Method for biological high-density synthesis of retinaldehyde and synthetic strain thereof

Abstract

The invention discloses a method for biological high-density synthesis of retinaldehyde and a synthetic strain thereof. The synthetic strain is obtained by expressing ATP citrate lyase ACL, acetyl-CoA synthase ACS, choline kinase CK, inositol polyphosphate kinase IPK and vitreoscilla hemoglobin VHb in host bacteria. Wherein the host bacterium is a pichia pastoris gene modified bacterial strain PP-B2C. The retinaldehyde production performance of the recombinant strain is verified on the basis of a precursor supply pathway, an IUP pathway and a dissolved oxygen level, and the retinaldehyde production capacity of the pichia pastoris is further improved. The construction method of the recombinant pichia pastoris is simple, the synthesis of retinaldehyde can be better promoted, and the retinaldehyde can be efficiently synthesized by the engineering strain by utilizing glucose and methanol through amplification fermentation of a 5L fermentation tank, so that the recombinant pichia pastoris is beneficial to industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a method for biosynthesizing retinal at high density and a synthetic strain thereof. BACKGROUND

[0002] Retinal is an active form of vitamin A, also known as vitamin A aldehyde. Its molecular formula is C 20 H 28 O, which is an important organic compound and plays a key role in the body, especially in visual function. The synthesis methods mainly include chemical synthesis and biological synthesis. The chemical synthesis of retinal has defects such as high cost, low purity and complex process, and the biological method uses microorganisms as hosts and utilizes renewable carbon sources for fermentation, thereby avoiding the use of complex catalytic steps in chemical synthesis and significantly reducing wastewater, waste gas and waste residue emissions.

[0003] Biological synthesis usually uses Saccharomyces cerevisiae or Yarrowia lipolytica as a host, and introduces related genes to construct a retinal synthesis pathway through genetic engineering means. Professor Hou Jin's team of Shandong University introduced retinol dehydrogenase BLH and beta-carotene-15,15'-oxygenase RHD12 into beta-carotene-producing Yarrowia lipolytica, and first obtained 38 mg / L of retinal. The yield of retinal in CN116144518B is only 1.2 g / L in a shake flask fermentation of Saccharomyces cerevisiae, and needs to be fed-batch fermented to increase to 5.89 g / L. In addition, the insufficient activity of key enzymes (such as beta-carotene oxygenase) may limit the conversion efficiency of precursors, and needs to be optimized through enzyme engineering (such as semi-rational modification). At present, further improving the yield of retinal is still the research direction to promote the industrial application of retinal. SUMMARY

[0004] The present application provides a method for biosynthesizing retinal, which further improves the yield of retinal by enhancing the precursor acetyl-CoA, increasing the isopentenol utilization pathway (IUP) or increasing the dissolved oxygen level in the synthetic strain.

[0005] A strain of retinal-producing recombinant Pichia pastoris with enhanced precursor acetyl-CoA, the recombinant Pichia pastoris is obtained by expressing ATP-citrate lyase ACL and acetyl-CoA synthase ACS derived from Yarrowia lipolytica in the host strain.

[0006] The host strain is a recombinant strain producing retinyl aldehyde, for example, a recombinant strain PP-B obtained by introducing into a Pichia pastoris GS115 strain an expression cassette of geranylgeranyl diphosphate synthase CrtE, phytoene synthase / phytoene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl CoA reductase tHMGR, and beta-carotene-15,15'-monooxygenase BCMO; the strain and its specific construction method have been disclosed in CN 118240674 A.

[0007] Or in the recombinant strain PP-B, the dehydrogenase Zwf1, phosphodehydrogenase Gnd2, NADH kinase Pos5 gene and beta-carotene-15,15'-monooxygenase derived from Saccharomyces cerevisiae are expressed. BB3-Z4-intE12 is used as a vector plasmid to construct a recombinant plasmid containing Gnd2, Pos5, Zwf1 genes, which is integrated into the intE12 site of the host strain PP-B genome to obtain the recombinant strain PP-BC; then BB3eH-14-BCMO is used as a vector plasmid to construct a recombinant plasmid containing the BCMO gene, which is integrated into the ENO site of PP-BC genome, and a recombinant Pichia pastoris strain PP-B2C producing retinyl aldehyde is obtained; the strain and its specific construction method have been disclosed in CN 120399915 A.

