The cross-coupling reaction is one of the most important chemical reactions in the modern organic chemistry. Biocatalysts capable of catalyzing C-C coupling reactions are desired in the chemical industry for sustainable development. Cytochrome P450 monooxygenases (P450s) have received considerable attention as biocatalysts capable of catalyzing such reactions. Here, we focused on actinomycete P450s, which have high homology with CYP158A2, involved in the oxidative C-C coupling reaction for fiaviolin dimerization in Streptomyces coelicolor A3(2). The screening of a chemical library composed of 426 aromatic compounds identified several combinations of P450s and their potential substrates. The type-1 difference spectrum indicated that the identified substrates bind to the active sites of a P450, named StV1 from Streptomyces violaceusniger. A redshift of the absorption maximum of the reaction products, together with LC-MS analysis suggested the presence of extended conjugate systems in the products through direct C-C coupling between two aromatic rings. The results demonstrated that actinomycete P450s have great potential to be utilized as biocatalysts for oxidative C-C coupling reactions and to facilitate the synthesis of diverse coupling products. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.