Hydrogen Photoproduction Is Attenuated by Disruption of an Isoamylase Gene in Chlamydomonas reinhardtii

Abstract

DNA insertional transformants of Chlamydomonas reinhardtii were screened chemochromically for attenuated H 2 production. One mutant, displaying low H 2 gas photoproduction, has a nonfunctional copy of a gene that shows high homology to the family of isoamylase genes found in several photosynthetic organisms. DNA gel blotting and gene complementation were used to link this isoamylase gene to previously characterized nontagged sta7 mutants. This mutant is therefore denoted sta7-10. In C. reinhardtii, the STA7 isoamylase gene is important for the accumulation of crystalline starch, and the sta7-10 mutant reported here contains <3% of the glucose found in insoluble starch when compared with wild-type control cells. Hydrogen photoproduction rates, induced after several hours of dark, anaerobic treatment, are attenuated in sta7 mutants. RNA gel blot analysis indicates that the mRNA transcripts for both the HydA1 and HydA2 [Fe]-hydrogenase genes are expressed in the sta7-10 mutant at greater than wild-type levels 0.5 h after anaerobic induction. However, after 1.5 h, transcript levels of both HydA1 and HydA2 begin to decline rapidly and reach nearly undetectable levels after 7 h. In wildtype cells, the hydrogenase transcripts accumulate more slowly, reach a plateau after 4 h of anaerobic treatment, and maintain the same level of expression for >7 h under anaerobic incubation. Complementation of mutant cells with genomic DNA corresponding to the STA7 gene restores both the starch accumulation and H 2 production phenotypes. The results indicate that STA7 and starch metabolism play an important role in C. reinhardtii H 2 photoproduction. Moreover, the results indicate that mere anaerobiosis is not sufficient to maintain hydrogenase gene expression without the underlying physiology, an important aspect of which is starch metabolism.

Rewiring hydrogenase-dependent redox circuits in cyanobacteria

“…To express hydrogenase maturation factors HydEF and HydG (29), we cloned these genes separately, placing each of them downstream of the S. elongatus psba1 promoter, which normally constitutively expresses the D 1 subunit of photosystem II (PSII). These maturation factors were then combined into a cassette and integrated into the genome at the previously defined neutral site 1 (30) (Fig.…”

Hydrogen Production in Chlamydomonas: Photosystem II-Dependent and -Independent Pathways Differ in Their Requirement for Starch Metabolism

“…Based on the observation that starchless C. reinhardtii mutants sta6 and sta7 are strongly affected in their ability to produce hydrogen, Posewitz et al (2004) proposed that starch metabolism plays a central role in C. reinhardtii hydrogen production. Actually, two different pathways can supply reductants (i.e.…”

Potential for hydrogen production with inducible chloroplast gene expression in Chlamydomonas

“…Further improvements of the system need to take into account that hydrogen production depends on other factors besides the turn-off of PSII activity, namely starch accumulation, which feeds electrons downstream of PSII into the photosynthetic electron transport chain and sustains respiration required for consuming the remaining O 2 produced by the residual activity of PSII (13)(14)(15). Additionally, there is competition of hydrogen production with other electron sinks such as the Calvin-Benson cycle and cyclic electron flow.…”

“…This process leads to anaerobiosis, which in turn induces the synthesis of hydrogenase. Moreover, sulfur starvation leads to the accumulation of carbohydrates, which is important for sustained hydrogen production in the long term (13,14). It also leads to the inhibition of the Calvin-Benson cycle and thereby removes an important electron sink, thus favoring hydrogen production (15).…”