[0008] As a preferred embodiment, the nucleotide sequences of the ATP-citrate lyase ACL and acetyl-CoA synthase ACS are shown in SEQ ID NO: 1-2, respectively.

[0009] A recombinant Pichia pastoris strain producing retinyl aldehyde through isopentenol utilization pathway (IUP), which is obtained by expressing choline kinase CK derived from Saccharomyces cerevisiae and myo-inositol polyphosphate kinase IPK derived from Arabidopsis thaliana in a host strain producing retinyl aldehyde;

[0010] A recombinant Pichia pastoris strain producing retinyl aldehyde through isopentenol utilization pathway (IUP), which is obtained by expressing choline kinase CK derived from Saccharomyces cerevisiae and myo-inositol polyphosphate kinase IPK derived from Arabidopsis thaliana in a host strain producing retinyl aldehyde through enhanced precursor acetyl-CoA.

[0011] As an optimized embodiment, the nucleotide sequences of the choline kinase CK and myo-inositol polyphosphate kinase IPK are shown in SEQ ID NO: 3-4, respectively.

[0012] A retinal-producing recombinant Pichia pastoris strain capable of increasing dissolved oxygen level, which is obtained by further expressing hemoglobin VHb derived from Vitreoscilla in a retinal-producing recombinant Pichia pastoris strain with enhanced precursor acetyl-CoA.

[0013] A retinal-producing recombinant Pichia pastoris strain capable of increasing dissolved oxygen level, which is obtained by expressing hemoglobin VHb derived from Vitreoscilla in a retinal-producing recombinant Pichia pastoris strain of IUP utilization pathway.

[0014] As an optimized embodiment, the nucleotide sequences of the hemoglobin VHb are respectively shown in SEQ ID NO: 5.

[0015] A high-retinal-producing recombinant Pichia pastoris strain, which is obtained by expressing ATP-citrate lyase ACL, acetyl-CoA synthase ACS, choline kinase CK, inositol polyphosphate kinase IPK and hemoglobin VHb in a host strain.

[0016] The host strain is Pichia pastoris genetically modified strain PP-B or PP-B2C.

[0017] The construction method of the above-mentioned recombinant Pichia pastoris strain comprises:

[0018] ACL and ACS co-expression recombinant plasmid is constructed by taking BB3rN-AC as a vector plasmid, and the recombinant plasmid is integrated into the RGI2 site of the PP-B2C genome to obtain the recombinant Pichia pastoris strain PP-B2C-CoA with enhanced precursor acetyl-CoA.

[0019] ACL, ACS, IPK and CK co-expression recombinant plasmid is constructed by taking BB3rN-AE as a vector plasmid, and the recombinant plasmid is integrated into the RGI2 site of the PP-BC genome to obtain the recombinant Pichia pastoris strain PP-B2CCI of isopentenol utilization pathway.

[0020] ACL, ACS, IPK, CK and VHb co-expression recombinant plasmid is constructed by taking BB3rN-AF as a vector plasmid, and the recombinant plasmid is integrated into the RGI2 site of the PP-B2C genome to obtain the recombinant Pichia pastoris strain PP-B2CCIV of isopentenol utilization pathway.

[0021] A method for biologically synthesizing retinal at high density, which comprises the following steps:

[0022] The strain was taken out from the preservation tube, inoculated into YPD test tube at 1-5% inoculation amount, and cultured at 28-32°C for 24h to obtain seed liquid;

[0023] The seed liquid was inoculated into fermentation medium at 1-10% inoculation amount, and cultured at 25°C with 220rpm shaking for 5 days.

[0024] The fermentation medium is 40g / L carbon source, 10g / L yeast extract and 20g / L tryptone.

[0025] As a preferred embodiment, the fermentation culture uses glucose or methanol as the carbon source.

