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

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.

#### **4.1. Screening of micro-organisms**

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 the growth of the microorganisms.

#### **4.2. Genome modifications**

**Figure 3.** Enzymes involved in nucleus degradation of AD.

**2.2. Biotransformation and substrates used**

118 Secondary Metabolites - Sources and Applications

**3.1. Substrate solubility**

*Rhodococcus, Streptomyces* and *Mycobacterium* sp*.* [2, 3].

**3. Factors affecting androstenedione production**

produced leading to a loss in yield by around 16.3 percent [12].

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,* 

Several researchers have reported their work in developing a microbial biotransformation process with high yield of AD with wide industrial applications [9–11]. Therefore, it was important to understand the factors affecting the actual biotransformation process. The basic and the most reported research is on low solubility of phytosterols in the aqueous media. This leads to low mass transfer rate and low substrate availability for conversion of phytosterol to AD. Oxygen transfer rate was also reported to be another critical parameter in the biotransformation of phytosterol [12]. Along with oxygen transfer rate (OTR) the effect of by-products such as 1,4-HBC formed during the bioprocess was found to affect the amount of the products

> 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


To enhance the production of AD from phytosterol ionic liquid-aqueous biphasic systems were established by Yuan et al. [19]. Ionic liquids (ILs) with different cations and anions provided distinct but favorable substrate solubilization and product distribution for two phase conversion. The results of this study showed that AD production reached 2.23 g L−1 after 5 days of biotransformation with substrate concentration of 5 g L−1. Further, ionic liquids which are easy to recycle produce negligible vapor pressure which indicates the industrial application of ILs in biphasic transformation process. Hence this strategy can be built up fur-

Microbial Biotransformation for the Production of Steroid Medicament

http://dx.doi.org/10.5772/intechopen.75149

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A recent strategy employed for enhanced AD production is the use of three-stage fermentation system. The well-known two-stage fermentation system uses a complex sugar (fructose in this case) as an initial carbon source followed by additional supplementation of simple sugar (glucose) which leads to elimination of the lag phase of the microorganism as well as an increase in biomass [21]. Shao et al. showed the effect of different carbon and energy sources on *Mycobacterium neoaurum* for enhancement of the desired steroid intermediate (AD/ADD) [13]. The researchers found that fructose acted as an optimal initial carbon source. Subsequent feeding by glucose maintained the metabolism of *mycobacterium neoaurum*. The researchers proposed three-stage fermentation by addition of phytosterol as third source and found enhanced ADD production (18.6 g/ l) which is reported to be the highest using *mycobacterium neoaurum*.

Gerber et al. developed a whole–cell system based on recombinant *Bacillus megaterium* which encodes CYP11A1, the enzyme responsible for side chain cleavage of sterols [4]. The microorganism's PHB granules, aggregates of bioplastic coated with a protein/phospholipid monolayer act as substrate storage entities. As described earlier, substrate solubility of sterols in microbial biotransformation process is one of the major obstacles; PHB granules increase the conversion rate by serving as substrate storage entity. This phenomenon leads to increase in the mass transfer thereby increasing the desired product. Microorganisms which code for PHB production or the organisms having the ability to produce PHB granules can be screened for the biotransformation of sterols. These organisms may help in substrate avail-

As per the available literature, there are number of strategies to increase the product yield. However, this area of research is still a hot topic as new strategies need to be developed which can lead to high yields of AD from cheaper substrates. Techniques involving industrially appropriate strains or addition of bioavailable catalysts to enhance the conversion or analysis of molecular pathway for generation of by-products. This may lead to development of a commercially cost effective process with high yields of the desired product. AD being one of the most important steroids which has high demand in pharmaceuticals as it is the precursor for

ability thereby enhancing the efficiency of biotransformation process.

ther for a more scaled up production.

**4.4. Three-stage fermentation system**

**4.5. Use of PHB granules**

**5. Conclusions**

**Table 1.** Microorganisms reported to have the ability to transform sterols to steroidal intermediates.

enzymes responsible for nucleus degradation of AD. To avoid the nucleus degradation genome modification or mutation studies can be performed. Wei et al. has demonstrated a study to increase the yield of AD or ADD by mutations in Ksh or KsdD genes [14]. Ksh enzyme has two subunits namely A and B known as monooxygenase and reductase respectively. As described by Wei et al., the whole genome sequencing of *Mycobacterium neoaurum* explains six distantly placed gene clusters encoding KsdD enzyme and two gene clusters encoding KshA and KshB each. It was found that NwIB-02 *Mycobacterium neoaurum* with mutation in KsdD gene accumulated AD as the main product. Besides that, double mutated *mycobacterium neoaurum* i.e. mutations in both KsdD and Ksh genes accumulated AD as the main product of microbial biotransformation of phytosterol. Cloning of any one of the genes encoding these enzymes into a more economically cultivable strain may lead to enhanced production of desired products.

#### **4.3. Liquid polymer-based systems**

As described earlier, low substrate solubility is one of the major obstacles in aqueous bioconversion systems involving hydrophobic compounds. Researchers derived an alternative of using organic solvents in the aqueous media to increase the substrate solubility and mass transfer. On the other hand, organic solvents are not environment friendly. An alternative to organic solvents are the use of supercritical fluids, liquid polymers, ionic liquids and natural oils [19, 20]. Such strategies also negate the drawbacks caused by organic solvents i.e. damaging effects on microbial cells and its hazardous nature. Carvalho et al. demonstrated the use of poly (methylphenylsiloxane) oil (Silicone B oil) for AD extraction. It provides a suitable media for sitosterol side chain cleavage. AD yields close to 10 mM were obtained in almost 4–5 days of incubation, for an initial substrate concentration of 12 mM (referred to the polymer/organic solvent phase), with a biocatalyst concentration of 5 mg dry cell weight/ml. The researchers concluded the use of silicone B oil as non-volatile and non-toxic for providing a sustainable environment for the microbial side chain cleavage of sitosterol, both in single liquid phase system and in oil: aqueous two liquid phase systems.

