Artificial covalent linkage of bacterial acyl carrier proteins for fatty acid production.

Acyl carrier proteins (ACPs) are essential to the production of fatty acids. In some species of marine bacteria, ACPs are arranged into tandem repeats joined by peptide linkers, an arrangement that results in high fatty acid yields. By contrast, Escherichia coli, a relatively low producer of fatty acids, uses a single-domain ACP. In this work, we have engineered the native E. coli ACP into tandem di- and tri-domain constructs joined by a naturally occurring peptide linker from the PUFA synthase of Photobacterium profundum. The size of these tandem fused ACPs was determined by size exclusion chromatography to be higher (21 kDa, 36 kDa and 141 kDa) than expected based on the amino acid sequence (12 kDa, 24 kDa and 37 kDa, respectively) suggesting the formation of a flexible extended conformation. Structural studies using small-angle X-ray scattering (SAXS), confirmed this conformational flexibility. The thermal stability for the di- and tri-domain constructs was similar to that of the unfused ACP, indicating a lack of interaction between domains. Lastly, E. coli cultures harboring tandem ACPs produced up to 1.6 times more fatty acids than wild-type ACP, demonstrating the viability of ACP fusion as a method to enhance fatty acid yield in bacteria.

Fused dimerization increases expression, solubility, and activity of bacterial dehydratase enzymes.

FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ-FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids.