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Figure Legend Fig. S1.

Heterologous expression system in Aspergillus oryzae for fungal biosynthetic gene clusters of secondary metabolites Applied Microbiological Biotechnology Kanae Sakai, 1 Hiroshi Kinoshita, 1 and Takuya Nihira 1,2*

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  1. Heterologous expression system in Aspergillus oryzae for fungal biosynthetic gene clusters of secondary metabolites Applied Microbiological Biotechnology Kanae Sakai,1 Hiroshi Kinoshita,1 and Takuya Nihira1,2* 1International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan 2MU-OU Collaborative Research Center for Bioscience and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., 10400 Bangkok, Thailand *Corresponding author. Tel.: +81-6-6879-7452; Fax: +81-6-6879-7454; E-mail: nihira@icb.osaka-u.ac.jp

  2. Figure Legend Fig. S1. Representative genotype of transformants. (a) Southern blot analysis of the MK transformants. Southern blot analysis was carried out against MluI-digested genomic DNA using the mokB fragment as the probe. The mokB fragment was amplified by PCR with primers mokB-f and mokB-r. Lanes C1, cosmid 12-33; C2, cosmid sCnDmokB; W, A. oryzae NS4 strain; M, λ-EcoT14I digest DNA marker; 1, M12-33-5; 2 to 4, candidate transformants (MK5-1,3,4); 5, M12-33-17; 6 to 9, candidate transformants (MK17-1, 2, 5, 6); 10, M12-33-18; 11, candidate transformants (MK18-1). The bands indicated by arrow were derived from cosmid 12-33. The band indicated by arrowhead was derived from sCnDmokB. (b) Southern blot analysis of the MK-L transformants. Southern blot analysis was carried out against SnaBI-digested genomic DNA using the AolaeA PCR fragment (amplified by AolaeA-3f: 5’-atggcctaatgtacgccc-3’, AolaeA-3r: 5’-ctcctgcagagtctcggat-3’) as the probe. Lanes V, pPTRI-laeA; M, λ-EcoT14I digest DNA marker; 1, M12-33-17 strain; 2, MK-L1; 3, MK-L2. The band indicated by arrow was derived from pPTRI-laeA. The band indicated by arrowhead was the signal of native laeA gene. (c) Southern blot analysis of KsMK transformants. Southern blot analysis was carried out against EcoRI-digested genomic DNA using the mokA or mokB2 PCR fragment (amplified by mokA-f, -r or mokB-2f, -2r primer sets) as the probe. Lanes: H, A. oryzae host strain (Ks31); 6, 21, candidate transformants (KsMK-6, 21). (d) Southern blot analysis of TQ transformants. Southern blot analysis was carried out against KpnI-digested genomic DNA using the tdiD PCR fragment as a probe. Lanes: V, TQ gene cluster expression vector sCsCTQ; M, λ-EcoT14 digest DNA marker; H, Ks31 host; 25, 33, 34,36, 39, 47, 52, 53, candidate transformants (KsTQ-25, 33, 34, 36, 39, 47, 52, 53). The arrowhead indicated the position of 9.4 kb. FIG. S2. 1H-NMR spectrum of MK purified A. oryzae transformant KsMK-25 FIG. S3. 1H-NMR spectrum of MJ purified A. oryzae transformant KsMK-25 FIG. S4. 1H-NMR spectrum of TQ purified A. oryzae transformant KsTQ-39

  3. FIG. S1 a C1 C2 M W 1 2 3 4 5 6 7 8 9 10 11 H 6 21 c b V M 1 2 3 mokA mokB2 d (probe) V H 25 33 34 36 39 47 52 53

  4. FIG. S2

  5. FIG. S3

  6. FIG. S4

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