Increased Cordycepin Production in Yarrowia lipolytica Using Combinatorial Metabolic Engineering Strategies

Abstract

As the first nucleoside antibiotic discovered in fungi, cordycepin, with its various biological activities, has wide applications. At present, cordycepin is mainly obtained from the natural fruiting bodies of Cordyceps militaris. However, due to long production periods, low yields, and low extraction efficiency, harvesting cordycepin from natural C. militaris is not ideal, making it difficult to meet market demands. In this study, an engineered Yarrowia lipolytica YlCor-18 strain, constructed by combining metabolic engineering strategies, achieved efficient de novo cordycepin production from glucose. First, the cordycepin biosynthetic pathway derived from C. militaris was introduced into Y. lipolytica. Furthermore, metabolic engineering strategies including promoter, protein, adenosine triphosphate, and precursor engineering were combined to enhance the synthetic ability of engineered strains of cordycepin. Fermentation conditions were also optimized, after which, the production titer and yields of cordycepin in the engineered strain YlCor-18 under fed-batch fermentation were improved to 4362.54 mg/L and 213.85 mg/g, respectively, after 168 h. This study demonstrates the potential of Y. lipolytica as a cell factory for cordycepin synthesis, which will serve as the model for the green biomanufacturing of other nucleoside antibiotics using artificial cell factories.

Overproduction of cordycepin in Saccharomyces cerevisiae by cordycepin synthase screening and metabolic engineering

“…Huo et al constructed a COR‐producing S. cerevisiae strain, which was fermented and optimized to reach 137.27 mg L −1 of COR 17 . Recently, a Y. lipolytica strain YlCor‐18 was constructed and COR content reached 4362.54 mg L −1 under fed‐batch fermentation 25 . In this work, S. cerevisiae was selected and engineered to produce the COR by introducing genes EA1 and EA2 , derived from E. atlantica , and combining strategies of metabolic pathway regulation and fermentation optimization and amplification to achieve 725.16 mg L −1 of COR yield, which was the highest yield of heterologous synthetic COR in S. cerevisiae reported so far.…”

“…Huo et al achieved heterologous production of COR by overexpressing CmCNS1 and CmCNS2 genes derived from C. militaris and the yield of COR reached 137.27 mg L −1 in a 5‐L fermenter by fermentation optimization in S. cerevisiae 17 . Song et al constructed an engineered strain YlCor‐18 by metabolic engineering, and the COR titer reached 4362.54 mg L −1 after 168 h fed‐batch fermentation in Yarrowia lipolytica 25 . As a traditional chassis strain, S. cerevisiae is considered an excellent host for the heterologous synthesis of natural products due to its powerful protein modification function, sophisticated genetic manipulation, rapid growth rate, and abundant cofactor content 26 .…”

“…17 Song et al constructed an engineered strain YlCor-18 by metabolic engineering, and the COR titer reached 4362.54 mg L À1 after 168 h fed-batch fermentation in Yarrowia lipolytica. 25 As a traditional chassis strain, S. cerevisiae is considered an excellent host for the heterologous synthesis of natural products due to its powerful protein modification function, sophisticated genetic manipulation, rapid growth rate, and abundant cofactor content. 26 These results indicate that the construction of COR yeast cell factory is very beneficial to the industrial production of COR.…”

“…Constructing COR synthesis pathway in yeast to produce COR has become the mainstream research direction and the COR synthesis pathway in yeast has been predicted in research. 25 in a 5-L fermenter by fermentation optimization in S. cerevisiae. 17 Song et al constructed an engineered strain YlCor-18 by metabolic engineering, and the COR titer reached 4362.54 mg L À1 after 168 h fed-batch fermentation in Yarrowia lipolytica.…”

Efficient de novo production of bioactive cordycepin by Aspergillus oryzae using a food-grade expression platform

“…Regarding high-cordycepin-producing strain, Y. lipolytica , a constitutive promoter (translation elongation factor 1; Tef1p ) was used to control cordycepin biosynthesis genes. In addition, a combinatorial approach, including enzyme fusion engineering, supply modular engineering, and fed-batch fermentation, was implemented to enhance cordycepin production [ 21 , 50 ]. Enzyme fusion engineering has been proposed as an efficient strategy to improve the interaction between two enzymes and increase the production of various metabolites [ 51 , 52 ].…”