Engineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 h when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 h. We also engineered a E. coli strain to produce 24 mg/L of prenol in an ethanol-containing medium, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds.

利用非常规底物的工程微生物可以创建短而有效的途径将底物转化为产品。在这项研究中，我们使用来自Dickeya zeae 的乙醛脱氢酶（由ada编码）和来自酿酒酵母的乙醇脱氢酶（由adh2编码）在大肠杆菌中设计并构建了一个两步异源乙醇利用途径（EUP）。这种EUP可以在不消耗ATP的情况下将乙醇转化为乙酰辅酶A，并且每分子乙醇产生两分子NADH。我们优化了这两个基因的表达，发现可以通过以特定顺序（ada-adh2) 与组成型启动子 (PgyrA)。当乙醇用作唯一碳源时，带有 EUP的工程大肠杆菌菌株在 96 小时内消耗了大约 8 g/L 的乙醇。随后，我们将 EUP 与聚羟基丁酸 (PHB) 的生物合成相结合，聚羟基丁酸 (PHB) 是一种源自乙酰辅酶 A 的可生物降解聚合物。携带 EUP 和 PHB 生物合成途径的工程大肠杆菌菌株在 96 小时内从 10 g/L 乙醇和 1 g/L 天冬氨酸家族氨基酸中产生 1.1 g/L PHB。我们还设计了一种大肠杆菌菌株，以在含乙醇的培养基中生产 24 mg/L 的异戊烯醇，支持将乙醇转化为不同类别的乙酰辅酶 A 衍生化合物的可行性。