Recombinant pichia pastoris strain with high astaxanthin yield as well as construction method and application of recombinant pichia pastoris strain

Abstract

The invention relates to a recombinant pichia pastoris strain capable of producing astaxanthin at high yield as well as a construction method and application of the recombinant pichia pastoris strain. The recombinant pichia pastoris strain is obtained by expressing ATP (adenosine triphosphate) citrate lyase (ACL) and acetyl-CoA synthase (ACS) or phosphoketolase (PK) and phosphotransacetylase (PTA), choline kinase (CK), inositol polyphosphate kinase (IPK) and vitreoscilla hemoglobin (VHB) in host bacteria. Wherein the host bacterium is a pichia pastoris gene modified strain PP-LC2. The astaxanthin production performance of the recombinant strain is verified on the basis of comparison of precursor supply pathways, IUP pathway construction and improvement of the oxygen supply capacity of the engineering strain, and the astaxanthin production capacity of the pichia pastoris is further improved. The construction method of the recombinant pichia pastoris is simple, the synthesis of astaxanthin can be better promoted, the engineering strain can efficiently synthesize the astaxanthin by utilizing methanol through amplification fermentation of a 5L fermentation tank, and industrial production is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, in particular to a recombinant Pichia pastoris with high astaxanthin production, a construction method thereof and an application thereof. Technical Background

[0002] Pichia pastoris has become a widely used expression system in biotechnology and industrial production due to its efficient protein expression, comprehensive post-translational modification capabilities, high-density fermentation characteristics, strict expression control system, and strong secretion capacity. Based on these advantages, Pichia pastoris shows great potential for the production of terpenoids such as carotenoids. Recent research has demonstrated significant progress in the synthesis of terpenoids such as β-carotene, lycopene, and astaxanthin, providing a solid foundation for further optimization and application. Compared with other microorganisms, Pichia pastoris offers safety and reliability, making it widely applicable in demanding industries such as food and pharmaceuticals. Furthermore, its natural ability to utilize methanol as a carbon source allows for simple culture conditions, effectively reducing production costs. Furthermore, its mature molecular manipulation technology platform strongly supports its further development and application, giving it broad prospects in biotechnology. By introducing β-carotene and astaxanthin synthesis modules into Pichia pastoris, heterologous synthesis of astaxanthin has been successfully achieved, providing a new approach for the industrial production of related compounds.

[0003] Prior art has genetically modified Pichia pastoris strains to produce astaxanthin-producing recombinant Pichia pastoris. However, the astaxanthin produced after the introduction of the product synthesis module is toxic to cells. The resulting cellular damage while increasing astaxanthin production also inhibits further growth, preventing further increases in astaxanthin production. Furthermore, CN 119120242 A provides astaxanthin-producing recombinant Pichia pastoris, which increases astaxanthin production in Pichia pastoris through genetic and metabolic engineering. Currently, further increasing astaxanthin production remains a research goal for promoting the industrialization of astaxanthin. Summary of the Invention

[0004] The first object of the present invention is to provide an astaxanthin-producing recombinant Pichia pastoris with enhanced supply of precursor acetyl-CoA.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A recombinant Pichia pastoris strain for producing astaxanthin with enhanced supply of precursor acetyl-CoA, the recombinant Pichia pastoris strain being obtained by expressing in a host bacterium ATP citrate lyase (ACL) and acetyl-CoA synthase (ACS) from Yarrowia lipolytica or phosphoketolase (PK) from Leuconostoc mesenteroides and phosphotransacetylase (PTA) from Clostridium coli.

[0007] Wherein, the host bacteria is the genetically modified Pichia pastoris PP-LC2.

[0008] As a preferred embodiment, the nucleotide sequences of the ATP citrate lyase ACL, acetyl-CoA synthase ACS, phosphoketolase PK and phosphotransacetylase PTA are shown in SEQ ID NOs: 1-4, respectively.

[0009] The second object of the present invention is to provide an astaxanthin-producing recombinant Pichia pastoris strain of isopentenol utilization pathway (IUP).

[0010] A recombinant astaxanthin-producing Pichia pastoris strain of isopentenol utilization pathway (IUP) was obtained by expressing choline kinase CK and inositol polyphosphate kinase IPK in the host bacteria.

[0011] Further preferably, the recombinant Pichia pastoris strain is obtained by expressing choline kinase CK and inositol polyphosphate kinase IPK in a host bacterium that enhances the precursor acetyl-CoA.

[0012] Through the action of two enzymes, CK and IPK, isopentenol can be converted into IPP in two steps, thereby increasing the content of IPP, the precursor of astaxanthin synthesis.

