Previously, we utilized in vitro evolution to alter the catalytic functions of several carotenoid enzymes and produce the novel carotenoids tetradehydrolycopene and torulene in Escherichia coli. Here we report on the successful extension of these pathways and the C30 carotenoid diaponeurosporene pathway with additional carotenoid genes. Extension of the known acyclic C30 pathway with C40 carotenoid enzymes - spheroidene monooxygenase and lycopene cyclase - yielded new oxygenated acylic products and the unnatural cyclic C30 diapotorulene, respectively. Extension of acyclic C40 pathways with spheroidene monooxygenase generated novel oxygenated carotenoids including the violet phillipsiaxanthin. Extension of the torulene biosynthetic pathway with carotene hydroxylase, desaturase, glucosylase, and ketolase yielded new torulene derivatives. These results demonstrate the utility of extending an in vitro evolved central metabolic pathway with catalytically promiscuous downstream enzymes in order to generate structurally novel compounds.
Bibliographical noteFunding Information:
The authors gratefully acknowledge support from the Defense Advanced Research Projects Agency (DARPA-BIOS N66001-02-1-8928), McKnight Foundation, David and Lucile Packard Foundation, and National Science Foundation (NSF MRI DBI-0079864).