Carotenoid phi cyclosynthase and application thereof in construction of engineering strain for synthesizing isospongiene

Abstract

The invention discloses carotenoid phi cyclization enzyme and application of the carotenoid phi cyclization enzyme in construction of engineering strains for synthesizing isospongiene. The bacterial strain is obtained by introducing expression cassettes of geranyl geranyl diphosphate synthase CrtE, phytoene synthetase / lycopene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl CoA reductase tHMGR and carotenoid [phi] cyclase CrtU into the pichia pastoris GS115, and is characterized in that the expression cassettes of the geranyl geranyl diphosphate synthase CrtE, the phytoene synthetase / lycopene cyclase CrtYB, the phytoene desaturase CrtI, the 3-hydroxy-3-methylglutaryl CoA reductase tHMGR and the carotenoid [phi] cyclase CrtU are used as raw materials; the coding gene sequence of the CrtE is as shown in SEQ ID No. 1; the sequence of the coding gene of the CrtYB is as shown in SEQ ID No. 2; the coding gene sequence of the CrtI is as shown in SEQ ID No. 3; the sequence of the coding gene of the tHMGR is as shown in SEQ ID No. 4; and the coding gene sequence of the CrtU is as shown in SEQ ID No. 5. According to the invention, the carotenoid phi cyclization enzyme is utilized for the first time to construct the pichia pastoris strain synthesized by the isocaponeic alkene, and the biosynthesis of the isocaponeic alkene in the yeast is realized.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and synthetic biology and relates to carotenoids The invention discloses a cyclosynthase and a protein encoded by the cyclosynthase and an application thereof, and specifically relates to an application in an engineering strain of Pichia pastoris for synthesizing isorenieratene. Background Art

[0002] Pichia pastoris is a methylotrophic yeast with strong respiratory metabolism, so it can grow to very high cell density in simple culture medium and can be easily expanded from shake flasks to large-scale bioreactors. The comprehensive characteristics of Pichia pastoris give this strain great potential in the production of compounds such as carotenoids. Recent studies have shown that Pichia pastoris is suitable for the production of natural products such as hyaluronic acid, carotenoids and sesquiterpenoid (+)-nokatone. Compared with other microorganisms, Pichia pastoris is safe and reliable and can be used in many industries such as food and medicine; the strain grows fast, has strong stress resistance, is simple to culture, and is suitable for industrialization; the molecular operation platform is mature and the genetic tools are complete, and the development prospects are broad.

[0003] Isospongene is a rare carotenoid with a benzene ring end group. The aromatic rings at both ends can form conjugated large π bonds with the symmetrical isoprene carbon chain. This structure gives it stronger stability. In addition to having high antioxidant capacity, isospongene can also effectively absorb ultraviolet radiation, so it has a cell photoprotective effect. As a carotenoid with a more stable structure and better functions, isospongene shows great application potential in functional foods and medicines. The biosynthesis of isospongene is mainly through carotenoids. Cyclosynthase (CrtU), catalyzed by β-carotene. Currently, isospongeline has only been detected in a few photosynthetic bacteria, Brevibacterium linum, Streptomyces and Rhodococcus, and the yield is low, so it is urgent to construct an engineered strain that can efficiently synthesize isospongeline. Summary of the invention

[0004] The purpose of the present invention is to provide a carotenoid Cyclosynthase genes and their encoded proteins;

[0005] Another object of the present invention is to carotenoids Cyclosynthase is used in the synthesis of isospongene;

[0006] Another object of the present invention is to construct a method for synthesizing isospongene engineered Pichia yeast;

[0007] Another object of the present invention is to provide a method for identifying isospongene;

[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0009] Carotenoids Cyclosynthase, whose amino acid sequence is shown in SEQ ID No.6.

[0010] Encoding carotenoids Cyclosynthase gene sequence.

[0011] Encoding carotenoids The nucleotide sequence of the cyclosynthase gene is shown in SEQ ID No. 5. The gene (from the start codon to the stop codon) is 1566 bp in length and encodes 521 amino acids.

[0012] Carotenoids Acquisition of cyclosynthase gene:

[0013] The target gene was found by genome comparison analysis. First, the genome of Rhodococcus aetherivorans was annotated by NCBI to find the putative carotenoids. Cyclosynthase gene, cloned from Rhodococcus etherivorans, the present invention confirms through functional verification that it has carotenoid Cyclosynthase, which has carotenoids The gene with cyclosynthase function is used for the subsequent construction of Pichia pastoris engineering strain for synthesizing isospongeline.

[0014] The carotenoids Application of cyclosynthase in the synthesis of isospongene.

[0015] The carotenoid The cyclosynthase gene is introduced into the host bacteria to produce isospongene by fermentation.

