Recombinant pichia pastoris strain for producing astaxanthin as well as construction method and application of recombinant pichia pastoris strain

Abstract

The invention relates to a recombinant pichia pastoris strain for producing astaxanthin as well as a construction method and application of the recombinant pichia pastoris strain. The recombinant pichia pastoris strain is obtained by expressing dehydrogenase Zwf1, phosphate dehydrogenase Gnd2, NADH kinase Pos5 and fusion protein CrtZ-Linker-CrtW of beta-carotene ketolase CrtW and beta-carotene hydroxylase CrtZ in host bacteria. Wherein the host bacterium is a pichia pastoris gene modified strain PP-L. The fermentation conditions of the recombinant strain are optimized based on cofactor engineering, and the astaxanthin production capacity of the pichia pastoris is further improved. The construction method of the recombinant pichia pastoris is simple, synthesis of astaxanthin can be better promoted, the engineering strain can efficiently synthesize astaxanthin by using glucose and methanol through fermentation optimization, and industrial production is facilitated.

Description

Technical Field

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

[0002] Pichia pastoris is a methylotrophic yeast with strong respiratory metabolism, so it can grow to very high cell densities in simple culture media and can also be easily expanded from shake flasks to large-scale bioreactors. Therefore, the comprehensive characteristics of Pichia pastoris give this strain great potential in the production of compounds such as carotenoids. Recent studies have demonstrated that Pichia pastoris is suitable for the production of natural products such as exogenous proteins, lycopene, 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 has a fast growth rate, strong stress resistance, simple cultivation, and is suitable for industrialization; the molecular operation platform is mature and the genetic tools are complete, and the development prospects are broad. Haematococcus pluvialis ( H. pluvialis ) is recognized as the most ideal source of natural astaxanthin in nature. Astaxanthin is a natural antioxidant and the most powerful antioxidant discovered so far. Its antioxidant capacity is 10 times that of natural β-carotene and 550 times that of vitamin E. Introducing exogenous β-carotene synthesis modules and astaxanthin synthesis modules into Pichia pastoris can enable it to heterologously synthesize astaxanthin.

[0003] Prior art has genetically modified Pichia pastoris to obtain recombinant Pichia pastoris that produces 3S, 3S' astaxanthin with the same configuration as Haematococcus pluvialis. However, the astaxanthin obtained after the introduction of the product synthesis module has a certain toxic effect on cells. The cell damage caused by the increase in astaxanthin production also inhibits the further increase of production, making it impossible to further increase the astaxanthin production. In addition, CN118146970A provides a recombinant Pichia pastoris that produces astaxanthin, and the astaxanthin production of Pichia pastoris is increased by genetic engineering means. At present, further increasing the astaxanthin production is still a research direction to promote the industrial application of astaxanthin. Summary of the invention

[0004] The first object of the present invention is to provide a recombinant Pichia pastoris strain producing astaxanthin.

[0005] To achieve the above object, the present invention adopts the following technical solution: A recombinant Pichia pastoris strain producing astaxanthin, wherein the recombinant Pichia pastoris strain is obtained by expressing dehydrogenase Zwf1, phosphate dehydrogenase Gnd2, NADH kinase Pos5, and fusion protein CrtZ-Linker-CrtW of β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ in a host bacterium; Wherein, the host bacteria is the genetically modified strain of Pichia pastoris PP-L.

[0006] As a preferred embodiment, the linker used in the fusion protein is GGGEKGGG or GGGSGGGS.

[0007] As a preferred embodiment, the nucleotide sequences of the dehydrogenase Zwf1, phosphate dehydrogenase Gnd2, NADH kinase Pos5, β-carotene ketolase CrtW, and β-carotene hydroxylase CrtZ are shown in SEQ ID NOs: 1-5, respectively.

[0008] The second object of the present invention is to provide a method for constructing the above-mentioned recombinant Pichia pastoris strain, comprising: Using BB3-Z4-intE12 as a vector plasmid, a co-expression recombinant plasmid of Zwf1, Gnd2, Pos5 and CrtZ-Linker-CrtW was constructed, and the recombinant plasmid was integrated into the intE12 site of the PP-L genome to obtain the recombinant Pichia pastoris strain.

[0009] The third object of the present invention is to provide the use of the above-mentioned recombinant Pichia pastoris strain in the fermentation production of astaxanthin.

