CN114317307A - Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield and construction method and application thereof - Google Patents
Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield and construction method and application thereof Download PDFInfo
- Publication number
- CN114317307A CN114317307A CN202111681773.7A CN202111681773A CN114317307A CN 114317307 A CN114317307 A CN 114317307A CN 202111681773 A CN202111681773 A CN 202111681773A CN 114317307 A CN114317307 A CN 114317307A
- Authority
- CN
- China
- Prior art keywords
- genetically engineered
- tlip
- plasmid
- engineered bacterium
- astaxanthin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- JEBFVOLFMLUKLF-IFPLVEIFSA-N Astaxanthin Natural products CC(=C/C=C/C(=C/C=C/C1=C(C)C(=O)C(O)CC1(C)C)/C)C=CC=C(/C)C=CC=C(/C)C=CC2=C(C)C(=O)C(O)CC2(C)C JEBFVOLFMLUKLF-IFPLVEIFSA-N 0.000 title claims abstract description 53
- 235000013793 astaxanthin Nutrition 0.000 title claims abstract description 53
- MQZIGYBFDRPAKN-ZWAPEEGVSA-N astaxanthin Chemical compound C([C@H](O)C(=O)C=1C)C(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)[C@@H](O)CC1(C)C MQZIGYBFDRPAKN-ZWAPEEGVSA-N 0.000 title claims abstract description 53
- 229940022405 astaxanthin Drugs 0.000 title claims abstract description 53
- 239000001168 astaxanthin Substances 0.000 title claims abstract description 53
- 241000894006 Bacteria Species 0.000 title claims abstract description 28
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 17
- 238000010276 construction Methods 0.000 title claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 41
- 230000004151 fermentation Effects 0.000 claims abstract description 41
- 238000000034 method Methods 0.000 claims abstract description 31
- 241000235015 Yarrowia lipolytica Species 0.000 claims abstract description 27
- 238000004519 manufacturing process Methods 0.000 claims abstract description 20
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 19
- 239000008103 glucose Substances 0.000 claims abstract description 19
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 10
- 230000008569 process Effects 0.000 claims abstract description 9
- 101150077059 TPS1 gene Proteins 0.000 claims abstract description 6
- 101150114010 ggs1 gene Proteins 0.000 claims abstract description 6
- 239000013612 plasmid Substances 0.000 claims description 54
- 239000012634 fragment Substances 0.000 claims description 30
- 238000012258 culturing Methods 0.000 claims description 23
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 20
- 239000001963 growth medium Substances 0.000 claims description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 17
- 101150050575 URA3 gene Proteins 0.000 claims description 15
- 230000014509 gene expression Effects 0.000 claims description 15
- 230000001131 transforming effect Effects 0.000 claims description 10
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 9
- 238000010367 cloning Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 241000588724 Escherichia coli Species 0.000 claims description 8
- 101150076348 FTO gene Proteins 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 241000282414 Homo sapiens Species 0.000 claims description 7
- 238000001976 enzyme digestion Methods 0.000 claims description 7
- 230000037361 pathway Effects 0.000 claims description 7
- 108020004705 Codon Proteins 0.000 claims description 6
- 241000168517 Haematococcus lacustris Species 0.000 claims description 6
- 241001489146 Mucor circinelloides Species 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 6
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 6
- 229960000723 ampicillin Drugs 0.000 claims description 6
- 241001272975 bacterium F1 Species 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 6
- 238000000576 coating method Methods 0.000 claims description 6
- 230000007547 defect Effects 0.000 claims description 6
- 238000010353 genetic engineering Methods 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 238000009630 liquid culture Methods 0.000 claims description 6
- XIXADJRWDQXREU-UHFFFAOYSA-M lithium acetate Chemical compound [Li+].CC([O-])=O XIXADJRWDQXREU-UHFFFAOYSA-M 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 241000948060 bacterium F2 Species 0.000 claims description 4
- 238000005457 optimization Methods 0.000 claims description 4
- KJTLQQUUPVSXIM-ZCFIWIBFSA-M (R)-mevalonate Chemical compound OCC[C@](O)(C)CC([O-])=O KJTLQQUUPVSXIM-ZCFIWIBFSA-M 0.000 claims description 3
- VWFJDQUYCIWHTN-YFVJMOTDSA-N 2-trans,6-trans-farnesyl diphosphate Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-YFVJMOTDSA-N 0.000 claims description 3
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 claims description 3
- VWFJDQUYCIWHTN-UHFFFAOYSA-N Farnesyl pyrophosphate Natural products CC(C)=CCCC(C)=CCCC(C)=CCOP(O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-UHFFFAOYSA-N 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 230000001851 biosynthetic effect Effects 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 238000011084 recovery Methods 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 238000009423 ventilation Methods 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 2
- 239000012533 medium component Substances 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 abstract description 4
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 abstract description 4
- 230000012010 growth Effects 0.000 abstract description 4
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 230000006698 induction Effects 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 9
- 235000021466 carotenoid Nutrition 0.000 description 6
- 235000011187 glycerol Nutrition 0.000 description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- 150000001747 carotenoids Chemical class 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 244000144974 aquaculture Species 0.000 description 2
- 238000009360 aquaculture Methods 0.000 description 2
- -1 carotenoid compound Chemical class 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000012262 fermentative production Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 101150054413 HXT2 gene Proteins 0.000 description 1
- 241000282620 Hylobates sp. Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101150014136 SUC2 gene Proteins 0.000 description 1
- 101100507949 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HXT2 gene Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000235013 Yarrowia Species 0.000 description 1
- 101100215634 Yarrowia lipolytica (strain CLIB 122 / E 150) XPR2 gene Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a genetically engineered bacterium capable of improving the biosynthesis yield of astaxanthin and a construction method and application thereof, wherein AtoB, tHMGR, IDI, GGS1, pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1, pEXP-CarB-tICL and pHXT2-FTO-tLIP are introduced into yarrowia lipolytica Polf. The invention has the advantages that: the effective conversion of thallus growth and product production in the microbial fermentation production process is realized by introducing the glucose concentration induction type promoter and combining RNA demethylase, the yield of astaxanthin produced by yeast fermentation is improved, the operation process is simplified, and the production cost is reduced.
Description
Technical Field
The invention relates to the technical field of construction of genetically engineered bacteria, in particular to a construction technology of genetically engineered bacteria for improving the yield of astaxanthin.
Background
Astaxanthin is a red ketonized carotenoid and has a strong antioxidant function. It is mainly used as feed additive for aquaculture to improve fish color. Astaxanthin is also increasingly used in the food and cosmetic fields because of its strong antioxidant function. In addition, astaxanthin has been shown to have anti-tumor, anti-inflammatory and immune system enhancing effects. Because of the functions of astaxanthin, the global annual demand of astaxanthin reaches 250 tons, the sales amount approaches $ 4.5 billion, and $ 25 billion is expected in 2025.
