Plasmid Copy Number Engineering Accelerates Fungal Polyketide Discovery upon Unnatural Polyketide Biosynthesis

Saccharomyces cerevisiae has been extensively used as a convenient synthetic biology chassis to reconstitute fungal polyketide biosynthetic pathways. Despite progress in refactoring these pathways for expression and optimization of the yeast production host by metabolic engineering, product yields often remain unsatisfactory. Such problems are especially acute when synthetic biological production is used for bioprospecting via genome mining or when chimeric fungal polyketide synthases (PKSs) are employed to produce novel bioactive compounds. In this work, we demonstrate that empirically balancing the expression levels of the two collaborating PKS subunits that afford benzenediol lactone (BDL)-type fungal polyketides is a facile strategy to improve the product yields. This is accomplished by systematically and independently altering the copy numbers of the two plasmids that express these PKS subunits. We applied this plasmid copy number engineering strategy to two orphan PKSs from genome mining where the yields of the presumed BDL products in S. cerevisiae were far too low for product isolation. This optimization resulted in product yield improvements of up to 10-fold, allowing for the successful isolation and structure elucidation of new BDL analogues. Heterocombinations of these PKS subunits from genome mining with those from previously identified BDL pathways led to the combinatorial biosynthesis of several additional novel BDL-type polyketides.