[0010] As a preferred embodiment, the recombinant Pichia pastoris strain is subjected to seed culture and fermentation culture to synthesize astaxanthin.

[0011] As a preferred embodiment, Fe is added during the fermentation of the recombinant Pichia pastoris strain. 2+ , sodium citrate, BHT, Tween-80 or more.

[0012] As a preferred embodiment, the sodium citrate is added at the beginning of fermentation; the Fe 2+ , BHT, and Tween-80 are added in the middle of fermentation.

[0013] As a preferred embodiment, the Fe 2+ The addition amount is 0.5-1.5mM, the addition amount of sodium citrate is 0.5g / L-3 g / L, the addition amount of BHT is 0.05%-0.25% v / v, and the addition amount of Tween-80 is 10-30%.

[0014] As a preferred embodiment, the fermentation culture uses glucose or methanol as a carbon source.

[0015] The recombinant Pichia pastoris of the present invention can synthesize astaxanthin from scratch using glucose, thus realizing the efficient synthesis of the natural product astaxanthin in Pichia pastoris. In addition, the present invention further improves the ability of Pichia pastoris to produce astaxanthin using exogenous additives: metal ions, sodium citrate, BHT, and Tween-80. The astaxanthin production of the PP-LC2 strain in a 5L fermenter fed-batch fermentation can reach 716.13 mg / L, and the astaxanthin content is 16.88 mg / g, laying a foundation for further realizing industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The plasmid is BB2-AB-pGAP-CrtZ-GGGEKGGG-CrtW-ScCYC1tt, the CrtZ-GGGEKGGG-CrtW promoter is pGAP, the terminator is ScCYC1tt, and the ampicillin gene carried by the plasmid is used as a selection marker for Escherichia coli DH5α.

[0017] Figure 2 The plasmid is BB2-AB-pGAP-CrtZ-GGGSGGGS-CrtW-ScCYC1tt, the CrtZ-GGGSGGGS-CrtW promoter is pGAP, the terminator is ScCYC1tt, and the ampicillin gene carried by the plasmid is used as a selection marker for Escherichia coli DH5α.

[0018] Figure 3 The plasmid is BB2-BC-pGPM1-Gnd2-RPP1Btt, the Gnd2 promoter is pGPM1, the terminator is RPP1Btt, and the ampicillin gene carried by the plasmid is used as a selection marker for Escherichia coli DH5α.

[0019] Figure 4 The plasmid is BB2-CD-pADH2-Pos5-RPS2tt, the Pos5 promoter is pADH2, the terminator is RPS2tt, and the ampicillin gene carried by the plasmid is used as a selection marker for Escherichia coli DH5α.

[0020] Figure 5 The plasmid is BB2-DE-pTEF1-Zwf1-RPL2Att, the Zwf1 promoter is pTEF1, the terminator is RPL2Att, and the ampicillin gene carried by the plasmid is used as a selection marker for Escherichia coli DH5α.

[0021] Figure 6The structure diagram of plasmid BB3-Z4-intE12-ZW1-Gnd2-Pos5-Zwf1 is shown. The bleomycin gene carried by the plasmid is used as a selection marker for Pichia pastoris.

[0022] Figure 7 The structure diagram of plasmid BB3-Z4-intE12-ZW2-Gnd2-Pos5-Zwf1 is shown. The bleomycin gene carried by the plasmid is used as a selection marker for Pichia pastoris.

[0023] Figure 8 This is a diagram of the structure of plasmid BB3-Z1-intE12-ZW.

[0024] Fig. 9 Comparison of astaxanthin production of recombinant bacteria PP-L, PP-L1, PP-L2, PP-L3, PP-LC1, and PP-LC2.

[0025] Fig.10 This is a graph showing the growth, astaxanthin production and content when 14 different metal ions were added to Pichia pastoris.

[0026] Fig.11 The growth rate (OD) of Pichia pastoris after adding different concentrations of sodium citrate 600 ), astaxanthin production (Astaxanthin) and content (Content) diagram.

[0027] Fig.12 This is a graph showing the growth, astaxanthin production and content of Pichia pastoris when different concentrations of BHT were added.

[0028] Fig.13 This is a graph showing the growth, astaxanthin production and content when different concentrations of Tween-80 were added to Pichia pastoris.

[0029] Fig.14 The growth, sugar consumption (Glucose), astaxanthin production and content of Pichia pastoris in a 5L fermenter with glucose as the carbon source.

[0030] Fig.15 The growth, methanol consumption, astaxanthin production and content, and β-carotene production of Pichia pastoris in a 5L fermenter with methanol as the carbon source. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0032] The original bacteria used in the examples is the Pichia pastoris PP-L strain, which is a modified strain of Pichia pastoris, obtained by introducing geranylgeranyl diphosphate synthase CrtE, phytoene synthase / lycopene cyclase CrtYB, phytoene desaturase CrtI and 3-hydroxy-3-methylglutaryl CoA reductase tHMGR, β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ fusion protein (linker amino acid sequence is GGGGGGGG) into Pichia pastoris GS115, and the strain and its specific construction method have been disclosed in CN118146970A. The Pichia pastoris GS115 strain can be purchased from commercial channels.

[0033] In the embodiment, the extraction and quantitative analysis steps of astaxanthin are as follows: 1. Extraction of astaxanthin (1) Take 1 mL of the mixed fermentation broth and centrifuge at 12,000 rpm for 5 min (wash twice with pure water).

[0034] (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.

[0035] (3) Add 4 mL of anhydrous ethanol.

[0036] (4) Centrifuge the sample at 12,000 rpm for 5 min. Transfer the supernatant to a new centrifuge tube and store in a dark place.

[0037] 2. Quantitative analysis of astaxanthin The concentration of astaxanthin was detected using high performance liquid chromatography.

[0038] 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 450 nm; the mobile phase was methanol and methyl tert-butyl ether; the flow rate was controlled at 1.0 mL / min; and the column temperature was 25°C.

[0039] Example 1 Amplification of genetic elements and preparation of target plasmids 1. Preparation of target gene According to the information provided by NCBI H. pluvialisThe nucleotide sequences of β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ are obtained by codon optimization. After the gene codons are optimized, the exogenous genes are better compatible with the Pichia pastoris chassis. The gene sequences are shown in SEQ ID No: 4-5, which are the same as the CrtW and CrtZ gene sequences introduced into PP-L.

[0040] According to the coding gene sequences of dehydrogenase Zwf1, phosphate dehydrogenase Gnd2 and NADH dehydrogenase Pos5 from Saccharomyces cerevisiae provided by NCBI, PCR amplification was performed using the Saccharomyces cerevisiae genome as a template. The gene sequences are shown in SEQ ID No: 1-3.

[0041] (II) Construction of recombinant plasmid 1. Using BB1-23 (Plasmid #98496) as a vector plasmid, a recombinant plasmid containing Gnd2, Pos5, Zwf1, and CrtZ-linker-CrtW was constructed. In this example, a recombinant plasmid CrtZ-linker-CrtW containing three different linkers was constructed, namely GGGGGGGG (consistent with PP-L), GGGEKGGG, and GGGSGGGS.

[0042] GenScript Biotech Inc. was commissioned to synthesize the CrtW and CrtZ gene sequences. The recombinant plasmids BB1-23-Gnd2, BB1-23-Pos5, and BB1-23-Zwf1 were amplified by PCR using the Saccharomyces cerevisiae genome as a template to obtain the Gnd2, Pos5, and Zwf1 gene sequences.

[0043] Each gene fragment was inserted into plasmid BB1-23 by the GoldenGate method to obtain recombinant plasmids BB1-23-CrtZ-GGGGGGGG-CrtW, BB1-23-CrtZ-GGGEKGGG-CrtW, BB1-23-CrtZ-GGGSGGGS-CrtW, BB1-23-Gnd2, BB1-23-Pos5 and BB1-23-Zwf1, as follows: CrtZ-GGGGGGGG-CrtW, using CrtZ-F and CrtZ-R1 as primers and the CrtZ gene sequence as a template, the CrtZ fragment was amplified; using CrtW-F1 and CrtW-R as primers and the CrtW gene sequence as a template, the CrtW fragment was amplified, and CrtZ and CrtW were connected by overlap extension PCR to obtain CrtZ-GGGEKGGG-CrtW. The primer sequences are shown in Table 6.

[0044] CrtZ-GGGEKGGG-CrtW, using CrtZ-F and CrtZ-R2 as primers and the CrtZ gene sequence as a template, amplify the CrtZ fragment; using CrtW-F2 and CrtW-R as primers and the CrtW gene sequence as a template, amplify the CrtW fragment, and connect CrtZ and CrtW by overlap extension PCR to obtain CrtZ-GGGEKGGG-CrtW. The primer sequences are shown in Table 6.

