CN-121975834-A - Yarrowia lipolytica engineering strain capable of producing beta-carotene at high yield and construction thereof

CN121975834ACN 121975834 ACN121975834 ACN 121975834ACN-121975834-A

Abstract

The invention provides a yarrowia lipolytica engineering strain for high-yield beta-carotene and construction thereof, belonging to the technical field of synthetic biology and metabolic engineering, the invention obtains the yarrowia lipolytica engineering strain which takes glucose as a substrate to efficiently biosynthesize beta-carotene by integrating multiple copies of geranylgeranyl pyrophosphate synthase genes, compartmentalization positioning expression beta-carotene synthesis pathway related enzyme genes, diacylglycerol acyltransferase genes, cell cycle regulation transcription factors, regulation oxidative stress related genes, regulation cell wall and membrane lipid steady state related genes and regulation exogenous protein folding and expression related genes. The engineering strain of yarrowia lipolytica constructed by the invention has high beta-carotene yield, the yield at the shake flask level can reach 3253.2mg/L, and the yield can be improved to 26.5g/L during 5L-scale fed-batch fermentation. The invention has great practical significance and economic value for the industrialization and commercialization of high-value terpenoid products.

Inventors

WU YUANQING
XU YONG
HUANG KE
YANG ZHIBIN
HU JIANGLIN
ZHU MIN

Assignees

河北维达康生物科技股份有限公司

Dates

Publication Date: 20260505
Application Date: 20260206

Claims (10)

1. A construction method of a yarrowia lipolytica engineering strain with high beta-carotene production is characterized in that yarrowia lipolytica is taken as an original strain, and a multicopy geranylgeranyl pyrophosphate synthase gene GPS, a bifunctional phytoene synthase/lycopene cyclase gene CarRP, a phytoene dehydrogenase gene CarB, an isopentenyl pyrophosphate isomerase gene IDI, a diacylglycerol acyltransferase gene DGA1 and a cell cycle regulatory transcription factor YALI1_A20855g are integrated into a genome of the original strain, so that the yarrowia lipolytica engineering strain with high beta-carotene production is obtained.
2. The construction method according to claim 1, wherein the nucleotide sequence of the GPS gene is shown in SEQ ID No.1, the nucleotide sequence of the CarRP gene is shown in SEQ ID No.2, the nucleotide sequence of the CarB gene is shown in SEQ ID No.3, and the IDI, DGA1, yali1_a20855g genes are all endogenous genes of yarrowia lipolytica.
3. The method of claim 1, wherein the CarRP, carB, GPS and IDI genes are located in the lipid droplets using the signal peptide OLE, the nucleotide sequence of which is shown in SEQ ID No. 84.
4. The construction method according to claim 1, wherein at least one of copper-zinc superoxide dismutase gene SOD1, copper-zinc superoxide dismutase gene SOD2, gene related to regulating cell wall and membrane lipid homeostasis yali1_f25035g and gene related to regulating foreign protein folding yali1_a00102g is integrated in the genome of the yarrowia lipolytica engineering strain, wherein yali1_f25035g and yali1_a00102g genes are yarrowia lipolytica endogenous genes.
5. The method of claim 1, wherein the SOD1 and SOD2 genes are endogenous yarrowia lipolytica genes.
6. The method of claim 1, wherein the integration site for integration of each gene into the genome of yarrowia lipolytica is selected from the group consisting of E12 site, F15 site, B3 site, A2 site, C3 site, C4 site, and A1 site.
7. The method of claim 1, wherein the starting strain is yarrowia lipolytica engineering strain YLCar.
8. An engineered strain of yarrowia lipolytica produced according to the construction method of any one of claims 1-7.
9. The use of an engineered strain of yarrowia lipolytica of claim 8 for the synthesis of beta-carotene.
10. A method for synthesizing beta-carotene, characterized in that the engineering strain of yarrowia lipolytica as claimed in claim 8 is fermented in a culture solution containing glucose to obtain a fermentation solution rich in beta-carotene, and the fermentation solution is purified.

