Evolution of the C ₃₀ Carotenoid Synthase CrtM for Function in a C ₄₀ Pathway

Abstract

The C 30 carotene synthase CrtM from Staphylococcus aureus and the C 40 carotene synthase CrtB from Erwinia uredovora were swapped into their respective foreign C 40 and C 30 biosynthetic pathways (heterologously expressed in Escherichia coli) and evaluated for function. Each displayed negligible ability to synthesize the natural carotenoid product of the other. After one round of mutagenesis and screening, we isolated 116 variants of CrtM able to synthesize C 40 carotenoids. In contrast, we failed to find a single variant of CrtB with detectable C 30 activity. Subsequent analysis revealed that the best CrtM mutants performed comparably to CrtB in an in vivo C 40 pathway. These mutants showed significant variation in performance in their original C 30 pathway, indicating the emergence of enzymes with broadened substrate specificity as well as those with shifted specificity. We discovered that Phe 26 alone determines the specificity of CrtM. The plasticity of CrtM with respect to its substrate and product range highlights the potential for creating further new carotenoid backbone structures.

Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

“…Green residues are involved in the first half-reaction (formation of PSPP), while blue residues form the pocket of the second half-reaction in which PSPP is rearranged. Red residues correspond by sequence alignment to F26 and W38 of CrtM, which were found to control the product size of that enzyme (213,214). Shown in yellow are the residues that align with mutations in CrtM that improve both its C 30 and C 40 carotenoid synthase activities.…”

“…Carotenoid synthases are quite specific with respect to product size, which reflects a strong preference for prenyl diphosphate substrates of a particular length. The C 40 carotenoid synthase CrtB does not detectably accept C 15 PP as a substrate, while the C 30 synthase CrtM accepts two molecules of C 20 PP to form C 40 carotenoids much less efficiently than it accepts two molecules of C 15 PP to form C 30 carotenoids (163,214). Even though various longer and shorter isoprenyl diphosphates are made by different organisms (146), no known carotenoids are derived from them.…”

“…Carotenoid desaturases which primarily catalyze two, three, four, and five desaturation steps are known; one-step and six-step desaturated carotenoids have been reported only as minor products in natural carotenogenic organisms. The ability of carotenoid desaturases to accept different substrates was demonstrated when C 30 and C 40 desaturases were tested in each other's pathway (163,214). C 40 desaturases from Erwinia (four-step enzymes), Rhodobacter (three-step), and Anabaena (two-step) are all active on C 30 substrates.…”

“…Using error-prone PCR to perform random mutagenesis, expressing the mutant genes with the remaining genes necessary to produce lycopene in E. coli, and screening to find red colonies, we identified single amino acid substitutions in the S. aureus C 30 carotenoid synthase CrtM (F26L or F26S) that confer the ability to make the C 40 backbone just as efficiently as CrtB (214). Repeating this experiment using a PCR method designed to guarantee that the libraries would be free from mutation at F26 allowed us to uncover two additional amino acid substitutions, W38C and E180G, that also confer C 40 function on CrtM (213).…”

“…In a heterologous host, however, its desaturation step number is less distinct. Several groups have reported that CrtN produces the four-step 4,4Ј-diapolycopene as a major product in an E. coli system (118,163,211,214). Desaturase step number can also be altered by manipulation of enzymes further downstream in the carotenoid pathway (69).…”