Highlight on Engineering Mycobacterium smegmatis for testosterone production

The development of male attributes such as external genitalia and secondary sexual characteristics is due to a hormone called testosterone produced in the testicles. This hormone is also responsible for the development and maintenance of muscle mass (Griggs et al., 1989), bone density, red blood cell counts (Bachman et al., 2014), supports sexual and reproductive function and contribute to a man’s sense of anger and vitality (Batrinos, 2012). While the underlying mechanism is not known, the production of testosterone in males gradually declines with age, beginning at around 30 (Morley and Perry, 2000). The decline is associated with a near-total lack of interest in sex, erectile dysfunction (Kratzik et al., 2005), diabetes (Barrett-Connor, 1992), Alzheimer’s disease (Moffat et al., 2004), cardiac failure (Kontoleon et al., 2003), hypercholesterolaemia (Haffner et al., 1993), osteoporosis (Campion and Maricic, 2003), frailty, obesity (Svartberg et al., 2004), hypertension (Phillips et al., 1993) and ischaemic heart disease (Barrett-Connor and Khaw, 1988). The idea of supplementing declining levels of testosterone to treat diseases was realized by the scientific community as early in the mid-1930s (Hamilton, 1937). In recent years, the use of testosterone therapy has become more widespread, but the only available synthetic form of testosterone is still expensive. Hence, a very little fraction of those affected are able to afford this therapy. Microbes are ubiquitous and humans have exploited their biological processes, especially through genetic manipulation for the production of hormones and antibiotics. In 1978, genes encoding human insulin was cloned and expressed in E. coli followed by cloning of growth hormone in 1979, which replaced the unhuman forms of both these hormones. However, until now, testosterone (TS) has been chemically produced from androst-4ene-3,17-dione (AD) (Ercoli and Ruggierii, 1953). Not only being expensive, the synthetic form has been reported to induce several side-effects such as allergy, nausea or vomiting, impotency, painful or difficult urination, high levels of calcium in the blood, mild truncal acne, weight gain (Matsumoto, 1990) and coronary heart disease (Tripathy et al., 1998). In mammals, 17-ketosteroid reductase (17b-HSD) enzyme catalyses the synthesis of TS from AD. There was a hunt for microbial sources – both bacterial and fungal enzymes (Donova et al., 2005) that could convert AD to TS in order to reduce the production cost and benefit patients allergic to the synthetic derivative of TS. The ability of microorganisms to reduce 17-ketoto 17b hydroxysteroids was first reported in Saccharomyces cerevisiae, during the transformation of androst-4-ene-3,17-dione to testosterone (Charney and Herzog, 1967). Subsequently, the ability to carry out 17b-reduction of AD was reported for a variety of microorganisms of different taxonomy, including Mycobacterium, Pediococcus, Brevibacterium, Bacillus, Arthrobacter, Lactobacillus and Nocardia (Wix et al., 1968; Uwajima et al., 1973; Mahato and Mukherjee, 1984; Dutta et al., 1992; Kumar et al., 2001). But, very few microbial 17b-OH SDHs were isolated and characterized. The most investigated enzyme is 3(17)bhydroxysteroid dehydrogenase (3(17) b-OH SDH) isolated from Comamonas testosteroni (earlier classified as Pseudomonas testosteroni) (Groman and Engel, 1977). Attempts were made to introduce the 17b-hydroxysteroid gene from Comamonas testosteroni in E. coli (known model system for biotechnological processes) (Pl esiat et al., 1991). However, substantially low uptake of AD across the cellular membrane by E. coli limited the production process instigating researchers to look for novel biological models for the industrial production of Received 2 November, 2016; revised 29 October, 2016; accepted 3 November, 2016. *For correspondence. E-mail [email protected]; Tel. +91-011-27666254; Fax +91-011-27666254. Microbial Biotechnology (2017) 10(1), 73–75 doi:10.1111/1751-7915.12466