position 43 to alanine in the wild-type gemmaene A synthase shown in SEQ ID NO.1; The two-point mutants are GAS6-12, a mutant from the variable Anabaena alginate-derived gemmaeene A synthase shown in SEQ ID NO.1, with an amino acid sequence formed by mutating leucine at position 43 to alanine and valine at position 164 to glycine, as shown in SEQ ID NO.35; or GAS6-13, a mutant with an amino acid sequence formed by mutating leucine at position 43 to alanine and tyrosine at position 44 to leucine, as shown in SEQ ID NO.

37.

2. The gene encoding the gemmaene A synthase mutant of claim 1.

3. The gene according to claim 2, characterized in that, The gene encoding mutant GAS6 is shown in SEQ ID NO.14, the gene encoding mutant GAS6-12 is shown in SEQ ID NO.36, and the gene encoding mutant GAS6-13 is shown in SEQ ID NO.

38.

4. A recombinant expression vector, characterized in that, It includes the gene encoding the gemmaene A synthase mutant as described in claim 2.

5. The method for constructing the recombinant expression vector according to claim 4, characterized in that, The specific steps are as follows: amplify the gene fragment encoding the gemmaene A synthase mutant and the vector backbone fragment respectively, then assemble the gene fragment encoding the gemmaene A synthase mutant and the vector backbone fragment, and transform E. coli to obtain the recombinant expression vector.

6. The recombinant expression vector according to claim 4, characterized in that, The plasmid vector Int1-TEF1p-AOX1t-donor, containing the gene encoding a gamma-aluminum A synthase mutant, as well as the TEF1 promoter and AOX1 terminator, was constructed through the following steps: the gene fragment encoding the gamma-aluminum A synthase mutant and the vector backbone fragment Int1-TEF1p-AOX1t were amplified separately, and then the gene fragment encoding the gamma-aluminum A synthase mutant and the vector backbone fragment Int1-TEF1p-AOX1t were assembled using a seamless cloning method. The assembly was then transformed into Escherichia coli DH5α strain to obtain the recombinant expression vector Int1-TEF1p-GAS-AOX1t-donor.

7. A Pichia pastoris engineered strain that produces high levels of β-elemene, characterized in that, The genome integrates the gene encoding the gemmaeene A synthase mutant as described in claim 2.

8. The method for constructing the engineered Pichia pastoris strain according to claim 7, characterized in that, The specific steps are as follows: The recombinant expression vector of claim 4 was amplified by PCR to form an expression cassette DNA fragment containing a germaxene A synthase mutant with a homologous arm. This fragment was then electroporated into Pichia pastoris strain GS115-Cas9 along with the corresponding guide RNA plasmid to obtain a Pichia pastoris engineered strain that produces high levels of β-elemene.

9. The application of the engineered Pichia pastoris strain according to claim 7 in the fermentation production of β-elemene.

10. The application according to claim 9, characterized in that, The specific method is as follows: The engineered Pichia pastoris seed culture was inoculated into YPD medium and fermented for 24-36 h. Then, 1 / 4 to 1 / 3 of the volume of the fermentation liquid was added for in-situ extraction with n-dodecane. After fermentation continued for 24-48 h, the organic phase was collected by centrifugation to obtain biosynthesized β-elemene.

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