Journal
GREEN CHEMISTRY
Volume 18, Issue 6, Pages 1774-1784Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c5gc02353j
Keywords
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Funding
- National Basic Research Program of China (973 Program) [2012CB725202]
- High-Tech Research and Development Programs of China [2011AA02A211, SS2015AA021004]
- National Natural Science Foundation of China [31570085]
- Jiangsu Province Science Fund for Distinguished Young Scholars [BK20150002]
- Fundamental Research Funds for the Central Universities [JUSRP51306A]
- 111 Project [111-2-06]
- Priority Academic Program Development of Jiangsu Higher Education Institutions
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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.
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