Optimization of the 11α-hydroxylation of steroid DHEA by solvent-adapted Beauveria bassiana

Article abstract of DOI:10.1080/10242422.2017.1289183

To extend the use of Beauveria bassiana for commercial applications, the optimization of reaction conditions and accurate prediction of biotransformation products are necessary. This work enhances the selective hydroxylation capacity of strain ATCC 7159, resulting in a cost effective and eco-friendly process for the synthesis of valuable 11α-hydroxy steroids. Our work establishes the biochemical pathway of DHEA to hydroxylated intermediates with strain ATCC 7159, and distinguishes the optimum conditions for reactor arrangements, substrate concentration, reaction temperature, and pH. Higher substrate conversion, selectivity, and yield of desired product was achieved with “resting cells.” Addition of higher volumes of growing medium relative to reaction buffer increases the reaction rate. When a diluted amount of substrate is used, a higher yield of 11α-hydroxy steroids is achieved. Also, reactions at 26 °C with pH ranges between 6.0 and 7.0 result in the highest conversion (70%) and the higher product yield (45.8%). B. bassiana has the capacity to metabolize DHEA and similar steroids in different reaction schemes, and has a promising future as biocatalyst to be used in the production of drug metabolites.

Full text of DOI:10.1080/10242422.2017.1289183

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Optimization of the 11α-hydroxylation of steroid
DHEA by solvent-adapted Beauveria bassiana
Richard Gonzalez, Felipe Nicolau & Tonya L. Peeples
To cite this article: Richard Gonzalez, Felipe Nicolau & Tonya L. Peeples (2017): Optimization of
the 11α-hydroxylation of steroid DHEA by solvent-adapted Beauveria bassiana, Biocatalysis and
Biotransformation, DOI: 10.1080/10242422.2017.1289183
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1289183
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Date: 06 March 2017, At: 01:27

Biocatalysis and Biotransformation, 2017; Early Online: 1–7
RESEARCH ARTICLE
Optimization of the 11a-hydroxylation of steroid DHEA by
solvent-adapted Beauveria bassiana
RICHARD GONZALEZ, FELIPE NICOLAU & TONYA L. PEEPLES
Department of Chemical and Biochemical Engineering, The University of Iowa, Iowa City, IA, USA
Abstract
To extend the use of Beauveria bassiana for commercial applications, the optimization of reaction conditions and accurate
prediction of biotransformation products are necessary. This work enhances the selective hydroxylation capacity of strain
ATCC 7159, resulting in a cost effective and eco-friendly process for the synthesis of valuable 11a-hydroxy steroids. Our
work establishes the biochemical pathway of DHEA to hydroxylated intermediates with strain ATCC 7159, and
distinguishes the optimum conditions for reactor arrangements, substrate concentration, reaction temperature, and pH.
Higher substrate conversion, selectivity, and yield of desired product was achieved with ‘‘resting cells.’’ Addition of higher
volumes of growing medium relative to reaction buffer increases the reaction rate. When a diluted amount of substrate is
used, a higher yield of 11a-hydroxy steroids is achieved. Also, reactions at 26 ꢀC with pH ranges between 6.0 and 7.0 result
in the highest conversion (70%) and the higher product yield (45.8%). B. bassiana has the capacity to metabolize DHEA and
similar steroids in different reaction schemes, and has a promising future as biocatalyst to be used in the production of drug
metabolites.
Keywords: Beauveria bassiana; DHEA; steroid biotransformation; biocatalytic reactions; process; biohydroxylations
Introduction
applications, to induce and improve biocatalytic
oxidations.
Beauveria bassiana is a whole-cell biocatalyst used in
a variety of chemical biotransformations (Huszcza
et al. 2005). This entomopathogenic fungus has been
applied globally as an environmentally friendly
mycoinsecticide (Pedrini et al. 2007), and is one of
the major biocontrol agents applied in the United
States (Huszcza et al. 2005; Faria and Wraight
2007). The popularity of this fungus as a biopesticide
has increased substantially in recent years, as exten-
sive research has enhanced its effectiveness (Lehr
2012). As a result, most work in understanding the
complex regulation of cellular functions has been in
the area of virulence enhancement for pest control.
