CN116426398B - Yarrowia lipolytica engineering bacterium and application thereof - Google Patents

Yarrowia lipolytica engineering bacterium and application thereof Download PDF

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CN116426398B
CN116426398B CN202310361322.8A CN202310361322A CN116426398B CN 116426398 B CN116426398 B CN 116426398B CN 202310361322 A CN202310361322 A CN 202310361322A CN 116426398 B CN116426398 B CN 116426398B
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yarrowia lipolytica
zmx1378
gene
engineering bacterium
astaxanthin
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CN116426398A (en
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吴伟斌
林博
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Guangdong Zhumei Biomedical Technology Co ltd
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Fujian Pingtan Baoshigui Biotechnology Partnership LP
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Abstract

The application discloses yarrowia lipolytica engineering bacteria and application thereof, belonging to the field of biotechnology; the preservation number is CCTCC NO: the yarrowia lipolytica engineering strain ZMW1378 of M2023294 is a yarrowia lipolytica engineering strain which is obtained by recombining and integrating mutation gene clusters expressing beta-carotene ketolase and beta-carotene hydroxylase modified by directed evolution on a genome of the yarrowia lipolytica, wherein the gene clusters are used for expressing phytoene synthase, lycopene cyclase, phytoene desaturase, diacylglycerol acyltransferase and hemoglobin in a multi-copy recombination manner, knocking out a glycerol-3-phosphate dehydrogenase gene GUT2 on the genome of the strain, and efficiently biosynthesizing astaxanthin by taking glucose as a substrate; the yarrowia lipolytica engineering bacteria constructed by the application can produce astaxanthin through glucose feedback feed-back fermentation, the yield can reach 3775.4mg/L, the production process is simple, and the method can be applied to large-scale production.

Description

Yarrowia lipolytica engineering bacterium and application thereof
Technical Field
The application relates to yarrowia lipolytica engineering bacteria and application thereof, and belongs to the technical field of microorganisms.
Background
Astaxanthin is the fat-soluble carotenoid compound with the strongest antioxidant activity in nature and is commonly found in various marine bacteria, algae and aquatic animals. Astaxanthin protects cells, lipids and membrane lipoproteins from oxidative damage; astaxanthin also has the characteristics of resisting aging, resisting inflammation, resisting cancer, inhibiting helicobacter pylori infection, activating immune response, improving memory and the like, has been widely applied to the fields of foods, medicines, health products, cosmetics, feeds and the like, has global annual demand of 250 tons, sales amount of 4.5 hundred million dollars, and is expected to reach 25 hundred million dollars by 2025, and has extremely high economic and application values.
Currently, most of astaxanthin used in the market is obtained by chemical synthesis or extraction from algae. The chemical synthesis of astaxanthin, due to its stereoisomers, cannot be absorbed and utilized by the human body and can only be used for coloring of animals and aquaculture. Although a variety of organisms are rich in astaxanthin, direct extraction has low production efficiency and does not have economic advantages. In the prior art, the haematococcus pluvialis is transformed for astaxanthin production, but the extraction process is complex due to long culture period, and the haematococcus pluvialis is not suitable for industrial production.
In recent years, construction of artificial cell factories to obtain astaxanthin by engineering microorganism chassis cells using synthetic biology methods and loading target optimized heterologous product synthesis pathways has attracted general attention of international researchers and gradually developed as an effective method for replacing chemical synthesis and natural extraction. In the prior art and patent, the synthesis of astaxanthin in microorganisms is realized by introducing a heterologous way of astaxanthin, hosts mainly comprise escherichia coli, saccharomyces cerevisiae, rhodotorula, and the like, but the engineering bacteria still have the problem of low astaxanthin yield. In addition, E.coli has safety problems caused by endotoxin, and is not sufficient for industrial production.
Yarrowia lipolytica contains abundant acetyl-CoA, a precursor raw material for the synthesis of carotenoid compounds, and has a large amount of lipids accumulated in the cells without endotoxin production, which makes yarrowia lipolytica an excellent chassis fungus for the biosynthesis of astaxanthin. Therefore, the development of the yarrowia lipolytica engineering bacteria with high astaxanthin yield has important application value. At present, some patents and documents realize the de novo synthesis of astaxanthin by introducing heterologous pathway genes into yarrowia lipolytica, mainly over-expressing related genes in metabolic pathways, but the introduction of exogenous genes causes the problems of metabolic burden on cell growth, increased oxygen demand, variable filamentous morphology of strains, product feedback inhibition and the like, so that the yield of yarrowia lipolytica engineering bacteria is lower. Solves the difficult problem that astaxanthin and metabolic intermediate products accumulate to inhibit the metabolism and growth of the strain in the fermentation production process of yarrowia lipolytica engineering bacteria, and has important significance for improving the production efficiency of astaxanthin.
Disclosure of Invention
In order to solve the problems in the prior art, the application provides a yarrowia lipolytica engineering bacterium ZMX1378, which can be used for efficiently synthesizing astaxanthin by taking glucose as a substrate, and the synthesized astaxanthin has high yield and high purity.
The technical scheme of the application is as follows:
one of the purposes of the present application is to provide a yarrowia lipolytica engineering bacterium ZMX, 1378, with a preservation number of CCTCC NO: yarrowia lipolytica engineering bacterium ZMX1378 of M2023294, and the preservation information of the yarrowia lipolytica engineering bacterium ZMX1378 of the present application is as follows:
strain name: yarrowia lipolytica
Latin name: yarrowia lipolytica
Strain number: ZMX1378
Preservation mechanism: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (cctccc)
Address: university of Chinese Wuhan
Preservation date: 2023, 3, 21;
wherein the yarrowia lipolytica engineering bacterium ZMX1378 genome is integrated with
The CrtYB-CrtI-DAG1-VHb gene cluster and the BsCrtW-PaCrtZ mutant gene cluster.
Further, the yarrowia lipolytica engineering bacterium ZMX1378 genome overexpresses the CrtYB gene and the CrtI gene, the CrtYB gene and the CrtI gene are derived from Phaffia rhodozyma Xanthophyllomyces dendrorhous, the CrtYB gene expresses phytoene synthase/lycopene cyclase, as shown in SEQ ID NO.1, and the CrtI gene expresses phytoene desaturase, as shown in SEQ ID NO. 2.
