11

From Soybean Phytosterols

to Steroid Hormones
Feng-Qing Wang, Kang Yao and Dong-Zhi Wei
State Key Laboratory of Bioreactor Engineering
School of Biotechnology, East China University of Science and Technology, Shanghai
China
1. Introduction
Phytosterols, which are structurally and physiologically similar to cholesterol, are a large
group of steroidal triterpenes. They are essential to maintaining normal function in plant
cell membranes. In recent years, some of their beneficial effects on human health have come
to light. Phytosterols can lower intestinal cholesterol absorption, thereby reducing serum
levels of low-density lipoproteins and the risk of atherosclerosis. The pharmaceutical
industry has a long history of converting phytosterols to therapeutic steroid hormones by
microbial transformation. One commercially available phytosterol product is a phytosterol
mixture extracted from soybean oil deodorizer distillate. Soybean phytosterols usually
include four sterols: -sitosterol, stigmasterol, campesterol, and brassicasterol, all of which
make good raw materials for the production of steroid hormones because of their typical A-
ring molecular structure with a 3-hydroxyl group and a 5,6-double bond (Fig. 1). Two
kinds of steroid hormone intermediates can be produced from soybean phytosterols
through microbial transformation. The first of these are the C19-steroids, which include
androsta-4-ene-3,17-dione (AD), androsta-1,4-dien-3,17-dione (ADD), 9-hydroxy-
androstra-4-ene-3,17-dione, and testosterone; the second are the C22-steroids, such as 20-
carboxy-pregna-l,4-dien-3-one and 20-hydroxymethylpregna-1,4-dien-3-one (Fig. 2). C19-
steroids are the products of complete side chain cleavage. They can be used as precursors to
almost all kinds of steroid hormones, including sex hormones, anabolic steroids, and even
adrenocortical hormones. C22-steroids are the products of truncated side chain. These make
good precursors to adrenocortical hormones. The chemical conversion of sapogenins to
steroids is a well-established alternative to microbial transformation of phytosterols to
steroids. This method has many shortcomings, however, such as higher costs, more steps,
low yield, the waste of land resources, and the destruction of wild plant resources. In light
of this, microbial conversion of soybean phytosterols to steroids shows great value in the
synthesis of steroid hormones.
Here, we summarize our knowledge of the occurrence of phytosterols in soybeans and the
technology that can be used to transform them from phytosterols to steroids through the
regulation and modification of microbial catabolism. Based on analysis of the metabolic
mechanisms of phytosterols and the bottlenecks inherent in the microbial transformation
process, we will also discuss areas for development and improvement.
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Fig. 1. Cholesterol and soybean phytosterols based on steroid skeleton
2. Soybean phytosterols
2.1 Soybean phytosterols and human health
Phytosterols are a group of steroid alcohols that occur naturally in plants. They are generally
isolated during the process of producing vegetable oils, especially soybean oil. Soybean oil
deodorizer distillate (SODD) is one main wastes of the soybean-processing process. It is rich
in phytosterols and has become the main source of commercial phytosterols (Ramamurthi &
McCurdy, 1993; Hirota, et al., 2003, Benites, et al., 2009). -sitosterol, stigmasterol,
campesterol, and brassicasterol are the four major types of soybean phytosterols soybean.
They differ in the double bond at C22 and the substituents at C24 (Fig. 1). Just like
cholesterol in animals, phytosterols regulate of the fluidity of plant cell membranes and
feature in cellular differentiation and proliferation (Benveniste, 1986, Piironen, et al., 2000).
Soybeans are a well-known health food, rich in quality protein, fatty acids, and other
healthy components, including phytosterols. Because most people enjoy taking in high-fat,
high-energy foods that raise the serum cholesterol level out of healthy range, dyslipidemia,
together with its accompanied syndromes, hyperlipidemia, adiposis, and cardiovascular
disease, are now threatening the health of citizens of the developed and developing world
(Kes Niemi & Miettinen, 1987; Genest, et al., 2003). In recent years, many studies have
indicated that phytosterols may be beneficial as food additives because they can lower the
absorption of cholesterol in intestines by 10% to 15% (Ostlund, et al., 2003; St-Onge, et al.,
2003). The FDA has stated that daily intake of moderate amounts of phytosterols can reduce
the risk of heart disease. The long-term intake of phytosterol-rich foods could efficiently
diminish plasma cholesterol and atherosclerotic risk. For patients who cannot tolerate
cholesterol-lowering drugs, it may be advisable to adopt a phytosterol-rich diet as a
substitute (Ostlund, et al., 2003).
2.2 Phytosterol biosynthesis
Like cholesterol, phytosterols are biosynthetically derived from squalene. However, the
synthesis of phytosterols involves a relatively complicated mechanism. In plants, the
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anabolism of phytosterols can be divided into two parts with squalene being the critical
point (Fig. 3) (Piironen, et al., 2000).


Fig. 2. Steroids derived from soybean phytosterols by microbial transformation
The pre-squalene pathway is a nucleus-accumulating process derived from isoprenoid,
using isopentenyl pyrophosphate (IPP) as the fundamental building block. It is carried out
by the mevalonic acid pathway (Guo, et al., 1995). It has been determined that the series of
enzymes involved in the pre-squalene pathway from acetyl coenzyme A to squalene, via the
formation of 3-hydroxymethyl-3-glutaryl coenzyme A (HMG) are mevolonate, isopentenyl
pyrophosphate (IPP), and farnesyl pyrophosphate (FPP). Within this process, 3-
hydroxymethyl-3-glutaryl coenzyme A reductase (HMGR) is one predominant but
controversial factor believed to determine the capability of phytosterol synthesis (Bach,
1986; Goldstein & Brown, 1990; Stermer, et al., 1994). Squalene synthesis was reported to be
involved in competition with sesquiterpene, which is regulated by sesquiterpene cyclase
(SC) (Vogeli & Chappell, 1988). The activation of sesterpene synthesis would
correspondingly result in the suppression of sterol accumulation. Consequently, the
inhibition of SC should be regarded as an important control mechanism for enhancing sterol
formation.
The post-squalene pathway mainly refers to the modification of branch chain on steroidal
compounds. Conversion of cycloartenol to -sitosterol and stigmasterol involves two
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methylation steps, whereas conversion to campesterol and brassicasterol involves one.
There are two different families of sterol methyl transferases (SMT 1 and SMT 2) known to
be responsible for biosynthesis of the 24-methyl and 24-ethyl sterols, respectively (Nes &
Venkatramesh, 1999; Bouvier-Nav, et al., 1998). SMT has been found to be associated with
the 5-sterol production. Most importantly, it has the highest degree of sterol specificity f
any compound involved in the pathway. In other words, the ultimate concentration ratio of
phytosterol components, campesterol/-sitosterol, primarily depends on SMT activity.




Fig. 3. Sterol synthesis in plants, yeasts and mammals.
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3. Conversion of soybean phytosterols to steroid hormones
3.1 Steroid hormones
Steroids are a class of endocrine hormones secreted by the sexual organs and the adrenal
cortex in animals. Sterols are closely related to the regulation of physiological effects and the
development of reproductive structures, bone, and brain. Many synthetic steroid hormones
have been developed and used as pharmaceuticals to cure diseases resulting from the
secretion defects of endocrine steroid hormones. Steroid hormones can be grouped into
three categories according to their physiological functions: corticosteroids, sex steroids, and
anabolic steroids. Corticosteroids can be further divided into two kinds: glucocorticoids and
mineralocorticoids. Glucocorticoids play a critical role in regulating many aspects of
metabolism and immune function. They also exhibit favorable anti-inflammatory potencies.
Mineralocorticoids help maintain blood volume and control renal excretion of electrolytes
(Craigie, et al., 2009). Common corticosteroids include C21-steroids, such as hydrocortisone
and prednisone. Sex steroids are a type of sex hormone. They include androgens, estrogens,
and progestagens, which affect the sex differences and support reproduction (Callewaert, et
al., 2010). Anabolic steroids are similar to androgens, interacting with androgen receptors to
increase muscle and bone synthesis (Kicman, 2008). Both sex and anabolic steroids have C19
structures. Aside from their endocrine roles, some steroids also perform antiviral,
anticancer, cholesterol management, cardiovascular, and neuroprotective functions (Wang,
et al., 2007; Garcia-Segura & Balthazart, 2009).
Usually, steroid hormones are synthesized by semi-synthesis using natural steroids as
starting materials. Sapogenins and phytosterols are two basic kinds of starting materials
commonly used for the production of steroids. These form two main technical routes in the
pharmaceutical industry (Fernandes, et al., 2003; Malaviya & Gomes, 2008). One is the
chemical transformation of sapogenins such as diosgenin, hecogenin and solasodine to C21
steroids such as 16-dexosy-prognenolone. The other is the microbial transformation of
phytosterols to C19 steroids or C22 steroids (Fig. 2). Compared to the chemical
transformation of sapogenins, the microbial transformation of phytosterols has the
advantage of a simple and environmental friendly process, a stable supply, and low costs.
3.2 Microbial transformation of phytosterols to valuable steroids
Early in the 1980s, the putative metabolic pathway for synthesizing sterols in
microorganisms had already been proposed according to the identification of metabolic
intermediates (Fig. 4). So far, the cleavage process of sterol nucleus has been extensively
studied, and many of the steps in its multi-step reactions have been identified. However
side chain cleavage has not been well studied until now. In 2007, van der Geize disclosed the
gene clusters involved in sterol catabolism from Rhodococcus sp. RHA1 and Mycobacterium
species, signifying a new starting point for the biotransformation of phytosterols (van der
Geize, et al., 2007).
As shown in Figure 4, the biotransformation of phytosterols is initiated by the oxidation of
3-hydroxyl-5-ene moiety of sterols to 4-ene-3-one moiety. This is carried out by cholesterol
oxidases (Cho) (Aparicio & Martin, 2008; Doukyu, 2009). Then the transformation process
bifurcates into two independent routes: steroid nucleus decomposition and side chain
cleavage (Szentirmai, 1990).
3-ketosteroid-9-hydroxylase (Ksh) and 3-ketosteroid-1-dehydrogenase (KstD) are the
enzymes responsible for the decomposition of the steroid nucleus, which acts independently
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and successively on rings A and B to form 1,4-dien-9-hydroxy-steroids, structurally
unstable chemicals that spontaneously evoke the opening of ring B (van der Geize, et al.,
2001, 2002a). Maintaining an intact steroid nucleus is key to producing valuable steroids
from the catabolism of phytosterols. Kshs and KstDs have attracted much attention for their
special roles in initiating cleavage of the steroid nucleus. One enzyme must be inactivated to
block the decomposition of the steroid nucleus for the development of steroid-producing
strains (van der Geize, et al., 2000, 2002a; Petrusma, et al., 2009). As shown in Figures 2 and 4,
the inactivation of Kshs, KstDs, or both can result in the production of C19 steroids such as
AD, ADD, and 9-OHAD. In theory, the production of AD can be achieved via the
inactivation of both Kshs and KstDs in a microbial cell, and the accumulation of ADD or 9-
OHAD can be achieved by disrupting the Ksh or KstD genes. Traditional mutagenesis-
screening techniques have long been employed as a practical means of producing C19-
steroid-producing strains (Donova, et al., 2005c; Huang, et al., 2006; Gulla, et al., 2010).
Nevertheless, C19 steroids such as AD, ADD, and testosterone are known to coexist in
products (Donova, et al., 2005c; Huang, et al., 2006). This is one of the more significant
deficiencies between the strains selected from random mutations; the subtle differences in
structure between C19 steroids make them difficult to separate in industrial applications.
This seriously increases production costs and limits the application of these mutant strains.
In addition, mutant strains have other deficiencies, including residual decomposition of the
steroid nucleus and reduced transformation efficiency, such as we observed in our study.
These deficiencies can be attributed to the complex metabolic processes of the sterols and
multi-isoenzymes of KstD and Ksh within microorganisms. These make it difficult to target
and block all of the KstD and Ksh genes without interfering with other important genes.
Genetic engineering can be considered a viable technique toward facilitating the
development of ideal steroid-producing strains. However, genetic engineering was limited
by the genetically undefined mechanisms of sterol catabolism until 2000 and 2002, when van
der Geize et al. first revealed the KstD and Ksh genes responsible for the catabolism of
sterols in Rhodococcus erythropolis SQ1 (van der Geize, et al., 2000, 2002a). This team went on
to describe the isoezymes of KstD and Ksh (van der Geize, et al., 2002b, 2008). Notably, in
2007, they revealed the existence of a gene cluster encoding sterol catabolism in Rhodococcus
and Mycobacterium species (van der Geize, et al., 2007). The research of van der Geize and co-
workers facilitated the development of ideal sterol-transformation strains for the synthesis
of valuable C19-steroids. Subsequently, targeted disruption or augmentation of KstDs and
Kshs was successfully used in the development of C19 steroid-producing strains (van der
Geize, et al., 2000; Wei, et al., 2010).
Cleavage of the sterol C17-side chain has been well depicted as similar to the fatty acid -
oxidation and the genes likely to be responsible for this process have been shown to be part
of the gene cluster responsible for sterol catabolism (Szentirmai, 1990; Van der Geize, et al.,
2007). However, the genetic definitions of these processes have yet to be outlined. Using
mutagenesis-screening techniques, the C17-side chain cleavage can be blocked to produce
products with truncated side chains, such as C22-steroids, which are valuable candidate
intermediates for the synthesis of steroid hormones (Fig. 2) (Szentirmai, 1990). C22-steroid-
producing strains are rarer than C19 strains, partly because of the complex enzyme systems
likely to be involved in side chain cleavage.
In the terms of practical applications, both C19-steroids and C22-steroids can be readily
used to produce higher value-added steroid hormones. The C19-steroids can be grouped by
pharmacological activity into anabolic and androgen steroids. These steroids are already
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237
used as the main precursors for all kinds of sex and anabolic hormones, largely because
their structures are already similar to those of the desired final products (Malaviya &
Gomes, 2008). The C19-steroids can also be used as precursors for the synthesis of C21
corticosteroids (Andrews, et al., 1996). C19-steroids are highly important precursors of
steroid hormones and can be used to synthesize almost all of the steroid hormones used in
clinical settings. C22-steroids show their advantage in the synthesis of C21 corticosteroids,
which can be achieved by simple oxidative decarboxylation (Tora & Ambrus, 1990).


Fig. 4. Catabolic pathway of phytosterols (with -sitosterol as substrate). (HSD represents
the enzyme 17-hydroxy steroid dehydrogenase.) (Kieslich, 1985; Szentirmai, 1990; van der
Geize, et al., 2007)
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4. Inherent problems and solutions in strain improvement
The microbial metabolism of sterols has already been shown to be a promising means of
preparing valuable steroids. During the second half of the twentieth century, many
microbial strains were developed and tailored to synthesize many kinds of C19 and C22
steroids using phytosterols as substrates (Szentirmai, 1990). So far, however, the production
of steroids from phytosterols by microbial transformation is still not widely used in the
pharmaceutical industry. This may be because of two inherent problems in the phytosterol
biotransformation process: (1) The low water solubility of phytosterols can lead to poor
bioavailability. (2) The final products are toxic to microbial cells. Many technological
strategies have been proposed to overcome these problems. These have been reviewed in
detail by Malaviya and Fernandes (Fernandes, et al., 2003; Malaviya & Gomes, 2008). Here,
we mainly focus on how to develop robust organisms that can withstand their own
products.
4.1 Low water solubility and adaptive mechanisms
Most steroids have solubility below 0.1 mM. Phytosterols are more hydrophobic, their water
solubility usually being no more than 1 M. The poor solubility of phytosterols seriously
retards their bioavailability because of inadequate mass transfer (Goetschel & Bar, 1992;
Phase & Patil, 1994; Malaviya & Gomes, 2008). Many efforts have been carried out to
improve the dispersity and dissolubility of phytosterols in reaction media. Among these,
surfactant-facilitated emulsification, favored for its practicality and effectiveness, is a
common means of enhancing the bioavailability of phytosterols (Fernandes, et al., 2003;
Malaviya & Gomes, 2008). There are other strategies, but many of these seem inadequate in
practice. For example, organic solvent facilitated dissolution significantly raises the
complexity of the process and production costs and also results in environmental pressures.
It is also not very productive.
