Evolution of a Pathway to Novel Long-Chain Carotenoids

Abstract

Using methods of laboratory evolution to force the C 30 carotenoid synthase CrtM to function as a C 40 synthase, followed by further mutagenesis at functionally important amino acid residues, we have discovered that synthase specificity is controlled at the second (rearrangement) step of the two-step reaction. We used this information to engineer CrtM variants that can synthesize previously unknown C 45 and C 50 carotenoid backbones (mono-and diisopentenylphytoenes) from the appropriate isoprenyldiphosphate precursors. With this ability to produce new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids by using carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and dioxygenases, from naturally occurring pathways.Carotenoids are natural pigments with important biological activities (4,10,16,17). Most are based on a 40-carbon (C 40 ) phytoene backbone produced by condensation of 2 molecules of geranylgeranyldiphosphate (GGDP; C 20 PP), a reaction catalyzed by the carotenoid synthase CrtB (Fig. 1). The vast majority of the Ͼ700 known carotenoids (9) arise as a result of different types and levels of modification of the C 40 backbone, catalyzed by promiscuous (downstream) carotenoid biosynthetic enzymes (5). A few bacteria, notably Staphylococcus and Heliobacterium spp. (23,24), have a C 30 pathway, which starts with the CrtM synthase-catalyzed condensation of 2 molecules of farnesyldiphosphate (FDP; C 15 PP) to form 4,4Ј-diapophytoene. Yet other bacteria (such as Corynebacterium and Halobacterium spp.) are known to accumulate C 50 carotenoids, but these longer-chain structures are biosynthesized starting from the C 40 structure by the addition of 2 C 5 (isoprene) units (14). Various longer isoprenyldiphosphates are made by different organisms (30) and are potential precursors for longerchain carotenoids. They are precursors to other biosynthetic pathways, however, and no known carotenoids are derived from them. Thus, carotenoid size is tightly controlled by the carotene synthase reaction (20,27).To create new pathways for the biosynthesis of carotenoids with backbones larger than C 40 , we focused on engineering the carotenoid synthase to accept longer diphosphate substrates. Very little is known of the structure or basis for the specificity of these membrane-associated enzymes. Using random mutagenesis and a functional complementation screen for C 40 synthase activity, however, we identified single-amino-acid substitutions in the C 30 synthase CrtM (F26L or F26S) that confer the C 40 function (27). By repeating this experiment with a random mutant library that was free from mutation at F26, we recently found two more amino acid substitutions, W38C and E180G, that confer the same phenotype (26). Upon further mutagenesis at these three residues, we show here that the specificity of the carotenoid synthase CrtM is controlled at the second (rearrangement) step of its two-step reaction. Furthermore, we have engineered synthase variants that can make ...

Mechanism of action and inhibition of dehydrosqualene synthase

“…6 A and B clearly suggesting that both cationic inhibitors act as transition state-reactive intermediate analogs which occupy the S2 site, mimicking a cyclopropyl carbocation species, rather than being isosteres for the farnesyl carbocation which forms in S1. In FPPS, the cationic or nitrogen-containing bisphosphonate class of inhibitors bind to the S1 site (18,21,22), but of course most of the binding energy there can be attributed to the bisphosphonateMg 2þ 3 interaction. In the cationic inhibitors here, no such metal ion chelation is present, and binding is dominated by hydrophobic interactions with, potentially, the ammonium groups (and the cationic reaction intermediates) also being stabilized by the protein's charge field, just as in FPPS (17).…”

Redesign, Reconstruction, and Directed Extension of the Brevibacterium linens C 40 Carotenoid Pathway in Escherichia coli

“…This combinatorial biosynthetic approach basically relies on the functional coordination of pathway enzymes from different sources in a heterologous host (5,19,35). Carotenogenic enzymes tend to be promiscuous in their substrate specificity (33) and show unexpected/hidden activities (20) when expressed in heterologous host microorganisms. One example is the unusual activity of diapophytoene desaturase CrtN in E. coli, which resulted in structurally novel compounds (20).…”

Directed Enzyme and Pathway Evolution

“…Directed evolution experiments on CrtM have resulted in the production of a variant that can now synthesize phytoene (C40 carotenoid) from two GGPPs [141]. Additional CrtM mutants were obtained that utilize farnesylgeranyl disphopshate (C25, produced from a mutant FPP synthase from B. stearothermophilus) to produce novel C45 and C50 carotenoids [142]. As with the C35 carotenoid, the C45 and C50 carotenoids served as substrates for phytoene desaturase CrtI, to produce additional new desaturated carotenoids [130].…”