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CN-122012264-A - Yarrowia lipolytica BS-3 and application thereof in resveratrol production

CN122012264ACN 122012264 ACN122012264 ACN 122012264ACN-122012264-A

Abstract

The invention relates to the technical field of microorganisms and fermentation, in particular to yarrowia lipolytica BS-3 and application thereof in resveratrol production. According to the invention, the yarrowia lipolytica Po1f is selected as a chassis strain, the metabolic pathway is modified, the synthesis pathway of coumaric acid is enhanced, the feedback inhibition of tyrosine is eliminated, the supply of malonyl CoA is enhanced, the exogenous protein related to the synthesis pathway of resveratrol is introduced, a basic strain for producing resveratrol is constructed, and ARTP mutagenesis is carried out on the strain to obtain a high-yield strain of resveratrol, namely the yarrowia lipolytica BS-3 of resveratrol. Then, the invention optimizes the fermentation process (optimizing the concentration of ammonium ions and the concentration of dissolved phosphorus in fermentation liquor) of the strain, and controls the oxygen supply control strategy through the specific oxygen consumption rate, so that the resveratrol yield of the strain can reach 36.9 g/L, and the conversion rate reaches 5.9%.

Inventors

  • SHI YANGHUI
  • ZHANG TIANYU
  • LI YONG
  • Lv Rongjian
  • GUI QIAN
  • WANG YONG
  • WANG LI

Assignees

  • 杭州精构生物科技有限责任公司

Dates

Publication Date
20260512
Application Date
20260305

Claims (10)

  1. 1. Yarrowia lipolytica (Yarrowia lipolytica) BS-3 capable of producing resveratrol with high yield, and is characterized in that the preservation number of the yarrowia lipolytica BS-3 is CGMCC No.39118.
  2. 2. Use of yarrowia lipolytica BS-3 of claim 1 for the preparation of a microbial preparation for the production of resveratrol.
  3. 3. A microbial preparation for producing resveratrol, wherein the microbial preparation comprises yarrowia lipolytica BS-3 of claim 1.
  4. 4. A microbial preparation according to claim 3, wherein the microbial preparation further comprises an adjuvant.
  5. 5. Use of yarrowia lipolytica BS-3 of claim 1 or a microbial preparation of claim 3 or 4 for the production of resveratrol.
  6. 6. A method for increasing resveratrol yield, comprising the step of fermenting and culturing yarrowia lipolytica BS-3 of claim 1.
  7. 7. The method according to claim 6, wherein ammonia water, a glucose solution, a potassium dihydrogen phosphate solution and an ammonium sulfate solution are fed during the fermentation culture.
  8. 8. The method according to claim 6, wherein the aeration rate of the fermentation culture is 6L/min, the stirring speed is 700-900 rpm, the pH is 5+ -0.1, the temperature is 30 ℃, the dissolved oxygen is 20%, the tank pressure is kept at 0.04-0.05 MPa, and the specific oxygen consumption rate is controlled between 1.5-3 mmol/g/h.
  9. 9. The method according to claim 7, wherein when the glucose in the culture medium used for fermentation culture is lower than 2g/L, glucose solution is fed, the initial feeding rate is 4g/L/h, and the concentration of residual sugar is controlled to be 0.5-2 g/L; In the fermentation culture process, the concentration of dissolved phosphorus in the fermentation liquor is kept between 200 and 400 mg/L; During the fermentation culture, the concentration of ammonium ions in the fermentation broth is kept between 3 and 6 g/L.
  10. 10. The method according to claim 7, wherein the glucose solution contains 60% glucose by mass, the potassium dihydrogen phosphate solution contains 140 g/L of potassium dihydrogen phosphate, and the ammonium sulfate solution contains 200 g/L of ammonium sulfate.

Description

Yarrowia lipolytica BS-3 and application thereof in resveratrol production Technical Field The invention relates to the technical field of microorganisms and fermentation, in particular to yarrowia lipolytica BS-3 and application thereof in resveratrol production. Background Resveratrol is a natural polyphenol compound, the chemical name of which is (E) -3,5, 4-trihydroxy stilbene, the resveratrol is white needle-like crystal in appearance, and is insoluble in water but soluble in organic solvents. It was originally isolated from the plant resveratrol and was later found in a variety of plants such as grape (especially grape skin), giant knotweed, peanut, mulberry, etc. Resveratrol is synthesized as an antitoxin when plants are stressed by fungal infection or ultraviolet radiation and the like. Resveratrol exists mainly in both trans and cis forms, wherein the physiological activity of trans isomer is stronger and more stable. The most interesting biological properties include antioxidant, anti-inflammatory, cardiovascular system protecting and potentially antitumor activities. Yarrowia lipolytica (Yarrowia lipolytica) is a microbial strain which is certified by the U.S. Food and Drug Administration (FDA) to be "recognized as safe" (GRAS), and has important application value in the field of industrial fermentation. The yeast is used to synthesize a variety of high value-added products including human milk oligosaccharides (e.g., 3-fucosyllactose), carotenoids, microbial oils and fats, nervonic acids, and other functional lipids. Meanwhile, the novel recombinant expression vector is a high-efficiency protein expression system, and can secrete a large amount of industrial enzymes such as lipase, protease and the like. The strain has a wide substrate utilization spectrum, can naturally utilize various carbon sources such as alkanes, glycerol, organic acids and the like, has excellent environmental robustness, and is resistant to extreme conditions such as high permeability, high salt, organic acids, various inhibitors and the like. In addition, the fermentation process is simple and convenient to regulate and control, easy to amplify and very suitable for large-scale industrial production In recent years, synthesis of resveratrol, a high value added product, by using metabolic engineering yarrowia lipolytica has become a research hotspot and has made remarkable progress. For example, the Danish university of technology was engineered to synthesize 12.4 g/L resveratrol with glucose as substrate in 90 hours, the Jiangnan university was engineered to reconstruct metabolic pathways in 2022 to increase the yield to 22.5 g/L in 140 hours, and the Hunan Hongjian Biotechnology Co., ltd., published patent in 2023 (CN 117625698A) was optimized by 50L scale process to reach 28.2 g/L in 162 hours. The prior researches mainly focus on genetic engineering of strains and optimization of basic fermentation parameters (such as carbon sources and feeding strategies). For the specific production host of yarrowia lipolytica, systematic process research of real-time monitoring and fine dynamic regulation and control of key parameters (such as ammonia nitrogen concentration, phosphate availability, dissolved oxygen and oxygen uptake rate and the like) in the fermentation process still belongs to the blank at present. The lack of this technology limits the further exploitation of the metabolic potential of cells and the stable and efficient operation of the production process. In view of the above, the present invention aims to fill the technical gap and provide a process control technology special for fermenting and producing resveratrol by yarrowia lipolytica. The process is characterized in that key parameters such as ammonia nitrogen, soluble phosphate (dissolved phosphorus), oxygen and the like in fermentation liquor are monitored on line and subjected to feedback control, and nutrition supply and culture environments are dynamically regulated and controlled, so that accurate guidance of cell metabolic flows is realized, and finally, the purposes of stable production and yield and conversion efficiency are achieved. Disclosure of Invention The invention aims to provide yarrowia lipolytica BS-3 and application thereof in resveratrol production, so as to solve the problems in the prior art. According to the invention, yarrowia lipolytica (Yarrowia lipolytica) Po1f is selected as a chassis strain, metabolic engineering is used as a basis, metabolic pathways of the strain are modified by a conventional genetic engineering technology means, the synthesis pathway of coumaric acid is enhanced, feedback inhibition of tyrosine is eliminated, malonyl CoA supply is enhanced, exogenous proteins related to the synthesis pathway of resveratrol are introduced, a basic strain for resveratrol production is constructed, and ARTP mutagenesis is carried out on the strain to obtain a resveratrol high-yield strain. The fermentation process (optimiza