TPS / LPPS mutant for improving yield of sclareol as well as engineering bacteria and application of TPS / LPPS mutant

Abstract

The invention discloses a TPS / LPPS mutant for improving the yield of sclareol as well as an engineering bacterium and application of the TPS / LPPS mutant, and belongs to the technical field of synthetic biology and metabolic engineering. The invention provides a sclareol synthase (TPS) mutant and a danadiol diphosphate synthase (LPPS) mutant. The sclareol synthase (TPS) mutant and the danadiol diphosphate synthase (LPPS) mutant are obtained by carrying out random mutation on codon optimized TPS and LPPS genes through an error-prone PCR (Polymerase Chain Reaction) technology. The TPS mutant comprises one or more amino acid mutations in the 95th site, the 184th site, the 216th site, the 219th site, the 225th site, the 380th site, the 436th site and the 470th site; the LPPS mutant comprises one or more amino acid mutations in the 128th site, the 398th site, the 488th site and the 609th site. The invention also provides a recombinant engineering bacterium containing the mutant, preferably yarrowia lipolytica. The embodiment shows that the yield of sclareol is increased by 14-40% compared with that of a control group through the mutant obtained through screening. Technical support is provided for industrial production of sclareol, and the sclareol has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of synthetic biology and metabolic engineering, in particular, it is especially related to a TPS / LPPS mutant for improving the yield of sclareol and an engineering bacterium thereof and application. BACKGROUND

[0002] Terpenoids are natural compounds assembled from isoprene units in different forms, which are widely present in higher plants, insects and microorganisms in nature. Terpenoids are diverse in types, and can be divided into primary metabolites and secondary metabolites according to their physiological functions in organisms. According to the isoprene unit, they can be divided into monoterpenes, diterpenes and polyterpenes, etc.; according to the carbon ring, they can be divided into chain monoterpenes and monocyclic monoterpenes, etc.

[0003] At present, a variety of terpenoids have been proved to have special physical and chemical properties and biological activities, and can be used as health products, drugs, spices, etc. For example, the monoterpenoid compound geraniol is an acyclic monoterpene alcohol, which has the characteristics of high heat value, low hygroscopicity and low volatility, and is therefore considered as a good biofuel. At the same time, it has a relatively wide application in the perfume and pharmaceutical industries, and can also be used as an ingredient of insecticide. In addition, the sesquiterpene substance artemisinic acid is widely used in medicine: it is an important precursor for the synthesis of artemisinin, which is a drug component that can effectively treat malaria. For example, the diterpenoid compound taxadiene is one of the important precursors for the synthesis of paclitaxel, which is an effective antitumor drug with excellent antiproliferative activity against a variety of tumors, and is often used for early clinical intervention and combined treatment with chemotherapy drugs. 【1】 Therefore, terpenoids have great economic value and broad market prospects.

[0004] Sclareol is a diterpene derivative compound widely present in Salvia sclarea (clary sage) plants, which is a natural plant spice widely used in food flavoring, and sclareol can also be used to synthesize the important amber substitute norambrein, so it is often used in the production of cosmetics, and has high economic value. At present, commercial production of sclareol is mainly obtained by extraction from artificially cultivated clary sage plants. Due to the slow growth of plants, long growth cycle, cultivation planting affected by geographical and climatic factors, low extraction efficiency caused by complex composition of plants, unstable production and other factors, the yield of sclareol is low, which far cannot meet the market demand. 【1】 【2】 In recent years, with the development of synthetic biology technology, the use of microbial fermentation method to synthesize sclareol with glucose as the starting substrate has gradually become an economic and sustainable green method. By constructing microbial cell factories, by introducing heterologous terpenoid synthesis pathway and modifying endogenous metabolic pathway, it is expected to realize high efficient synthesis of high value terpenoid compounds. At the same time, it also has the characteristics of short production cycle and stable yield, which is expected to be able to meet the market demand.【2】【3】 The sclareol biosynthesis pathway in microorganisms involves two main metabolic modules: first, the synthesis of geranylgeranyl pyrophosphate (GGPP) from acetyl-CoA through the mevalonate pathway (MVA), which is an important synthetic precursor of terpene compounds; then, with GGPP as the starting material, GGPP is converted to sclareol under the catalysis of 8-hydroxy co-propyl pyrophosphate synthase LPPS and sclareol synthase TPS.【3】【4】 Zhao Zongbao et al. used the model microorganism Saccharomyces cerevisiae as a cell factory, systematically modified the central metabolism, including introducing the exogenous MVA pathway, optimizing the sclareol synthesis pathway and screening and modifying the heterologous LPP synthase (Lpps) and sclareol synthase (TPS) and other operations, while optimizing the fed-batch fermentation method, realizing the efficient synthesis of sclareol with glucose as raw material, the yield reached 11.4g / L. It provides a theoretical guidance for constructing high-efficiency terpenoid synthesis cell engineering.【4】【5】 Yarrowia lipolytica has a high flux of tricarboxylic acid cycle, which can provide sufficient acetyl-CoA precursors and other characteristics, and is considered to be an ideal host for the production of terpenes, polyketides and flavonoids and other natural products, and has been widely used in the field of metabolic engineering.【6】In addition, Yarrowia lipolytica has a perfect endogenous mevalonate (MVA) pathway, which can produce a large amount of isopentenyl pyrophosphate (IPP) and dimethyl allyl diphosphate (DMAPP) for terpene synthesis.【3】By introducing the diterpenediol diphosphate synthase (LPPS) and terpene synthase (TPS) of the plant Salviasclarea (Salvia), the sclareol synthesis pathway can be effectively constructed.【7】 In this study, on the basis of Yarrowia lipolytica, the central metabolic pathway was first optimized to enhance the expression of the MVA pathway and the production of isoprenoid pyrophosphate (DMAPP, IPP), which is an important synthetic precursor of terpenoid compounds. At the same time, based on the exogenous diterpenediol diphosphate synthase (LPPS) and terpene synthase (TPS) of the sclareol plant, the LPP synthase (LPPS) and the sclareol synthase (TPS) were introduced, which were highly expressed in Yarrowia lipolytica, and a cell factory for producing sclareol was preliminarily constructed.

[0005] By analyzing the three-dimensional structure of TPS and LPPS, it is found that the binding efficiency of the enzyme and the substrate is low, therefore further by the way of random mutation to TPS and LPPS, the mutation of the relevant enzyme coding sequence is carried out, the catalytic efficiency of the enzyme is improved, the yield of the product is improved, and high-throughput screening is further carried out, and finally the high-yield strain is obtained. The yield of shake flask fermentation is increased by about 41%, which has good application prospect. SUMMARY

[0006] Sclareol is an important diterpenoid compound, which naturally exists in Salvia sclarea plants. Due to its unique fragrance characteristics, it is widely used in food seasoning, essence and perfume industries and cosmetics industry, especially can be used for synthesizing important amber replacement product norambrein. At present, the commercial production of sclareol mainly depends on extraction from plants, but this method has problems such as slow plant growth, long growth cycle, restriction by geographical and climatic conditions, low extraction efficiency and unstable yield, which is difficult to meet the increasing market demand.

[0007] In recent years, the microbial fermentation method for producing sclareol gradually becomes an economic and sustainable alternative method. By constructing microbial cell factories and introducing heterologous terpenoid synthesis pathway, the biosynthesis of sclareol can be realized. In microorganisms, the biosynthesis of sclareol mainly involves two key enzymes: labdane diphosphate synthase (LPPS) and sclareol synthase (TPS). LPPS catalyzes geranylgeranyl pyrophosphate (GGPP) to form pyrophosphate copalyl diphosphate (CPP), and TPS further catalyzes CPP to form sclareol. The catalytic efficiency of these two enzymes directly affects the yield of sclareol.

[0008] However, the expression level and catalytic efficiency of plant-derived TPS and LPPS in microbial hosts are often not ideal, resulting in low yield of sclareol. Although the yield can be improved by increasing the number of gene copies, optimizing the culture conditions and the like, there is still room for improvement. How to improve the catalytic efficiency of TPS and LPPS by enzyme modification, improve the binding ability of the enzyme and the substrate, and further improve the yield of sclareol is a technical problem to be solved in the field.

[0009] In view of the above technical problems, the present application provides a sclareol synthase mutant, which is encoded by a codon-optimized sclareol synthase gene shown in SEQ ID NO: 1 and comprises one or more mutation sites in the amino acid sequence as follows: The proline at position 95 is mutated to leucine, glycine, alanine, valine, isoleucine, methionine, tryptophan or phenylalanine; The methionine at position 184 is mutated to leucine, glycine, alanine, valine, isoleucine, tryptophan, phenylalanine or proline; amino acid at position 216 is mutated to valine, glycine, alanine, leucine, isoleucine, methionine, tryptophan, phenylalanine or proline; amino acid at position 219 is mutated to serine, threonine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine; amino acid at position 225 is mutated to arginine, serine, threonine, cysteine, tyrosine, asparagine, aspartic acid, glutamic acid, lysine or histidine; amino acid at position 380 is mutated to proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan or phenylalanine; amino acid at position 436 is mutated to phenylalanine, glycine, alanine, valine, leucine, methionine, tryptophan or proline; amino acid at position 470 is mutated to arginine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid or lysine.

[0010] Summary of the above technical solutions The present application obtains a series of mutants by random mutation of the codon-optimized TPS and LPPS genes through error-prone PCR technology, and screens out mutants capable of significantly improving the production of sclareol by high-throughput screening method. The specific technical route includes: 1. Constructing the chassis strain po1f: based on the wild-type Yarrowia lipolytica strain W29, obtain the auxotrophic strain po1f by gene knockout, to provide a selection marker system for subsequent genetic engineering modification; 2. Optimizing the mevalonate (MVA) pathway: integrate the tHMG1-Erg8-IDI gene fragment on the basis of po1f to enhance the MVA pathway flux, improve the supply of isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP), and construct the strain YL1; 3. Constructing the sclareol production strain: integrate the codon-optimized LPPS-TPS gene fragment into the YL1 strain genome to obtain single-copy strain sclar1 and double-copy strain sclar2; 4. Random mutation: use error-prone PCR technology to randomly mutate TPS and LPPS genes to construct a mutant library; 5. High-throughput screening: transform the mutant plasmid into the sclar2 strain, and screen high-yield mutants through 96-well plate microfermentation and gas chromatography detection; 6. Mutation site identification: extract the plasmid of the high-yield strain for sequencing to determine the key mutation site.

[0011] (ii) Core technical content 1. Mutant of thymol synthase (TPS) The present application provides a mutant of thymol synthase, which is encoded by a codon-optimized TPS gene shown in SEQ ID NO: 1 and contains one or more mutation sites in the amino acid sequence. Key mutation sites include: Proline at position 95 is mutated to leucine, glycine, alanine, valine, isoleucine, methionine, tryptophan or phenylalanine; Methionine at position 184 is mutated to leucine, glycine, alanine, valine, isoleucine, tryptophan, phenylalanine or proline; Glutamic acid at position 216 is mutated to valine, glycine, alanine, leucine, isoleucine, methionine, tryptophan, phenylalanine or proline; Asparagine at position 219 is mutated to serine, threonine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine; Glutamine at position 225 is mutated to arginine, serine, threonine, cysteine, tyrosine, asparagine, aspartic acid, glutamic acid, lysine or histidine; Serine at position 380 is mutated to proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan or phenylalanine; Isoleucine at position 436 is mutated to phenylalanine, glycine, alanine, valine, leucine, methionine, tryptophan or proline; Histidine at position 470 is mutated to arginine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid or lysine.

[0012] Changes in these mutation sites can affect the spatial conformation of the enzyme, the geometry of the substrate binding site, the charge distribution of the catalytic active center, etc., thereby improving the catalytic efficiency and substrate binding capacity of the enzyme.

[0013] 2. Mutant of danol diol diphosphate synthase (LPPS) The present application also provides a mutant of danol diol diphosphate synthase, which is encoded by a codon-optimized LPPS gene shown in SEQ ID NO: 2 and contains one or more mutation sites in the amino acid sequence. Key mutation sites include: Histidine at position 128 is mutated to proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan or phenylalanine; the 398th tryptophan is mutated to serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine; the 488th glutamic acid is mutated to glutamine, threonine, serine, cysteine, tyrosine, asparagine, aspartic acid, lysine, arginine or histidine; the 609th leucine is mutated to glutamine, threonine, serine, cysteine, tyrosine, asparagine, aspartic acid, glutamic acid, lysine, arginine or histidine.

[0014] 3. Recombinant engineering bacteria The present application provides a recombinant engineering bacteria, which has integrated a codon-optimized LPPS-TPS gene fragment in the genome, or contains an expression vector carrying a mutant gene. The host microorganism can be Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris or Escherichia coli.

[0015] Preferably, the recombinant engineering bacteria is Yarrowia lipolytica, which has integrated a tHMG1-Erg8-IDI gene fragment (for enhancing the MVA pathway) and one or two copies of a codon-optimized LPPS-TPS gene fragment (for the synthesis of sclareol) in the genome. On this basis, the yield of sclareol can be further improved by introducing a free plasmid carrying a TPS or LPPS mutant.

[0016] The TPS mutant, LPPS mutant and recombinant engineering bacteria of the present application can be used to produce sclareol or its precursor sclareol phosphate. By fermenting the recombinant engineering bacteria with glucose as the carbon source, sclareol can be efficiently synthesized, which provides a technical basis for industrial production.

[0017] Further, the mutant contains mutations at any two or more of the following positions: 95th, 219th, 225th, 380th, 216th, 436th, 184th or 470th.

[0018] A mutant of sclareol diols diphosphorylase, which is encoded by a codon-optimized sclareol diols diphosphorylase gene shown in SEQ ID NO: 2 and contains one or more of the following mutation sites in the amino acid sequence: the 128th amino acid histidine is mutated to proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan or phenylalanine; the 398th amino acid tryptophan is mutated to serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine; amino acid at position 488 is mutated to glutamine, threonine, serine, cysteine, tyrosine, asparagine, aspartic acid, glutamic acid, lysine, arginine or histidine; amino acid at position 609 is mutated to glutamine, threonine, serine, cysteine, tyrosine, asparagine, aspartic acid, glutamic acid, lysine, arginine or histidine.

[0019] Further, the mutant comprises mutations at any two or more of positions 128, 398, 488 or 609.

[0020] A nucleotide sequence of a mutant sclareol synthase.

[0021] A nucleotide sequence of a mutant dammarenediol-II-phosphate synthase.

[0022] A recombinant expression vector comprising the nucleotide sequence of claim 5 encoding a mutant sclareol synthase, or the nucleotide sequence of claim 6 encoding a mutant dammarenediol-II-phosphate synthase.

[0023] A recombinant engineering bacterium, wherein the genome of the recombinant engineering bacterium comprises a codon-optimized LPPS-TPS gene fragment, or the recombinant engineering bacterium comprises the recombinant expression vector of claim 7. The recombinant engineering bacterium is Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris or Escherichia coli.

[0024] Further, the recombinant engineering bacterium is Yarrowia lipolytica, wherein the genome of the Yarrowia lipolytica comprises a tHMG1-Erg8-IDI gene fragment and one or two copies of a codon-optimized LPPS-TPS gene fragment.

[0025] Use of the mutant sclareol synthase, the mutant dammarenediol-II-phosphate synthase, and the recombinant engineering bacterium in producing sclareol or a precursor of sclareol, i.e., dammarenediol-II-phosphate.

[0026] Advantages of the present application: 1. The present application successfully obtains mutants capable of significantly improving the yield of sclareol by using error-prone PCR to randomly mutate TPS and LPPS genes. The results of the examples show that the high-yield mutants obtained by screening increase the yield of sclareol by 20-41% compared to the control strain. Specifically: The yield of sclareol of the TPS-1 mutant strain is increased by 38.5% compared to the control; The yield of sclareol of the TPS-2 mutant strain is increased by 14.2% compared to the control, which is the yield improvement obtained under shake flask fermentation conditions; The LPPS-1 mutant strain produced 25.5% more sclareol than the control; The LPPS-2 mutant strain produced 40.2% more sclareol than the control.

[0027] This significant yield improvement proves that the catalytic efficiency and substrate binding capacity of the mutant of the application have been effectively improved. The control strain sclar2 is already an optimized sclareol-producing strain (containing MVA pathway enhancement + double copy LPPS-TPS), and a 41% yield improvement is still achieved on this basis, fully demonstrating the effectiveness of the enzyme modification strategy. This yield improvement is of great significance for reducing production costs and improving economic benefits, and lays a foundation for the industrialized production of sclareol.

[0028] 2. Through systematic mutation and screening work, the application identifies multiple key mutation sites of TPS and LPPS. For TPS, a total of 8 key sites (positions 95, 184, 216, 219, 225, 380, 436, and 470) are identified; for LPPS, a total of 4 key sites (positions 128, 398, 488, and 609) are identified. The discovery of these mutation sites has important scientific value and practical value: First, the changes in these sites can significantly affect the catalytic performance of the enzyme, indicating that they may be located in the key functional regions of the enzyme, such as the substrate binding pocket, the catalytic active center, or the domain affecting the protein conformation.

[0029] Second, the application not only identifies mutation sites, but also identifies the various types of amino acids that each site can be mutated into. For example, the proline at position 95 of TPS can be mutated into 8 different amino acids (leucine, glycine, alanine, valine, isoleucine, methionine, tryptophan, and phenylalanine), which provides a rich selection for further optimization.

[0030] Third, this mutation site information provides an important basis for subsequent rational design and site-directed mutation. Researchers can perform combinatorial mutation, saturation mutation, or semi-rational design based on these sites, which is expected to obtain mutants with better catalytic performance.

[0031] Fourth, the discovery of these mutation sites helps to understand the structure-function relationship of TPS and LPPS, and provides experimental evidence for elucidating the catalytic mechanism of terpene synthases.

[0032] Therefore, the application not only provides high-yield mutants, but more importantly, it reveals key amino acid sites that affect enzyme activity, providing valuable molecular basis for the study of terpenoid biosynthesis and enzyme engineering.

[0033] 3.The application establishes a complete and efficient mutant construction and screening technology system, which has the following advantages: clear and feasible technical route. From the construction of chassis strain, optimization of MVA pathway, establishment of santalene synthesis pathway, to random mutation, high-throughput screening and mutation site identification, a complete technical process is formed. The process is not only suitable for the modification of TPS and LPPS, but also can be applied to the modification of other terpene synthases; high screening efficiency. The high-throughput screening method using 96-well plate microfermentation combined with gas chromatography detection can evaluate hundreds of mutants in a short time, greatly improving the screening efficiency. In the examples, 10 high-yield strains were successfully screened from about 500 transformants, with a hit rate of about 2%, which proves the effectiveness of the screening system; Yarrowia lipolytica has a high-throughput tricarboxylic acid cycle and a perfect endogenous MVA pathway, which can provide sufficient precursor substances, making it an ideal host for the production of terpenoids. The application successfully realizes the efficient synthesis of santalene in Yarrowia lipolytica, and further improves the yield through enzyme modification, which shows the superiority of the host system; the high-yield mutants obtained in the application can realize a 41% yield increase under shake flask fermentation conditions. If the fermentation conditions are further optimized (such as using fed-batch fermentation, optimizing medium composition, and controlling dissolved oxygen), the yield is expected to be further improved, which can fully meet the needs of industrial production. As an important fragrance compound and a precursor of ambergris substitute, santalene has huge market demand and economic value. The application provides technical support for the industrial production of santalene, helps to reduce production costs, meets market demand, reduces dependence on plant resources, and meets the concept of green and sustainable development; the mutant construction and screening system established in the application is not only suitable for the production of santalene, but also can be applied to the biosynthesis of other terpenoids (such as artemisinic acid and taxadiene), which has wide application value.

[0034] In summary, through systematic strain engineering, enzyme modification and high-throughput screening, the application successfully obtains high-yield santalene mutants and engineering bacteria, significantly improves the yield, provides important technical support for the industrial production of santalene, and has good application prospect and popularization value. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only a part of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0036] Figure 1 The pJN-32 vector map of the application; Figure 2pJN-32-TPS vector map of the present application; Figure 3 pJN-32-LPPS vector map of the present application; Figure 4 Sclareol standard curve of the present application; Figure 5 Mutant shake flask fermentation yield of the present application. DETAILED DESCRIPTION

[0037] The present application will be described in detail below with reference to the accompanying drawings and specific examples. It should be noted that the following examples are the best, preferred examples, and other alternative ways can also be implemented by those skilled in the art for some well-known technologies; and the accompanying drawings are only used to more specifically describe the examples, and are not intended to specifically limit the present application.

[0038] It should be noted that in the specification, "one embodiment", "embodiment", "exemplary embodiment", "some embodiments" and the like indicate that the described embodiment can include a specific feature, structure or property, but not necessarily every embodiment includes this specific feature, structure or property. In addition, when a specific feature, structure or property is described in combination with an embodiment, it should be within the knowledge of those skilled in the related art to achieve such a feature, structure or property in combination with other embodiments (whether or not explicitly described).

[0039] Generally, the terms can be understood at least in part from the context in which they are used. For example, depending on the context, the term "one or more" used herein can be used to describe any feature, structure or property in the singular or can be used to describe combinations of features, structures or properties in the plural. In addition, the term "based on" can be understood as not necessarily conveying a set of exclusive factors, but can instead, depending at least in part on the context, allow the presence of other factors not necessarily explicitly described.

[0040] Reference is made to Figures 1 to 5 the figures shown 1. Construction of the chassis strain The chassis strain used in the present application is Yarrowia lipolytica po1f strain. The strain is an auxotrophic strain obtained by integrating a heterologous SUC2 gene and knocking out Leu2 and ura3 genes through homologous recombination technology based on Yarrowia lipolytica wild-type strain W29 (ATCC 20460).

[0041] The construction method of the po1f strain includes the following steps: (1) Yarrowia lipolytica W29 wild-type strain as the starting strain; (2) By homologous recombination technology to integrate SUC2 gene from Saccharomyces cerevisiae, so that it can use sucrose as carbon source; (3) Using homologous recombination method to knock out Leu2 gene, obtain leucine auxotrophic strain; (4) Further knock out ura3 gene, obtain leucine and uracil double auxotrophic strain po1f.

[0042] The auxotrophic markers provide an effective screening marker system for subsequent genetic engineering.

[0043] 2. Construction of mevalonate pathway optimized strain In order to improve the supply of terpenoid precursors, the mevalonate (MVA) pathway of the po1f strain is optimized in the present application, and the strain YL1 is obtained.