The impact of virulence enhancement on oxidative
biocatalysis has not been fully explored. To this end,
we are examining pesticide-related strategies,
designed to enhance the efficacy of biopesticide
In extending the use of B. bassiana for commercial
application, optimization of reaction conditions and
accurate prediction of biotransformation products
are necessary. A well-characterized biocatalyst has
transformative potential to overcome technology
gaps for selective bio-oxidations of commercial
interest. One area where fungal transformations
have been actively applied is in bioconversion of
steroids, which represent one of the largest sectors in
pharmaceutical industry with a market value over
$10 billion (Morgan and Moynihan 2000). In our
research, B. bassiana was applied in selective oxida-
tions for the synthesis of steroids to increase the
application as a practical biocatalyst and to further
improve oxidative selectivity. The rationale for suc-
cessful biotransformation arises from the potential
induction of oxidative enzymes in environments that
mimic natural conditions for B. bassiana.
Correspondence: Tonya L. Peeples, 4133 Seamans Center, The University of Iowa, Iowa City, IA 52242-1527, USA. Tel: 1-319-384-2681. E-mail: tonya-
peeples@uiowa.edu
(Received 8 March 2016; accepted 27 January 2017)
ISSN 1024-2422 print/ISSN 1029-2446 online ß 2017 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2017.1289183

2
R. Gonzalez et al.
When B. bassiana is cultivated in the presence of
and corn steep liquor (CSL) were purchased from
Sigma Aldrich.
n-alkanes such as n-hexadecane (n-C16), the gene
expression of (8) cytochrome (CYP) P450 mono-
oxygenase enzymes is induced (Pedrini et al. 2007,
2010). The importance of CYP enzymes in the
conversion of chemicals to valuable intermediates is
well known (Holland et al. 1999; Pfeifer and
Khachatourians 1989). These enzymes have a tre-
mendous potential in drug development (Kumar
2010). In our previous work, CYP’s potential was
presumably targeted with the design of a biotrans-
formation process involving n-C16 for the possible
induction of CYP genes (Gonzalez et al. 2015). In
this work, the previous process has been further
characterized and optimized for selective oxidation.
These efforts will lead to more cost effective and eco-
friendly production of 11a-hydroxy steroid deriva-
tives that cannot be easily synthesized by solely
chemical methods (Holland and Weber 1999;
Hanson and Hunter 2003; Kolek et al. 2008, 2009;
Huang et al. 2010).
Media preparation
n-alkane medium (NM): This medium was prepared
by mixing 0.4 g KH2PO4, 1.4 g Na2HPO4, 0.6 g
MgSO4.7H2O,
1.0 g
KCl,
and
0.7 g
of
NH4NO3.7H2O per liter of distilled water, at
pH 5. After sterilization, the medium was supple-
mented with n-hexadecane (n-C16) (10% v/v)
(Crespo et al. 2000). Glycerol medium (GM): This
medium was prepared by mixing 20 g of corn steep
liquor and 12.6 mL of glycerol per liter of water, at
pH 7. Buffer solution: This solution was prepared by
mixing 2.09 g Na(NH4) HPO4ꢁ4H2O and 1.74 g K2
HPO4 per liter of distilled water, at pH 7. When the
solution was at room temperature, 5 mL of 2 M
dextrose was added. The dextrose was sterilized by
passing it through 0.45 mm filter during the addition
to the buffer (Osorio-Lozada et al. 2008).
Our interest is to characterize the system and
identify reaction conditions that improve the hydrox-
ylation of unfunctionalized carbons during the
preparation of steroids. The catalytic activity of
B. bassiana was studied with dehydroepiandroster-
one (DHEA). DHEA is a steroid hormone produced
by adrenal glands, is the most abundant circulating
hormone in humans, and functions as a precursor to
potent androgens like androstenedione, testosterone,
estrone and estradiol (Boron and Boulpaep).
Previous studies showed that the reaction pathway
of DHEA derivatives varies with biocatalytic strain,
substrate and reaction parameters, which currently
yields poor amounts of valuable 11a-hydroxy inter-
Inoculum growth on n-alkanes
Cells were harvested (10% v/v) from Potato Dextrose
Broth (PDB) directly into NM (Phase 1). These cells
were grown and adapted to n-C16 for 15 days at
250 RPM and 26 ꢀC. Another 10% v/v inoculum
from this culture was used to repeat the process and
inoculate a new NM. Multiple generations (61) have
grown successfully using n-alkanes as carbon source
from June 2011 to April 2014.