At present, astaxanthin is mainly obtained by chemical synthesis, but the chemically synthesized astaxanthin is a racemic mixture, cannot be absorbed and utilized by human bodies, and only can be used for coloring animals and aquaculture. Although astaxanthin is synthesized by various biological methods, the problems of low production efficiency and no competitive economy generally exist, and compared with the method, the microbial fermentation method is not limited by raw materials, and the production process is green and clean, so that the method has obvious advantages. Yarrowia lipolytica (Yarrowia lipolytica) is an excellent strain of the bacterium for synthesizing carotenoids, contains abundant acetyl-CoA as a precursor material of a synthetic carotenoid compound, and accumulates a large amount of lipids in cells to provide storage space for lipid-soluble carotenoid compounds.
Since the production of target products by microorganisms is often performed by exogenous metabolic pathways, which imposes a burden on cell growth, it has been a problem faced by the biosynthetic industry. The prior solutions have been to design different temperature sensitive promoters, to switch the fermentation process from the strain growth phase to the product accumulation phase by changing the fermentation temperature, and to promote this switch by microbial cell density sensing. Most strains only grow in a specific temperature interval, cannot grow across the temperature interval, and are denseness-sensitiveThe sensitivity is not high, and signal paths for releasing and receiving the induction factors are additionally designed, so that the growth of the strain is influenced, the metabolic burden of the strain is increased, and the practical application is limited by the problems. The HXT2 upstream promoter of yeast cell can inhibit or open the transcription of downstream genes according to the high or low glucose concentration (
et al, 1996). Glucose is a common carbon source for fermentative production, and fermentative production of carotenoids requires high concentrations of glucose during the biomass production phase and only low concentrations of glucose during the product production phase (Gao et al, 2017). N6-adenylate methylation (m6A) modification is a common post-transcriptional reversible modification of mRNA. m6A methylases and demethylases regulate RNA processing and metabolism by methylation and demethylation, and promote gene transcriptional expression by promoting chromosome opening (Chandola et al, 2014; Yu et al, 2021). Therefore, the method has important significance for solving the problem that the target product is accumulated to inhibit the growth of the strain in the fermentation production process of the carotenoid so as to improve the yield of the target product.
Disclosure of Invention
The invention aims to provide a genetically engineered bacterium capable of improving astaxanthin biosynthesis yield and a construction method and application thereof, so as to solve the problem of low astaxanthin production yield of the existing microorganisms.
In order to achieve the purpose, the invention adopts the following technical scheme:
a genetic engineering bacterium capable of improving the biosynthesis yield of astaxanthin is a strain F2 obtained by introducing AtoB, tHMGR, IDI, GGS1, pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1, pEXP-CarB-tICL and pHXT2-FTO-tLIP into yarrowia lipolytica Polf;
wherein the sequence of pHXT2 is shown in SEQ ID NO: 34; the sequence of FTO is shown in SEQ ID NO. 35.
The invention also provides a construction method of the genetic engineering bacteria capable of improving the biosynthesis yield of the astaxanthin, which comprises the following steps:
(1) constructing a plasmid pMA containing pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1 and pEXP-CarB-tICL and a plasmid pMB containing pHXT 2-FTO-tLIP;
(2) introducing mevalonate pathway and farnesyl pyrophosphate pathway-related genes AtoB, tHMGR, IDI and GGS1 into yarrowia lipolytica Polf to obtain strain F0;
(3) transforming the strain F0 by the plasmid pMA after enzyme digestion to obtain a genetically engineered bacterium F1;
(4) and transforming the plasmid pMB subjected to enzyme digestion into the genetically engineered bacterium F1 to obtain a genetically engineered bacterium F2.
Further, the procedure for constructing plasmid pMA containing pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1 and pEXP-CarB-tICL includes:
extracting genome DNA of Saccharomyces cerevisiae CEN.PK2-1C and yarrowia lipolytica Polf;
PCR amplifying URA3 gene by using primer loxP-URA3-F/loxP-URA3-R and yarrowia lipolytica Polf genome DNA as template, and amplifying URA3 gene segment by using primer URA3-F and URA3-R and using the PCR product as template; plasmid pUC19 was digested simultaneously with Nde I and Kpn I, and the URA3 gene fragment was cloned into pUC19 between Nde I and Kpn I by seamless cloning to construct plasmid pUC19URA 3;
amplifying pFBA, tXPR2, pGPD, tLIP, pTEF, pEXP and tICL fragments by using primers pFBA-F/pFBA-R, tXPR2-F/tXPR2-R, pGPD-F/pGPD-R, tLIP-F/tLIP-R, pTEF-F/pTEF-R, pEXP-F/pEXP-R and tICL-F/tICL-R respectively and taking yarrowia lipolytica Polf genomic DNA as a template; amplifying a CYC1 fragment by using a primer CYC1-F/CYC1-R and yeast CEN. PK2-1C genomic DNA as a template;
after codon optimization of CarRP and CarB derived from Mucor circinelloides (Rhizomucor circinelloides) and CrtZ and CrtW genes derived from Haematococcus pluvialis (Haematococcus pluvialis), the codon-optimized CarRP, CarB, CrtZ and CrtW fragments were amplified using the primers CarRP-F/CarRP-R, CarB-F/CarB-R, CrtZ-F/CrtZ-R and CrtW-F/CrtW-R, respectively, as templates;
splicing four expression frames of pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1 and pEXP-CarB-tICL, carrying out double digestion, purification and recovery on a plasmid pUC19URA3 by using BamH I and Hind III, and then assembling a digested plasmid skeleton and the four expression frames to construct a plasmid pMA.
Further, the construction process of the plasmid pMB containing pHXT2-FTO-tLIP comprises the following steps:
extracting yarrowia lipolytica Polf genomic DNA;
PCR amplifying URA3 gene by using primer loxP-URA3-F/loxP-URA3-R and yarrowia lipolytica Polf genome DNA as template, and amplifying URA3 gene segment by using primer URA3-F and URA3-R and using the PCR product as template; plasmid pUC19 was digested simultaneously with Nde I and Kpn I, and the URA3 gene fragment was cloned into pUC19 between Nde I and Kpn I by seamless cloning to construct plasmid pUC19URA 3;
pHXT2 and tLIP fragments were amplified using yarrowia lipolytica Polf genomic DNA as template with primers pHXT2-F/pHXT2-R and tLIP-F/tLIP-R1, respectively;
FTO gene from human (Homo sapiens) is optimized by codon and used as a template, and a primer FTO-F/FTO-R is used for amplifying an optimized FTO gene fragment;
splicing pHXT2-FTO-tLIP expression frame, purifying and recovering plasmid pUC19URA3 by double enzyme digestion with BamH I and Hind III, and assembling the digested plasmid skeleton and pHXT2-FTO-tLIP expression frame to construct plasmid pMB.
Further, the sequence of pHXT2 obtained by amplifying the pHXT2 fragment is shown as SEQ ID NO: 34.
Further, the FTO sequence obtained by amplifying the optimized FTO gene fragment is shown as SEQ ID NO. 35.
