Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI, 53706, USA.
DOE Center for Advanced Bioenergy and Bioproducts Innovation, University of Wisconsin-Madison, Madison, WI, 53706, USA.
Nat Commun. 2022 Mar 25;13(1):1619. doi: 10.1038/s41467-022-29218-3.
Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA's that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.
微生物脂质代谢是生产油脂化学品的有吸引力的途径。主要策略集中在异源硫酯酶上，以合成所需链长的脂肪酸。为了将酸转化为油脂化学品（例如脂肪酸醇、酮），需要将缩小的脂肪酸池重新激活为辅酶 A 硫酯，每次重新激活需要消耗一个 ATP——如果酰基链可以直接从 ACP 转移到 CoA-硫酯，就可以节省这笔费用。在这里，我们通过异源表达 PhaG 展示了这种替代酰基转移酶策略，PhaG 是一种最初在假单胞菌中发现的酶，它可以在酰基载体蛋白和辅酶 A 硫酯形式之间转移 3-羟基酰基链，用于生成聚羟基烷酸单体。我们使用它来创建酰基辅酶 A 的池，这些酰基辅酶 A 可以被重新定向到油脂化学品产品中。通过生物勘探、诱变和代谢工程，我们开发了三株大肠杆菌，能够生产超过 1g/L 的中链游离脂肪酸、脂肪酸醇和甲基酮。