Further, the genome of the yarrowia lipolytica engineering bacterium ZMX1378 overexpresses the DAG1 gene, and the DAG1 gene is derived from yarrowia lipolytica Yarrowia lipolytica and expresses diacylglycerol acyltransferase as shown in SEQ ID No. 3.
Furthermore, the genome of the yarrowia lipolytica engineering bacterium ZMX1378 is subjected to over-expression of a VHb gene, the VHb gene is derived from Vitreoscilla, and the expressed hemoglobin is shown as SEQ ID NO. 4.
Further, the yarrowia lipolytica engineering bacterium ZMX1378 genome overexpresses a BsCrtW mutant gene, the BsCrtW mutant gene is derived from Brevundimonas sp.marine, and expresses beta-carotene ketolase, as shown in SEQ ID No.5, wherein A at position 12 is mutated to C, G at position 241 is mutated to A, and G at position 485 is mutated to T.
Further, the genome of the yarrowia lipolytica engineering bacterium ZMX1378 overexpresses a Pacrtz mutant gene, the Pacrtz mutant gene is derived from Pantoea ananatis and expresses beta-carotene hydroxylase, as shown in SEQ ID No.6, and A at 147 is mutated into C and G at 392 is mutated into A.
Further, the glycerol-3-phosphate dehydrogenase GUT2 site is knocked out on the genome of the yarrowia lipolytica engineering bacterium ZMX1378.
The second purpose of the application is to provide a construction method of yarrowia lipolytica engineering bacterium ZMX1378 for biosynthesis of astaxanthin, which comprises the following steps:
(1) Introducing the CrtYB-CrtI-DAG1-VHb gene cluster into a yarrowia lipolytica basal disc strain through a multicopy plasmid pINA1312, and screening to obtain a yarrowia lipolytica engineering bacterium ZM072 for synthesizing astaxanthin precursor beta-carotene;
(2) Knocking out the GUT2 gene of the glycerol-3-phosphate dehydrogenase on the genome of the ZM072 in a gene recombination mode to obtain yarrowia lipolytica engineering bacterium ZM072 (delta GUT 2);
(3) The BsCrtW-PaCrtZ gene cluster is subjected to co-directed evolution transformation, mutant library genes of the BsCrtW-PaCrtZ gene cluster are introduced into the yarrowia lipolytica strain ZM072 (delta GUT 2) through single copy plasmid pINA1269, and the yarrowia lipolytica engineering bacterium ZMX1378 for efficiently synthesizing astaxanthin is obtained through high-throughput screening.
Wherein the yarrowia lipolytica based on fungus can be yarrowia lipolytica ATCC MAY-2613.
The third purpose of the application is to synthesize astaxanthin by using glucose as a substrate by the yarrowia lipolytica engineering bacterium ZMX1378.
The fourth object of the present application is to provide a method for synthesizing astaxanthin by using glucose as a substrate by yarrowia lipolytica engineering bacterium ZMX, wherein the yarrowia lipolytica engineering bacterium ZMX is cultured to obtain seed liquid; inoculating the seed liquid into a fermentation culture medium, and performing glucose feedback feed-back fermentation, wherein the pH value in the fermentation process is controlled to be 5.6-6.0, the temperature in the fermentation process is 28-30 ℃, and the fermentation time is preferably 96-128h; the concentration of the glucose feedback feed is controlled to be 0.2-2g/L, and astaxanthin is obtained through glucose feedback feed fermentation.
Compared with the prior art, the application has the beneficial effects that:
(1) On the basis of introducing astaxanthin anabolism key enzyme genes CrtYB and CrtI into yarrowia lipolytica, the application uses an in-vitro directional transformation technology to simultaneously carry out directional transformation mutation on a key gene cluster BsCrtW-PaCrtZ on an astaxanthin anabolism path as a whole, optimizes the expression level and enzyme activity proportion of exogenously introduced key genes BsCrtW and PaCrtZ, optimizes astaxanthin metabolic flow, reduces strain metabolism burden, obtains high-yield astaxanthin excellent strain ZMX1378 through high-throughput screening, and effectively improves the synthesis efficiency of astaxanthin;
(2) According to the application, on the basis of introducing a preferable astaxanthin synthesis module gene into yarrowia lipolytica engineering bacteria, the transparent vibrio hemoglobin VHb gene is introduced, so that the oxygen utilization rate of the strain and the expression efficiency of astaxanthin anabolism key enzyme are improved, and the energy consumption and the production cost are reduced;
(3) According to the application, on the basis of introducing astaxanthin optimized synthesis module genes and hemoglobin genes into the yarrowia lipolytica engineering bacteria, the yarrowia lipolytica diacylglycerol acyltransferase DAG1 gene is overexpressed, the glycerol-3-phosphate dehydrogenase GUT2 gene for regulating liposome degradation on the genome of the engineering bacteria is knocked out, the lipid content of the engineering bacteria is improved, the storage space of astaxanthin is increased, and thus the astaxanthin production capacity of the engineering bacteria is improved.
(4) The yarrowia lipolytica engineering bacterium ZMX1378 provided by the application can be used for fermenting and producing astaxanthin, and the yield of astaxanthin in fermentation broth reaches 3237.2mg/L at a fermentation scale of 30 tons, so that the yarrowia lipolytica engineering bacterium has high industrial application value.
Drawings
FIG. 1 is a schematic diagram of the structure of recombinant plasmid pINA1312-CrtYB-CrtI-DAG1-VHb in example 2;
FIG. 2 is a schematic diagram of the structure of the recombinant plasmid pINA1269-BsCrtW-PaCrtZ of example 4;
FIG. 3 is a graph of astaxanthin production by glucose feedback fed-batch fermentation with yarrowia lipolytica ZMX001 and ZMX1378 of the 50L fermentor of example 5;
FIG. 4 is a graph of astaxanthin production by 30T fermenter yarrowia lipolytica ZMX1378 glucose feedback fed-batch fermentation of example 6.
Detailed Description
The application is further described below in connection with the preferred embodiments, and neither the endpoints of the ranges disclosed in the application nor any of the values are limited to the precise range or value, and such range or value should be understood to include values near the range or value; for numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the examples below, "codon optimization" refers to the redesign of a gene using preferred codons and avoiding low or rare codons. Each organism exhibits some degree of codon usage difference or preference, and those most frequently utilized are preferred codons.
The molecular biology experiments in the following examples include plasmid construction, digestion, ligation, competent cell preparation, transformation, medium configuration, etc., and are mainly performed with reference to the "molecular cloning Experimental guidelines" (third edition). The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers.
