Efficient Bioconversion of Echinocandin B to Its Nucleus by

Overexpression of Deacylase Genes in Different Host Strains

Lei Shao,a Jian Li,a Aijuan Liu,a Qing Chang,a,b Huimin Lin,a Daijie Chena
State Key Lab of New Drugs and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, Shanghai, Chinaa; College of Life Sciences, East China Normal
University, Shanghai, Chinab

Anidulafungin, which noncompetitively inhibits ␤-(1,3)-D-glucan synthase in fungal cell wall biosynthesis, is the newest anti-
fungal drug to be developed. Echinocandin B deacylase from Actinoplanes utahensis NRRL 12052 catalyzes the cleavage of the
linoleoyl group of echinocandin B, a key step in the process of manufacturing anidulafungin. Unfortunately, the natural yield of
echinocandin B nucleus is low. In our study, the echinocandin B deacylase gene was systematically overexpressed by genetic en-
gineering in its original producer, A. utahensis, and in the heterologous hosts Streptomyces lividans TK24 and Streptomyces al-
bus. The introduction of additional copies of the gene, under the control of PermE* or its native promoter, into hosts showed
significant increases in its transcription level and in the efficiency of the bioconversion of echinocandin B to its nucleus. The con-
ditions for the cultivation and bioconversion of A. utahensis have been optimized further to improve production. As a result,
while the wild-type strain initially produced 0.36 g/liter, a concentration of 4.21 g/liter was obtained after the generation of a
strain with additional copies of the gene and further optimization of the reaction conditions. These results are useful for enhanc-
ing echinocandin B nucleus production in A. utahensis. Our study could enable the engineering of commercially useful echino-
candin B nucleus-overproducing stains.

F ungal infections are being seen in ever-increasing numbers,

largely because of the increase in the size of the population at
risk over the past 20 years. This population includes cancer pa-
tients, transplant recipients, and other individuals receiving im-
munosuppressive treatment. These people are at greater risk than
others owing to their weakened immune systems and the chronic
nature of diseases (1–3). The increased incidence of invasive fun-
gal infections has created a major challenge for health care profes-
sionals. Since cell walls are present in fungal cells but absent in
animal cells, the fungal cell wall perhaps represents the ideal target
for the therapeutic treatment of fungal pathogens in humans (4,
5). The development of echinocandins, the first class of antifun-
gals to target the fungal cell wall, was a milestone in antifungal
chemotherapy (6, 7). Three semisynthetic echinocandin deriva-
tives have been developed for clinical use: caspofungin, micafun-
gin, and anidulafungin (8). Their strengths include low toxicity,
rapid fungicidal activity against most isolates of Candida spp., and
predictable, favorable kinetics allowing once-a-day dosing. Besides
Candida spp., their inhibitory spectrum includes Aspergillus spp. and
Pneumocystis jirovecii, but not Cryptococcus neoformans (9, 10).
Echinocandin B (ECB), obtained by the fermentation of Asper-
gillus nidulans and Aspergillus rugulosus, is known as one of the
natural cyclic hexapeptides that have a linoleoyl side chain, which
inhibits a crucial enzyme in fungal cell wall biosynthesis, ␤-(1,3)-
D-glucan synthase (11). ECB can be modified by enzymatic deacy-
lation to a cyclic hexapeptide without a linoleoyl side chain and by
subsequent chemical reacylation to generate a few therapeutic an-
tifungal agents for clinical practice, such as anidulafungin (12–
15). A deacylase from Actinoplanes utahensis NRRL 12052 cata-
lyzes the cleavage of the linoleoyl side chain from ECB (Fig. 1), an
essential reaction for the three subsequent synthetic steps (16). The
enzyme is a membrane-associated heterodimer composed of 63-kDa
and 18- to-20-kDa subunits, and the expression of its activity is not
affected by any cofactors, metal ion chelators, or reducing agents. In
addition to that of ECB, this deacylase mediates the cleavage of acu-
leacin A, FR901379, various semisynthetic ECB derivatives, dapto-
mycin and its three derivatives, teicoplanin, pseudomycin A, and cap-
saicins (17, 18). Thus, it may become increasingly significant as a
pharmaceutical biocatalyst.
However, enzymatic deacylation was rate-limiting when con-
ducted with whole cells of A. utahensis. The low bioconversion
yield was presumably related to inadequate production of the ECB
deacylase. Because of the need for improved antifungal agents for the
treatment of systemic fungal infections and the broad specificity of
the enzyme, we were interested in constructing engineered strains
with high production of the enzyme and in developing a better bio-
conversion method to further improve the efficiency of bioconver-
sion of ECB. Here we describe the cloning of the gene encoding ECB
deacylase, under the control of its native promoter or a PermE* pro-
moter, into the original deacylase-producing strain and two Strepto-
myces strains by ⌽C31-directed site-specific recombination in order
to understand the effects of the promoters and gene dosage on the
efficiency of the bioconversion of ECB to the ECB nucleus, particu-
larly with regard to its potential biotechnological application.
MATERIALS AND METHODS
Bacterial strains, plasmids, and reagents. The bacterial strains and plas-
mids used in this paper are listed in Table 1. Streptomyces lividans TK24,
Streptomyces albus, and A. utahensis NRRL 12052 were obtained from our
Received 10 September 2012 Accepted 20 November 2012
Published ahead of print 7 December 2012
Address correspondence to Lei Shao, shaolei00@gmail.com, or Daijie Chen,
chen_daijie@yahoo.com.
L.S., J.L., and A.L. contributed equally to this work.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.02792-12

