Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. This discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.

中文翻译：

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在细菌中分解代谢乙酰丙酸的代谢途径。

微生物可以分解多种有机化合物，因此具有进行许多与工业相关的生物转化的潜力。实现生物精炼策略潜力的一个障碍是我们对代谢途径的不完全了解，包括那些可用于吸收天然丰富或容易产生的原料的途径。例如，乙酰丙酸（LA）是一种碳源，可以很容易地作为木质纤维素生物质的脱水产物获得，并且可以用作某些细菌的唯一碳源。然而，LA分解代谢的遗传学和结构仍然未知。在这里，我们报告的鉴定和表征的七基因操纵子，使恶臭假单胞菌KT2440的LA分解代谢。当该路径被纯化的蛋白质重建后，我们观察到了四个酰基辅酶A中间体的形成，包括一个独特的4-磷戊酰基辅酶A和先前观察到的3-羟基戊酰基辅酶A产物。使用适应性进化，我们获得了具有fadE和atoC功能缺失的大肠杆菌LS5218突变体，当它表达恶臭假单胞菌操纵子中的五种酶时，它们能够在LA上稳定生长。这一发现将使人们能够更有效地利用生物质水解产物和代谢工程技术，以使用LA作为原料进行生物转化。普蒂达操纵子。这一发现将使人们能够更有效地利用生物质水解产物和代谢工程技术，以使用LA作为原料进行生物转化。普蒂达操纵子。这一发现将使人们能够更有效地利用生物质水解产物和代谢工程技术，以使用LA作为原料进行生物转化。