Biotransformation procedure with resting cells
ˇ
mediates (Capek et al 1966; Protiva et al. 1968;
Phase 1 cultures (10% v/v inoculum) were washed
twice with sterile water and transferred to 200 mL of
GM. The flask was incubated at 250 RPM and 26 ꢀC
Huszcza et al. 2005; Xiong et al. 2006; Swizdor et al.
2011). Our work establishes the biochemical route
from DHEA to hydroxylated intermediates with
strain ATCC 7159, and defines the reactor arrange-
ment, substrate concentration, temperature and pH
for optimal production of these steroids.
for
3 days. This culture was centrifuged at
5000 RPM for 10 minutes; cells were washed with
buffer (3 ꢂ 50 mL) and re-suspended in 200 mL of
buffer. An amount of 1 mL of an ethanolic solution
of DHEA (200 mg/mL) was added to the resting
cells. The flask was incubated at 250 RPM and 26 ꢀC
for 7 days.
Materials and methods
Microorganism
Beauveria bassiana was purchased from the American
Type and Culture Collection (ATCC). This strain,
ATCC 7159, was isolated from a laboratory con-
taminant and routinely grown on potato dextrose
broth (PDB). n-Alkane solvents and salts were
purchased from Fisher Scientific. House deionized
water was further purified for experimental work
Biotransformation procedure with growing cells
A slant of B. bassiana ATCC-7159, kept in potato
dextrose agar, was transferred to 25 mL of GM. The
flask was incubated at 250 RPM and 26 ꢀC for
3 days. This culture was transferred into 200 mL of
GM. The flask was incubated at 250 RPM and 26 ꢀC
for 1 day. An amount of 1 mL of ethanolic solution of
DHEA (200 mg/mL) was added to this culture.
using
Ultrapure water purification system. DHEA, PDB
a
Thermo Fisher Barnstead Nanopure

Optimization of DHEA hydroxilation by B. bassiana
3
The flask was incubated at 250 RPM and 26 ꢀC for
7 days.
silica gel column chromatography (2 cm ꢂ 10 cm)
eluting with a gradient of chloroform/methanol
(12:1, v/v). Compounds were visualized by spraying
TLC plates with a solution of phosphomolybdic
acid (1:10, v/v) and heating at 105 ꢀC until
Biotransformation procedure with different volume
ratios
color
developed.
High-Performance
Liquid
Chromatography (HPLC) was performed with a
Shimadzu instrument (Riverwood Drive Columbia,
Phase 1 cultures (10% v/v inoculum) were washed
twice with sterile water and transferred into flasks
containing 25 mL, 50 mL, 75 mL, and 100 mL of
GM. These flasks were incubated at 250 RPM and
26 ꢀC for 3 days. These cultures were centrifuged
and washed following subsection 2.4 procedure, and
re-suspended in 25 mL of buffer, respectively.
DHEA (25 mg) dissolved in ethanol was added to
each flask and incubated at 250 RPM and 26 ꢀC for
7 days.
MD) equipped with
a C18 column (4.6 mm
ꢂ125 mm). Isocratic column elution was monitored
by a photodiode array detector. The wavelength was
set at 250 nm, and a methanol–water (60:40, v/v)
mobile phase was eluted at a flow rate 0.5 mL/min.
The 1H NMR Spectra were recorded at room
temperature on a 300 MHz Bruker Avance spec-
trometer (Billerica, MA) using deuterated chloro-
form (CDCl3) as a solvent and tetramethylsilane as
an internal standard.
Biotransformation procedure for the effect of initial
substrate concentration (DHEA)
Results
Phase 1 cultures (10% v/v inoculum) were washed
twice with sterile water and transferred to 7 flasks
containing 100 mL of GM. These flasks were
incubated at 250 RPM and 26 ꢀC for 3 days. These
cultures were centrifuged and washed following
subsection 2.4 procedure, and re-suspended in
100 mL of buffer. An amount of 1 mL of ethanolic
solution of DHEA (0.25 mg/mL, 0.52 mg/mL,
0.83 mg/mL, 1 mg/mL, 1.33 mg/mL, 2 mg/mL,
2.5 mg/mL) was added to each flask respectively
and incubated at 250 RPM and 26 ꢀC for 7 days.
