Biosynthesis of Odd-Carbon Unsaturated Fatty Dicarboxylic Acids Through Engineering the HSAF Biosynthetic Gene in Lysobacter enzymogenes.

Abstract

Fatty dicarboxylic acids (FDCA) are useful as starting materials or components for plastics, polyesters, nylons, and fragrances. Most of the commercially available FDCA contain an even number of carbons, and there remain few sustainable methods for production of FDCA with an odd number of carbons (o-FDCA). In this work, we explored a novel biosynthetic route to unsaturated o-FDCA. The approach was based on genetic modifications of hsaf pks-nrps, encoding a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) in Lysobacter enzymogenes, an environmental bacterium emerging as a new biocontrol agent. This single-module PKS-NRPS catalyzes the biosynthesis of lysobacterene A, a polyene-containing precursor of the antifungal natural product Heat-Stable Antifungal Factor (HSAF). We genetically removed the NRPS module from this gene and generated a new strain of L. enzymogenes, in which the PKS module was fused to the thioesterase domain of hsaf pks-nrps. The chimeric gene was verified by DNA sequencing, and its expression in L. enzymogenes was confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The total fatty acids were extracted, esterified, and analyzed by GC-MS. The results showed that the engineered strain produced new fatty acids that were absent in the wild type. The main product was identified as hepta-2,4-dienedioic acid, an unsaturated o-FDCA. This work sets the foundation to explore a sustainable and environment-friendly approach toward unsaturated o-FDCA, which could be used as precursors for new compounds that can serve as versatile feedstock for industrial materials.