초록
<P>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 17β-hydroxysteroid dehydrogenase type 3 (17β-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 17β-HSD3 and realized its functional expression in <I>Pichia pastoris</I> GS115. The engineered <I>P. pastoris</I>/17β-HSD3 cells exhibited good selectivity for the efficient transformation of AD to TS. Moreover, <I>Saccharomyces cerevisiae</I> glucose-6-phosphate dehydrogenase (G6PDH) was introduced to strengthen the NADPH regeneration system into the pathway from AD to TS by <I>P. pastoris</I>/17β-HSD3. By optimization of the transformation conditions from AD to TS and applying the fed-batch strategy, the co-expressed system <I>P. pastoris</I>/17β-HSD3-G6PDH produced TS of 11.6 g L<SUP>−1</SUP>, which is the highest reported yield using a bioconversion method. Compared with the ever highest reported production (≤1.7 g L<SUP>−1</SUP>), 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 <I>P. pastoris</I> harboring 17β-HSD3 and G6PDH could be a promising candidate to produce TS in the pharmaceutical industry. The <I>P. pastoris</I> system co-expression target enzyme and the cofactor regeneration enzyme may be helpful for enhancing the production of other steroids.</P><BR><BR><P>Graphic Abstract</P><P>Co-expressing human 17β-hydroxysteroid dehydrogenase type 3 and <I>S. cerevisiae</I> glucose 6-phosphate dehydrogenase for testosterone production.<BR><IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c5gc02353j'><BR></P>