34NISR Research GRANT
2009 Research Grant

Comprehensive Survey of Cytochrome P450 Function fromAspergillus oryzae

Hirofumi ICHINOSE

gested to be useful for bioindustrial applications.

Faculty of Agriculture, Kyushu UniversityMethods
Research aims
Cytochromes P450 (P450s) constitute a large superfamily of heme-containing monooxygenases, which are distributed in a wide variety of organisms1). The vast majority ofP450s are thought to have specifically emerged and individually diversified during evolution of each organism, implying P450s play crucial roles in specialization to meet themetabolic requirements of each organism, especially secondary metabolic pathways such as detoxification of xenobiotics and synthesis of secondary metabolites. As well as
the biological impacts of P450s, it is of great interest to utilize their catalytic functions in biotechnology processes.
Within the last few years, the sequence database of P450s
has enlarged exponentially, and continues to increase. Although a compilation of P450 sequences is compulsory toincrease understanding of metabolic diversity and evolutionary history of living organisms, their biological rolesand functions are still poorly understood.
A wide variety of filamentous fungi are used to produce
economically valuable consumer items. The filamentous
fungus Aspergillus oryzae is one of the most widely usedmicroorganisms, and has been used for more than 1000
years in Japanese fermentation industries to produce indigenous products such as sake (rice wine), miso (soybeanpaste), and shoyu (soy sauce). Besides fermentation technologies, many application studies have been focused on
the utilization of A. oryzae for production of recombinant
enzymes and primary and secondary metabolites. Recently,
the whole genomic sequence of A. oryzae (strain RIB40)
was determined and made available to the public2). The increased gene number in A. oryzae is mainly due to a gain ofextra genes involved in secondary metabolic pathways, including P450s, suggesting that A. oryzae has unique metabolic processes that are absent in other Aspergillus species.
In the present study, we explored the molecular diversityof A. oryzae P450s (AoCYPs) by bioinformatic genome
analysis and experimental validation. Furthermore, we developed a comprehensive functional screening system ofAoCYPs in which 121 isoforms were co-expressed withyeast NADPH-P450 reductase in Saccharomyces cerevisiae.
The functional screening system promises rapid and comprehensive characterization of the catalytic potential of AoCYPs. Using the functional screening system, a series ofcatalytic properties of AoCYPs were characterized and sug-

