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

Saccharomyces cerevisiae has beenextensively used as a convenient synthetic biology chassis to reconstitutefungal polyketide biosynthetic pathways. Despite progress in refactoringthese pathways for expression and optimization of the yeast productionhost by metabolic engineering, product yields often remain unsatisfactory.Such problems are especially acute when synthetic biological productionis used for bioprospecting via genome mining or when chimeric fungalpolyketide synthases (PKSs) are employed to produce novel bioactivecompounds. In this work, we demonstrate that empirically balancingthe expression levels of the two collaborating PKS subunits that affordbenzenediol lactone (BDL)-type fungal polyketides is a facile strategyto improve the product yields. This is accomplished by systematicallyand independently altering the copy numbers of the two plasmids thatexpress these PKS subunits. We applied this plasmid copy number engineeringstrategy to two orphan PKSs from genome mining where the yields ofthe presumed BDL products in S. cerevisiae werefar too low for product isolation. This optimization resulted in productyield improvements of up to 10-fold, allowing for the successful isolationand structure elucidation of new BDL analogues. Heterocombinationsof these PKS subunits from genome mining with those from previouslyidentified BDL pathways led to the combinatorial biosynthesis of severaladditional novel BDL-type polyketides.