[0026] Further preferably, fed-batch fermentation is used, and the initial carbon source concentration in the fermentation tank is 40g / L; when the carbon source is about to be depleted, feeding is performed to maintain the carbon source concentration in the fermentation tank below 10g / L, so that the carbon source can be fermented, and the carbon source concentration is greater than 0.

[0027] In the fed-batch fermentation, the fermentation temperature is 25°C, the rotation speed is 500rpm, and the dissolved oxygen is maintained at 40%.

[0028] Beneficial effects:

[0029] The recombinant Pichia pastoris of the application can synthesize retinyl aldehyde from scratch using glucose or methanol, realizing efficient synthesis of the natural product retinyl aldehyde in Pichia pastoris. In addition, by constructing the precursor acetyl-CoA synthesis pathway and the IUP pathway, the supply of precursors acetyl-CoA and FPP is enhanced, the synthesis of retinyl aldehyde precursors is improved, thereby promoting the accumulation of retinyl aldehyde. In addition, Pichia pastoris needs a large amount of oxygen to participate in high-density fermentation, and at the same time, 15,15'-β-carotene monooxygenase also needs oxygen to participate in the catalysis of the conversion of β-carotene to retinyl aldehyde, and the introduction of hemoglobin from Methylosinus trichrome can obviously improve this problem. The yield of retinyl aldehyde can reach 171.61mg / L using glucose as the carbon source in the continuous feeding fermentation of the PP-B2CCIV strain in a 5L fermentation tank, and the yield of retinyl aldehyde reaches 56.35mg / L using methanol as the carbon source, laying a foundation for further industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Figure 3 is a structural diagram of plasmid BB3rN-AC-ACL-ACS, which carries a nourseothricin gene used as a selection marker for Pichia pastoris.

[0031] Figure 2 Figure 4 is a structural diagram of plasmid BB3rN-AE-ACL-ACS-IPK-CK, which carries a nourseothricin gene used as a selection marker for Pichia pastoris.

[0032] Figure 3 Figure 1 is a structural diagram of plasmid BB3rN-AF-ACL-ACS-IPK-CK-VHB, which carries a nourseothricin gene as a selection marker for Pichia pastoris.

[0033] Figure 4 Figure 4 is a production yield diagram of retinyl aldehydes produced by different engineered strains.

[0034] Figure 5 Figure 5 is a production yield diagram of retinyl aldehydes produced by engineered strain PP-B2CCIV in a 5L bioreactor using glucose as a carbon source.

[0035] Figure 6 Figure 6 is a production yield diagram of retinyl aldehydes produced by engineered strain PP-B2CCIV in a 5L bioreactor using methanol as a carbon source. DETAILED DESCRIPTION

[0036] The technical solutions of the present application are further described below in combination with the accompanying drawings and examples.

[0037] The original strain used in the examples is Pichia pastoris PP-B2C strain, which is a Pichia pastoris engineered strain obtained by introducing geranylgeranyl diphosphate synthase CrtE, phytoene synthase / phytoene cyclase CrtYB, phytoene desaturase CrtI, 3-hydroxy-3-methylglutaryl CoA reductase tHMGR, two copies of beta-carotene-15,15'-monooxygenase BCMO, dehydrogenase Zwf1, phosphogluconate dehydrogenase Gnd2, and NADH dehydrogenase Pos5 into Pichia pastoris GS115 strain, which can be purchased from commercial channels.

[0038] The PP-B strain and its specific construction method have been disclosed in CN 118240674 A. The Pichia pastoris GS115 strain can be purchased from commercial channels.

[0039] The method for constructing the P. pastoris PP-B2C strain is as follows: BB3-Z4-intE12 is used as a carrier plasmid to construct a recombinant plasmid derived from S. cerevisiae Gnd2, Pos5 and Zwf1, and the recombinant plasmid is integrated into the intE12 site of the PP-B genome; and an engineering recombinant P. pastoris strain PP-BC is obtained. The dehydrogenase Zwf1, the phosphodehydrogenase Gnd2 and the NADH dehydrogenase Pos5 of S. cerevisiae are obtained to obtain the engineering strain PP-B2C. The coding gene sequences of the dehydrogenase Zwf1 (Gene ID: 855480), the phosphodehydrogenase Gnd2 (Gene ID: 855480) and the NADH dehydrogenase Pos5 (Gene ID: 855913) from S. cerevisiae are provided on NCBI. BB3eH-14-BCMO is used as a carrier plasmid to construct a BCMO recombinant plasmid, and the recombinant plasmid is integrated into the ENO site of the above-mentioned recombinant P. pastoris strain genome to obtain a recombinant P. pastoris strain PP-B2C with increased copy number of beta-carotene-15, 15'-monooxygenase.

