Effects of modulation of pentose-phosphate pathway on biosynthesisof ansamitocins in Actinosynnema pretiosum

Ansamitocins, produced by Actinosynnema pretiosum, are a group of maytansinoid antibiotics that block the assembly of tubulin into functional microtubules. The precursors of ansamitocin biosynthesis are generally derived from the Embden-Meyerhof-Parnas (EMP) pathway and the tricarboxylic acid cycle. In this study, central carbon flux distributions were analyzed by C-13-based flux analysis to reveal the contribution of individual central carbon metabolism pathways. To direct more carbon flux into ansamitocin biosynthesis, pentose phosphate (PP) pathway only and the combination of PP pathway and Entner-Doudoroff (ED) pathway were weakened, respectively. Ansamitocin P-3 (AP-3) productions by both kinds of pathways weakened mutant strains were significantly enhanced in chemically defined medium. In order to draw metabolic flux to the biosynthesis of ansamitocins more efficiently, heterologous phosphoglucomutase was subsequently overexpressed based on a mutant strain with combinational regulation of PP pathway and ED pathway. More fluxes were successfully directed into the UDP-glucose synthetic pathway and the AP-3 production was further improved in this case, reaching approximately 185 mg/L in fermentation medium. It was demonstrated that eliminating the bypass pathways and favoring the precursor synthetic pathway could effectively improve ansamitocin production by A. pretiosum, suggesting a promising role of metabolic strategy in improving secondary metabolite production. (C) 2016 Elsevier B.V. All rights reserved.

安丝菌素由珍贵束丝放线菌（Actinosynnema pretiosum）产生，是一类美登素类抗生素，可阻止微管蛋白组装成功能性微管。安丝菌素生物合成的前体通常来源于糖酵解途径（Embden - Meyerhof - Parnas，EMP）和三羧酸循环。在本研究中，通过基于碳 - 13的通量分析来分析中心碳通量分布，以揭示各个中心碳代谢途径的贡献。为了使更多的碳通量进入安丝菌素的生物合成，分别削弱了磷酸戊糖（PP）途径以及磷酸戊糖途径和2 - 酮 - 3 - 脱氧 - 6 - 磷酸葡萄糖酸（Entner - Doudoroff，ED）途径的组合。在化学成分确定的培养基中，这两种途径被削弱的突变株的安丝菌素P - 3（AP - 3）产量均显著提高。为了更有效地将代谢通量引向安丝菌素的生物合成，随后在一个对磷酸戊糖途径和2 - 酮 - 3 - 脱氧 - 6 - 磷酸葡萄糖酸途径进行组合调控的突变株基础上，过量表达异源磷酸葡萄糖变位酶。在这种情况下，更多的通量成功地被导向尿苷二磷酸葡萄糖合成途径，并且AP - 3的产量进一步提高，在发酵培养基中达到约185mg/L。研究表明，消除旁路途径并有利于前体合成途径可以有效提高珍贵束丝放线菌的安丝菌素产量，这表明代谢策略在提高次生代谢产物产量方面具有良好的应用前景。（C）2016爱思唯尔有限公司。保留所有权利。