[0016] A strain producing isospongene, wherein geranylgeranyl diphosphate synthase CrtE, phytoene synthase / lycopene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl CoA reductase tHMGR and carotenoids are introduced into Pichia pastoris The expression cassette of the cyclosynthetic enzyme CrtU is obtained; the coding gene sequence of the CrtE is shown in SEQ ID No.1; the coding gene sequence of the CrtYB is shown in SEQ ID No.2; the coding gene sequence of the CrtI is shown in SEQ ID No.3; the coding gene sequence of the tHMGR is shown in SEQ ID No.4; and the coding gene sequence of the CrtU is shown in SEQ ID No.5.

[0017] In the present invention, the genes of geranylgeranyl diphosphate synthase CrtE, phytoene synthase / lycopene cyclase CrtYB, and phytoene desaturase CrtI are derived from Xanthophyllomyces denrorhous; the gene of 3-hydroxy-3-methylglutaryl CoA reductase is derived from Saccharomyces cerevisiae; the carotenoid The cyclosynthase gene CrtU is derived from Rhodococcus aetherivorans.

[0018] The Pichia pastoris is Pichia pastoris GS115.

[0019] As a preferred embodiment, the promoter of the expression cassette is the pGPM1 promoter, pPDC1 promoter, pMDH3 promoter, pADH2 promoter or pGAP promoter of Pichia pastoris; the terminator is the ScCYC1tt terminator, RPP1Btt terminator, RPS2tt terminator, RPL2Att terminator or RPS3tt terminator of Pichia pastoris.

[0020] As a preferred embodiment, the recombinant Pichia pastoris also expresses a marker gene; the marker gene is selected from the kanamycin sulfate encoding gene expression cassette of plasmid BB3aK-AF.

[0021] Further preferably, the CrtI promoter is pGPM1, and the terminator is ScCYC1tt; the crtE promoter is pPDC1 promoter, and the terminator is RPP1Btt; the crtYB promoter is pMDH3, and the terminator is RPS2tt; the tHMGR promoter is pADH2, and the terminator is RPL2Att; the CrtU promoter is pGAP, and the terminator is RPS3tt. The kanamycin sulfate gene carried by the plasmid is used as a screening marker for Pichia pastoris.

[0022] Another object of the present invention is to provide a method for constructing the above strain, wherein the expression cassettes of CrtE, CrtYB, CrtI and tHMGR are introduced into the Pichia pastoris GS115 in the form of a plasmid and integrated into the genome of the Pichia pastoris strain.

[0023] include:

[0024] Inserting the gene fragments of CrtE, CrtYB, CrtI, tHMGR and CrtU into plasmid BB1-23 by GoldenGate method to obtain plasmids BB1-23-CrtE, BB1-23-CrtYB, BB1-23-CrtI, BB1-23-tHMGR and BB1-23-CrtU;

[0025] The plasmid BB1-23-CrtI, plasmid BB1-12-pGPM1, and plasmid BB1-34-ScCYC1tt were inserted into plasmid BB2-AB by the GoldenGate method to obtain plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt;

[0026] The plasmid BB1-23-CrtE, plasmid BB1-12-pPDC1, and plasmid BB1-34-RPP1Btt were inserted into plasmid BB2-BC by the GoldenGate method to obtain plasmid BB2-BC-pPDC1-CrtE-RPP1Btt;

[0027] The plasmid BB1-23-CrtYB, plasmid BB1-12-pMDH3 and plasmid BB1-34-RPS2tt were inserted into plasmid BB2-CD by GoldenGate method to obtain plasmid BB2-CD-pMDH3-CrtYB-RPS2tt;

[0028] The plasmid BB1-23-tHMGR, plasmid BB1-12-pADH2 and plasmid BB1-34-RPL2Att were inserted into plasmid BB2-DE by GoldenGate method to obtain plasmid BB2-DE-pADH2-tHMGR-RPL2Att;

[0029] The plasmid BB1-23-CrtU, plasmid BB1-12-pGAP, and plasmid BB1-34-RPS3tt were inserted into plasmid BB2-EF by GoldenGate method to obtain plasmid BB2-EF-pGAP-CrtYB-RPS3tt;

[0030] The plasmids BB2-AB-pGPM1-CrtI-ScCYC1tt, BB2-BC-pPDC1-CrtE-RPP1Btt, BB2-CD-pMDH3-CrtYB-RPS2tt, BB2-DE-pADH2-tHMGR-RPL2Att and BB2-EF-pGAP-CrtYB-RPS3tt were inserted into the plasmid BB3aK-AF by the GoldenGate method to obtain the plasmid BB3aK-AF-L8CrtU;

[0031] The plasmid BB3aK-AF-L8CrtU was introduced into Pichia pastoris GS115 to obtain a recombinant strain.

