Redesign, reconstruction, and directed extension of the Brevibacterium linens C40 carotenoid pathway in Escherichia coli.
In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY(2) and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed beta-carotene pathway synthesized an unexpected 7,8-dihydro-beta-carotene in addition to beta-carotene. Extension of the beta-carotene pathway with the B. linens beta-ring desaturase CrtU and Pantoea beta-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature.
Department of Molecular Science and Technology, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon 443-749, South Korea.
Se Hyeuk
SH
Kim
Yun Hee
YH
Park
Claudia
C
Schmidt-Dannert
Pyung Cheon
PC
Lee
D053898:Biosynthetic Pathways / Q000235:genetics*; D001951:Brevibacterium / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D002338:Carotenoids / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering; D005827:Genetics, Microbial; D007218:Industrial Microbiology / Q000379:methods*; D020636:Pantoea / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D016992:Rhodobacter / Q000201:enzymology / Q000235:genetics
D011994:Recombinant Proteins; D002338:Carotenoids