**2. Synthesis of androstenedione from phytosterols**

AD is synthesized in the body by adrenal cortex and gonads stimulating formation of testosterone or estradiol. Adrenocorticotropic hormone (ACTH) stimulates the steroid synthesis pathway from cholesterol leading to formation of AD. Phytosterol being similar to cholesterol is able to stimulate the formation of AD by its side-chain cleavage. (Elaboration of the pathway in **Figure 2**).

As shown in the **Figure 2**, CYP11A1 is an enzyme that is responsible for side chain cleavage of the sterol resulting in AD formation.

#### **2.1. Chemical synthesis**

progesterone, cortisone, cortisol, prednisone and prednisolone [1]. The steroid pharmaceuticals are of great importance for their role in the management of human fertility, osteoporosis, menopause and blood pressure regulation [2, 3]. Commercially, steroid production represents one of the largest sectors of medical products manufactured by the pharmaceutical industry [4]. Production of AD and androstadienedione (ADD) exceeds 1000 tons per year in the world. Therefore, production of AD on a large scale with cost effective process becomes an area of demanding research in biotechnology. Apart from the broadly used natural compounds, phytosterols gained an increasing importance as raw materials for the synthesis of steroidal drugs such as pregnenolone, boldenone, androstenedione and androstadienedione. The utilization of cholesterol and phytosterol as the sole carbon source by *Mycobacterium* sp. for growth and proliferation led to a serious development in microbial biotransformation

Phytosterols are thoroughly widespread in plants and are similar to cholesterol in terms of physiological functions and structure. Phytosterols differ from cholesterol by having a methyl or ethyl group at C-24 (**Figure 1**). Phytosterols participate in essential cellular processes since they modulate permeability and fluidity of membranes. In addition, they are precursors for the synthesis of steroid hormones and are involved in plant defense mechanisms. β-Sitosterol, campesterol, and stigmasterol are the main phytosterols found in plants (**Figure 1**). The most important phytosterols, β-sitosterol (C-29 carbon skeleton), campesterol (C-28), and stigmasterol (C-29), contribute up to 98% of all the phytosterols found in plants [5]. As cholesterol acts as a starting material for steroid production by adrenal cortex and gonads, phytosterol can act as a starting material for AD production by many microorganisms which are able to utilize

**Figure 1.** (A) Structure of cholesterol, (B) structure of campesterol, (C) structure of β-sitosterol, (D) structure of

processes for production of large number of steroidal compounds [1].

phytosterols as carbon and energy source.

116 Secondary Metabolites - Sources and Applications

stigmasterol.

Marsh demonstrated a chemical synthesis of AD (aromatase inhibitors) which involves multiple steps [6]. AD can be produced chemically by addition of thiol reagents to appropriate dienone intermediates. C19 steroids should retain the steroidal nucleus for their effective functions. However, the steroidal nucleus is sensitive to a temperature leading to its degradation [7]. Steroidogenic enzymes were also sensitive to endocrine-disrupting chemicals (EDCs) [8]. Apart from the above mentioned limitations, chemical synthesis leads to added costs and lower yield of AD with lengthy and sensitive procedures. Additionally, chemical synthesis of AD also requires use of toxic/harmful chemicals which affects the environment. Hence, there is a need to develop a process which is environment friendly and cost effective. Microbial biotransformation of phytosterol fulfills the need.

**Figure 2.** Side chain cleavage of sterol leading to AD formation.

#### **2.2. Biotransformation and substrates used**

Sterol rich substrates are used to obtain steroidal intermediates by biotransformation. There are a number of organisms known to transform phytosterols to AD such as *Aspergillus, Arthrobacter, Bacillus, Brevibacterium, Chryseobacterium, Fusarium, Gordonia, Nocardia, Pseudomonas, Rhodococcus, Streptomyces* and *Mycobacterium* sp*.* [2, 3].

**3.2. Nucleus degradation**

The change in the steroidal nucleus of AD is one of the major obstacles reported in the microbial biotransformation of phytosterols. After formation of AD during microbial biotransformation, further degradation of AD takes place producing either ADD or 9-hydroxy androstenedione (9-OHAD) [7]. In a phenomenon which was coined as nucleus degradation by the researchers, two major enzymes have been implicated so far: 3-ketosteroid-1,2-dehydrogenase (KsdD) was found to be responsible for AD to ADD conversion and hence lead to low yield of AD. 3-ketosteroid-9-hydroxylase (Ksh), was observed to be responsible for AD to

Microbial Biotransformation for the Production of Steroid Medicament

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9-OHAD conversion, leading to nucleus degradation and low yield of AD (**Figure 3**).

mation of phytosterol is a temperature sensitive process.

**4.1. Screening of micro-organisms**

the growth of the microorganisms.

**4.2. Genome modifications**

**4. Techniques to enhance production of androstenedione**

Xu et al. demonstrated that the enzymes involved in nucleus degradation of AD are temperature sensitive [7]. 3-ketosteroid-1,2-dehydrogenase (KsdD) and 3-ketosteroid-9-hydroxylase (Ksh) are enzymes sensitive at 37°. These enzymes break the B ring of steroidal nucleus which leads to degradation of the steroid. Hence it can be concluded that the microbial biotransfor-

As described earlier, to develop a process which is industrially applicable for high yields of AD, different techniques need to be incorporated. In the following section, we discuss the effective techniques to enhance AD production; thus making the process more economical.

In the process of microbial biotransformation, selection of the microorganisms is a very crucial step. Microorganisms which are able to utilize sterols as their sole energy and carbon source are reported to have the ability to produce AD as an intermediate in the sterol degradation pathway [13]. Such microorganisms e.g., *Mycobacterium* sp.*, Mycobacterium smegmatis, Rhodococcus* sp.*, Deinococcus radiodurans, Pseudomonas* sp. have been documented to have an ability to produce AD or ADD. **Table 1** illustrates the organisms which are able to utilize sterols as their sole carbon and energy source to produce steroidal intermediates, AD, ADD, 9-hydroxy AD or 9-hydroxy ADD and also the various strategies implemented for enhancing

As mentioned in the earlier sections, researchers have demonstrated the pathways followed by microorganisms for biotransformation of phytosterol, the enzymes responsible for AD production and its nucleus degradation. Various studies have been performed on genome sequencing and proteomics to determine the sequence of the genes involved in the side chain cleavage of phytosterol. CYP11A1 in *Mycobacterium neoaurum* was reported to be involved in side chain cleavage of phytosterol leading to formation of AD [14]. Further, as described in Section 3.2, 3-ketosteroid-1,2-dehydrogenase (KsdD) and 3-ketosteroid-9-hydroxylase (Ksh) are the
