Fatty dicarboxylic acids (FDCA), are useful as starting materials or components for plastics, polyesters, nylons and fragrances. Many industrial materials employ fatty dicarboxylic acids with an odd number of carbons (odd-carbon FDCA) as raw materials; at present, these can only be synthesized from petrochemical feedstocks using the chemical methods. Production of FDCAs using metabolic engineering of microorganisms would provide a more sustainable process. However, the FDCAs that are available through biosynthetic pathways have even-numbered chains. In this research work we have engineered a new strain of Lysobacter enzymogenes (Le) which can produce 34 ng/ml odd-carbon diunsaturated FDCA. This unsaturated FDCA can be reduced to produce saturated odd chain FDCA or can be used to generate functionalized FDCAs. To accomplish the proposed goal, we have genetically modified the polyketide synthase-nonribosomal peptide synthase-thioesterase (PKS-NRPS-TE) gene of wild type Le. NRPS domains of this gene were clipped off so that thioesterase can immediately follow the polyketide synthase (PKS) domains. This recombinant gene was then transformed into the Le where homologous recombination occurred, which was confirmed by different methods. Hence a new strain of Le was successfully engineered. Fatty acids from the wild type as well as from the engineered strain were extracted, derivatized into methyl esters and analyzed by gas chromatography–mass spectrometry (GCMS). While analyzing the EIC for masses corresponding to the mass of expected products, a peak in the EIC for mass 184 [di-unsaturated C7 FDCA, (2E, 4E)-hepta-2, 4-dienedioic acid] was observed only in the engineered strain but not in the wild type. This is a biosynthetically produced novel compound which can be used for various industrial purposes.

Production of odd-carbon diunsaturated dicarboxylic acid through engineering the HSAF synthase gene in Lysobacter enzymogenes

ACS Spring Meeting 2021

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