Molecular characterization and cDNA isolation of AoCYPs
Possible coding sequences for AoCYPs were searchedagainst the A. oryzae genome database at the National Institute of Technology and Evaluation (NITE) based upon sequence similarity to known P450s3). To evaluate annotation
accuracy, we identified the P450 signature sequence (F-x-xG-x-x-x-C-x-G) in the heme-binding domain, the E-x-x-R
motif in the K-helix, a conserved Thr in the center of the Ihelix, and the hydrophobic transmembrane domain (TMD)
at the N-terminal region. For gene amplification by RTPCR, total RNA was isolated from the fungal cells grown insynthetic culture media containing 1% glucose and 1.2 mM
ammonium tartrate4,5). PCR amplifications were carried out
using gene-specific primers designed to anneal to 5- and3-untranslated regions; basically, to the 20–30 bp upstream
or downstream flanking sequence from the putative startand stop codons. The amplified cDNAs were cloned into
pUC18 plasmid and sequenced with an automated DNASequencer.
Construction of a functional screening system
The isolated cDNA of AoCYPs were cloned into yeast
expression plasmid pGYR, which allows co-expression ofNADPH-cytochrome P450 reductase and P450s6). The expression plasmid harboring cDNA was transformed into S.
cerevisiae AH22. A positive transformant of S. cerevisiae
was selected on synthetic dextrose (SD) agar plate (2% glucose, 0.67% yeast nitrogen base without amino acid,20 mg/L L-histidine, 1.5% agar). A single colony of transformant was then inoculated in 4SD liquid medium(0.5 ml) and simultaneously grown using 96-deep-wellplates. After 4-days incubation, transformants were harvested by centrifugation and resuspended in 2 ml potassium
phosphate buffer (10 mM, pH 7.0) containing 10% glycerol. The 96-well plates were stored at 80°C until use.
Then, 20 ml solution containing transformants were simultaneously inoculated into 0.5 mL of 4SD liquid media
supplemented with 0.5 mM substrate. The plates were further incubated (12,000 rpm, 28 °C) for bioconversion. After
2-days incubation, reactions were stopped by an addition of
methanol/acetone (0.5 ml). After removal of cell debris,
metabolic products were analyzed by HPLC. If necessary,the metabolic products were extracted by ethyl acetate, purified by preparative HPLC, and analyzed by GC-MS, LC-35Fig. 1. Transcriptomic survey of AoCYPs encoded on chromosome VI.
A; Effects of culture conditions on gene expression of AoCYPs. RT-PCR was carried out using total RNA extracted from A. oryzae grown for 5-day in YPDliquid culture medium (Lane 1 to 10) or 10-day in a liquid culture medium (Lane 11 to 20). cDNA fragments; CYP51F4 (Lane 1 and 11), CYP58F1 (Lane 2and 12), CYP58G1 Lane (Lane 3 and 13), CYP505C3 (Lane 4 and 14), CYP531E1 (Lane 5 and 15), CYP5080E1 (Lane 6 and 16), CYP5087B1 (Lane 7 and17), CYP5106A1 (Lane 8 and 18), CYP5107A1 (Lane 9 and 19), and CYP5114A1 (Lane 10 and 20).
B; Time course of gene expression in a nitrogen-limited synthetic liquid culture medium. RT-PCR was performed with total RNA individually
extracted from A. oryzae grown for 5-, 10-, 18-, or 21-day in a synthetic liquid culture medium.

Fig. 2. Functional screening of AoCYP catalyzing genistein conversion.
Metabolic products from S. cerevisiae expressing CYP57B3 were analyzed by HPLC. 8-OH, 6-OH, and 3-OH indicate 8-hydroxygenistein, 6hydroxygenistein, and 3-hydroxygenistein, respectively.
Catalytic activity of CYP57B3 against genistein.

MS, and/or NMR.

Fig. 3.

Results

in 13 genes, suggesting that they are possibly pseudogenesthat have originated from gene reorganizations and/or single mutations during fungal evolution. Therefore, 142 AoCYPs from the A. oryzae genome were selected for furtherinvestigation.
To elucidate transcriptional capability of AoCYPs, weperformed gene amplification by RT-PCR. Expression profiles of genes involved in secondary metabolic systems ofA. oryzae are very likely to be affected by cultivation conditions. Therefore, we used YPD and synthetic liquid culture
media for fungal growth4,5). Figure 1 shows expression profiles of AoCYPs encoded on chromosome VI. When fungi
were grown in YPD liquid culture medium, many genes
were not amplified by RT-PCR, whereas only some genes
were confirmed to be expressed (Fig. 1A). In contrast, a series of AoCYP genes were strongly expressed when A.
oryzae was grown in a nutrient-limited synthetic liquid culture medium (Fig. 1A), suggesting that transcriptional regulation of AoCYP responds to nutrient limitation or starvation. Although AoCYPs encoded on chromosome VI

Molecular characterization and cDNA isolation of AoCYPs
The whole-genome sequence of A. oryzae strain RIB40
was released recently. According to the public database,
there are several candidate genes assigned to P450s. However, some candidates have low sequence similarity toknown genes for P450s but significantly higher similarity toother proteins, suggesting that annotational errors may beinvolved (data not shown). Several candidates have unexpected truncations of their N- and/or C-terminal sequence(s). Therefore, we further refined gene annotation accuracy
based on the following sequence features: (i) conservationof F-x-x-G-x-x-x-C-x-G in the heme-binding domain, (ii)conservation of E-x-x-R in the K-helix, (iii) A/G-G-x-x-T
at the center of the I-helix, and (iv) a hydrophobic transmembrane domain (TMD) at the N-terminal region. Aftersearching the database, 155 putative genes of P450 wereidentified from the whole-genome sequence. However, sequence deletions and/or inframe stop codon(s) were found36
Table 1.

