Isolation and characterization of a synthetic gene and a genomic clone encoding acyl carrier protein from Escherichia coli
Rawlings, Merriann
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https://hdl.handle.net/2142/20849
Description
Title
Isolation and characterization of a synthetic gene and a genomic clone encoding acyl carrier protein from Escherichia coli
Author(s)
Rawlings, Merriann
Issue Date
1993
Doctoral Committee Chair(s)
Cronan, John E.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Microbiology
Language
eng
Abstract
Acyl carrier protein (ACP) is a required cofactor for the synthesis and subsequent metabolism of fatty acids in Escherichia coli. Previous work has suggested that DNA segments encoding ACP were somehow toxic to E. coli. To investigate this possibility, a synthetic gene encoding ACP was assembled using a novel $lacZ\ \alpha$-complementation test. When provided with the sequences necessary for transcription and translation, the gene was expressed at high levels. Despite the already functional excess of ACP in wild-type cells its overexpression was indeed lethal, resulting in a decreased cellular growth rate and a precipitous drop in cell viability. One of the most obvious differences between ACP overproducing and wild-type strains was the accumulation of apo-ACP by the former cells. Normally apo-ACP is not detected in vivo. A genomic clone encoding ACP has also been isolated and sequenced. The ACP gene (called acpP) was located on the genetic map between fabF and fabD which encode two fatty acid biosynthetic enzymes, 3-ketoacyl-ACP synthase II and malonyl CoA-ACP transacylase, respectively. An open reading frame between acpP and fabD encodes a 26.5-kDa protein that has significant sequence identity ($>$40%) with a plant 3-ketoacyl-ACP reductase and thus is believed to encode the same enzyme in E. coli. This gene (called fabG) is cotranscribed with acpP. Thus, the gene encoding ACP is located within a cluster of fatty acid biosynthetic genes.
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