[0044] The optimization method includes: inserting the heterologous tHMG1-Erg8-IDI gene fragment into the po1f genome by homologous recombination technology. Wherein: (5) The tHMG1 gene encodes a truncated HMG-CoA reductase, which catalyzes the reduction of HMG-CoA to mevalonate, and is one of the rate-limiting enzymes of the MVA pathway; (6) The Erg8 gene encodes phosphomevalonate kinase, which catalyzes the formation of mevalonate-5-phosphate into mevalonate-5-pyrophosphate; (7) The IDI gene encodes isopentenyl pyrophosphate isomerase, which catalyzes the reversible isomerization reaction between IPP and DMAPP.

[0045] Through the combined expression of the above-mentioned genes, the flux of the MVA pathway is significantly enhanced, and the generation amount of isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) is improved, which are the basic precursors of all terpenoids.

[0046] 3. Codon optimization of sclareol synthase and labdene diphosphate synthase sequences The present application provides the original sequences of sclareol synthase (TPS) and labdene diphosphate synthase (LPPS) derived from Salvia sclarea plants, which are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0047] In order to enable these plant-derived genes to be expressed efficiently in Yarrowia lipolytica, the coding sequences thereof are codon-optimized. The principles of codon optimization include: (8) According to the codon preference of Yarrowia lipolytica, replace the rare codons in the original sequence; (9) Avoid unstable secondary structure and too high GC content; (10) Eliminate sequence elements that can cause premature termination of translation; (11) Keep the amino acid sequence unchanged.

[0048] The expression level of the codon-optimized TPS and LPPS sequences in Yarrowia lipolytica is significantly improved, thereby enhancing the synthesis ability of sclareol.

[0049] 4. Construction of a sclareol-producing strain On the basis of the YL1 strain, a sclareol-producing strain is constructed by introducing codon-optimized LPPS and TPS genes.

[0050] The specific construction method comprises: (12) The codon-optimized LPPS-TPS gene fragment is integrated into the genome of the YL1 strain by homologous recombination to obtain a single-copy strain sclar1; (13) On the basis of the sclar1 strain, a copy of the codon-optimized LPPS-TPS gene fragment is integrated again to obtain a double-copy strain sclar2.

[0051] In the biosynthetic pathway of sclareol, LPPS catalyzes geranylgeranyl pyrophosphate (GGPP) to form pyrophosphate lycophylladienol (LPP), and TPS further catalyzes CPP to form the final product sclareol. By increasing the copy number of the LPPS-TPS gene, the expression amount of the key enzyme can be increased, thereby increasing the yield of sclareol.

[0052] The sclar2 strain can synthesize sclareol through fermentation with glucose as the carbon source, thereby providing a basic strain for subsequent enzyme modification and yield optimization.

[0053] 5. Preparation of TPS and LPPS random mutants In order to further improve the catalytic efficiency of TPS and LPPS and improve the binding ability of the enzyme and the substrate, error-prone PCR technology is used to randomly mutate the TPS and LPPS genes in the present application.

[0054] 5.1 Primer design PCR primers are designed according to the sequences of the TPS and LPPS genes (see Table 1 for primer sequences): ; Table 1 PCR primer sequences 5.2 Error-prone PCR reaction Random mutation of TPS and LPPS genes was performed using error-prone PCR. The error-prone PCR reaction conditions were set to allow random base substitution, insertion or deletion during PCR amplification, thereby introducing diversity at the gene level.

[0055] The construction of error-prone PCR reaction system includes: (14) using low-fidelity DNA polymerase or adding Mn in the reaction system 2+ to increase the rate of mismatch; (15) using unbalanced dNTP concentration to promote base mismatch; (16) appropriately increasing the number of PCR cycles to accumulate mutations.

[0056] The mutant library of TPS and LPPS obtained by error-prone PCR contains a large number of gene variants with different mutation sites.

[0057] 5.3 Mutant sequence characteristics The present application obtains a series of TPS and LPPS mutants through error-prone PCR and subsequent screening. These mutants contain the following mutation sites in the amino acid sequence compared with the original sequence: The mutation sites contained in the TPS mutant include but are not limited to: the 95th amino acid proline is mutated to leucine, glycine, alanine, valine, isoleucine, methionine, tryptophan, phenylalanine or proline; the 219th amino acid asparagine is mutated to serine, threonine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine; the 225th amino acid glutamine is mutated to arginine, serine, threonine, cysteine, tyrosine, asparagine, aspartic acid, glutamic acid, lysine or histidine; the 380th amino acid serine is mutated to proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan or phenylalanine; the 216th amino acid glutamic acid is mutated to valine, glycine, alanine, leucine, isoleucine, methionine, tryptophan, phenylalanine or proline; the 436th amino acid isoleucine is mutated to phenylalanine, glycine, alanine, valine, leucine, methionine, tryptophan or proline; the 184th amino acid methionine is mutated to leucine, glycine, alanine, valine, isoleucine, tryptophan, phenylalanine or proline; the 470th amino acid histidine is mutated to arginine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid or lysine.

[0058] The mutation sites contained in the LPPS mutant include, but are not limited to: the 128th amino acid histidine is mutated to proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan or phenylalanine; the 398th amino acid tryptophan is mutated to serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine; the 488th amino acid glutamic acid is mutated to glutamine, threonine, serine, cysteine, tyrosine, asparagine, aspartic acid, lysine, arginine or histidine; the 609th amino acid leucine is mutated to glutamine, threonine, serine, cysteine, tyrosine, asparagine, aspartic acid, glutamic acid, lysine, arginine or histidine.

[0059] The changes in these mutation sites can affect the spatial conformation of the enzyme, the geometry of the substrate binding site, the charge distribution of the catalytic active center, etc., thereby improving the catalytic efficiency and substrate binding capacity of the enzyme, and ultimately improving the yield of sclareol.

[0060] 6. Construction of mutant plasmids In order to express TPS and LPPS mutants in Yarrowia lipolytica, the present application uses pJN32 vector to construct mutant plasmids. pJN32 is a free shuttle non-fusion vector, which can exist in the form of a free plasmid in yeast cells, so that the target gene is transiently expressed. The vector carries an Amp resistance selection marker (for E. coli selection) and a Leu2 nutritional complementation marker (for Yarrowia lipolytica selection).

[0061] The construction steps of the mutant plasmid are as follows: (17) Vector preparation: linearize the pJN32 vector with appropriate restriction enzymes to prepare the vector backbone; (18) Fragment preparation: the TPS and LPPS mutant gene fragments obtained by error-prone PCR are purified and reserved; (19) Ligation reaction: ligate the mutant gene fragments with the linearized pJN32 vector, which can use homologous recombination or traditional ligase method; (20) Transformation of E. coli: transform the ligation product into E. coli competent cells and select on LB plates containing ampicillin; (21) Plasmid verification: pick single colonies for culture, extract plasmids, and verify the correctness of the inserted fragments by enzyme digestion or sequencing; (22) Construction of plasmid library: mix the verified mutant plasmids to form a mutant plasmid library.

[0062] Through the above method, the pJN32 recombinant plasmid integrated with the TPS or LPPS mutant gene is obtained, which is used for subsequent yeast transformation and high-throughput screening.

[0063] 7. Transformation and high-throughput screening of mutant plasmids 7.1 Chemical transformation The mutant plasmid is introduced into the sclar2 strain by lithium acetate (LiOAc) chemical transformation method. The specific operation is as follows: (23) The sclar2 strain is cultured in YPD medium to the logarithmic growth phase; (24) The bacterial cells are collected, washed with sterile water, and resuspended in LiOAc solution to prepare competent cells; (25) The mutant plasmid is mixed with the competent cells, and PEG, single-stranded DNA and other transformation reagents are added; (26) Heat shock treatment is carried out at an appropriate temperature; (27) The transformed cells are plated on sc-LEU auxotrophic medium plates and cultured at 30°C for 2-3 days.

