Abstract
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.
Highlights
Acyl carrier proteins (ACPs) are essential to the production of fatty acids
The ACP2 and ACP3 fragments were made by overlap PCR, using primers that were complementary to both the E. coli gene encoding ACP and to the P. profundum gene encoding the peptide that links the ACPs from the polyunsaturated fatty acids (PUFA) synthase (Fig. S1)
We purified the proteins by a combination of nickel-affinity purification and size exclusion chromatography, resulting in highly pure elution fractions with yields of 15, 24, and 18 mg of pure protein per liter of culture, for ACP1, ACP2, and ACP3, respectively
Summary
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. We have engineered the native E. coli ACP into tandem diand 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. Our results show the feasibility of enhancing fatty acid production by genetically fusing ACP domains together into multi-domain proteins
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