1126 aem.asm.org Applied and Environmental Microbiology p. 1126 –1133 February 2013 Volume 79 Number 4
 ECB Bioconversion by Overexpression of Deacylase

FIG 1 ECB deacylase-catalyzed reaction.

laboratory. Biochemicals, chemicals, media, restriction enzymes, and other
molecular biological reagents were from standard commercial sources.
DNA isolation, manipulation, and sequencing. DNA isolation and
manipulation were performed by standard methods (21). PCR amplifica-
tions were conducted on an authorized Thermal Cycler (Eppendorf AG,
Hamburg, Germany) using PrimerSTAR HS DNA polymerase (TaKaRa).
Primer synthesis and DNA sequencing were carried out at Shanghai In-
vitrogen Biotechnology Co.
Plasmid construction. To express the ECB deacylase gene under the
control of a PermE* promoter, a 2.8-kb DNA fragment that contains only
the gene encoding ECB deacylase was amplified by PCR from A. utahensis
NRRL 12052 genomic DNA using primers 5=-AAAGAATTCGTGCGGG
CCTGAAA-3= and 5=-AAATCTAGAGACTGCGTGAGTTCTGC-3= and
was cloned into the pSP72 vector, yielding pYG2001. The identity of the
PCR product with the gene encoding ECB deacylase (GenBank accession
number BD226911) was confirmed by sequencing. A 0.5-kb fragment
containing a PermE* promoter was PCR amplified from pYG1003 using
primers 5=-AAAAGATCTTCTAGAAGCCCGACCCGAGCA-3= and 5=-A
AAGAATTCTCCGGAGGTCGCACC-3= and was cloned into the BglII/
EcoRI site of pYG2001, yielding pYG2002. (Underlined letters in primer
sequences represent restriction sites.) The 3.3-kb XbaI/XbaI fragment
containing the gene coding for ECB deacylase and a PermE* promoter from
pYG2002 was inserted into the XbaI site of the pSET152 vector, yielding
pYG2003 for the expression of the gene encoding ECB deacylase under the
control of a PermE* promoter by a ⌽C31-directed site-specific recombina-
tion event.
For the expression of the gene encoding ECB deacylase under the
control of its native promoter, a 4.0-kb DNA fragment that contains the
gene encoding ECB deacylase with approximately 1 kb of upstream se-
quence and 0.5 kb of downstream sequence was amplified from A. uta-
hensis NRRL 12052 genomic DNA by PCR using primers 5=-ATAGAATT
CCGTGCCCAGCTGTTC-3= and 5=-AAATCTAGAGACTGCGTGAGT

TABLE 1 Bacterial strains and plasmids used in this study

Source or
Strain or plasmid Description reference
Strains
E. coli
DH5␣ Host for general cloning Invitrogen
ET12567(pUZ8002) Donor strain for conjugation between E. coli and Actinomyces 3
A. utahensis ECB deacylase-producing strain NRRL 12052
DYG2001 A. utahensis derivative with two copies of the ECB deacylase gene (second copy under the control of PermE*) This work
DYG2002 A. utahensis derivative with two copies of the ECB deacylase gene under the control of its native promoter This work
DYG2003 A. utahensis derivative with three copies of the ECB deacylase gene (second and third copies under the This work
control of PermE*)
S. lividans TK24 19
DYG2004 S. lividans derivative with a single copy of the ECB deacylase gene under the control of PermE* This work
DYG2005 S. lividans derivative with a single copy of the ECB deacylase gene under the control of its native promoter This work
DYG2006 S. lividans derivative with two copies of the ECB deacylase gene under the control of PermE* This study
S. albus 19
DYG2007 S. albus derivative with a single copy of the ECB deacylase gene under the control of PermE* This work
DYG2008 S. albus derivative with a single copy of the ECB deacylase gene under the control of its native promoter This work
DYG2009 S. albus derivative with two copies of the ECB deacylase gene under the control of PermE* This work

Plasmids
pSP72 E. coli subcloning vector Promega
pSET152 E. coli replicon, Streptomyces ⌽C31 attachment site; Aprr 16
pYG1003 2.0-kb fragment containing PermE*-controlled aveBIV in pSET152 20
pYG2001 2.8-kb PCR fragment containing the ECB deacylase gene in pSP72 This work
pYG2002 3.3-kb PCR fragment containing a PermE*-controlled ECB deacylase gene in pSP72 This work
pYG2003 3.3-kb PCR fragment containing a PermE*-controlled ECB deacylase gene in pSET152 This work
pYG2004 4.0-kb PCR fragment containing the ECB deacylase gene and its 5= and 3= regulatory sequences in pSP72 This work
pYG2005 4.0-kb PCR fragment containing the ECB deacylase gene and its 5= and 3= regulatory sequences in pSET152 This work
pYG2006 3.3-kb PCR fragment containing a PermE*-controlled ECB deacylase gene in pSP72 This work
pYG2007 Two copies of a 3.3-kb PCR fragment containing a PermE*-controlled ECB deacylase gene in pYG2003 This work