[0013] As an optimized embodiment, the choline kinase CK is derived from Saccharomyces cerevisiae, and the inositol polyphosphate kinase IPK is derived from Arabidopsis thaliana;

[0014] The nucleotide sequences of the choline kinase CK and inositol polyphosphate kinase IPK are shown in SEQ ID NOs: 5-6, respectively.

[0015] The third object of the present invention is to provide a recombinant Pichia pastoris strain that can enhance the production of astaxanthin and enhance molten oxygen.

[0016] A recombinant Pichia pastoris strain with enhanced thiolysis and production of astaxanthin was obtained by expressing the hemoglobin VHB from Vitreoscilla in the host bacteria.

[0017] More preferably, the isopentenol utilization pathway (IUP) is constructed by expressing the hemoglobin VHB derived from Vitreoscilla in the host bacteria.

[0018] As an optimized embodiment, the nucleotide sequence of the hemoglobin VHB is shown in SEQ ID NO: 7.

[0019] More preferably, a recombinant Pichia pastoris strain with high astaxanthin production, wherein the supply of the precursor acetyl-CoA is enhanced, the isopentenol utilization pathway is increased, and the molten oxygen is enhanced, is obtained by expressing ATP citrate lyase ACL, acetyl-CoA synthase ACS, choline kinase CK from Saccharomyces cerevisiae, inositol polyphosphate kinase IPK from Arabidopsis thaliana, and hemoglobin VHB from Vitreoscilla in the host bacteria.

[0020] A fourth object of the present invention is to provide a method for constructing the above-mentioned recombinant Pichia pastoris strain, comprising:

[0021] Using BB3rN-AC as a vector plasmid, a recombinant plasmid for co-expression of ACL and ACS was constructed, and the recombinant plasmid was integrated into the RGI2 site of the PP-LC2 genome to obtain the recombinant Pichia pastoris strain PP-BC-CoA that enhances the precursor acetyl-CoA;

[0022] Using BB3rN-AE as a vector plasmid, a co-expression recombinant plasmid of ACL, ACS, IPK, and CK was constructed, and the recombinant plasmid was integrated into the RGI2 site of the PP-LC2 genome to obtain the recombinant Pichia pastoris strain PP-BC-CoA-IUP of the isopentenol utilization pathway.

[0023] Using BB3rN-AF as a vector plasmid, a co-expression recombinant plasmid of ACL, ACS, IPK, CK, and VHB was constructed, and the recombinant plasmid was integrated into the RGI2 site of the PP-LC2 genome to obtain the recombinant Pichia pastoris strain PP-BC-CoA-IUP-VHB of the isopentenol utilization pathway.

[0024] As a preferred embodiment, the fermentation culture uses glucose or methanol as a carbon source.

[0025] Application of the above recombinant Pichia pastoris strain in astaxanthin production.

[0026] The applications specifically include:

[0027] The recombinant strain was taken out from the cryopreservation tube, inoculated into a YPD test tube at a 1-10% inoculation rate, and cultured at 25-30° C. for 24 hours to obtain a seed solution;

[0028] The seed liquid is inoculated into a fermentation medium at an inoculation rate of 1 to 10%, and cultured at 25 to 30° C. with shaking for 3 to 8 days; the carbon source of the fermentation medium is glucose or methanol.

[0029] The strain can ferment and produce astaxanthin using methanol as the sole carbon source.

[0030] Fermentation medium formula: 10g / L yeast powder, 20g / L peptone, 20g / L glucose or methanol.

[0031] Beneficial effects:

[0032] The recombinant Pichia pastoris of the present invention can synthesize astaxanthin de novo using glucose or methanol, achieving efficient synthesis of the natural product astaxanthin in Pichia pastoris. Furthermore, by constructing the precursor acetyl-CoA synthesis pathway and the IUP pathway, the present invention enhances the precursor supply and improves the astaxanthin production capacity of Pichia pastoris. Furthermore, astaxanthin synthesis requires the participation of large amounts of oxygen. By expressing hemoglobin, the intracellular dissolved oxygen is increased, further enhancing the astaxanthin production capacity of Pichia pastoris. The PP-LC2-CoA-IUP-VHB strain can achieve an astaxanthin yield of 205.43 mg / L and an astaxanthin content of 5.20 mg / g in continuous fed-batch fermentation in a 5L fermentor using methanol as the sole carbon source, laying the foundation for further industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The diagram shows the structure of plasmid BB3rN-AC-ACL-ACS, which carries the nourseothricin gene used as a selection marker for Pichia pastoris.

