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 <i>Serratia marcescens</i> 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 <i>S. marcescens</i> 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 <i>Pseudomonas putida</i> KT2440 chassis strain yielded a maximum prodigiosin titer of 665 mg/L in shake-flask cultures, significantly outperforming production levels of the native <i>S</i>. <i>marcescens</i> 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.