The total gene synthesis, primer synthesis and sequencing in the following examples were performed by Nanjinouzan biotechnology Co., ltd.
YPD slant culture medium in the following examples comprises 20g/L peptone, 10.0g/L yeast extract, 20.0g/L glucose, 20g/L agar;
the seed culture medium comprises 20g/L peptone, 10.0g/L yeast extract powder and 20.0g/L glucose;
the fermentation medium comprises 20g/L glucose, 10g/L (NH) 4 ) 2 SO 4 ，5g/L KH 2 PO 4 ，5g/LMgSO 4 10ml/L vitamin, 10ml/L trace metal salt; wherein, the trace metal salt solution comprises the following components: 5g/L ZnSO 4 ，0.3g/L MnCl 2 ，0.3g/L CuSO 4 ，0.5g/L CoCl 2 ，0.5g/L Na 2 MoO 4 ，2g/LCaCl 2 ，2g/L FeSO 4 The method comprises the steps of carrying out a first treatment on the surface of the Wherein the vitamin solution comprises the following components: 0.1g/L biotin, 2g/L calcium pantothenate, 2g/L nicotinic acid, 20g/L inositol, 2g/L thiamine hydrochloride, 0.5g/L pyridoxal phosphate, 0.1g/L para-aminobenzoic acid.
In the present application, yarrowia lipolytica (ATCC No. MAY-2613, available from ATCC), plasmid pCRISPRy1 (available from Addgene Inc.), E.coli JM109 E.coli DH 5. Alpha. (available from Shanghai) and plasmid pINA1269 were prepared according to the preparation method described in (Madzak C, treaton B, blancin-Roland S.Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the Yeast Yarrowia Lipolytica J Mol Biotechnol.2000Apr;2 (2): 207-16) paper, and plasmid pINA1312 was prepared according to the preparation method described in (Nicaud JM, madzak C, van Broek P, gysler C, duboc P, niedenberer P, galidin C.protein expression and secretion in the Yeast Yarrowia Liptica.FEMS Yeast Res.2002Aug;2 (3): 371).
EXAMPLE 1 construction of engineering bacterium ZM072 for synthesizing astaxanthin precursor beta-carotene yarrowia lipolytica
1. Construction of recombinant plasmid pINA1312-CrtYB-CrtI-DAG 1-VHb:
(1) Synthesis of genetic elements and primers: artificially synthesizing nucleotide sequences from a rhodotorula phytoene synthase/lycopene cyclase encoding gene CrtYB and a phytoene desaturase encoding gene CrtI provided on NCBI, from a yarrowia lipolytica diacylglycerol acyltransferase encoding gene DAG1 and from a Vitreoscilla hemoglobin encoding gene VHb, wherein the nucleotide sequences obtained by codon optimization are respectively shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and the synthetic genes are respectively connected into pUC19 plasmid vector frameworks to obtain pUC19-CrtYB, pUC19-CrtI, pUC19-DAG1 and pUC19-VHb plasmids, and primers used in example 1 are shown in Table 1;
TABLE 1 primer sequences used in example 1
(2) Construction of recombinant plasmid pINA1312-CrtYB-CrtI-DAG 1-VHb:
constructing a phytoene synthase/lycopene cyclase coding gene CrtYB expression module, using PmeI-CYB-up and KpnI-CYB-down as primers and pUC19-CrtYB as a template, and amplifying PCR fragments containing Pme I cleavage sites, crtYB and KpnI cleavage sites; double enzyme digestion is carried out on pINA1312 plasmid by using Pml I and Kpn I, the PCR fragment carrying the Pme I blunt end site and Kpn I enzyme digestion site is subjected to enzyme ligation, and is transferred into escherichia coli to obtain correctly-ligated positive clones, so that pINA1312-hp4d-CrtYB-xpr2t plasmid is obtained, and then the Pml I-CYB-up and CYBI-down are used as primers for amplification to obtain hp4d-CrtYB-xpr2t fragment;
constructing a phytoene desaturase coding gene CrtI expression module, using PmeI-CI-up and KpnI-CI-down as primers and pUC19-CrtI as a template, and amplifying a PCR fragment containing Pme I cleavage sites, crtI and KpnI cleavage sites; double enzyme digestion is carried out on pINA1312 plasmid by using Pml I and Kpn I, enzyme ligation is carried out on the PCR fragment carrying the Pme I blunt end site and Kpn I enzyme digestion site, and the PCR fragment is transferred into escherichia coli for screening to obtain positive clones which are correctly connected, so that pINA1312-hp4d-CrtI-xpr2t plasmid is obtained, and then CYBI-up and IDAG-down are used as primers for amplification to obtain hp4d-CrtI-xpr2t fragment;
constructing a diacylglycerol acyltransferase coding gene DAG1 expression module, using PmeI-DAG-up and KpnI-DAG-down as primers and pUC19-DAG1 as a template, and amplifying a PCR fragment containing PmeI cleavage sites, DAG1 and KpnI cleavage sites; double digestion is carried out on pINA1312 plasmid by using Pml I and Kpn I, the PCR fragment carrying Pme I blunt end site and Kpn I digestion site is connected by enzyme, and then the PCR fragment is transferred into escherichia coli for screening to obtain positive clone with correct connection, thus obtaining
pINA1312-hp4d-DAG1-xpr2t plasmid, and then using IDAG-up and DVHB-down as primers to amplify and obtain hp4d-DAG1-xpr2t fragment;
constructing a hemoglobin coding gene VHb expression module, using PmeI-VHb-up and KpnI-VHb-down as primers and pUC19-VHb as a template, and amplifying a PCR fragment containing PmeI cleavage sites, VHb and KpnI cleavage sites; double enzyme digestion is carried out on pINA1312 plasmid by using Pml I and Kpn I, enzyme ligation is carried out on the PCR fragment carrying the Pme I blunt end site and Kpn I enzyme digestion site, and the PCR fragment is transferred into escherichia coli for screening to obtain positive clones which are correctly connected, thus obtaining pINA1312-hp4d-VHb-xpr2t plasmid, and then the Hp4d-VHb-xpr2t fragment is obtained by taking DVHB-up and BamHI-VHb-down as primers for amplification;
