Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

Abstract

Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed

Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

“…Carotenoids are commercially used as food colorants and animal feed supplements, and more recently, they are being used as nutraceuticals and cosmetic and pharmaceutical compounds (2). The increasing industrial importance of carotenoids has led to renewed efforts in developing bioprocesses for large-scale production of a range of carotenoids, including lycopene, ␤-carotene, and other more structurally diverse carotenoids (3)(4)(5)(6).…”

Adaptive evolution of fish hatching enzyme: one amino acid substitution results in differential salt dependency of the enzyme

“…For example, (i) GAG (Val) to GTG (Glu) substitution in the sixth codon of human β-globin gene results in sickle cell disease (Ingram, 1956;Ingram, 1957); (ii) single amino acid substitutions in carotenoid biosynthetic enzymes alter substrate specificity and reaction selectivity to produce a variety of carotenoids (Umeno et al, 2005); and (iii) a single amino acid change (S84C) of short wavelength-sensitive pigment 1 (SWS1) is responsible for transformation to UV-sensitive pigments in birds (Yokoyama et al, 2000). The present study provides a good example, showing that only one non-synonymous substitution causes the environmental adaptation of the gene.…”

Extraction of nutraceuticals from Spirulina (blue‐green alga): A bioorganic chemistry practice using thin‐layer chromatography

“…3) [7]. Among this general class of carotenes, b-carotene is Violaxanthin is a naturally occurring xanthophyl pigment with an orange color that is found in a variety of plants, including algae and cyanobacteria, and it is biosynthesized from zeaxanthin by epoxidation [8].…”