Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan.
Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, Miyazaki, Japan.
Appl Environ Microbiol. 2021 May 26;87(12):e0261720. doi: 10.1128/AEM.02617-20.
An outer membrane -type cytochrome (OmcZ) in Geobacter sulfurreducens is essential for optimal current production in microbial fuel cells. OmcZ exists in two forms, small and large, designated OmcZ and OmcZ, respectively. However, it is still not known how these two structures are formed. A mutant with a disruption of the GSU2075 gene encoding a subtilisin-like serine protease (designated for the mcrotease), which is located downstream of , produced low currents at a level similar to that of the -deficient mutant strain. Biochemical analyses revealed that the mutant accumulated OmcZ and did not produce OmcZ, which is thought to be a mature form that is essential for the extracellular electron transfer to the electrode. A heterologous expression system cell lysate from an Escherichia coli strain producing OzpA cleaved OmcZ and generated OmcZ as the proteolytic product. Among the culture supernatant, loosely bound outer surface, and intracellular protein fractions from wild-type G. sulfurreducens, only the culture supernatant protein fraction showed OmcZ cleavage activity, indicating that the mature form of OmcZ, OmcZ, can be produced outside the cells. These results indicate that OzpA is an essential protease for current production via the maturation of OmcZ, and OmcZ is the key to the extracellular electron transfer to electrodes. This proteolytic maturation of OmcZ is a unique regulation among known -type cytochromes in G. sulfurreducens. Microbial fuel cells are a promising technology for energy generation from various waste types. However, the molecular mechanisms of microbial extracellular electron transfer to the electrode need to be elucidated. G. sulfurreducens is a common key player in electricity generation in mixed-culture microbial fuel cell systems and a model microorganism for the study of extracellular electron transfer. Outer membrane -type cytochrome OmcZ is essential for an optimal current production by G. sulfurreducens. OmcZ proteolytic cleavage occurs during maturation, but the underlying mechanism is unknown. This study identifies a subtilisin-like protease, OzpA, which plays a role in cleaving OmcZ and generating the mature form of OmcZ (OmcZ). OzpA is essential for current production and, thus, the proteolytic maturation of OmcZ. This is a novel regulation of the -type cytochrome for G. sulfurreducens extracellular electron transfer. This study also provides new insights into the design strategy and development of microbial extracellular electron transfer for an efficient energy conversion from chemical energy to electricity.
在产电菌 Geobacter sulfurreducens 中，一种外膜细胞色素（OmcZ）对于微生物燃料电池中最佳电流产生是必需的。OmcZ 存在两种形式，小和大，分别命名为 OmcZ 和 OmcZ。然而，目前尚不清楚这两种结构是如何形成的。一个破坏编码枯草溶菌素样丝氨酸蛋白酶（命名为 mcrotease）的 GSU2075 基因的突变体，该蛋白酶位于基因下游，产生的电流水平与缺陷突变体相似。生化分析表明，突变体积累 OmcZ 但不产生 OmcZ，这被认为是一种成熟形式，对于将电子从细胞外转移到电极至关重要。一个异源表达系统的大肠杆菌细胞裂解物能够切割 OmcZ 并生成 OmcZ 作为蛋白水解产物。在野生型 G. sulfurreducens 的培养物上清液、松散结合的外表面和细胞内蛋白馏分中，只有培养物上清液蛋白馏分显示 OmcZ 切割活性，表明 OmcZ 的成熟形式 OmcZ 可以在细胞外产生。这些结果表明，OzpA 是一种必需的蛋白酶，通过 OmcZ 的成熟来产生电流，OmcZ 是电子从细胞外向电极转移的关键。这种 OmcZ 的蛋白水解成熟是 G. sulfurreducens 中已知的 -型细胞色素的一种独特调节。微生物燃料电池是一种很有前途的技术，可以从各种废物类型中产生能量。然而，微生物细胞外电子向电极的转移的分子机制仍需阐明。G. sulfurreducens 是混合培养微生物燃料电池系统中发电的常见关键参与者，也是研究细胞外电子转移的模式微生物。外膜型细胞色素 OmcZ 对于 G. sulfurreducens 产生最佳电流是必需的。OmcZ 的蛋白水解切割发生在成熟过程中，但潜在的机制尚不清楚。本研究鉴定了一种枯草溶菌素样蛋白酶 OzpA，它在 OmcZ 的切割和生成成熟形式 OmcZ（OmcZ）中发挥作用。OzpA 对于电流产生是必需的，因此，OmcZ 的蛋白水解成熟是必需的。这是 G. sulfurreducens 细胞外电子转移的 -型细胞色素的一种新的调节。本研究还为微生物细胞外电子转移的设计策略和开发提供了新的见解，以实现从化学能到电能的高效能量转换。
