Efficient microbial synthesis of chemicals requires the coordinated supply of precursors and cofactors to maintain cell growth and product formation. Substrates with different entry points into the metabolic network have different energetic and redox statuses. Generally, substrate cofeeding could bypass the lengthy and highly regulated native metabolism and facilitates high carbon conversion rate. Aiming to efficiently synthesize the high-value rose-smell 2-phenylethanol (2-PE) in Y. lipolytica, we analyzed the stoichiometric constraints of the Ehrlich pathway and identified that the selectivity of the Ehrlich pathway and the availability of 2-oxoglutarate are the rate-limiting factors. Stepwise refactoring of the Ehrlich pathway led us to identify the optimal catalytic modules consisting of l-phenylalanine permease, ketoacid aminotransferase, phenylpyruvate decarboxylase, phenylacetaldehyde reductase, and alcohol dehydrogenase. On the other hand, mitochondrial compartmentalization of 2-oxoglutarate inherently creates a bottleneck for efficient assimilation of l-phenylalanine, which limits 2-PE production. To improve 2-oxoglutarate (aKG) trafficking across the mitochondria membrane, we constructed a cytosolic aKG source pathway by coupling a bacterial aconitase with a native isocitrate dehydrogenase (ylIDP2). Additionally, we also engineered dicarboxylic acid transporters to further improve the 2-oxoglutarate availability. Furthermore, by blocking the precursor-competing pathways and mitigating fatty acid synthesis, the engineered strain produced 2669.54 mg/L of 2-PE in shake flasks, a 4.16-fold increase over the starting strain. The carbon conversion yield reaches 0.702 g/g from l-phenylalanine, 95.0% of the theoretical maximal. The reported work expands our ability to harness the Ehrlich pathway for production of high-value aromatics in oleaginous yeast species.

中文翻译：

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重构解脂耶氏酵母中高产2-苯基乙醇的Ehrlich途径。

化学品的有效微生物合成需要前体和辅助因子的协调供应，以维持细胞生长和产物形成。具有不同进入代谢网络入口点的底物具有不同的能量和氧化还原状态。通常，底物共同进料可以绕开冗长且高度受控的天然代谢，并促进高碳转化率。为了有效地合成解脂耶氏酵母中的高价值玫瑰味2-苯基乙醇（2-PE），我们分析了Ehrlich途径的化学计量约束，并确定了Ehrlich途径的选择性和2-氧戊二酸酯的可用性限速因素。Ehrlich途径的逐步重构使我们确定了由L-苯丙氨酸通透酶，酮酸转氨酶，苯丙酮酸脱羧酶，苯乙醛还原酶和醇脱氢酶。另一方面，2-氧戊二酸酯的线粒体区室化固有地产生了有效吸收1-苯丙氨酸的瓶颈，这限制了2-PE的生产。为了改善2-氧戊二酸（aKG）跨线粒体膜的运输，我们通过将细菌乌头酸酶与天然异柠檬酸脱氢酶（ylIDP2）偶联来构建胞质aKG源途径。此外，我们还设计了二羧酸转运蛋白，以进一步提高2-氧戊二酸的利用率。此外，通过阻断前体竞争途径并减轻脂肪酸合成，工程菌株在摇瓶中产生了2669.54 mg / L的2-PE，比起始菌株增加了4.16倍。碳转化率达到0。来自1-苯丙氨酸的702g / g，为理论最大值的95.0％。报道的工作扩大了我们利用Ehrlich途径在油质酵母物种中生产高价值芳烃的能力。