Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 2437 Pammel Drive, Molecular Biology Building, rm 4210, Ames, IA, 50011, USA.
Appl Microbiol Biotechnol. 2017 Dec;101(23-24):8431-8441. doi: 10.1007/s00253-017-8586-9. Epub 2017 Oct 26.
The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min mg. FabD R117A maintained K values for holo-ACP (~ 40 μM) and displayed small changes in K for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.
发酵系统生产新型生物可再生化学品的商业影响将随着合成新分子的酶的扩展而蓬勃发展。尽管脂肪酸生物合成存在一定程度的天然变异性，但通过酶工程获得的分子空间是无限的。原核脂肪酸生物合成酶在功能化酰基辅酶 A（ACP）上构建碳链，该 ACP 提供溶解性、稳定性和与合成酶相互作用的支架。在这里，我们确定大肠杆菌中的丙二酰辅酶 A（CoA）/全酰基辅酶 A（ACP）转酰基酶（FabD）是一种用于工程改造以多样化微生物脂肪酸生物合成的平台酶。FabD R117A 变体产生了用于脂肪酸生物合成的新型 ACP 基引物和延伸单元。与野生型酶高度特异性地使用丙二酰-CoA 生成丙二酰-ACP 不同，R117A 变体从全酰基-ACP 和相应的酰基辅酶 A 合成了乙酰-ACP、琥珀酰-ACP、异丁酰-ACP、2-丁烯酰-ACP 和 β-羟基丁酰-ACP 等，比活度为 3.7 至 120 nmol min mg。与丙二酰-CoA（80 ± 30 μM）相比，FabD R117A 对全酰基-ACP 的 K 值 (~40 μM) 和对乙酰乙酰-CoA（110 ± 30 μM）和乙酰-CoA（200 ± 70 μM）的 K 值变化较小。FabD R117A 代表了一种新型催化剂，可合成广泛的酰基-酰基-ACP。
