New Insights on Steroid Biotechnology

Abstract

Nowadays steroid manufacturing occupies a prominent place in the pharmaceutical industry with an annual global market over $10 billion. The synthesis of steroidal active pharmaceutical ingredients (APIs) such as sex hormones (estrogens, androgens, and progestogens) and corticosteroids is currently performed by a combination of microbiological and chemical processes. Several mycobacterial strains capable of naturally metabolizing sterols (e.g., cholesterol, phytosterols) are used as biocatalysts to transform phytosterols into steroidal intermediates (synthons), which are subsequently used as key precursors to produce steroidal APIs in chemical processes. These synthons can also be modified by other microbial strains capable of introducing regio- and/or stereospecific modifications (functionalization) into steroidal molecules. Most of the industrial microbial strains currently available have been improved through traditional technologies based on physicochemical mutagenesis and selection processes. Surprisingly, Synthetic Biology and Systems Biology approaches have hardly been applied for this purpose. This review attempts to highlight the most relevant research on Steroid Biotechnology carried out in last decades, focusing specially on those works based on recombinant DNA technologies, as well as outlining trends and future perspectives. In addition, the need to construct new microbial cell factories (MCF) to design more robust and bio-sustainable bioprocesses with the ultimate aim of producing steroids à la carte is discussed.

Keywords: actinobacteria; metabolic engineering; microbial cell factory; pharmaceuticals; steroid; systems biology.

Figure 1

Chemical structures of relevant steroidal compounds containing the gonane tetracyclic skeleton. Structures of basic precursors, key intermediates (synthons) and active pharmaceutical ingredients (APIs) are shown. AD, 4-androstene-3,17-dione; ADD, 1,4-androstadiene-3,17-dione; 9OH-AD, 9α-hydroxy-4-androstene-3,17-dione; 4-HBC, 22-hydroxy-23,24-bisnorchol-4-ene-3-one; 1,4-HBC, 22-hydroxy-23,24-bisnorchol-1,4-diene-3-one; 9OH-4-HBC, 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one; 11OH-AD, 11α-hydroxy-4-androstene-3,17-dione; DHEA, 3β-hydroxy-5-androstene-7-one, dehydroepiandrosterone; TS, testosterone.

Figure 2

Classification of the microbial bioprocesses for steroid synthesis described to date: (i) production of steroidal intermediates from natural sterols (e.g., bioconversion of phytosterols into ADD); (ii) functionalization of steroidal molecules (e.g., bioconversion of AD into ADD); (iii) de novo biosynthesis of steroids (e.g., biosynthesis of hydrocortisone from non-steroidal substrates). Several microbial strains, isolated from natural sources and improved by conventional mutagenesis or designed by using recombinant DNA technologies, are used as microbial cell factories (MCF) to produce key steroidal intermediates (synthons). The resulting synthons are subsequently modified by chemical steps or additional bioconversion processes to synthetize final steroidal active pharmaceutical ingredients (APIs).

Figure 3

Scheme of the rational construction of mycobacterial strains producing steroidal intermediates from natural sterols by metabolic engineering approaches. Based on the knowledge of the catabolic pathway of sterols in actinobacteria, the redirection of metabolic flux toward the accumulation/synthesis of C-19 (red) or C-22 (gray) steroidal intermediates can be addressed via single/multiple gene deletions. The metabolic flux can be also redirected to the synthesis of new steroidal compounds by incorporating heterologous enzymatic activities (pink) (e.g., 17β-hydroxysteroid dehydrogenase, 17β-HSD). kstD, 3-ketosteroid-Δ¹-dehydrogenase; ksh, 3-ketosteroid-9α-hydroxylase; hsd4A, hydroxysteroid dehydrogenase. Abbreviations of metabolites: ADD, 1,4-androstadiene-3,17-dione; AD, 4-androstene-3,17-dione; 9OH-AD, 9α-hydroxy-4-androstene-3,17-dione; TS, 17β-hydroxy-4-androstene-3,17-dione; testosterone; 1,4-HBC, 22-hydroxy-23,24-bisnorchol-1,4-diene-3-one; 4-HBC, 22-hydroxy-23,24-bisnorchol-4-ene-3-one; 9OH-4-HBC, 9,22-dihydroxy- 23,24-bisnorchol-4-en-3-one.

Figure 4

Microbial functionalization of steroidal molecules of industrial relevance. (A) Δ1-Dehydrogenation of 3-ketosteroids: conversion of cortisone into prednisone. (B) 9α-Hydroxylation of 3-ketosteroids: conversion of progesterone into 9α-hydroxy-progesterone. (C) 11α-Hydroxylation of 3-ketosteroids: conversion de AD into 11α-hydroxy-AD. (D) Baeyer-Villiger oxidation: conversion of ADD into testolactone. KstD, 3-ketosteroid-Δ¹-dehydrogenase, KSH, 3-ketosteroid-9α-hydroxylase, P450, 11α-hydroxylase, cytochrome P450, BMVO, Baeyer-Villiger monooxygenase.

Figure 5

Current challenges of steroid biotechnology. New microbial cell factories are needed to design new industrial bioprocesses for the production of steroids à la carte, i.e., more robust one-step biotechnological processes with higher substrate conversion yields and product selectivity. Using multiple currently available methodologies, at least the following challenges should be addressed: (i) construction of a second generation of mycobacterial strains for the production of steroidal intermediates from sterols; (ii) identification of new microbial chassis and steroid-modifying enzymatic activities for the functionalization of steroidal molecules; (iii) de novo biosynthesis of steroids from non-steroidal substrates in mycobacteria.