Cloning, nucleotide sequence, and expression of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase

Abstract

We report the cloning and nucleotide sequence of the gene encoding malonyl cocnzymc A-acyl carrier protein transacylasc of Esciwrichiu co/i. Malonyl transacylasc has been overexpressed 1.55.fold compared to a wild-type strain, Overexpression of this enzyme alters the fatty acid composition of a wild-type E. coli strain; increased amounts of ris-vacccnate arc incorporated into the mcmbranc phospholipids. Mulonyl transacylase; Malonyl-CoA: Acyl transferase; Fatty acid biosynthesis 1. 1NTRODUCTION Malonyl coenzyme A-acyl carrier protein trans-acylase (malonyl transacylase) catalyzes a key reaction of fatty acid synthesis in bacteria and plants, the conversion of malonyl-CoA to malonyl-acyl carrier protein (ACP). Malonyl-ACP then acts as the two carbon donor in the elongation steps of fatty acid synthesis. A similar reaction occurs in fatty acid synthesis in fungi and mammals except that both the malonyl transacylase and ACP moieties are domains of the polyfunctional fatty acid synthases of these organisms. E. co/i malonyl transacylase has been purified [1,2], and aspects of the enzymatic mechanism studied [3]. Mutants defective in the enzyme have also been isolated [4], In this paper, we report the cloning, DNA sequence, and overexpression of malonyl transacylase. We also report the effects of overexpression on the fatty acid composition of the membrane phospholipids of E. co/i, 2. MATERIALS AND METHODS The bacterial strains used were all derivatives of E. ru/i K-l?. Strain L48 (also called LA2-89) [4], carries a temperature-sensitive lesion in the&&I gene resulting in a malonyl transacylaseol'abnormal thermo-lability. recA derivatives of this strain wrrc made tither by PI transduction or bacterial conjugation using a T~rrfO element closely linked to rccAf. Standard recombinant DNA methods were used as were standard plasmids and host strains. DNA sequencing was done using the Sequenasc kits from United States Bibchemicals with both single-stranded and double-stranded templates. The sequence reported was obtained by complete sequencing of both DNA strands and was independently obtained in both laboratories. Protein cxpres-C~r-esfi~rrlen~ &ifzrz. sion studies were done using either the T7 polymerase system (for the truncated protein) [S], or the f~ promoter of plasmid pCKRlOl [6]. 3. RESULTS 3.1. Sequejrce of fabD The fobD gene was isolated from two different sources; lambda miniset phase 235 of the Kohara library [7] and from a minibank of E. co/i DNA fragments in plasmid pACYCl84 [El. In both cases, the gene was isolated by complementation (or recombinational repair) of theJrbl> strain LA2-89 which encodes a temperature sensitive enzyme. The gene carried by a235 was further localized by subcloning into high copy number plasmids. The minimal clone conferring growth at 42°C was sequenced and found to encode a truncated version of FabD lacking the last 21 amino acids. N-terminal sequencing of the truncated protein gave the sequence TQFAFVFPGQ, and thus the initiation me-thionine is the most upstream of the three in-fr...

Formation of catalytically active S‐acetyl(malonate decarboxylase) requires malonyl‐coenzyme A

“…Hence, the evolutionary origin for MdcH may be a gene operating in the synthesis of polyacetate compounds. Recently, the fabD gene of E. coli has been cloned [26,27] and the structure of its gene product determined [28]. The active site of the enzyme is related to a/b hydrolases where SerHis and Asp/Glu operate together as a charge relay system.…”

Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components ofMycobacterium tuberculosis Fatty Acid Synthase II

“…Liquid cultures of recombinant E. coli were grown in LB broth at 37°C with either 150 g/ml ampicillin or 25 g/ml kanamycin, according to the selection marker present on the plasmid. Strain LA2-89, a thermosensitive E. coli fabD mutant (fabD89), was a generous gift from A. R. Stuitje (16). E. coli C41(DE3), which expresses the T7 RNA polymerase, was used as a host for the overproduction of M. tuberculosis AcpM and mtFabD and was kindly provided by B. Miroux and J. E. Walker (19).…”

“…fatty acid or polyketide synthases has demonstrated that a serine, such as Ser-92 in the E. coli FabD protein (16,22,25), is the active nucleophilic residue of these enzymes. During its transfer from malonyl-CoA to ACP, the malonyl moiety is transiently attached to this serine to form a stable malonyl-serine enzyme intermediate (26).…”

“…We have recently shown that mtFabH, a ␤-ketoacyl-ACP synthase, forms a pivotal link between the type I and type II FAS elongation systems in M. tuberculosis. Indeed, mtFabH uses lauroyl-CoA (C 12 ) and myristoyl-CoA (C 14 ) to generate myristoyl-AcpM (C 14 ) and palmitoyl-AcpM (C 16 ), respectively, the preferred substrates of the FAS-II system (13). We have also demonstrated that the ␤-ketoacyl-ACP synthase A (KasA) belongs to the FAS-II sys-tem and catalyzes the first reaction of the elongation step by condensing a two-carbon unit from malonate (malonyl-AcpM) to a pre-existing carbon chain esterified to the phosphopantetheine moiety of AcpM.…”

Modification of Brassica napus seed oil by expression of the Escherichia coli fabH gene, encoding 3-ketoacyl-acyl carrier protein synthase III

“…The following primers were used: (1) 5' -GAATTGCCGCTCGTCTAGAATCTG-3 ' which introduces a Xba I site 130 bp upstream of the starting ATG of the coding region [ 31 ]; (2) 5' -GCGGATCCGAGAACCCTGTCCAG-GG-3', which introduces a Bam HI site at the 3' end, beyond the stop codon, 40 bp into the coding region of the E. coIifabD gene [32]. The following thermal cycle was used: denaturation at 94 ° C for 30 s; annealing at 52 ° C for 60 s; and extension at 72 °C for 90 s, repeated 30 times.…”