The mechanism of sterol uptake by microbial cells has already been demonstrated: during
the transformation process, microbial cells adhere to the surfaces of sterol particles, forming
stable agglomerates. Sterol uptake takes place via the direct contact between the cells and
the sterol particles (Atrat, et al., 1991; Goetschel & Bar, 1992; Fernandes, et al., 2003). This is
an adaptive mechanism that allows microorganisms to use minimally water-soluble
hydrocarbons as carbon sources. Like phytosterols, many polycyclic aromatic hydrocarbons
(PAH), such as naphthalene and anthracene, also exhibit extremely low water solubility and
are also poorly bioavailable (Johnsen & Karlson, 2004; Harms, et al., 2010a, 2010b). Many
microorganisms have been shown to use these hydrophobic hydrocarbons as carbon sources
through similar evolutionary adaptations (Miyata, et al., 2004; Heipieper, et al., 2010; Parales
& Ditty, 2010; Harms, et al., 2010a). Generally, the microorganisms adapted to use
hydrophobic hydrocarbons have the following physiological properties: (1) lipophilic cell
walls and adaptive changes in the surface properties allowing direct adhesion to the
hydrophobic substrates, (2) high affinity uptake systems such as active transporters and
membrane-associated enzymes for initial degradation, (3) the ability to excrete
biosurfactants or bioemulsifiers in order to increase the bioavailability of hydrophobic
hydrocarbons (Wick, et al., 2002; Heipieper, et al., 2010; Miyata, et al., 2004; Parales & Ditty,
2010; Johnsen & Karlson, 2004; Perfumo, et al., 2010). Several studies regarding adaptive
responses to PAH have been carried out with Gram-positive bacteria belonging to the
mycolate-containing bacteria families, including Mycobacterium, Rhodococcus,
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Corynebacterium, and Nocardia. Interestingly, mycolate-containing actinobacteria are also
used in the transformation of phytosterols (Heipieper, et al., 2010; Kim, et al., 2010).
Although these adaptive properties have not been well studied, some studies have shown
that the similar adaptive responses play key roles in the transformation process of
phytosterols (Atrat, et al., 1991; Rajkhowa, et al., 2000; Fernandes, et al., 2003).
4.1.1 Cell envelope and adaptive changes affecting bioavailability
The mycolate-containing actinobacteria are well known as hydrophobic organisms. They
have complex and extremely lipophilic cell envelopes containing large amounts of long-
chained mycolic acids specific to this group of bacteria. Mycolic acids may reach up to 60%
of the dry mass of the cell walls of some actinobacteria and typically range in size from 30
90 carbon atoms. These are often modified in structure by alkylation and hydroxylation and
cross-linked to polysaccharide components, forming an exceptionally thick and rigid
envelope. This envelope is of particular interest with respect to adaptations allowing the
microorganism to degrade a broad range of hydrophobic solid compounds (Korycka-
Machala, et al., 2001; Heipieper, et al., 2010; Kim, et al., 2010). Many researches have also
found that bacterial hydrophobic envelopes play a critical role in enhancing the uptake
capacity of phytosterols (Atrat, et al., 1991; Rajkhowa, et al., 2000; Fernandes, et al., 2003).
Mycolate-containing bacteria are able to change their physiochemical surface properties by
modifying the composition of their envelopes (Wick, et al., 2002; Falkinham, 2009; Heipieper,
et al., 2010). In the case of mycobacteria, anthracene-grown cells respond to the low
bioavailability of their hydrophobic substrate by modifying their cell envelopes via the
synthesis of mycolic acids with longer alkyl chains. This significantly increases the
hydrophobicity of the outer membrane, leading to up to 70-fold stronger adhesion to
hydrophobic surfaces than glucose-grown cells (Wick, et al., 2002). Although the adaptive
changes of the cell envelope relevant to phytosterol-transforming activity are not well
understood, cell wall lipid content has been proved to more than double in the presence of
sterols. High lipid content and long-chain fatty acids in the envelope of sterol-grown cells is
believed to contribute to the high adherence activity of these cells to sterols (Rajkhowa, et al.,
2000). It is conceivable that enhancing the hydrophobicity of the cell envelope by strain
development based on natural adaptive mechanisms may be an effective means of
overcoming the problem of low bioavailability.
It has been noted that the superficial envelope of mycolate-containing actinobacteria does
not always seem to be advantageous with regard to phytosterol bioavailability. Some
researchers have shown that the cell envelope might be a barrier to permeability, which is
disadvantageous for phytosterol uptake and biotransformation. This can be improved by
compounds such as vancomycin, glycine, lecithin, protamine, polymixin B nonapeptide,
bacitracin, and ethambutol (Fernandes, et al., 2003; Malaviya & Gomes, 2008). Vancomycin,
glycine, and bacitracin are effective reagents, affecting the peptidoglycan layer of the cell
membrane. Ethambutol is can disrupt the biosynthesis of arabinogalactan in the cell
envelope. Lecithin and protamine have been shown to alter the fatty acid profile of the cell
envelope. Structurally speaking, in the envelope of mycolate-containing actinobacteria,
mycolic acids attach to the 5'-hydroxyl groups of D-arabinose residues of arabinogalactan
and form a mycolyl-arabinogalactan-peptidoglycan complex, becoming barriers to
permeability for exogenous chemicals (Draper, 1998). The chemicals listed above have been
found to affect the outer part (mycolic acid layer) of the cell envelope. This may motivate
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research into enhancing the bioavailability and conversion productivity of phytosterols
through strain improvement focusing on a structurally changed envelope.
4.1.2 Affinity uptake of sterols
In 1991, Atrat et al. described a model of flexible multi-component mesophase (FMCM) on
the cellular uptake of sterol substrates via direct contact between cells and the substrate
particles based on observation by freeze-fracture electron microscopy. In this model, they
assumed that a FMCM exists between cells and sterol particles and that this FMCM
mediates sterol uptake, forming a sharp concentration gradient to drive transport. At the
same time, they considered that there should be channels composed of sterol-binding or
steroid-transforming proteins connecting the cytoplasm with the cell surface to reinforce the
transport of sterols across the thick cell walls (Atrat, et al., 1991).
