Yeast As a Promising Heterologous Host for Steroid Bioproduction
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Journal of Industrial Microbiology & Biotechnology https://doi.org/10.1007/s10295-020-02291-7 METABOLIC ENGINEERING AND SYNTHETIC BIOLOGY - MINI REVIEW Yeast as a promising heterologous host for steroid bioproduction Shanhui Xu1 · Yanran Li1 Received: 30 April 2020 / Accepted: 6 July 2020 © Society for Industrial Microbiology and Biotechnology 2020 Abstract With the rapid development of synthetic biology and metabolic engineering technologies, yeast has been generally con- sidered as promising hosts for the bioproduction of secondary metabolites. Sterols are essential components of cell mem- brane, and are the precursors for the biosynthesis of steroid hormones, signaling molecules, and defense molecules in the higher eukaryotes, which are of pharmaceutical and agricultural signifcance. In this mini-review, we summarize the recent engineering eforts of using yeast to synthesize various steroids, and discuss the structural diversity that the current steroid- producing yeast can achieve, the challenge and the potential of using yeast as the bioproduction platform of various steroids from higher eukaryotes. Keywords Yeast · Steroids · Biotransformation · De novo synthesis · Metabolic engineering Introduction as the medicinally important steroidal alkaloids and steroidal saponins [8]. Sterols are essential membrane components that regulate Intriguingly, some phytosteroids exhibit promising anti- membrane fuidity and permeability in eukaryotic organ- viral activities via diferent mechanisms, which make them isms [1]. Animals generally synthesize cholesterol, fungi intriguing lead structures for novel antiviral drug discovery produce ergosterol, and plants utilize an array of sterols with and development [9–12]. Plants also produce ecdysteroid β-sitosterol and campesterol as the most common ones [1]. analogs, phytoecdysteroids, as natural defense mechanism Despite the essential roles in membrane modulation, sterols against phytophagous insects [4]. Moreover, steroids are also exhibit important bioactivities. For example, due to the an important pharmaceutical medication, with the global structural similarity to cholesterol, phytosterols can reduce steroid market projected to reach $17 billion in 2025 [13]. cholesterol absorption in digestive system and reduce the Steroid drugs generally fall into two categories: anabolic risk of heart disease [2, 3]. In higher eukaryotes, sterols are and corticosteroids. Anabolic steroids are involved in the also precursors to a wide array of specialized metabolites treatment of muscle loss or late puberty [14]; while corti- with broad structural diversity and biological activities. costeroids can be used as anti-infammatory drugs or allergic In insects, cholesterol is converted into insect steroid hor- treatment [15]. During the recent outbreak of COVID-19, mones, such as ecdysteroids that can be potentially utilized methylprednisolone (median price $16.03/80 mg [16]), as anabolic steroids [4]. In human, cholesterol is the precur- a corticosteroid, was reported to have positive efects on sor to an array of steroid hormones such as progesterone and a faster improvement of the clinical symptoms caused by vitamin D [5, 6]. In plants, campesterol is the main precursor COVID-19 [17]. More recently, another corticosteroid, of the ubiquitous phytohormone brassinosteroids [7]; cho- dexamethasone (median price $12.30/4 mg from www. lesterol, although exhibits very low abundance in plants, is GoodR x.com), has been found to able to reduce the death the precursor to a broad spectrum of phytochemicals, such by roughly one-third of the patients on ventilators [18]. However, most of these specialized steroids are not natively produced at a high level, due to the nature of their * Yanran Li biological functions. Similar to other specialized metabo- [email protected] lites, the structural complexity also makes the chemical 1 Department of Chemical and Environmental Engineering, synthesis of these compounds challenging and expensive 900 University Avenue, Bourns Hall, Suite A220, Riverside, [19]. Recent development of synthetic biology provides an CA 92521, USA Vol.:(0123456789)1 3 Journal of Industrial Microbiology & Biotechnology alternative sourcing strategy of these molecules through bio- especially in the model organism Saccharomyces cerevi- technological production in the fast-growing, fermentable siae. The biosynthesis of ergosterol in S. cerevisiae can be microorganisms, such as yeast and Escherichia coli. The divided into three modules [22]: (1) biosynthesis of meva- structural and biosynthetic similarity of ergosterol in yeast lonate (MVA) from acetyl-CoA, (2) conversion of MVA into to the other eukaryotic sterols highlights the