초록
<P><B>Abstract</B></P> <P>In this study, we metabolically engineered <I>Corynebacterium glutamicum</I> to produce triacylglycerols (TAGs) by completing and constraining a <I>de novo</I> TAG biosynthesis pathway. First, the plasmid pZ8_TAG4 was constructed which allows the heterologous expression of four genes: three (<I>atf1</I> and <I>atf2,</I> encoding the diacylglycerol acyltransferase; <I>pgpB,</I> encoding the phosphatidic acid phosphatase) to complete the TAG biosynthesis pathway<I>,</I> and one gene (<I>tadA</I>) for lipid body assembly. Second, we applied four metabolic strategies to increase TAGs accumulation: (i) boosting precursor supply by heterologous expression of <I>tesA</I> (encoding thioesterase to form free fatty acid to reduce the feedback inhibition by acyl-ACP) and <I>fadD</I> (encoding acyl-CoA synthetase to enhance acyl-CoA supply), (ii) reduction of TAG degradation and precursor consumption by deleting four cellular lipases (<I>cg0109</I>, <I>cg0110</I>, <I>cg1676</I> and <I>cg1320</I>) and the diacylglycerol kinase (<I>cg2849</I>), (iii) enhancement of fatty acid biosynthesis by deletion of <I>fasR</I> (<I>cg2737</I>, 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 (<I>pqo</I>) and lactic acid production (<I>ldh</I>) pathways. The final strain (<I>CgTesRtcEfasEbp</I>/pZ8_TAG4) achieved a 7.5% yield of total fatty acids (2.38±0.05g/L intracellular fatty acids and 0.64±0.09g/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.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Corynebacterium glutamicum</I> was engineered as a microbial lipids producer. </LI> <LI> Fourteen genes have been altered to complete and optimize the <I>de novo</I> TAG biosynthesis pathway in <I>Corynebacterium glutamicum</I>. </LI> <LI> A complete TAG biosynthesis pathway is necessary but not sufficient for a non-native oleaginous microorganism to produce TAGs. </LI> <LI> The enhancement of fatty acid synthesis and the elimination of by-products have a strong synergistic effect on lipid production. </LI> <LI> The highest reported fatty acids production in an engineered non-oleaginous bacterium. </LI> </UL> </P>