In 2007 and 2008, van der Geize, Mohn, and their colleagues disclosed and substantiated the
idea that there is a steroid uptake system in the gene cluster encoding sterol catabolism (van
der Geize, et al., 2007; Mohn, et al., 2008). The steroid uptake system is a kind of mce4
("mammalian cell entry") locus encoded by an operon including 10 genes conserved in
diverse mycolate-containing actinobacteria. This locus is composed of two transmembrane
permease (supAB) genes and eight mce4 protein (mce4ABCDEFHI) genes (Mohn, et al.,
2008). The 10 genes have been shown to encode components of complex ATP-binding
cassette ABC transporters. The deletion and inhibition of these genes has been shown to
result in an absence of sterol uptake. Additionally, transcriptional analyses have indicated
that the operon was up-regulated 4.0-fold during growth on cholesterol, relative to growth
on pyruvate. This strongly supports the idea that these 10 genes are specifically involved in
sterol uptake. Ultimately, Mohn speculated that the proteins encoded by the mce4 loci might
form a complex of ABC uptake transporters in the membrane of mycolate-containing
bacteria to mediate the movement of the sterol substrates across the thick, hydrophobic cell
envelope (Mohn, et al., 2008). This research on the biological function of the mce4 system
provides us with new insight into the mechanism of active transport of phytosterols in the
microbial transformation process.
Atrat also speculated that steroid-transforming proteins, especially extracellular enzymes,
such as cholesterol oxidase (3-hydoxysteroid dehydrogenase), might make up part of the
FMCM structure and stimulate sterols transport (Atrat, et al., 1991). Cholesterol oxidases are
extracellular flavoenyzmes that catalyze the oxidation and isomerization of cholesterol to
cholest-4-en-3-one (cholestenone or ketocholesterol), which is in charge of the first
compulsory step in bacterial sterol catabolic pathways that transform sterols into sterones
(Kreit & Sampson, 2009). These enzymes often occur in secreted and cell-surface-associated
forms. This is closely related to their role in oxidizing exogenous sterols. Cholesterol
oxidases are in a class of interfacial enzymes that catalyze reactions with membrane-soluble
substrates. An enzyme is catalytically active at the contact membrane interface of cholesterol
or other sterols (Sampson & Kwak, 2008). In a recent study, we identified two cholesterol
oxidases, ChoM1 and ChoM2, from a gene cluster encoding sterol degradation in a
Mycobacterium strain. ChoM1, a class I cholesterol oxidase, does not occur as a secreted form,
while ChoM2, a class II cholesterol oxidase, can be secreted into the medium. The roles of
these two ChoMs in the utilization of phytosterols have been demonstrated by genetic
inactivation, which showed that both ChoM1 and ChoM2 are necessary for the
Mycobacterium strain to use phytosterols as carbon sources. Notably, the deletion of ChoM2
led to the Mycobacterium strain failing to survive on a medium with phytosterols as the sole
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carbon source. Both enzymes could enhance transformation efficiency by augmenting their
activity in an ADD-producing strain. We also found Mycobacterium strains to make use of
sterones to a greater degree than sterols. Cholesterol and phytosterols are indispensable to
cellular membranes in animals and plants, respectively. They can exist in the plasma and
mimic membranes. They function in altering the properties of lipids in membranes to
maintain membrane integrity and fluidity. However, many studies have indicated that
cholestenone dose not have any effect to change the properties of membrane lipids and is
not stable in the membrane (Bacia, et al., 2005; Beattie, et al., 2005; Lintker, et al., 2009). The
differences in intra-membrane behavior between sterols and sterones and the physiological
functions of cholesterol oxidases imply that cholesterol oxidases may be part of the sterol
transport process. Although further investigation is still needed to confirm their specific
roles on cellular sterol uptake, there is little doubt that the augmentation of cholesterol
oxidases will improve phytosterol-transforming strains.
4.1.3 Excretion of biosurfactants and bioemulsifiers and bioaccessibility and
bioavailability of phytosterols
The bioaccessibility of hydrophobic hydrocarbons is considered an important part of their
bioavailability. It depends on the contact surface area between microbial cells and substrates
(Harms, et al., 2010b). The smaller the particle size, the greater this area will be.
Experimental observation has indicated that the most favorable arrangement to sterol
absorption is substrate particles that are very similar in size to the intended microbial cells
(Atrat, et al., 1991). Although the size of the phytosterol particles can be affected by
mechanical attrition, the hydrophobic phytosterols tend to self-aggregate in water to form
agglomerates. This seriously limits their bioaccessibility and bioavailability. In most cases,
surfactant-facilitated emulsification is used to inhibit the aggregation of phytosterols and
keep the phytosterol particles homodisperse in the reaction medium. However, synthetic
surfactants and emulsifiers are generally toxic to bacteria. They can solubilize cell
membranes and induce enzymatic disorders, leading to necrosis and cell lysis (Li & Chen,
2009). In recent years, biosurfactants and bioemulsifiers have emerged as alternatives to
synthetic surfactants and emulsifiers. They have attracted attention for their
biocompatibility with cells. In natural environments, many bacteria secrete biosurfactants or
bioemulsifiers to render minimally water-soluble carbon sources accessible (Li & Chen,
2009, Perfumo, et al., 2010). Mycolate-containing actinobacteria such as Rhodococcus,
Corynebacterium, Mycobacterium, and Nocarida have a catabolic capacity for phytosterols and
have been shown to have the ability to produce biosurfactants (Perfumo, et al., 2010). From
this, it can be concluded that the selection and development of organisms with a robust
ability to produce biosurfactants or bioemulsifiers may be an alternative to synthetic
surfactants.