Structural identification of metabolites
Incubation of DHEA with B. bassiana ATCC 7159
gave two metabolites that were separated by chro-
matography on silica (see Figure 1). The first
metabolite was identified as androstenediol (I) by
1
comparison of its NMR data to that of DHEA. H
NMR spectrum of I had a new resonance signal at
dH 3.64 ppm (t) consistent with the addition of a
proton during the reduction of C-17 ketone. The
main metabolite, 3b,11a,17b-trihydroxyandrost-5-
ene (II) was identified by comparison of its NMR
data to that of compound I. 1H NMR spectrum of II
had a new resonance signal at dH 3.93 ppm (dt)
consistent with substitution at an equatorial proton.
Downfield b-carbon shifts in the 13C NMR spectra
for C-9 (D 7.9 ppm) and C-12 (D 18.4 ppm)
confirmed hydroxylation at C-11.
Biotransformation procedure for temperature-pH
effect
Phase 1 cultures (10% v/v inoculum) were washed
twice with sterile water and transferred to flasks
containing 25 mL of GM. These flasks were incu-
bated at 250 RPM and 26 ꢀC for 3 days. These
glycerol-grown cultures were washed following sub-
section 2.4 procedure, and re-suspended in 25 mL of
buffer. DHEA (25 mg) dissolved in ethanol was
added to each flask, and the pH values for buffer
solutions in each flask were 5, 6, 7, 8, and 9. There
were five flasks for each pH value, and they were
incubated separately at 15 ꢀC, 20 ꢀC, 26 ꢀC, 30 ꢀC,
and 35 ꢀC at 250 RPM for 7 days.
Androstenediol (I): GCMS 290.22 m/z (lit.
290.44). 1H NMR (CDCl3) ꢀH: 0.88 (3H, s, 18-
H), 1.02 (3H, s, 19-H), 3.54 (1H, m, 3a-H), 3.67
(1H, t, J ¼ 8.4 Hz, 17a-H), 5.35 (1H, d, J ¼ 5.1 Hz,
6-H). 13C NMR (CDCl3): ꢀ (ppm): 143.5 (C-5),
124.0 (C-6), 84.5 (C-17), 74.4 (C-3), 54.0 (C-14),
52.9 (C-9), 45.4 (C-13), 44.9 (C-4), 39.9 (C-1),
39.4 (C-12), 39.2 (C-10), 34.6 (C-8), 34.1 (C-7),
34.1 (C-2), 33.2 (C-16), 26.1 (C-15), 23.3 (C-11),
22.1 (C-19), 13.6 (C-18). Rf in ethyl acetate/
chloroform (3:7): 0.65; HPLC Rt: 5.23 min.
Isolation and identification of products
3b,11a,17b-trihydroxyandrost-5-ene (II): GCMS
306.22 m/z (lit. 306.43). 1H NMR (CDCl3) ꢀH: 0.81
(3H, s, 18-H), 1.21 (3H, s, 19-H), 3.55 (1H, m, 3a-
H), 3.63 (1H, t, J ¼ 8.5 Hz, 17a-H), 3.93 (1H, dt,
J ¼ 4.5 Hz, J ¼ 12.0 Hz, 11b-H), 5.33 (1H, d,
J ¼ 5 Hz, 6-H). 13C NMR (CDCl3): ꢀ (ppm): 145.8
After biotransformation, the buffer was centrifuged
and steroids were extracted from the liquid super-
natant with ethyl acetate (3 ꢂ 50 mL). After removal
of the solvent, metabolites were analyzed with Thin
Layer Chromatography (TLC) and separated by

4
R. Gonzalez et al.
Figure 1. The 11a-hydroxylation of DHEA by B. bassiana.
selectivity and yield of desired 11a-hydroxy II
product was 54.3% and 34.2%, respectively. In
experiments with growing cells, 38% of the substrate
was metabolized. Isolated products included:
53.2 mg of I and 22.8 mg II. The selectivity and
yield of desired 11a-hydroxy II product was 40.4%
and 15.3%, respectively.
