Pichia pastoris endogenous constitutive promoter independent of culture medium carbon source and application of pichia pastoris endogenous constitutive promoter

Abstract

The invention discloses a pichia pastoris endogenous constitutive promoter independent of a culture medium carbon source and application of the pichia pastoris endogenous constitutive promoter. The constitutive promoter is a 6-phosphogluconate dehydrogenase gene promoter, and the nucleotide sequence of the constitutive promoter is as shown in SEQ ID No. 1. The promoter provided by the invention shows constitutive expression in culture media taking glucose, glycerol and methanol as carbon sources, and the transcription intensities in the three culture media are similar. The promoter overcomes the problem that the activity of an existing pichia pastoris constitutive promoter is easily influenced by a carbon source component of a culture medium, and provides a reliable regulation element for stable expression of a target gene in pichia pastoris. The expression plasmid constructed by using the gene can realize stable expression of a target gene in pichia pastoris, and has a good application prospect in the biotechnical fields of heterologous synthesis pathway construction and the like.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering and biotechnology, and in particular to an endogenous constitutive promoter of Pichia pastoris that is independent of a culture medium carbon source and an application thereof. Background Art

[0002] Pichia pastoris is a methylotrophic yeast with advantages such as efficient methanol utilization, high-density fermentation, and low secreted protein background. It is widely used in heterologous protein expression, biofuel synthesis, and biomanufacturing of small molecule compounds. In these biosynthetic processes, promoters, as the core elements of gene transcription regulation, determine the initiation and intensity of gene expression, thereby affecting the synthesis efficiency of the target product.

[0003] Among the reported Pichia promoters, inducible promoters such as the alcohol oxidase 1 (AOX1) promoter have high transcription levels under methanol induction conditions, but they are highly dependent on methanol. Therefore, during the fermentation process, it is necessary to accurately control the amount and time of methanol addition and maintain a stable methanol concentration, which is a relatively complicated operation (New Biotechnology, 2013, 25; 30 (4): 385-404.). If the methanol concentration changes, the addition is delayed or interrupted, etc., it may cause a significant change in the gene expression level, which will adversely affect the stability and yield of the product synthesis.

[0004] Although existing constitutive promoters do not require inducers, they often show carbon source preference. For example, the most commonly used glyceraldehyde-3-phosphate dehydrogenase promoter in Pichia pastoris (P GAP ), showing high transcriptional activity in a medium with glucose as the carbon source, but its activity decreased by one-third to two-thirds in a medium with glycerol or methanol as the carbon source (Gene, 1997, 186(1):37-44.). When the Pichia cell factory constructed based on this type of constitutive promoter switches between different carbon sources to optimize cell growth and product synthesis, it is difficult to ensure that the transcription level of its downstream genes remains consistent throughout the process. Therefore, it is of great application value to discover and identify endogenous constitutive promoters of Pichia that are independent of the carbon source of the culture medium. Summary of the invention

[0005] In order to address the shortcomings and deficiencies of the prior art, the present invention aims to provide a Pichia pastoris endogenous constitutive promoter that is independent of the carbon source of the culture medium and its application.

[0006] According to a first aspect of an embodiment of the present application, a Pichia pastoris endogenous constitutive promoter that is independent of a culture medium carbon source is provided, and the DNA sequence of the Pichia pastoris endogenous constitutive promoter is shown as SEQ ID No.1.

[0007] Furthermore, the Pichia pastoris endogenous constitutive promoter is located in the 5' non-coding region before the start codon ATG of gene PAS_chr3_0277 (NCBI GeneID: 8200105), and the DNA sequence length is 500 bases. For the convenience of subsequent description, the above promoter is named P 0277 , the corresponding gene was named 0277.

[0008] According to a second aspect of an embodiment of the present application, there is provided a use of the Pichia pastoris constitutive promoter described in the first aspect in an expression cassette, wherein the expression cassette comprises the Pichia pastoris endogenous constitutive promoter described in the first aspect, a structural gene or a regulatory gene connected downstream thereof, and a terminator.

