Negative Regulation Of Escherichia Coli BCCP On AccBC Operon Transcription

Fatty acids are one of the most critical components of cell membrane, closelyconcerned with biological growth and metabolism. Fatty acids as well as theirderivtives are widely used in food, pharmaceuticals, chemical engineering etc.Echerichia coli, as a mode microorganism, has very known genetic background andgene manipulation, which is familiar to all. It is an ideal carrier for studying themechanism of fatty acid metabolism. The synthetase of bacteria differs from that ofmammals, belonging to Type II fatty acid synthesis system. Each single reaction iscatalyzed by a certain enzyme and Acyl carrier protein (ACP) is used as the carrier inthe synthesis process. Encoding genes of Acetyl-CoA carboxylase(ACC) inEcherichia coli regulate the synthesis of fatty acids. In the pathway of fatty acids,with bicarbonate as carboxyl source and ATP, ACC is responsible for catalyzing theformation of malonyl CoA from Acetyl-CoA.The synthesis of malonyl CoA in Echerichia coli consists of two steps, and theoverall process involves the participation of four genes: accA, accB, accC and accD.The first step is that the carboxylation of the biotin part of biotin carboxyl carrierprotein (BCCP) is catalyzed by biotin carboxylase (BC; AccC); The second step is thecarboxyl groups produced in the above process are transferred from BCCP toAcetyl-CoA catalyzed by carboxyltransferase consisting of AccA and AccD. Thus far,there have been many reports on the research of fatty acids, which used Echerichiacoli as the Type II template.The present research was focused on the negative regulation of accBC operon inthe fatty acid synthesis of Echerichia coli. accBC genes of3000bp were amplifiedfrom E. coli using PCR. In the presence of accBC::lux fusion, the research on the transcriptional fusion of accBC promoters showed that the overexpression of AccBand AccC made the fusion reporter activity decrease markedly.The overexpression of AccD didn’t have any effect on the activity. Northernblotting results showed the overexpression of AccB was the cellular signal forautoregulation. The overexpression of68amino-terminal amino acids of AccB playeda critical role in negatively regulating accBC operons.In the present work, accB from Bacillus subtilis was expressed in Echerichiacoli(ΔaccB), which complemented the AccB activity but didn’t cause negativeregulation to accBC transcription. The chimeric protein carrying44amino-terminal44amino acids was fused into the carboxyl-terminal biotinylation domain of Bacillussubtilis AccB. The resulting not only complemented AccB activities but also causednegative regulation to accBC transcription in Echerichia coli. accA and accB wereknocked out and this revealed that the absence of both accA and accB resulted in thestable expression of accBC. Malonyl CoA synthase gene matABC from fromRhizobium trifolii complemented the function of Acetyl-CoA carboxylase, whichallowed the absence of ACC in the process of fatty acid synthesis.