Biotransformation with different volume ratio
Table 1 shows the results from 7-day biotransform-
ations of 25 mg of DHEA in 25 mL of resting cells
with different GM to buffer ratio (see Table 1).
Figure 3 shows the effect of ‘‘volume ratio’’ in the
yield of desired product II. Experiments were per-
formed in triplicate.
Figure 2. Higher amount of steroids were synthesized with
‘‘Resting cells’’, and the selectivity and yield of desired 11a-
hydroxy II was increased as well.
(C-5), 125.1 (C-6), 85.1 (C-17), 75.5 (C-3), 72.6
(C-11), 60.8 (C-9), 57.8 (C-12), 54.7 (C-14), 47.4
(C-13), 46.5 (C-4), 43.3 (C-1), 42.4 (C-10), 36.5
(C-2), 35.8 (C-8), 35.3 (C-7), 33.7 (C-16), 27.2 (C-
15), 22.4 (C-19), 15.5 (C-18). Rf in ethyl acetate/
chloroform (3:7): 0.44; HPLC Rt: 8.31 min.
Biotransformation for biocatalyst saturation effect
Figures 4 show the results of 7-day biotransform-
ations with different concentrations of DHEA in
100 mL of resting cells. Values for reaction rates,
conversion, selectivity and yield were determined for
each condition. Experiments were performed in
triplicate.
Determination of the metabolic pathway in the
reaction
To establish the pathway of DHEA derivatives, the
composition of mixtures sampled after various trans-
formation periods was analyzed. Results indicate that
the first stage in the reaction was the reduction of the
C-17 ketone in DHEA. The resulting 3,17-hydroxy
derivative was further metabolized through 11a-
hydroxylation; these derivatives did not appear to
be further metabolized to other compounds (see
Figure 1). In all cases of transformations, the 3,17-
hydroxy derivative was detected before the 11a-
hydroxy product. Time course studies previously
published by our group confirmed this reaction
mechanism (Gonzalez et al. 2015).
Biotransformation for temperature-pH effect
Figure 5 shows the results of 7-day biotransform-
ations of 25 mg of DHEA in 25 mL of resting cells
with temperatures and pH between 15–35 ꢀC and
5–9, respectively. Values for conversion, yield and
selectivity were determined for each condition.
Discussion
B. bassiana strain ATCC 7159 transformed 3-
hydroxy-17-oxo DHEA to the 3,17-hydroxy steroid
(I). Analysis of composition of the product mixtures
as a function of reaction time indicates that I
undergoes an 11a-hydroxylation to synthesize the
3,11,17-hydroxy steroid (II). Other by-products
were not detected.
Biotransformation with resting and growing cells
After 7-day biotransformations of 200 mg of DHEA,
experiments with resting cells resulted in 63%
substrate conversion (See Figure 2). Isolated prod-
ucts included: 66mg of androstenediol (I) and 38 mg
of 3b,11a,17b-trihydroxyandrost-5-ene (II). The
The biotransformation of DHEA was carried out
under different fermentation conditions using
arrangements of ‘‘growing cells’’ and ‘‘resting

Optimization of DHEA hydroxilation by B. bassiana
5
Table 1. Biotransformation of 25 mg of DHEA with different volumes of growth medium GM and buffer solution.
Cells grown on
glycerol GM (mL)
Re-suspended
in Buffer (mL)
Ratio
DHEA
Conversion (%)
Selectivity of II
(%)
I (mg)
II (mg)
25
50
75
100
25
25
25
25
1
2
3
4
65.1 ꢃ 3.3
67 ꢃ 4.0
71 ꢃ 5.1
72 ꢃ 5.7
8.1 ꢃ 0.9
9.7 ꢃ 1.0
10.2 ꢃ 1.2
10.2 ꢃ 1.4
5.7 ꢃ 0.6
7.0 ꢃ 0.7
7.5 ꢃ 0.9
7.7 ꢃ 1.0
35.3 ꢃ 3.7
67.8 ꢃ 2.8
68.9 ꢃ 3.3
69.6 ꢃ 5.7
competition between cells production and biotrans-
formation could be one of the reasons for such a low
amount of (I) and (II) formed during ‘‘growing’’
condition. This limitation can be overcome by
increasing initial DHEA concentrations since the
presence of a competitive inhibitor can be overcome
by high substrate concentrations (Reed et al. 2010).