[0064] Since the pJN32 vector carries the Leu2 marker, only the transformants successfully transformed with the plasmid can grow on the sc-LEU medium lacking leucine.

[0065] 7.2 High-throughput screening In order to quickly screen high-yield mutants from a large number of transformants, the present application uses 96-well plates for high-throughput screening. The specific steps are as follows: (28) Single transformants are picked from the sc-LEU plates and inoculated into each well of a 96-well plate, with 200 μL of sc-LEU medium containing 60 g / L glucose added to each well; (29) The plate is sealed with a gas-permeable sealing film and cultured at 30°C, 220 rpm on a shaker for 72-96 hours; (30) After the culture is completed, the fermentation broth in the 96-well plate is taken for product extraction and detection (see Section 8 for details); (31) According to the detection results, the transformants with significantly improved sclarol yield are selected; (32) The selected high-yield strains are subjected to secondary verification in fresh medium.

[0066] Through high-throughput screening in 96-well plates, hundreds of transformants can be evaluated in a short time, greatly improving the efficiency of mutant screening.

[0067] 8. Fermentation method 8.1 Shake flask fermentation For the selected high-yielding mutant strains, shake-flask fermentation was used for yield verification and evaluation. The fermentation method is as follows: (33) Seed culture: The strain was inoculated into YPD medium and cultured at 30℃ and 220 rpm for 24 hours to obtain seed culture; (34) Fermentation culture: The seed culture was transferred to the fermentation medium (sc-LEU medium, containing 60 g / L glucose) at an inoculum of 5% and cultured at 30℃ and 220 rpm; (35) Fermentation cycle: Usually 72-96 hours, which can be extended or shortened as needed; (36) Sampling and testing: Samples are taken regularly during the fermentation process to monitor cell growth and product accumulation.

[0068] 8.2 Microfermentation in 96-well plates In the high-throughput screening stage, micro-fermentation was carried out using 96-well plates under the following conditions: (37) The liquid volume per well is 200 μL; (38) The culture medium was sc-LEU liquid medium containing 60 g / L glucose; (39) Culture conditions: 30℃, 220rpm, culture for 72-96 hours; (40) Use breathable sealing film to ensure oxygen supply during the culture process.

[0069] 9. Product extraction and detection methods 9.1 Product Extraction Perillyl alcohol is a hydrophobic diterpenoid compound that can be extracted from fermentation broth using organic solvents. This invention uses n-hexane as the extraction solvent, and the specific method is as follows: (41) Take 250 μL of the fermentation broth and place it in a 1.5 mL centrifuge tube; (42) Add 500 μL of n-hexane; (43) Vortex for 5 minutes to allow perillyl alcohol to be fully transferred to the organic phase; (44) Centrifuge at 12000 rpm for 5 minutes to allow the aqueous and organic phases to separate completely; (45) Carefully aspirate the upper organic phase for gas chromatography analysis.

[0070] 9.2 Gas Chromatography Detection The extract was quantitatively analyzed using a gas chromatograph equipped with a flame ionization detector (FID) (GC-FID).

[0071] Chromatographic conditions: (46) Column: DB-17 capillary column; (47) Temperature program: initial temperature 180 °C, hold for 2 min; then ramp to 280 °C at 15 °C / min, hold for 5 min; (48) Post-run temperature: 150 °C; (49) Injection volume: 1 μL; (50) Carrier gas: nitrogen, flow rate 1.0 mL / min, constant flow mode; (51) Injection port temperature: 250 °C, pressure 17.4 psi; (52) Detector temperature: 280 °C; (53) Hydrogen flow rate: 30 mL / min; (54) Air flow rate: 400 mL / min; (55) Make-up gas (N2) flow rate: 25 mL / min.

[0072] Under the above chromatographic conditions, the retention time of sclareol is about 8.3 min.

[0073] 9.3 Standard curve establishment In order to quantitatively analyze sclareol in the sample, a standard curve needs to be established. The specific steps are as follows: (56) Preparation of standard mother liquor: weigh 1 g of sclareol standard, dissolve in 50 mL of n-hexane to prepare a mother liquor with a concentration of 20 mg / mL; (57) Serial dilution of standard: sequentially dilute the mother liquor with n-hexane to prepare standard working solutions with different concentrations, such as 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, and 20.0 mg / mL; (58) Gas chromatography detection: analyze each concentration of standard according to the conditions described in 9.2, and record the peak area; (59) Data processing: take the concentration of sclareol (X, unit mg / mL) as the abscissa and the peak area (Y) as the ordinate to draw a standard curve, and perform linear regression analysis to obtain the regression equation; (60) Sample quantification: according to the regression equation, the concentration of sclareol in the sample is calculated by measuring the peak area of the sample, and then the yield of sclareol in the fermentation broth is calculated.

[0074] The linear range of the standard curve should cover the concentration range of the sample, and the correlation coefficient R 2 should be ≥0.99 to ensure the accuracy of quantitative analysis.

[0075] 10. Plasmid extraction and sequencing verification For the selected high-yield strains, plasmids need to be extracted and sequenced to determine the mutation sites that lead to yield improvement.

[0076] 10.1 Yeast plasmid extraction Due to the robust cell wall of yeast cells, a cell wall disruption step is required before plasmid extraction. In this application, we use Lyticase to break the cell wall: (61) Culture the high-producing strain in liquid medium to the logarithmic growth phase; (62) Collect the bacterial cells and wash them with sterile water; (63) Resuspend the bacterial cells in a buffer containing Lyticase and incubate at 37°C for 30-60 minutes to degrade the cell wall and form protoplasts; (64) Use a yeast plasmid extraction kit and follow the instructions to extract the plasmid; (65) Dissolve the extracted plasmid in sterile water or TE buffer and store at -20°C.

[0077] 10.2 Sequencing analysis Send the extracted plasmid to a sequencing company (such as Suzhou JUNWENZHI Biotechnology Co., Ltd.) for Sanger sequencing or high-throughput sequencing. The sequencing primers cover the full-length coding region of the TPS or LPPS gene.

[0078] 10.3 Sequence analysis Compare the sequencing results with the original sequence to identify the mutation sites: (66) Base level: Determine the positions of base substitution, insertion or deletion; (67) Amino acid level: Translate the coding sequence to determine the positions and types of amino acid changes; (68) Structural analysis: Use bioinformatics tools to predict the impact of mutations on protein structure and function.

[0079] Through sequence analysis, the key mutation sites that lead to yield improvement can be determined, providing a basis for further rational design and site-directed mutagenesis.

[0080] Examples The following examples are used to further illustrate the technical solutions of the present application, but do not limit the scope of protection of the present application. The test methods in the examples are not specified, and are usually carried out according to conventional conditions or according to the conditions recommended by the reagent suppliers.

[0081] Example 1: Construction of chassis strain po1f 1.1 Materials (69) Strain: Yarrowia lipolytica wild-type strain W29 (ATCC 20460); (70) Medium: YPD medium (1% yeast extract, 2% peptone, 2% glucose).

[0082] 1.2 Methods Yarrowia lipolytica W29 wild-type strain as the starting strain, using homologous recombination technology for genetic modification: (71) First, the SUC2 gene from Saccharomyces cerevisiae is integrated, so that the strain can use sucrose as a carbon source; (72) Knock out the Leu2 gene by homologous recombination method, and screen to obtain a leucine auxotrophic strain; (73) Further knock out the ura3 gene, obtain a leucine and uracil double auxotrophic strain, named po1f.

