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Chemistry 256 Name: Exercise 1: Switchgrass ethanol The use of corn to produce ethanol for biofuels raises significant economic and ethical issues; a plant like switchgrass, which is not used for food, may provide an alternate source of ethanol. Of course, one of the reasons switchgrass is not used for food is that its cellulose is made of arabinose units, rather than glucose units. The fermentation bacteria typically do not process arabinose (shown below) into ethanol. E. coli is capable of fermenting arabinose to ethanol, but it doesn’t naturally tolerate high ethanol levels. Z. mobilis does tolerate high ethanol levels, but can’t ferment arabinose. Deanda, Zhang, Eddy, and Picataggio (1996) combined the most useful features of both bacteria by introducing the genes in E. coli that code for arabinosefermenting enzymes into Z. mobilis. 1. Why not put the ethanol-tolerance genes into E. coli instead? The five E. coli genes inserted into the Z. mobilis genome are: • araA which codes for L-arabinose isomerase, which interconverts L-arabinose and Lribulose • araB which codes for L-ribulokinase, which uses ATP to phosphorylate L-ribulose at C5 • araD which codes for L-ribulose 5-phosphate isomerase, which interconverts Lribulose 5-phosphate and L-xylulose 5-phosphate • talB which codes for transaldolase • tktA which codes for transketolase These five genes allowed the entry of arabinose into the pentose phosphate pathway, where it was converted to glucose-6-phosphate and then fermented to ethanol. 2. The three ara enzymes eventually converted arabinose into which sugar? 3. The product from question 2 feeds into the pathway shown on page 507. Combining the five E. coli enzymes listed above with the enzymes in the pentose phosphate pathway, describe the overall path for the fermentation of six arabinose molecules to ethanol. I’ve started it off for you (Ar = arabinose). Six Ar → Six Ru → Six Ru5P → 4. What is the stoichiometry of the fermentation of six arabinose molecules to ethanol and CO2? How many ATP and NADH molecules will this reaction generate? Hint: count carbons. Further hint: The figure on page 507 is not quantitative, so be careful. 5. Another sugar commonly found in plants is xylose: What additional enzyme(s) would you need to have the modified Z. mobilis from the previous questions convert xylose into ethanol? Don’t name the enzyme(s) (for it (they) may not exist), just state the reaction(s) to be catalyzed.