Escuela de Ingeniería Química, Universidad del Valle, Cali, A.A. 25360, Colombia.
Chair of Bioprocess Engineering, Department of Biotechnology, Technische Universität Berlin, Ackerstr. 76, ACK 24, 13355, Berlin, Germany.
Appl Microbiol Biotechnol. 2018 May;102(9):4009-4023. doi: 10.1007/s00253-018-8841-8. Epub 2018 Mar 9.
Clavulanic acid (CA) is produced by Streptomyces clavuligerus (S. clavuligerus) as a secondary metabolite. Knowledge about the carbon flux distribution along the various routes that supply CA precursors would certainly provide insights about metabolic performance. In order to evaluate metabolic patterns and the possible accumulation of tricarboxylic acid (TCA) cycle intermediates during CA biosynthesis, batch and subsequent continuous cultures with steadily declining feed rates were performed with glycerol as the main substrate. The data were used to in silico explore the metabolic capabilities and the accumulation of metabolic intermediates in S. clavuligerus. While clavulanic acid accumulated at glycerol excess, it steadily decreased at declining dilution rates; CA synthesis stopped when glycerol became the limiting substrate. A strong association of succinate, oxaloacetate, malate, and acetate accumulation with CA production in S. clavuligerus was observed, and flux balance analysis (FBA) was used to describe the carbon flux distribution in the network. This combined experimental and numerical approach also identified bottlenecks during the synthesis of CA in a batch and subsequent continuous cultivation and demonstrated the importance of this type of methodologies for a more advanced understanding of metabolism; this potentially derives valuable insights for future successful metabolic engineering studies in S. clavuligerus.
克拉维酸（CA）是由链霉菌属（S. clavuligerus）产生的一种次级代谢产物。了解为 CA 前体提供各种途径的碳通量分布情况，无疑会深入了解代谢性能。为了评估 CA 生物合成过程中的代谢模式和三羧酸（TCA）循环中间产物的可能积累情况，我们以甘油为主要底物进行了分批和随后的连续培养，且进料速率逐渐降低。利用这些数据，我们在计算机上探索了链霉菌属中的代谢能力和代谢中间产物的积累情况。当甘油过剩时，克拉维酸会积累，但随着稀释率的降低，其含量会逐渐下降；当甘油成为限制因素时，CA 的合成就会停止。我们观察到链霉菌属中琥珀酸、草酰乙酸、苹果酸和乙酸的积累与 CA 产量之间存在很强的相关性，并且还使用通量平衡分析（FBA）来描述网络中的碳通量分布。这种结合了实验和数值方法的方法还确定了分批和随后的连续培养过程中 CA 合成过程中的瓶颈，并证明了这种方法对于更深入了解代谢的重要性；这可能为未来在链霉菌属中的成功代谢工程研究提供有价值的见解。