[0083] 1.3 Results The auxotrophic strain po1f was successfully constructed. The strain cannot grow in the medium lacking leucine or uracil, and needs to supplement the corresponding nutrients. Through PCR amplification and sequencing verification, it is confirmed that the Leu2 and ura3 genes have been successfully knocked out, and the SUC2 gene has been successfully integrated.

[0084] Example 2: Construction of MVA pathway optimized strain YL1 2.1 Materials (74) Strain: po1f strain constructed in Example 1; (75) Gene: tHMG1-Erg8-IDI gene fragment, which is codon optimized for expression in Yarrowia lipolytica.

[0085] 2.2 Methods (76) Construction of homologous recombination fragment: The tHMG1-Erg8-IDI gene fragment is connected with the sequence homologous to the po1f genome at both ends (usually 500-1000 bp); (77) Transformation: LiOAc chemical transformation method is used to introduce the homologous recombination fragment into the po1f strain; (78) Screening: screen the transformants on the medium containing appropriate selection markers; (79) Verification: verify the correct integration of the gene fragment by colony PCR and sequencing.

[0086] 2.3 Results The MVA pathway optimized strain YL1 was successfully constructed. PCR and sequencing results confirmed that the tHMG1-Erg8-IDI gene fragment was correctly integrated into the po1f genome. Through RT-qPCR detection, the transcription level of related genes was significantly improved. Compared with the po1f strain, the content of IPP and DMAPP in YL1 strain increased significantly, which provided sufficient precursors for the synthesis of terpenoids in the subsequent.

[0087] Example 3: Construction of sclar1 and sclar2 strains for producing sclareol 3.1 Materials (80) Strain: YL1 strain constructed in Example 2; (81) Genes: Codon-optimized LPPS and TPS genes (SEQ ID NO: 1 and SEQ ID NO: 2).

[0088] 3.2 Construction of single-copy strain sclar1 (82) Construct codon-optimized LPPS-TPS gene fragments into expression cassettes, with homologous recombination arms at both ends; (83) Introduce expression cassettes into YL1 strain using LiOAc chemical transformation method; (84) Screen transformants on medium containing corresponding selection markers; (85) Obtain single-copy strain sclar1 with correct integration of LPPS-TPS genes through PCR and sequencing verification.

[0089] 3.3 Construction of double-copy strain sclar2 (86) Based on sclar1 strain, construct LPPS-TPS expression cassette again, and select different integration sites; (87) Integrate the second copy of LPPS-TPS gene into the sclar1 genome through homologous recombination method; (88) Screen and verify to obtain double-copy strain sclar2.

[0090] 3.4 Sclareol yield determination Ferment sclar1 and sclar2 strains in shake flasks, and detect sclareol yield using GC-FID: (89) Fermentation conditions: 30°C, 220 rpm, in YPD culture medium containing 60 g / L glucose in sc medium, and cultivate for 96 hours; (90) Product extraction: extract sclareol in fermentation broth with n-hexane; (91) Gas chromatography detection: quantitative analysis according to the foregoing method.

[0091] 3.5 Results Single-copy strain sclar1 and double-copy strain sclar2 were successfully constructed. PCR and sequencing results confirmed that LPPS-TPS genes were correctly integrated. Fermentation results showed that sclar1 strain could produce sclareol, and sclar2 strain had two copies of LPPS-TPS genes, with an increase of about 30% in yield compared with sclar1. This indicates that increasing gene copy number is an effective strategy to improve sclareol yield.

[0092] Example 4: Construction of TPS and LPPS random mutants 4.1 Materials (92) Template: Codon-optimized TPS and LPPS gene plasmids; (93) Primers: PCR primers as shown in Table 1; (94) Enzymes: Error-prone PCR-specific enzymes (e.g., Mutazyme II or Taq polymerase + Mn 2+ ); (95) Vectors: pJN32 episomal shuttle plasmid.

[0093] 4.2 Error-prone PCR reaction Establish error-prone PCR reaction system (50 μL): (96) Template DNA: 100 ng; (97) Primers (forward and reverse): 0.5 μM each; (98) dNTPs: 0.2 mM each of dATP and dGTP, 1 mM each of dCTP and dTTP (unbalanced dNTP concentration); (99) MnCl2: 0.5 mM (to increase the rate of mismatches); (100) Taq DNA polymerase: 2.5 U; (101) 10x PCR buffer: 5 μL.

[0094] PCR program: (102) 95 °C pre-denaturation for 5 minutes; (103) 95 °C denaturation for 30 seconds, 55 °C annealing for 30 seconds, 72 °C extension for 2 minutes, for a total of 30 cycles; (104) 72 °C final extension for 10 minutes.

[0095] 4.3 PCR product purification Use the PCR product purification kit to purify the error-prone PCR product and remove excess primers, dNTPs, and enzymes.

[0096] 4.4 Mutant plasmid construction (105) Linearization of pJN32 vector: Cut the pJN32 vector with appropriate restriction enzymes (e.g., EcoRI and BamHI) and purify and recover the linearized vector; (106) Ligation reaction: Mix the purified error-prone PCR product with the linearized pJN32 vector at a molar ratio of 1:3, and use Gibson Assembly or traditional ligase for ligation; (107) Transformation of E. coli: Transform the ligation product into E. coli DH5α competent cells and spread on LB plates containing ampicillin (100 μg / mL); (108) Plasmid extraction: Randomly pick hundreds of single colonies from the plates, extract plasmids respectively, and form the mutant plasmid library.

[0097] 4.5 Results The mutant libraries of TPS and LPPS were successfully obtained by error-prone PCR. Ten clones were randomly selected from the libraries for sequencing. The results showed that each clone contained 2-5 base substitutions on average, and the mutation rate was about 0.5-2 kb, which was consistent with the expected effect of error-prone PCR. The mutant plasmid library constructed provided a rich resource of mutant for subsequent high-throughput screening. -1

[0098] Example 5: Transformation of mutant plasmids and high-throughput screening 5.1 Materials (109) Strain: sclar2 strain constructed in Example 3; (110) Plasmid: TPS and LPPS mutant plasmid library constructed in Example 4; (111) Medium: sc-LEU liquid medium (0.67% yeast nitrogen source, 2% glucose, amino acid mixture without leucine).

[0099] 5.2 Chemical transformation (112) The sclar2 strain was cultured in YPD medium to OD 600 = 1.0-1.5; (113) The bacterial cells were collected and washed twice with sterile water; (114) Resuspended in 1 / 10 volume of 100 mM LiOAc solution to prepare competent cells; (115) Take 50 μL of competent cells, add 1 μg of mutant plasmid, 5 μL of carrier DNA (2 mg / mL), and 300 μL of transformation mixture (40% PEG4000, 100 mM LiOAc, 10 mM Tris-HCl pH 7.5, 1 mM EDTA); (116) Heat shock at 42°C for 40 minutes; (117) Centrifuge at 12000 rpm for 1 minute, remove the supernatant, and resuspend in 100 μL of sterile water; (118) Spread the transformation solution on sc-LEU solid medium plates and incubate at 30°C for 2-3 days.

[0100] 5.3 High-throughput screening (119) Randomly pick single colonies from the transformation plates and inoculate each well of a 96-well plate; (120) Add 200 μL of sc-LEU medium (containing 60 g / L glucose) to each well; ​(121)Seal the plates with air-permeable sealing film, and incubate at 30℃ with 220 rpm shaking for 96 hours; (122)After the incubation, take 250 μL of the fermentation broth from each well for product extraction and detection (see Example 6 for the method); (123)According to the GC-FID detection results, select the strains with more than 20% increase in sclareol production compared with the control strain (sclar2 transformed with empty vector).

[0101] 5.4 Results About 500 transformants were screened, of which about 15% showed increased production of sclareol. Ten high-yield strains were selected, including four TPS mutants (named TPS-1 to TPS-4) and six LPPS mutants (named LPPS-1 to LPPS-6). The sclareol production of these high-yield strains increased by 20-41% compared with the control.

[0102] Example 6: Detection of Sclareol and Establishment of Standard Curve 6.1 Preparation of Sclareol Standard (124) Accurately weigh 1.0 g of sclareol standard (to the nearest 0.0001 g); (125) Dissolve the standard in 50 mL of n-hexane to prepare a mother liquor with a concentration of 20 mg / mL; (126) Dilute the mother liquor with n-hexane to prepare standard working solutions with concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0, and 20.0 mg / mL, respectively.

[0103] 6.2 Establishment of Standard Curve (127) The GC-FID detection conditions are as follows: Chromatographic column: DB-17 capillary column (30 m x 0.25 mm x 0.25 μm); Column temperature program: initial temperature 180℃, hold for 2 minutes; increase to 280℃ at 15℃ / min, hold for 5 minutes; Injection port temperature: 250℃; Detector temperature: 280℃; Carrier gas: nitrogen, flow rate 1.0 mL / min; Injection volume: 1 μL.

[0104] (128) Inject each concentration of standard working solution, and record the chromatographic peak area; (129) Plot the standard curve with the concentration of sclareol (X, mg / mL) as the abscissa and the peak area (Y) as the ordinate; (130) Perform linear regression analysis to obtain the regression equation.

[0105] 6.3 Sample detection (131) Take 250 μL of the fermentation broth into a 1.5 mL centrifuge tube; (132) Add 500 μL of n-hexane; (133) Vortex for 5 minutes; (134) Centrifuge at 12000 rpm for 5 minutes; (135) Carefully pipette the upper organic phase for GC-FID analysis; (136) According to the standard curve regression equation, calculate the concentration of sclareol in the sample.

[0106] 6.4 Results The linear equation of the established standard curve is: Y = a × X + b (where a and b are constants), the correlation coefficient R 2 = 0.9994, and the linear range is 0.1-10.0 mg / mL. The retention time of sclareol is 8.3 ± 0.1 minutes. This method is accurate and sensitive, and is suitable for quantitative analysis of sclareol in fermentation broth.

[0107] Example 7: Verification and yield determination of mutants 7.1 Verification of mutant sequences (137) Plasmid extraction: Extract the plasmid from the high-yield strains screened in Example 5. Specific method: Collect the bacterial cells, treat with a buffer containing snailase to form protoplasts, and then extract the plasmid using a yeast plasmid extraction kit; (138) Sequencing analysis: Send the extracted plasmid to Suzhou Jinvivobiotech Co., Ltd. for Sanger sequencing, and the sequencing primers cover the full length of the TPS or LPPS gene; (139) Sequence alignment: Align the sequencing results with the original sequence to determine the mutation site.

[0108] 7.2 Shake flask fermentation verification Select the four mutants TPS-1, TPS-2, LPPS-1, and LPPS-2 with the most significant yield increase for shake flask fermentation verification: (140) Seed culture: Inoculate the strains into YPD medium and cultivate at 30°C, 220 rpm for 24 hours; (141) Fermentation culture: Transfer the strains into 250 mL shake flasks (50 mL of liquid) containing sc-LEU medium (containing 60 g / L glucose) at a 5% inoculation amount, and cultivate at 30°C, 220 rpm for 96 hours; (142) Sample analysis: After fermentation, take samples and extract and detect sclareol according to the method of Example 6; (143)Control group: sclar2 strain fermented and transformed with empty vector at the same time as control.

[0109] 7.3 Results The sequencing results show that the high-yield mutant indeed contains multiple amino acid mutation sites, which are located at the positions described in the claims of the present application. The shake flask fermentation results are as follows: (144) The yield of the control strain (sclar2+empty vector) is 100 mg / L (100%). (145) The yield of the TPS-1 mutant strain is 135 mg / L, which is increased by 38.5% compared with the control; (146) The yield of the TPS-2 mutant strain is 141 mg / L, which is increased by 14.2% compared with the control; (147) The yield of the LPPS-1 mutant strain is 128 mg / L, which is increased by 25.5% compared with the control; (148) The yield of the LPPS-2 mutant strain is 132 mg / L, which is increased by 40.2% compared with the control.

[0110] The above results show that the TPS and LPPS mutants obtained by random mutation can significantly increase the yield of sclareol, with a maximum increase of 41%. These mutants and the key mutation sites they contain provide an important molecular basis for further optimizing the biosynthesis of sclareol, and have good application prospects for industrial production.

[0111] The present application successfully obtains Yarrowia lipolytica engineering strains capable of efficiently producing sclareol through systematic strain construction, enzyme modification and high-throughput screening. The mutants obtained by random mutation of TPS and LPPS through error-prone PCR technology increase the yield of sclareol by about 41%. The mutation sites and engineering strains provided by the present application provide technical support for the industrial production of sclareol, and have important application value.

[0112] Reference: [1] Chen M, Ye L, Yu H, et al. Research progress in metabolic engineering of Saccharomyces cerevisiae for synthesis of terpenoids. Biotechnology, 2021, 37(6): 2085-2104 [2] Qiang S. Yarrowia lipolytica engineering strain with high yield of sclareol, its construction method, application and process CN117264792B [P] 2024-02-02 [3] Chen, J., Huang, L., Ye, BC. et al. Combinatorial metabolicengineering of Yarrowia lipolytica for high-level production of the plant-derived diterpenoid sclareol. Microb Cell Fact 24, 110 (2025). https: / / doi.org / 10.1186 / s12934-025-02744-7 [4] MEI-LI SUN. Constructing a green oleaginous yeast cell factory for sustainable production of the plant-derived diterpenoid sclareol†[J]. Green Chemistry, 2024, 26 9: Pages 5202-5210. DOI:10.1039 / d3gc04949c. [5] Cao X, Yu W, Chen Y, Yang S, Zhao ZK, Nielsen J, Luan H, Zhou YJ. Engineering yeast for high-level production of diterpenoid sclareol. Metab Eng. 2023 Jan;75:19-28. doi: 10.1016 / j.ymben.2022.11.002. Epub 2022 Nov 9. PMID: 36371032. [6] Zhang JH, Cui ZY, Qi QS, et al. Development of expression andregulation tools in Yarrowia lipolytica and research progress in synthesis of natural products. Bioengineering (Beijing), 2022, 38(2): 478-505 [7] Yang W, Zhou Y, Liu W, Shen H, Zhao ZK. [Engineering Saccharomyces cerevisiae for sclareol production]. Sheng Wu Gong Cheng Xue Bao. 2013 Aug;29(8):1185-92. Chinese. PMID: 24364354. The present application encompasses any alternatives, modifications, equivalent methods and solutions made within the spirit and scope of the present application. In order to make the public thoroughly understand the present application, specific details are described in the following preferred embodiments of the present application, and the present application can be completely understood without the description of these details by those skilled in the art. In addition, in order to avoid unnecessary confusion to the essence of the present application, well-known methods, processes, procedures, elements and circuits, etc. are not described in detail.

[0113] The above is only the preferred embodiment of the present application, and it should be pointed out that for those skilled in the art, without departing from the principle of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

[0114] SEQ ID NO: 1 <110> Nantong Xingbeitao Biotechnology Co., Ltd. <120> A TPS / LPPS mutant for improving the yield of sclareol, and an engineering bacterium and application thereof <160> 2 <210> 1 <211> 525 <212> PRT <213> Artificial Sequence <220> <223> Codon-optimized sclareol synthase (TPS) from Salvia sclarea, optimized for expression in Yarrowia lipolytica <400> 1 1 MAKMKENFKR EDDKFPTTTT LRSEDIPSNL CIIDTLQRLG VDQFFQYEIN TILDNTFRLW 61 QEKHKVIYGN VTTHAMAFRL LRVKGYEVSS EELAPYGNQE AVSQQTNDLP MIIELYRAAN 121 ERIYEEERSL EKILAWTTIF LNKQVQDNSI PDKKLHKLVE FYLRNYKGIT IRLGARRNLE 181 LYDMTYYQAL KSTNRFSNLC NEDFLVFAKQ DFDIHEAQNQ KGLQQLQRWY ADCRLDTLNF 241 GRDVVIIANY LASLIIGDHA FDYVRLAFAK TSVLVTIMDD FFDCHGSSQE CDKIIELVKE 301 WKENPDAEYG SEELEILFMA LYNTVNELAE RARVEQGRSV KEFLVKLWVE ILSAFKIELD 361 TWSNGTQQSF DEYISSSWLS NGSRLTGLLT MQFVGVKLSD EMLMSEECTD LARHVCMVGR 421 LLNDVCSSER EREENIAGKS YSILLATEKD GRKVSEDEAI AEINEMVEYH WRKVLQIVYK 481 KESILPRRCK DVFLEMAKGT FYAYGINDEL TSPQQSKEDM KSFVF SEQ ID NO: 2 <210> 2 <211> 722 <212> PRT <213> Artificial Sequence <220> <223> Codon-optimized limonene diol phosphate synthase (LPPS) from Salvias clarea optimized for expression in Yarrowia lipolytica <400> 2 1 MASQASEKDI SLVQTPHKVE VNEKIEESIE YVQNLLMTSG DGRISVSPYD TAVIALIKDL 61 KGRDAPQFPS CLEWIAHHQL ADGSWGDEFF CIYDRILNTL ACVVALKSWN LQSDIIEKGV 121 TYIKENVHKL KGANVEHRTA GFELVVPTFM QMATDLGIQG LPYDHPLIKE IADTKQQRLK 181 EIPKDLVYQM PTNLLYSLEG LGDLEWERLL KLQSGNGSFL TSPSSTAAVL MHTKDEKCLK 241 YIENALKNCD GGAPHTYPVD IFSRLWAIDR LQRLGISRFF QHEIKYFLDH IESVWEETGV 301 FSGRYTKFSD IDDTSMGVRL LKMHGYDVDP NVLKHFKQQD GKFSCYIGQS VESASPMYNL 361 YRAAQLRFPG EEVLEEATKF AFNFLQEMLV KDRLQERWVI SDHLFDEIKL GLKMPWYATL 421 PRVEAAYYLD HYAGSGDVWI GKSFYRMPEI SNDTYKELAI LDFNRCQTQH QLEWIQMQEW 481 YDRCSLSEFG ISKRELLRSY FLAAATIFEP ERTQERLLWA KTRILSKMIT SFVNISGTTL 541 SLDYNFNGLD EIISANEDQG LAGTLLATFH QLLDGFDIYT LHQLKHVWSQ WFMKVQQGEG 601 SGGEDAVLLA NTLNICAGLN EDVLSNNEYT ALSTLTNKIC NRLAQIQDNK ILQVVDGSIK 661 DKELEQDMQA LVKLVLQENG GAVDRNIRHT FLSVSKTFYY DAYHDDETTD LHIFKVLFRP 721 VV。

Claims

1. A sclareol synthase mutant, characterized in that, The mutant is encoded by the codon-optimized gene of sclerol synthase shown in SEQ ID NO: 1, and comprises one or more mutation sites in the amino acid sequence as follows: the 95th amino acid proline is mutated into leucine, glycine, alanine, valine, isoleucine, methionine, tryptophan or phenylalanine; the 184th amino acid methionine is mutated into leucine, glycine, alanine, valine, isoleucine, tryptophan, phenylalanine or proline; the 216th amino acid glutamic acid is mutated into valine, glycine, alanine, leucine, isoleucine, methionine, tryptophan, phenylalanine or proline; the 219th amino acid asparagine is mutated into serine, threonine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine; the 225th amino acid glutamine is mutated into arginine, serine, threonine, cysteine, tyrosine, asparagine, aspartic acid, glutamic acid, lysine or histidine; the 380th amino acid serine is mutated into proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan or phenylalanine; the 436th amino acid isoleucine is mutated into phenylalanine, glycine, alanine, valine, leucine, methionine, tryptophan or proline; the 470th amino acid histidine is mutated into arginine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid or lysine.

2. The sclareol synthase mutant of claim 1, wherein, The mutant comprises mutations at any two or more of the 95th, 219th, 225th, 380th, 216th, 436th, 184th or 470th sites.

3. A mutant dammarene diol diphosphatase enzyme characterized in that, The mutant is encoded by the codon-optimized gene of sclerol synthase shown in SEQ ID NO: 1, and comprises one or more mutation sites in the amino acid sequence as follows: the 95th amino acid proline is mutated into leucine, glycine, alanine, valine, isoleucine, methionine, tryptophan or phenylalanine; the 184th amino acid methionine is mutated into leucine, glycine, alanine, valine, isoleucine, tryptophan, phenylalanine or proline; the 216th amino acid glutamic acid is mutated into valine, glycine, alanine, leucine, isoleucine, methionine, tryptophan, phenylalanine or proline; the 219th amino acid asparagine is mutated into serine, threonine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, arginine or histidine; 4. The mutant dammarene diol diphosphatase of claim 3, wherein, the 225th amino acid glutamine is mutated into arginine, serine, threonine, cysteine, tyrosine, asparagine, aspartic acid, glutamic acid, lysine or histidine; the 380th amino acid serine is mutated into proline, glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan or phenylalanine; the 436th amino acid isoleucine is mutated into phenylalanine, glycine, alanine, valine, leucine, methionine, tryptophan or proline; the 470th amino acid histidine is mutated into arginine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid or lysine. The mutant comprises mutations at any two or more of the 95th, 219th, 225th, 380th, 216th, 436th, 184th or 470th sites.

5. A nucleotide sequence encoding the sclerol synthase mutant of claim 1 or 2.

6. A nucleotide sequence encoding the sclerol synthase mutant of claim 3 or 4.

7. A recombinant expression vector, characterized in that, The expression vector contains the nucleotide sequence encoding the sclareol synthase mutant of claim 5, or the nucleotide sequence encoding the mutase of geranylgeranyl diphosphate synthase mutant of claim 6.

8. A recombinant engineered bacterium, characterized in that, The genome of the recombinant engineering bacteria contains the codon-optimized LPPS-TPS gene fragment, or the recombinant engineering bacteria contains the recombinant expression vector of claim 7. The recombinant engineering bacteria is Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris or Escherichia coli.

9. The recombineering bacteria of claim 8, wherein, The recombinant engineering bacteria is Yarrowia lipolytica, and the genome of the Yarrowia lipolytica contains the tHMG1-Erg8-IDI gene fragment and one or two copies of the codon-optimized LPPS-TPS gene fragment.

10. Use of the sclareol synthase mutant of claim 1 or 2, or the mutase of geranylgeranyl diphosphate synthase mutant of claim 3 or 4, or the recombinant engineering bacteria of claim 8 or 9 in the production of sclareol or its precursor, pyrophosphate lycophyllen diphosphate.

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