[0040] In the examples, the extraction and quantitative analysis steps of retinal are as follows:

[0041] 1. Extraction of retinal

[0042] (1) 1 mL of the mixed fermentation broth was centrifuged at 12000 rpm for 5 min (washed twice with pure water).

[0043] (2) After controlling the water content, the sample was resuspended in 2 mL of dimethyl sulfoxide (DMSO) (preheated at 60°C) and uniformly shaken on a vortex shaker, and then placed in a 55°C water bath for 15 min.

[0044] (3) 4 mL of anhydrous ethanol was added.

[0045] (4) The sample was centrifuged at 12000 rpm for 5 min. The supernatant was transferred to a new centrifuge tube and stored in the dark.

[0046] 2. The concentration of retinal was detected by high performance liquid chromatography.

[0047] The liquid chromatograph used in the present study is an Agilent Technologies 1200 Infinity series; the chromatographic column is an AcclaimTM120 C18 chromatographic column; the ultraviolet absorption wavelength is 325 nm; the mobile phase is methanol and acetonitrile (95:5); the flow rate control is 1.0 mL / min; and the column temperature is 40°C.

[0048] Example 1. Amplification of gene elements and preparation of target plasmid

[0049] (I) Preparation of target genes

[0050] According to the coding gene sequence of ATP-citrate lyase and acetyl-CoA synthase from Yarrowia lipolytica provided on NCBI, PCR amplification was carried out on the Saccharomyces cerevisiae genome as a template, and the gene sequence is shown in SEQ ID No: 1-2.

[0051] According to the coding gene sequence of choline kinase from Saccharomyces cerevisiae provided on NCBI, PCR amplification was carried out on the Saccharomyces cerevisiae genome as a template, and the gene sequence is shown in SEQ ID No: 3; the myo-inositol polyphosphate kinase from Arabidopsis thaliana was synthesized by Kinsbio Biotech Co., Ltd. and codon optimized, and the gene sequence is shown in SEQ ID No: 4.

[0052] According to the coding gene sequence of hemoglobin VHb from Vitreoscilla provided on NCBI, the gene sequence was synthesized by Kinsbio Biotech Co., Ltd. and codon optimized, and the gene sequence is shown in SEQ ID No: 5.

[0053] (II) Construction of recombinant plasmids

[0054] 1. Using BB1-23 (Plasmid #98496) as a vector plasmid, a recombinant plasmid containing ACL, ACS, IPK, CK and VHb was constructed.

[0055] The IPK and VHb gene sequences were synthesized by Kinsbio Biotech Co., Ltd.; 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 recombinant plasmid BB1-23-CK was PCR amplified using the Saccharomyces cerevisiae genome as a template to obtain the CK gene sequence.

[0056] Each gene fragment was inserted into plasmid BB1-23 by Golden Gate method to obtain recombinant plasmids BB1-23-ACL, BB1-23-ACS, BB1-23-IPK, BB1-23-CK and BB1-23-VHb, as follows:

[0057] ACL-F and ACL-R were used as primers to amplify the ACL fragment using the Yarrowia lipolytica genome as a template, and the primer sequences are shown in Table 4.

[0058] ACS-F and ACS-R were used as primers to amplify the ACS fragment using the Yarrowia lipolytica genome as a template, and the primer sequences are shown in Table 4.

[0059] IPK-F and IPK-R were used as primers to amplify the IPK fragment using the IPK gene sequence as a template, and the primer sequences are shown in Table 4.

[0060] CK-F and CK-R as primers, Saccharomyces cerevisiae genome as template, CK fragment was amplified, and the primer sequences are shown in Table 4.

