Improving 3’-Hydroxygenistein Production in Recombinant Pichia … · 2016-03-21 · Improving...
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A 2016⎪Vol. 26⎪No. 0
J. Microbiol. Biotechnol. (2016), 26(3), 498–502http://dx.doi.org/10.4014/jmb.1509.09013 Research Article jmbReview
Improving 3’-Hydroxygenistein Production in Recombinant Pichiapastoris Using Periodic Hydrogen Peroxide-Shocking StrategyTzi-Yuan Wang1†, Yi-Hsuan Tsai2†, I-Zen Yu2, and Te-Sheng Chang2*
1Biodiversity Research Center, Academia Sinica, Taipei 115, Taiwan, Republic of China2Department of Biological Sciences and Technology, National University of Tainan, Tainan, 702, Taiwan, Republic of China
Orobol (3’-hydroxygenistein) was first purified from
Orobus tuberuosus in 1939 [2], and later from Calophyllum
polyanthum [9], Erycibe expansa [10], Sophora japonica (Sophorae
Flos) [3], and Eclipta prostrata [13]. This compound has also
been isolated from the fermentation broth of Aspergillus
niger [15] and from tempeh, a fermented soybean food [6].
Multiple bioactivities related to 3’-hydroxygenistein have
already been reported, including antiproliferative activity
toward T47D tumorigenic breast epithelial cells [11, 16],
HIV-1 integrase inhibitory activity [13], anti-inflammatory
activity [14], hepatoprotective activity [10], and anti-
melanogenesis activity [4], making 3’-hydroxygenistein
industrially important. However, 3’-hydroxygenistein rarely
occurs in nature [1]. Therefore, to overcome the natural
scarcity of 3’-hydroxygenistein, the recombinant Pichia
pastoris X-33 was constructed to produce detectable 3’-
hydroxygenistein from genistein (Figs. 1A and 1B) [4]. The
recombinant strain, harboring a fusion gene (CYP57B3-
sCPR) composed of the CYP57B3 gene from Aspergillus
oryzae and a cytochrome reductase gene (sCPR) from
Saccharomyces cerevisiae, is able to produce 3.5 mg/l of
3’-hydroxygenistein in a 5 L fermenter. However, since
this amount is still insufficient for industrial application,
improving the 3’-hydroxygenistein production by P. pastoris
has become an advanced research topic.
Based on the known antioxidant properties of carotenoids,
Reyes et al. [12] successfully obtained a 3-fold increase in
carotenoid production in an engineered S. cerevisiae strain
via periodic hydrogen peroxide treatment. Therefore,
this study initially assayed the antioxidant activity of 3’-
hydroxygenistein using a 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical-scavenging activity assay [5]. The results
showed that the IC50 value of 3’-hydroxygenistein (3.9 µM)
for DPPH radical-scavenging activity was lower than that
of ascorbic acid (8.9 µM) and over 400-fold lower than that
of its precursor, genistein (>1,600 µM) (Fig. 1C). Thus, since
the results revealed that 3’-hydroxygenistein is a potent
antioxidant, this encouraged application of the same
above-mentioned “hydrogen peroxide-shocking” strategy
[12] to improve the 3’-hydroxygenistein production in the
X-33 recombinant.
Three parallel cultures (P1 to P3) of the recombinant X-33
were initiated for 20 ml cultivations in YPD medium (1%
yeast extract, 2% peptone, and 2% dextrose), containing
Received: September 4, 2015
Revised: December 11, 2015
Accepted: December 14, 2015
First published online
December 23, 2015
*Corresponding author
Phone: +886-6-2602137;
Fax: +886-6-2606153;
E-mail: [email protected]
†These authors contributed
equally to this work.
pISSN 1017-7825, eISSN 1738-8872
Copyright© 2016 by
The Korean Society for Microbiology
and Biotechnology
3’-Hydroxygenistein can be obtained from the biotransformation of genistein by the
engineered Pichia pastoris X-33 strain, which harbors a fusion gene composed of CYP57B3 from
Aspergillus oryzae and a cytochrome P450 oxidoreductase gene (sCPR) from Saccharomyces
cerevisiae. P. pastoris X-33 mutants with higher 3’-hydroxygenistein production were selected
using a periodic hydrogen peroxide-shocking strategy. One mutant (P2-D14-5) produced
23.0 mg/l of 3’-hydroxygenistein, representing 1.87-fold more than that produced by the
recombinant X-33. When using a 5 L fermenter, the P2-D14-5 mutant produced 20.3 mg/l of 3’-
hydroxygenistein, indicating a high potential for industrial-scale 3’-hydroxygenistein
production.
