Question

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?

(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 fermented to ethanol.

(c) The three ara enzymes eventually converted arabinose into which sugar?

(d) The product from part (c) feeds into the pentose phosphate shunt. Combining the five E. coli enzymes listed above with the enzymes of this pathway, describe the overall pathway for the fermentation of 6 molecules of arabinose to ethanol.

(e) 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 if Z. mobilis can use the Embden-Meyerhof-Parnas (EMP) pathway?

(f) Z. mobilis uses the Entner-Doudoroff pathway for ethanol fermentation. 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. Although this 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.

Answer 1

a) There are numerous genes that work together to provide ethanol tolerance, as compared to number of gene required to break down arabinose to a sugar that can enter teh metabolism cycle.

b) araA: L-arabinose isomerase for the conversion of L-arabinose to L-ribulose
araB: L-ribulokinase for the phosphorylation of L-ribulose
araD: L-ribulose-5-phosphate epimerase for the interconversion of L-ribulose-5-phosphate to L-xylulose-5-phosphate
talB: transaldolase
tklA: transketolase

c) The 3 ara enzymes convert arabinose into L-xylulose-5-phosphate

d)