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

Abstract

ORCID ID: 0000-0001-5600-4076 (W.Y.)Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

“…Complementation was performed with either FDX5 cDNA or gDNA under the control of the PSAD promoter in the pGEM T-easy vector (Promega). Transformation was performed by introducing 1 μg DNA of plasmids pJM43Ble-FDX5cDNA and pJM43Ble-FDX5gDNA (both linearized with NotI) into fdx5 (in CC-124 background) by electroporation (0.8 kv, 25 uF) using a BioRad GenePulser II Electroporator (Bio-Rad), as previously described (35). Transformants were selected on solid TAP medium containing 100 μg/mL ampicillin, 5 μg/mL paromomycin (for original fdx5 mutation), and 6 μg/mL Zeocin (for the introduced FDX5 gene) and then selected for growth in the dark.…”

Concerted Up-regulation of Aldehyde/Alcohol Dehydrogenase (ADHE) and Starch in Chlamydomonas reinhardtii Increases Survival under Dark Anoxia

“…Although the presence of a PFL and PTA-ACK in mitochondria has been confirmed by biochemical and molecular approaches (12,24), the attempts to detect an ADHE have failed as yet (12,33) (Fig. 4).…”

The Involvement of Hydrogen-producing and ATP-dependent NADPH-consuming Pathways in Setting the Redox Poise in the Chloroplast of Chlamydomonas reinhardtii in Anoxia

“…The first conditions that come to mind are fluctuations in light intensity, but hypoxia or even anoxia is also frequently encountered by photosynthetic microbes found in soils or ponds, a biotope they usually share with dense microbial communities comprising nonphotosynthetic organisms who actively consume oxygen as they respire (1). Chlamydomonas reinhardtii is remarkably equipped with a battery of fermentative pathways allowing it to metabolize pyruvate into lactate or even ethanol, formate, acetate, and hydrogen (2)(3)(4)(5). The transition from aerobiosis to hypoxia or anoxia, which occurs in natural habitat at twilight and at night when photosynthetic oxygen evolution switches off, is accompanied by a massive metabolic remodeling that involves transcriptional regulation and the onset of dedicated metabolic pathways (6 -10), and prominent among them is, in C. reinhardtii, the hydrogen production pathway (2,11).…”