Multidimensionally engineered Escherichia coli for efficient gadusol biosynthesis with high-throughput quantitative analysis

Abstract

Gadusol, as a cyclohexenone compound, has attracted intensive attention because of its sunscreen and antioxidant properties. However, its limited availability, especially from microbial cell factories, hinders its applications. Here, we systematically engineered Escherichia coli for the efficient biosynthesis of gadusol. First, the gadusol biosynthesis pathway was reprogrammed in E. coli, yielding an initial titer of 45.2 mg/l. Then, a robust colorimetric assay was developed for high-throughput quantification of gadusol. Through a small RNA library screening approach, the pivotal regulatory targets were rapidly identified and regulated, resulting in 700 mg/l of gadusol. Next, after directed evolution of the committed enzyme transketolase A and optimization of the availability of cofactors NAD+ and adenosylmethionine, the titer of gadusol was increased to 965 mg/l. Eventually, by applying a fed-batch fermentation strategy, the production of gadusol reached 4.2 g/l. The results provide a comprehensive framework for rapid screening and construction of gadusol microbial cell factories.