In ‘‘resting cells’’, the buffer in which the biotrans-
formation occurs contains a very diluted concentra-
tion of nutrients that in comparison to the
concentration of substrate.
Experiments in resting cells with different GM/
Buffer ratios (GM commonly used for cell growth and
buffer for reaction) revealed the optimum ratio for
DHEA conversion. In most cases, a ratio of 1 is used,
but our research explored the effects of a higher ratio
on efficacy and product distribution (Osorio-Lozada
et al. 2008). In Table 1, results showed that higher
volumes of GM compared to buffer (higher ratios)
increase the amount of DHEA derivatives, substrate
conversion, selectivity and desired product yield.
Higher volume ratios increase the value of these
parameters to a similar shape of a negative exponen-
tial curve, which suggests the saturation of the
proteins responsible for the reaction (see Figures 3
and 4). The biotransformation of 1.0 mg/mL with
volume ratio 1 and 4 resulted in 65.1% and 69.63%
selectivity of desired II respectively. A similar value in
selectivity (between 65 and 69%) was observed for all
experiments. The amount of nutrients increased with
the volume of growth media. With higher amount of
nutrients in the media (particularly glycerol), the cells
could concentrate on dividing more new cells and at
the same time expressing more CYP’s in a larger
environment. This was observed in the increase of the
conversion, even though the amount of re-suspended
volume remains the same (See Table 1). However, the
constant volume of suspended buffer did not confirm
that there was the same amount of biomass in each
flask. This means that increasing the amount of cells
and biomass with volume ratio, will not affect reaction
selectivity. Interestingly, there was a large enhance-
ment in the conversion of DHEA from 54.6 to 72.0%.
There was also a remarkable increment in the yield of
desired product II from 35.3 to 49.7%. This improve-
ment might be explained by assuming that the
Figure 3. Effect of high volumes of glycerol medium GM
compared to buffer (higher ratios) in biotransformations.
Figure 4. Effect of initial substrate concentration in the reaction.
cells.’’ When 1.0 mg/mL of DHEA was subject to
both configurations, 63% of the substrate was
converted to products with ‘‘resting cells’’ and 38%
with ‘‘growing cells’’. In addition, higher amounts of
steroids I and II were synthesized with ‘‘resting
cells’’, and the selectivity and yield of desired 11a-
hydroxy II increased as well (see Figure 2). The
observed differences of conversion might be attrib-
uted to just the competitive inhibition between
substrate and components of the medium in ‘‘grow-
ing cells.’’ In this environment, enzymes may pref-
erentially catalyze the medium instead of reacting
with the substrate (Kim et al. 2002). Cells may
preferentially consume the nutrients within the
medium so that cells could continuously reproduce
the
enzymes
for
biotransformation.
Such

6
R. Gonzalez et al.
Figure 5. Effect of temperature and pH on DHEA Yield (A), Selectivity (B) and Conversion of 11a-hydroxy steroid (II) (C).
expression of oxidative enzymes such as CYP is
responsible for the yield increase, which is propor-
tional to the amount of biomass produced with GM.
Data shows that higher GM/Buffer ratios mean will
result in dense concentration of cells expressing CYP
proteins for the biotransformation of DHEA.
temperature and pH values. Experiments at initial
pH value ranges between 6.0 and 7.0, and mid
temperatures (20–30 ꢀC), high conversion (70%),
yield (45.8%) and selectivity (67.8%) was achieved.
At pH 5, the conversion of DHEA was similar (61%)
to pH 7 and the yield of desired steroid II decreased
considerably (31%). At high pH 8 and 9, low
conversion of substrate was obtained (17–28%) and
very low yield of II (1–7%). Poor yield was also
obtained in experiments with low (15–20 ꢀC) and
high temperatures (35 ꢀC). Interestingly, the changes
in temperature and pH do not induce the synthesis of
new steroids besides I and II, but have a considerable
effect on the efficiency of B. bassiana. It seems that
26 ꢀC and pH range 6–7 are the most suitable values
to synthesize desired steroid II (see Figure 5). This
fact is supported by Daniel and Danson (2010) who
confirmed that these parameters have an essential
effect on CYP activity at mild environments.