February 2013 Volume 79 Number 4 aem.asm.org 1127

Shao et al.
TCTGC-3= and was cloned into the pSP72 vector, yielding pYG2004. The
identity of the PCR product with the gene encoding ECB deacylase was
also confirmed by sequencing. The 4.0-kb EcoRI/XbaI fragment from
pYG2004 was inserted into the corresponding sites of pSET152, yielding
pYG2005.
In order to express two copies of the gene encoding ECB deacylase
under the control of a PermE* promoter, another 3.3-kb DNA fragment
with terminal EcoRV/EcoRV sites containing the gene encoding ECB de-
acylase and a PermE* promoter was amplified by PCR from pYG2002
using primers 5=-AAAGATATCAGCCCGACCCGAGCA-3= and 5=-AAA
GATATCGACTGCGTGAGTTCTGC-3= and was cloned into the pSP72
vector, yielding pYG2006. The identity of the PCR product with the gene
encoding ECB deacylase was confirmed. The 3.3-kb EcoRV/EcoRV frag-
ment containing the gene encoding ECB deacylase and a PermE* pro-
moter from pYG2006 was inserted into the corresponding site of the re-
combinant vector pYG2003, yielding the last vector, pYG2007, containing
two deacylase gene expression cassettes. The correct directions of the gene
encoding ECB deacylase and the PermE* promoter in the pYG2003 or
pYG2007 vector were verified by restriction enzyme digestion.
Conjugation and generation of recombinant strains. For overex-
pression and heterologous expression of the ECB deacylase gene in A.
utahensis NRRL 12052 and the two Streptomyces strains (S. lividans TK24
and the S. albus strain), expression vectors pYG2003, pYG2005, and
pYG2007 were each introduced into hosts by intergeneric conjugation
from Escherichia coli ET12567(pUZ8002) according to the standard pro-
cedure (19, 22). Transformants that were resistant to apramycin were
identified as the recombinant strains, whose genomic DNAs were inte-
grated with the deacylase gene and the apramycin resistance gene by
⌽C31-directed site-specific recombination. The genotypes of the recom-
binant strains were further confirmed by PCR amplification with the vec-
tor-specific primer pair M13-47 and RV-M.
Culture growth and deacylation procedure. Wild-type and recombi-
nant strains were grown on agar plates with a medium consisting of 2%
soluble starch, 0.05% NaCl, 0.05% K2HPO4·3H2O, 0.1% KNO3, 0.05%
MgSO4·7H2O, 0.001% FeSO4·7H2O, and 2% agar powder (pH 7.4) at
28°C for sporulation. For the fermentation of A. utahensis NRRL 12052,
an agar piece around 1 cm2 was inoculated into a 250-ml flask containing
50 ml of a seed medium consisting of 2.5% sucrose, 2.0% oatmeal, 0.25%
yeast powder, 0.1% K2HPO4, 0.05% KCl, 0.05% MgSO4·7H2O, and
0.0002% FeSO4·7H2O and was incubated at 28°C and 220 rpm for 3 days.
A 250-ml flask containing 50 ml of fresh fermentation medium, consisting
of 2% sucrose, 1% peanut meal, 0.1% KH2PO4, and 0.025%
MgSO4·7H2O, was then inoculated with 5 ml of the seed culture, and
incubation was continued at 28°C and 220 rpm for 4 days. For the fer-
mentation of S. lividans TK24 and S. albus, an agar piece around 1 cm2 was
inoculated into a 250-ml flask containing 50 ml of a seed medium con-
sisting of 1.0% glucose, 0.5% yeast powder, and 1% peptone and was
incubated at 30°C and 220 rpm for 30 h. A 250-ml flask containing 50 ml
of fresh fermentation medium consisting of 2.5% glucose, 1% bean flour,
0.3% NaCl, and 0.3% CaCO3 was then inoculated with 1 ml of the seed
culture, and incubation was continued at 30°C and 220 rpm for 2 days.
The wet mycelia (15 g) were harvested by centrifugation, washed twice
with double-distilled water, and then resuspended in a 50-ml biotransfor-
mation reaction mixture containing 0.1 M Na2HPO4/NaH2PO4 buffer
(pH 6.8) in the presence of 2% dimethyl sulfoxide (DMSO). The enzy-
matic reaction was initiated by addition of the substrate ECB (1.5 g/liter)
and was continued at 30°C and 220 rpm for 5 h before sample analysis.
Different experiments were subsequently conducted to optimize the
cultivation medium of A. utahensis by changing the carbon and nitrogen
sources and their proportions. Maltose, sucrose, glycerol, lactose, and
soybean oil were used individually as the carbon source, at concentrations
of 20 g/liter, instead of glucose. Cottonseed meal, soybean powder, and
oatmeal were used individually as the nitrogen source, at concentrations
of 10 g/liter, instead of peanut meal. The optimum concentration of su-
