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Abstract：
Isoprenoids are a diverse family of compounds that are synthesized from two isomeric compounds, isopentenyl diphosphate and dimethylallyl diphosphate. In most bacteria, isoprenoids are produced from the essential methylerythritol phosphate (MEP) pathway. The terminal enzymes of the MEP pathway IspG and IspH are [4Fe-4S] cluster proteins, and in Zymomonas mobilis, the substrates of IspG and IspH accumulate in cells in response to O2, suggesting possible lability of their [4Fe-4S] clusters. Here, we show using complementation assays in Escherichia coli that even under anaerobic conditions, Z. mobilis IspG and IspH are not as functional as their E. coli counterparts, requiring higher levels of expression to rescue viability. A deficit of the sulfur utilization factor (SUF) Fe-S cluster biogenesis pathway did not explain the reduced function of Z. mobilis IspG and IspH since no improvement in viability was observed in E. coli expressing the Z. mobilis SUF pathway or having increased expression of the E. coli SUF pathway. Complementation of single and double mutants with various combinations of Z. mobilis and E. coli IspG and IspH indicated that optimal growth required the pairing of IspG and IspH from the same species. Furthermore, Z. mobilis IspH conferred an O2-sensitive growth defect to E. coli that could be partially rescued by co-expression of Z. mobilis IspG. In vitro analysis showed O2 sensitivity of the [4Fe-4S] cluster of both Z. mobilis IspG and IspH. Altogether, our data indicate an important role of the cognate protein IspG in Z. mobilis IspH function under both aerobic and anaerobic conditions.
OBJECTIVE: Isoprenoids are one of the largest classes of natural products, exhibiting diversity in structure and function. They also include compounds that are essential for cellular life across the biological world. In bacteria, isoprenoids are derived from two precursors, isopentenyl diphosphate and dimethylallyl diphosphate, synthesized primarily by the methylerythritol phosphate pathway. The aerotolerant Z. mobilis has the potential for methylerythritol phosphate pathway engineering by diverting some of the glucose that is typically efficiently converted into ethanol to produce isoprenoid precursors to make bioproducts and biofuels. Our data revealed the surprising finding that Z. mobilis IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway in part to evade the oxygen sensitivity of IspH.
OBJECTIVE: Isoprenoids are one of the largest classes of natural products, exhibiting diversity in structure and function. They also include compounds that are essential for cellular life across the biological world. In bacteria, isoprenoids are derived from two precursors, isopentenyl diphosphate and dimethylallyl diphosphate, synthesized primarily by the methylerythritol phosphate pathway. The aerotolerant Z. mobilis has the potential for methylerythritol phosphate pathway engineering by diverting some of the glucose that is typically efficiently converted into ethanol to produce isoprenoid precursors to make bioproducts and biofuels. Our data revealed the surprising finding that Z. mobilis IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway in part to evade the oxygen sensitivity of IspH.
摘要：
类异戊二烯是由两种异构化合物合成的多种化合物家族，异戊烯二磷酸和二甲基烯丙基二磷酸。在大多数细菌中，类异戊二烯由必需的甲基赤藓糖醇磷酸(MEP)途径产生。MEP途径IspG和IspH的末端酶是[4Fe-4S]簇蛋白，在运动发酵单胞菌中，响应O2，IspG和IspH的底物在细胞中积累，表明它们的[4Fe-4S]簇可能不稳定。这里,我们表明，即使在厌氧条件下，在大肠杆菌中使用互补测定，Z.mobilisIspG和IspH的功能不如其大肠杆菌对应物，需要更高水平的表达来拯救生存能力。硫利用因子(SUF)Fe-S簇生物生成途径的缺陷并不能解释Z.mobilisisIspG和IspH的功能降低,因为在表达Z.mobilisisSUF途径或具有增加的大肠杆菌SUF途径表达的大肠杆菌中未观察到活力的改善。用Z.mobilis和大肠杆菌IspG和IspH的各种组合补充单突变体和双突变体表明最佳生长需要来自相同物种的IspG和IspH的配对。此外，Z.mobilisIspH赋予大肠杆菌O2敏感的生长缺陷，可以通过共表达Z.mobilisIspG部分挽救。体外分析显示Z.mobilisIspG和IspH的[4Fe-4S]簇的O2敏感性。总之,我们的数据表明，在有氧和厌氧条件下，同源蛋白IspG在Z.mobilisIspH功能中的重要作用。
目的：类异戊二烯是最大的一类天然产物，在结构和功能上表现出多样性。它们还包括对整个生物世界的细胞生命至关重要的化合物。在细菌中，类异戊二烯衍生自两种前体，异戊烯二磷酸和二甲基烯丙基二磷酸，主要由甲基赤藓糖醇磷酸途径合成。耐氧Z.mobilis具有通过将一些通常有效转化为乙醇的葡萄糖转移以产生类异戊二烯前体以制备生物产品和生物燃料而进行甲基赤藓糖醇磷酸途径工程改造的潜力。我们的数据揭示了令人惊讶的发现,即需要共同优化Z.mobilisisIspG和IspH以改善经由赤藓糖醇磷酸甲酯途径的通量,部分地规避IspH的氧敏感性。
目的：类异戊二烯是最大的一类天然产物，在结构和功能上表现出多样性。它们还包括对整个生物世界的细胞生命至关重要的化合物。在细菌中，类异戊二烯衍生自两种前体，异戊烯二磷酸和二甲基烯丙基二磷酸，主要由甲基赤藓糖醇磷酸途径合成。耐氧Z.mobilis具有通过将一些通常有效转化为乙醇的葡萄糖转移以产生类异戊二烯前体以制备生物产品和生物燃料而进行甲基赤藓糖醇磷酸途径工程改造的潜力。我们的数据揭示了令人惊讶的发现,即需要共同优化Z.mobilisisIspG和IspH以改善经由赤藓糖醇磷酸甲酯途径的通量,部分地规避IspH的氧敏感性。