[0032] The BB2-AB-pGPM1-CrtI-ScCYC1tt, BB2-BC-pPDC1-CrtE-RPP1Btt, BB2-CD-pMDH3-CrtYB-RPS2tt, BB2-DE-pADH2-tHMGR-RPL2Att, and BB2-EF-pGAP-CrtYB-RPS3tt plasmids were constructed by the GoldenGate method using Bpi1 enzyme and T4 ligase.

[0033] The BB3aK-AF-L8CrtU plasmid was constructed by the GoldenGate method using Bsa1 enzyme and T4 ligase.

[0034] Application of recombinant strains producing isospongene in the production of carotenoids.

[0035] The recombinant strain is used in producing isospongene.

[0036] The application comprises: inoculating the recombinant bacteria into a nutrient medium at 25 to 35° C., fermenting and culturing for 3 to 8 days, and obtaining a fermentation product;

[0037] The nutrient medium is: 10 g / L glucose, 20 g / L peptone and 10 g / L yeast powder;

[0038] The strain culture temperature is preferably 30° C., and the fermentation time is 4 to 5 days.

[0039] In a 50 ml shake flask fermentation, the highest isosponge yield of 159.41 mg / L and the isosponge content of 9.43 mg / g could be produced.

[0040] The fermentation product is extracted with dimethyl sulfoxide and acetone to obtain isospongene.

[0041] The extraction method includes the following steps: taking 2 ml of fermentation liquid, centrifuging at 12000 rpm for 3 min, washing twice with ultrapure water, discarding the supernatant, adding 2 ml of dimethyl sulfoxide, shaking, bathing at 55°C for 15 min, adding 2 ml of acetone, standing at 55°C in a water bath away from light for 15 min, and 12000 rpm for 3 min to take the supernatant for the next step of detection.

[0042] The present invention also provides a method for identifying the product of the recombinant strain and its yield;

[0043] The fermentation product is extracted with dimethyl sulfoxide and acetone to obtain isospongene, and the product isospongene is qualitatively detected by LC-MS analysis and its yield is detected by high performance liquid chromatography.

[0044] The liquid phase conditions are:

[0045] Chromatographic column: C30 chromatographic column; UV absorption wavelength of 450nm; injection volume of 10uL; mobile phase A is methanol and B is methyl tert-butyl ether, flow rate: 1mL / min; elution procedure: first stage: use a mixed solution of mobile phase A and mobile phase B for elution for 30 minutes, wherein the volume percentage of mobile phase A changes from 95% to 70%, and the volume percentage of mobile phase B changes from 5% to 30%; second stage: use a mixed solution of mobile phase A and mobile phase B for elution for 20 minutes, wherein the volume percentage of mobile phase A changes from 70% to 50%, and the volume percentage of mobile phase B changes from 30% to 50%; third stage: use a mixed solution of mobile phase A and mobile phase B for elution for 10 minutes, wherein the volume percentage of mobile phase A changes from 50% to 95%, and the volume percentage of mobile phase B changes from 50% to 5%.

[0046] The mass spectrometry conditions were:

[0047] Acquisition mode: MSE (low energy / high energy switching scan); electrospray positive ion / negative ion scanning respectively; capillary voltage 2KV; cone voltage 40V; nebulizer gas temperature: 450℃; nebulizer gas flow rate 900L / h; cone backflush gas 50L / h; ion source temperature 115℃; scanning range 50~1000m / z; scanning speed 0.2s; collision energy 6eV / 20~45eV; online lock mass; 250pg / uL leucine enkephalin continuous injection, flow rate 10ul / min; acquisition interval 0.5s; acquisition time 0.5s.

[0048] Beneficial effects:

[0049] 1. This invention is the first to prove the verification of carotenoids in Pichia pastoris The function of the cyclosynthetic enzyme CrtU is a gene with a new function. The full length (from the start codon to the stop codon) is 1566bp and can encode 521 amino acids.

[0050] 2. The present invention realizes the de novo synthesis of isospongene using glucose by using engineered Pichia pastoris for the first time. The cyclosynthase CrtU was used to construct a strain capable of synthesizing isospongene de novo, which is the first report of synthesizing isospongene in a non-natural strain.

[0051] 3. The present invention provides a method for extracting and detecting isospongene in a recombinant Pichia pastoris strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the structure diagram of plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt, the CrtI promoter is pGPM1, the terminator is ScCYC1tt, and the ampicillin gene carried by the plasmid is used as a screening marker for Escherichia coli DH5α.