crose or cottonseed meal was determined by dosing different amounts of
1128 aem.asm.org
sucrose (2 to 6%) or cottonseed meal (1 to 5%). The optimum pH for
biotransformation was determined at 30°C. The optimum temperature
for biotransformation was determined at pH 6.8. Ethanol was used as the
cosolvent instead of DMSO. The optimum concentration of the ECB sub-
strate was determined by dosing different amounts of ECB (1.5 to 12
g/liter) under the optimized conditions.
Analytical methods for the detection of ECB and the ECB nucleus.
The reaction was interrupted by the addition of methanol. After low-
speed centrifugation to remove precipitated proteins and mycelia, the
activity of the deacylase was determined by monitoring the formation of
the cyclic hexapeptide (the ECB nucleus) by high-performance liquid
chromatography (HPLC) with an Agilent C18 column (250 by 4.6 mm;
Agilent, Palo Alto, CA). The column was equilibrated with 92% solvent A
(H2O containing 2 mg/ml ammonium acetate) and 8% solvent B (60%
CH3CN containing 2 mg/ml ammonium acetate), and the analytical method
was developed with the following program: from 0 min to 15 min, a linear
gradient from 92% A– 8% B to 2% A–98% B; from 15 min to 25 min, a linear
gradient from 2% A–98% B to 8% A–92% B; and from 25 min to 32 min, a
constant proportion of 92% A– 8% B. HPLC was carried out at a flow rate of
0.8 ml/min with UV detection at 222 nm using an Agilent 1120 HPLC system
(Agilent Technologies, Palo Alto, CA). The identities of compounds were
confirmed by liquid chromatography-mass spectrometry (LC-MS) analysis
performed on an LCMS-2010A system (Shimadzu, Japan).
Assay of transcript levels by RT-PCR amplification. Total RNAs of
three wild-type strains and nine recombinant strains were isolated from
mycelia in the fermentation cultures. An additional purification step was
carried out by using TRIzol reagent (catalog no. 15596-026; Invitrogen).
To obtain cDNAs, DNase treatment and reverse transcription were per-
formed by using random primers (catalog no. C1181; Promega) and
Moloney murine leukemia virus (M-MLV) reverse transcriptase (catalog
no. M1701; Promega) according to the manufacturer’s instructions. The
transcript levels of the ECB deacylase gene were assayed on a Rotor-Gene
RG-3000A instrument (Corbett Research Co., Australia), using real-time
quantitative PCR and the 2⫺⌬⌬CT method (23). Reverse transcription-
PCR (RT-PCR) amplification was performed on each 25 ␮l of the mixture
(consisting of 1 ␮g/ml of template cDNA, 2⫻ SYBR Premix [Shanghai
Xinghan Sci-Tech Co., Shanghai, China] and 0.4 ␮M forward and reverse
primers) with the following program: 95°C for 3 min, followed by 30
cycles of 94°C for 20 s, 60°C for 20 s, and 72°C for 15 s. For assessment of
the transcript level of the ECB deacylase gene, the 244-bp internal frag-
ment was amplified from cDNA of the recombinant strains and wild-type
A. utahensis NRRL 12052 using primers 5=-CGCATGTACGAGGACGTC
AC-3= and 5=-GCACAGCGGGGACGCTGA-3=. For assessment of the
transcript level of the 16S rRNA gene (as a control), the 242-bp internal
fragments were amplified from cDNA of S. lividans TK24 and its recom-
binant strains using primers 5=-GCAATCTGCCCTTCACTCTG-3= and
5=-TATTCCCCACTGCTGCCTCC-3=; the 285-bp internal fragments
were amplified from cDNA of S. albus and its recombinant strains using
primers 5=-GCCGATACTGACGCTGAGGA-3= and 5=-GGACCCTGTC
TCCAGAGTTTTC-3=; and the 255-bp internal fragments were amplified
from cDNA of A. utahensis NRRL 12052 and its recombinant strains using
primers 5=-GGAGGCAGCAGTGGGGAAT-3= and 5=-TGAGCCTCGGG
ATTTCACATT-3=.
RESULTS AND DISCUSSION
Qualitative and quantitative analysis of ECB and the ECB nu-
cleus. ECB and its nucleus were extracted from a bioconversion
mixture, and their identities were determined according to the
method described above. As shown in Fig. 2, analysis of the sam-
ples revealed that the product and the remaining substrate ECB
had the same retention times as those of the standard ECB nucleus
(6.75 min) and ECB (22.80 min), respectively. Their identities
were further confirmed by electrospray ionization-quadrupole
time of flight (ESI–Q-TOF)–MS analysis, showing (M ⫹ Na)⫹
ions at m/z 820.40, consistent with the molecular formula
Applied and Environmental Microbiology