[0009] Furthermore, the process of constructing a limonene biosynthesis pathway based on the Pichia pastoris constitutive promoter includes:

[0010] The Pichia pastoris constitutive promoter P 0277 and PAS_chr1-4_0510 promoter P 0510 , constitutive strong promoter P GAP and P TEF1 The truncated limonene synthase gene CltLS2, the NPP synthase gene SlNDPS1, and the acetyl-CoA synthase mutant gene SeACS were inserted into L641P and upstream of the isopentenyl pyrophosphate isomerase gene ScIDI1. GAP Insert the truncated HMG-CoA reductase 1 gene SctHMG1 upstream to construct five expression cassettes P 0277 -CltLS2-T AOX1 , P 0510 -SlNDPS1-T AOX1 , P GAP -SeACS L641P -T CYC1 , P TEF1 -ScIDI1-T AOX1 , P GAP -SctHMG1-T CYC1 ;

[0011] P 0277 -CltLS2-T AOX1 and P 0510 -SlNDPS1-T AOX1 Inserted into the neutral site PNSI-2 of Pichia pastoris, P GAP -SeACS L641P -T CYC1 Inserted into the neutral site PNSI-16 of Pichia pastoris, PTEF1 -ScIDI1-T AOX1 and P GAP -SctHMG1-T CYC1 Insertion into the neutral site Int12 of Pichia pastoris resulted in an engineered strain carrying an expression cassette related to the limonene biosynthesis pathway.

[0012] The promoter P provided by the present invention 0277 It is constitutively expressed in glucose, glycerol and methanol culture media, and the transcription intensity is similar in the three culture media. This carbon source-independent and transcriptionally stable characteristic can make the growth of Pichia pastoris cells and protein expression orderly coordinated, avoiding expression disorders caused by changes in the carbon source of the culture medium, and has application advantages in the heterologous expression of environmentally sensitive or complex proteins. Therefore, the promoter P provided by the present invention 0277 It can be used to construct expression cassettes and corresponding plasmids, and enriches the constitutive promoter library that can be used to construct natural product synthesis pathways.

[0013] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0015] Figure 1 The growth curves of Pichia pastoris GS115 WT strain in YPD, BMGY and BMMY media;

[0016] Figure 2 The relative transcription level bar graph of gene 0277 and GAP when the Pichia pastoris GS115 WT strain was cultured in YPD and BMGY medium for 8 h, 24 h, 48 h, and in BMMY medium for 4 h, 16 h, 48 h, 72 h, and 96 h (ACT1 is the internal reference gene);

[0017] Figure 3 To carry P 0277 -CltLS2-T AOX1 and P 0510 -SlNDPS1-T AOX1 Map of plasmid PNSI2-CltLS2-SlNDPS1 expressing cassette;

[0018] Figure 4 To carry P GAP -SeACS L641P -T CYC1 Plasmid PNSI16-SeACS expressing cassette L641P Atlas;

[0019] Figure 5 To carry P TEF1 -ScIDI1-T AOX1 and P GAP -SctHMG1-T CYC1 Map of plasmid Int12-tHMG1-IDI1 expressing cassette;

[0020] Figure 6 It is the gas chromatography result of the product of the engineered strain Pplim1h fermentation for 72 hours, lim-non is the negative control, and lim-standard is the limonene standard control;

[0021] Figure 7 The mass spectrometry results of limonene obtained by fermenting the engineered strain Pplim1h for 72 hours, lim-standard represents the limonene standard;

[0022] Figure 8 This is the transcription level of exogenous genes for limonene synthesis in the engineered strain Pplim1h. DETAILED DESCRIPTION

[0023] Here, exemplary embodiments are described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application.

[0024] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0025] In the following examples, unless otherwise specified, all reagents and consumables involved are purchased from common reagent manufacturers in the field. Similarly, unless otherwise specified, the experimental methods and technical means used are of the types commonly used in the field. Among them, the detailed steps of plasmid extraction, DNA fragment recovery and ligation, RNA extraction and reverse transcription and other related operations can refer to the instructions provided with the corresponding kits. For details on the preparation of culture media, cultivation of yeast cells, etc., please refer to Invitrogen TM The company's operating manual "EasySelect TMPichia ExpressionKit" (manual source URL: https: / / assets.fishersci.com / TFS-Assets / LSG / manuals / easyselect_man.pdf).

