Heterologous Production of Levopimaradiene in Saccharomyces cerevisiae.

Abstract

Levopimaradiene (LP) is a precursor of the important anticancer compound ginkgolide. However, the current low synthetic yield in yeast limits the progress of the microbial ginkgolide synthesis pathway. In order to increase the synthetic flux of LP in S. cerevisiae, we first overexpressed the fusion protein of Bts1p-Erg20p(F96C) in a geranylgeranyl diphosphate (GGPP)-enhanced strain. The LP concentration was 20.36 mg L^-1 when the T79LPS^M593I/Y700F gene was integrated. The overexpression of a series of genes in the mevalonate (MVA) pathway led to a significant increase in the LP yield, reaching 59.37 mg L^-1. Next, the spheroplast protein Y (SPY) tag was fused to the N-terminus of LP synthase, which increased the yield of LP to 82.21 mg L^-1. In order to consume the accumulated precursor GGPP and balance the expression levels of BTS1 (encoding geranylgeranyl diphosphate synthase) and LPS (encoding levopimaradiene synthase) genes, the expression copy numbers of BTS1 and LPS genes were regulated using scaffold protein technology. Subsequently, an LP yield of 215.50 mg L^-1 was achieved via fed-batch fermentation in a 5-L bioreactor, which represents the highest reported level in S. cerevisiae currently. This lays the foundation for advancing the heterologous synthesis of ginkgolides and provides a reference for the efficient synthesis of natural products in S. cerevisiae.