Overproduction of Acetyl-CoA Carboxylase Activity Increases the Rate of Fatty Acid Biosynthesis in Escherichia coli

Mark S Davis,José Solbiati,John E Cronan

doi:10.1074/jbc.m004756200

Abstract

Acetyl-CoA carboxylase (ACC) catalyzes the first committed step of the fatty acid synthetic pathway. Although ACC has often been proposed to be a major rate-controlling enzyme of this pathway, no direct tests of this proposal in vivo have been reported. We have tested this proposal in Escherichia coli. The genes encoding the four subunits of E. coli ACC were cloned in a single plasmid under the control of a bacteriophage T7 promoter. Upon induction of gene expression, the four ACC subunits were overproduced in equimolar amounts. Overproduction of the proteins resulted in greatly increased ACC activity with a concomitant increase in the intracellular level of malonyl-CoA. The effects of ACC overexpression on the rate of fatty acid synthesis were examined in the presence of a thioesterase, which provided a metabolic sink for fatty acid overproduction. Under these conditions ACC overproduction resulted in a 6-fold increase in the rate of fatty acid synthesis.

Highlights

Fatty acids are an essential component of the cellular membranes of all living organisms excepting the Archaea
We assayed the relative production of the four Acetyl-CoA carboxylase (ACC) subunits by labeling induced cultures with a mixture of 35S-labeled methionine and cysteine in the presence of rifampicin, a specific inhibitor of E. coli RNA polymerase, such that only gene products expressed from a T7 promoter are 35S-labeled [21]
Low levels of malonyl-CoA in wild type E. coli cells were reported by Heath and Rock [19], who reported accumulation of much larger pools of malonyl-CoA when fatty acid synthesis is blocked by addition of cerulenin

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains and Media—Strain BL21(DE3) containing DE3, a prophage carrying the T7 RNA polymerase gene [21], was used for. Plasmid Constructions—Plasmid pMSD1 was constructed by inserting a 800-base pair pLS182 [10] KpnI-SalI DNA fragment containing the accB gene into the same sites of pFN476 [22]. Plasmid pMSD4 resulted from insertion of a 4.2-kilobase pair XbaI-SalI accD DNA fragment from pPJ10 into the same sites of pFN476. Plasmid pMSD6 was made by inserting the 2.8-kilobase pair accBC SacI-XbaI DNA fragment of pLS182 [10] between the same sites of pFN476. A 4.2kilobase pair XbaI-SalI DNA fragment of pPJ10 containing the accD gene and the downstream folC gene was inserted into the same sites of pMSD6 to give plasmid pMSD7 (accBCD plus folC). As described previously [23], following induction the cells were treated with trichloroacetic acid; a mixture of unlabeled CoA, malonyl-CoA, and acetyl-CoA were added as internal standards (these were detected by UV absorption); and the CoA compounds were separated and quantitated. Assay of Lipid Synthesis—Labeling of lipids with [1-14C]acetate and thin layer chromatography were performed as described previously [27], followed by PhosphorImager analysis

RESULTS AND DISCUSSION

CONCLUSIONS

Methods