Engineering Pseudomonas putida for production of 3-hydroxyacids using hybrid type I polyketide synthases

Engineered type I polyketide synthases (T1PKSs) are a potentially transformative platform for the biosynthesis of small molecules. Due to their modular nature, T1PKSs can be rationally designed to produce a wide range of bulk or specialty chemicals. While heterologous PKS expression is best studied in microbes of the genus <i>Streptomyces</i>, recent studies have focused on the exploration of non-native PKS hosts. The biotechnological production of chemicals in fast growing and industrial relevant hosts has numerous economic and logistic advantages. With its native ability to utilize alternative feedstocks, <i>Pseudomonas putida</i> has emerged as a promising workhorse for the sustainable production of small molecules. Here, we outline the assessment of <i>P. putida</i> as a host for the expression of engineered T1PKSs and production of 3-hydroxyacids. After establishing the functional expression of an engineered T1PKS, we successfully expanded and increased the pool of available acyl-CoAs needed for the synthesis of polyketides using transposon sequencing and protein degradation tagging. This work demonstrates the potential of T1PKSs in <i>P. putida</i> as a production platform for the sustainable biosynthesis of unnatural polyketides.