Department of Biology and Biological Engineering, Division of Industrial Biotechnology, Chalmers University of Technology, Gothenburg, Sweden.
Department of Chemistry and Chemical Engineering, Division of Chemistry and Biochemistry, Gothenburg, Sweden.
PLoS One. 2018 Feb 23;13(2):e0193503. doi: 10.1371/journal.pone.0193503. eCollection 2018.
The biobased production of adipic acid, a precursor in the production of nylon, is of great interest in order to replace the current petrochemical production route. Glucose-rich lignocellulosic raw materials have high potential to replace the petrochemical raw material. A number of metabolic pathways have been proposed for the microbial conversion of glucose to adipic acid, but achieved yields and titers remain to be improved before industrial applications are feasible. One proposed pathway starts with lysine, an essential metabolite industrially produced from glucose by microorganisms. However, the drawback of this pathway is that several reactions are involved where there is no known efficient enzyme. By changing the order of the enzymatic reactions, we were able to identify an alternative pathway with one unknown enzyme less compared to the original pathway. One of the reactions lacking known enzymes is the reduction of the unsaturated α,β bond of 6-amino-trans-2-hexenoic acid and trans-2-hexenedioic acid. To identify the necessary enzymes, we selected N-ethylmaleimide reductase from Escherichia coli and Old Yellow Enzyme 1 from Saccharomyces pastorianus. Despite successful in silico docking studies, where both target substrates could fit in the enzyme pockets, and hydrogen bonds with catalytic residues of both enzymes were predicted, no in vitro activity was observed. We hypothesize that the lack of activity is due to a difference in electron withdrawing potential between the naturally reduced aldehyde and the carboxylate groups of our target substrates. Suggestions for protein engineering to induce the reactions are discussed, as well as the advantages and disadvantages of the two metabolic pathways from lysine. We have highlighted bottlenecks associated with the lysine pathways, and proposed ways of addressing them.
生物基己二酸的生产是一个很有吸引力的课题，因为它可以替代目前的石化生产路线，用于生产尼龙的前体。富含葡萄糖的木质纤维素原料具有很高的潜力来替代石化原料。已经提出了许多代谢途径，用于将葡萄糖微生物转化为己二酸，但在工业应用可行之前，还需要提高产率和浓度。一种提议的途径始于赖氨酸，它是一种由微生物从葡萄糖工业生产的必需代谢物。然而，该途径的缺点是有几个反应涉及到没有已知的高效酶。通过改变酶反应的顺序，我们能够确定与原始途径相比，少了一个未知酶的替代途径。缺乏已知酶的反应之一是 6-氨基-trans-2-己烯酸和 trans-2-己二烯二酸的不饱和 α,β 键的还原。为了鉴定必需的酶，我们从大肠杆菌中选择了 N-乙基马来酰亚胺还原酶，从酿酒酵母中选择了 Old Yellow Enzyme 1。尽管成功地进行了基于计算机的对接研究，其中两个目标底物都可以适应酶的口袋，并且预测了与两个酶的催化残基的氢键，但没有观察到体外活性。我们假设缺乏活性是由于天然还原醛和我们目标底物的羧酸盐之间的电子吸电能力的差异所致。讨论了诱导反应的蛋白质工程建议，以及从赖氨酸出发的两种代谢途径的优缺点。我们强调了与赖氨酸途径相关的瓶颈，并提出了解决这些瓶颈的方法。
