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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.

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Figure 1: C40 carotenoid biosynthesis branches into a variety of pathways to acyclic and cyclic carotenoids for which biosynthetic genes from bacteria have been cloned (for a review see refs 15, 17).
Figure 2: HPLC analysis of carotenoid extracts of E. coli transformants carrying plasmids pAC-crtEEU-crtBEU and (A) pUC-crtIEU expressing the wild-type phytoene desaturase; (B) pUC-I14 expressing desaturase mutant I14; (C) pUC-I25 expressing desaturase mutant I25.
Figure 3: Cell pellets of E. coli transformants expressing wild-type and mutant cyclases.
Figure 4: HPLC analysis of carotenoid extracts of E. coli transformants carrying plasmids (A) pAC-crtEEU-crtBEU-I14 and pUC-Y2 expressing desaturase mutant I14 together with cyclase mutant Y2 and (B) pAC-crtEEU-crtBEU-crtIEU and pUC-Y2 expressing wild-type desaturase together with cyclase mutant Y2.

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Acknowledgements

We are grateful to Dr. Peter G. Green (Environmental Analysis Center, Caltech) for technical support and helpful discussions on mass analysis. C.S.-D. was supported by the Deutsche Forschungsgemeinschaft (DFG) and D.U. by the Japan Society for the Promotion of Science (JSPS).

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Correspondence to Claudia Schmidt-Dannert or Frances H. Arnold.

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Schmidt-Dannert, C., Umeno, D. & Arnold, F. Molecular breeding of carotenoid biosynthetic pathways. Nat Biotechnol 18, 750–753 (2000). https://doi.org/10.1038/77319

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