[0061] VHb-F and VHb-R as primers, Saccharomyces cerevisiae genome as template, VHb fragment was amplified, and the primer sequences are shown in Table 4.

[0062] The amplified ACL, ACS, IPK, CK and VHb fragments were recovered, and agarose gel electrophoresis was used for purification and recovery.

[0063] GoldenGate assembly was performed using Bsal enzyme and T4 ligase from Shanghai Biyun Tian Biotechnology Co., Ltd., and the reaction system was as follows:

[0064] Table 1

[0065]

[0066]

[0067] The circular recombinant vector was transformed into E. coli DH5a competent cells, and positive recombinant plasmids BB1-23-ACL, BB1-23-ACS, BB1-23-IPK, BB1-23-CK and BB1-23-VHb were obtained by kanamycin sulfate-resistant plate screening and colony PCR and sequencing verification.

[0068] 2. Construction of ACL, ACS, IPK, CK and VHb expression cassette

[0069] The plasmid BB1-23-ACL and the plasmid BB1-12-pGAP, the plasmid BB1-34-RPS25Att were inserted into the plasmid BB2-AB by GoldenGate method using Bpil enzyme and T4 ligase to obtain the plasmid BB2-AB-pGAP-ACL-RPS25Att.

[0070] The plasmid BB1-23-ACS and the plasmid BB1-12-pMDH3, the plasmid BB1-34-RPS2tt were inserted into the plasmid BB2-BC by GoldenGate method using Bpil enzyme and T4 ligase to obtain the plasmid BB2-BC-pMDH3-ACS-RPS2tt.

[0071] The plasmid BB1-23-IPK and the plasmid BB1-12-pADH2, the plasmid BB1-34-RPS2tt were inserted into the plasmid BB2-CD by GoldenGate method using Bpil enzyme and T4 ligase to obtain the plasmid BB2-CD-pADH2-IPK-RPS2tt.

[0072] Plasmid BB1-23-CK was inserted into plasmid BB2-DE by Golden Gate method using Bpil enzyme and T4 ligase with plasmid BB1-12-pGPM1, plasmid BB1-34-RPL2Att, to obtain plasmid BB2-DE-pGPM1-CK-RPL2Att.

[0073] Plasmid BB1-23-VHb was inserted into plasmid BB2-EF by Golden Gate method using Bpil enzyme and T4 ligase with plasmid BB1-12-pGCW14, plasmid BB1-34-RPS17Btt, to obtain plasmid BB2-EF-pGCW14-VHb-RPS17Btt.

[0074] The construction process of recombinant plasmid BB2-AB-pGAP-ACL-RPS25Att is as follows:

[0075] Golden Gate assembly was performed using Bpil enzyme and T4 ligase of Nanjing Fumax Biotechnology Co., Ltd., and the reaction system was as follows:

[0076] Table 2

[0077]

[0078]

[0079] The circular recombinant vector was transformed into E. coli DH5a competent cells, screened by ampicillin-resistant plates, and verified by colony PCR and sequencing, to obtain positive recombinant plasmid BB2-AB-pGAP-ACL-RPS25Att.

[0080] The construction process of recombinant plasmid BB2-BC-pMDH3-ACS-RPS2tt is as follows:

[0081] The reaction system and Table 2 are basically the same, only the plasmids are replaced by BB2-BC, BB1-12-pMDH3, BB1-23-ACS and BB1-34-RPS2tt.

[0082] The circular recombinant vector was transformed into E. coli DH5a competent cells, screened by ampicillin-resistant plates, and verified by colony PCR and sequencing, to obtain positive recombinant plasmid BB2-BC-pMDH3-ACS-RPS2tt.

[0083] The construction process of recombinant plasmid BB2-CD-pADH2-IPK-RPS2tt is as follows:

[0084] The reaction system is basically the same as Table 2, only the plasmid is replaced by BB2-CD, BB1-12-pADH2, BB1-23-IPK and BB1-34-RPS2tt.

[0085] The circular recombinant vector is transformed into E. coli DH5a competent cells, screened by ampicillin-resistant plate, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid BB2-CD-pADH2-IPK-RPS2tt.

