Fluorescence-activated cell sorting and random mutagenesis for enhanced pyrroloquinoline quinone accumulation by Hyphomicrobium denitrificans

Evolutionary engineering requires the development of high-throughput screening techniques to identify and isolate target products or phenotypes of interest from extensive libraries. This study aimed to establish a high-throughput screening strategy for high-yielding single-cell strains of pyrroloquinoline quinone (PQQ) and combined it with random mutagenesis approaches to enhance PQQ accumulation. Initially, the fluorescence properties of PQQ were analyzed for the first time, leading to the development of a fluorescence-based detection method for PQQ and a fluorescence-activated cell sorting-based screening approach for high-yield strains of PQQ. Secondly, the library constructed via random mutagenesis confirmed the feasibility of the screening methodology, resulting in the identification of a mutant exhibiting a 1.9-fold enhancement in PQQ titer. Ultimately, omics tools found multiple candidate genes for improving PQQ production. In summary, the high-throughput screening model established provides practical guidance for the screening of other natural products.