[0045] CrtZ-GGGSGGGS-CrtW, using CrtZ-F and CrtZ-R3 as primers and the CrtZ gene sequence as a template, amplify the CrtZ fragment; using CrtW-F3 and CrtW-R as primers and the CrtW gene sequence as a template, amplify the CrtW fragment, and connect CrtZ and CrtW by overlap extension PCR to obtain CrtZ-GGGSGGGS-CrtW. The primer sequences are shown in Table 6.

[0046] The Gnd2 fragment was amplified using Gnd2-F and Gnd2-R as primers and the Saccharomyces cerevisiae genome as a template. The primer sequences are shown in Table 6.

[0047] The Pos5 fragment was amplified using Pos5-F and Pos5-R as primers and the Saccharomyces cerevisiae genome as a template. The primer sequences are shown in Table 6.

[0048] The Zwf1 fragment was amplified using Zwf1-F and Zwf1-R as primers and the Saccharomyces cerevisiae genome as a template. The primer sequences are shown in Table 6.

[0049] The PCR enzyme used for amplification is Phanta Max Super-Fidelity DNA Polymerase from Nanjing Novozymes Biotechnology Co., Ltd. The system is as follows: Table 1

[0050] The amplified CrtZ-GGGGGGGG-CrtW, CrtZ-GGGEKGGG-CrtW, CrtZ-GGGSGGGS-CrtW, Gnd2, Pos5, and Zwf1 fragments were recovered and purified and recovered by agarose gel electrophoresis.

[0051] GoldenGate assembly was performed using Bsa1 enzyme and T4 ligase from Shanghai Biotech Biotechnology Co., Ltd. The reaction system was as follows: Table 2

[0052] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmids BB1-23-CrtZ-GGGGGGGG-CrtW, BB1-23-CrtZ-GGGEKGGG-CrtW, BB1-23-CrtZ-GGGSGGGS-CrtW, BB1-23-Gnd2, BB1-23-Pos5, and BB1-23-Zwf1 were obtained through plate screening for kanamycin sulfate resistance and verification by colony PCR and sequencing.

[0053] 2. Construct expression cassettes of CrtZ-GGGEKGGG-CrtW, CrtZ- GGGSGGGS -CrtW, Gnd2, Pos5, and Zwf1.

[0054] Plasmids BB1-23-CrtZ-GGGEKGGG-CrtW and BB1-23-CrtZ-GGGSGGGS-CrtW were respectively inserted into plasmid BB2-AB (Plasmid #98534) with plasmid BB1-12-pGAP (Plasmid #98495) and plasmid BB1-34-ScCYC1tt (Plasmid #98497) by the GoldenGate method using Bpi1 enzyme and T4 ligase to obtain plasmids BB2-AB-pGAP-CrtZ-GGGEKGGG-CrtW-ScCYC1tt and BB2-AB-pGAP-CrtZ-GGGSGGGS-CrtW-ScCYC1tt; Plasmid BB1-23-Gnd2, plasmid BB1-12-pGPM1, and plasmid BB1-34-RPP1Btt were inserted into plasmid BB2-BC (Plasmid #98535) using the GoldenGate method using Bpi1 enzyme and T4 ligase to obtain plasmid BB2-BC-pGPM1-Gnd2-RPP1Btt; Plasmid BB1-23-Pos5, plasmid BB1-12-pADH2, and plasmid BB1-34-RPS2tt were inserted into plasmid BB2-CD (Plasmid #98536) by GoldenGate method using Bpi1 enzyme and T4 ligase to obtain plasmid BB2-CD-pADH2-Pos5-RPS2tt; Plasmid BB1-23-Zwf1, plasmid BB1-12-pADH2, and plasmid BB1-34-RPL2Att were inserted into plasmid BB2-DE (Plasmid#98537) using the GoldenGate method using Bpi1 enzyme and T4 ligase to obtain plasmid BB2-DE-pTEF1-Zwf1-RPL2Att; the structure of the recombinant plasmid is shown in Figures 1 to 5 .