[0086] The construction process of the recombinant plasmid BB2-DE-pGPM1-CK-RPL2Att is as follows:

[0087] The reaction system is basically the same as Table 2, only the plasmid is replaced by BB2-DE, BB1-12-pGPM1, BB1-23-CK and BB1-34-RPL2Att.

[0088] The circular recombinant vector is transformed into E. coli DH5a competent cells, screened by ampicillin-resistant plate, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid BB2-DE-pGPM1-CK-RPL2Att.

[0089] The construction process of the recombinant plasmid BB2-EF-pGCW14-VHb-RPS17Btt is as follows:

[0090] The reaction system is basically the same as Table 2, only the plasmid is replaced by BB2-EF, BB1-12-pGCW14, BB1-23-VHb and BB1-34-RPS17Btt.

[0091] The circular recombinant vector is transformed into E. coli DH5a competent cells, screened by ampicillin-resistant plate, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid BB2-EF-pGCW14-VHb-RPS17Btt.

[0092] 3. With BB3rN-AC (Plasmid #98551) as the vector, a co-expression recombinant plasmid of ACL and ACS is constructed:

[0093] The plasmids BB2-AB-pGAP-ACL-RPS25Att and BB2-BC-pMDH3-ACS-RPS2tt are inserted into the plasmid BB3rN-AC by Golden Gate method using Bsal enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AC-ACL-ACS.

[0094] The structure of the recombinant plasmid BB3rN-AC-ACL-ACS is shown in Figure 1 , and the construction process is as follows:

[0095] GoldenGate assembly using Bsal enzyme and T4 ligase, the reaction system as follows:

[0096] Table 3

[0097] System 10 μL BB3rN-AC 1 μL BB2-AB-pGAP-ACL-RPS25Att 1 μL BB2-BC-pMDH3-ACS-RPS2tt 1 μL Bsa1 0.5 μL BSA 1 μL T4 ligase 0.5 μL T4 buffer 1 μL ddH2O 4 μL

[0098] The circular recombinant vector was transformed into E. coli DH5a competent cells, screened by nourseothricin-resistant plates and verified by colony PCR and sequencing, and the positive recombinant plasmid BB3rN-AC-ACL-ACS was obtained.

[0099] 4. BB3rN-AE (Plasmid #98553) was used as a vector to construct the expression cassette of ACL, ACS, IPK and CK.

[0100] The 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 the plasmid BB3rN-AE by GoldenGate method using Bsal enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AE-ACL-ACS-IPK-CK.

[0101] The construction process of the recombinant plasmid BB3rN-AE-ACL-ACS-IPK-CK is as follows:

[0102] The reaction system and Table 3 are basically the same, only the plasmids are replaced by BB3rN-AC, BB2-AB-pGAP-ACL-RPS25Att, BB2-BC-pMDH3-ACS-RPS2tt, BB2-CD-pADH2-IPK-RPS2tt and BB2-DE-pGPM1-CK-RPL2Att.

[0103] The circular recombinant vector was transformed into E. coli DH5a competent cells, screened by nourseothricin-resistant plates and verified by colony PCR and sequencing, and the positive recombinant plasmid BB3rN-AE-ACL-ACS-IPK-CK was obtained.

[0104] 5. BB3rN-AE (Plasmid #98553) was used as a vector to construct the expression cassette of ACL, ACS, IPK, CK and VHb.

[0105] 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-AE by Golden Gate method using Bsal enzyme and T4 ligase to obtain the recombinant plasmid BB3rN-AF-ACL-ACS-IPK-CK-VHb.

[0106] The construction process of the recombinant plasmid BB3rN-AF-ACL-ACS-IPK-CK-VHb is as follows:

[0107] The reaction system is basically the same as that in Table 3, except that the plasmids are replaced by BB3rN-AC, 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.

[0108] The circular recombinant vector was transformed into E. coli DH5a competent cells, screened by a plate resistant to nourseothricin, and verified by colony PCR and sequencing to obtain the positive recombinant plasmid BB3rN-AF-ACL-ACS-IPK-CK-VHb.

