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Last Name Last name:First name: Metabolic Pathway Engineering Problem Set 5 Engineering a Fermentation System: Fermentation of plant matter to produce ethanol for fuel is one potential method for reducing the use of fossil fuels and thus the CO2 emissions that lead to global warming. Many microorganisms can break down cellulose then ferment the glucose to ethanol. However, many potential cellulose sources, including agricultural residues and switchgrass, also contain substantial amounts of arabinose, which is not as easily fermented. Escherichia coli is capable of fermenting arabinose to ethanol, but it is not naturally tolerant of high ethanol levels, thus limiting its utility for commercial ethanol production. Another bacterium, Zymomonas mobilis, is naturally tolerant of high levels of ethanol but cannot ferment arabinose. Deanda, Zhang, Eddy, and Picataggio (1996) described their efforts to combine the most useful features of these two organisms by introducing the E. coli genes for the arabinose-metabolizing enzymes into Z. mobilis. (a) Why is this a simpler strategy than the reverse: engineering E. coli to be more ethanol-tolerant? Stated another way, why is increasing ethanol tolerance in a microbe more challenging than introducing the ability to catabolize arabinose? (b) Deanda and colleagues inserted five E. coli genes into the Z. mobilis genome. Name the five genes, their protein products, and the chemical transformation that they catalyze individually. The five E. coli genes inserted into Z. mobilis allowed the entry of arabinose into the nonoxidative phase of the pentose phosphate pathway, where it was converted to glucose-6-phosphate and glyceraldehyde-3-phosphate and fermented to ethanol. In the Discussion section of the article, the authors state: In the overall fermentation reaction, 3 mol of L-arabinose is converted to 5 mol of ethanol. Neglecting the NAD(P)H balance, the stoichiometry can be shown by the following equation: 3 L-arabinose + 3 ADP + 3 Pi ---> 5 ethanol + 5 CO2 + 3 ATP + 3H2O. The theoretical ethanol yield based on this stoichiometry is 0.51 g of ethanol per g of L-arabinose or 1.67 mol of ethanol per mol of L-arabinose. In this new pathway, the net ATP yield from 3 mol of L-arabinose is 2 mol less than that postulated for conventional L-arabinose fermentation through a combination of pentose phosphate and Embden-Meyerhoff-Parnas pathways. The following questions [(c)-(f)] aim to explain the authors’ rationale. (c) The three ara enzymes eventually converted arabinose into which sugar? (d) The product from part (c) feeds into the pentose phosphate shunt. The five E. coli enzymes introduced into Z. mobilis allow conversion of 6 molecules of L-arabinose into how many molecules of fructose-6-phosphate and glyceraldehyde-3-phosphate? Explain and write the overall reaction. Be sure to include any energy expenditure. (e) Z. mobilis uses the Entner-Doudoroff pathway for ethanol fermentation (Fig. 3 of the article). As a result, the expected ATP yield is only 1 ATP per molecule of arabinose. Explain why conversion of 6 molecules of arabinose to ethanol and carbon dioxide would only yield 6 molecules of ATP. Please write out the reaction at each stage as well as the net reaction, balancing the stoichiometry. (f) If Z. mobilis can use the Embden-Meyerhof-Parnas (EMP) pathway, what is the stoichiometry of the fermentation of 6 molecules of arabinose to ethanol and CO2? How many ATP molecules would you expect this reaction to generate? Although the ED pathway generates less ATP per arabinose molecule and is less beneficial for the bacterium, it is better for ethanol production. Why? (g) The genome of Z. mobilis was not sequenced when this work was originally conducted. Use IMG or BLAST (or MetaCyc) to find whether the following enzymes are encoded in the Z. mobilis ZM4 genome and provide the locus tag / gene name (beginning with “ZMO”) of each, if present: transaldolase, transketolase, phosphoglucose isomerase (glucose-6-phosphate isomerase), phosphofructokinase, fructose-bisphosphate aldolase, and triosephosphate isomerase. Explain why Z. mobilis cannot use the EMP pathway to degrade glucose. (h) Pseudomonas aeruginosa also uses the Entner-Doudoroff pathway to degrade glucose. Use IMG or BLAST (or MetaCyc) to identify which enzymes in the EMP pathway are missing from P. aeruginosa PAO1. (i) Biochemical experiments performed by Robertson and McCullough (1968) suggested that Brucella abortus uses the pentose phosphate pathway, instead of the EMP or Entner-Doudoroff pathway, to degrade glucose. The labeling and enzymological results, obtained with B. abortus strain 19 grown in rich media in shaken culture, suggested that key enzymes are missing. Use IMG or BLAST (or MetaCyc) to identify which enzymes in the EMP and ED pathways are missing from B. abortus S19. Is there a discrepancy from experimental results? Explain. Last name ___________________Problem Set 5 - Page 1 of 2