[0055] The construction process of recombinant plasmids BB2-AB-pGAP-CrtZ-GGGEKGGG-CrtW-ScCYC1tt and BB2-AB-pGAP-CrtZ-GGGSGGGS-CrtW-ScCYC1tt is as follows: GoldenGate assembly was performed using Bpi1 enzyme and T4 ligase from Nanjing Formase Biotechnology Co., Ltd. The reaction system was as follows: Table 3

[0056] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmids BB2-AB-pGAP-CrtZ-GGGEKGGG-CrtW-ScCYC1tt and BB2-AB-pGAP-CrtZ-GGGSGGGS-CrtW-ScCYC1tt were obtained by plate screening of ampicillin resistance and verification by colony PCR and sequencing. The plasmid structures are shown in Figure 1 , 2 .

[0057] The construction process of the recombinant plasmid BB2-BC-pGPM1-Gnd2-RPP1Btt is as follows: The reaction system was basically the same as that in Table 3, except that the plasmids were changed to BB2-BC, BB1-12-pGPM1, BB1-23-Gnd2, and BB1-34-RPP1Btt.

[0058] 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-pGPM1-Gnd2-RPP1Btt. The plasmid structure is shown in Figure 3 .

[0059] The construction process of the recombinant plasmid BB2-CD-pADH2-Pos5-RPS2tt is as follows: The reaction system was basically the same as that in Table 3, except that the plasmids were replaced with BB2-CD, BB1-12-Padh2, BB1-23-Pos5, and BB1-34-RPS2tt.

[0060] 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-pADH2-Pos5-RPS2tt. The plasmid structure is shown in Figure 4 .

[0061] The construction process of the recombinant plasmid BB2-DE-pTEF1-Zwf1-RPL2Att is as follows: The reaction system was basically the same as that in Table 3, except that the plasmids were changed to BB2-DE, BB1-12-pTEF1, BB1-23-Zwf1, and BB1-34-RPL2Att.

[0062] 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-DE-pTEF1-Zwf1-RPL2Att. The plasmid structure is shown in Figure 5 .

[0063] 3. Using BB3-Z4-intE12 as a vector, construct the co-expression recombinant plasmids of CrtZ-GGGEKGGG-CrtW / CrtZ-GGGGGGGG-CrtW and Gnd2, Pos5, and Zwf1.

[0064] ① Construction of BB3-Z4-intE12 Using BB3aK-AE (Plasmid #98532) as a template, the selection marker G418 geneticin was replaced with bleomycin, and the insertion site AOXTT was replaced with intE12.

[0065] ② Construction of BB3-Z4-intE12-ZW1-Gnd2-Pos5-Zwf1 and BB3-Z4-intE12-ZW2-Gnd2-Pos5-Zwf1 recombinant plasmids The plasmids BB2-AB-pGAP-CrtZ-GGGEKGGG-CrtW-ScCYC1tt and BB2-AB-pGAP-CrtZ-GGGSGGGS-CrtW-ScCYC1tt were respectively inserted into the plasmid BB3-Z4-intE12 with the plasmids BB2-BC-pGPM1-Gnd2-RPP1Btt, BB2-CD-pADH2-Pos5-RPS2tt and BB2-DE-pTEF1-Zwf1-RPL2Att by the Goldengate method using Bsa1 enzyme and T4 ligase to obtain the recombinant plasmids BB3-Z4-intE12-ZW1-Gnd2-Pos5-Zwf1 and BB3-Z4-intE12-ZW2-Gnd2-Pos5-Zwf1. The only difference between the two recombinant plasmids is the inserted linker.

[0066] The structures of the recombinant plasmids BB3-Z4-intE12-ZW1-Gnd2-Pos5-Zwf1 and BB3-Z4-intE12-ZW2-Gnd2-Pos5-Zwf1 are shown in Figure 6 , 7 , and its construction process is as follows: GoldenGate assembly was performed using Bsa1 enzyme and T4 ligase. The reaction system was as follows: Table 4

[0067] The circular recombinant vector was transformed into Escherichia coli DH5α competent cells, and the positive recombinant plasmids BB3-Z4-intE12-ZW1-Gnd2-Pos5-Zwf1 and BB3-Z4-intE12-ZW2-Gnd2-Pos5-Zwf1 were obtained through plate screening for bleomycin resistance and verification by colony PCR and sequencing.

[0068] 4. Using BB3-Z1-intE12 as a vector, construct the expression cassettes of CrtZ-GGGGGGGG-CrtW, CrtZ-GGGEKGGG-CrtW, and CrtZ-GGGSGGGS-CrtW.

