During anoxia, the green alga Chlamydomonas reinhardtii activates various ill-defined fermentation pathways leading to the production of hydrogen, formate, acetate and ethanol. A bioinformatic survey was performed to look for C. reinhardtii genes potentially involved in fermentation that could compete for carbon and reductant, with a view to increasing H2 yields by metabolic engineering. Antibodies were raised against the predicted NAD+-dependent D-lactate dehydrogenase (LDH), pyruvate decarboxylase (PDC3), pyruvate-formate lyase (PFL1), pyruvate synthase/pyruvate:ferredoxin oxidoreductase (PFOR), pyruvate dehydrogenase E1 α components (PDC1 and PDC2) and a bifunctional acetaldehyde/alcohol dehydrogenase (ADH1). These antibodies were used to improve metabolic models by providing information about localisation, extending previous studies to include analysis of the cytoplasmic fraction and putative LDH. Additionally, it was demonstrated that the majority of enzymes are constitutively present, even during photoautotrophic growth where active lactate, formate and ethanol production pathways were found.
Artificial microRNA technology was used to knockdown PFL1, whose gene product catalyses the conversion of pyruvate to acetyl-CoA and formate. PFL1-knockdown had no impact upon H2 evolution during sulphur-deprivation, but caused a decrease in the excretion of formate and ethanol, also revealing a pathway for production of 3-hydroxybutyrate, a metabolite not previously detected in this alga. Additionally amiRNA knockdown was used to provide direct evidence for a PDC3 catalysed pathway during sulphur-deprivation. PDC3-knockdown led to the re-direction of flux via PFL1 and a decrease in H2 production rates. Inhibiting both the PFL1 and PDC3 catalysed pathways did not improve upon wild type yields of H2 but instead re-directed carbon flux to the production of lactate and alanine.
Overall these data show amiRNA is an effective method for down regulating metabolic pathways controlled by PFL1 and PDC3. However if H2 yields in C. reinhardtii are to be significantly improved by metabolic engineering it will likely require the knockdown of multiple fermentative pathways