[0109] Table 4 primer sequences

[0110]

[0111]

[0112] Example 2 Construction of recombinant bacteria

[0113] 1. Construction of recombinant bacteria PP-BC-CoA

[0114] The plasmid BB3rN-AC-ACL-ACS of the gene expression cassette was introduced into P. pastoris PP-B2C to obtain the recombinant bacteria PP-B2C-CoA.

[0115] 2. Construction of recombinant bacteria PP-B2CCI

[0116] The plasmid BB3rN-AE-ACL-ACS-IPK-CK of the gene expression cassette was introduced into P. pastoris PP-B2C-CoA to obtain the recombinant bacteria PP-B2CCI.

[0117] 3. Construction of recombinant strain PP-B2CCIV

[0118] The plasmid BB3rN-AF-ACL-ACS-IPK-CK-VHb of the gene expression cassette was introduced into the P. pastoris PP-B2CCI to obtain the recombinant strain PP-B2CCIV.

[0119] The specific method is as follows:

[0120] 1. The competent cells were prepared from the overnight culture of the engineered P. pastoris in YPD liquid medium (containing 2% peptone, 1% yeast extract and 2% glucose).

[0121] 2. The BB3rN-AC-ACL-ACS / BB3rN-AE-ACL-ACS-IPK-CK / BB3rN-AF-ACL-ACS-IPK-CK-VHb was introduced into the corresponding P. pastoris competent cells by using electroporator for homologous recombination.

[0122] 3. The positive clones with correct PCR identification were selected on the YPD selection plate with the addition of bleomycin, and single colonies were grown in 2-3 days. The positive clones were named as the recombinant strains PP-B2C-CoA / PP-B2CCI / PP-B2CCIV.

[0123] Example 3. Application of the recombinant strains in the production of retinol

[0124] The original strain PP-B2C and the recombinant strains PP-B2C-CoA, PP-B2CCI and PP-B2CCIV in Example 2 were used to produce retinol.

[0125] The specific method is as follows: the strain was taken from the stock tube, inoculated into YPD test tube at 1% inoculation amount, and cultured at 30°C for 24h to obtain the seed liquid;

[0126] The seed liquid was inoculated into 50mL fermentation medium (40g / L glucose, 10g / L yeast extract and 20g / L tryptone) at 1% inoculation amount, and cultured at 25°C with 220rpm shaking for 5 days. 40g / L glucose was added at the 24th hour as the sole carbon source. The retinol yield of the recombinant strains PP-B2C, PP-B2C-CoA, PP-B2CCI and PP-B2CCIV was 40.98mg / L, 48.15mg / L, 52.84mg / L and 64.12mg / L, respectively. Figure 4 Compared with the starting strain, the retinol yield of PP-B2C-CoA, PP-B2CCI and PP-B2CCIV was increased by 17.50%, 28.94% and 56.47%, respectively.

[0127] Example 4. High density fermentation of recombinant strain PP-B2CCIV for the production of retinyl aldehyde

[0128] 1. Seed culture

[0129] a. Primary seed culture: The recombinant strain PP-B2CCIV was inoculated into YPD tubes at 1% inoculation volume from the frozen tube and incubated at 30°C, 200 rpm for 24 hours as the primary seed culture. The YPD medium contains 2% peptone, 1% yeast extract and 2% glucose.

[0130] b. Secondary seed culture: The primary seed culture was inoculated into new seed medium at 10% inoculation volume and incubated at the same condition as a to obtain the seed culture for fermentation.

[0131] 2. Fed-batch fermentation

[0132] The seed culture was inoculated into a 5L fermentor containing fermentation medium (40g / L glucose, 20g / L peptone, 10g / L yeast extract) and the recombinant strain PP-B2CCIV was cultivated in the 5L fermentor with continuous glucose feeding. The fermentation temperature was 25°C, 500 rpm and the dissolved oxygen was maintained at 40%. The initial glucose concentration in the fermentor was 40g / L and glucose was fed when the glucose was almost depleted to maintain the glucose concentration at about 10g / L. The cell dry weight was measured every 12 hours and the retinyl aldehyde was extracted from the fermentation broth for the determination of the retinyl aldehyde concentration. The OD 600 of the cell reached 289.35 and the retinyl aldehyde production reached 171.61 mg / L with the maximum of 51.65 mg / L of extracellular retinyl aldehyde production Figure 5 . In addition, when methanol was used as the carbon source to replace glucose, the OD 600 of the cell reached 207.2 and the retinyl aldehyde production reached 56.35 mg / L with the maximum of 9.15 mg / L of extracellular retinyl aldehyde production Figure 6 . This is the highest production of retinyl aldehyde using methanol as the sole carbon source.