The biocatalyst performance in biotransformation
processes greatly depends on the initial substrate
concentration (Cook et al. 2014). Substrate conver-
sion, selectivity of B. bassiana and yield of desired
product are also sensitive to initial DHEA concen-
tration. To better understand these parameters,
experiments were performed with different initial
concentrations of DHEA ranging from 0.25 mg/mL
(0.86 mM) up to 2.5 mg/mL (8.66 mM). Using a
diluted amount of substrate (0.52 mg/mL) results in
a desired product yield of 36.3%, which is higher
than the 11.3% yield obtained at high concentrations
of DHEA (2.5 mg/mL). This presumably results
from the interaction of large amount of biocatalyst
relative to a small concentration of substrate. If the
initial concentration of substrate is chosen within an
intermediate range (0.83–1.0 mg/mL), considerable
rate of reaction, high conversion, selectivity and
yield, could be achieved.
Conclusions
B. bassiana may have very useful prospects in the
pharmaceutical industry because it leads to high
yields of valuable 11a-hydroxy intermediates. The
optimization of reaction parameters will facilitate
significant advancement in the application of
B. bassiana to multiple reactions of commercial
interest (Hou et al. 2014). Two types of oxidative
It has been shown that pH and temperature of a
reaction greatly affects B. bassiana efficiency and
product distribution (Xiong et al. 2006). In these
experiments, the change in conversion, yield, and
selectivity was constant through the different

Optimization of DHEA hydroxilation by B. bassiana
7
Gonzalez R, Nicolau F, Peeples T. 2015. n-Alkane solvent-
enhanced biotransformation of steroid DHEA by B. bassiana as
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ation of 3’,17’- and 3’,17’-dihydroxy-5’-androstanes by
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tions. Curr Opin Biotechnol 11:547–553.
Holland HL, Morris TA, Nava PJ, Zabic M. 1999. A new
paradigm for Biohydroxylation by Beauveria bassiana ATCC
7159. Tetrahedron 55:7441–7460.
Hou C, Qin G, Liu T, Geng T, Gao K, Pan Z, Qian H, Guo X.
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products were observed when DHEA was subject to
biotransformation with B. bassiana strain ATCC
7159 under different reactor configurations, volume
of growth medium, substrate concentration, tem-
perature and pH of reaction. Desired steroid II was
synthesized more efficiently in a resting cell arrange-
ment with a GM/Buffer ratio of 4, with moderate
DHEA concentration (0.83–1.0 g/L), and a mild
environment (26 ꢀC, pH 7). These results show the
ability of B. bassiana to adapt and metabolize a
substrate in different biotransformation settings.
More importantly, these results suggest that this
fungus presents a promising future as biocatalyst to
be used for enhancement in the production of drug
metabolites.
Huang LH, Li J, Xu G, Zhang X-H, Wang Y-G, Yin Y-L,
Liu H-M. 2010. Biotransformation of dehydroepiandrosterone
(DHEA) with Penicillium griseopurpureum Smith and
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The authors would like to acknowledge the support
from the Department of Education Graduate
Assistantships in Areas of National Need
(GAANN) program and the University of Iowa
College of Engineering. Authors would like to
thank Dr. Mani V. Subramanian, Dr. Sujit
Mohanty and Dr. Ryan Summers for the quantifica-
tion of steroids with HPLC. Chris Allara for
performing temperature-pH experiments and Dr.
Horacio Olivo for sharing his knowledge on steroids.
´
Kolek T, Szpineter A, Swizdor A. 2008. Baeyer-Villiger oxidation
of DHEA, pregnenolone, and androstenedione by Penicillium
lilacinum AM111. Steroids 73:1441–1445.
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Kolek T, Szpineter A, Swizdor A. 2009. Studies on Baeyer-
Villiger oxidation of steroids: DHEA and pregnenolone
D-lactonization pathways in Penicillium camemberti AM83.
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biotechnology, medicine and bioremediation. Expert Opin
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Declaration of interest
Osorio-Lozada A, Tovar-Miranda R, Olivo H. 2008.
Biotransformation
The authors report no conflicts of interest. The
authors alone are responsible for the content and
writing of this article.
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