NEWS

A few days ago, the China National Light Industry Council held a scientific and technological achievement appraisal meeting on the "synthetic biology manufacturing technology of resveratrol" in Jinshi, Hunan Province. The project was jointly completed by the team of academician Chen Jian from the Future food science Center of Jiangnan University and Hunan Hongjian Biotechnology Co., Ltd. The experts of the Appraisal Committee listened to the project research work and technical report, and reviewed the relevant appraisal data. After inquiry and discussion, they agreed that the overall technology of the "synthetic biology manufacturing technology of resveratrol" project has reached the international leading level, and the research results will play a positive role in promoting the application of synthetic biotechnology to the production of natural products.

Resveratrol is a type of stilbene polyphenol compound with physiological activities such as scavenging free radicals, antioxidant activity, and cardiovascular protection. It has broad application prospects in fields such as medicine, functional foods, and cosmetics. At present, resveratrol is mainly produced using plant extraction methods, which heavily relies on natural plant resources and is constrained by factors such as low extraction efficiency, high cost, and limited sources, making it unable to meet the growing market demand. With the continuous development of synthetic biotechnology, on the basis of precise analysis of the biosynthetic pathways of astragalus compounds such as resveratrol, the efficient heterologous synthesis of astragalus compounds by microorganisms is highly anticipated. Previously, multiple teams both domestically and internationally have conducted research on natural enzyme mining, pathway assembly, metabolic regulation, and fermentation optimization, but the production has been consistently low, making it difficult to meet industrial applications.

Group photo of Academician Chen Jian of Jiangnan University and Mr. Li Hongbing, Chairman of Hongjian

At present, resveratrol is mainly extracted from the plant Polygonum cuspidatum. Due to environmental factors, the yield of plant extraction is low and cannot meet the global market demand. According to Future Market Insights, the market size of resveratrol is approximately $97.7 million, and it is expected to grow at a compound annual growth rate of 8-10% from 2023 to 2030.

Although resveratrol has such a huge market, due to low plant extraction rates, low chemical synthesis efficiency, and low microbial synthesis yield, it cannot meet market requirements, resulting in a consistently high price of resveratrol. Taking the application of resveratrol in the cosmetics industry as an example, although resveratrol is a promising antioxidant, its high price has led to high prices for downstream end products.

How to open up new avenues and develop low-cost production technologies for resveratrol is the fundamental way to solve this problem. To this end, Academician Chen Jian's team has focused on researching and developing key technologies for the production of resveratrol through fermentation, with funding from national key research and development programs, innovation research group projects of the National Natural Science Foundation of China, and outstanding youth funds of the National Natural Science Foundation of China.

Professor Zhou Jingwen, the project research team, introduced the research and development work to the expert group at the meeting. On the basis of the previous research work, after 10 years of hard work, the research team has carried out a number of research work on the manufacturing of resveratrol synthetic biology in depth.

First, the synthetic biology manufacturing of resveratrol in Escherichia coli. The recombinant Escherichia coli for synthesis of p-coumaric acid was constructed through genetic engineering, and the high-throughput screening model for p-coumaric acid was established. Combined with the directed evolution strategy, the activity of tyrosine ammonia lyase was improved, and the yield of p-coumaric acid was increased by 20%. The De novo synthesis synthesis of resveratrol in Escherichia coli was achieved by modular assembly of resveratrol pathway, balancing malonic acid assimilation pathway and resveratrol synthesis pathway. This result provides a reference for combining genetic engineering modification strategies and high-throughput screening strategies to improve the efficiency of resveratrol synthesis in model microorganisms.

The second is the synthetic biology manufacturing of resveratrol in Saccharomyces cerevisiae. Taking the model eukaryotic Saccharomyces cerevisiae as the starting strain, through metabolic engineering transformation, the precursor supply was enhanced and the bypass competition was weakened. Exploring the effect of transport proteins on the accumulation of coumaric acid and enhancing the accumulation of precursor coumaric acid. Balance gluconeogenesis pathway and shikimic acid pathway, strengthen shikimic acid pathway related genes, enhance shikimic acid flux, and compartmentalize tyrosine ammonia lyase to achieve efficient accumulation of vanillic acid from precursors. Finally, by randomly integrating 4CL and STS related genes through TY transposition, a maximum yield of 88.37 mg/L of resveratrol in brewing yeast was achieved. This result provides ideas for the production of resveratrol in eukaryotic microorganisms and demonstrates that eukaryotic microorganisms have more advantages than prokaryotic microorganisms.

The third is the efficient synthesis of resveratrol through metabolic remodeling in lipolytic yeast. Starting from unconventional yeast lipolytic Arlo's yeast, different resveratrol synthesis genes were screened, and the shikimic acid pathway was strengthened. The fusion expression of coumarin CoA ligase and stilbene synthase was achieved, and the synergistic expression of tyrosine and phenylalanine ammonia lyase pathways increased precursor supply, ultimately redistributing carbon metabolism flow, achieving efficient accumulation of coumarin acid and resveratrol. These results indicate that unconventional yeast lipolytic yeast is an excellent host for the production of resveratrol. Starting from the strain of Lactobacillus lipolyticus, which enhances the synthesis of coumaric acid by its precursor, the efficient synthesis of resveratrol was achieved by integrating key rate limiting step genes using a multi-copy integration tool and enhancing the supply of malonyl coenzyme A.

The fourth is to optimize the fermentation process based on the study of microbial physiological characteristics. Referring to the known stoichiometric relationships of biochemical reactions, as well as the metabolic pathways and physiological metabolic characteristics of lipolytic yeast, the fermentation process was optimized to keep lipolytic yeast in yeast form during the fermentation process, achieving high production of resveratrol and laying the foundation for industrial production.

The fifth is to expand the scale of resveratrol fermentation industry and extract products. On the basis of the above research, the optimization control strategy of the fermentation process was used to control the environmental factors in the step-by-step amplification process of the high-yield resveratrol fermentation process of the lipolytic Arlo yeast. The industrial scale fermentation experiment was successfully achieved, and the yield, conversion rate, and production intensity were further improved. Resveratrol was extracted from fermentation broth using recyclable solvents, and the process conditions were systematically optimized, including pretreatment of fermentation broth, screening of decolorizing agents, and optimization of extraction conditions. The efficient separation and high-purity extraction of resveratrol were successfully achieved.

On site of expert appraisal meeting for resveratrol

The appraisal committee believes that the project has constructed an efficient resveratrol synthesis cell factory through research on synthetic gene screening, enhancement of shikimic acid pathway, reshaping of central carbon metabolism flow, optimization of key gene expression intensity, construction of multi copy integration tools, and high-throughput screening of high-yield strains in lipolytic Arlo yeast. The production, conversion rate, and production intensity of resveratrol in a 30 ton fermentation tank have reached international standards, and the separation and high-purity extraction of resveratrol produced by fermentation have been achieved.

Experts point out that this project has significant practical significance in achieving the industrial production of resveratrol through fermentation, breaking the monopoly of foreign companies on the production of resveratrol through synthetic biotechnology, and comprehensively improving the technical level of the resveratrol fermentation industry. It is recommended to further accelerate the promotion and application of project technology.