Keywords: 3’-Hydroxygenistein, orobol, CYP57B3, Pichia pastoris
499 Wang et al.
J. Microbiol. Biotechnol.
100 µg/ml zeocin, 250 µM δ-aminolevulinic acid, and
500 µM genistein (Fig. 1). After 48 h of incubation, 0.1 ml of
each culture was mixed with 0.9 ml of glycerol as a stock
and stored at −80°C. A 0.5 ml sample from each culture was
also mixed with 0.5 ml of methanol for 3’-hydroxygenistein
analysis using ultra-performance liquid chromatography
(UPLC). Meanwhile, another 0.2 ml from each culture was
mixed with hydrogen peroxide and reacted at 25°C for
30 min, after which 0.01 ml of the hydrogen peroxide
mixture was inoculated into another 20 ml of fresh YPD
medium and cultivated following the method of Reyes et al.
[12]. This culture process was continued until the cultured
yeast stopped growing. Additionally, for comparison, two
cultures (P1 and P2) were initially treated with 250 mM
and then with 500 mM hydrogen peroxide after 20 days,
whereas P3 was initially treated with 500 mM hydrogen
peroxide. After the periodic hydrogen peroxide-shocking
treatment, six subcultures (as indicated by the arrows in
Fig. 2) that showed hyperproduction of 3’-hydroxygenistein
were selected to isolate the best mutant. The −80°C stocks
of the six selected subcultures were revived on YPD plates
by incubation at 28°C for 48 h. Five colonies were then
randomly selected from each subculture and cultured in
20 ml of a YPD medium as seed cultures. After 48 h of
incubation, each seed culture was transferred into three
20 ml batch cultures. Following a 72-h incubation, the 3’-
hydroxygenistein production was determined using UPLC.
The highest producer among the five colonies from each of
the six subcultures was then selected for a final comparison.
Fig. 2D shows that all the candidates produced significantly
higher concentrations of 3’-hydroxygenistein than the
original recombinant X-33, where P2-D14-5 showed the
highest 3’-hydroxygenistein production.
We then identified the genotype and phenotype differences
between the recombinant X-33 and P2-D14-5 (Fig. 3). In the
genotype analysis, the fusion gene CYP57B3-sCPR was
amplified with primers (forward, 5’-ATGATAGGGACG
GTCTTGGAC-3’; reverse, 5’-GACATCTTCTTGGTATCT
Fig. 1. 3’-Hydroxygenistein production from biotransformation of genistein by recombinant X-33.
(A) UPLC profile of fermentation broth after 72 h of cultivation of recombinant X-33 in a 5 L fermenter. The operational conditions for the UPLC
analysis were as previously described [4]. (B) Diagram of the biotransformation. (C) DPPH radical-scavenging activity of two soy isoflavones and
ascorbic acid. The DPPH scavenging activity was determined as previously described [5]. The IC50 values represent the concentrations required for
50% DPPH radical-scavenging activity. The mean (n = 3) is shown, and the SD is represented by error bars.
Improving 3'-Hydroxygenistein Production in P. pastoris 500
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ACCTG-3’) from the isolated genomic DNA and sequenced
(Genomics, Taipei, Taiwan). For the quantitative polymerase
chain reaction analysis, the oligonucleotide primers used
were for CYP57B3 (forward, 5’-GACACCATGACTGCT
GAAGG-3’; reverse, 5’-TTGAGGTACGGCATCTCTTG-3’)
and for the yeast actin ACT1 gene, as an internal control
(forward, 5’- GCGAATACTACGCGAGATGA-3’; reverse,
5’-CCTCAGTTTCCTGCTCCTTC-3’). The relative expression
of the CYP57B3 gene was normalized to that of the
ACT1 gene (∆Ct) and quantified using the ∆∆Ct relative
quantification method. The relative mRNA fold was
expressed as (mutant/parent) = 2[-∆∆Ct]. The genotype analysis
revealed that the CYP57B3-sCPR fusion gene sequences
were identical (data not shown), and no significant expression
differences were observed between the two strains after a
24-h incubation (Fig. 3A). Meanwhile, the phenotype
analysis revealed that the 3’-hydroxygenistein production
at 72 h of cultivation was 1.87-fold higher in P2-D14-5
(23.0 mg/l) than in the recombinant X-33 (12.3 mg/l);
however, the growth rates of the two strains were the same
(0.19 gl-1h-1) (Fig. 3B). At present, there is no clear explanation
for the time delay observed between the mRNA expression
and 3’-hydroxygenistein production, although this may
reflect differences in protein synthesis or trafficking. In
addition, both strains showed higher hydrogen peroxide
resistance when cultivated in the presence of genistein for
Fig. 2. Periodic hydrogen peroxide-shocking of recombinant X-33.
