Proteome and transcriptional analysis of ethanol-grown Sulfolobus solfataricus P2 reveals ADH2, a potential alcohol dehydrogenase.
Sulfolobus solfataricus P2 was shown to survive on ethanol at various concentrations (0.08-3.97% w/v) as the sole carbon source. The highest ethanol consumption rate was 15.1 mg/L/hr (via GC-MS analysis) in cultures grown on 0.79% w/v ethanol. In vivo metabolic labeling, using 13C universally labeled ethanol, provided evidence for both ethanol uptake and metabolic utilization. Results obtained from isobaric mass tag-facilitated shotgun proteomics (iTRAQ) indicate that on average, 21 and 31% of the 284 proteins identified (with > or = 2 MS/MS) are increased and decreased expression in ethanol cultures compared to glucose control cultures. Preliminary analysis shows >2-fold increase of the zinc-dependent alcohol dehydrogenase, ADH-10 (SSO2536), and the putative ADH-2 (SSO0764) in both translational and transcriptional data (using quantitative RT-PCR), suggesting both proteins are integral to ethanol metabolism. Evidence that ethanol was catabolised into central metabolism via acetyl-CoA intermediates was further indicated by another >2-fold increase in protein expression levels of various acetyl-CoA synthetases. The decreased expression (>2-fold) of isocitrate dehydrogenase at the protein level suggests that the ethanol grown cultures shifted toward the glyoxylate cycle. Subsequently, the activity of ADH-2 was confirmed by overexpression in Escherichia coli, with the resultant purified in vitro enzyme exhibiting an activity that increased with temperature up to 95 degrees C, and giving a specific activity of 1.05 U/mg.
Chong
Poh Kuan
PK
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom.
Burja
Adam M
AM
Radianingtyas
Helia
H
Fazeli
Alireza
A
Wright
Phillip C
PC
D000426:Alcohol Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology; D000431:Ethanol / Q000378:metabolism*; D007700:Kinetics; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D048229:Sulfolobus solfataricus / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology; D053719:Tandem Mass Spectrometry; D014158:Transcription, Genetic