Further, the operation process of the step (3) is as follows:
digesting plasmid pMA for 1 hour by NotI, recovering a fragment containing no escherichia coli replicon and ampicillin resistance genes by agarose gel electrophoresis, then transforming a linearized pMA into a strain F0 by a lithium acetate method, coating an SD-URA defect culture medium, selecting 120 transformants with the deepest orange red color after 2-3 days, respectively inoculating into 10ml of liquid YPD40 culture medium, culturing for 72 hours at 200rpm and 30 ℃, detecting the yield of astaxanthin, and obtaining a genetically engineered bacterium F1 with the highest yield;
and (4) digesting the plasmid pMB for 1 hour by using Not I, recovering a fragment which does Not contain an escherichia coli replicon and an ampicillin resistance gene by agarose gel electrophoresis, then transforming a linearized pMB into a strain F1 by using a lithium acetate method, coating an SD-URA defect culture medium, selecting 120 transformants with the deepest orange red color after 2-3 days, respectively inoculating into 10ml of liquid YPD40 culture medium, culturing at 200rpm and 30 ℃ for 72 hours, detecting the yield of astaxanthin, and obtaining the genetically engineered bacterium F2 with the highest yield, and storing in a glycerol tube at-80 ℃.
Further, the YPD40 medium components included 1% yeast extract, 2% peptone and 4% glucose in terms of mass fraction.
The invention also provides an application of the genetic engineering bacteria capable of improving the biosynthesis yield of the astaxanthin in astaxanthin production, which comprises the following steps:
1) culturing a primary seed solution: taking a strain F2 stored in a glycerol tube at the temperature of-80 ℃, drawing a line on a solid YPD plate, culturing for 24 hours at the temperature of 30 ℃, inoculating a single colony of the strain F2 on the solid YPD plate into a 50ml conical flask containing 10ml of YPD liquid culture medium, and culturing for 24 hours at the temperature of 30 ℃ and the rotating speed of a shaking table of 220 rpm;
2) culturing a secondary seed solution: transferring the primary seed solution into a 300ml conical flask containing 100ml YPD liquid culture medium according to the inoculation amount of 10 wt%, culturing for 8 hours at the temperature of 30 ℃ at the rotating speed of a shaking table of 220rpm until the OD600 of the cells reaches 10 to obtain a secondary seed solution;
3) the secondary seed solution was transferred to a 5L fermentor containing 2L fermentation medium for fermentation at 10% wt inoculum size.
More preferably, the detailed process of step 3) is: transferring the secondary seed liquid into a 5L fermentation tank containing 2L of fermentation medium according to the inoculation amount of 10% by weight for fermentation, culturing under the conditions that the initial medium consists of 30g/L of yeast extract, 60g/L of peptone and 100g/L of glucose, the fermentation temperature is 30 ℃, the stirring speed is 300-800rpm, the ventilation quantity is 4L/min, the pH value is 6.8 and the dissolved oxygen is more than 25%, supplementing 200ml of 600g/L glucose once when the fermentation time is 48 hours, and feeding 600g/L glucose according to 10 ml/hour flow when the fermentation time is 72 hours until the fermentation time is 240 hours.
Compared with the prior art, the invention has the following advantages:
by utilizing the characteristic that a yeast HXT2 gene promoter inhibits or activates downstream gene expression along with high or low glucose concentration, the gene expression activity is enhanced by combining RNA demethylase FTO, and by introducing a glucose concentration induction type promoter and combining RNA demethylase, the contradiction that the growth of thalli and the production of products cannot be effectively converted in the microbial fermentation production process is well solved, the yield of astaxanthin produced by yeast fermentation is improved, the operation process is simplified, and the production cost is reduced.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention:
FIG. 1 is a schematic of plasmid pMA;
FIG. 2 is a schematic of plasmid pMB;
FIG. 3 is a graph showing the results of fermentation tank production of astaxanthin by the astaxanthin-producing strains (F1 and F2) of the present invention.
Detailed Description
The present invention will be described in detail with reference to the drawings and specific embodiments, which are illustrative of the present invention and are not to be construed as limiting the present invention.
The yarrowia lipolytica strain of the invention is Polf (ATCC No. MAY-2613; genotype MATAura3-302 leu2-270 XPR-322axp2-deltaNU49 XPR2:: SUC2, available from ATCC), the restriction enzymes used were purchased from ThermoFisher, the reagents used for plasmid extraction and gel recovery, the reagents used for PCR and seamless cloning, and the E.coli (DH 5. alpha.) for molecular cloning were purchased from Nanjing Hooker Biotech, Inc., and the reagents used for Saccharomyces cerevisiae, yarrowia lipolytica and E.coli genomic DNA extraction were purchased from Qiagen.
The primers used in the examples are shown in table 1:
TABLE 1 primer List
Example 1 astaxanthin Synthesis pathway plasmid construction
Saccharomyces cerevisiae CEN. PK2-1C and yarrowia lipolytica Polf genomic DNA were extracted using the Blood & Cell Culture DNA Mini Kit from Qiagen.
PCR amplifying URA3 gene by using primer loxP-URA3-F/loxP-URA3-R and yarrowia lipolytica Polf genome DNA as template, and amplifying URA3 gene segment by using primer URA3-F and URA3-R and using the PCR product as template; plasmid pUC19 was digested simultaneously with Nde I and Kpn I and the URA3 gene fragment was cloned into pUC19 between Nde I and Kpn I by using a seamless Cloning (Novozan Clonexpress Ultra One Step Cloning Kit) to construct plasmid pUC19URA 3.
The base sequence of the loxP-URA3-F is shown in SEQ ID NO. 1; the base sequence of loxP-URA3-R is shown in SEQ ID NO. 2; the base sequence of URA3-F is shown in SEQ ID NO. 3; the base sequence of URA3-R is shown in SEQ ID NO. 4.
(1) Construction of plasmid pMA
Amplifying pFBA, tXPR2, pGPD, tLIP, pTEF, pEXP and tICL fragments by using primers pFBA-F/pFBA-R, tXPR2-F/tXPR2-R, pGPD-F/pGPD-R, tLIP-F/tLIP-R, pTEF-F/pTEF-R, pEXP-F/pEXP-R and tICL-F/tICL-R respectively and taking yarrowia lipolytica Polf genomic DNA as a template; amplifying a CYC1 fragment by using a primer CYC1-F/CYC1-R and yeast CEN. PK2-1C genomic DNA as a template;
CarRP and CarB derived from Mucor circinelloides (Rhizomucor circinelloides) and CrtZ and CrtW genes derived from Haematococcus pluvialis (Haematococcus pluvialis) are subjected to codon optimization treatment by Nanjing Kinski technology Co., Ltd, and the CarRP, CarB, CrtZ and CrtW fragments are amplified by using the optimized genes as templates respectively as primers CarRP-F/CarRP-R, CarB-F/CarB-R, CrtZ-F/CrtZ-R;
the base sequence of pFBA-F is shown as SEQ ID NO. 23; the base sequence of pFBA-R is shown as SEQ ID NO. 24; the base sequence of tXPR2-F is shown as SEQ ID NO. 27; the base sequence of tXPR2-R is shown as SEQ ID NO. 28; the base sequence of pGPD-F is shown as SEQ ID NO: 29; the base sequence of pGPD-R is shown as SEQ ID NO. 30; the base sequence of tLIP-F is shown as SEQ ID NO. 9; the base sequence of tLIP-R is shown as SEQ ID NO. 10; the base sequence of pTEF-F is shown as SEQ ID NO. 11; the base sequence of pTEF-R is shown as SEQ ID NO 12; the base sequence of pEXP-F is shown as SEQ ID NO: 17; the base sequence of pEXP-R is shown as SEQ ID NO. 18; the base sequence of tICL-F is shown as SEQ ID NO. 21; the base sequence of tICL-R is shown in SEQ ID NO. 22; the base sequence of CYC1-F is shown as SEQ ID NO. 15; the base sequence of CYC1-R is shown as SEQ ID NO. 16; the base sequence of CarRP-F is shown in SEQ ID NO. 31; the base sequence of CarRP-R is shown in SEQ ID NO: 32; the base sequence of CarB-F is shown in SEQ ID NO: 19; the base sequence of CarB-R is shown as SEQ ID NO. 20; the base sequence of CrtZ-F is shown as SEQ ID NO. 25; the base sequence of CrtZ-R is shown as SEQ ID NO. 26; the base sequence of CrtW-F is shown as SEQ ID NO. 13; the base sequence of CrtW-R is shown as SEQ ID NO. 14;
the promoter, the gene and the terminator are spliced into four expression frames of pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1 and pEXP-CarB-tICL by overlap PCR, the plasmid pUC19URA3 is purified and recovered by double enzyme digestion of BamHI and Hind III, and the digested plasmid framework and the four expression frames are assembled to construct plasmid pMA by Gibbon ligation (Novozan ClonExpress Ultra One Step Cloning Kit). Plasmid pMA is shown in FIG. 1.
