44NISR Research GRANT
2010 Young Investigator Research Grant

Metabolic Engineering of Aspergillus niger for production ofaromatic compounds
Takasumi HATTORI
Research Collaboration and Promotion Center, Waseda University

Research aims
A filamentous fungus Aspergillus niger is capable ofhigh-yield production of citric acid from molasses andother raw materials containing saccharides, and widely used
for industrial production of citric acid, which reaches 1.6
million tons per year in the world1). On the other hand,aromatic compounds are important precursors of many
useful polymers, pharmaceuticals, and agrochemicals, etc.
Presently, the aromatic compounds are mostly preparedfrom petroleum. However, in near future, production of aromatic compounds from renewable raw materials would behighly desirable from the view point of building a low-carbon society. In this study, we examine the possibility that
aromatic compounds can be produced from glucose, a renewable carbon source, in A. niger, using fungal type III
polyketide synthases.
Methods
Two fungal type III polyketide synthase (PKS) genes,
AB206758 (csyA)2) and XM_001390834 (An-csyA), were
amplified by RT-PCR from Aspergillus oryzae RIB40 andA. niger NRRL328, respectively. These genes are cloned
into the pET-21a() vector (Merck, Darmstadt, Germany),and Escherichia coli BL21 (DE3) was transformed with therecombinant plasmids. The recombinant PKS proteins wereproduced by culturing the transformed E. coli strains for 16
hours at 16°C. For transformation of A. niger, a pyrG  mutant of citric acid-producing A. niger strain WU-2223L wasco-transformed with each of the PSK genes cloned in thepNAN8142 vector3) and a fragment of pyrG gene by protoplast-PEG method. The standard conditions for citric acidproduction were essentially the same as described previously4).
The reaction mixture for the assay of PKS contains
125 mg/l of purified PKS obtained through Ni2-affinity

Fig. 1chromatography, 0.3 mM starter substrate, and 0.7 mM malonyl-CoA in 50 mM K2HPO4-KH2PO4 buffer (pH 7.0). The
reaction was performed at 30°C for 16 hours.
The extracellular metabolites and reaction products wereextracted from supernatant by ethylacetate-HCl, dried, resuspened in methanol, and analyzed by HPLC. The ODS100V column (Tosoh, Tokyo, Japan) and PDA (SPDM20A, Shimadzu, Kyoto, Japan) or ESI-IT-MS (LCQDeca, ThermoFishier, MA, USA) detectors were used for
the HPLC analyses.
The intracellular metabolome analysis was performed at
Human Metabolome Technology Inc., Tsuruoka, Japan.
Results
In this study, we focused on PKS as a key enzyme for
production of aromatic compounds (Fig. 1), and cDNAs ofA. oryzae PKS gene (csyA) and its homologue from A.
niger (An-csyA) were cloned for overexpression of thegenes. The deduced amino acid sequence of the An-csyAgene product contains the residues and active centers thatare conserved among PKSs (Fig. 2). To examine the enzymatic activity of the An-cysA gene product, the recombinant
An-CsyA protein was incubated with each of fifteen different fatty acyl-CoAs (a starter substrate) and malonyl-CoA(an elongation substrate). The LC-MS/MS analysis of extracted reaction mixture revealed structures of reaction
products as shown in Table 1. The reaction products werepyrone-type polyketides, which are made up of a moleculeof a starter substrate (C2–C12 fatty acyl-CoA) and 2–5
molecules of the elongation substrate (malonyl-CoA).
Therefore, An-CsyA is a novel type III PKS capable ofusing a smaller starter substrate, a property that is suitable
for the aromatic compounds synthesis5).
The genes, An-csyA and csyA, were introduced into A.
niger WU-2223L and placed under the control of a promoter for high level expression. The transcripts of An-csyA

Synthesis of aromatic compounds from renewable glucose by A. niger.

