Filling Knowledge Gaps in Biological Networks: integrating global approaches to understand H2 metabolism in Chlamydomonas reinhardtii - Final Report

[摘要] The green alga Chlamydomonas reinhardtii (Chlamydomonas) has numerous genes encoding enzymes that function in fermentative pathways. Among these genes, are the [FeFe]-hydrogenases, pyruvate formate lyase, pyruvate ferredoxin oxidoreductase, acetate kinase, and phosphotransacetylase. We have systematically undertaken a series of targeted mutagenesis approaches to disrupt each of these key genes and omics techniques to characterize alterations in metabolic flux. Funds from DE-FG02-07ER64423 were specifically leveraged to generate mutants with disruptions in the genes encoding the [FeFe]-hydrogenases HYDA1 and HYDA2, pyruvate formate lyase (PFL1), and in bifunctional alcohol/aldehyde alcohol dehydrogenase (ADH1). Additionally funds were used to conduct global transcript profiling experiments of wildtype Chlamydomonas cells, as well as of the hydEF-1 mutant, which is unable to make H2 due to a lesion in the [FeFe]-hydrogenase biosynthetic pathway. In the wildtype cells, formate, acetate and ethanol are the dominant fermentation products with traces of CO2 and H2 also being produced. In the hydEF-1 mutant, succinate production is increased to offset the loss of protons as a terminal electron acceptor. In the pfl-1 mutant, lactate offsets the loss of formate production, and in the adh1-1 mutant glycerol is made instead of ethanol. To further probe the system, we generated a double mutant (pfl1-1 adh1) that is unable to synthesize both formate and ethanol. This strain, like the pfl1 mutants, secreted lactate, but also exhibited a significant increase in the levels of extracellular glycerol, acetate, and intracellular reduced sugars, and a decline in dark, fermentative H2 production. Whereas wild-type Chlamydomonas fermentation primarily produces formate and ethanol, the double mutant performs a complete rerouting of the glycolytic carbon to lactate and glycerol. Lastly, transcriptome data have been analysed for both the wildtype and hydEF-1, that correlate with our observed fermentative metabolites. Intriguingly, over half of the most differentially regulated genes are of unknown function.

[发布日期] 2011-06-30

[学科分类] 再生能源与代替技术

[关键词] Chlamydomonas;Hydrogen;Fermentation;Metabolic Engineering.