Molecular breeding of carotenoid biosynthetic pathways

Abstract

The burgeoning demand for complex, biologically active molecules for medicine, materials science, consumer products, and agrochemicals is driving efforts to engineer new biosynthetic pathways into microorganisms and plants. We have applied principles of breeding, including mixing genes and modifying catalytic functions by in vitro evolution, to create new metabolic pathways for biosynthesis of natural products in Escherichia coli. We expressed shuffled phytoene desaturases in the context of a carotenoid biosynthetic pathway assembled from different bacterial species and screened the resulting library for novel carotenoids. One desaturase chimera efficiently introduced six rather than four double bonds into phytoene, to favor production of the fully conjugated carotenoid, 3, 4,3',4'-tetradehydrolycopene. This new pathway was extended with a second library of shuffled lycopene cyclases to produce a variety of colored products. One of the new pathways generates the cyclic carotenoid torulene, for the first time, in E. coli. This combined approach of rational pathway assembly and molecular breeding may allow the discovery and production, in simple laboratory organisms, of new compounds that are essentially inaccessible from natural sources or by synthetic chemistry.

Complete genome sequence of the myxobacterium Sorangium cellulosum

“…6). The carotenoids produced by S. cellulosum So ce56 have not been identified to date, but comparison to known carotenoid biosynthetic pathways 28 suggests that the strain should assemble phytoene, and further convert it into phytofluene, z-carotene and neurosporene. There also appear to be genes present for the further transformation of neurosporene to unknown products.…”

Erratum: correction: Enabling the chemistry of life

“…The biological selection methods are sufficiently powerful that one can find outcomes that are very rare biologically in a short space of time. A good example is the recent report 34 of expression of a functional carotenoid biosynthetic pathway in E. coli by selecting for bacteria that become red. The continuing progress in biological production of polyhydroxyalkanoate polymers with controlled sizes and properties 35 by engineering the respective polymerases increases the likelihood of economically viable production of these biodegradable plastics by biocatalysis.…”

Laboratory-Directed Protein Evolution

“…It is also a popular tool for accelerated adaptation of protein functions (e.g., stability, specificity, or affinity) in extreme conditions such as unusual temperatures and organic solvents (198,204,221,222,(327)(328)(329)(330), as well as for improvement of recombinant protein biosynthesis (152,185). Directed evolution has also given rise to altered specificities and activities of enzymes (113-115, 126, 141, 294, 337), enhanced intramolecular interactions (292), modified protein-protein interaction (180), and altered metabolic pathways (263). In the following sections we present some examples of the applications of these technologies.…”

“…The pathway of a chosen mutant was further modified by introducing a library of shuffled cyclase genes. The engineering of the carotenoid pathway represents a fine example of how directed evolution can be used to redesign a complex pathway (68,147,167,175,176,178,205,206,257,262,263,305,320,324).…”