Chong Poh Kuan, Burja Adam M, Radianingtyas Helia, Fazeli Alireza, Wright Phillip C
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom.
J Proteome Res. 2007 Oct;6(10):3985-94. doi: 10.1021/pr070232y. Epub 2007 Sep 7.
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.
嗜热栖热菌P2已被证明能够在不同浓度（0.08 - 3.97% w/v）的乙醇作为唯一碳源的条件下存活。在以0.79% w/v乙醇培养的菌液中，最高乙醇消耗速率为15.1 mg/L/小时（通过气相色谱 - 质谱分析）。使用13C全标记乙醇进行的体内代谢标记，为乙醇摄取和代谢利用提供了证据。等压质量标签辅助鸟枪法蛋白质组学（iTRAQ）获得的结果表明，与葡萄糖对照培养物相比，在乙醇培养物中鉴定出的284种蛋白质（≥2次串联质谱）平均有21%和31%的表达量增加和减少。初步分析显示，在翻译和转录数据（使用定量逆转录 - 聚合酶链反应）中，锌依赖性乙醇脱氢酶ADH - 10（SSO2536）和假定的ADH - 2（SSO0764）的表达量增加了2倍以上，表明这两种蛋白质对于乙醇代谢至关重要。各种乙酰辅酶A合成酶的蛋白质表达水平进一步增加了2倍以上，这进一步表明乙醇通过乙酰辅酶A中间体被分解代谢进入中心代谢。蛋白质水平上异柠檬酸脱氢酶表达量的降低（>2倍）表明，以乙醇生长的培养物转向了乙醛酸循环。随后，通过在大肠杆菌中过表达证实了ADH - 2的活性，所得的体外纯化酶活性随温度升高至95摄氏度而增加，比活性为1.05 U/mg。