4.2 Toxic effects of steroid products on microorganisms
The cellular toxicity of steroids to microbial cells is one of the major factors limiting the
productivity of conversion from phytosterols to steroid products. Some steroid products,
such as AD and ADD, even at low concentrations, are believed to inhibit cell growth and
respiration and suppress the enzyme activity in the sterol degradation pathway, causing
low product yield (Zeillinger & Spona, 1986; Perez, et al., 2003; Donova, 2007). Many
attempts to overcome this problem are being investigated by segregating steroid products
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from the reaction media through in situ product recovery and by developing mutants with
improved tolerances to the steroid products (Perez, et al., 2003; Malaviya & Gomes, 2008).
4.2.1 In situ product recovery
In situ product recovery is a common and effective means of reducing the negative effects of
toxic products on microbial cells. Some special adsorbents, such as amberlite XAD-7 resin
for AD and ADD, are often added to the reaction media to increase the yield of AD and
ADD by absorbing steroid products (Huang, et al., 2006; Malaviya & Gomes, 2008). Positive
results have also been obtained by using polymers as reservoirs, reducing the concentration
of steroid products in the reaction medium. For example, poly dimethyl siloxane and
cyclodextrins have been used to recover ADD and AD from the reaction medium. This can
improve transformation productivity. In addition, aqueous or organic-aqueous two-phase
systems are also clean and effective tools for in situ product recovery. In two-phase systems,
the water-immiscible organic phase acts as reservoir for toxic products. The significantly
higher solubility of sterols in the organic phase allows the system to instantaneously recover
toxic steroid products during biotransformation. For a more detailed discussion of in situ
steroid recovery, we refer to the review by Malaviya and Gomes (2008).
4.2.2 Development of mutants with increased resistance to steroid products
The developing mutants with improved tolerance to toxic products may be a more effective
means of solving the problem of toxic products than product sequestration. Perez et al.
demonstrated that high concentrations of androstanes could be used to isolate tolerant
mutants and increase androstane yield. In that case, colonies growing at least 1 mg/ml ADD
in culture medium after nitrosoguanidine mutagenesis showed improved ADD yield, going
from 18.81 mg/ml to 38.99 mg/ml (Perez, et al., 2003). So far, the actual nature of the toxicity
of steroids to microbial cells has not been elucidated. Similarly, the mechanisms by which
microbial cells become resistant remain unknown. Even so, robust tolerance to toxic steroid
products is one of the most important characteristics of improved phytosterol-transforming
strains.
4.2.3 Eflux transporters for steroid products
In natural environments, tolerance to toxins is important for survival. In the case of
phytosterol-transforming mycobacteria, there are two common mechanisms involved in
resistance to toxicity: the rigid envelope acts as a barrier to extracellular toxins and the active
multidrug efflux pumps remove intracellular toxins (Nikaido, 1994; De Rossi, et al., 2006). In
the phytosterol transformation process, most of the reactions occur intracellularly.
Therefore, it is reasonable to conclude that multidrug efflux pumps play a role in resistance
to toxic steroid products. Genes encoding drug efflux transporters have been identified in
several mycobacterial species, including M. fortuitum, M. tuberculosis, and M. smegmatis
(Ainsa, et al., 1998; Li, et al., 2004). These genes, which exist in the form of multi-copy
plasmids or replicates in the genome, encode efflux proteins that transport tetracycline,
fluoroquinolones, and other compounds outwards. Because steroid compounds resemble
tetracycline and fluoroquinolones, it is reasonable that some non-specific efflux transporters
may transport steroids outside the cell. These efflux transporters should be investigated for
applicability to the development of phytosterol-transforming organisms resistant to toxic
products.
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4.3 Development of ideal organisms
Strain performance plays a central role in the commercial development of microbial
conversion systems. In essence, the abovementioned problems can be attributed to deficient
microbial strains. The fundamental solution is to develop robust, efficient, highly productive
microbial strains. Random mutagenesis is the conventional breeding approach and has been
the main means of developing custom phytosterol-transforming strains (Egorova, et al.,
2002; Donova, et al., 2005a; Sripalakit, et al., 2006). However, strains surviving multiple
rounds of mutation are genetically undefined and vulnerable to further changes. Directed
genetic modification may be a more feasible option for subsequent isolation of mutant
strains with desired functions. Rational metabolic engineering provides a more effective and
well-defined alternative to strain development than random mutagenesis. Strategies for
developing the desired organisms by metabolic engineering can be summarized as follows:
(1) inhibition of B-ring cleavage to maintain the steroidal nucleus by inactivating the key
enzymes KstDs and Kshs; (2) overexpression of genes encoding rate-limiting reactions and
blockage of downstream and branched reactions to increase the accumulation of desired
product and reduce the production of unwanted by-products; (3) enhancement of cellular
uptake of phytosterols by increasing the activity of active transporters and/or the
permeability of the cell wall; (4) enhancement of cellular tolerance to toxic products and
efflux capacity of toxic products.
Ideal microorganisms should have the following characteristics:
(1) Lack of pathogenicity, ease of culture
Many organisms that can transform phytosterols into valuable steroids have been isolated.
These include mycobacteria, which seem perform the best. This is why most of the microbial
strains used in industrial and academic research are members of the genus Mycobacterium
(Atrat, et al., 1991; Fernandes, et al., 2003; Perez, et al., 2006). However, mycobacteria have
two serious deficiencies. First, many Mycobacterium species are known to be stubborn
pathogens. These include M. tuberculosis and M. leprae. Many other Mycobacterium species,
although generally non-pathogenic, have been shown to be opportunistic pathogens
(Malaviya & Gomes, 2008; Cassidy, et al., 2009; van Ingen, et al., 2009). For example M.
fortuitum can cause local cutaneous disease, osteomyelitis, and joint infections (Wallace, et
al., 1983). The other problem is that mycobacteria are difficult to culture because of their
complicated physiological states, rod-coccus growth cycle, slow growth rate, and special
nutritional requirements (Falkinham, 2009; Kim, et al., 2010). In addition, the process by
which mycobacteria convert phytosterols to steroid hormones is complicated and can last
more than a week. This reduces its economic benefit. For these reasons, it may be best to
select safer and more rapidly growing organisms, such as those in the genera Arthrobacter,
Bacillus, Corynebacterium, Brevibcterium, Rhodoccocus, Norcardia, and others.
(2) Ease of modification
Unfortunately, genetic manipulation techniques suitable to most of the organisms with
proven capacity for phytosterols conversion lags behind those that work on other bacterial
species. This makes it difficult to improve these organisms by means of metabolic
engineering. For example, it is difficult to perform genetic recombination on mycobacteria
because of the high rate of illegitimate recombination and the lack of universal tools among
Mycobacterium species (Kim, et al., 2010). This has hindered the progress of strain
improvement. In addition, phytosterol metabolism in microbes is a complex process. To
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build an ideal strain, many metabolic reactions and physiological properties will need to be
modified and regulated. An ideal phytosterol-transforming organism, therefore, must be
easy to be modified, preferably by relatively simple genetic recombination techniques.
(3) Single-product production
Most phytosterol-transforming strains can transform phytosterols into many structurally
similar products, such as the C19 steroids AD, ADD, and their derivatives, at the same time
(Egorova, et al., 2002; Donova, et al., 2005a, 2005b, 2005c; Molchanova, et al., 2007). Because
the differences in the structures of these products are very subtle, it is difficult to separate
and purify them. This significantly complicates the production process and increases
production costs (Molchanova, et al., 2007). An ideal microbial strain, therefore, must be able
to transform phytosterols into only a single desired product.
(4) Powerful phytosterol uptake capacity
The extreme hydrophobicity of phytosterols seriously limits their bioavailability. As
mentioned above, most of the strategies that enhance phytosterol uptake increase the
complexity of the production process and add to its costs. However, many microorganisms
already have numerous physiological adaptations that allow them to take up highly
hydrophobic compounds, such as phytosterols. These adaptations can be further improved
to enhance the organisms' phytosterol uptake capacity. The following strategies should be
attempted: i) Increase the direct contact between microbial cells and phytosterol particles by
changing the physiochemical properties of the cell envelope. ii) Enhance the active transport
capacity for phytosterols by augmenting the activity of mce4 transporters. iii) Overexpress
cholesterol oxidases. iv) Increase the bioaccessibility of phytosterols by reinforceing the
excretion of biosurfactants and/or bioemulsifiers.
(5) Strong tolerance to toxic products
Although several methods of sequestering toxic steroids from the reaction media have been
tested, the development of mutants with improved tolerance to steroid products may be a
more effective way to bypass the toxic effects of steroid products on microbial cells
(Molchanova, et al., 2007; Perez, et al., 2003; Malaviya & Gomes, 2008). An ideal organism
should have physiological functions that reduce or eliminate the harmful effects of toxic
products. Such organisms may be developed using strategies that allow the selection of
evolutionary mutants by continuous, prolonged exposure to high concentrations of toxic
products and augment efflux transporters.
5. The future of phytosterol production
Phytosterol catabolism in microbes is extremely intricate. It involves not only dozens of
specific biocatalytic reactions but also cellular properties catering to phytosterol metabolism,
such as phytosterol uptake systems and resistance to toxic products. Currently, only the
genes directly encoding sterol catabolism were found as a cluster of 51 genes in Rhodococci
and 80 to 82 genes in mycobacteria (van der Geize, et al., 2007). Most of which have not been
well characterized in function until now. The most urgent task is to clearly characterize and
elucidate the mechanisms of phytosterol catabolism and the cellular properties related to
phytosterol uptake, tolerance to toxic products, and global regulations.
Along with gradual in-depth research on the mechanisms by which phytosterols are
transformed into steroid hormones, metabolic engineering may be useful to the
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development ideal microbial "factories" for the production of desired steroid hormones from
phytosterols. To meet the basic standards depicted in section 4.3, strain improvement by
metabolic engineering should include three steps:
The first step is to rationally modify the catabolic phytosterol pathway toward formation of
the target product, avoiding cleavage of the steroid nucleus and formation of any
byproducts. The cleavage of the steroid nucleus can be prevented by disrupting the key
enzymes in steroid B-ring cleavage, KstDs and Kshs (Fig. 2). The accumulation of
byproducts can be eliminated by blocking side reactions and amplifying rate-limiting
reactions. To produce ADD, for example, Kshs should be blocked and KstDs should be
activated (Fig. 5). Several similar projects have already been successful in Rhodococcus
species and Mycobacterium (van der Geize, et al., 2007; Wei, et al., 2010).
The second step should focus on economically feasible productivity. The strains developed
in the first stage may be limited in efficiency. To increase their economic benefit, they should
be reconstructed to avoid limitations in productivity. The phytosterol uptake capacity of the
organisms should be enhanced by augmenting sterol transporters such as Mce proteins, and
reducing the barrier effect of the cell envelope (Mohn, et al., 2008; Korycka-Machala, et al.,
2005; Hoffmann, et al., 2008). The tolerance of the microbial cells to toxic products should be
improved. This may be achieved by enhancing the cells' ability to expel active products by
augmenting or rationally designing specific efflux pumps or by reducing the sensibility of
the strain to the toxic products. Negative regulators and feedback inhibitors limiting the
phytosterol metabolism may also be of use and should be examined and deregulated. For
example two TetR-type transcriptional regulators control sterol utilization in mycobacteria
(Kendall, et al., 2010).
