Hydrocortisone is an effective anti-inflammatory drug and also an important intermediate for synthesis of other steroid drugs. The filamentous fungus Absidia orchidis is renowned for biotransformation of acetylated cortexolone through 11β-hydroxylation to produce hydrocortisone. However, due to the presence of 11α-hydroxylase in A. orchidis, the 11α-OH by-product epi-hydrocortisone is always produced in a 1:1?M ratio with hydrocortisone. In order to decrease epi-hydrocortisone production, Saccharomyces cerevisiae was engineered in this work as an alternative way to produce hydrocortisone through biotransformation. Through transcriptomic analysis coupled with genetic verification in S. cerevisiae, the A. orchidis steroid 11β-hydroxylation system was characterized, including a cytochrome P450 enzyme CYP5311B2 and its associated redox partners cytochrome P450 reductase and cytochrome b5. CYP5311B2 produces a mix of stereoisomers containing 11β- and 11α-hydroxylation derivatives in a 4:1?M ratio. This fungal steroid 11β-hydroxylation system was reconstituted in S. cerevisiae for hydrocortisone production, resulting in a productivity of 22?mg/L·d. Protein engineering of CYP5311B2 generated a R126D/Y398F variant, which had 3 times higher hydrocortisone productivity compared to the wild type. Elimination of C20-hydroxylation by-products and optimization of the expression of A. orchidis 11β-hydroxylation system genes further increased hydrocortisone productivity by 238% to 223?mg/L·d. In addition, a novel steroid transporter ClCDR4 gene was identified from Cochliobolus lunatus, overexpression of which further increased hydrocortisone productivity to 268?mg/L·d in S. cerevisiae. Through increasing cell mass, 1060?mg/L hydrocortisone was obtained in 48?h and the highest productivity reached 667?mg/L·d. This is the highest hydrocortisone titer reported for yeast biotransformation system so far.,Copyright ? 2019 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.