recovering the hp4d-CrtYB-xpr2t, hp4d-CrtI-xpr2t, hp4d-DAG1-xpr2t and hp4d-VHb-xpr2t fragments containing homologous fragments, carrying out enzyme digestion and purification recovery on a plasmid pINA1312 by pml I, and integrating the expression module into the pINA1312 plasmid by the Gibson assembly (Norwei pran ClonExpress Ultra One Step Cloning Kit) technology to obtain a pINA1312-CrtYB-CrtI-DAG1-VHb recombinant plasmid;
2. construction of beta-carotene yarrowia lipolytica engineering bacterium ZM072 for synthesizing astaxanthin precursor and shake flask screening
(1) Construction of engineering bacteria ZM072 for producing beta-carotene yarrowia lipolytica
The method comprises the steps of adopting yarrowia lipolytica Yarrowia lipolytica ATCC MYA-2613 as host bacteria, namely, sclerotium rolfsii, carrying out digestion on the pINA1312-CrtYB-CrtI-DAG1-VHb recombinant plasmid obtained in the previous step by using Not I to obtain linear plasmid vectors carrying 4 expression cassettes of hp4d-CrtYB-xpr2t, hp4d-CrtI-xpr2t, hp4d-DAG1-xpr2t and hp4d-VHb-xpr2t, and integrating the linear vectors carrying the 4 expression cassettes into Zeta sites in yarrowia lipolytica MYA-2613 by a lithium acetate method; the lithium acetate conversion method comprises the following steps: culturing yarrowia lipolytica in 2mL YPD liquid medium for 18 hr, adding 200 μL into fresh YPD liquid medium, culturing for 4-5 hr, centrifuging at 6000rpm at room temperature for 5min to collect thallus, and sterilizing with ddH 2 O re-suspending the thalli, centrifuging again to collect thalli, and discarding the supernatant; then 1mL of 100mM pre-cooled lithium acetate at 4 ℃ is added into the centrifuged thalli, the thalli are collected by centrifugation at 6000rpm after being subjected to standing treatment at room temperature for 5min, and the preparation of yarrowia lipolytica competent cells is completed; the transformation mixed system comprises 240 mu LPEG (50% W/v), 36 mu L of 1.0M lithium acetate solution, 10 mu L of salmon sperm ss-DNA and linearized vector integration fragments, respectively, adding about 400ng of equivalent amount, filling sterile water to 360 mu L, sequentially adding the above-mentioned materials into competent cells, vortex shaking 1min,30 ℃ water bath for 30min,42 ℃ water for 30min, then centrifuging at 6000rpm for 5min, adding 1mL of YPD liquid culture medium after supernatant removal, culturing at 30 rpm for 2h to recover thalli, centrifuging at 6000rpm for 5min, washing 2 times with sterile water after supernatant removal, finally re-suspending with 100 mu L of sterile water, performing coating screening in SD solid culture medium, culturing at 30 ℃ for 48h, primarily selecting reddish or red transformant single colony on a flat plate by a visual inspection method, respectively inoculating into a test tube containing 4mL of YPD liquid culture medium, activating and culturing at 220rpm for 24h at 30 ℃, activating a glycerol freezing tube at-80 ℃ and extracting a part of the required genome by utilizing the bacterial liquid; secondly, using the extracted yeast genome as a template, carrying out PCR verification by using PmlI-CYB-up and BamHI-VHb-down primers, and verifying positiveThe transformant strain of (2) is a successfully constructed engineering strain of yarrowia lipolytica for the heterologous synthesis of beta-carotene;
(2) Shake flask screening of yarrowia lipolytica engineered strain ZM072 for producing beta-carotene
Shake flask fermentation screening: the YPD slant culture medium is used for culturing and fermenting yarrowia lipolytica engineering strains, 300 engineering strains which are dark and positive are selected from YPD plates, respectively inoculated into 10mL of YPD liquid culture medium for activating seed liquid, then transferred to 250mL of shake flask for loading 30mL of YPD liquid culture medium, shake-cultured for 4 days at 30 ℃ and 220rpm, and then detected for beta-carotene;
the detection method comprises the following steps: cultured yarrowia lipolytica cells were collected by centrifugation and resuspended in 0.7mL DMSO, then an equal volume of acetone was added, incubated at 55 ℃ for 10min, at 45 ℃ for 15min, and finally the sample was centrifuged at 12000×g for 5min; the supernatant containing beta-carotene was analyzed using an Shimadzu LC-20AT high performance liquid chromatograph equipped with a 450nm variable wavelength detector and a XDBC18 column (Eclipse, USA) used, with mobile phases of methanol, acetonitrile and methylene chloride (42:42:16), flow rates of 1.0mL/min, column temperatures of 30 ℃;
after the selected 300 dark positive strains are cultured by the fermentation mode, the detection results of 5 strains ZM011, ZM025, ZM072, ZM111, ZM211 and control strain MYA-2613 with higher beta-carotene yield are shown in Table 2;
TABLE 2 screening results for engineering bacteria of yarrowia lipolytica producing beta-carotene
Strain name MYA-2613 ZM011 ZM025 ZM072 ZM111 ZM211
Beta-carotene production (mg/L) 0 52.3 62.1 78.5 72.6 70.3
In conclusion, yarrowia lipolytica engineering bacterium ZM072 is selected.