45Fig. 2 Comparison of the deduced amino acid sequences of A. niger An-CysA and other type III PKSs. G.h 2PS, G. hybrida 2PS; M.s CHS, M. sativa
CHS; N.c ORAS, N. crassa ORAS; R.p ALS, R. palmatum ALS; A.a OKS, A. arborescens OKS; A.n AncsyA, A. niger An-CsyA. The critical active-site
residue 197 is marked in bold number, the active center (Cys164, Phe215, His303, Asn336) are marked in italicized numbers, and the residues lining the active site (Gly256, Phe265, Ser338) are marked in underlined numbers.
Table 1.

Polyketides synthesized by reactions with An-CsyA

Starter substrateSide chain

Triketide pyrone1)

Tetraketide pyrone2)

Pentaketide pyrone3)

Hexaketide pyrone

Acetyl-CoA
Acetoacetyl-CoA
Malonyl-CoA
Methylmalonyl-CoA
Isobutyryl-CoA
Benzoyl-CoA
Butyryl-CoA
Hexanoyl-CoA
Octanoyl-CoA
Decanoyl-CoA
Lauroyl-CoA
Myristoyl-CoA

RCH3RCH3RCH3RCH(CH3)COOH
RCH(CH3)2RC6H5R(CH2)2CH3R(CH2)4CH3R(CH2)6CH3R(CH2)8CH3R(CH2)10CH3R(CH2)12CH3
4)4),5)
or cysA were detected in transformants by RT-PCR analysis.
The transformants were then cultivated under the conditions for citric acid production for 7 days, and the extracellular metabolites were extracted and analyzed. However, wedetected no remarkable accumulation of polyketides in thecells (data not shown).
The metabolome analysis of A. niger WU-2223L cells
after 7 days cultivation under the conditions for citric acidproduction revealed the intracellular accumulation ofacetyl-CoA (0.3 nmol/g-wet cell). But malonyl-CoA, which
can serves as an elongation substrate in An-CysA-mediatedreaction, was not detected in the cells.

Conclusion
In this study, we examined the properties of a novel type
III PKS from A. niger (An-CysA). The An-CysA exhibited
activities towards many different fatty acyl-CoAs and produced pyrone-type polyketides made up of a molecule ofC2–C12 fatty acyl-CoA and 2–5 molecules of malonylCoA. The simple overexpression of these PKS genes in A.
niger did not resulted in the production of polyketides
under the conditions described previously for citric acidproduction.
The pyrone-type polyketides are precursors of resolcinoltype polyketides, which are aromatic compounds. At pres-46

ent, we are attempting to modify the metabolic pathways inA. niger to increase intracellular levels of malonyl-CoA for
production of polyketides.
References1)

2)

Kirimura, K., Honda, Y., and Hattori, T. (2011) Citric Acid, p. 135–
142. In Moo-Young, M., Butler, M., Webb, C., Moreira, A., Grodzinski, B., Cui, Z. F., and Agathos, S. (eds.), Comprehensive Biotechnology, 2 nd ed. Elsevier
Seshime, Y., Juvvadi, P. R., Kitamoto, K., Ebizuka, Y., Nonaka, T.,and Fujii, I. (2010) Aspergillus oryzae type III polyketide synthase

CsyA is involved in the biosynthesis of 3,5-dihydroxybenzoic acid.
Bioorg. Med. Chem. Lett. 20: 4785–4788.
3) Minetoki, T., Kumagai, C., Gomi, K., Kitamoto, K., and Takahashi,K. (1998) Improvement of promoter activity by the introduction ofmultiple copies of the conserved region III sequence, involved in theefficient expression of Aspergillus oryzae amylase-encoding genes.
Appl. Microbiol. Biotechnol. 50: 459–467.
4) Hattori, T., Kino, K., and Kirimura, K. (2009) Regulation of alternative oxidase at the transcription stage in Aspergillus niger under theconditions of citric acid production. Curr. Microbiol. 58: 321–325.
5) Hansen, C. A., and Frost. J. W. (2002) Deoxygenation of polyhydroxybenzenes: an alternative strategy for the benzene-free synthesis ofaromatic chemicals. J. Am. Chem. Soc. 124: 5926–5927.