Identification of the Elusive Pyruvate Reductase ofChlamydomonas reinhardtiiChloroplasts

Abstract

Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including d-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to d-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD+-dependent d-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a ‘lactate valve’ for the export of lactate to the mitochondrion for oxidation by cytochrome-dependent d-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm.

Dynamic metabolic profiling together with transcription analysis reveals salinity-induced starch-to-lipid biosynthesis in alga Chlamydomonas sp. JSC4

“…Next, transcription of genes involved in lipid synthesis11121314151617 was measured. Determination of the mRNA expression involved in lipid synthesis showed that PFOR mRNA under 2% SS increased approximately 10.5-fold from Day 3 to Day 5 and then remained constant; these levels are much higher than under 0% SS.…”

“…1); the genes encoding phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), ADP-glucose phosphorylase (AGPP), α-amylase (AMY) and starch phosphorylase (SP) are related to starch biosynthesis and degradation1011. Also, the genes encoding pyruvate dehydrogenase (PDH), pyruvate-ferredoxin oxidoreductase (PFOR), acetyl-CoA (AcCoA) carboxylase (ACC), pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALDH), AcCoA synthetase (ACS), and glycerol 3-phosphate dehydrogenase (GPDH) are related to lipid biosynthesis11121314151617. The compartmentalization of carbon metabolism in C. reinhardti was reviewed by Johnson et al .…”

Modulation of TOR kinase activity in Chlamydomonas reinhardtii: Effect of N-starvation and changing carbon pool

“…Additionally, glucose supplemented TP Agar (1% Bacto Agar) plates were used to grow cells in light in presence of glucose at various concentrations. Optical density of cells was measured as absorbance at 750 nm to get and estimate of cell density and number as shown previously (Burgess et al , 2016).…”

Microalgal hydrogen production research

“…A third approach to microalgal H 2 production encompasses the light-independent fermentative pathway that can generate various small organic molecules such as formate, acetate, and ethanol along with H 2 [51]. This relies on the pyruvate-ferredoxin oxidoreductase (PFOR) enzyme in a process similar to the one observed in obligate anaerobic microorganisms (Fig.…”

“…It was also reported that the pyruvate-formate-lyase (PFL) enzyme in C. reinhardtii could catalyze a reaction that oxidizes pyruvate to formate, while generating acetyl-CoA [55,56]. Although other metabolic routes exist, ethanol can be generated via the acetaldehyde dehydrogenase (AcDH)/alcohol dehydrogenase (ADH) pathway [51,57], while phosphate acetyl transferase (PAT) together with acetate kinase (ACK) contribute to acetate production from acetyl-CoA [51,58]. In comparison with the aforementioned light-dependent pathways, the lightindependent fermentative pathway generates relatively smaller amounts (traces) of H 2 in the microalgal cells [18,27].…”