Example 2 knockout of the glycerol-3-phosphate dehydrogenase GUT2 Gene on the yarrowia lipolytica engineering bacterium ZM072 genome
1. Construction of targeting Gene GUT2 Gene knockout plasmid pCRISPRy1-GUT2
To further increase the accumulation level of yarrowia lipolytica engineering bacteria ZM072 grease, GUT2 gene (GB accession number: YALI0B13970 g) encoding glycerol-3-phosphate dehydrogenase on the targeted shear genome was selected and constructed; the construction of the pCRISPRy1-GUT2 plasmid was described using the plasmid pCRISPRy1 (available from Addgene Corp.) with reference to the paper (Schwartz CM, hussain MS, blenner M, wheeldon I. Synthetic RNA Polymerase IIIPromoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica. ACS synthetic biol.2016Apr 15;5 (4): 356-9.); transferring the pCRISPRyl plasmid into DH5 alpha competence of the escherichia coli, transferring the transformant into LB medium containing 100 mug/ml ampicillin, culturing overnight, and extracting the pCRISPRyl plasmid by using an axygen plasmid extraction kit; the pCRISPRyl plasmid is subjected to single enzyme digestion of AvrII and then is subjected to gel electrophoresis purification and recovery to be used as a carrier; annealing to obtain double-stranded DNA of 60bp by using the synthesized DNA oligonucleotide single-stranded primer; the digested pCRISPRyl vector DNA recovered from the above gel was subjected to Gibson assembly (Gibson DG, young L, chuang RY, venter JC, hutchison CA 3rd,SmithHO.Enzymatic assembly of DNA molecules up to several hundred kilobases.Nat Methods.2009May;6 (5): 343-5) with 60bp double-stranded DNA to obtain pCRISPRy1-GUT2 plasmid for GUT2 interrupt inactivation;
2. transcriptional expression of sgrnas in CRISPR/Cas9 systems
The single-stranded guide sgrnas used in this example were present as expression vectors, and the double-stranded DNA and N20 sequences as RNA transcription templates are shown in table 3 below;
TABLE 3 primer sequences for CRISPR/Cas9 System
3. Plasmid pCRISPRy1-GUT2 transformed yarrowia lipolytica ZM072 and screening
Marking yarrowia lipolytica ZM072 strain on YPD solid culture medium, culturing at 30deg.C for 2 days, selecting single colony, transferring into test tube containing 4mL YPD liquid culture medium, culturing at 30deg.C at 220rpm overnight, transferring into 250mL shake flask containing 25mL YPD liquid culture medium, culturing at 30deg.C at 220rpm for 4-6 h, culturing to OD 600 0.8 to 1.0, and the bacterial liquid is used for preparing conversion competence; competent preparation and transformation were performed using Frozen-EZ Yeast Transformation II Kit, and with strict reference to the instructions for use, the transformed product was coated with SC-leu (glucose 20g/L, YNB basic nitrogen source 1.7g/L, leucine and uracil 50mg/L each), incubated at 30℃for 4 days, and colony PCR screening of monoclonal transformants was performed using the verification primers GUT2-up, GUT2-down, to obtain yarrowia lipolytica engineering strain ZM072 (. DELTA.GUT2).
EXAMPLE 3 construction of yarrowia lipolytica engineering ZMX001 for heterologous astaxanthin synthesis and shake flask screening
1. Construction of recombinant plasmid pINA1269-BsCrtW-PaCrtZ
Synthesis of genetic elements: artificial synthesis of nucleotide sequence based on marine beta-carotene ketolase encoding gene BsCrtW provided on NCBI and beta-carotene hydroxylase encoding gene from Pantoea ananatis
The nucleotide sequence of PaCrtZ is shown in SEQ ID NO.5 and SEQ ID NO.6, synthetic genes are respectively connected into pUC19 plasmid vector frameworks to obtain pUC19-BsCrtW and pUC19-PaCrtZ plasmids, and the primers are shown in Table 4;
TABLE 4 primer sequences used in example 4
Construction of recombinant plasmid pINA 1269-BsCrtW-PaCrtZ: constructing a beta-carotene ketolase encoding gene BsCrtW expression module, using PmeI-HCW-up and KpnI-HCW-down as primers and pUC19-CrtYB as a template, and amplifying a PCR fragment containing PmeI cleavage sites, bsCrtW and KpnI cleavage sites; double enzyme digestion is carried out on pINA1296 plasmid by using Pml I and Kpn I, the PCR fragment carrying the Pme I blunt end site and Kpn I enzyme digestion site is subjected to enzyme ligation, and the PCR fragment is transferred into escherichia coli for screening to obtain positive clones which are correctly connected, so that pINA1269-hp4d-BsCrtW-xpr2t plasmid is obtained, and then the Pml I-HCW-up and CWZ-down are used as primers for amplification to obtain hp4d-BsCrtW-xpr2t fragment;
constructing a beta-carotene hydroxylase coding gene PaCrtZ expression module, using PmeI-CZ-up and KpnI-CZ-down as primers, pUC19-PaCrtZ as a template, and amplifying a PCR fragment containing Pme I cleavage sites, paCrtZ and Kpn I cleavage sites; double enzyme digestion is carried out on pINA1269 plasmid by using Pml I and Kpn I, the PCR fragment carrying the Pme I blunt end site and Kpn I enzyme digestion site is subjected to enzyme ligation, and the PCR fragment is transferred into escherichia coli for screening to obtain positive clones which are correctly connected, so that pINA1269-hp4d-PaCrtZ-xpr2t plasmid is obtained, and then CWZ-up and BamHI-PacrtZ-down are used as primers for amplification to obtain hp4d-PacrtZ-xpr2t fragment;
recovering the constructed hp4d-BsCrtW-xpr2t and hp4d-PaCrtZ-xpr2t fragments, carrying out enzyme digestion and purification recovery on plasmid pINA1269 by using pml I, and integrating the expression module into the pINA1269 plasmid by using the Gibson assembly (Norwegian ClonExpress Ultra One Step Cloning Kit) technology to obtain pINA1269-BsCrtW-PaCrtZ;
2. construction of astaxanthin-producing yarrowia lipolytica engineering bacteria and shake flask verification
Construction of yarrowia lipolytica engineering ZMX001 for heterologous astaxanthin synthesis:
the preparation method comprises the steps of adopting yarrowia lipolytica engineering bacteria ZM072 (delta GUT 2) obtained in the embodiment 3 as host bacteria, carrying out enzyme digestion on pINA1269-BsCrtW-PaCrtZ recombinant plasmids obtained in the previous step by using Not I to obtain linear plasmid vectors carrying 2 expression cassettes of hp4d-BsCrtW-xpr2t and hp4d-PacrtZ-xpr2t, integrating the linear vectors carrying the 2 expression cassettes into yarrowia lipolytica engineering bacteria ZM072 (delta GUT 2) by the lithium acetate method, carrying out coating screening in a culture medium under the condition of 30 ℃, culturing for 48 hours, preliminarily selecting single colony of red transformants on a flat plate by a visual inspection method, respectively inoculating the single colony into a test tube containing 4 YPD liquid culture medium, carrying out activation culture for 24 hours at 30 ℃ and 220rpm/min, preserving the activation bacterial liquid at-80 ℃ by using a glycerol freezing tube, extracting a required genome by using a part of bacterial liquid, and carrying out PCR (i.e., carrying out verification that the extracted genome is a positive template of the yeast strain, namely, the strain is a yeast strain which is a positive template for the successful strain of the yarrowia lipolytica, namely, crlW-HCdZ;
and (3) verifying yarrowia lipolytica engineering bacteria ZMX001 shake flask:
shaking and fermenting: for shake flask single fermentation, firstly picking up ZMX001 strain from a flat plate for monoclonal, inoculating into 10mL of YPD liquid culture medium for activating seed liquid, transferring the activated seed liquid to 250mL of shake flask for loading 30mL of YPD liquid culture medium, shaking and culturing at 220rpm for 4 days at 30 ℃ and then detecting astaxanthin;
the detection method comprises the following steps: cultured yarrowia lipolytica engineering ZMX cells were collected by centrifugation and resuspended in 0.7mL DMSO, then an equal volume of acetone was added, incubated at 55 ℃ for 10min, then at 45 ℃ for 15min, and finally the sample was centrifuged at 12000×g for 5min; the Detector was an ultraviolet Detector, a Waters2489 UV/Vis Detector (Waters Corp., USA) from Waters company, a chromatographic column C18 was used for astaxanthin production, the specific model was BDS HYPERSIL C (150 mm. Times.4.6 mm,5 μm, thermo), the measurement wavelength was 470nm, the flow rate was 1mL/min, the column temperature was set to 25 ℃, the column was rinsed for half an hour with mobile phase A pure methanol before use, and the mobile phase used for astaxanthin production was solvent B: acetonitrile: water (9:1); solvent C: methanol to isopropanol (3:2), the measurement conditions are set as follows: 0-15min,0-90% B;15-30min,90% B;30-35min,90-0% B; culturing strain ZMX001 in the fermentation mode described above was performed at 35-55min 0% B, and finally an astaxanthin yield of 53.2mg/L was detected.
Example 4 construction and validation of astaxanthin-producing yarrowia lipolytica engineering ZMX1378
1. Error-prone PCR (polymerase chain reaction) transformation of BsCrtW-PaCrtZ gene cluster
Taking plasmid pINA1269-BsCrtW-PaCrtZ as a template, carrying out random mutation on hp4d-BsCrtW-xpr2t-hp4d-PaCrtZ-xpr2t tandem genes by changing magnesium ion concentration and dNTP concentration and carrying out error-prone PCR, wherein a PCR product is a mutation library of a gene cluster hp4d-BsCrtW-xpr2t-hp4d-PacrtZ-xpr 2;
error-prone PCR System (100. Mu.L): dATP, dCTP, dTTP, dGTP concentration is 80mM/mL, mgCl 2 The concentration of the primer(s) (the sequence shown in underline is the cleavage recognition site) was 40mM/mL, and the concentration of the primer(s) (the sequence shown in underline is the cleavage recognition site) was 10. Mu. Mol/L; the PCR procedure was: pre-denaturation at 98℃for 2min; denaturation at 94℃for 1min, annealing at 56℃for 1min, extension at 72℃for 2min, reaction for 35 cycles; finally, the mixture is extended for 10min at 72 ℃;
2. BsCrtW-PaCrtZ Gene Cluster DNA shuffling
PCR amplification is carried out by taking the amplification product of the error-prone PCR as a DNA reorganization template, pfu as DNA polymerase, bsCrtW-up and PaCrtZ-down as primers, and the obtained PCR amplification product is the hp4d-BsCrtW-xpr2t-hp4d-PacrtZ-xpr2 mutant library after DNA reorganization:
PCR procedure:
94 ℃ 1min and 53 ℃ 3min;72 ℃,20s,10 cycles;
94 ℃ 1min,55 ℃ 3min 20s;72 ℃,20s,20 cycles;
94 ℃ 1min,54 ℃ 3min35s;72 ℃,20s,40 cycles;
the temperature is kept at 72 ℃ for 20min to ensure that the extension is complete.
The PCR system is as follows: dNTP concentration is 80mM/mL, upstream primer concentration and downstream primer concentration are 10 mu mol/L, and error-prone PCR product final concentration is 200ng;
3. construction of a pool of synthetic astaxanthin yarrowia lipolytica mutants
The hp4d-BsCrtW-xpr2t-hp4d-PaCrtZ-xpr2 mutant library after DNA reorganization is digested by restriction enzymes Pml I and BamH I, and then is connected with pINA1269-BsCrtW-PaCrtZ plasmid digested by the same restriction enzymes, the connection product is transformed into Escherichia coli JM109, and the Escherichia coli JM109 bacterial library containing pINA1269-BsCrtW-PacrtZ mutant is obtained by expansion culture, and then the mutant plasmid library is extracted from the Escherichia coli JM109 bacterial library; the pINA1269-BsCrtW-PaCrtZ mutant plasmid library obtained in the last step is digested by Not I to obtain a linear mutant plasmid library carrying 2 series expression cassettes of hp4d-BsCrtW-xpr2t and hp4d-PaCrtZ-xpr2t, the linear carrier mutant library carrying 2 expression cassettes is integrated into yarrowia lipolytica engineering bacterium ZM072 (delta GUT 2) by the lithium acetate method, the coating is carried out in a culture medium, the culture condition is 30 ℃, and the culture is carried out for 48 hours to obtain the yarrowia lipolytica mutant library strain for synthesizing astaxanthin;
4. screening of high-yield astaxanthin yarrowia lipolytica engineering bacterium ZMX1378
The method comprises the steps of (1) primarily selecting darker monoclonals of yarrowia lipolytica engineering bacteria containing pINA1269-BsCrtW-PacrtZ mutants on a flat plate through a visual inspection method, culturing the monoclonals in a 96-well plate of YPD culture medium at 30 ℃ and at 150rpm for 72 hours, detecting astaxanthin, screening out nearly ten thousand mutant strains to obtain a strain with the astaxanthin yield of 1.2mg/L in the 96-well plate, and naming the strain as yarrowia lipolytica ZMX1378;
5. shaking flask verification of yarrowia lipolytica engineering bacterium ZMX1378
The ZMX1378 strain is picked from a flat plate for monoclonal, inoculated into 10mL of YPD liquid culture medium for activating seed liquid, then transferred to a 250mL shake flask for loading 30mL of YPD liquid culture medium, shake-cultured for 4 days at 30 ℃ and 220rpm, and then astaxanthin detection is carried out. The cultured yarrowia lipolytica engineering bacteria ZMX1378 cells were collected by centrifugation and resuspended in 0.7mL DMSO, then an equal volume of acetone was added, incubated for 10min at 55 ℃, then 15min at 45 ℃, and finally the samples were centrifuged at 12000×g for 5min; according to the detection method, the astaxanthin yield of 102.2mg/L is finally detected;
6. sequencing of the hp4d-BsCrtW-xpr2t-hp4d-PaCrtZ-xpr2 mutant Gene of yarrowia lipolytica ZMX1378
The genome of the high-yield astaxanthin yarrowia lipolytica ZMX1378 is used as a template, and is sent to Shanghai biological engineering Co-Ltd for sequencing through primers BsCrtW-up and PaCrtZ-Down, and the result shows that 5 gene mutations are generated on the hp4d-BsCrtW-xpr2T-hp4d-PacrtZ-xpr2 gene cluster, the A at the 12 th position on the BsCrtW gene sequence is mutated into C, the G at the 241 th position is mutated into A, and the G at the 485 th position is mutated into T; a at position 147 of the PaCrtZ gene sequence is mutated to C and G at position 392 is mutated to A.
EXAMPLE 6 50L fermentation production of astaxanthin by yarrowia lipolytica ZMX001 and ZMX1378
1. First-order seed liquid culture of yarrowia lipolytica ZMX001 and ZMX1378
(1) The yarrowia lipolytica ZMX001 and ZMX1378 monoclonal are respectively inoculated on YPD slant culture medium and cultured for 48h (48 h + -2 h in practical application) at 30 ℃ (30 ℃ + -1 ℃ in practical application);
(2) The yarrowia lipolytica ZMX and ZMX inclined planes prepared in the step (1) are respectively resuspended by 10mL of sterile water, and then inoculated into a seed shake flask (5L seed shake flask liquid filling amount is 1L) filled with a first-stage seed shake flask YPD liquid medium in an inoculum size of 1% (volume percent) (1% + -0.5% in practical application), and cultured for 24 hours (24 hours+ -2 hours in practical application) at 30 ℃ (30 ℃ + -1 ℃ in practical application) to obtain a first-stage seed culture solution;
2. secondary seed liquid culture of yarrowia lipolytica ZMX001 and ZMX1378
The primary seed culture solution prepared by the method is inoculated with 2L of secondary seeds in a seed transferring amount of 5 percent (volume percent) (5 percent plus or minus 1 percent in practical application)In a 5L fermentation tank of YPD liquid culture medium, culturing at 30deg.C (30deg.C+ -1deg.C in practical application) for 22h (22h+ -2h in practical application), and dissolving oxygen concentration of 40% (40% -80% in practical application) to obtain OD 600 About 2 (2+/-0.5 in practical application);
3. yarrowia lipolytica ZMX001 and ZMX1378 glucose feedback feed fermentation astaxanthin production
The secondary seed culture solution prepared by the method is inoculated into a 50L fermentation tank filled with 18L fermentation medium according to the transplanting amount of 10% (volume percent) (10% +/-2% in practical application), the culture is carried out for 72-96h at the temperature of 30+/-2 ℃, the pH value in the fermentation process is controlled to be 5.6-6.0, the dissolved oxygen concentration can be 10-20%, and when the glucose content in the fermentation process is lower than 2g/L, 600g/L of glucose aqueous solution is automatically added into the fermentation tank, so that the glucose content of a fermentation system is kept between 0.2 and 2 g/L;
4. detection of astaxanthin content in the obtained fermentation broth
Cultured yarrowia lipolytica engineering bacteria ZMX001 and ZMX1378 cells were collected by centrifugation and resuspended in 0.7mL DMSO, respectively, then an equal volume of acetone was added, incubated at 55 ℃ for 10min, and at 45 ℃ for 15min, finally the sample was centrifuged at 12000×g for 5min, the Detector was an ultraviolet Detector, a Waters2489 UV/Vis Detector (Waters corp., USA) from Waters company, the column of column C18 was chosen for astaxanthin assay, specific model BDS HYPERSIL C18 (150 mm×4.6mm,5 μm, thermo), the assay wavelength was 470nm, the column temperature was set at 25 ℃, the column was rinsed half an hour with mobile phase a pure solvent, and the mobile phase used to detect astaxanthin production was solvent B: acetonitrile: water (9:1); solvent C: methanol to isopropanol (3:2), the measurement conditions are set as follows: 0-15min,0-90% B;15-30min,90% B;30-35min,90-0% B;35-55min 0% B; the fermentation process curves of the yarrowia lipolytica engineering bacteria ZMX and ZMX1378 are shown in fig. 3, the maximum astaxanthin concentration of the fermentation liquid of the yarrowia lipolytica engineering bacteria ZMX1378 is 3775.4mg/L, the astaxanthin concentration of the fermentation liquid of the control strain ZMX001 is 732.7mg/L, and the astaxanthin yield of the yarrowia lipolytica engineering bacteria ZMX1378 is improved by about 415.2%.
Example 5 production of astaxanthin by 30 ton fermentors of yarrowia lipolytica engineering ZMX1378
1. First-stage seed liquid culture of yarrowia lipolytica engineering bacterium ZMX1378
(1) Selecting 5-10 yarrowia lipolytica engineering bacteria ZMX1378 monoclonal and respectively inoculating the monoclonal bacteria into eggplant type bottles of 100mL YPD slant culture medium, and culturing for 48h (48 h + -2 h in practical application) at 30 ℃ (30 ℃ + -1 ℃ in practical application);
(2) 5 yarrowia lipolytica engineering bacteria ZMX1378 eggplant type bottles prepared in the step (1) are respectively resuspended by 20mL of sterile water, and then inoculated into 5L seed shake flasks (the liquid filling amount of 5L seed shake flasks is 1L) filled with 1L of first-stage seed shake flask YPD liquid medium in 1 percent by volume percent (1% + -0.5% in practical application), and cultured for 24h (24 h+ -2 h in practical application) at 30 ℃ (30 ℃ + -1 ℃ in practical application) to obtain first-stage seed culture liquid;
2. yarrowia lipolytica engineering bacterium ZMX1378 secondary seed liquid culture
Transferring 5L primary seed liquid prepared in the steps into a 200L fermentation tank filled with 100L secondary seed YPD liquid medium at 5% (volume percentage) (5% + -1% in practical application), culturing at 30deg.C (30 ± 3 ℃ in practical application) for 24h (24 h+ -2 h in practical application), and dissolving oxygen concentration of 40% (40% -80% in practical application) to obtain OD 600 About 2 (2+/-0.5 in practical application);
3. yarrowia lipolytica engineering bacterium ZMX1378 three-stage seed liquid culture
The 100L secondary seed liquid prepared in the steps is inoculated into a 2T fermentation tank filled with 1000L of three-stage seed YPD liquid medium in a seed transferring amount of 10% (volume percent) (5% +/-1% in practical application), and is cultured for 24 hours (24 hours+/-2 hours in practical application) at a temperature of 30 ℃ (30+/-3 ℃ in practical application), wherein the dissolved oxygen concentration can be 40% (40% -80% in practical application), so that three-stage seed liquid with an OD600 of about 3 (3+/-0.5 in practical application) is obtained;
4. yarrowia lipolytica engineering bacterium ZMX1378 for producing shikimic acid by glucose feedback feed supplement fermentation in 30T fermentation tank
The three-stage seed culture solution prepared in the steps is inoculated into a 30T fermentation tank filled with a 10T fermentation medium according to the transplanting amount of 10% (volume percent) (15% +/-2% in practical application), the culture is carried out for 72-96h at the temperature of 30+/-2 ℃, the pH value in the fermentation process is controlled to be 5.6-6.0, the dissolved oxygen concentration can be 10-30%, and when the glucose content in the fermentation process is lower than 2g/L, 600g/L of glucose aqueous solution is automatically added from the fermentation tank, so that the glucose content of a fermentation system is kept between 0.2 and 2 g/L;
5. detection of astaxanthin content produced by yarrowia lipolytica engineering bacterium ZMX1378 in 30T fermentation tank
The process curve of the fermentation production of astaxanthin by yarrowia lipolytica engineering bacterium ZMX1378 in a 30T fermentation tank is shown in figure 4, and the astaxanthin in the fermentation liquid is detected according to the astaxanthin detection method, and the result shows that the concentration of the astaxanthin in the fermentation liquid of ZMX1378 in the 30T fermentation tank is 3237.2mg/L; at present, no report on large-scale fermentation production of astaxanthin acid by using yarrowia lipolytica engineering bacteria with glucose as a carbon source exists in China, most of the report stays in a laboratory stage, and a large gap exists between industrial application.
The foregoing examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the foregoing examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present patent should be made in the equivalent manner, and are included in the scope of the present patent.

Claims (9)

1. Yarrowia lipolytica engineering bacterium ZMX1378 is characterized in that: the preservation number is CCTCCNO: yarrowia lipolytica engineering bacteria ZMX1378 of M2023294; the yarrowia lipolytica engineering ZMX1378 genome integrates the CrtYB-CrtI-DAG1-VHb gene cluster and the BsCrtW-PaCrtZ mutant gene cluster.
2. The yarrowia lipolytica engineering bacterium ZMX1378 of claim 1, wherein: the genome of the yarrowia lipolytica engineering bacterium ZMX1378 overexpresses the CrtYB gene and the CrtI gene, the CrtYB gene and the CrtI gene are derived from Phaffia rhodozyma Xanthophyllomyces dendrorhous, the CrtYB gene is shown as SEQ ID NO.1, and the CrtI gene is shown as SEQ ID NO. 2.
3. The yarrowia lipolytica engineering bacterium ZMX1378 of claim 1, wherein: the genome of the yarrowia lipolytica engineering bacterium ZMX1378 overexpresses the DAG1 gene, and the DAG1 gene is derived from yarrowia lipolytica Yarrowia lipolytica as shown in SEQ ID NO. 3.
4. The yarrowia lipolytica engineering bacterium ZMX1378 of claim 1, wherein: the yarrowia lipolytica engineering bacterium ZMX1378 genome overexpresses a VHb gene, and the VHb gene is derived from Vitreoscilla hyaline and is shown as SEQ ID NO. 4.
5. The yarrowia lipolytica engineering bacterium ZMX1378 of claim 1, wherein: the yarrowia lipolytica engineering bacterium ZMX1378 genome overexpresses a BsCrtW mutant gene, and the BsCrtW mutant gene is derived from a gene shown as SEQ ID NO.5 in Brevundimonas sp.
6. The yarrowia lipolytica engineering bacterium ZMX1378 of claim 2, wherein: the genome of the yarrowia lipolytica engineering bacterium ZMX1378 overexpresses a PaCrtZ mutant gene, the PaCrtZ mutant gene is derived from a gene shown as SEQ ID NO.6 in Pantoea ananatis, A at 147 th position is mutated into C, and G at 392 th position is mutated into A.
7. The yarrowia lipolytica engineering bacterium ZMX1378 of claim 1, wherein: the gene locus of the glycerol-3-phosphate dehydrogenase GUT2 is knocked out on the genome of the yarrowia lipolytica engineering bacterium ZMX1378.
8. The method for efficiently synthesizing astaxanthin by using glucose as a substrate by yarrowia lipolytica engineering bacterium ZMX1378 as claimed in claim 1.
9. A method for synthesizing astaxanthin by taking glucose as a substrate by yarrowia lipolytica engineering bacterium ZMX1378, which is characterized by comprising the following steps of: culturing the yarrowia lipolytica engineering bacterium ZMX1378 of claim 1 to obtain a seed solution; inoculating the seed liquid into a fermentation culture medium, and performing glucose feedback feed-back fermentation, wherein the pH value in the fermentation process is controlled to be 5.6-6.0, the temperature in the fermentation process is 28-30 ℃, and the fermentation time is 96-128h; the concentration of the glucose feedback feed is controlled to be 0.2-2g/L, and astaxanthin is obtained through glucose feedback feed fermentation.
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CN114214218A (en) * 2021-12-24 2022-03-22 华东理工大学 Engineering bacterium for producing astaxanthin and preparation method and application thereof
CN114317307A (en) * 2021-12-30 2022-04-12 广州智特奇生物科技股份有限公司 Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield and construction method and application thereof
CN115029257A (en) * 2022-05-05 2022-09-09 南京工业大学 Recombinant yarrowia lipolytica for producing beta-carotene and construction method and application thereof

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US8846374B2 (en) * 2006-12-12 2014-09-30 E I Du Pont De Nemours And Company Carotenoid production in a recombinant oleaginous yeast

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Publication number Priority date Publication date Assignee Title
CN114214218A (en) * 2021-12-24 2022-03-22 华东理工大学 Engineering bacterium for producing astaxanthin and preparation method and application thereof
CN114317307A (en) * 2021-12-30 2022-04-12 广州智特奇生物科技股份有限公司 Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield and construction method and application thereof
CN115029257A (en) * 2022-05-05 2022-09-09 南京工业大学 Recombinant yarrowia lipolytica for producing beta-carotene and construction method and application thereof

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