Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H2 production

Abstract

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.

Hypoxic survival requires a 2-on-2 hemoglobin in a process involving nitric oxide

“…The physiological role of HYDA2 is unclear. A mutant deficient for HYDA1 has much lower H 2 -production rates in the light whereas dark H 2 production is not impaired in this strain (24). It might be speculated that HYDA2 becomes more important during fermentative H 2 generation in the dark.…”

“…Transcription of the HYDA1 gene, encoding the major [FeFe]-hydrogenase isoform of C. reinhardtii (24,25) and inducible by anaerobiosis (26), was similar in strains amiTHB8-1 and -2 vs. strain CC-124 (Fig. 4C).…”

“…4C) compared with wild-type amounts. HYDA2 encodes a second [FeFe]-hydrogenase HYDA2 of C. reinhardtii (25), which contributes to about 25% of maximal hydrogenase enzyme activity in anaerobic algal cells (24). The physiological role of HYDA2 is unclear.…”

A novel screening method for hydrogenase-deficient mutants in Chlamydomonas reinhardtii based on in vivo chlorophyll fluorescence and photosystem II quantum yield

“…HydA2 [18,19], HydA1 being responsible for the major hydrogenase activity under anaerobic conditions [20,21]. To date, two assembly factors (HydEF and HydG) belonging to the "Sadenosylmethionine radical" protein super-family have been proved to be required for the proper maturation of the H-cluster of [FeFe] hydrogenase [22].…”

Alternative Acetate Production Pathways inChlamydomonas reinhardtiiduring Dark Anoxia and the Dominant Role of Chloroplasts in Fermentative Acetate Production

“…Aspects of Chlamydomonas metabolism appear to have significant flexibility, as established by physiological/metabolic studies with wild type and mutants aberrant for aspects of fermentation metabolism Gibbs, 1984, 1985;Kreuzberg, 1984;Gibbs et al, 1986;Ohta et al, 1987;Hemschemeier and Happe, 2005;Atteia et al, 2006;Mus et al, 2007;Dubini et al, 2009;Timmins et al, 2009;Grossman et al, 2011;Philipps et al, 2011;Burgess et al, 2012;Catalanotti et al, 2012Catalanotti et al, , 2013Magneschi et al, 2012;Meuser et al, 2012;Yang et al, 2014; Figure 1), global examination of gene expression as cells acclimate to anoxic conditions (Mus et al, 2007), and analysis of the Chlamydomonas genome Merchant et al, 2007). The two dominant fermentative pathways that putatively consume pyruvate under anoxic, laboratory conditions involve the enzymes pyruvate formate lyase (PFL1) and pyruvate ferredoxin oxidoreductase (PFR1).…”