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Engineering Plant Cytochrome P450s for Self-sufficiency in Escherichia coli


The objective of this work was to engineer plant Cytochrome P450s to be self-sufficient in Escherichia coli, as a proof-of-concept of a novel protein engineering platform, termed ProtoVitro. The P450 reaction of interest was the hydroxylation of limonene for its potential to produce cancer therapeutics.1 The reaction can be performed naturally by plant P450s but not bacterial ones,2 so we created 28 fusion protein variants that each contained a plant P450 heme domain and either a bacterial or eukaryotic reductase domain. The heme domains were selected based on previously observed activity on limonene, and the reductase domains were selected as the maximally informative set from a list of thousands of prokaryotic and eukaryotic sequences. The rapid prototyping of diverse sequences facilitated by ProtoVitro allowed us to identify optimal protein variants in a more robust fashion than alternative protein engineering methods.

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