FIG 2 HPLC analysis of ECB nucleus production in a reaction system with
whole cells of control strain A. utahensis NRRL 12052. Standard ECB (p) and
ECB nucleus () were used as controls. (I) Standard ECB; (II) standard ECB
nucleus; (III) A. utahensis NRRL 12052.
C34H51N7O15 for the ECB nucleus, and (M ⫹ Na)⫹ ions at m/z
1,082.61, consistent with the molecular formula C52H81N7O16 for
ECB. The concentration of the ECB nucleus in the reaction mix-
ture was calculated according to the standard curve of the refer-
ence ECB nucleus. The methods described here for qualitative and
quantitative analysis of ECB and the ECB nucleus were applied to
FIG 3 Genotypes and phenotypes of the recombinant strains. (A) Predicted fragment sizes for PCR analysis. Each wavy line indicates the DNA fragment of the
vector integrated by ⌽C31-directed site-specific recombination. (B) HPLC analysis of ECB nucleus production in a reaction system with ECB (p) and the ECB
nucleus ().
February 2013 Volume 79 Number 4
ECB Bioconversion by Overexpression of Deacylase
the biotransformation reaction mixture of A. utahensis, S. lividans,
S. albus, and the recombinant strains obtained in this study.
Overexpression of the ECB deacylase gene in A. utahensis
NRRL 12052. Enzymes can be overproduced by increasing the
gene copy number. To investigate the effect of deacylase gene dos-
age, pYG2003 and pYG2007, which contain one and two deacylase
gene expression cassettes, respectively, under the control of a
PermE* promoter, were constructed in pSET152 and were intro-
duced into A. utahensis NRRL 12052 by a ⌽C31-directed site-specific
recombination event, yielding recombinant strains DYG2001, con-
taining two copies of the deacylase gene, and DYG2003, containing
three copies of the deacylase gene. To express deacylase under the
control of the native promoter, pTG2005 was constructed in
pSET152 and was introduced into A. utahensis NRRL 12052, yielding
recombinant strain DYG2002, which contained two copies of the
deacylase gene (Fig. 3A). The 3.3-kb, 4.0-kb, and 6.6-kb fragments
could be amplified separately from genomic DNAs of the recombi-
nant strains mentioned above by PCR using primers M13-47 and
RV-M. Sequencing showed that these recombinant strains had the
designed genotypes (Fig. 4). Besides this, there were no apparent dif-
ferences in growth characteristics or morphology.
Based on these results, we determined the bioconversion effi-
ciency of each recombinant strain. As shown in Fig. 5, the effi-
ciency of bioconversion of ECB was doubled in DYG2001 and
DYG2002. These observations may mean that insufficient ECB
deacylase activity was present in vivo. This idea is supported by
other results. Even higher bioconversion efficiency was observed for
DYG2003, containing three copies of the deacylase gene (Fig. 5). In
aem.asm.org 1129
Shao et al.

