초록
<P><B>Abstract</B></P> <P>As a bioactive triterpenoid, squalene is widely used in the food industry, cosmetics, and pharmacology. Squalene’s major commercial sources are the liver oil of deep-sea sharks and plant oils. In this study, we focused on the enhancement of squalene biosynthesis in <I>Yarrowia lipolytica</I>, with particular attention to the engineering of acetyl-CoA metabolism based on genome-scale metabolic reaction network analysis. Although the overexpression of the rate-limiting endogenous <I>ylHMG1</I> (3-hydroxy-3-methylglutaryl-CoA reductase gene) could improve squalene synthesis by 3.2-fold over that by the control strain, the availability of the key intracellular precursor, acetyl-CoA, was found to play a more significant role in elevating squalene production. Analysis of metabolic networks with the newly constructed genome-scale metabolic model of <I>Y. lipolytica</I> iYL_2.0 showed that the acetyl-CoA pool size could be increased by redirecting carbon flux of pyruvate dehydrogenation towards the ligation of acetate and CoA or the cleavage of citrate to form oxaloacetate and acetyl-CoA. The overexpression of either acetyl-CoA synthetase gene from <I>Salmonella enterica</I> (<I>acs*</I>) or the endogenous ATP citrate lyase gene (<I>ylACL1</I>) resulted in a more than 50% increase in the cytosolic acetyl-CoA level. Moreover, iterative chromosomal integration of the <I>ylHMG1</I>, <I>asc*</I>, and <I>ylACL1</I> genes resulted in a significant improvement in squalene production (16.4-fold increase in squalene content over that in the control strain). We also found that supplementation with 10 mM citrate in a flask culture further enhanced squalene production to 10 mg/g DCW. The information obtained in this study demonstrates that rationally engineering acetyl-CoA metabolism to ensure the supply of this key metabolic precursor is an efficient strategy for the enhancement of squalene biosynthesis.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The engineering of acetyl-CoA metabolism was based on genome-scale metabolic reaction network analysis. </LI> <LI> Metabolically engineered <I>Yarrowia lipolytica</I> exhibited significant improvement in squalene production. </LI> <LI> 10 mg/g DCW of squalene was achieved in engineered <I>Yarrowia lipolytica</I> in YPD medium supplemented with citrate. </LI> </UL> </P>