Efficient testosterone production by engineered Pichia pastoris co-expressing human 17 beta-hydroxysteroid dehydrogenase type 3 and Saccharomyces cerevisiae glucose 6-phosphate dehydrogenase with NADPH regeneration

Traditionally, testosterone (TS), an important hormone drug and precursor for the synthesis of other steroids, was chemically produced. Recently, TS has been prepared through side-chain degradation of some sterols (cholesterol or phytosterol) using microbial fermentation methods. However, the TS production is at a low level, and the biotransformation process is long and with many by-products formed. NADPH-dependent 17 beta-hydroxysteroid dehydrogenase type 3 (17 beta-HSD3) from human testis catalyzes the conversion of 4-androstene-3,17-dione (AD) to TS. In this work, we optimized the gene codons of human 17 beta-HSD3 and realized its functional expression in Pichia pastoris GS115. The engineered P. pastoris/ 17 beta-HSD3 cells exhibited good selectivity for the efficient transformation of AD to TS. Moreover, Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase (G6PDH) was introduced to strengthen the NADPH regeneration system into the pathway from AD to TS by P. pastoris/17 beta-HSD3. By optimization of the transformation conditions from AD to TS and applying the fed-batch strategy, the co-expressed system P. pastoris/17 beta-HSD3-G6PDH produced TS of 11.6 g L-1, which is the highest reported yield using a bioconversion method. Compared with the ever highest reported production (<= 1.7 g L-1), our production was improved by about 7-fold. More importantly, no by-products were detected during the whole bioconversion process. This study indicated that the recombinant P. pastoris harboring 17 beta-HSD3 and G6PDH could be a promising candidate to produce TS in the pharmaceutical industry. The P. pastoris system co-expression target enzyme and the cofactor regeneration enzyme may be helpful for enhancing the production of other steroids.