(2) Construction of plasmid pMB
pHXT2 and tLIP fragments were amplified using yarrowia lipolytica Polf genomic DNA as template with primers pHXT2-F/pHXT2-R and tLIP-F/tLIP-R1, respectively;
FTO gene originated from human (Homo sapiens) is subjected to codon optimization treatment by Nanjing Kingsley science and technology Co., Ltd, and FTO gene fragment is amplified by using the optimized gene as a template by using a primer FTO-F/FTO-R.
The base sequence of pHXT2-F is shown in SEQ ID NO: 5; the base sequence of pHXT2-R is shown in SEQ ID NO 6; the base sequence of FTO-F is shown as SEQ ID NO. 7; the base sequence of FTO-R is shown as SEQ ID NO. 8; the base sequence of tLIP-F is shown as SEQ ID NO. 9; the base sequence of tLIP-R1 is shown in SEQ ID NO: 33.
The sequence of the amplified pHXT2 is shown in SEQ ID NO: 34:
AGAAGAAGCAGGACTACTTTTTCGAGATCAGAGGCGAGCTTGATCGGGAGTTCAAGGAGGTTTTCTACAGACTACACGAGCATCTTGTCTACAAGTTCCATCTCTTCTGGAAGCATGGTGTGCAGGAGAAGAAGATTCAGAGCGTTCTGCAGACTGACAACCACCTGAAGATCTTCCTGGAGAAATACGACGCAAACCTGCGACGGGGAGACTGGAAATAAAACATGAGAGACACACACGTGCAACACACAAACATTAGTTAATTGATTCATCTGATCTGCAAAAACATGTTGGTGATGGTTAAGAGTCACATAACAAGCAGGGTCCATGCACTCGCTTTCTTACATAGCACCATAGAATACGGTGTTGTCGGCATTATCAAGACAGAATTTACTTTGGTGATGTTATCACATGAGCCGGGGGGCATAGGGTGGGCACTCGTACTCTATGCGTTATTGGTTGCAGACCCATAATCTCAAGGGCGTTCCAGAACATACACTATGCGACCGCAGGGACCCTCGATATGGACGATGAATAGTATGGCAACATAAACCAGAAACTATTATATAGAGATGATGGGGGCGACATTTCGGTAATTACGTTCATCCACCTTCGATC
the sequence of the FTO obtained by amplification is shown as SEQ ID NO:
ATGAAGCGAACTCCCACCGCCGAAGAACGGGAGCGAGAGGCCAAAAAGCTGCGGCTGCTCGAAGAACTCGAGGACACGTGGCTCCCATATCTCACCCCCAAGGACGATGAGTTCTACCAGCAGTGGCAGCTCAAGTACCCTAAACTGATTCTGAGAGAAGCAAGCTCAGTTTCTGAGGAGCTCCACAAGGAGGTTCAGGAGGCGTTTCTGACGCTCCATAAGCACGGATGTCTGTTTAGAGATCTGGTGCGAATCCAGGGAAAGGATCTTTTGACCCCGGTATCGCGGATTCTCATCGGAAACCCGGGCTGTACCTACAAGTATCTCAACACCCGTCTCTTCACCGTCCCCTGGCCCGTCAAGGGCTCCAACATCAAGCATACAGAGGCAGAGATTGCCGCGGCTTGTGAGACTTTCTTGAAGCTCAACGACTATCTTCAGATAGAGACCATTCAGGCCTTAGAAGAGTTGGCAGCCAAGGAGAAGGCCAACGAGGATGCCGTGCCTCTTTGCATGTCTGCTGATTTCCCACGAGTCGGTATGGGTTCGTCATACAACGGTCAGGACGAGGTGGATATCAAGTCTCGCGCTGCCTACAATGTGACTCTGCTGAACTTTATGGACCCCCAGAAAATGCCCTACCTGAAAGAGGAGCCCTACTTTGGAATGGGCAAGATGGCTGTTTCATGGCACCACGATGAAAACCTCGTGGATAGATCCGCAGTTGCTGTCTACTCTTACTCGTGCGAGGGACCTGAGGAGGAAAGTGAGGACGACTCCCATCTTGAGGGGCGAGACCCCGACATCTGGCACGTGGGCTTCAAGATTTCGTGGGACATTGAAACGCCTGGTCTTGCCATCCCCCTACACCAGGGCGACTGCTACTTCATGCTGGACGATCTAAATGCGACACATCAACATTGTGTGCTGGCCGGAAGTCAACCTCGATTCAGCTCGACCCACAGAGTGGCTGAATGCTCCACTGGAACATTGGACTACATCCTGCAACGGTGCCAACTGGCTCTGCAGAATGTCTGTGATGACGTTGACAACGATGATGTGTCCCTTAAGAGCTTTGAGCCTGCTGTCCTAAAGCAGGGCGAGGAAATCCACAACGAAGTCGAGTTTGAGTGGCTGAGACAGTTCTGGTTCCAAGGTAACCGATACCGACGATGCACCGACTGGTGGTGCCAGCCCATGGCCCAGCTGGAGGCACTGTGGAAGAAGATGGAGGGTGTGACCAATGCCGTTCTCCACGAGGTCAAACGCGAGGGCTTACCGGTGGAGCAACGAAACGAGATCCTGACTGCCATTCTGGCGTCTCTTACGGCCCGTCAGAACCTACGTCGAGAATGGCATGCTCGCTGTCAGTCTCGAATCGCTCGGACTTTGCCAGCTGACCAGAAGCCCGAGTGTCGACCTTACTGGGAGAAGGACGACGCCTCCATGCCTCTGCCCTTTGACTTGACAGACATTGTCTCCGAGCTGCGAGGCCAGCTGCTTGAAGCAAAGCCTTGA
pHXT2, FTO and tLIP were spliced into pHXT2-FTO-tLIP expression cassette by overlap PCR, plasmid pUC19URA3 was recovered by double digestion with BamHI and HindIII, and plasmid pMB was constructed by assembling the digested plasmid backbone and pHXT2-FTO-tLIP expression cassette by Gibson ligation (NuoWei Zan Clonexpress Ultra One Step Cloning Kit). Plasmid pMB is shown in FIG. 2.
