Supplemental Materials
for
Characterization of negative regulatory genes for the biosynthesis
of rapamycin in Streptomyces rapamycinicus and its application
for improved
Young Ji Yoo1, Jae-yeon Hwang1, Hea-luyung Shin1, Heqing Cui1, Jinwon Lee2, and Yeo
Joon Yoon1*
1
Department of Chemistry and Nano Science, Ewha Womans University, Ewha Global Top5
Research Program, Seoul 120-750, Republic of Korea.
2
Department of Chemical and Biomolecular Engineering, Sogang University, Seoul, 121-742,
Republic of Korea.
+82-2-3277-4082
+82-2-3277-3419
joonyoon@ewha.ac.kr
1
Table S1 Primers used for RT-PCR.
Primer
Sequence (5’-3’)
Description
rapYF
GACAGCGCAGCAGAAGAAG
Forward primer for rapY
rapYR
GGCCCTGTATGACCAGTACG
Reverse primer for rapY
rapRF
CCTACGACGTGCTGATCCTC
Forward primer for rapR
rapRR
GGTAACCCTCCCCCTTGTC
Reverse primer for rapR
rapSF
CTTCTCCGCCCTCTTTCTG
Forward primer for rapS
rapSR
CTGGCTCTCGAAGGACTTCTC
Reverse primer for rapS
rapBF
TACCGGTGACGGTGTCATCT
Forward primer for rapB
rapBR
GTGAAGTCGAATCTGGGTCAT
Reverse primer for rapB
rapAF
CGTCCGCTGTTCGTGTTAT
Forward primer for rapA
rapAR
GTGTGCAAATGTCCGTATCG
Reverse primer for rapA
rapPF
AGGTGGTGCTGTCCCACTAC
Forward primer for rapP
rapPR
GGACGAGAGTGAACTGATCCA
Reverse primer for rapP
rapCF
TGATGGCTGGTCGTTCACTA
Forward primer for rapC
rapCR
AGGAGTTCGGTGATGATTTCC
Reverse primer for rapC
rapHF
GTGGGCCAGTTGCAGATAGT
Forward primer for rapH
rapHR
GCGGACTGATCAACGATGT
Reverse primer for rapH
rapGF
GGTACGGATGTACGTCGTGAA
Forward primer for rapG
rapGR
GTGCGTAAGGAGACGTTGAGT
Reverse primer for rapG
rapXF
GCATTTGTCCTGCTCGTGA
Forward primer for rapX
rapXR
AAGGACATCGACTACGCCTTC
Reverse primer for rapX
rapWF
CGAGTCGTACGGTGATCTTGT
Forward primer for rapW
rapWR
CAAGGACGAGGCCAAAAAG
Reverse primer for rapW
rapVF
CACCTTGAGGTGATTCCTGTC
Forward primer for rapV
rapVR
GGCCAGAAAGTCCAAGAGC
Reverse primer for rapV
rapUF
AGTTGTTCTCGCCCTTGGT
Forward primer for rapU
rapUR
CCGAAGACGCTGATCATTATT
Reverse primer for rapU
rapTF
ACGCCAGATAGCAGATGTTGT
Forward primer for rapT
rapTR
TAGTGTCGTCCGTCCTCGAAT
Reverse primer for rapT
rapQF
GTCACCCCTACGACGTACTGA
Forward primer for rapQ
2
rapQR
GTCGATGGTGAAACCGTTCT
Reverse primer for rapQ
rapNF
CCATCACGACTTCCTGTGAGT
Forward primer for rapN
rapNR
GAGATCCACCCCGAGTACAAC
Reverse primer for rapN
rapMF
CCCACTTCAGTTCGGGTATTT
Forward primer for rapM
rapMR
GTGTGCACGATTCCGAGTT
Reverse primer for rapM
rapKF
GAACTACGTCAGCGGAATCAA
Forward primer for rapK
rapKR
GGTGGCGTACATAGACCTTCA
Reverse primer for rapK
rapJF
GACATGGAGAGCATCAAGAGG
Forward primer for rapJ
rapJR
GTACCCGAAAGCGAAGTTGT
Reverse primer for rapJ
rapIF
CGCAGTTCCTCTACCAGGAG
Forward primer for rapI
rapIR
CACATCGATCTTGAGCAGTCC
Reverse primer for rapI
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Table S2 Sequence homology of RapH, RapG, RapR, RapS, RapY, and RapX in the rapamycin
biosynthetic gene clusters
Rapamycin producing strains
Protein
Streptomyces
rapamycinicus
Streptomyces
iranensis
Percent identity/similarity
(aa residues, GenBank accession no.)