potential of farnesyl pyrophosphate (FPP) and 3) ergosterol biosynthesis using yeast as the bioproduction host for sterols and deriva- from FPP. The third module can be further divided into (3-1) tives [20]. The previous metabolic engineering eforts in lanosterol biosynthesis from FPP, (3-2) zymosterol biosyn- establishing steroid production in yeast have been very well thesis from lanosterol and (3-3) ergosterol biosynthesis from reviewed in Wriessnegger’s 2013 review [20]. In this review, zymosterol. Ergosterol, cholesterol, and the major phytos- we will mainly discuss the eforts in engineered steroid bio- terols, campesterol and β-sitosterol, are diferent in three production in yeast since 2013 and focus on the metabolic aspects: saturation of C7–C8 bond in B-ring, saturation of engineering strategies, and the challenges and potential bio- C22–C23 bond, and the alkane group at C24 in the side synthetic capacities of yeast-based steroid bioproduction. chain (Fig. 1). Sterol biosynthesis is highly conserved in eukaryotes [23]. The synthesis of ergosterol, cholesterol, and phytos- Ergosterol biosynthesis in yeast terols diverges from lanosterol, and shares a number of downstream intermediates. For example, zymosterol and Ergosterol, similar to cholesterol in animals and phytosterols episterol in the ergosterol synthesis (submodule 3-3) can in plants, is the most abundant sterol in fungi and plays an also be found in animals and plants, respectively [8, 24]. essential role in maintaining physicochemical properties of The convergency of the eukaryotic sterol structures and bio- plasma membrane and involves in protein sorting and pro- synthesis makes it possible to redirect the biosynthesis of tein receptor regulation [21]. The biosynthesis of ergosterol ergosterol to cholesterol and phytosterols in yeast. The late- exists in almost all yeast species and is well characterized, stage biosynthesis of ergosterol from zymosterol (module Fig. 1 Ergosterol synthesis and sterols from mammals and plants. The endogenous ergosterol biosynthesis in yeast is highlighted in yellow. The three diferent sheds of yellow represented three modules of ergosterol synthesis. Major phytosterols that have been synthesized or possibly synthe- sized in yeast are highlighted in green, and cholesterol is highlighted in blue. The char- acteristic structural features of sterols from diferent eukaryotic organisms are highlighted in pink. The enzymes marked in red represent the ones that require NADPH/NADP+, and the ones that require ATP are marked in blue 1 3 Journal of Industrial Microbiology & Biotechnology 3-3) involves fve ERG enzymes, ERG2-6. ERG6 is the Steroid hydroxylation usually involves 7α-, 11α-, 11β-, ∆24-sterol C-methyltransferase, converting zymosterol into 14α-hydroxylases, while dehydrogenation is generally fecosterol [25]. Fecosterol is then converted into episterol by catalyzed by 3β-, 5α-HSDs. Biotransformation of andros- ERG2 through the isomerization of C8–C9 double bond to tenedione (AD) to testosterone (TS) by S. cerevisiae was C7–C8 position [25]. ERG3 is the C5 desaturase that con- discovered as early as in 1937 [33], with the same function verts episterol into ergosta-5,7,24(28)-trienol [25]. ERG5 discovered in Schizosaccharomyces pombe later [34]. The is the C-22 sterol desaturase that catalyzes the conversion biotransformation of steroid drugs by microbial strains was from ergosta-5,7,24(28)-trienol to Ergosta-5,7,22,24(28)- intensively studied in the 1990s, but limited to strain min- tetraenol [25]. ERG4 is the ∆24-sterol reductase that reduces ing without comprehensive enzyme characterizations [32, C24–C28 double bond to synthesize ergosterol [25]. The 35]. However, the wild-type strains are limited to specifc corresponding genes (erg2-6) are all identifed as nones- types of reactions, which cannot be utilized to catalyze sential genes to yeast viability [26]. Previous investigations biotransformation beyond native catalytic capacity. For on the efects of erg gene deletions suggest that ERG2-6 example, although S. cerevisiae was reported to be able enzymes can accept a broad range of sterol structures, which to catalyze the reduction at C3, C20 and C17 of C21 and indicates the broad substrate promiscuities of ERG2-6 and C19 steroids [36], it cannot catalyze the desired hydroxyla- that these enzymes may not follow a specifc order to aford tion of steroids for the synthesis of hydrocortisone, which the synthesis of ergosterol [27]. For example, the disruption requires the C11-hydroxylation. It is important to charac- of erg6 accumulates not only zymosterol, but also cholesta- terize the enzymes responsible for the bioconversion in the 7,24-dienol, cholesta-5,7,24-trienol and cholesta-5,7,22,24- corresponding microbial strains, and thus enable the estab- tetraenol, which indicates