The third step is to comprehensively reprogram phytosterol-transforming organisms based
on systems biology. In brief, the metabolic flux from phytosterols to steroid products should
be comprehensively investigated, modified, and optimized based on industrial
requirements. Approaches to this include transcriptomics, proteomics, metabolomics, and
computational modeling.
Microorganisms with good phytosterol conversion capacities may be not good candidates for
modification by metabolic engineering. This is partly because of the complexity of
physiological process of reprogramming the cell. Recently, metabolic engineering has become
capable of creating novel, finely controlled metabolic and regulatory circuits that can produce
desired products effectively. Reconstructing the transformation pathway from phytosterols to
steroid homones in a heterologous host organism with suitable physiological trains, then, may
an alternative to developing engineered organisms. In 2003, Szczebara and co-workers
described a process that rerouted ergosterol biosynthesis in Saccharomyces cerevisiae to
compatible brassicasterol and campesterol, two ingredients in the four soy phytosterols (Fig. 1)
and then extended the pathway to produce hydrocortisone by mimicking hydrocortisone
biosynthesis seen in the mammalian adrenocortex. In other words, an artificial, fully self-
sufficient biosynthetic pathway was successfully built in a microbial host, suggesting that the
production of steroid hormones may also take place in this simple, environmentally friendly,
low-cost manner (Szczebara, et al., 2003). This kind of revolutionary approach to the
production of steroid hormones may be the future of the steroid pharmaceutical industry.
The integrated reconstruction of the phytosterol transformation system in microbes may
become a reality in the near future, most likely in one of two ways.
The first way would be to rebuild the phytosterol transformation system in robust hosts that
would be superior to existing phytosterol-transforming organisms in tolerance to high
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concentrations of steroid hormones, phytosterol uptake capacity, simplicity of gene
manipulation, and cell cultivation. This would need to be founded on a comprehensive
understanding of the processes by which phytosterols are converted to products, including
the degradation of C17-side chains. In order to facilitate the assembly and control of this
system, the process of design and reconstruction should based on synthetic biology,
standardizing the gene parts required, designing controllable and efficient gene circuits,
developing functional modules, and integrating and optimizing the rebuilt system in the
selected host. Therefore, a novel engineered organism with ideal phytosterol-transforming
properties may be developed (Fig. 5A).


Fig. 5. Transformation pathway of phytosterols in S. cerevisiae.
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The second way would be to learn from the works of Szczebara and Dumas and reconstitute
the phytosterol conversion system in yeast (Szczebara, et al., 2003; Dumas, et al., 2006). Yeast
is a proven producer of endogenous sterols, such as ergosterol and ergosta-5,7-dienol.
Ergosterol and ergosta-5,7-dienol are similar to the precursors of phytosterols, and they can
be converted to brassicasterol and campesterol by C7-reductases from plants (Hartmann,
2003; Szczebara, et al., 2003). Brassicasterol and campesterol are two components of the four
main phytosterols found in soybeans (Fig. 1). Therefore, the endogenous sterol biosynthesis
seen in yeast may be rerouted to produce soybean phytosterols by the introduction of C7-
reductase from a plant, such as the soybean (Fig. 4C). In this way, the conversion pathway
of soybean phytosterols to C19 steroids or C22 steroids can be reassembled (Fig. 4A) and
then linked to the natural ergosterol biosynthetic pathway in yeast. This would result in
steroid hormones in self-sufficient in yeast fed on a simple carbon source, such as glucose or
ethanol (Fig. 4). The benefits of this method are that the many reactions involved in
producing C19 or C22-steroids from phytosterols would be integrated in a single
fermentation step, avoiding the need for the production or addition of phytosterols. Such a
simple production process would revolutionize the synthesis of steroid hormones in
industry and significantly lower production costs and environmental impact. Nevertheless,
there are also some drawbacks to this technique. One important issue is the inherent
difficulties of the yeast system to adapt to the exogenous biocoversion process, such as the
toxicity of the steroids to the yeast organism. Much more additional research must be
performed to improve the physiological properties of yeast before it can become an
industrially advantageous means of producing steroid products.
6. Aknowlegements
The work was supported by the National Basic Research Program of China (2009CB724703)
and the Fundamental Research Funds for the Central Universities and China Postdoctoral
Science Foundation ( 20100470757).
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Soybean and Health
Edited by Prof. Hany El-Shemy
ISBN 978-953-307-535-8
Hard cover, 502 pages
Publisher InTech
Published online 12, September, 2011
Published in print edition September, 2011
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Worldwide, soybean seed proteins represent a major source of amino acids for human and animal nutrition.
Soybean seeds are an important and economical source of protein in the diet of many developed and
developing countries. Soy is a complete protein, and soy-foods are rich in vitamins and minerals. Soybean
protein provides all the essential amino acids in the amounts needed for human health. Recent research
suggests that soy may also lower risk of prostate, colon and breast cancers as well as osteoporosis and other
bone health problems, and alleviate hot flashes associated with menopause. This volume is expected to be
useful for student, researchers and public who are interested in soybean.
How to reference
In order to correctly reference this scholarly work, feel free to copy and paste the following:
Feng-Qing Wang, Kang Yao and Dong-Zhi Wei (2011). From Soybean Phytosterols to Steroid Hormones,
Soybean and Health, Prof. Hany El-Shemy (Ed.), ISBN: 978-953-307-535-8, InTech, Available from:
http://www.intechopen.com/books/soybean-and-health/from-soybean-phytosterols-to-steroid-hormones