To enhance the production of AD from phytosterol ionic liquid-aqueous biphasic systems were established by Yuan et al. [19]. Ionic liquids (ILs) with different cations and anions provided distinct but favorable substrate solubilization and product distribution for two phase conversion. The results of this study showed that AD production reached 2.23 g L−1 after 5 days of biotransformation with substrate concentration of 5 g L−1. Further, ionic liquids which are easy to recycle produce negligible vapor pressure which indicates the industrial application of ILs in biphasic transformation process. Hence this strategy can be built up further for a more scaled up production.

#### **4.4. Three-stage fermentation system**

A recent strategy employed for enhanced AD production is the use of three-stage fermentation system. The well-known two-stage fermentation system uses a complex sugar (fructose in this case) as an initial carbon source followed by additional supplementation of simple sugar (glucose) which leads to elimination of the lag phase of the microorganism as well as an increase in biomass [21]. Shao et al. showed the effect of different carbon and energy sources on *Mycobacterium neoaurum* for enhancement of the desired steroid intermediate (AD/ADD) [13]. The researchers found that fructose acted as an optimal initial carbon source. Subsequent feeding by glucose maintained the metabolism of *mycobacterium neoaurum*. The researchers proposed three-stage fermentation by addition of phytosterol as third source and found enhanced ADD production (18.6 g/ l) which is reported to be the highest using *mycobacterium neoaurum*.

#### **4.5. Use of PHB granules**

enzymes responsible for nucleus degradation of AD. To avoid the nucleus degradation genome modification or mutation studies can be performed. Wei et al. has demonstrated a study to increase the yield of AD or ADD by mutations in Ksh or KsdD genes [14]. Ksh enzyme has two subunits namely A and B known as monooxygenase and reductase respectively. As described by Wei et al., the whole genome sequencing of *Mycobacterium neoaurum* explains six distantly placed gene clusters encoding KsdD enzyme and two gene clusters encoding KshA and KshB each. It was found that NwIB-02 *Mycobacterium neoaurum* with mutation in KsdD gene accumulated AD as the main product. Besides that, double mutated *mycobacterium neoaurum* i.e. mutations in both KsdD and Ksh genes accumulated AD as the main product of microbial biotransformation of phytosterol. Cloning of any one of the genes encoding these enzymes into a more economically cultivable strain may lead to enhanced production of desired products.

**Organism Product Strategy Reference** *Mycobacterium neoaurum* AD, ADD Three-stage fermentation [13]

*Nocardioides simplex* AD, ADD, T Selective media containing Hp-β- CD [15] *Mycobacterium smegmatis* AD, ADD Selective media containing Hp-β- CD [16] *Mycobacterium tuberculosis* AD, ADD Selective media containing Hp-β- CD [17] *Moraxella ovis* AD, ADD Natural medium containing rice bran oil [11] *Deinococcus radiodurans,* AD, ADD Natural medium containing rice bran oil [11] *Corynebacterium equi* AD, ADD Natural medium containing rice bran oil [11] *Corynebacterium urealyticum* AD, ADD Natural medium containing rice bran oil [11] *Mycobacterium smegmatis* AD, ADD Selective media [18]

8- hydroxyquinoline

[10]

*Pseudomonas aeruginosa* AD,ADD, B Selective media containing

120 Secondary Metabolites - Sources and Applications

AD: Androstenedione, ADD: androstadienedione, T: Testosterone, B: Boldenone.

**Table 1.** Microorganisms reported to have the ability to transform sterols to steroidal intermediates.

As described earlier, low substrate solubility is one of the major obstacles in aqueous bioconversion systems involving hydrophobic compounds. Researchers derived an alternative of using organic solvents in the aqueous media to increase the substrate solubility and mass transfer. On the other hand, organic solvents are not environment friendly. An alternative to organic solvents are the use of supercritical fluids, liquid polymers, ionic liquids and natural oils [19, 20]. Such strategies also negate the drawbacks caused by organic solvents i.e. damaging effects on microbial cells and its hazardous nature. Carvalho et al. demonstrated the use of poly (methylphenylsiloxane) oil (Silicone B oil) for AD extraction. It provides a suitable media for sitosterol side chain cleavage. AD yields close to 10 mM were obtained in almost 4–5 days of incubation, for an initial substrate concentration of 12 mM (referred to the polymer/organic solvent phase), with a biocatalyst concentration of 5 mg dry cell weight/ml. The researchers concluded the use of silicone B oil as non-volatile and non-toxic for providing a sustainable environment for the microbial side chain cleavage of sitosterol, both in single liquid phase

**4.3. Liquid polymer-based systems**

system and in oil: aqueous two liquid phase systems.

Gerber et al. developed a whole–cell system based on recombinant *Bacillus megaterium* which encodes CYP11A1, the enzyme responsible for side chain cleavage of sterols [4]. The microorganism's PHB granules, aggregates of bioplastic coated with a protein/phospholipid monolayer act as substrate storage entities. As described earlier, substrate solubility of sterols in microbial biotransformation process is one of the major obstacles; PHB granules increase the conversion rate by serving as substrate storage entity. This phenomenon leads to increase in the mass transfer thereby increasing the desired product. Microorganisms which code for PHB production or the organisms having the ability to produce PHB granules can be screened for the biotransformation of sterols. These organisms may help in substrate availability thereby enhancing the efficiency of biotransformation process.
