Download - 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 Nihira1,2*
1International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan2MU-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: [email protected]
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
FIG. S1
mokA
mokB2
(probe)
c6H 21
a
b MV 1 2 3
C1 C2 WM 1 2 3 4 5 6 7 8 9 10 11
V 25 33 34 36 39 47 52 53H
d
FIG. S2
FIG. S3
FIG. S4