Improved linalool production in Saccharomyces cerevisiae by combining directed evolution of linalool synthase and overexpression of the complete mevalonate pathway

摘要

With special fragrance and various biological properties, linalool has wide applications in food, pharmaceuticals and cosmetics industries. Microbial biosynthesis has become a promising approach to linalool production, however its efficiency is limited by the limited precursor supply and poor efficiency of linalool synthase. With the goal of enhancing heterologous linalool production in Saccharomyces cerevisiae, protein engineering and metabolic engineering were simultaneously employed to solve these bottlenecks. Linalool-producing yeast was first constructed by introduction of t670McLIS selected out from a collection of linalool synthases. The efficiency of linalool biosynthesis was then improved by strengthening the supply of geranyl pyrophosphate (GPP) as precursor via overexpression of the complete mevalonate (MVA) pathway and a GPP synthase variant. To accelerate the conversion of the accumulated GPP to linalool, t670McLIS was engineered by means of directed evolution after development of a competition-based color-indicated high-throughput screening method. Expression of the t670McLIS variant in the engineered S. cerevisiae with enhanced precursor supply yielded 53.14 mg/L of linalool in biphasic shake-flask culture. This study delivered an efficient linalool-producing yeast cell factory and meanwhile provided efficient strategies for biosynthesis of other valuable monoterpenes.