FIG 4 PCR analysis with genomic DNA from S. lividans TK24, S. albus, A. utahensis NRRL 12052, or mutants as the template. By use of primers M13-47
(5=-CGCCAGGGTTTTCCCAGTCACGAC-3=) and RV-M (5=-GAGCGGATAACAATTTCACACAGG-3=), the genotypes of the recombinant strains were con-
firmed by PCR to be well in agreement with their designed genetic patterns, demonstrating that the ⌽C31-directed site-specific recombination event had taken
place. Lane 1, no product for wild-type A. utahensis; lane 2, 3.3-kb product for DYG2001; lane 3, 4-kb product for DYG2002; lane 4, 6.6-kb product for DYG2003;
lane 5, no product for wild-type S. lividans TK24; lane 6, 3.3-kb product for DYG2004; lane 7, 4-kb product for DYG2005; lane 8, 6.6-kb product for DYG2006;
lane 9, no product for wild-type S. albus; lane 10, 3.3-kb product for DYG2007; lane 11, 4.0-kb product for DYG2008; lane 12, 6.6-kb product for DYG2003. All
PCR products were sequenced to confirm each mutation.

our study, we found a decline in the level of the ECB substrate
in the control system containing only the substrate and the
bioconversion buffer, while the ECB nucleus product could be
accumulated stably. In the A. utahensis NRRL 12052 biocon-
version system, we found both the ECB nucleus product and
the ECB substrate, while no visible ECB substrate was detected
in the three recombinant strains upon HPLC analysis, as shown
in Fig. 3B.
Another interesting question is the means of integrating the
deacylase gene into A. utahensis and Streptomyces. As has been
observed previously with 4=-epidaunorubicin production in
Streptomyces coeruleorubidus (20), the ECB deacylase gene could
be stably integrated into the genomes of host strains by a ⌽C31-
directed site-specific recombination event. Thus, it is not neces-
sary to use a culture medium with apramycin resistance for the
fermentation of the recombinant strains. Such a medium without
apramycin helps cut the cost of ECB nucleus production on an
industrial scale.
Heterologous expression of the ECB deacylase gene con-
trolled by different promoters. Streptomyces spp. are well-known
producers of biologically active compounds and enzymes (24–
26). Techniques for the cultivation of these organisms on an in-
dustrial scale are well established, so these species are especially
attractive as hosts for heterologous products (27). Different spe-
cies of Streptomyces were screened as heterologous expression
hosts by using the same bioconversion method as A. utahensis. For
the two species examined, S. lividans (strain TK24) and S. albus, no
ECB nucleus or ECB from whole cells was detected in the reaction
mixtures. The PermE* promoter has been used frequently as a
strong constitutive promoter for native and heterologous genes in

FIG 5 Production of the ECB nucleus and ratio of the target gene transcript level to the transcript level of 16S rRNA, used as a control (relative mRNA), in
reaction systems with whole cells of the different strains. Error bars represent standard deviations. For each strain, the experiment was carried out independently
three times.