(A to C) Hydrogen peroxide used (■ ) and 3’-hydroxygenistein production (●) profiles of three independent cultivations. (D) 3’-
Hydroxygenistein production by recombinant X-33 and isolated hyperproducing mutants after 72-h incubation in shaking flasks (arrows indicated
in panels A to C). The mean (n = 3) is shown, and the SD is represented by error bars.
501 Wang et al.
J. Microbiol. Biotechnol.
72 h than when cultured without genistein (Fig. 3C).
Therefore, our results indicated that the ability to produce
3’-hydroxygenistein made the strains more resistant to
hydrogen peroxide. Lee et al. [7, 8] also found that periodic
hydrogen peroxide-shocking up-regulated the expression
of genes involved in the aromatic compound-degrading
pathway and led to the accumulation of intracellular
antioxidant aromatic compounds, thereby increasing the
reactive oxygen species-scavenging activity and hydrogen
peroxide stress tolerance. Owing to the potent antioxidant
properties of 3’-hydroxygenistein (Fig. 1C), the accumulation
of 3’-hydroxygenistein in the present study also increased
the hydrogen peroxide stress tolerance (Figs. 3B and 3C).
Thus, the involvement of the P2-D14-5 mutations in oxidation-
reduction-related genes and adjusting the oxidation state to
increase 3’-hydroxygenistein production needs to be
investigated further. Therefore, future studies will focus on
the genome and/or transcriptome of P2-D14-5 to elucidate
the molecular mechanisms regulating 3’-hydroxygenistein
production in Pichia mutants.
In a scale-up experiment, P2-D14-5 was cultivated in a
5-L fermenter (Fig. 4). The resulting cell density in the
fermenter was 2-fold higher than that in flask culture
(Fig. 3B), but the maximal 3’-hydroxygenistein production
(20.3 mg/l) in the fermenter was slightly lower than that
observed in the flask (23.0 mg/l; Fig. 3B). Hence, the optimal
bioreactor operating parameters affecting productivity
remain to be established.
In summary, this study identified 3’-hydroxygenistein as
Fig. 3. Comparative analysis between recombinant X-33 and
P2-D14-5.
(A) Relative mRNA level of the CYP57B3-sCPR fusion gene. The
primer efficiency was 95% for the actin gene and 85% for the
CYP57B3-sCPR gene. The mean (n = 3) is shown, and the SD is
represented by error bars. (B) 3’-Hydroxygenistein production (bar)
and cell growth (curve) of recombinant X-33 (open) and P2-D14-5
(gray) cultured in shaking flasks. (C) Hydrogen peroxide resistance
determined by spot assays. The cells were cultivated with or without
genistein for 72 h. After hydrogen peroxide-shocking treatment, the
treated cells were series-diluted and spotted on YPD plates.
Fig. 4. 3’-Hydroxygenistein production (bar) and cell growth
(curve) profiles of P2-D14-5 in a 5 L fermenter.
The fermentation process was carried out as previously described [4],
with the YPD medium replacing the yeast nitrogen base medium. The
mean (n = 3) is shown, and the SD is represented by error bars.
Improving 3'-Hydroxygenistein Production in P. pastoris 502
March 2016⎪Vol. 26⎪No. 3
a powerful antioxidant with an IC50 value of 3.9 µM for
DPPH radical-scavenging activity, and demonstrated
that 3’-hydroxygenistein production could be successfully
increased 1.87-fold in a P. pastoris mutant when using a
periodic hydrogen peroxide-shocking strategy.
Acknowledgments
This study was funded by the Ministry of Science and
Technology of Taiwan (MOST 103-2221-E-024-009-MY2).
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