[0034] Figure 2 The diagram shows the structure of plasmid BB3rN-AC-PTA-PK. The nourseothricin gene carried by this plasmid is used as a selection marker for Pichia pastoris.

[0035] Figure 3 The plasmid BB3rN-AC-IPK-CK was constructed. The nourseothricin gene carried by the plasmid is used as a selection marker for Pichia pastoris.

[0036] Figure 4 The structure of plasmid BB3rN-14*-VHb is shown. The nourseothricin gene carried by this plasmid is used as a selection marker for Pichia pastoris.

[0037] Figure 5 The diagram shows the structure of plasmid BB3rN-AE-ACL-ACS-IPK-CK. The nourseothricin gene carried by this plasmid is used as a selection marker for Pichia pastoris.

[0038] Figure 6 The diagram shows the structure of plasmid BB3rN-AF-ACL-ACS-IPK-CK-VHB. The nourseothricin gene carried by this plasmid is used as a selection marker for Pichia pastoris.

[0039] Figure 7 This is a graph showing the astaxanthin production produced by the engineered strain.

[0040] Figure 8 is the OD of the engineered strains PP-LC2-CoA1-IUP and PP-LC2-CoA1-IUP-VHB 600 picture.

[0041] Figure 9 This is a fermentation diagram of the engineered strain PP-LC2-CoA1-IUP-VHB in a 5L fermentor using methanol as the sole carbon source. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0043] The original strain used in the examples was the Pichia pastoris PP-LC2 strain, a modified Pichia pastoris strain obtained by introducing geranylgeranyl diphosphate synthase CrtE, phytoene synthase / lycopene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl CoA reductase tHMGR, β-carotene ketolase CrtW, β-carotene hydroxylase CrtZ, dehydrogenase Zwf1, phosphate dehydrogenase Gnd2, and NADH kinase Pos5 into Pichia pastoris GS115. The strain and its construction method have been disclosed in CN 119120242 A. The Pichia pastoris GS115 strain is commercially available.

[0044] In the embodiment, the extraction and quantitative analysis steps of astaxanthin are as follows:

[0045] 1. Extraction of astaxanthin

[0046] (1) Take 1 mL of the mixed fermentation broth and centrifuge at 12000 rpm for 5 min (wash twice with pure water).

[0047] (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.

[0048] (3) Add 4 mL of anhydrous ethanol.

[0049] (4) Centrifuge the sample at 12,000 rpm for 5 minutes. Transfer the supernatant to a new centrifuge tube and store in the dark.

[0050] 2. Quantitative analysis of astaxanthin The concentration of astaxanthin was determined using high performance liquid chromatography.

[0051] The liquid chromatography model used in this study was Agilent Technologies 1200 Infinity series; the chromatographic column was: AcclaimTM120 C30 chromatographic column; the ultraviolet absorption wavelength was 450nm; the mobile phase was methanol and methyl tert-butyl ether; the flow rate was controlled at 1.0mL / min; and the column temperature was 25℃.

[0052] Example 1 Amplification of genetic elements and preparation of target plasmids

[0053] (1) Preparation of target genes

[0054] Based on the gene sequences encoding ATP-citrate lyase and acetyl-CoA synthase from Yarrowia lipolytica provided by NCBI, PCR amplification was performed using the Saccharomyces cerevisiae genome as a template. The gene sequences are shown in SEQ ID Nos: 1-2. Phosphoketolase from Leuconostoc mesenteroides and phosphotransacetylase from Clostridium coli were commissioned to GenScript Biotech Co., Ltd. for synthesis and codon optimization. The gene sequences are shown in SEQ ID Nos: 3-4.

[0055] Based on the gene sequence encoding choline kinase from Saccharomyces cerevisiae provided by NCBI, PCR amplification was performed using the Saccharomyces cerevisiae genome as a template, and the gene sequence is shown in SEQ ID No: 5. Inositol polyphosphate kinase from Arabidopsis thaliana was commissioned to be synthesized by GenScript Biotech Co., Ltd. and codon-optimized, and the gene sequence is shown in SEQ ID No: 6.

[0056] The hemoglobin from Vitreoscilla provided by NCBI was commissioned to be synthesized by GenScript Biotech Co., Ltd. and codon optimized. The gene sequence is shown in SEQ ID No: 7.

[0057] (2) Construction of recombinant plasmid

[0058] 1. Using BB1-23 (Plasmid #98496) as a vector plasmid, a recombinant plasmid containing ACL, ACS, PTA, PK, IPK, CK, and VHB was constructed. The plasmid structure is shown in Figure 1-6.

[0059] GenScript Biotech Co., Ltd. was commissioned to synthesize the PTA, PK, IPK, and VHB gene sequences. The recombinant plasmids BB1-23-ACL and BB1-23-ACS were PCR amplified using the Yarrowia lipolytica genome as a template to obtain the ACL and ACS gene sequences. The BB1-23-CK was PCR amplified using the Saccharomyces cerevisiae genome as a template to obtain the CK gene sequence.

[0060] Each gene fragment was inserted into plasmid BB1-23 by the GoldenGate method to obtain recombinant plasmids BB1-23-ACL, BB1-23-ACS, BB1-23-PTA, BB1-23-PK, BB1-23-IPK, BB1-23-CK and BB1-23-VHB, as follows:

[0061] ACL-F and ACL-R were used as primers, and the Yarrowia lipolytica genome was used as a template. The primer sequences are shown in Table 2.

[0062] ACS-F and ACS-R were used as primers, and the Yarrowia lipolytica genome was used as a template. The primer sequences are shown in Table 2.

[0063] PTA-F and PTA-R were used as primers, and the PTA gene sequence was used as a template. The primer sequences are shown in Table 2.

[0064] PK-F and PK-R were used as primers and the PK gene sequence was used as template. The primer sequences are shown in Table 2.

[0065] IPK-F and IPK-R were used as primers, and the IPK gene sequence was used as a template. The primer sequences are shown in Table 2.

[0066] CK-F and CK-R were used as primers and the Saccharomyces cerevisiae genome was used as a template. The primer sequences are shown in Table 2.

[0067] VHB-F and VHB-R were used as primers, and the VHB gene sequence was used as a template. The primer sequences are shown in Table 2.

[0068] The PCR enzyme used for amplification was Phanta Max Super-Fidelity DNA Polymerase from Nanjing Novozymes Biotechnology Co., Ltd. The amplified ACL, ACS, PTA, PK, IPK, CK, and VHB fragments were recovered and purified by agarose gel electrophoresis.

[0069] GoldenGate assembly was performed using Bsa1 enzyme and T4 ligase from Shanghai Beyotime Biotechnology Co., Ltd. The circular recombinant vectors were transformed into competent Escherichia coli DH5α cells, screened for kanamycin sulfate resistance on plates, and verified by colony PCR and sequencing to obtain positive recombinant plasmids BB1-23-ACL, BB1-23-ACS, BB1-23-PTA, BB1-23-PK, BB1-23-IPK, BB1-23-CK, and BB1-23-VHB.

[0070] 2. Construction of expression cassettes for ACL, ACS, PTA, PK, IPK, CK, and VHB

[0071] Plasmid BB1-23-ACL, plasmid BB1-12-pGAP, and plasmid BB1-34-RPS25Att were inserted into plasmid BB2-AB using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-AB-pGAP-ACL-RPS25Att;

[0072] Plasmid BB1-23-ACS, plasmid BB1-12-pMDH3, and plasmid BB1-34-RPS2tt were inserted into plasmid BB2-BC using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-BC-pMDH3-ACS-RPS2tt;

[0073] Plasmid BB1-23-PK, plasmid BB1-12-pGAP, and plasmid BB1-34-RPS25Att were inserted into plasmid BB2-AB using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-AB-pGAP-PK-RPS25Att;

[0074] Plasmid BB1-23-PTA, plasmid BB1-12-pMDH3, and plasmid BB1-34-RPS2tt were inserted into plasmid BB2-BC using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-BC-pMDH3-PTA-RPS2tt;

[0075] Plasmid BB1-23-IPK, plasmid BB1-12-pADH2, and plasmid BB1-34-RPS2tt were inserted into plasmid BB2-CD using the GoldenGate method with Bpi1 enzyme and T4 ligase to obtain plasmid BB2-CD-pADH2-IPK-RPS2tt;

[0076] Plasmid BB1-23-CK, plasmid BB1-12-pGPM1 and plasmid BB1-34-RPL2Att were inserted into plasmid BB2-DE by GoldenGate method using Bpi1 enzyme and T4 ligase to obtain plasmid BB2-DE-pGPM1-CK-RPL2Att.

[0077] Plasmid BB1-23-VHB, plasmid BB1-12-pGCW14 and plasmid BB1-34-RPS17Btt were inserted into plasmid BB2-EF by GoldenGate method using Bpi1 enzyme and T4 ligase to obtain plasmid BB2-EF-pGCW14-VHB-RPS17Btt.

[0078] The construction process of the recombinant plasmid BB2-AB-pGAP-ACL-RPS25Att is as follows:

[0079] GoldenGate assembly was performed using Bpi1 enzyme and T4 ligase from Nanjing Formase Biotechnology Co., Ltd. The reaction system was as follows:

[0080] Table 1

[0081] system 10 μL BB2-AB 1 μL BB1-12-pGAP 1 μL BB1-23-ACL 1 μL BB1-34-RPS25Att 1 μL Bpi1 0.5μL BSA 1 μL T4 ligase 0.5μL T4buffer 1 μL distilled water 3μL

[0082] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmids BB2-AB-pGAP-ACL-RPS25Att were obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0083] The construction process of the recombinant plasmid BB2-BC-pMDH3-ACS-RPS2tt is as follows:

[0084] The reaction system was basically the same as that in Table 1, except that the plasmids were replaced with BB2-BC, BB1-12-pMDH3, BB1-23-ACS, and BB1-34-RPS2tt.

[0085] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB2-BC-pMDH3-ACS-RPS2tt was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0086] The construction process of the recombinant plasmid BB2-AB-pGAP-PK-RPS25Att is as follows:

[0087] The reaction system was basically the same as that in Table 1, except that the plasmid was replaced with BB1-23-PK.

[0088] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB2-CD-pADH2-IPK-RPS2tt was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0089] The construction process of the recombinant plasmid BB2-BC-pMDH3-PTA-RPS2tt is as follows:

[0090] The reaction system was basically the same as that in Table 1, except that the plasmids were replaced with BB2-BC, BB1-12-pMDH3, BB1-23-PTA, and BB1-34-RPS2tt.

[0091] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB2-BC-pMDH3-PTA-RPS2tt was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0092] The construction process of the recombinant plasmid BB2-CD-pADH2-IPK-RPS2tt is as follows:

[0093] The reaction system was basically the same as that in Table 1, except that the plasmids were replaced with BB2-CD, BB1-12-pADH2, BB1-23-IPK, and BB1-34-RPS2tt.

[0094] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB2-CD-pADH2-IPK-RPS2tt was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0095] The construction process of the recombinant plasmid BB2-DE-pGPM1-CK-RPL2Att is as follows:

[0096] The reaction system was basically the same as that in Table 1, except that the plasmids were replaced with BB2-DE, BB1-12-pGPM1, BB1-23-CK, and BB1-34-RPL2Att.

[0097] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB2-DE-pGPM1-CK-RPL2Att was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0098] The construction process of the recombinant plasmid BB2-EF-pGCW14-VHB-RPS17Btt is as follows:

[0099] The reaction system was basically the same as that in Table 1, except that the plasmids were replaced with BB2-EF, BB1-12-pGCW14, BB1-23-VHB, and BB1-34-RPS17Btt.

[0100] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB2-EF-pGCW14-VHB-RPS17Btt was obtained by plate screening for ampicillin resistance and verification by colony PCR and sequencing.

[0101] 3. Using BB3rN-AC (Plasmid #98551) as a vector, construct the expression cassettes of ACL and ACS.

[0102] The plasmids BB2-AB-pGAP-ACL-RPS25Att and BB2-BC-pMDH3-ACS-RPS2tt were inserted into the plasmid BB3rN-AC using the GoldenGate method with Bsa1 enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AC-ACL-ACS.

[0103] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB3rN-AC-ACL-ACS was obtained through plate screening for nourseothricin resistance and verification by colony PCR and sequencing.

[0104] 4. Using BB3rN-AC (Plasmid #98551) as a vector, construct the expression cassettes of PK and PTA.

[0105] The plasmids BB2-AB-pGAP-PK-RPS25Att and BB2-BC-pMDH3-PTA-RPS2tt were inserted into the plasmid BB3rN-AC using the GoldenGate method with Bsa1 enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AC-PK-PTA.

[0106] The construction process of the recombinant plasmid BB3rN-AC-PK-PTA is as follows:

[0107] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB3rN-AC-PK-PTA was obtained by plate screening for nourseothricin resistance and verification by colony PCR and sequencing.

[0108] 5. Using BB3rN-AC (Plasmid #98551) as a vector, construct the expression cassettes of IPK and CK.

[0109] The plasmids BB2-CD-pADH2-IPK-RPS2tt and BB2-DE-pGPM1-CK-RPL2Att were inserted into the plasmid BB3rN-AC using the GoldenGate method with Bsa1 enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AC-IPK-CK.

[0110] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB3rN-AC-IPK-CK was obtained by plate screening for nourseothricin resistance and verification by colony PCR and sequencing.

[0111] 6. Use BB3rN-14* (Plasmid#98550) as a vector to construct the VHB expression cassette.

[0112] Plasmids BB1-23-pGAP, BB1-23-VHB and BB1-34-RPS25Att were inserted into plasmid BB3rN-14* by the GoldenGate method using Bpi1 enzyme and T4 ligase to obtain recombinant plasmid BB3rN-14*-VHB.

[0113] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB3rN-14*-VHB was obtained through plate screening for nourseothricin resistance and verification by colony PCR and sequencing.

[0114] 7. Using BB3rN-AE (Plasmid #98553) as a vector, construct expression cassettes for ACL, ACS, IPK, and CK.

[0115] Plasmids BB2-AB-pGAP-ACL-RPS25Att, BB2-BC-pMDH3-ACS-RPS2tt, BB2-CD-pADH2-IPK-RPS2tt, and BB2-DE-pGPM1-CK-RPL2Att were inserted into plasmid BB3rN-AE using the GoldenGate method with Bsa1 enzyme and T4 ligase to generate the recombinant plasmid BB3rN-AE-ACL-ACS-IPK-CK. The circular recombinant vectors were transformed into competent Escherichia coli DH5α cells, and positive recombinant plasmids BB3rN-AE-ACL-ACS-IPK-CK were obtained by plate selection for nourseothricin resistance, colony PCR, and sequencing.

[0116] 8. Using BB3rN-AF (Plasmid #98554) as a vector, construct expression cassettes for ACL, ACS, IPK, CK, and VHB.

[0117] The plasmids BB2-AB-pGAP-ACL-RPS25Att, BB2-BC-pMDH3-ACS-RPS2tt, BB2-CD-pADH2-IPK-RPS2tt, BB2-DE-pGPM1-CK-RPL2Att and BB2-EF-pGCW14-VHB-RPS17Btt were inserted into the plasmid BB3rN-AF using the GoldenGate method with Bsa1 enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AE-ACL-ACS-IPK-CK-VHB.

[0118] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmid BB3rN-AE-ACL-ACS-IPK-CK-VHB was obtained through plate screening for nourseothricin resistance and verification by colony PCR and sequencing.

[0119] Table 2 Primer sequences

[0120]

[0121]

[0122] Example 2 Construction of recombinant bacteria

[0123] 1. Construction of recombinant PP-LC2-CoA1 or PP-LC2-CoA2

[0124] The plasmid BB3rN-AC-ACL-ACS or BB3rN-AC-PK-PTA containing the gene expression cassette was introduced into Pichia pastoris PP-LC2 to obtain the recombinant bacteria PP-LC2-CoA1 or PP-LC2-CoA2.

[0125] 2. Construction of recombinant strain PP-LC2-IUP

[0126] The plasmid BB3rN-AC-IPK-CK containing the gene expression cassette was introduced into Pichia pastoris PP-LC2 to obtain the recombinant strain PP-LC2-IUP.

[0127] 3. Construction of recombinant strain PP-LC2-VHB

[0128] The plasmid BB3rN-14*-VHB containing the gene expression cassette was introduced into Pichia pastoris PP-LC2 to obtain the recombinant strain PP-LC2-VHB.

[0129] 4. Construction of recombinant PP-LC2-CoA1-IUP

[0130] The plasmid BB3rN-AC-ACL-ACS-IPK-CK containing the gene expression cassette was introduced into Pichia pastoris PP-LC2-CoA1 to obtain the recombinant bacteria PP-LC2-CoA1-IUP.

[0131] 5. Construction of recombinant bacteria PP-LC2-CoA1-IUP-VHB

[0132] The plasmid BB3rN-AC-ACL-ACS-IPK-CK-VHB containing the gene expression cassette was introduced into Pichia pastoris PP-LC2-CoA1-IUP to obtain the recombinant bacteria PP-LC2-CoA1-IUP-VHB.

[0133] The specific method is as follows:

[0134] ① Competent cells were prepared by culturing the engineered Pichia pastoris overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose).

[0135] ② Use an electroporator to introduce BB3rN-AC-ACL-ACS / BB3rN-AC-PK-PTA / BB3rN-AC-IPK-CK / BB3rN-14*-VHB / BB3rN-AC-ACL-ACS-IPK-CK / BB3rN-AC-ACL-ACS-IPK-CK-VHB into the corresponding Pichia pastoris competent cells for homologous recombination.

[0136] ③ Screen on YPD screening plates containing bleomycin. Single colonies will grow in 2 to 3 days. The correct positive clone identified by PCR will be named the recombinant bacteria PP-LC2-CoA1 / PP-LC2-CoA2 / PP-LC2-IUP / PP-LC2-VHB / PP-LC2-CoA1-IUP / PP-LC2-CoA1-IUP-VHB.

[0137] Example 3 Application of recombinant bacteria in the production of astaxanthin

[0138] The engineered strains were cultured using the original bacteria PP-LC2 and the recombinant bacteria PP-LC2-CoA1, PP-LC2-CoA2, PP-LC2-IUP, PP-LC2-VHB, PP-LC2-CoA1-IUP and PP-LC2-CoA1-IUP-VHB in Example 2 to produce astaxanthin.

[0139] The specific method is as follows: take out the strain from the seed tube, inoculate it into a YPD test tube with a 1% inoculum, and culture it at 25℃ for 24 hours to obtain the seed solution;

[0140] The seed liquid was inoculated at an inoculum size of 1% into 50 mL of fermentation medium (20 g / L glucose, 10 g / L yeast extract and 20 g / L peptone), and cultured at 25°C and 220 rpm for 5 days. 40 g / L glucose was added at 24 h. With glucose as the sole carbon source, the astaxanthin production of the recombinant strains PP-LC2, PP-LC2-CoA1, PP-LC2-CoA2, PP-LC2-IUP, PP-LC2-VHB, PP-LC2-CoA1-IUP and PP-LC2-CoA1-IUP-VHB were 127.82 mg / L, 158.76 mg / L, 141.66 mg / L, 148.32 mg / L, 153.98 mg / L, 171.98 mg / L and 208.78 mg / L, respectively. Figure 7). The above data show that increasing the supply of precursors, whether increasing intracellular acetyl-CoA or the direct precursor FPP for terpenoid synthesis, enhances the synthesis of astaxanthin in the engineered strains to varying degrees; and the introduction of Vitreoscilla hemoglobin mainly increases the production of astaxanthin by increasing the dissolved oxygen in the cells, thereby promoting the conversion of β-carotene to astaxanthin. At the same time, we measured the OD of the engineered strains PP-LC2-CoA1-IUP and PP-LC2-CoA1-IUP-VHB. 600 , it was found that the OD of the engineered strain after the introduction of Vitreoscilla hemoglobin 600 From 46.80 to 57.09, an increase of 21.99% ( Figure 8 For the intracellular product astaxanthin, increasing biomass can significantly increase astaxanthin production. This strategy can also provide a reference for the production of other intracellular compounds.

[0141] The present invention introduces an exogenous acetyl-CoA synthesis pathway and screens out the optimal acetyl-CoA synthesis pathway by comparing two different acetyl-CoA synthesis pathways, thereby strengthening the metabolic flow toward astaxanthin synthesis. In addition, the introduction of the artificially constructed IUP pathway enhances carbon flux by exogenously adding isopentenol, thereby simplifying the astaxanthin synthesis pathway. In addition, astaxanthin synthesis requires high dissolved oxygen. The introduction of the exogenous hemoglobin gene can increase the dissolved oxygen of the engineered cells and promote astaxanthin synthesis.

[0142] Example 4 Fermentation of astaxanthin by recombinant bacteria PP-LC2-CoA1-IUP-VHB

[0143] ①Seed liquid culture:

[0144] a. Primary seed solution: A 1% inoculum of the recombinant strain PP-LC2-CoA1-IUP-VHB was taken from a cryopreserved tube and inoculated into a YPD tube at 30°C and cultured at 200 rpm for 24 hours as a primary seed solution; the YPD medium contained 2% peptone, 1% yeast extract, and 2% glucose;

[0145] b. Secondary seed solution: Take a seed solution at a 10% inoculum size and inoculate it into a new seed culture medium, and culture it at a constant temperature under the same conditions as a to obtain a seed solution for fermentation culture.

[0146] ② Fed-batch fermentation

[0147] The seed solution obtained from the seed culture was inoculated into a 5L fermentor containing fermentation medium (20g / L methanol, 10g / L yeast extract, and 20g / L peptone). The recombinant strain PP-LC2-CoA1-IUP-VHB was subjected to batch fermentation in the 5L fermentor at a temperature of 25°C, 500rpm, and 40% dissolved oxygen. The initial methanol concentration in the fermentor was 20g / L. When the methanol was about to be exhausted, methanol was added to maintain the methanol concentration in the fermentor below 5g / L. Samples were taken every 12 hours to determine the dry cell weight, extract astaxanthin from the fermentation broth, and determine the astaxanthin content. 600 Reached 259.2, astaxanthin production reached 205.43mg / L, and astaxanthin content was 5.20mg / g ( Figure 9 ), which is the highest astaxanthin production by Pichia pastoris using methanol as the sole carbon source.