[0133] The present application increases the synthesis of precursors by introducing an exogenous acetyl-CoA synthesis pathway, thereby strengthening the metabolic flow to the synthesis of retinal; in addition, the introduction of the artificially constructed IUP pathway enhances the carbon flux by adding isopentenol exogenously, thereby simplifying the synthesis path of retinal. In addition, Pichia pastoris belongs to aerobic yeast, and a large amount of oxygen is needed in the fermentation process, and high-density fermentation often leads to insufficient oxygen supply, and the introduction of Azotobacter vinlandii VHb successfully solves this problem. At the same time, the key enzyme 15,15'-beta-carotene monooxygenase of the retinal synthesis pathway also needs the participation of oxygen to convert beta-carotene into retinal. Finally, the production capacity of the strain is comprehensively evaluated in a 5L fermenter by continuous feeding fermentation, and the OD 600 and retinal production of the recombinant strain in the fermenter are detected, which is expected to further improve the yield of retinal in a larger volume fermenter, laying a foundation for subsequent industrialization.

Claims

1. A recombinant Pichia pastoris strain producing high yield of retinal, characterized in that, The recombinant Pichia pastoris strain is obtained by expressing ATP-citrate lyase (ACL), acetyl-CoA synthase (ACS), choline kinase (CK), inositol polyphosphate kinase (IPK) and hemoglobin VHb in a host strain; The host strain is a recombinant strain producing retinoids.

2. The recombinant Pichia pastoris of claim 1, characterized in that, The ATP-citrate lyase ACL and the acetyl-CoA synthase ACS are derived from Yarrowia lipolytica Yarrowia lipolytica The coding gene of the ATP-citrate lyase is shown as SEQ ID No: 1; the coding gene of the acetyl-CoA synthase is shown as SEQ ID No:

2.

3. The recombinant Pichia pastoris of claim 1, wherein, The myo-inositol polyphosphate kinase IPK is derived from Arabidopsis thaliana Arabidopsis thaliana , the encoding gene is shown as SEQ ID No: 3; the choline kinase CK is derived from Saccharomyces cerevisiae Saccharomyces cerevisiae , the encoding gene is shown as SEQ ID No:

4.

4. The recombinant Pichia pastoris of claim 1, wherein, The hemoglobin VHb is derived from Vitreoscilla Vitreoscilla The coding gene of the hemoglobin VHb is shown as SEQ ID No:

5.

5. Use of the recombinant Pichia pastoris strain of any one of claims 1-4 in the fermentation production of retinoids.

6. Use according to claim 5, characterized in that, The method comprises the following steps: (1) culturing the recombinant strain of any one of claims 1-4 in YPD medium to obtain a fermentation product; (2) extracting the fermentation product with dimethyl sulfoxide and ethanol to obtain retinoids.

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

8. A method for high-density biological synthesis of retinaldehyde, characterized in that, Culturing the recombinant Pichia pastoris strain of any one of claims 1-4 to synthesize retinoids.

9. The method for high-density biosynthesis of retinaldehyde according to claim 8, characterized in that, After seed culture of the recombinant Pichia pastoris strain, the seed liquid is inoculated into a fermenter for continuous feeding fermentation, the initial carbon source concentration in the fermenter is 40 g / L, and when the carbon source is about to be depleted, feeding is carried out to maintain the carbon source concentration in the fermenter at 1-10 g / L.

10. The method for high-density biosynthesis of retinaldehyde according to claim 8, characterized in that, The fermentation temperature is 25°C, the rotation speed is 500 rpm, and the dissolved oxygen is maintained at 40%.

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