Department of Civil and Environmental Engineering, Colorado School of Mines, Golden, CO 80401, USA.
Biochem Biophys Res Commun. 2012 Jan 13;417(2):704-9. doi: 10.1016/j.bbrc.2011.12.002. Epub 2011 Dec 8.
Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H(2) chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H(2) production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H(2) photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H(2) production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H(2)-photoproduction rates did not exceed those of wild-type cells.
莱茵衣藻（整个文本中均使用“莱茵衣藻”）编码两个[FeFe]-氢化酶，分别命名为 HYDA1 和 HYDA2。虽然 HYDA1 被认为是主要的氢化酶，但 HYDA2 的作用尚不清楚。为了研究每种氢化酶的单独功能，并为未来的生物工程提供一个平台，我们分离了莱茵衣藻 hydA1-1、hydA2-1 单突变体和 hydA1-1 hydA2-1 双突变体。我们使用反向遗传学筛选鉴定了一个 HYDA2 插入突变体，随后对 hydA2-1 菌株进行诱变，并结合 H2 化学传感器表型筛选分离 hydA1-1 hydA2-1 突变体。hydA1-1 hydA2-1 突变体与野生型细胞的遗传杂交使我们能够分离出单突变体 hydA1-1。在每种突变基因型中均测定了发酵、光合和体外氢化酶活性。令人惊讶的是，对 hydA1-1 和 hydA2-1 单突变体以及 HYDA1 和 HYDA2 拯救的 hydA1-1 hydA2-1 突变体的分析表明，两种氢化酶都能够催化发酵或光合作用途径产生 H2。突变体和互补菌株的生理学表明，HYDA2 对 H2 光生产的贡献约为 HYDA1 的 25%，这与在 hydA1-1 突变体中测量的体外氢化酶活性水平相似。有趣的是，在互补了 HYDA1 的 hydA1-1 hydA2-1 菌株中观察到体外和发酵 H2 产生活性增强，而最大 H2 光生产速率并未超过野生型细胞。
