CN-119913054-B - Microorganism with increased production level of p-coumaric acid or p-coumaric acid derivative, construction method and application thereof

Abstract

The invention discloses a microorganism with improved production level of p-coumaric acid or p-coumaric acid derivatives, a construction method and application thereof, which comprises the steps of modifying a genetic target related to a p-coumaric acid metabolic pathway of the microorganism, wherein the target is one or a combination of two or more of 4-coumaroyl-CoA ligase, hydroxycinnamoyl-CoA hydratase lyase, hydroxycinnamoyl-CoA dehydrogenase, beta-ketoacyl-CoA thiolase and 3- (aryl) acrylate reductase, and the modification is to remove part or all of the activity of the genetic target related to the p-coumaric acid metabolic pathway. The invention reduces the degree of microorganism degradation of the p-coumaric acid by modifying the related genetic targets of the p-coumaric acid metabolic pathway, thereby improving the yield and/or the production efficiency of the p-coumaric acid and the p-coumaric acid derivatives produced by microorganisms.

Inventors

• WANG GUOKUN
• ZHOU HONGYI
• SHI ZHIHUI
• HE HAILIN
• ZHANG ZHAO

Assignees

• 中国科学院天津工业生物技术研究所

Dates

Publication Date

20260512

Application Date

20250115

Claims (3)

1. 1. A microorganism having an increased level of p-coumaric acid or p-coumaric acid derivative production, characterized in that: the genetic target related to the coumaric acid metabolic pathway of the microorganism is modified, the microorganism is yarrowia lipolytica (Yarrowia lipolytica), the modification method is to knock out the coding genes of the amino acid sequences shown in SEQ ID NO. 2 and SEQ ID NO. 5, the amino acid sequence shown in SEQ ID NO. 2 is hydroxycinnamoyl-CoA hydratase lyase, and the amino acid sequence shown in SEQ ID NO. 5 is beta-ketoacyl-CoA thiolase.
2. 2. A method of microbial construction with increased production of coumaric acid or coumaric acid derivatives, characterized in that the construction is performed according to the content of claim 1.
3. 3. Use of a microorganism according to claim 1 or a construction method according to claim 2 for increasing the yield of p-coumaric acid and resveratrol, comprising the steps of: Culturing the modified yarrowia lipolytica strain in a culture medium for 2-8 days, and harvesting p-coumaric acid and/or resveratrol from the culture; the culture medium comprises a carbon source and a nitrogen source, and can be specifically one or more of the following combinations: (i) The carbon source is selected from fermentable carbon sources including glucose, fructose, galactose, xylose and sucrose, or non-fermentable carbon sources including ethanol, acetic acid and acetate, glycerol, lactate, methanol and formic acid; (ii) The nitrogen source is selected from organic nitrogen sources or inorganic nitrogen sources, wherein the organic nitrogen sources comprise peptone, yeast powder and corn steep liquor, and the inorganic nitrogen sources comprise ammonium sulfate, ammonium chloride, nitrate and ammonia water; (iii) Biomass containing carbon and nitrogen sources, including agricultural, industrial and forestry residues and waste materials.

Description

Microorganism with increased production level of p-coumaric acid or p-coumaric acid derivative, construction method and application thereof Technical Field The invention belongs to the technical field of biology, and relates to a microorganism with an improved production level of p-coumaric acid or p-coumaric acid derivatives, and a construction method and application thereof. Background P-coumaric acid (p-Coumaric acid, p-CA) is a compound having various pharmacological actions such as antibacterial, anti-inflammatory and antioxidant effects, and is an important precursor metabolite of various compounds including phenolic acids (representative substances such as caffeic acid and ferulic acid), flavonoids (representative substances such as naringenin and catechin), stilbenes (representative substances such as resveratrol and pinosylvin) coumarin (representative substances such as umbelliferone and scopoletin). At present, the production of p-coumaric acid and p-coumaric acid derivatives mainly depends on plant extraction, but the economic crops used as extraction raw materials have limited planting area, longer culture period and are easily influenced by climate and season changes, and the sufficient and stable supply of the p-coumaric acid and the derivatives thereof cannot be ensured. Compared with the production mode of plant extraction, the method for producing the coumaric acid and the derivatives thereof by constructing a microbial cell factory is more environment-friendly and has more stable yield. The synthesis biology realizes the construction of an artificial biological system through bioengineering design, promotes the revolutionary development of the biosynthesis of natural products, provides a new strategy for stably producing the natural products, and is a research hotspot in recent years. The production of coumaric acid and its derivatives is realized by a microbial cell factory, and the key is the construction of high-yield strains. Exogenous synthesis approaches are introduced into the strain through genetic transformation tools, so that p-coumaric acid and derivatives thereof can be synthesized by microorganisms in a heterologous manner, and iterative design and optimization of a metabolic network of the strain are still required to realize high yield. Researchers have obtained a series of microbial cell factories for efficient heterologous synthesis of p-coumaric acid and its derivatives in the last 20 years by optimizing strategies such as source and expression of exogenous pathway genes, and improving precursor metabolite supply. The biodegradation of p-coumaric acid is an important factor restricting efficient production of p-coumaric acid and its derivatives by host microorganisms, and for example, yarrowia lipolytica, an important host for the production of polyphenols, was reported to have a high capacity to degrade p-coumaric acid and its derivatives, thereby limiting the yield improvement of p-coumaric acid and its derivatives, in the research of S-ez-S-ez et al （Sáez-Sáez, J., Wang, G., Marella, E. R., Sudarsan, S., Cernuda Pastor, M.,&Borodina, I. (2020). Engineering the oleaginous yeastYarrowialipolyticafor high-level resveratrol production. Metabolic engineering, 62, 51–61. ） and Zhu et al （Zhu, J., Yang, S., Cao, Q., Li, X., Jiao, L., Shi, Y.,Yan, Y., Xu, L., Yang, M., Xie, X., Madzak, C.,&Yan, J. (2024). EngineeringYarrowialipolyticaas a Cellulolytic Cell Factory for Production of p-Coumaric Acid from Cellulose and Hemicellulose. Journal ofagricultural and food chemistry, 72(11), 5867–5877.） on the construction of cell factories for the synthesis of p-coumaric acid and its derivatives. However, only a few p-coumarate degrading enzymes in other species are reported at present, and the process of metabolizing p-coumarate by various microorganisms including yarrowia lipolytica and the genes involved in this process are unknown. The metabolic pathway and related genes of the p-coumaric acid are identified and identified, and the strain is subjected to corresponding genetic engineering modification, so that the degradation consumption of the strain to the p-coumaric acid is reduced and eliminated, and the key for improving the level of the p-coumaric acid and the p-coumaric acid derivatives synthesized by microbial cells is provided. Disclosure of Invention It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below. It is another object of the present invention to provide a microorganism having an improved level of p-coumaric acid or p-coumaric acid derivatives, and a method for constructing the same and use thereof, by modifying a genetic target related to a p-coumaric acid metabolic pathway, to reduce the degree of degradation of p-coumaric acid by the microorganism, thereby improving the yield and/or production efficiency of p-coumaric acid and p-coumaric acid derivatives produced