[0053] Figure 2 This is the structure diagram of plasmid BB2-BC-pPDC1-CrtE-RPP1Btt. The promoter of crtE is the pPDC1 promoter, the terminator is RPP1Btt, and the ampicillin gene carried by the plasmid is used as a screening marker for Escherichia coli DH5α.

[0054] Figure 3 This is the structure diagram of plasmid BB2-CD-pMDH3-CrtYB-RPS2tt. The promoter of crtYB is pMDH3, the terminator is RPS2tt, and the ampicillin gene carried by the plasmid is used as a screening marker for Escherichia coli DH5α.

[0055] Figure 4 This is the structure diagram of plasmid BB2-DE-pADH2-tHMGR-RPL2Att. The promoter of HMGR is pADH2, the terminator is RPL2Att, and the ampicillin gene carried by the plasmid is used as a screening marker for Escherichia coli DH5α.

[0056] Figure 5 The structure diagram of plasmid BB2-EF-pGAP-CrtU-RPS3tt is shown. The promoter of crtU is pGAP, the terminator is RPS3tt, and the ampicillin gene carried by the plasmid is used as a screening marker for Escherichia coli DH5α.

[0057] Figure 6 This is the structure diagram of plasmid BB3aK-AF-L8CrtU. The CrtI promoter is pGPM1, and the terminator is ScCYC1tt; the crtE promoter is pPDC1 promoter, and the terminator is RPP1Btt; the crtYB promoter is pMDH3, and the terminator is RPS2tt; the tHMGR promoter is pADH2, and the terminator is RPL2Att; the CrtU promoter is pGAP, and the terminator is RPS3tt. The kanamycin sulfate gene carried by this plasmid is used as a screening marker for Pichia pastoris.

[0058] Figure 7 Liquid phase assay for the production of isospongene by recombinant Pichia pastoris.

[0059] Figure 8Mass spectrometry analysis of isospongene produced by recombinant Pichia pastoris.

[0060] Fig. 9 Yield of isospongene produced by recombinant Pichia pastoris in shake flask fermentation. DETAILED DESCRIPTION

[0061] The present invention will be further described below in conjunction with specific embodiments.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0063] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0064] The original strain of Pichia pastoris used in the examples is the Pichia pastoris GS115 strain, which can be purchased from commercial channels.

[0065] The primer sequences used in the examples are shown in Table 1 below.

[0066] Table 1 Primer sequences required for construction

[0067]

[0068] Example 1 Amplification of genetic elements and preparation of target plasmids

[0069] (I) Preparation of target gene

[0070] According to the nucleotide sequence of crtE, a gene encoding geranylgeranyl diphosphate synthase from X.denrorhous, provided on NCBI, the nucleotide sequence of crtYB, a gene encoding phytoene synthase / lycopene cyclase, and the nucleotide sequence of crtI, a gene encoding phytoene desaturase, after codon optimization, the gene sequences obtained are shown in SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3. After the gene codons are optimized, the exogenous genes are more compatible with the Pichia pastoris chassis. According to the 3-hydroxy-3-methylglutaryl CoA reductase gene sequence from Saccharomyces cerevisiae provided on NCBI, PCR amplification was performed using the Saccharomyces cerevisiae genome as a template, and the gene sequence of tHMGR is shown in SEQ ID No.4. According to the genome of Rhodococcus etherovorans uploaded by the applicant on NCBI, the putative carotenoids annotated were found. Cyclosynthase gene, cloned from Rhodococcus etherivorans, has been confirmed to have carotenoid Cyclosynthase, which has carotenoids The gene of cyclosynthetic enzyme function is used for the subsequent construction of the Pichia yeast engineering strain for synthesizing isospongene. The nucleic acid sequence of the cyclosynthetic enzyme is shown in SEQ ID No.5, and the amino acid sequence is shown in SEQ ID No.5.

[0071] (II) Construction of recombinant plasmid

[0072] 1. Construction of recombinant plasmids BB1-23-CrtI, BB1-23-CrtE and BB1-23-CrtYB: GenScript Biotech Co., Ltd. was commissioned to synthesize the crtI, crtE and crtYB gene sequences; the recombinant plasmid BB1-23-tHMGR was amplified by PCR using the Saccharomyces cerevisiae genome as a template to obtain the tHMGR gene sequence; the recombinant plasmid BB1-23-CrtU was amplified by PCR using the genome of R. aetherivorans N1 as a template to obtain the crtU gene sequence, and each gene fragment was inserted into plasmid BB1-23 by the GoldenGate method to obtain recombinant plasmids BB1-23-CrtI, BB1-23-CrtE, BB1-23-CrtYB, BB1-23-tHMGR and BB1-23-CrtU. The structures of the recombinant plasmids are shown in Figures 1 to 5 .

