Understanding fermentation metabolism and H2 production in Chlamydomonas reinhardtii through the analysis of mutants

Claudia Catalanotti1, Wenqiang Yang1, Alexandra Dubini2, Venkataramanan Subramanian2,3, Florence Mus1, Matthew C. Posewitz3, Michael Seibert2, and Arthur R. Grossman1

1) Carnegie Institution for Science, Stanford, CA 2) National Renewable Energy Laboratory, Golden, CO 3) Colorado School of Mines, Golden, CO

Chlamydomonas reinhardtii has a complex anaerobic metabolic network that can be induced in the dark to produce various fermentation products including H2. To characterize the fermentation pathways and the production of H2 in this alga, mutants defective for specific fermentation reactions were generated and are currently being analyzed. Two strategies were used in the generation of mutants: insertional mutagenesis and TILLING (Targeted Induced Local Lesions IN Genomes). Among the mutants isolated by insertional mutagenesis was one with a lesion in pyruvate formate lyase (pfl1), while the TILLING approach yielded five different mutant alleles of PFR, with three having non-synonymous substitutions that are likely to impact the activity of the encoded enzyme. The pfl mutant was shown to have an insertion in the 7th exon of the gene. As a result, this strain appears to accumulate no PFL protein. Furthermore, metabolite analyses have demonstrated that the mutant is unable to accumulate formate under anoxic conditions, while both low levels of ethanol and acetate are synthesized. Most intriguing is the finding that the pfl strain exhibits no H2 production and an increase in the accumulation of lactic acid; anaerobic conditions does not trigger the production of detectable levels of lactate in the parental strain. These results suggest that specific features that regulate fermentative pathways in Chlamydomonas are aberrant in a mutant that is unable to make PFL. We will discuss this work in the context of regulatory pathways that control fermentation metabolism in algae.

e-mail address of presenting author: ccatal1@stanford.edu