High-level prodigiosin production in Pseudomonas putida enabled by combinatorial metabolic engineering.

Prodigiosin, a bioactive tripyrrole pigment, exhibits a broad spectrum of biological activities-including antimicrobial, anticancer, and antimalarial properties-thereby holding significant promise for use in pharmaceutical applications and industrial biotechnology. In this study, three isolates of Serratia marcescens were recovered from the cabbage rhizosphere. Genomic analysis revealed a highly conserved prodigiosin biosynthetic gene cluster embedded within the chromosomes of all three isolates. Though prodigiosin production was detected in these three S. marcescens isolates, the relatively low yield severely limits the feasibility of its large-scale production. To address this issue, we employed a stepwise strategy involving heterologous expression, promoter engineering, genome-wide transposon mutagenesis, and optimization of fermentation media with the aim to achieve high-level prodigiosin production. The introduction of an engineered prodigiosin gene cluster into a tailored Pseudomonas putida KT2440 chassis strain yielded a maximum prodigiosin titer of 665 mg/L in shake-flask cultures, significantly outperforming production levels of the native S. marcescens isolates. When cultured in a small-scale stirred-tank bioreactor, the engineered strain further elevated the prodigiosin yield to 1161 mg/L. Our study presents a robust platform for prodigiosin overproduction, which can be adapted to improve the titers of other prodiginine family compounds.