[0069] ① Construction of BB3-Z1-intE12 BB3aK-14 * (Plasmid#98529) was used as a template, the selection marker G418 geneticin was replaced by bleomycin, and the insertion site AOXTT was replaced by intE12.

[0070] ② Construction of BB3-Z1-intE12-ZW1, BB3-Z1-intE12-ZW2 or BB3-Z1-intE12-ZW3 recombinant plasmids The plasmids BB1-23-CrtZ-GGGGGGGG-CrtW, BB1-23-CrtZ-GGGEKGGG-CrtW, and BB1-23-CrtZ-GGGSGGGS-CrtW were respectively inserted into the plasmid BB3-Z1-intE12 with BB1-12-pGAP and BB1-34-ScCYC1tt by the GoldenGate method using Bpi1 enzyme and T4 ligase to obtain recombinant plasmid BB3-Z1-intE12-ZW1 (containing CrtZ-GGGGGGGG-CrtW), recombinant plasmid BB3-Z1-intE12-ZW2 (containing CrtZ-GGGEKGGG-CrtW), and recombinant plasmid BB3-Z1-intE12-ZW3 (containing CrtZ-GGGSGGGS-Crt). The structures of the recombinant plasmids BB3-Z1-intE12-ZW1, BB3-Z1-intE12-ZW2 or BB3-Z1-intE12-ZW3 are shown in Figure 8 The specific construction process is as follows: GoldenGate assembly was performed using Bpi1 enzyme and T4 ligase. The reaction system was as follows: Table 5

[0071] The circular recombinant vector was transformed into E. coli DH5α competent cells, and positive recombinant plasmids BB3-Z1-intE12-ZW1, BB3-Z1-intE12-ZW2 and BB3-Z1-intE12-ZW3 were obtained by plate screening of bleomycin resistance and verification by colony PCR and sequencing. The plasmid structures are shown in Figure 8 .

[0072] Table 6 Primer sequence list

[0073] Example 2 Construction of recombinant bacteria 1. Construction of recombinant bacteria PP-L1, PP-L2, PP-L3, PP-LC1, and PP-LC2 Plasmids containing the gene expression cassettes of CrtZ-GGGGGGGG-CrtW, CrtZ-GGGEKGGG-CrtW, CrtZ-GGGSGGGS-CrtW, CrtZ-GGGEKGGG-CrtW-Gnd2-Pos5-Zwf1, and CrtZ-GGGSGGGS-CrtW-Gnd2-Pos5-Zwf1 were respectively cloned into BB3-Z1-intE12-ZW1, BB3-Z1-intE12-ZW2 / BB3-Z1-intE12-ZW3, BB3-Z4-intE12-ZW1-Gnd2-Pos5-Zwf1, and BB3-Z4- intE12-ZW2-Gnd2-Pos5-Zwf1 was introduced into Pichia pastoris PP-L, and the CrtZ-GGGGGGGG-CrtW, CrtZ-GGGEKGGG-CrtW, CrtZ-GGGSGGGS-CrtW, CrtZ-GGGEKGGG-CrtW-Gnd2-Pos5-Zwf1, and CrtZ-GGGSGGGS-CrtW-Gnd2-Pos5-Zwf1 expression cassettes were integrated into the intE12 site of the Pichia pastoris PP-L genome to obtain recombinant bacteria PP-L1, PP-L2, PP-L3, PP-LC1, and PP-LC2.

[0074] The specific method is as follows: ① Competent cells were prepared by culturing the engineered Pichia pastoris PP-L overnight in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose).

[0075] ② Use an electroporator to introduce B3-Z4-intE12-ZW1-Gnd2-Pos5-Zwf1 / BB3-Z4-intE12-ZW2-Gnd2-Pos5-Zwf1 into Pichia pastoris competent cells for homologous recombination.

[0076] ③ Screening was performed on YPD screening plates with bleomycin added. Single colonies grew out in 2 to 3 days. The correct positive clones identified by PCR were named recombinant bacteria PP-LC1 / PP-LC2.

[0077] Preparation method of competent cells of Pichia pastoris: (1) Grow yeast cells to OD 600 0.8~1.0; (2) Centrifuge at 4000 rpm for 5 min at room temperature; (3) Discard the supernatant and resuspend the bacterial slurry in 9 mL BEDS solution and 1 mL 1 M threitol; (4) Incubate on ice for 5 min; (5) Centrifuge at 4000 rpm for 5 min at room temperature; (6) Discard the supernatant and resuspend the bacterial sludge in 1 mL of BEDS solution; (7) Aliquot into 1.5 mL EP tubes and store at -80°C.

