There is a huge variety of naturally occurring isoprenoids, some of which are medically or commercially important and produced industrially. The production methods include in vitro enzymatic synthesis, which is an expensive and low-yield process. In $1999,$ Wang, Oh, and Liao reported their experiments to engineer the easily grown bacterium $E .$ coli to produce large amounts of astaxanthin, a commercially important isoprenoid. Astaxanthin is a red-orange carotenoid pigment (an antioxidant) produced by marine algae. Marine animals such as shrimp, lobster, and some fish that feed on the algae get their orange color from the ingested astaxanthin. Astaxanthin is composed of eight isoprene units; its molecular formula is $\mathrm{C}_{40} \mathrm{H}_{52} \mathrm{O}_{4}$. (EQUATION CAN'T COPY)
(a) Circle the eight isoprene units in the astaxanthin molecule. Hint: Use the projecting methyl groups as a guide. Astaxanthin is synthesized by the pathway shown on the next page, starting with $\Delta^{3}$ isopentenyl pyrophosphate (IPP). Steps $1$ and $2$ are shown in Figure $21-36,$ and the reaction catalyzed by IPP isomerase is shown in Figure $21-35$.
(c) Briefly describe the chemical transformation in step $5$
(d) The synthesis of cholesterol (Fig. $21-37$ ) includes a cyclization (ring closure) that requires a net oxidation by $\mathrm{O}_{2}$. Does the cyclization in step $\mathrm{O}$ of the astaxanthin synthetic pathway require a net oxidation of the substrate (lycopene)? Explain your reasoning.
E. coli does not make large quantities of many isoprenoids, and does not synthesize astaxanthin. It is known to synthesize small amounts of IPP, DMAPP, geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. Wang and colleagues cloned several of the $E .$ coli genes that encode enzymes needed for astaxanthin synthesis, in plasmids that allowed their overexpression. These genes included idi, which encodes IPP isomerase, and $i s p A,$ which encodes a prenyl transferase that catalyzes steps 1 and 2. To engineer an $E .$ coli capable of the complete astaxanthin pathway, Wang and colleagues cloned several genes from other bacteria into plasmids that would allow their overexpression in $E .$ coli. These genes included $c r t E$ from Erwinia uredovora, which encodes an enzyme that catalyzes step $3$ and $c r t B, c r t I, c r t Y, c r t Z,$ and $c r t W$ from Agrobacterium aurantiacum, which encode enzymes for steps 4, 5, 6, 7, and 8, respectively.
(EQUATION CAN'T COPY) The investigators also cloned the gene $g p s$ from Archaeoglobus fulgidus, overexpressed this gene in $E .$ coli, and extracted the gene product. When this extract was reacted with $\left[^{14} \mathrm{C}\right] \mathrm{IPP}$ and $\mathrm{DMAPP}$ or geranyl pyrophosphate or farnesyl pyrophosphate, only 14 $C-$ labeled geranylgeranyl pyrophosphate was produced in all cases.
(e) Based on these data, which step(s) in the pathway are catalyzed by the enzyme encoded by gps? Explain your reasoning. Wang and coworkers then constructed several $E$. coli strains overexpressing different genes, and measured the orange color of the colonies (wild-type $E .$ coli colonies are offwhite) and the amount of astaxanthin produced (as measured by its orange color). Their results are shown below (ND indicates not determined). (TABLE CAN'T COPY)
(f) Comparing the results for strains 1 through 4 with those for strains 5 through 8 , what can you conclude about the expression level of an enzyme capable of catalyzing step $3$ of the astaxanthin synthetic pathway in wild-type $E .$ coli? Explain your reasoning.
(g) Based on the data above, which enzyme is rate-limiting in this pathway, IPP isomerase or the enzyme encoded by $i d i ?$ Explain your reasoning.
(h) Would you expect a strain overexpressing $\mathrm{crtBIZYW}$, gps, and crtE to produce low $(+),$ medium $(++),$ or high $(+++)$ levels of astaxanthin, as measured by its orange color? Explain your reasoning.