Systems-level Analysis of Oxygen Exposure in Zymomonas mobilis: Implications for Isoprenoid Production

Zymomonas mobilis is an aerotolerant anaerobe and prolific ethanologen with attractive characteristics for industrial bioproduct generation. However, there is currently insufficient knowledge of the impact that environmental factors have on flux through industrially relevant biosynthetic pathways. Here, we examine the effect of oxygen exposure on metabolism and gene expression in Z. mobilis by combining targeted metabolomics, mRNA sequencing, and shotgun proteomics. We found that exposure to oxygen profoundly influenced metabolism, inducing both transient metabolic bottlenecks and long-term metabolic remodeling. In particular, oxygen induced a severe but temporary metabolic bottleneck in the methyl erythritol 4-phosphate pathway for isoprenoid biosynthesis caused by oxidative damage to the iron-sulfur co-factors of the final two enzymes of the pathway. This bottleneck was resolved with minimal changes in expression of isoprenoid biosynthetic enzymes. Instead, it was associated with pronounced upregulation of enzymes related to iron-sulfur cluster maintenance and biogenesis (i.e., flavodoxin reductase and the suf operon). We also detected major changes in glucose utilization in the presence of oxygen. Specifically, we observed increased gluconate production following exposure to oxygen, accounting for 18% of glucose uptake. Our results suggest that under aerobic conditions, electrons derived from the oxidation of glucose to gluconate are diverted to the electron transport chain where they can minimize oxidative damage by reducing reactive oxygen species such as H2O2. This model is supported by the simultaneous upregulation of three membrane-bound dehydrogenases, cytochrome c peroxidase, and a cytochrome bd oxidase following exposure to oxygen. Oxygen exposure timecourse. Z. mobilis cultures grown under anaerobic conditions were transferred to aerobic conditions during mid-log phase. A time zero sample was taken just before transfer. mRNA extractions were performed at 5, 15, 30, 45, 60, and 120 minutes after transfer to oxygen. 4 replicates for samples transferred to aerobic conditions (O2). Control samples were also collected from cultures that remained under anaerobic conditions at 60 and 120 minutes after the time zero sample. 3 replicates for anaerobic controls (An).

运动发酵单胞菌（Zymomonas mobilis）是一类耐氧厌氧菌，同时也是高产乙醇菌株，具备工业生物制品生产的优良特性。然而目前我们对环境因素如何影响工业相关生物合成途径的代谢流的认知仍存在不足。本研究结合靶向代谢组学（targeted metabolomics）、mRNA测序（mRNA sequencing）及鸟枪蛋白质组学（shotgun proteomics），探究了氧气暴露对运动发酵单胞菌代谢与基因表达的影响。研究发现，氧气暴露会显著调控菌体代谢，诱导产生瞬时代谢阻滞与长期代谢重编程。其中，异戊二烯生物合成的甲基赤藓糖醇4-磷酸（methyl erythritol 4-phosphate）途径中，途径最后两个酶的铁硫辅基（iron-sulfur co-factors）受到氧化损伤，会引发严重但短暂的代谢阻滞。该代谢阻滞并未通过上调异戊二烯生物合成酶的表达得以缓解，反而与铁硫簇维持与生物合成相关酶（即黄素氧还蛋白还原酶及suf操纵子）的显著上调密切相关。本研究还检测到氧气存在下葡萄糖利用的显著变化：具体而言，氧气暴露后葡萄糖酸的产量显著上升，其占葡萄糖摄取量的18%。研究结果表明，在有氧条件下，葡萄糖氧化为葡萄糖酸所产生的电子会被分流至电子传递链，通过还原过氧化氢（H₂O₂）等活性氧物种以减轻氧化损伤。该模型得到了以下实验现象的支持：氧气暴露后，三种膜结合脱氢酶、细胞色素c过氧化物酶（cytochrome c peroxidase）及细胞色素bd氧化酶（cytochrome bd oxidase）的表达同时上调。氧气暴露时间进程实验：将厌氧培养至对数中期的运动发酵单胞菌培养液转接至有氧环境，于转接前采集时间零点样本。有氧组（O₂）于转接后5、15、30、45、60及120分钟时取样提取mRNA，每组设置4次生物学重复。同时设置厌氧对照组（An），于时间零点后的60、120分钟从维持厌氧环境的培养液中取样，每组设置3次生物学重复。