Example 2 construction of astaxanthin-producing Strain
Strain F0 was obtained by introducing mevalonate pathway and farnesyl pyrophosphate pathway-related genes AtoB, tHMGR, IDI and GGS1 into yarrowia lipolytica Polf.
Cutting plasmid pMA by Not I for 1 hour, carrying out agarose gel electrophoresis to recover a fragment containing no escherichia coli replicon and ampicillin resistance genes, then transforming a linearized pMA into a strain F0 by a lithium acetate method, coating an SD-URA defect culture medium, selecting 120 transformants with the deepest orange red color after 2-3 days, respectively inoculating into 10ml of liquid YPD40 culture medium, and culturing at 200rpm and 30 ℃ for 72 hours at the mass fraction of YPD40 culture medium components including 1% of yeast extract, 2% of peptone and 4% of glucose to detect the yield of astaxanthin, so as to obtain a strain F1 with the highest yield and preserving glycerol tubes at-80 ℃ for later use.
Cutting plasmid pMB with NotI for 1 hour, carrying out agarose gel electrophoresis to recover a fragment containing no Escherichia coli replicon and ampicillin resistance genes, then transforming a linearized pMB into a strain F1 by a lithium acetate method, coating an SD-URA defect culture medium, selecting 120 transformants with the deepest orange red color after 2-3 days, respectively inoculating into 10ml of liquid YPD40 culture medium, culturing for 72 hours at 200rpm and 30 ℃, detecting the yield of astaxanthin, obtaining a strain F2 with the highest yield, and storing in a glycerol tube at-80 ℃ for later use.
EXAMPLE 3 tank fermentation of astaxanthin
The strain F1 and the strain F2 prepared in example 2 were fermented in 5L fermentors, respectively, to produce astaxanthin. The method comprises the following steps:
1) culturing a primary seed solution: taking a strain F1/a strain F2 stored in a glycerin tube at the temperature of minus 80 ℃ to draw lines on a solid YPD plate and culture the strains at the temperature of 30 ℃ for 24 hours, inoculating a single colony of the strain F1/the strain F2 on the solid YPD plate into a 50ml conical flask containing 10ml of YPD liquid culture medium, and culturing the strains at the temperature of 30 ℃ and the rotating speed of a shaking table of 220rpm for 24 hours;
2) culturing a secondary seed solution: transferring the primary seed solution into a 300ml conical flask containing 100ml YPD liquid culture medium according to the inoculation amount of 10% (wt), culturing for 8 hours at the temperature of 30 ℃ and the rotating speed of a shaking table of 220rpm until the OD600 of the cells reaches 10 to obtain a secondary seed solution;
3) transferring the secondary seed liquid into a 5L fermentation tank containing 2L of fermentation medium according to the inoculation amount of 10 percent (wt) for fermentation, culturing under the conditions that the initial medium consists of 30g/L of yeast extract, 60g/L of peptone and 100g/L of glucose, the fermentation temperature is 30 ℃, the stirring speed is 300-800rpm, the ventilation quantity is 4L/min, the pH value is 6.8 and the dissolved oxygen is more than 25 percent, supplementing 200ml of 600g/L of glucose once when the fermentation time is 48 hours, and feeding 600g/L of glucose according to 10 ml/hour flow when the fermentation time is 72 hours until the fermentation time is 240 hours.
Astaxanthin production was measured and recorded for different incubation times for strain F1, strain F2.
Astaxanthin production determination method:
1) 1mL of the suspension was centrifuged to remove the supernatant, and the pellet was resuspended in 0.7mL of DMSO.
2) After incubation at 55 ℃ for 10min, an equal volume of 0.7mL of acetone was added.
3) Incubate at 45 ℃ for 15min, and centrifuge at 12000rpm for 5 min.
4) The supernatant was transferred to a new EP tube and the astaxanthin production was measured by HPLC at 475nm (standard samples were dissolved in acetone and plotted as standard curves).
The results are shown in FIG. 3: after 264 hours of fermentation, the yield of the astaxanthin in the fermentation liquor of the strain F1 is 358mg/L, the yield of the astaxanthin in the fermentation liquor of the strain F2 is obviously improved, the yield of the astaxanthin in the fermentation liquor of the strain F2 reaches 763mg/L, and the yield of the astaxanthin in the fermentation liquor of the strain F2 is increased by more than one time compared with the yield of the astaxanthin in the fermentation liquor of the strain F1.
The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to the present invention.
Sequence listing
<110> Guangzhou Zhiteqi Biotechnology GmbH
<120> genetic engineering bacterium capable of improving astaxanthin biosynthesis yield and construction method and application thereof
<130> 2021
<160> 35
<170> SIPOSequenceListing 1.0
<210> 1
<211> 60
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
atcgcttcgg ataactcctg ctatacgaag ttatacgaat tcgcgcccag agagccattg 60
<210> 2
<211> 63
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
ataacttcgt atagcataca tcatacgaag ttattctgaa ttccgagaaa cacaacaaca 60
tgc 63
<210> 3
<211> 48
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
attgtactga gagtgcacca gcggccgcat cgcttcggat aactcctg 48
<210> 4
<211> 45
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
aggtcgactc tagaggatcc ccggataact tcgtatagca tacat 45
<210> 5
<211> 42
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
cgaagttatc cggggatcca gaagaagcag gactactttt tc 42
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
gatcgaaggt ggatgaacgt 20
<210> 7
<211> 35
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
ttcatccacc ttcgatcatg aagcgaactc ccacc 35
<210> 8
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
gtaaagagtg ataaatagct caaggctttg cttcaagcag 40
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
gctatttatc actctttaca ac 22
<210> 10
<211> 37
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
ccgccaaccc ggtctctgac ccttcgtggg tctcaat 37
<210> 11
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
agagaccggg ttggcggc 18
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
