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To make wider the practical use of enzymes for chemical industry, it is important to diversify available reaction types by developing multi-component enzymatic processes, which are functional combination of enzymatic reactions and complex biological systems providing energy or reducing power for the reaction. Recombinant microorganisms expressing the genes coding the desired enzymes were constructed and their potentials were evaluated in combination with their accessory components such as cofactor generation system, energy generation system, and inhibitor decomposing system. In this talk, the examples of multi- component enzymatic systems were presented in the application of stereospecific and regiospecific alcohol synthesis together with enzyme screening in microbial novel metabolisms. (1) Dioxygenase process: We obtained novel 2-oxoglutarate-dependent amino acid dioxygenases and applied them to hydroxy amino acid production. As an 2-oxoglutarate generation system, TCA-cycle of E. coli was metabolically engineered and the modified E. coli cells were used as a host cell for dioxygenase expression. As a result, a practical process for optically pure 4- hydroxyisoleucine production was established. (2) Aldolase process: Deoxyriboaldolase (DERA) from Klebsiella pneumoniae was overexpressed in E. coli. The E. coli cells produced 2-deoxyribose 5-phosphate (DR5P), a key intermediate for 2’-deoxyribonucleoside production, from acetaldehyde and fructose 1,6-diphosphate (FDP). Coupling with an efficient ATP-generating system for FDP production from glucose through partial reactions of alcoholic fermentation by baker’s yeast enabled direct production of DR5P from glucose and acetaldehyde. (3) Cytochrome P450 monooxygenase process: A mutant cytochrome P450 BM-3, F87V, catalyzed regioselective hydroxylation of various phenolic compounds. To enhance this activity, P450-stabilizing/activating factors were screened. Addition of superoxide dismutase to the P450 BM-3 reaction mixture greatly increased NADPH-consumption rate, slightly increased coupling efficiency, and resulted in acceleration of the reaction rate. (4) Enzyme screening in novel microbial metabolisms: Enzymes useful for multi- component enzyme systems were found through the analysis of novel microbial metabolisms of anaerobic fatty acid transformation, hydroxy amino acid metabolisms and oxidative pyrimidine metabolisms and applied for the production of useful chemicals.