[0148] By comparing different acetyl-CoA synthesis pathways, the present invention selected a synthesis route that can significantly increase the product yield. Subsequently, the artificially constructed IUP synthesis pathway was introduced, and isopentenol was used as a substrate to achieve a two-step synthesis of the common precursor IPP of terpenes. In addition, Pichia pastoris is an aerobic bacterium. At the same time, the synthesis process of astaxanthin also requires the participation of oxygen. The introduction of Vitreoscilla hemoglobin can significantly increase the biomass of the strain, alleviate the oxygen deficiency caused by high biomass, and significantly promote the yield of the engineered strain. Finally, continuous fed-batch fermentation was carried out in a 5L fermentor, and the production capacity of the strain was comprehensively evaluated. The OD value of the recombinant strain in the fermentor was measured. 600 Through the detection of astaxanthin production conditions, it is expected that the production of astaxanthin can be further increased in larger fermentation tanks, laying the foundation for subsequent industrialization.

Claims

1. A recombinant Pichia pastoris strain with high astaxanthin production, characterized in that: The supply of the precursor acetyl-CoA is enhanced in the recombinant Pichia pastoris strain by expressing ATP citrate lyase ACL, acetyl-CoA synthase ACS or phosphoketolase PK, phosphotransacetylase PTA in the host strain.

2. A recombinant Pichia pastoris strain with high astaxanthin production, characterized in that: The isopentenol utilization pathway is increased in the recombinant Pichia pastoris strain, which is obtained by expressing choline kinase CK derived from Saccharomyces cerevisiae and inositol polyphosphate kinase IPK derived from Arabidopsis thaliana in the host bacteria.

3. A recombinant Pichia pastoris strain with high astaxanthin production, characterized in that: The enhanced molten oxygen in the recombinant Pichia pastoris strain is achieved by expressing hemoglobin VHB derived from Vitreoscilla in the host bacteria.

4. A recombinant Pichia pastoris strain with high astaxanthin production, characterized in that: The recombinant Pichia pastoris strain enhances the supply of precursor acetyl-CoA, increases the isopentenol utilization pathway, and simultaneously enhances molten oxygen, and is obtained by expressing ATP citrate lyase ACL, acetyl-CoA synthase ACS, choline kinase CK from Saccharomyces cerevisiae, inositol polyphosphate kinase IPK from Arabidopsis thaliana, and hemoglobin VHB from Vitreoscilla in the host bacteria.

5. The recombinant Pichia pastoris according to claim 1, wherein The ATP citrate lyase ACL and acetyl-CoA synthase ACS are derived from Yarrowia lipolytica Yarrowia lipolytica , the gene sequence is shown in AEQID No: 1-2; the phosphoketolase PK is derived from Leuconostoc mesenteroides, and the phosphotransacetylase PTA is derived from Clostridium coli. The encoding gene of phosphoketolase PK is shown in SEQ ID No: 3; the encoding gene of phosphotransacetylase PTA is shown in SEQ ID No:

4.

6. The recombinant Pichia pastoris according to claim 4, wherein The gene encoding the inositol polyphosphate kinase is shown in SEQ ID No: 5; the gene encoding the choline kinase is shown in SEQ ID No: 6; the hemoglobin VHB is derived from Vitreoscilla, and the gene encoding the hemoglobin is shown in SEQ ID No:

7.

7. The method for constructing a recombinant Pichia pastoris strain with high astaxanthin production according to claim 4, characterized in that: Using BB3rN-AF as a vector plasmid, a co-expression recombinant plasmid of ACL, ACS, IPK, CK, and VHB was constructed, and the recombinant plasmid was integrated into the RGI2 site of the PP-LC2 genome to obtain a recombinant Pichia pastoris strain with high astaxanthin production.

8. Use of the recombinant Pichia pastoris strain according to any one of claims 1 to 6 in the fermentation production of astaxanthin.

9. The use according to claim 8, characterized in that The recombinant Pichia pastoris strain is subjected to seed culture and fermentation culture to synthesize astaxanthin.

10. The use according to claim 9, characterized in that The fermentation culture uses glucose or methanol as a carbon source.

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