Producing natural drugs such as Taxol by engineered microbes have attracted extensive attention in recent years. This work focused on how to design and construct artificial yeast for the production of taxadien-5α-ol

which was the first hydroxylated product catalyzed by cytochrome P450 enzymes in Taxol biosynthesis pathway. Based on combinatorial design

three hydroxylase genes derived from different yew subspecies and two reductase genes from different organisms with N-terminal truncatd with various length were tested simultaneously to investigate the fitness between the genes and the chassis. Two kinds of promoters were used for regulating the cytochrome P450 reductase genes. In total

72 kinds of combinations were constructed and then integrated into the chromosome of the taxadiene producing strain. 48 taxadine-5α-ol producing strains were obtained out of 72 combinations by co-expression of the hydroxylase and the reductase. The highest titer was 67.3 μg·L-1. This was the first time to realize the biosynthesis of taxadine-5α-ol form glucose in Saccharomyces cerevisiae. The result provided a good reference for the research of the catalytic reaction mediated by cytochrome P450 enzymes in microbes through the interactions between modules and chassis by combinatorial design.