Plants are light-driven "green" factories able to synthesize more than 200,000 different bioactive natural products, many of which are high-value products used as drugs (e.g., artemisinin, taxol, and thapsigargin). In the formation of natural products, cytochrome P450 (P450) monooxygenases play a key role in catalyzing regio- and stereospecific hydroxylations that are often difficult to achieve using the approaches of chemical synthesis. P450-catalyzed monooxygenations are dependent on electron donation typically from NADPH catalyzed by NADPH-cytochrome P450 oxidoreductase (CPR). The consumption of the costly cofactor NADPH constitutes an economical obstacle for biotechnological in vitro applications of P450s. This bottleneck has been overcome by the design of an in vitro system able to carry out light-driven P450 hydroxylations using photosystem I (PSI) for light harvesting and generation of reducing equivalents necessary to drive the P450 catalytic cycle. The in vitro system is based on the use of isolated PSI and P450 membrane complexes using ferredoxin as an electron carrier. The turnover rate of the P450 in the light-driven system was 413 min(-1) compared to 228 min(-1) in the native CPR-catalyzed system. The use of light as a substitute for costly NADPH offers a new avenue for P450-mediated synthesis of complex bioactive natural products using in vitro synthetic biology approaches.