[0078] BEDS solution: 10 mM bicine-NaOH, pH 8.3; 3% (v / v) ethylene glycol and 1 M sorbitol.

[0079] Example 3 Application of recombinant bacteria in the production of astaxanthin The original bacteria PP-L and the recombinant bacteria PP-L1, PP-L2, PP-L3, PP-LC1 and PP-LC2 in Example 2 were used for the cultivation of engineered bacteria to produce astaxanthin.

[0080] The specific method is as follows: take out the strain from the seed tube, inoculate it into a YPD test tube with a 1% inoculation amount, and culture it at 30℃ for 24h to obtain the seed solution; The seed liquid was inoculated into 50 mL fermentation medium (20 g / L glucose, 10 g / L yeast extract and 20 g / L tryptone) at an inoculum rate of 1%, and cultured at 25°C and 220 rpm for 5 days. 20 g / L glucose was added at 24 h. With glucose as the only carbon source, the astaxanthin production of recombinant bacteria PP-L, PP-L1, PP-L2, PP-L3, PP-LC1 and PP-LC2 was 124.33 mg / L, 124.67 mg / L, 128.30 mg / L, 129.87 mg / L, 132.67 mg / L and 136.33 mg / L, respectively. The results showed that increasing the intracellular NADPH content can promote the accumulation of astaxanthin in Pichia pastoris ( Fig. 9 ), and different linkers affect the production of astaxanthin.

[0081] Example 4 Different additives promote the production of astaxanthin by recombinant Pichia pastoris The recombinant strain PP-LC2 was used as the product synthesis strain 1. Effects of different types of metal ions on astaxanthin production by Pichia pastoris.

[0082] The specific method is as follows: take out the strain from the seed tube, inoculate it into a YPD test tube with a 1% inoculation amount, and culture it at 30°C for 24 hours to obtain the seed solution; The seed solution was inoculated into 50 mL fermentation medium (20 g / L glucose, 10 g / L yeast extract and 20 g / L tryptone) at a 1% inoculum, and cultured at 25°C and 220 rpm for 5 days. At 24 h, 20 g / L glucose and 1 mM different metal ions (including Ca 2+ 、Co 2+ Mg 2+ , Fe 2+ , Cu 2+ , Ba 2+ 、Zn 2+ NH4 + , Cu + 、Ni 2+ 、Na + , Mn 2+ , Fe 3+ , K + ) were used to investigate their effects on the production of astaxanthin by Pichia pastoris. The results showed that Fe 2+ Able to increase astaxanthin production to 141.4 mg / L ( Fig.10 ). Fe 2+ Can increase the production of intracellular astaxanthin, possibly due to Fe 2+ It can undergo Fenton reaction with intracellular H2O2 to produce oxygen free radicals, promoting the synthesis of astaxanthin.

[0083] 2. Effect of different concentrations of sodium citrate on astaxanthin production by Pichia pastoris.

[0084] At 0 h, 0.5 g / L to 3 g / L sodium citrate was added, and the other culture methods were consistent with the culture with metal ions added in Example 4. The results showed that the addition of 2 g / L sodium citrate could increase the astaxanthin production to 144.08 mg / L ( Fig.11 ). It is speculated that after sodium citrate enters the cells, it can be used by the cells to generate acetyl-CoA, the precursor of astaxanthin, thereby promoting the synthesis of intracellular astaxanthin.

[0085] 3. Effects of different concentrations of 2,6-di-tert-butyl-4-methylphenol (BHT) on the production of astaxanthin by Pichia pastoris.

[0086] At 24 h, 0.05%-0.25% [v / v] BHT was added, and the other culture methods were consistent with the culture with metal ions added in Example 4. The results showed that the addition of 0.1% BHT could increase the astaxanthin production to 152.39 mg / L ( Fig.12 ).

[0087] 4. Effect of different concentrations of Tween-80 on the production of astaxanthin by Pichia pastoris.

[0088] At 24 h, 1%-30% Tween-80 was added, and the other culture methods were consistent with the culture with metal ions added in Example 4. The results showed that the addition of 15% Tween-80 could promote the accumulation of astaxanthin to 178.99 mg / L. Fig.13 ) Example 5 Fermentation production of astaxanthin by recombinant strain PP-LC2 ① Seed liquid culture: a. Primary seed solution: Take the recombinant strain PP-LC2 from the cryopreservation tube at a 1% inoculation amount and inoculate it into a YPD test tube at 30°C and 200 rpm for 24 hours as the primary seed solution; the YPD medium contains 2% peptone, 1% yeast extract and 2% glucose; b. Secondary seed solution: Take the primary seed solution and inoculate it into a new seed culture medium at a 10% inoculation rate, and culture it at a constant temperature under the same conditions as a to obtain the seed solution for fermentation culture.

[0089] ② Fed-batch fermentation The seed liquid obtained from the seed culture was inoculated into a 5L fermenter filled with fermentation medium. The recombinant strain PP-LC2 was subjected to batch fed fermentation in a 5L fermenter at 25°C, 500 r, and the dissolved oxygen was maintained at 40%. The initial glucose concentration in the fermenter was 40 g / L. When the glucose was about to be exhausted, glucose was added to maintain the glucose concentration in the fermenter below 10 g / L. Samples were taken every 12 h to determine the cell dry weight, and astaxanthin was extracted from the fermentation broth to determine the astaxanthin content. At the beginning of the fermentation, 2 g / L of sodium citrate was added, and 1 mM Fe was added at 24 h. 2+ , 0.1% BHT and 15% Tween-80, bacterial OD 600 reached 101.52, the astaxanthin production reached 716.13 mg / L, and the astaxanthin content was 16.88 mg / g ( Fig.14 In addition, methanol was used as the carbon source instead of glucose, and the bacterial OD 600 It can reach 90.18, the astaxanthin production reaches 116.58 mg / L, and the content is 4.64 mg / g ( Fig.15 ).

[0090] The present invention improves the ability of Pichia pastoris to produce astaxanthin by increasing the level of intracellular NADPH. The engineered strain is optimized for fermentation under different conditions to enhance the metabolic flux of product synthesis. Finally, the production capacity of the strain is comprehensively evaluated by batch feeding fermentation in a 5L fermenter. The OD value of the recombinant strain in the fermenter is measured. 600 Through the detection of astaxanthin production, 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 producing astaxanthin, characterized in that: The recombinant Pichia pastoris strain is obtained by expressing dehydrogenase Zwf1, phosphate dehydrogenase Gnd2, NADH kinase Pos5, and fusion protein CrtZ-Linker-CrtW of β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ in a host bacterium; Wherein, the host bacteria is the genetically modified strain of Pichia pastoris PP-L.

2. The recombinant Pichia pastoris strain according to claim 1, characterized in that The linker used in the fusion protein is GGGEKGGG or GGGSGGGS.

3. The recombinant bacterium according to claim 1, characterized in that The nucleotide sequences of the dehydrogenase Zwf1, phosphate dehydrogenase Gnd2, NADH kinase Pos5, β-carotene ketolase CrtW, and β-carotene hydroxylase CrtZ are shown in SEQ ID NOs: 1-5, respectively.

4. The method for constructing the recombinant Pichia pastoris strain according to any one of claims 1 to 3, characterized in that: include: Using BB3-Z4-intE12 as a vector plasmid, a co-expression recombinant plasmid of Zwf1, Gnd2, Pos5 and CrtZ-Linker-CrtW was constructed, and the recombinant plasmid was integrated into the intE12 site of the PP-L genome to obtain the recombinant Pichia pastoris strain.

5. Use of the recombinant Pichia pastoris strain according to any one of claims 1 to 3 in the fermentation production of astaxanthin.

6. The use according to claim 5, characterized in that: The recombinant Pichia pastoris strain is subjected to seed culture and fermentation culture to synthesize astaxanthin.

7. The use according to claim 6, characterized in that: Add Fe during the fermentation of the recombinant Pichia pastoris strain 2+ , sodium citrate, BHT, Tween-80 or more.

8. The use according to claim 7, characterized in that: The sodium citrate is added at the beginning of fermentation; the Fe 2 + , BHT, and Tween-80 are added in the middle of fermentation.

9. The use according to claim 7, characterized in that: The Fe 2+ The addition amount is 0.5-1.5mM, the addition amount of sodium citrate is 0.5 g / L-3 g / L, the addition amount of BHT is 0.05%-0.25% v / v, and the addition amount of Tween-80 is 10-30%.

10. The use according to claim 6, characterized in that: The fermentation culture uses glucose or methanol as a carbon source.

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