[0073] The crtI fragment was amplified using crtI-F and crtI-R as primers and the crtI gene sequence as a template. The primer sequences are shown in Table 1.

[0074] The crtE fragment was amplified using crtE-F and crtE-R as primers and the crtE gene sequence as a template. The primer sequences are shown in Table 1.

[0075] The crtYB fragment was amplified using crtYB-F and crtYB-R as primers and the crtYB gene sequence as a template. The primer sequences are shown in Table 1.

[0076] The tHMGR fragment was amplified using tHMGR-F and tHMGR-R as primers and the tHMGR gene sequence as a template. The primer sequences are shown in Table 1.

[0077] The crtU fragment was amplified using crtU-F and crtU-R as primers and the crtU gene sequence as a template. The primer sequences are shown in Table 1.

[0078] The PCR enzyme used for amplification was Phanta Max Super-Fidelity DNA Polymerase from Nanjing Novozymes Biotechnology Co., Ltd. The system is shown in Table 2.

[0079] Table 2

[0080] system 50μL Phanta Max Super-Fidelity DNA Polymerase 1μL buffer 25μL Distilled water 20μL DNTP 1μL Quote 1μL quoted below 1μL template 1μL

[0081] The amplified crtI, crtE, crtYB, tHMGR and CrtU fragments were recovered and purified and recovered by agarose gel electrophoresis.

[0082] Amplification Procedure:

[0083]

[0084] GoldenGate assembly was performed using Bsa1 enzyme and T4 ligase from Shanghai Biotech Biotechnology Co., Ltd. The reaction system is shown in Table 3.

[0085] Table 3

[0086]

[0087]

[0088] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmids BB1-23-CrtI, BB1-23-CrtE, BB1-23-CrtYB, BB1-23-tHMGR and BB1-23-CrtU were obtained through plate screening for kanamycin sulfate resistance and verification by colony PCR and sequencing.

[0089] 2. The recombinant plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt was obtained by inserting plasmid BB1-23-CrtI, plasmid BB1-12-pGPM1 and plasmid BB1-34-ScCYC1tt into plasmid BB2-AB using Bpi1 enzyme and T4 ligase through the GoldenGate method to obtain plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt; the recombinant plasmid BB2-BC-pPDC1-CrtE-RPP1Btt was obtained by inserting plasmid BB1-23-CrtI, plasmid BB1-12-pGPM1 and plasmid BB1-34-ScCYC1tt into plasmid BB2-AB using Bpi1 enzyme and T4 ligase. BB1-23-CrtE, plasmid BB1-12-pPDC1, and plasmid BB1-34-RPP1Btt were inserted into plasmid BB2-BC by GoldenGate method using Bpi1 enzyme and T4 ligase to obtain plasmid BB2-BC-pPDC1-CrtE-RPP1Btt; recombinant plasmid BB2-CD-pMDH3-CrtYB-RPS2tt was obtained by plasmid BB1-23-CrtYB, plasmid BB1-12-pMDH3, plasmid BB1-34-RP S2tt was inserted into plasmid BB2-CD by GoldenGate method using Bpi1 enzyme and T4 ligase to obtain plasmid BB2-CD-pMDH3-CrtYB-RPS2tt; recombinant plasmid BB2-DE-pADH2-tHMGR-RPL2Att was inserted into plasmid BB1-23-tHMGR, plasmid BB1-12-pADH2, and plasmid BB1-34-RPL2Att by GoldenGate method using Bpi1 enzyme and T4 ligase 2-DE, and obtained plasmid BB2-DE-pADH2-tHMGR-RPL2Att; recombinant plasmid BB2-EF-pGAP-CrtU-RPS3tt was obtained by inserting plasmid BB1-23-CrtU, plasmid BB1-12-pGAP, and plasmid BB1-34-RPS3tt into plasmid BB2-CD by GoldenGate method using Bpi1 enzyme and T4 ligase, and obtained plasmid BB2-EF-pGAP-CrtU-RPS3tt; the structure of the recombinant plasmid is shown in Figures 1 to 5 .

[0090] The construction process of the recombinant plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt is as follows:

[0091] GoldenGate assembly was performed using Bpi1 enzyme and T4 ligase from Nanjing Formase Biotechnology Co., Ltd. The reaction system is shown in Table 4.

[0092] Table 4

[0093]

[0094]

[0095] The circular recombinant vector was transformed into E. coli DH5α competent cells, screened by ampicillin resistance plate and verified by colony PCR and sequencing to obtain the positive recombinant plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt. The plasmid structure is shown in Figure 1 .

