acetobutylicium fabZ. The methyl esters of fatty acids were obtained from the phospholipids as described in Methods. Lane 1 is the methyl esters of the wild type E. coli strainMG1655. Lane 2 is the esters of strain CY57 carrying vector pBAD24. Lane 3 is the esters of strain CY57 carrying pHW22 which encodes the C. acetobutylicium fabZ labeled in the absence of induction. Lane 4 is the esters of strain

CY57 (pHW22) following arabinose induction. Labels are as in Fig. 2. In vitro assay of C. acetobutylicium FabZ and FabF1 activities To allow direct assay of C. acetobutylicium FabF1 and FabZ activities we expressed the proteins in E. coli to facilitate their purification. [35S]Methionine labeling Thiazovivin in vitro showed that strain BL21 (DE3) carrying plasmids encoding either C. acetobutylicium fabF1 or fabZ under control of a phage T7 promoter expressed proteins of the expected sizes this website (Fig. 6A). However, the expression level of the FabZ protein was so low that it was not detected upon staining the SDS gels (Fig. 6B). We attributed this poor expression to the fact that the C. acetobutylicium FabZ gene contains 24 find more codons that correspond to nonabundant (rare) tRNA species in E. coli.

We therefore changed these codons to synonymous codons that correspond to abundant E. coli tRNA species thereby resulting in a modified gene we call fabZm. Plasmid pHW74m (which encoded the His-tagged fabZm under T7 promoter control) abundantly expressed a protein with an apparent mass of 17 kDa (Fig. 6B) in good agreement with the expected value for the His6-tagged protein (17.5 kDa). The His6-tagged FabZ protein was purified to essential homogeneity using nickel-chelate chromatography (Fig. 6B). We also purified the N-terminally His6-tagged versions of C. Terminal deoxynucleotidyl transferase acetobutylicium FabF1 and the E. coli fatty acid biosynthetic proteins FabD, FabG, FabA, FabZ,

FabB and FabI plus the Vibrio harveyi AasS acyl-ACP synthetase [18] by nickel-chelate chromatography. AasS was used to synthesize the 3-hydroxydecanoyl-ACP substrate whereas the other enzymes were used to assemble a defined in vitro fatty acid synthesis system in which the activities of E. coli FabA and C. acetobutylicium FabZ or E. coli FabB and C. acetobutylicium FabF1 could be directly compared. In reactions containing FabA 3-hydroxydecanoyl-ACP was converted to a mixture of trans-2 and cis-3-decenoyl-ACPs as expected from prior work [19, 20]. E. coli FabB is unable to elongate trans-2-decenoyl-ACP, but elongates the cis-3 species to 3-keto-cis-5-dodecenoyl-ACP in the presence of malonyl-ACP [20]. This product is then reduced by FabG and dehydrated by FabA to form trans-2-cis-5-dodecadienoyl-ACP[20]. The trans-2-cis-5-dodecadienoyl-ACP product accumulates because the reaction mixtures lacked enoyl-ACP reductase which precluded further elongations [20].