Genetic circuits that encode extracellular electron transfer (EET) pathways allow the intracellular state of Escherichia coli to be electronically monitored and controlled. However, relatively low electron flux flows through these pathways, limiting the degree of control by these circuits. Since the EET pathway is composed of multiple multiheme cytochromes c (cyts c) from Shewanella oneidensis MR-1, we hypothesized that lower expression levels of cyt c may explain this low EET flux and may be caused by the differences in the cyt c maturation (ccm) machinery between these two species. Here, we constructed random mutations within ccmH by error-prone PCR and screened for increased cyt c production. We identified two ccmH mutants, ccmH-132 and ccmH-195, that exhibited increased heterologous cyt c expression, but had different effects on EET. The ccmH-132 strain reduced WO3 nanoparticles faster than the parental control, whereas the ccmH-195 strain reduced more slowly. The same trend is reflected in electrical current generation: ccmH-132, which has only a single mutation from WT, drastically increased current production by 77%. The percentage of different cyt c proteins in these two mutants suggests that the stoichiometry of the S. oneidensis cyts c is a key determinant of current production by Mtr-expressing E. coli. Thus, we conclude that modulating cyt c maturation effectively improves genetic circuits governing EET in engineered biological systems, enabling better bioelectronic control of E. coli.

中文翻译：

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修改细胞色素c的成熟度可以提高工程大肠杆菌的生物电子性能。

编码细胞外电子转移（EET）途径的遗传电路可以对大肠杆菌的细胞内状态进行电子监测和控制。但是，相对较低的电子通量流经这些路径，从而限制了这些电路的控制程度。由于EET途径是由Shewanella oneidensis MR-1的多种多血红素细胞色素c（cyts c）组成的，因此我们假设cyt c的较低表达水平可能解释了这种低EET通量，并且可能是由于cyt c成熟的差异所致（ ccm）这两个物种之间的机械。在这里，我们通过易错PCR在ccmH内构建了随机突变，并筛选了增加的cyt c产量。我们鉴定了两个ccmH突变体ccmH-132和ccmH-195，它们显示出增加的异源cyt c表达，但对EET的影响不同。ccmH-132菌株还原WO3纳米颗粒的速度比亲代对照快，而ccmH-195菌株还原速度更慢。在电流产生中也反映了相同的趋势：ccmH-132（仅从WT发生了一次突变），使电流产生急剧增加了77％。在这两个突变体中不同的cyt c蛋白的百分比表明，拟南芥cyt c的化学计量是表达Mtr的大肠杆菌当前产量的关键决定因素。因此，我们得出结论，调节细胞成熟可以有效地改善工程化生物系统中控制EET的遗传电路，从而实现对大肠杆菌的更好生物电子控制。仅来自WT的一个突变的ccmH-132，使电流产生急剧增加了77％。在这两个突变体中不同的cyt c蛋白的百分比表明，拟南芥cyt c的化学计量是表达Mtr的大肠杆菌当前产量的关键决定因素。因此，我们得出结论，调节细胞成熟可以有效地改善工程化生物系统中控制EET的遗传电路，从而实现对大肠杆菌的更好生物电子控制。仅来自WT的一个突变的ccmH-132，使电流产生急剧增加了77％。在这两个突变体中不同的cyt c蛋白的百分比表明，拟南芥cyt c的化学计量是表达Mtr的大肠杆菌当前产量的关键决定因素。因此，我们得出结论，调节细胞成熟可以有效地改善工程化生物系统中控制EET的遗传电路，从而实现对大肠杆菌的更好生物电子控制。