Actinoplanes
N902-109
RapH
100/100
(872, CAA60471.1)
91/93
(901, CDR13506.1)
60/70
(889, AGL12177.1)
RapG
100/100
(330, CAA60472.1)
94/95
(330, CDR13486.1)
-
RapR
100/100
(220, CAA60455.1)
96/97
(210, CDR13598.1)
-
RapS
100/100
(399, CAA60456.1)
94/96
(396, CDR13597.1)
-
RapY
100/100
(204, CAA60451.1)
83/90
(184, CDR15877.1)
-
RapX
100/100
(235, CAA60452.1)
94/96
(234, CDR13601.1)
-
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Table S3 The amino acid sequences of rapY and rapS genes. The deleted sequences are
underlined, and the conserved regions are indicated by colored. (green: HTH-motif; blue:
histidine kinase domain; red: histidine kinase-like ATPase domain).
Gene Name
Protein Sequence
rapY
MTAQQKKGRPATGGAALRQRVTEAITEAFAELADA
GYARMSMESVARRAGVGKAALYRRWPSKQAMVTE
LIRGKVTDLPPTPATGALRTDLRELLTTFRGQLANPL
LARIAAGLLAEASHDDALAEGLYTGVTAPRRAAAHA
ILRGAIDRGELPPGLDLDLGTDLLIAPLAFRVLVIQG
RSDDEYLETLTNAIEAALRAAVR
rapS
MRLNPWALLSRLPFRARLTAAFSALFLIAGIALLAFV
VLLARHGTEQQAQGISVTYGDVPSGSGAPMGPVRPT
RPNLTQRAPGDVAMFQKIDQTVRAVQDTALRQMVL
W S A V G L L A MA L L A G V L G W W L A G R A L R P V A S M T E T A
RRISEQSLHQRLALTGPDDELHLADTFDTMLDRLEK
SFESQRRFVANASHELKTPLTVQRTSLQVGLADPLPE
GLADVREDLLTANHEAEQLINGLLLLARSDRGLEKT
QTMDVAAIVRLVSTGLTPLATKNGVRIDLDADTPLA
VPGDPVLLRHLLTNLVRNAIQYNHPGGHVRIRLDTP
TVTVTNTGPHVSPEQIPDLFEPFHRLDGDRTATTGHG
LGLSIAHSIANAHHATLTAQPGTEGGLTLTLRFPCRR
5
FIG S1 Organization of rapamycin biosynthetic gene clusters. (a) S. rapammycinicus
ATCC29253. (b) Streptomyces iranensis. (c) Actinoplanes sp. N902-109.
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FIG S2 Construction and verification of overexpression mutants. (a) Expression plasmids were
constructed in pSET152 derivative containing an ermE* promoter with PacI and XbaI (insert:
rapY or rapS or rapR or rapX). For southern hybridization, ermE* promoter was used as a
probe and the genomic DNAs were digested by KpnI and XbaI. (b) Overexpression of rapY,
rapR and rapS in S. rapamycinicus, respectively, lane 1, DIG labled marker; lane 2, rapR
overexpression mutants; lane 3, rapY overexpression mutants; lane 4, rapS overexpression
mutants. (c) Complementation of ΔrapY and ΔrapS by pRAPY and pRAPS, respectively, lane1,
ΔrapY; lane 2, ΔrapS; lane 3-4, ΔrapY/pRAPY; lane 5-6, ΔrapS/pRAPS; lane 7, DIG labeled
marker. (d) Overexpression of rapX in S. rapamycinicus and ΔrapS strains, lane 1, wild-type
strain as a control; lane 2, ΔrapS; lane 3, WT/pRAPX; lane 4, ΔrapS/pRAPX; lane 4, DIG
labeled marker.
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FIG S3 Construction and verification of rapY in-frame deletion in S. rapamycinicus. (a)
Schematic representation of rapY in-frame deletion by homologous recombination. (b)
Southern hybridization analysis. Genomic DNA restricted with kpnI from wild-type (lane 1),
single-crossover mutant (lane 10), and wild-type revertant (lane 2, 3, 4, 7, 8, 9). Genomic DNA
restricted with KpnI and XbaI from double-crossover ΔrapY mutant (lane 6). Molecular weight
marker (lane 5). The HindIII-XbaI fragment of LA was used as a probe.
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FIG S4 Construction and verification of rapS in-frame deletion in S. rapamycinicus. (a)
Schematic representation of rapS in-frame deletion by homologous recombination. (b)
Southern hybridization analysis. Genomic DNA restricted with PvuII and NotI from wild-type
(lane 8), single-crossover mutant (lane 9), double-crossover ΔrapS mutant (lane 1), and wildtype revertant (lane 2, 3, 6, 7) strains. Lane 4 was not detected. DIG labeled marker (lane 5).
The XbaI-EcoRI fragment of RA was used as a probe.
9