Metabolic engineering Corynebacterium glutamicum to produce triacylglycerols

• Corynebacterium glutamicum was engineered as a microbial lipids producer.
• Fourteen genes have been altered to complete and optimize the de novo TAG biosynthesis pathway in Corynebacterium glutamicum.
• A complete TAG biosynthesis pathway is necessary but not sufficient for a non-native oleaginous microorganism to produce TAGs.
• The enhancement of fatty acid synthesis and the elimination of by-products have a strong synergistic effect on lipid production.
• The highest reported fatty acids production in an engineered non-oleaginous bacterium.

In this study, we metabolically engineered Corynebacterium glutamicum to produce triacylglycerols (TAGs) by completing and constraining a de novo TAG biosynthesis pathway. First, the plasmid pZ8_TAG4 was constructed which allows the heterologous expression of four genes: three (atf1 and atf2, encoding the diacylglycerol acyltransferase; pgpB, encoding the phosphatidic acid phosphatase) to complete the TAG biosynthesis pathway, and one gene (tadA) for lipid body assembly. Second, we applied four metabolic strategies to increase TAGs accumulation: (i) boosting precursor supply by heterologous expression of tesA (encoding thioesterase to form free fatty acid to reduce the feedback inhibition by acyl-ACP) and fadD (encoding acyl-CoA synthetase to enhance acyl-CoA supply), (ii) reduction of TAG degradation and precursor consumption by deleting four cellular lipases (cg0109, cg0110, cg1676 and cg1320) and the diacylglycerol kinase (cg2849), (iii) enhancement of fatty acid biosynthesis by deletion of fasR (cg2737, TetR-type transcriptional regulator of genes for the fatty acid biosynthesis), and (iv) elimination of the observed by-product formation of organic acids by blocking the acetic acid (pqo) and lactic acid production (ldh) pathways. The final strain (CgTesRtcEfasEbp/pZ8_TAG4) achieved a 7.5% yield of total fatty acids (2.38±0.05 g/L intracellular fatty acids and 0.64±0.09 g/L extracellular fatty acids) from 4% glucose in shake flasks after process optimization. This corresponds to maximum intracellular fatty acids content of 17.8±0.5% of the dry cell.