Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase.
The expression of a plant (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (BTE) cDNA in Escherichia coli results in a very high level of extractable medium-chain-specific hydrolytic activity but causes only a minor accumulation of medium-chain fatty acids. BTE's full impact on the bacterial fatty acid synthase is apparent only after expression in a strain deficient in fatty acid degradation, in which BTE increases the total fatty acid output of the bacterial cultures fourfold. Laurate (12:0), normally a minor fatty acid component of E. coli, becomes predominant, is secreted into the medium, and can accumulate to a level comparable to the total dry weight of the bacteria. Also, large quantities of 12:1, 14:0, and 14:1 are made. At the end of exponential growth, the pathway of saturated fatty acids is almost 100% diverted by BTE to the production of free medium-chain fatty acids, starving the cells for saturated acyl-ACP substrates for lipid biosynthesis. This results in drastic changes in membrane lipid composition from predominantly 16:0 to 18:1. The continued hydrolysis of medium-chain ACPs by the BTE causes the bacterial fatty acid synthase to produce fatty acids even when membrane production has ceased in stationary phase, which shows that the fatty acid synthesis rate can be uncoupled from phospholipid biosynthesis and suggests that acyl-ACP intermediates might normally act as feedback inhibitors for fatty acid synthase. As the fatty acid synthesis is increasingly diverted to medium chains with the onset of stationary phase, the rate of C12 production increases relative to C14 production. This observation is consistent with activity of the BTE on free acyl-ACP pools, as opposed to its interaction with fatty acid synthase-bound substrates.
Voelker
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Calgene, Inc., Davis, California 95616.
Davies
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D003001:Cloning, Molecular; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D064429:Fatty Acid Synthases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis*; D005231:Fatty Acids, Unsaturated / Q000096:biosynthesis; D008055:Lipids / Q000737:chemistry; D010743:Phospholipids / Q000096:biosynthesis; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D008055:Lipids; D010743:Phospholipids; D011994:Recombinant Proteins; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II; C018748:oleoyl-(acyl-carrier-protein) hydrolase