De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica

Abstract

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. <i>Yarrowia lipolytica</i> stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer <i>Y. lipolytica</i> for resveratrol production. The resveratrol biosynthetic pathway was integrated into <i>Y. lipolytica</i> by adding genes encoding tyrosine ammonia lyase from <i>Rhodotorula glutinis</i>, 4-coumarate CoA ligase from <i>Nicotiana tabacum</i>, and stilbene synthase from <i>Vitis vinifera</i>. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from <i>Arabidopsis thaliana</i>, malonyl-CoA synthase, and a malonate transporter protein from <i>Bradyrhizobium diazoefficiens</i>. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of <i>Y. lipolytica</i> as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.