[0096] The construction process of the recombinant plasmid BB2-BC-pPDC1-CrtE-RPP1Btt is as follows:

[0097] GoldenGate assembly was performed using Bpi1 enzyme and T4 ligase from Nanjing Formase Biotechnology Co., Ltd. The reaction system was basically the same as that in Table 3, except that the plasmids were changed to BB2-BC, BB1-12-pPDC1, BB1-23-CrtE, and BB1-34-RPP1Btt.

[0098] The circular recombinant vector was transformed into E. coli DH5α competent cells, screened by ampicillin resistance plate and verified by colony PCR and sequencing to obtain the positive recombinant plasmid BB2-BC-pPDC1-CrtE-RPP1Btt. The plasmid structure is shown in Figure 2 .

[0099] The construction process of the recombinant plasmid BB2-CD-pMDH3-CrtYB-RPS2tt is as follows:

[0100] GoldenGate assembly was performed using Bpi1 enzyme and T4 ligase from Nanjing Formase Biotechnology Co., Ltd. The reaction system was basically the same as that in Table 3, except that the plasmids were replaced with BB2-CD, BB1-12-pMDH3, BB1-23-CrtYB, and BB1-34-RPS2tt. The circular recombinant vector was transformed into E. coli DH5α competent cells, screened by ampicillin resistance plate, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid BB2-CD-pMDH3-CrtYB-RPS2tt. The plasmid structure is shown in Figure 3 .

[0101] The construction process of the recombinant plasmid BB2-DE-pADH2-tHMGR-RPL2Att is as follows:

[0102] GoldenGate assembly was performed using Bpi1 enzyme and T4 ligase from Nanjing Formase Biotechnology Co., Ltd. The reaction system was basically the same as that in Table 3, except that the plasmids were replaced with BB2-DE, BB1-12-pADH2, BB1-23-tHMGR, and BB1-34-RPL2Att. The circular recombinant vector was transformed into E. coli DH5α competent cells, and the positive recombinant plasmid BB2-DE-pADH2-tHMGR-RPL2Att was obtained by plate screening of ampicillin resistance and verification by colony PCR and sequencing. The plasmid structure is shown in Figure 4 .

[0103] The construction process of the recombinant plasmid BB2-EF-pGAP-CrtU-RPS3tt is as follows:

[0104] GoldenGate assembly was performed using Bpi1 enzyme and T4 ligase from Nanjing Formase Biotechnology Co., Ltd. The reaction system was basically the same as that in Table 3, except that the plasmids were replaced with BB2-EF, BB1-12-pGAP, BB1-23-CrtU, and BB1-34-RPS3tt. The circular recombinant vector was transformed into E. coli DH5α competent cells, screened by ampicillin resistance plate, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid BB2-EF-pGAP-CrtU-RPS3tt. The plasmid structure is shown in Figure 5 .

[0105] 3. The recombinant plasmid BB3aK-AE-CrtIEYBtHMGR was constructed by inserting plasmids BB2-AB-pGPM1-CrtI-ScCYC1tt, BB2-BC-pPDC1-CrtE-RPP1Btt, BB2-CD-pMDH3-CrtYB-RPS2tt, BB2-DE-pADH2-tHMGR-RPL2Att and BB2-EF-pGAP-CrtU-RPS3tt into plasmid BB3aK-AF by GoldenGate method using Bsa1 enzyme and T4 ligase to obtain plasmid BB3aK-AF-L8CrtU, thus completing the construction of recombinant plasmid BB3aK-AF-L8CrtU. The structure of recombinant plasmid BB3aK-AF-L8CrtU is shown in Figure 6 .

[0106] The construction process of the recombinant plasmid BB3aK-AF-L8CrtU is as follows:

[0107] GoldenGate assembly was performed using Bsa1 enzyme and T4 ligase, and the reaction system is shown in Table 5.

[0108] Table 5

[0109] system 10μL BB3aK-AE 1μL BB2-AB-pGPM1-CrtI-ScCYC1tt 1μL BB2-BC-pPDC1-CrtE-RPP1Btt 1μL BB2-CD-pMDH3-CrtYB-RPS2tt 1μL BB2-DE-pADH2-tHMGR-RPL2Att 1μL BB2-EF-pGAP-CrtU-RPS3tt 1μL Bsa1 0.5μL BSA 1μL T4 Ligase 0.5μL T4buffer 1μL Distilled water 2μL

[0110] The circular recombinant vector was transformed into E. coli DH5α competent cells, screened by kanamycin sulfate plate resistance and verified by colony PCR and sequencing to obtain the positive recombinant plasmid BB3aK-AF-L8CrtU. The plasmid structure is shown in Figure 5 .

[0111] Example 2 Construction of recombinant bacteria

[0112] Construction of recombinant strain PP-L8U

[0113] The plasmid BB3aK-AF-L8CrtU containing the crtI-crtYB-crtE-tHMGR-crtU gene expression cassette was introduced into Pichia pastoris, and the crtI-crtYB-crtE-tHMGR-crtU expression cassette was integrated into the genome EGI2 site to obtain the recombinant bacteria PP-L8U. The specific method is as follows:

[0114] ① Prepare competent cells by culturing the original Pichia pastoris overnight in YPD liquid medium (containing 2% peptone, 1% yeast powder and 2% glucose).

[0115] ② Use an electroporator to introduce BB3aK-AE-CrtIEYBtHMGR into Pichia pastoris competent cells for homologous recombination.

[0116] ③ Screening was performed on YPD screening plates with kanamycin sulfate added. Single colonies grew out in 2 to 3 days. The positive clones identified correctly by PCR were named recombinant bacteria PP-L8U.

[0117] Preparation method of competent cells of Pichia pastoris:

[0118] 1. Grow yeast cells to OD 600 0.8~1.0;

[0119] 2. Centrifuge at 4000 rpm for 5 min at room temperature;

[0120] 3. Discard the supernatant and resuspend the bacterial sludge in 9 mL BEDS solution and 1 mL 1 M threitol;

[0121] 4. Incubate on ice for 5 minutes;

[0122] 5. Centrifuge at 4000 rpm for 5 min at room temperature;

[0123] 6. Discard the supernatant and resuspend the bacterial sludge in 1 mL of BEDS solution;

[0124] 7. Package into 1.5mL EP tubes and store at -80℃.

[0125] BEDS solution: 10 mM bicine-NaOH, pH 8.3; 3% (v / v) ethylene glycol and 1 M sorbitol.

[0126] Example 3 Application of recombinant bacteria in the production of isospongene

[0127] 1. Cultivation of engineered bacteria

[0128] A single colony was picked from the plate and inoculated into YPD medium. After culturing at 30°C for 24 hours, it was inoculated into a fermentation shake flask at a 1% inoculation rate and cultured at 30°C, 180 rpm for 120 hours.

[0129] 2. Extraction of isospongene

[0130] (1) Take 1 mL of the mixed fermentation broth and centrifuge at 12000 rpm for 3 min (wash twice with pure water).

[0131] (2) After draining the water, resuspend the solution in 2 mL of dimethyl sulfoxide (DMSO) (preheated at 60°C) and shake it evenly on a vortex shaker. Then place it in a 55°C water bath for 15 min.

[0132] (3) Add 2 mL of acetone and incubate in a 55°C water bath for 15 min.

[0133] (4) Centrifuge the sample at 12,000 rpm for 3 min. Transfer the supernatant to a new centrifuge tube and store in a dark place.

[0134] During the shake flask fermentation, starting from the 24th hour, samples were taken every day to detect the production of isospongene by the strain. In the 50 ml shake flask fermentation, the highest isospongene production of 159.41 mg / L and the isospongene content of 9.43 mg / g ( Fig. 9 ).

[0135] Quantitative Analysis of Isospongene

[0136] The liquid chromatography model used in this study is Agilent Technologies 1200 Infinity series; the chromatographic column is: C30 chromatographic column; UV absorption wavelength is 450nm; mobile phase is methanol and methyl tert-butyl ether; flow rate is controlled at 1.0mL / min; column temperature is 25℃. Figure 7 This is the HPLC detection chart of isospongene of the recombinant strain.

[0137] LC-MS Detection of Isospongene

[0138] The LC-MS model used in this study was Acquity I-class ultra-high performance liquid chromatography and VION ion mobility quadrupole time-of-flight mass spectrometer, and the mass spectrometry conditions were as follows: acquisition mode: MSE (low energy / high energy switching scan); electrospray positive ion / negative ion scanning respectively; capillary voltage 2KV; cone voltage 40V; nebulizer gas temperature: 450℃; nebulizer gas flow rate 900L / h; cone backflush gas 50L / h; ion source temperature 115℃; scan range 50-1000m / z; scan speed 0.2s; collision energy 6eV / 20-45eV; online lock mass; 250pg / uL leucine enkephalin continuous injection, flow rate 10ul / min; acquisition interval 0.5s; acquisition time 0.5s. Figure 8 This is the mass spectrometry identification result of isospongene.

[0139] SEQ ID No.1

[0140]

[0141]

[0142] SEQ ID No.2

[0143]

[0144] SEQ ID No.3

[0145]

[0146]

[0147] SEQ ID No.4

[0148]

[0149] SEQ ID No.5

[0150]

[0151]

[0152] SEQ ID No.6

[0153]

Claims

1. Carotenoid φ cyclosynthase, the amino acid sequence of which is shown in SEQ ID No.

6.

2. The gene sequence encoding the carotenoid φ cyclosynthase according to claim 1, characterized in that: The gene sequence is shown as SEQ ID No.

5.

3. Use of the carotenoid φ cyclosynthase according to claim 1 in the synthesis of isospongene.

4. A recombinant strain for synthesizing isospongene, characterized in that: The recombinant strain is obtained by introducing expression cassettes of geranylgeranyl diphosphate synthase CrtE, phytoene synthase / lycopene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl CoA reductase tHMGR and carotenoid φ cyclase CrtU into Pichia pastoris; the coding gene sequence of CrtE is shown in SEQ ID No.1; the coding gene sequence of CrtYB is shown in SEQ ID No.2; the coding gene sequence of CrtI is shown in SEQ ID No.3; the coding gene sequence of tHMGR is shown in SEQ ID No.4; and the coding gene sequence of CrtU is shown in SEQ ID No.

5.

5. The recombinant strain according to claim 4, characterized in that The promoter of the expression cassette is the pGPM1 promoter, pPDC1 promoter, pMDH3 promoter, pADH2 promoter or pGAP promoter of Pichia pastoris; the terminator is the ScCYC1tt terminator, RPP1Btt terminator, RPS2tt terminator, RPL2Att terminator or RPS3tt terminator of Pichia pastoris.

6. The recombinant strain according to claim 4, characterized in that The recombinant Pichia pastoris also expresses a marker gene; the marker gene is selected from the kanamycin sulfate encoding gene expression cassette of plasmid BB3aK-AF.

7. The method for constructing the recombinant strain according to any one of claims 4 to 6, characterized in that: The expression cassettes of CrtE, CrtYB, CrtI and tHMGR are introduced into the Pichia pastoris GS115 in the form of plasmids and integrated into the genome of the Pichia pastoris strain.

8. The construction method according to claim 7, characterized in that: include: Inserting the gene fragments of CrtE, CrtYB, CrtI, tHMGR and CrtU into plasmid BB1-23 by GoldenGate method to obtain plasmids BB1-23-CrtE, BB1-23-CrtYB, BB1-23-CrtI, BB1-23-tHMGR and BB1-23-CrtU; The plasmid BB1-23-CrtI, plasmid BB1-12-pGPM1, and plasmid BB1-34-ScCYC1tt were inserted into plasmid BB2-AB by the GoldenGate method to obtain plasmid BB2-AB-pGPM1-CrtI-ScCYC1tt; The plasmid BB1-23-CrtE, plasmid BB1-12-pPDC1, and plasmid BB1-34-RPP1Btt were inserted into plasmid BB2-BC by the GoldenGate method to obtain plasmid BB2-BC-pPDC1-CrtE-RPP1Btt; The plasmid BB1-23-CrtYB, plasmid BB1-12-pMDH3 and plasmid BB1-34-RPS2tt were inserted into plasmid BB2-CD by GoldenGate method to obtain plasmid BB2-CD-pMDH3-CrtYB-RPS2tt; The plasmid BB1-23-tHMGR, plasmid BB1-12-pADH2 and plasmid BB1-34-RPL2Att were inserted into plasmid BB2-DE by GoldenGate method to obtain plasmid BB2-DE-pADH2-tHMGR-RPL2Att; The plasmid BB1-23-CrtU, plasmid BB1-12-pGAP, and plasmid BB1-34-RPS3tt were inserted into plasmid BB2-EF by the GoldenGate method to obtain plasmid BB2-EF-pGAP-CrtYB-RPS3tt; The plasmids BB2-AB-pGPM1-CrtI-ScCYC1tt, BB2-BC-pPDC1-CrtE-RPP1Btt, BB2-CD-pMDH3-CrtYB-RPS2tt, BB2-DE-pADH2-tHMGR-RPL2Att and BB2-EF-pGAP-CrtYB-RPS3tt were inserted into the plasmid BB3aK-AF by the GoldenGate method to obtain the plasmid BB3aK-AF-L8CrtU; The plasmid BB3aK-AF-L8CrtU was introduced into Pichia pastoris GS115 to obtain a recombinant strain.

9. The construction method according to claim 7, characterized in that: The gene fragments of CrtE, CrtYB, CrtI, tHMGR and CrtU were inserted into plasmid BB1-23 by the GoldenGate method using Bsa1 enzyme and T4 ligase.

10. Use of the recombinant strain according to any one of claims 4 to 6 in the production of isospongene.

11. The use according to claim 9, characterized in that: The recombinant strain is cultured on a nutrient medium to obtain a fermentation product containing isospongene, and the product isospongene is separated.

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