Description

Yarrowia lipolytica engineering strain capable of producing beta-carotene at high yield and construction thereof Technical Field The invention belongs to the technical field of synthetic biology and metabolic engineering, and particularly relates to a yarrowia lipolytica engineering strain with high beta-carotene yield, a construction method thereof and application thereof in preparing beta-carotene. Background Beta-carotene is one of the carotenoids, an orange-colored fat-soluble compound, which is also the most commonly occurring natural pigment in nature. Beta-carotene, which can be used as a precursor of vitamin A, is a type A total nutrient food enhancer identified by the food additive joint expert committee of the world health organization and the joint national food and agriculture organization, and has been approved for use by 52 countries and regions in the world. Beta-carotene has important physiological functions of resisting oxidation, protecting eyesight, resisting aging, resisting tumor, resisting osteoporosis, resisting radiation, preventing arteriosclerosis, improving immunity and the like, and has wide application prospect in the fields of foods, medicines, health care products and the like. At present, the production method of carotenoid such as beta-carotene mainly comprises a chemical synthesis method, a natural extraction method and a microorganism heterologous synthesis method, wherein the chemical synthesis method has the defects of complex process, high energy consumption, high pollution and the like, and in addition, the content of the beta-carotene in plants is low, so that large-scale plant extraction cannot be performed. Compared with other methods, the method has the advantages that the microorganism chassis cells are genetically engineered by a synthetic biological method, and a heterologous synthetic way is introduced, so that the biosynthesis of beta-carotene is realized, and the safety and the economy are higher. Yarrowia lipolytica has a broad substrate availability and is also a recognized safety strain. The strain contains rich acetyl-CoA, provides precursors for the synthesis of carotene compounds, and simultaneously has a large amount of accumulated lipid in cells to provide storage space for the carotene compounds, so yarrowia lipolytica can be selected as a dominant chassis strain for realizing the biosynthesis of beta-carotene. However, it should be noted that the present production of beta-carotene using yarrowia lipolytica has the technical problems of low substrate conversion rate, low beta-carotene yield, etc. Disclosure of Invention In view of the technical problems in the background art, the invention aims to provide a yarrowia lipolytica engineering strain with high yield of beta-carotene and a construction method thereof, so as to solve the problems of low substrate conversion rate, low beta-carotene yield and the like in the existing biosynthesis of beta-carotene by yarrowia lipolytica. In order to achieve the above purpose, the present invention adopts the following technical scheme: In a first aspect, the invention provides a construction method of a yarrowia lipolytica engineering strain with high beta-carotene yield, which comprises the steps of taking yarrowia lipolytica as a starting strain, integrating a multicopy geranylgeranyl pyrophosphate synthase gene GPS, a bifunctional phytoene synthase/lycopene cyclase gene CarRP, a phytoene dehydrogenase gene CarB, an isopentenyl pyrophosphate isomerase gene IDI, a diacylglycerol acyltransferase gene DGA1 and a cell cycle regulatory transcription factor YALI1_A20855g into a genome of the starting strain, so as to obtain the yarrowia lipolytica engineering strain with high beta-carotene yield. In the construction methods of the invention, the corresponding genes can be integrated into the yarrowia lipolytica genome using prior art techniques (e.g., CRISPR/Cas9 localization integration methods). Preferably, in the above construction method, the GPS gene is double-copied. Preferably, in the above construction method, the GPS gene is derived from Archaeoglobus fulgidus (thermophilic anaerobic archaebacteria), the nucleotide sequence of which is optimized and then integrated into the genome of yarrowia lipolytica, and in some embodiments of the invention, the optimized GPS gene sequence is shown in SEQ ID NO. 1. Preferably, in the above construction method, carRP gene and CarB gene are both derived from Mucor circinelloides (Mucor circinelloides), and the nucleotide sequences of both should be optimized and integrated into the genome of yarrowia lipolytica, and in some embodiments of the invention, the optimized CarRP gene sequence is shown in SEQ ID NO.2, and the optimized CarB gene sequence is shown in SEQ ID NO. 3. Preferably, in the above construction method, the IDI, DGA1, YALI1_A20855g genes are all yarrowia lipolytica endogenous genes. In some embodiments of the invention, the IDI gene sequence is as shown