[0026] Description of the experimental materials used in the following examples:

[0027] Pichia pastoris GS115 WT strain was purchased from Invitrogen TM Company; Saccharomyces cerevisiae BY4741 WT strain and DH5α WT strain were purchased from Beijing Huayueyang Biological Company.

[0028] pPIC9 plasmid was purchased from Invitrogen TM Company; pGGA plasmid was purchased from New England Biolabs; the plasmid pZ-panARS-hCas9-sgRNA carrying the CRISPR / Cas system has been published in the literature (World Journal of Microbiology and Biotechnology, 2019, 35(6):79.).

[0029] Strong constitutive promoter P GAP and P TEF1 Sequence amplification from Invitrogen TM The company's commercial plasmid pGAPZ A.

[0030] The limonene synthase gene CltLS2 and the NPP synthase gene SlNDPS1 are derived from plasmid pCS13 and have been published in the literature (ACS Synthetic Biology, 2019, 8(5):968-975.); the acetyl-CoA synthase mutant gene SeACS L641P Derived from plasmid pPDH4, it has been published in the literature (Biochemical Engineering Journal, 2021, 176: 108155.); the truncated HMG-CoA reductase 1 gene tHMG1 and isopentenyl pyrophosphate isomerase gene IDI1 sequences were amplified from the Saccharomyces cerevisiae BY4741 WT genome, and the specific sequences have been published in the literature (Bioresources and Bioprocessing, 2023, 10(1): 38.).

[0031] The Cre / loxp recycling system has been published in the literature (Scientific Reports, 2017, 7(1): 11113.).

[0032] Integration sites PNSI-2 and PNSI-16 have been disclosed in the literature (Nucleic Acids Research, 2021, 49(13):7791-7805.); integration site Int12 has been disclosed in the literature (ACS Synthetic Biology, 2022, 11(2):623-633.).

[0033] Example 1: P 0277 Transcription activity assay in three culture media

[0034] In order to detect the characteristics of the promoter of the present invention in different culture media, the GS115 WT strain was fermented in YPD, BMGY and BMMY liquid culture media, respectively, and the transcription level of the gene 0277 in different culture environments and different growth stages was detected.

[0035] The specific operations are as follows:

[0036] (1) Determination of growth curve

[0037] A. Activation of bacterial strains: Take out the GS115 WT strain stored in glycerol from the -80°C refrigerator, streak it on a YPD solid plate, and culture it in a 30°C incubator for 2-3 days until a single colony grows.

[0038] B. Seed solution preparation: Select a robust GS115 WT single clone and inoculate it into a test tube containing 5 mL of YPD liquid medium, and culture it at 30°C and 220 rpm with shaking overnight.

[0039] C. Inoculation and culture in YPD and BMGY medium: Take appropriate amount of the above seed solution and inoculate into 250mL shake flasks containing 50mL YPD and BMGY medium respectively. The initial OD 600 About 0.2. Culture continuously at 30℃ and 220rpm for 48h, take samples at intervals and draw a growth curve.

[0040] D. Inoculation and culture in BMMY medium: When the bacterial OD in BMGY medium 600 When it reaches 2-6, collect the total OD 600 Transfer 50 μL of bacterial suspension to a sterilized 50 mL centrifuge tube, collect the bacteria by centrifugation at 5000 rpm, wash twice with BMMY medium, and transfer to 50 mL BMMY medium containing 100 μL biotin solution and 500 μL methanol. Cultivate continuously at 30°C and 220 rpm for 96 h, add 500 μL methanol every 24 h, take samples at intervals, and draw growth curves.

[0041] Results: Pichia pastoris GS115 WT strain entered the stationary phase after growing in YPD medium for about 36 hours. 600 32 to 34. After growing in BMGY medium for about 40 hours, the bacterial OD 600 The growth of GS115 WT strain in BMMY medium containing 1% methanol had no obvious logarithmic growth phase and plateau phase. The OD of the bacteria after 96 hours of culture was 600 About 8. See the growth curve Figure 1 .

[0042] (2) Detection of 0277 gene transcription level

[0043] A. RNA sample collection: According to the growth curves of GS115 WT strain in the above three culture media, 8h, 24h, and 48h were selected as sampling points in YPD and BMGY culture media, and 4h, 16h, 48h, 72h, and 96h were selected as sampling points in BMMY culture media to cover the entire growth cycle of the strain in the three culture media. In the three culture media, the total cell mass was collected as OD 600 ~10 yeast cells. Centrifuge at 12000 rpm and discard the supernatant.

[0044] B. RNA extraction: According to the instructions of OMEGA's yeast RNA extraction kit (OMEGA bio-tek), total RNA of yeast cell samples was extracted, and then the integrity of total RNA was detected by 1% (w / v) agarose gel electrophoresis, and the concentration of total RNA was measured by a micro spectrophotometer (Nano-400A).

[0045] C. Genomic DNA digestion and reverse transcription: Reference IIQ RT SuperMix for qPCR (Novagen) instructions.

[0046] D. RT-qPCR analysis: Primers were set near the 3' end of the gene ORF, and the size of the amplified product was 100-200 bp. For specific operation methods, please refer to the instruction manual of ChamQ Blue Universal SYBR qPCR Master Mix (Novozymes).

[0047] E. Taking ACT1 (NCBI Gene ID: 8200200) as the internal reference gene, the transcription level of 0277 relative to the internal reference gene was calculated by the ΔCt value, and the control gene was GAP (NCBI Gene ID: 8198905).

[0048] Results: In YPD, BMGY and BMMY culture media, the gene 0277 corresponding to the endogenous promoter of the present invention showed constitutive expression at different culture time points, and the transcription level was about twice that of the internal reference gene, and there was no carbon source preference. Therefore, the promoter of the present invention showed good application potential. Specific results are shown in Figure 2 .

[0049] Example 2: Construction of a plasmid carrying an expression cassette related to the limonene synthesis pathway

[0050] The constitutive promoter P shown in SEQ ID No.1 0277 、PAS_chr1-4_0510 promoter P 0510 , constitutive strong promoter P GAP and P TEF1 The truncated limonene synthase gene CltLS2, the NPP synthase gene SlNDPS1, and the acetyl-CoA synthase mutant gene SeACS were inserted into L641P and upstream of the isopentenyl pyrophosphate isomerase gene ScIDI1. GAP Insert the truncated HMG-CoA reductase 1 gene SctHMG1 upstream to construct five expression cassettes P 0277 -CltLS2-T AOX1 , P 0510 -SlNDPS1-T AOX1 , P GAP -SeACS L641P -T CYC1 , P TEF1 -ScIDI1-T AOX 1. P GAP -SctHMG1-T CYC1 Then, the expression cassettes were assembled into three plasmids to obtain plasmids PNSI2-CltLS2-SlNDPS1, PSNI16-SeACS L641P and Int12-tHMG1-IDI1. The specific operations are as follows:

[0051] (1) Use plasmid extraction kit (Novozyme) to extract pGGA and pPIC9 plasmids;

[0052] (2) Extraction of genomes of Pichia pastoris GS115 WT strain and Saccharomyces cerevisiae BY4741 WT strain:

[0053] Collect 1mL of the above cells cultured overnight in YPD medium, collect the precipitate after high-speed centrifugation, add 50mg of acid-washed glass sand, then add 200μL of yeast lysis solution, 200μL of phenol:chloroform:isoamyl alcohol (25:24:1) mixture and 200μL of 1×TE buffer, and vortex on a vortex for 90s. After centrifugation at 12000rpm for 10min, aspirate the supernatant and transfer it to an EP tube containing 2.5 volumes of pre-cooled anhydrous ethanol and 0.1 volumes of sodium acetate solution (pH 5.2), invert and mix. Centrifuge at 12000rpm for 2min, wash with 70% (v / v) ethanol, dry at room temperature, and resuspend with 200μL 1×TE.

[0054] (3) Expression cassette construction

[0055] P 0277 , P 0510 , P GAP and P TEF1 Insert CltLS2, SlNDPS1, SeACS L641P and ScIDI1, etc. GAP Inserted upstream of the SctHMG1 gene to construct five expression cassettes P 0277 -CltLS2-T AOX1 , P 0510 -SlNDPS1-T AOX1 , P GAP -SeACS L641P -T CYC1 , P TEF1 -ScIDI1-T AOX1 , P GAP -SctHMG1-T CYC1 .

[0056] (4) Plasmid construction

[0057] A. Donor plasmid construction:

[0058] The pGGA plasmid was digested with XhoI / NotI to obtain the vector fragment, and then the 1000bp upstream and downstream homology arms of PNSI-2 or PNSI-16 were added on both sides, and the above fragments were assembled by seamless cloning to obtain PNSI2-helper and PNSI16-helper plasmids. The pPIC9 plasmid was PCR amplified to obtain the vector fragment, and then the 1000bp upstream and downstream homology arms of Int12 were added on both sides, and P was added between the homology arms. TEF1 and T AOX1 , the above fragments were assembled by seamless cloning to obtain the Int12-helper plasmid. The sgRNA sequence corresponding to the integration site was referred to the corresponding reference.

[0059] B. Construction of PNSI2-CltLS2-SlNDPS1:

[0060] The plasmid PNSI2-helper was digested with KpnI / BamHI to obtain the vector fragment, and then the Cre / loxp recovery system sequence with FluR resistance marker (SEQ ID No.3) and CltLS2 expression cassette (P 0277 -CltLS2-T AOX1 ) sequence and SlNDPS1 expression cassette (P 0510 -SlNDPS1-T AOX1 ) sequence. The fragments were purified and assembled by seamless cloning to obtain the expression plasmid PNSI2-CltLS2-SlNDPS1 ( Figure 3 ).

[0061] C. PSNI16-SeACS L641P Build:

[0062] The PNSI16-helper plasmid, the Cre / loxp recovery system sequence with the bleomycin resistance marker, and the SeACS L641P Expression cassette (P GAP -SeACS L641P -T CYC1 ) sequence. The individual fragments were purified and assembled by seamless cloning to obtain plasmid PNSI16-SeACS L641P ( Figure 4 ).

[0063] D. Construction of Int12-tHMG1-IDI1:

[0064] The Int12-helper plasmid, the Cre / loxp recovery system sequence containing the bleomycin resistance marker, and the tHMG1 expression cassette (P GAP -SctHMG1-T CYC1 ) and IDI1 expression cassette (P TEF1 -ScIDI1-T AOX1 ). The fragments were purified and assembled by seamless cloning to obtain plasmid Int12-tHMG1-IDI1 ( Figure 5 ).

[0065] Example 3: Construction of a limonene heterologous synthesis engineered strain

[0066] Using Pichia pastoris GS115 with the HIS4 gene complemented as the starting strain, the exogenous genes required for the limonene biosynthesis pathway were inserted into the neutral sites of PNSI-2, PNSI-16 and Int12 through the CRISPR / Cas9 system, and the resistance marker was recovered through the Cre / loxp system to obtain the Pplim1h engineered strain. The strain was fermented in a shake flask in BMMY medium, and the limonene production and the transcription level of the exogenous gene were detected.

[0067] The specific operations are as follows:

[0068] (1) Use plasmid extraction kit (Novozyme) to extract plasmids PNSI2-CltLS2-SlNDPS1 and PSNI16-SeACS L641P and Int12-tHMG1-IDI1.

[0069] (2) Amplify plasmids PNSI2-CltLS2-SlNDPS1 and PSNI16-SeACS respectively L641P and Int12-tHMG1-IDI1, and a gene fragment with homology arms inserted into the PNSI-2, PSNI-16, and Int12 sites, i.e., a homologous recombination fragment, was obtained.

[0070] (3) The homologous recombination fragment and the corresponding sgRNA plasmid were electroporated into GS115-HIS4 cells.

[0071] (4) Add 1 mL of pre-cooled 1 M sorbitol and YPD mixed solution (v:v=1:1) to (3), incubate at 30°C for 2 h, and then spread the bacterial solution on a resistant YPD plate and culture for 2-3 days. After screening the positive clones, transfer them to YPM liquid culture medium and culture for 2 days. Recover the resistance marker by methanol induction, and finally obtain the limonene-synthesizing strain Pplim1h without resistance marker.

[0072] (5) Two-phase fermentation in BMMY liquid medium: The Pplim1h clone was selected and inoculated into BMMY medium containing 10% (v / v) n-dodecane according to the method in Example 1. The culture was continued at 30°C and 220 rpm for 72 h, and 500 μL of methanol was added every 24 h. RNA samples were collected to measure the transcription level of the exogenous gene.

[0073] (6) Detection of limonene production: After the fermentation, the fermentation liquid was centrifuged at 4°C and 5000 rpm for 10 min, the upper organic phase was taken, and then centrifuged at 8000 rpm for 1 min. The upper organic phase was aspirated and filtered with an organic filter membrane with a pore size of 0.22 μm. The obtained sample was diluted 3 times with ethyl acetate and analyzed by gas chromatography-mass spectrometry (Agilent GC-MS / MS 8890+7010CGC). The detection conditions were: a gas chromatography system equipped with an Agilent HP-5 column (30m×320μm×0.25μm), an injection volume of 1μL, a split ratio of 1:5, 60°C for 3 min, heated to 280°C at 20°C / min, run for 5 min, and the injection port temperature was 250°C.

[0074] Results: Pplim was fermented in BMMY medium for 1 h in two phases. After 72 h of culture, GC-MS was used to determine the presence of limonene in the sample (the results of gas chromatography and mass spectrometry are shown in Figure 6 and Figure 7 ), the average yield was 3.99 mg / L, and the transcription levels of each exogenous gene in the limonene biosynthesis pathway were as follows Figure 8 As shown by P 0277 The transcription level of CltLS2 activated reached about 2 times of the internal reference at 24h, 48h and 72h. 0277 It has a stable transcription level and can be used together with other reported constitutive promoters to construct heterologous synthetic pathways of natural products including but not limited to limonene, thus enriching the constitutive promoter library of Pichia pastoris.

[0075] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the contents disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art that are not disclosed in the present application.

Claims

1. A constitutive promoter of Pichia pastoris that is independent of the carbon source of the culture medium, characterized in that: The DNA sequence of the Pichia pastoris endogenous constitutive promoter is shown in SEQ ID No.

1.

2. The Pichia pastoris endogenous constitutive promoter according to claim 1, characterized in that The Pichia pastoris endogenous constitutive promoter is located in the 5' non-coding region before the start codon ATG of gene PAS_chr3_0277.

3. Use of the Pichia pastoris constitutive promoter according to claim 1 in an expression cassette, characterized in that: The expression cassette comprises the endogenous constitutive promoter of Pichia pastoris as claimed in claim 1, a structural gene or a regulatory gene connected downstream thereof, and a terminator.

4. The use according to claim 3, characterized in that: The process of constructing a limonene synthesis pathway based on the Pichia pastoris constitutive promoter includes: The Pichia pastoris constitutive promoter P 0277 and PAS_chr1-4_0510 promoter P 0510 , constitutive strong promoter P GAP and P TEF1 The truncated limonene synthase gene CltLS2, the NPP synthase gene SlNDPS1, and the acetyl-CoA synthase mutant gene SeACS were inserted into L641P and upstream of the isopentenyl pyrophosphate isomerase gene ScIDI1. GAP Insert the truncated HMG-CoA reductase 1 gene SctHMG1 upstream to construct five expression cassettes P 0277 -CltLS2-T AOX1 , P 0510 -SlNDPS1-T AOX1 , P GAP -SeACS L641P -T CYC1 , P TEF1 -ScIDI1-T AOX1 , P GAP -SctHMG1-T CYC1 ; P 0277 -CltLS2-T AOX1 and P 0510 -SlNDPS1-T AOX1 The expression cassette was inserted into the neutral site PNSI-2 of Pichia pastoris. GAP -SeACS L641P -T CYC1 The expression cassette was inserted into the neutral site PNSI-16 of Pichia pastoris. TEF1 -ScIDI1-T AOX1 and P GAP -SctHMG1-T CYC1 The expression cassette was inserted into the neutral site Int12 of Pichia pastoris to obtain an engineered strain carrying the limonene biosynthesis pathway.

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