Catalytic potentials of AoCYPs against various compounds
Substrate

AoCYP

7-Ethoxycoumarin

CYP57B3, CYP5061B5, CYP5075A1,CYP5078A5, CYP531E1, CYP620H9,
CYP5080B2,
CYP57B3
CYP57B3
CYP57B3, CYP62C2
CYP5061B5, CYP5068A2, CYP62A1,
CYP65AC2, CYP630A3

Genistein
Daizein
Naringenin
Testosterone

showed different time course of gene expression, a significant expression level of AoCYPs appeared after 5 days incubation and continued until 21 days incubation (Fig. 1B).
Therefore, amplification and isolation of cDNAs by RTPCR were carried out using a RNA cocktail which was prepared by mixing total RNA obtained from 5-, 10-, 18-, and21-day-old mycelia grown in a synthetic liquid culture
medium. Using an RT-PCR technique, we determined transcriptional capabilities of 133 AoCYPs experimentally. To
our knowledge, this is the first report of experimental validation of AoCYPs expression, and our results provide evidence that a series of P450s can be expressed in ascomycetous fungi. So far, we isolated 121 full-length cDNAs
encoding a mature open reading frame.
Functional Survey of AoCYPs
The isolated cDNA of 121 AoCYPs were cloned intoyeast expression plasmid and transformed into S.
cerevisiae. To elucidate heterologous expression of recombinant AoCYPs, carbon monoxide (CO) difference spectraof each transformant were analyzed. After 2–4 days incubation in SD media, typical CO-difference spectra for P450
species were observed with a number of transformants7),
indicating that certain recombinant AoCYP enzymes weresuccessfully expressed in S. cerevisiae. Based upon CO-difference spectra, we confirmed significant expression levelsfor 70 AoCYPs. Then we initiated functional screening ofAoCYPs using a series of substrates. To develop a functional screening system, each transformant was separatelyinoculated in 4SD liquid medium (0.5 ml) and simultaneously grown using 96-deep-well plates. Transformants
grown in the 96-well plates could be easily replicated andused for further investigations. Then, transformants in the96-well plates were inoculated in 4SD liquid medium(0.5 mL) containing substrates and further incubated forbioconversion. When genistein was utilized as a substrate,
significant product formations were achieved by S. cerevisiae expressing CYP57B3 (Fig. 2). No reaction proceededwith the recombinant cell expressing cytochrome P450 oxidoreductase without AoCYPs, indicating that the conver-sion of genistein was achieved by an AoCYP-dependent reaction. Using 1H-NMR analysis, the products were identified as 8-hydroxy-, 6-hydroxy-, and 3-hydroxygenistein.
To the best of our knowledge, this is the first report describing catalytic function of CYP57B3 converting genistein toits hydroxylated products. Since isoflavonoids, includinghydroxygenistein, are known to show pharmacological andbiological activities, further experimental efforts would beencouraged to utilize CYP57B3 for industrial production ofvalue-added isoflavonoids. Moreover, it has been known
that hydroxygenisteins is involved in fermented soybean
product8). Thus, CYP57B3 plays important roles in production of hydroxygenisteins during soybean fermentation. In
this study, a series of catalytic potentials of AoCYPs wereelucidated using various compounds (Table 1). Productidentification and further screening are now under way.
Conclusion
We developed a functional screening system of AoCYPs
in which 121 isoforms were co-expressed with yeast
NADPH-P450 reductase in Saccharomyces cerevisiae.
Using the screening system, novel catalytic functions ofAoCYPs were experimentally elucidated. The functional
screening system promises rapid and comprehensive characterization of the catalytic potential of AoCYPs. Thoroughunderstanding of AoCYP functions will open the door foradvanced fungal biology and biotechnology.
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