Heterologous Production of a Novel Cyclic Peptide Compound, KK-1, in Aspergillus oryzae

Abstract

A novel cyclic peptide compound, KK-1, was originally isolated from the plant-pathogenic fungus Curvularia clavata. It consists of 10 amino acid residues, including five N-methylated amino acid residues, and has potent antifungal activity. Recently, the genome-sequencing analysis of C. clavata was completed, and the biosynthetic genes involved in KK-1 production were predicted by using a novel gene cluster mining tool, MIDDAS-M. These genes form an approximately 75-kb cluster, which includes nine open reading frames, containing a non-ribosomal peptide synthetase (NRPS) gene. To determine whether the predicted genes were responsible for the biosynthesis of KK-1, we performed heterologous production of KK-1 in Aspergillus oryzae by introduction of the cluster genes into the genome of A. oryzae. The NRPS gene was split in two fragments and then reconstructed in the A. oryzae genome, because the gene was quite large (approximately 40 kb). The remaining seven genes in the cluster, excluding the regulatory gene kkR, were simultaneously introduced into the strain of A. oryzae in which NRPS had already been incorporated. To evaluate the heterologous production of KK-1 in A. oryzae, gene expression was analyzed by RT-PCR and KK-1 productivity was quantified by HPLC. KK-1 was produced in variable quantities by a number of transformed strains, along with expression of the cluster genes. The amount of KK-1 produced by the strain with the greatest expression of all genes was lower than that produced by the original producer, C. clavata. Therefore, expression of the cluster genes is necessary and sufficient for the heterologous production of KK-1 in A. oryzae, although there may be unknown factors limiting productivity in this species.

Keywords: Aspergillus oryzae; antifungal activity; gene cluster; heterologous production; non-ribosomal peptide.

FIGURE 1

Chemical structure of KK-1 identified in the fungus Curvularia clavata. Constituent amino-acid units are separated by the dotted lines: Pip, pipecolic acid residue (non-proteinogenic amino acid); Val, valine residue; Asp, aspartic acid residue; Ile, isoleucine residue; Gly, glycine residue; Tyr, tyrosine residue.

FIGURE 2

Strategy for reconstruction of the NRPS gene in Aspergillus oryzae. The first step (top) is the introduction of the front half of the NRPS gene into the A. oryzae genome. The second step (middle) is the strategy used for marker recycling using the Cre/loxP system. The third step (bottom) is integration of the rear half of the NRPS gene into the A. oryzae NRPSfh strain, resulting in reconstruction of the NRPS gene in A. oryzae.

FIGURE 3

Expression of the NRPS gene in A. oryzae. (A) Arrows indicate regions of primer binding for qRT-PCR analyses. (B) Expression of the NRPS gene in the A. oryzae CNT strain and in strains NRPS-402 (402) and NRPS-403 (403) grown for 24 h in YPM medium. qRT-PCR was used to determine the levels of transcription of the NRPS gene by using primer sets A–C. Each value represents the ratio of expression to that of the histone H4 gene. Error bars represent standard deviations (three biological and two technical replicates). ND, not detected.

FIGURE 4

Strategy for introducing all genes involved in KK-1 biosynthesis. Shown is the cloning strategy used to introduce the seven cluster genes into the plasmid pA3AXPC, along with the resulting vectors, pA09OMT, pA0302, and pA678.

FIGURE 5

Expression of all genes introduced into A. oryzae. Expression of the genes required for KK-1 biosynthesis in A. oryzae strains 3, 9, 47, 53, 54, and 56 grown for 24 h in YPM medium. qRT-PCR was used to determine the levels of transcription of the indicated genes by using gene-specific primers. Each value represents the ratio of expression to that of the histone H4 gene in each strain. Error bars represent standard deviations (three biological and two technical replicates). ND, not detected.

FIGURE 6

KK-1 production, and confirmation of production, in A. oryzae. (A,B) HPLC UV spectra of (A) extract of supernatant from culture of the A. oryzae CNT strain and (B) extract of supernatant from culture of strain 54, into which all genes were successfully introduced. UV spectrum of the peak indicated by the arrow is identical to that of standard KK-1 derived from Curvularia clavata. (C,D) Extracted ion chromatograms (m/z 1113). (C) Standard KK-1 derived from Curvularia clavata. (D) Extract of supernatant from culture of A. oryzae strain 54. (E,F) Mass spectra of KK-1. (E) Standard KK-1 derived from Curvularia clavata. (F) Extract of supernatant from culture of A. oryzae strain 54.