1130 aem.asm.org Applied and Environmental Microbiology


FIG 6 Effects of temperature, pH, and the concentration of the ECB substrate on the biotransformation reaction. (A) Temperature course of the biotransfor-
mation reaction at pH 6.8. (B) pH course of the biotransformation reaction at 30°C. (C) Effects of different concentrations of the ECB substrate under optimized
conditions (pH 4 and 25°C). Error bars represent standard deviations.
Streptomyces species and related bacteria (28). To compare the
expression levels of the ECB deacylase gene under the control of its
native promoter and under the control of a PermE* promoter, the
genes were cloned into pSET152 and were then transformed into
the two Streptomyces species by a ⌽C31-directed site-specific re-
combination event to generate four new strains: DYG2004,
DYG2005, DYG2007, and DYG2008. Another expression plas-
mid, pYG2007, with two ECB deacylase gene expression cassettes
controlled by PermE*, was constructed and was introduced into S.
lividans TK24 and S. albus, respectively, to yield DYG2006 and
DYG2009, containing two copies of the deacylase gene (Fig. 3A).
The genotypes of these recombinant strains were verified by PCR
amplification, whereas no PCR products were isolated from the
wild-type strains, as expected (Fig. 4). Subsequently, the biocon-
version efficiencies of these recombinant strains were determined,
and each reaction mixture was analyzed by HPLC. HPLC analysis
revealed that the bioconversion efficiencies of these recombinant
strains were enhanced over that of the original strain, A. utahensis
NRRL 12052. More importantly, the bioconversion efficiencies of
the recombinant strains DYG2004 and DYG2007, containing
PermE*-controlled ECB deacylase, were higher than those of
strains DYG2005 and DYG2008, in which the ECB deacylase gene
was controlled by its native promoter. Strains DYG2006 and
DYG2009, containing two copies of the deacylase gene, have
higher bioconversion efficiencies than strains with one deacylase
February 2013 Volume 79 Number 4
ECB Bioconversion by Overexpression of Deacylase
gene copy (Fig. 5). These findings demonstrated that PermE* was
superior to the native promoter in the production of ECB deacyl-
ase for heterologous expression or overexpression and that there
was potentially an enhancement of bioconversion efficiency and
an improvement in the production of ECB deacylase due to the
increase in gene copy number. As has been observed previously,
the Streptomyces genome may contain multiple pseudo-attB sites.
However, the frequency of integration of pSET152 into the ⌬attB
strain with additional pseudo-attB sites is reduced approximately
300-fold (29). In our study, there are strong positive correlations
between the gene copy numbers of the recombinant strains and
the transcript levels of the ECB deacylase gene. Therefore, there
may be only one integrated gene. Of all the recombinant
strains, DYG2003 from A. utahensis NRRL 12052 showed the
highest bioconversion efficiency. A possible reason is that the
ECB deacylase gene can be expressed better in A. utahensis
NRRL 12052 and that DYG2003 contains more deacylase gene
copies than NRRL 12052.
Optimal conditions for biotransformation of A. utahensis.
For the cultivation of A. utahensis and its recombinant strains, the
new fermentation medium formulation based on 3.0% sucrose,
2.0% cottonseed meal, 0.12% K2HPO4·3H2O, and 0.05%
KH2PO4·3H2O was a good alternative solution for the pigmenta-
tion of strain DYG2003 (unpublished data). As a result, the
growth rate and mycelium volume of A. utahensis and its recom-
aem.asm.org 1131
Shao et al.
binant strains increased dramatically. Under the new culture con-
ditions, the concentration of mycelium in the fermentation broth
was 30% higher than that under the original conditions in the
same fermentation time. For the biotransformation reaction, the
optimal temperature was 25°C and the optimal pH was 4.5 (Fig.
6A and B). The optimum concentration of ECB in the reaction
system was detected by additions of different ECB quantities un-
der the optimized conditions (pH 4 and 25°C) for further evalu-
ation of the bioconversion efficiency of the recombinant strain
DYG2003 and the original strain A. utahensis NRRL 12052. With
whole cells of NRRL 12052, the production of the ECB nucleus in
reaction mixtures could rise as high as 4.21 g/liter when a suitable
concentration of ECB (8 g/liter) was added to the reaction system
(Fig. 6C). Additionally, ethanol is more economical and beneficial
than DMSO as the cosolvent used in the reaction system to sim-
plify the separation and purification of the product.
Assay of the transcript levels of the gene by RT-PCR amplifi-
cation. To assess the effects of different promoters and gene doses
on the increase in the amount of enzyme produced, RT-PCR am-
plification for analysis of the transcription levels of the ECB deac-
ylase gene was performed on the total RNAs extracted from cells of
each strain investigated in this study. The transcript levels of the
gene fragment that encodes 16S rRNA served as a control. As
shown in Fig. 5, the introduction of one or two additional copies
of the ECB deacylase gene caused significant increases in the tran-
script level of the gene. As expected, no ECB deacylase mRNA was
detected for wild-type S. lividans and S. albus. The ratios of the
transcript levels of the ECB deacylase gene under the control of
PermE* in the S. lividans and S. albus heterologous expression
hosts to the transcript level of the control 16S rRNA gene were as
high as 0.66 and 0.87, respectively, while those of the gene con-
trolled by its native promoter were as high as 0.46 and 0.84, re-
spectively. Duplication and triplication of the gene resulted in
increases as high as 2.00-fold and 6.94-fold in A. utahensis NRRL
12052. These results indicate that increasing the copy number
of the gene encoding ECB deacylase leads to a significant in-
crease in the transcript level of this gene, especially when it is
controlled by the PermE* promoter. The transcript levels of the
gene meet the expectation of increased efficiency of bioconver-
sion of ECB to the ECB nucleus in recombinant strains (men-
tioned above).
The deacylation of ECB is the starting point for the semi-
synthesis of anidulafungin. The current annual production of
anidulafungin is on the order of tons, and anidulafungin is
widely used as an important anti-infective drug. Synthesis of
the ECB nucleus by chemical deacylation is extremely difficult,
and the biotransformation method described here avoids this
problem. The findings of this study could enable the engineer-
ing of commercially useful ECB nucleus-overproducing
strains.
ACKNOWLEDGMENTS
We thank Jia Zeng and S. Gabrielle Gladstone, Department of Biological
Engineering, Utah State University, who helped with editing.
This work was supported in part by grants from the National Nat-
ural Science Foundation of China (grants 81172962, 30801449, and
81072557), the Science and Technology Commission of Shanghai Munic-
ipality (grant 11QB1406300), and the Ministry of Science and Technology
of China (grant 2009ZX09301-007).
1132 aem.asm.org
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