ctgcggttag tactgcaaaa 20
<210> 13
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
ctttttgcag tactaaccgc agatgcacgt ggcctctgct 40
<210> 14
<211> 38
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
gacataacta attacatgat taggccagag cggggacc 38
<210> 15
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
tcatgtaatt agttatgtc 19
<210> 16
<211> 37
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
aacgggcgcc aaactccgca aattaaagcc ttcgagc 37
<210> 17
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
ggagtttggc gcccgttt 18
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
tgctgtagat atgtcttgtg 20
<210> 19
<211> 37
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
caagacatat ctacagcaat gtccaagaag cacattg 37
<210> 20
<211> 37
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
ttttgctaaa caaactgctt agatgacgtt agagttg 37
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 21
gcagtttgtt tagcaaaata 20
<210> 22
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
gaccatgatt acgccaagct ttgtatgatt gatgttacta c 41
<210> 23
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
acgaagttat ccggggatcc tgcacccaac aataaatggg 40
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
ctgggttagt ttgtgtagag 20
<210> 25
<211> 38
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 25
ctacacaaac taacccagat gctgtctaag ctgcagtc 38
<210> 26
<211> 37
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
caggccatgg aggtacttat cgcttagacc agtccag 37
<210> 27
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
gtacctccat ggcctgtcc 19
<210> 28
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
ggccgattca tttcaaccca tctcacttgc gtatgtatgg 40
<210> 29
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
ggttgaaatg aatcggccg 19
<210> 30
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
tgttgatgtg tgtttaatt 19
<210> 31
<211> 36
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
ttaaacacac atcaacaatg ctgctgacct acatgg 36
<210> 32
<211> 38
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
taaagagtga taaatagctt agatggtgtt caggtttc 38
<210> 33
<211> 40
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
ccatgattac gccaagcttg acccttcgtg ggtctcaatg 40
<210> 34
<211> 618
<212> DNA
<213> yarrowia lipolytica Polf (yarrowia)
<400> 34
agaagaagca ggactacttt ttcgagatca gaggcgagct tgatcgggag ttcaaggagg 60
ttttctacag actacacgag catcttgtct acaagttcca tctcttctgg aagcatggtg 120
tgcaggagaa gaagattcag agcgttctgc agactgacaa ccacctgaag atcttcctgg 180
agaaatacga cgcaaacctg cgacggggag actggaaata aaacatgaga gacacacacg 240
tgcaacacac aaacattagt taattgattc atctgatctg caaaaacatg ttggtgatgg 300
ttaagagtca cataacaagc agggtccatg cactcgcttt cttacatagc accatagaat 360
acggtgttgt cggcattatc aagacagaat ttactttggt gatgttatca catgagccgg 420
ggggcatagg gtgggcactc gtactctatg cgttattggt tgcagaccca taatctcaag 480
ggcgttccag aacatacact atgcgaccgc agggaccctc gatatggacg atgaatagta 540
tggcaacata aaccagaaac tattatatag agatgatggg ggcgacattt cggtaattac 600
gttcatccac cttcgatc 618
<210> 35
<211> 1518
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
atgaagcgaa ctcccaccgc cgaagaacgg gagcgagagg ccaaaaagct gcggctgctc 60
gaagaactcg aggacacgtg gctcccatat ctcaccccca aggacgatga gttctaccag 120
cagtggcagc tcaagtaccc taaactgatt ctgagagaag caagctcagt ttctgaggag 180
ctccacaagg aggttcagga ggcgtttctg acgctccata agcacggatg tctgtttaga 240
gatctggtgc gaatccaggg aaaggatctt ttgaccccgg tatcgcggat tctcatcgga 300
aacccgggct gtacctacaa gtatctcaac acccgtctct tcaccgtccc ctggcccgtc 360
aagggctcca acatcaagca tacagaggca gagattgccg cggcttgtga gactttcttg 420
aagctcaacg actatcttca gatagagacc attcaggcct tagaagagtt ggcagccaag 480
gagaaggcca acgaggatgc cgtgcctctt tgcatgtctg ctgatttccc acgagtcggt 540
atgggttcgt catacaacgg tcaggacgag gtggatatca agtctcgcgc tgcctacaat 600
gtgactctgc tgaactttat ggacccccag aaaatgccct acctgaaaga ggagccctac 660
tttggaatgg gcaagatggc tgtttcatgg caccacgatg aaaacctcgt ggatagatcc 720
gcagttgctg tctactctta ctcgtgcgag ggacctgagg aggaaagtga ggacgactcc 780
catcttgagg ggcgagaccc cgacatctgg cacgtgggct tcaagatttc gtgggacatt 840
gaaacgcctg gtcttgccat ccccctacac cagggcgact gctacttcat gctggacgat 900
ctaaatgcga cacatcaaca ttgtgtgctg gccggaagtc aacctcgatt cagctcgacc 960
cacagagtgg ctgaatgctc cactggaaca ttggactaca tcctgcaacg gtgccaactg 1020
gctctgcaga atgtctgtga tgacgttgac aacgatgatg tgtcccttaa gagctttgag 1080
cctgctgtcc taaagcaggg cgaggaaatc cacaacgaag tcgagtttga gtggctgaga 1140
cagttctggt tccaaggtaa ccgataccga cgatgcaccg actggtggtg ccagcccatg 1200
gcccagctgg aggcactgtg gaagaagatg gagggtgtga ccaatgccgt tctccacgag 1260
gtcaaacgcg agggcttacc ggtggagcaa cgaaacgaga tcctgactgc cattctggcg 1320
tctcttacgg cccgtcagaa cctacgtcga gaatggcatg ctcgctgtca gtctcgaatc 1380
gctcggactt tgccagctga ccagaagccc gagtgtcgac cttactggga gaaggacgac 1440
gcctccatgc ctctgccctt tgacttgaca gacattgtct ccgagctgcg aggccagctg 1500
cttgaagcaa agccttga 1518
Claims (10)
1. A genetic engineering bacterium capable of improving astaxanthin biosynthesis yield is characterized in that:
the genetic engineering bacteria are strains F2 obtained by introducing AtoB, tHMGR, IDI, GGS1, pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1, pEXP-CarB-tICL and pHXT2-FTO-tLIP into yarrowia lipolytica Polf;
wherein the sequence of pHXT2 is shown in SEQ ID NO: 34; the sequence of FTO is shown in SEQ ID NO. 35.
2. The method for constructing a genetically engineered bacterium according to claim 1, which is capable of increasing the biosynthesis yield of astaxanthin, comprising:
the method comprises the following steps:
(1) constructing a plasmid pMA containing pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1 and pEXP-CarB-tICL and a plasmid pMB containing pHXT 2-FTO-tLIP;
(2) introducing mevalonate pathway and farnesyl pyrophosphate pathway-related genes AtoB, tHMGR, IDI and GGS1 into yarrowia lipolytica Polf to obtain strain F0;
(3) transforming the strain F0 by the plasmid pMA after enzyme digestion to obtain a genetically engineered bacterium F1;
(4) and transforming the plasmid pMB subjected to enzyme digestion into the genetically engineered bacterium F1 to obtain a genetically engineered bacterium F2.
