PDF(Pubmed)
Abstract：
To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering remain scarce. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous xylose isomerase pathway remained unresolved. Here, we elucidate the xylose metabolism and establish a foundation for further engineering followed by ALE. First, native glycolysis is derepressed by deleting the local transcriptional regulator gene hexR. We then enhance the pentose phosphate pathway by implanting exogenous transketolase and transaldolase into two lag-shortened strains and allow ALE to finetune the rewired metabolism. Subsequent multilevel analysis and reverse engineering provide detailed insights into the parallel paths of bacterial adaptation to the non-native carbon source, highlighting the enhanced expression of transaldolase and xylose isomerase along with derepressed glycolysis as key events during the process.
摘要：
为了将微生物细胞工厂的底物范围扩大到可再生底物，合理的遗传干预通常与适应性实验室进化(ALE)相结合。然而,通过理性代谢工程启动的全面研究能够全面了解适应过程仍然很少。工业主力恶臭假单胞菌被设计为利用非天然糖D-木糖，但是它通过外源木糖异构酶途径同化到细菌生化网络中仍未解决。这里,我们阐明了木糖的代谢，并为ALE的进一步工程奠定了基础。首先,通过删除局部转录调节基因hexR来抑制天然糖酵解。然后，我们通过将外源转酮醇酶和转醛缩酶植入两个滞后缩短的菌株中来增强戊糖磷酸途径，并使ALE可以改善重新连接的代谢。随后的多水平分析和逆向工程提供了对细菌适应非天然碳源的平行路径的详细见解。强调转醛缩酶和木糖异构酶的表达增强以及抑制糖酵解是该过程中的关键事件。