3. The method for constructing a genetically engineered bacterium capable of increasing the biosynthesis yield of astaxanthin according to claim 2, wherein the genetically engineered bacterium comprises:
the process of constructing plasmid pMA containing pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1 and pEXP-CarB-tICL includes:
extracting genome DNA of Saccharomyces cerevisiae CEN.PK2-1C and yarrowia lipolytica Polf;
PCR amplifying URA3 gene by using primer loxP-URA3-F/loxP-URA3-R and yarrowia lipolytica Polf genome DNA as template, and amplifying URA3 gene segment by using primer URA3-F and URA3-R and using the PCR product as template; plasmid pUC19 was digested simultaneously with Nde I and Kpn I, and the URA3 gene fragment was cloned into pUC19 between Nde I and Kpn I by seamless cloning to construct plasmid pUC19URA 3;
amplifying pFBA, tXPR2, pGPD, tLIP, pTEF, pEXP and tICL fragments by using primers pFBA-F/pFBA-R, tXPR2-F/tXPR2-R, pGPD-F/pGPD-R, tLIP-F/tLIP-R, pTEF-F/pTEF-R, pEXP-F/pEXP-R and tICL-F/tICL-R respectively and taking yarrowia lipolytica Polf genomic DNA as a template; amplifying a CYC1 fragment by using a primer CYC1-F/CYC1-R and yeast CEN. PK2-1C genomic DNA as a template;
after codon optimization of CarRP and CarB derived from Mucor circinelloides (Rhizomucor circinelloides) and CrtZ and CrtW genes derived from Haematococcus pluvialis (Haematococcus pluvialis), the codon-optimized CarRP, CarB, CrtZ and CrtW fragments were amplified using the primers CarRP-F/CarRP-R, CarB-F/CarB-R, CrtZ-F/CrtZ-R and CrtW-F/CrtW-R, respectively, as templates;
splicing four expression frames of pFBA-CrtZ-tXPR2, pGPD-CarRP-tLIP, pTEF-CrtW-CYC1 and pEXP-CarB-tICL, carrying out double digestion, purification and recovery on a plasmid pUC19URA3 by using BamH I and Hind III, and then assembling a digested plasmid skeleton and the four expression frames to construct a plasmid pMA.
4. The method for constructing a genetically engineered bacterium capable of increasing the biosynthesis yield of astaxanthin according to claim 2, wherein the genetically engineered bacterium comprises:
the construction process of the plasmid pMB containing pHXT2-FTO-tLIP is as follows:
extracting yarrowia lipolytica Polf genomic DNA;
PCR amplifying URA3 gene by using primer loxP-URA3-F/loxP-URA3-R and yarrowia lipolytica Polf genome DNA as template, and amplifying URA3 gene segment by using primer URA3-F and URA3-R and using the PCR product as template; plasmid pUC19 was digested simultaneously with Nde I and Kpn I, and the URA3 gene fragment was cloned into pUC19 between Nde I and Kpn I by seamless cloning to construct plasmid pUC19URA 3;
pHXT2 and tLIP fragments were amplified using yarrowia lipolytica Polf genomic DNA as template with primers pHXT2-F/pHXT2-R and tLIP-F/tLIP-R1, respectively;
FTO gene from human (Homo sapiens) is optimized by codon and used as a template, and a primer FTO-F/FTO-R is used for amplifying an optimized FTO gene fragment;
splicing pHXT2-FTO-tLIP expression frame, purifying and recovering plasmid pUC19URA3 by double enzyme digestion with BamH I and Hind III, and assembling the digested plasmid skeleton and pHXT2-FTO-tLIP expression frame to construct plasmid pMB.
5. The method for constructing a genetically engineered bacterium capable of increasing the biosynthesis yield of astaxanthin according to claim 4, wherein the genetically engineered bacterium comprises:
the sequence of pHXT2 obtained by amplifying the pHXT2 fragment is shown as SEQ ID NO: 34.
6. The method for constructing a genetically engineered bacterium capable of increasing the biosynthesis yield of astaxanthin according to claim 4, wherein the genetically engineered bacterium comprises:
the FTO sequence obtained by amplifying the optimized FTO gene fragment is shown as SEQ ID NO. 35.
7. The method for constructing a genetically engineered bacterium capable of increasing the biosynthesis yield of astaxanthin according to claim 2, wherein the genetically engineered bacterium comprises:
the operation process of the step (3) is as follows:
digesting plasmid pMA for 1 hour by NotI, recovering a fragment containing no escherichia coli replicon and ampicillin resistance genes by agarose gel electrophoresis, then transforming a linearized pMA into a strain F0 by a lithium acetate method, coating an SD-URA defect culture medium, selecting 120 transformants with the deepest orange red color after 2-3 days, respectively inoculating into 10ml of liquid YPD40 culture medium, culturing for 72 hours at 200rpm and 30 ℃, detecting the yield of astaxanthin, and obtaining a genetically engineered bacterium F1 with the highest yield;
and (4) digesting the plasmid pMB for 1 hour by using Not I, recovering a fragment which does Not contain an escherichia coli replicon and an ampicillin resistance gene by agarose gel electrophoresis, then transforming a linearized pMB into a strain F1 by using a lithium acetate method, coating an SD-URA defect culture medium, selecting 120 transformants with the deepest orange red color after 2-3 days, respectively inoculating into 10ml of liquid YPD40 culture medium, culturing at 200rpm and 30 ℃ for 72 hours, detecting the yield of astaxanthin, and obtaining the genetically engineered bacterium F2 with the highest yield, and storing in a glycerol tube at-80 ℃.
8. The method for constructing a genetically engineered bacterium capable of increasing the biosynthesis yield of astaxanthin according to claim 7, wherein the genetically engineered bacterium comprises:
the YPD40 medium components comprise 1% of yeast extract, 2% of peptone and 4% of glucose in percentage by mass.
9. The use of the genetically engineered bacterium of claim 1 for increasing the biosynthetic yield of astaxanthin in astaxanthin production, wherein:
the method comprises the following steps:
1) culturing a primary seed solution: taking a strain F2 stored in a glycerol tube at the temperature of-80 ℃, drawing a line on a solid YPD plate, culturing for 24 hours at the temperature of 30 ℃, inoculating a single colony of the strain F2 on the solid YPD plate into a 50ml conical flask containing 10ml of YPD liquid culture medium, and culturing for 24 hours at the temperature of 30 ℃ and the rotating speed of a shaking table of 220 rpm;
2) culturing a secondary seed solution: transferring the primary seed solution into a 300ml conical flask containing 100ml YPD liquid culture medium according to the inoculation amount of 10 wt%, culturing for 8 hours at the temperature of 30 ℃ at the rotating speed of a shaking table of 220rpm until the OD600 of the cells reaches 10 to obtain a secondary seed solution;
3) the secondary seed solution was transferred to a 5L fermentor containing 2L fermentation medium for fermentation at 10% wt inoculum size.
10. The use of the genetically engineered bacterium according to claim 9 for increasing the biosynthetic yield of astaxanthin in astaxanthin production, wherein:
the detailed process of the step 3) is as follows: transferring the secondary seed liquid into a 5L fermentation tank containing 2L of fermentation medium according to the inoculation amount of 10% by weight for fermentation, culturing under the conditions that the initial medium consists of 30g/L of yeast extract, 60g/L of peptone and 100g/L of glucose, the fermentation temperature is 30 ℃, the stirring speed is 300-800rpm, the ventilation quantity is 4L/min, the pH value is 6.8 and the dissolved oxygen is more than 25%, supplementing 200ml of 600g/L glucose once when the fermentation time is 48 hours, and feeding 600g/L glucose according to 10 ml/hour flow when the fermentation time is 72 hours until the fermentation time is 240 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111681773.7A CN114317307B (en) | 2021-12-30 | 2021-12-30 | Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield as well as construction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111681773.7A CN114317307B (en) | 2021-12-30 | 2021-12-30 | Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield as well as construction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114317307A true CN114317307A (en) | 2022-04-12 |
| CN114317307B CN114317307B (en) | 2024-05-10 |
Family
ID=81023461
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111681773.7A Active CN114317307B (en) | 2021-12-30 | 2021-12-30 | Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield as well as construction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114317307B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116426398A (en) * | 2023-04-06 | 2023-07-14 | 福建平潭宝世贵生物科技合伙企业(有限合伙) | Yarrowia lipolytica engineering bacterium and application thereof |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005062923A2 (en) * | 2003-12-24 | 2005-07-14 | Massachusetts Institute Of Technology | Gene targets for enhanced carotenoid production |
| KR20090046376A (en) * | 2007-11-06 | 2009-05-11 | 부경대학교 산학협력단 | Yeast transformed with carotenoid biosynthesis genes and method of high astaxanthin production using the same |
| CN103865818A (en) * | 2012-12-07 | 2014-06-18 | 上海来益生物药物研究开发中心有限责任公司 | Construction method of genetically engineered bacterium for producing astaxanthin |
| US20150315550A1 (en) * | 2012-12-20 | 2015-11-05 | Dsm Ip Assets B.V. | Carotene hydroxylases and their use for producing carotenoids |
| CN106318878A (en) * | 2015-07-06 | 2017-01-11 | 中国科学院青岛生物能源与过程研究所 | Red yeast engineering bacterium for high-yield production of astaxanthin and construction method thereof |
| WO2019210268A2 (en) * | 2018-04-27 | 2019-10-31 | The Broad Institute, Inc. | Sequencing-based proteomics |
| CN111321087A (en) * | 2020-02-21 | 2020-06-23 | 华东理工大学 | Yarrowia lipolytica gene engineering bacterium for producing β -carotene and application thereof |
| EP3702468A1 (en) * | 2019-03-01 | 2020-09-02 | Jennewein Biotechnologie GmbH | Fermentative production of carbohydrates by microbial cells utilizing a mixed feedstock |
| CN113151340A (en) * | 2020-11-25 | 2021-07-23 | 广州智特奇生物科技股份有限公司 | Genetic engineering bacterium for increasing yield of beta-carotene and application thereof |
-
2021
- 2021-12-30 CN CN202111681773.7A patent/CN114317307B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005062923A2 (en) * | 2003-12-24 | 2005-07-14 | Massachusetts Institute Of Technology | Gene targets for enhanced carotenoid production |
| KR20090046376A (en) * | 2007-11-06 | 2009-05-11 | 부경대학교 산학협력단 | Yeast transformed with carotenoid biosynthesis genes and method of high astaxanthin production using the same |
| CN103865818A (en) * | 2012-12-07 | 2014-06-18 | 上海来益生物药物研究开发中心有限责任公司 | Construction method of genetically engineered bacterium for producing astaxanthin |
| US20150315550A1 (en) * | 2012-12-20 | 2015-11-05 | Dsm Ip Assets B.V. | Carotene hydroxylases and their use for producing carotenoids |
| CN106318878A (en) * | 2015-07-06 | 2017-01-11 | 中国科学院青岛生物能源与过程研究所 | Red yeast engineering bacterium for high-yield production of astaxanthin and construction method thereof |
| WO2019210268A2 (en) * | 2018-04-27 | 2019-10-31 | The Broad Institute, Inc. | Sequencing-based proteomics |
| EP3702468A1 (en) * | 2019-03-01 | 2020-09-02 | Jennewein Biotechnologie GmbH | Fermentative production of carbohydrates by microbial cells utilizing a mixed feedstock |
| CN111321087A (en) * | 2020-02-21 | 2020-06-23 | 华东理工大学 | Yarrowia lipolytica gene engineering bacterium for producing β -carotene and application thereof |
| CN113151340A (en) * | 2020-11-25 | 2021-07-23 | 广州智特奇生物科技股份有限公司 | Genetic engineering bacterium for increasing yield of beta-carotene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| LARISSA RIBEIRO RAMOS TRAMONTIN 等: "Enhancement of Astaxanthin Biosynthesis in Oleaginous Yeast Yarrowia lipolytica via Microalgal Pathway", MICROORGANISMS ., vol. 7, no. 10 * |
| 谢贵各;朱丽;蒋宇;戈梅;倪孟祥;: "产虾青素酿酒酵母工程菌的构建", 中国医药生物技术, no. 06 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116426398A (en) * | 2023-04-06 | 2023-07-14 | 福建平潭宝世贵生物科技合伙企业(有限合伙) | Yarrowia lipolytica engineering bacterium and application thereof |
| CN116426398B (en) * | 2023-04-06 | 2023-11-28 | 福建平潭宝世贵生物科技合伙企业(有限合伙) | Yarrowia lipolytica engineering bacterium and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114317307B (en) | 2024-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106701606B (en) | Genetic engineering candida utilis capable of degrading and utilizing kitchen waste and construction method thereof | |
| CN102102086B (en) | L-lactate dehydrogenase gene-deleted engineering bacterium and construction method and application thereof | |
| CN112831427B (en) | Yarrowia lipolytica for high yield of beta-carotene and application thereof | |
| CN109207373B (en) | Microbial strain for high yield of citric acid and method for producing citric acid by fermenting starch sugar through microbial strain | |
| CN110982721B (en) | Method for improving yield of saccharomyces cerevisiae metabolites | |
| CN114317307B (en) | Genetically engineered bacterium capable of improving astaxanthin biosynthesis yield as well as construction method and application thereof | |
| CN112899210B (en) | Method for improving validamycin fermentation level by enhancing positive regulatory protein gene expression | |
| CN108641992B (en) | Engineering bacterium for producing riboflavin at high temperature and construction method and application thereof | |
| CN112608936B (en) | Promoter for regulating and controlling expression of exogenous gene of yeast, regulating and controlling method and application thereof | |
| CN116751731B (en) | Fermentation process for large-scale plasmid production | |
| CN101613707B (en) | Method for producing glutathione by use of metabolic engineering bacteria | |
| CN111500479B (en) | Construction and application of non-methanol-induced dual-promoter pichia pastoris engineering bacteria | |
| CN111484942A (en) | Method for producing adipic acid by using saccharomyces cerevisiae | |
| CN1191369C (en) | Process for producing adenosylmethionine by metabolic engineering bacteria | |
| CN115806922A (en) | Genetically engineered strain of zymomonas mobilis and application thereof | |
| CN112961815B (en) | Genetic engineering bacterium for high yield of tetrahydropyrimidine and application thereof | |
| CN105062981A (en) | Pyruvate carboxylase mutant N315F with improved enzymatic activity and application of pyruvate carboxylase mutant N315F | |
| CN118256529A (en) | Recombinant bacterium for producing canthaxanthin and construction method and application thereof | |
| CN114606146B (en) | Yeast for producing D-limonene and application thereof | |
| CN104403956A (en) | Construction and application of xylitol high-temperature and high-yield engineered strains | |
| CN115058350B (en) | Method for improving S-adenosylmethionine yield by introducing potassium ion transporter | |
| CN112501043B (en) | Saccharomyces cerevisiae engineering bacterium for producing geranylgeraniol and construction method and application thereof | |
| CN110551769B (en) | Production method of phloroglucinol | |
| CN118086079A (en) | Yeast capable of tolerating low pH value and application thereof | |
| CN115975835A (en) | Pichia pastoris engineering bacteria for high yield of glucose oxidase and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |