Supplementary Table 1Bacterial Strains and Plasmids Used in This Study

Strains/plasmids / Description / Reference/source
Strains
A. orientalis dA9 / Derivative of A. orientalis ATCC43491
with disrupted NRPS producing no vancomycin but more ECO-0501 / This work
S. venezuelaeATCC 15439 / picromycin-producing strain / This work
E. coli DH5α / supE44 ΔlacU169(φ80lacZΔM15) / TIANGEN
E. coli DH5α/BT340 / E. coli DH5α containing FLP recombinase
plasmid / Gust et al. 2003
E. coli ET12567(pUZ8002) / hsdR17 recA1endA1gyrA96thi-1 relA1dam-13::Tn9(Cmr) dcm-6 hsdM;
harbors conjugative plasmidpUZ8002; Cmr, Kmr / Gust et al. 2003
BW25113(pIJ790) / BW25113 containing λ RED recombination plasmid pIJ790 / Gust et al. 2003
Plasmids
pIJ773 / pBluescript KS (+) containing the
apramycin resistance gene aac(3)IV
and the oriT of plasmid RP4, flanked by FRT sites / Gust et al. 2003
pIJ778 / pBluescript KS (+) containing the
spectinomycin/streptomycin resistance
gene aadA and the oriT of plasmid RP4,
flanked by FRT sites / Gust et al. 2003
cosNL31C / SuperCos1 with part of ECO-0501 biosynthesis gene cluster containing ECO-orf27 / This work
pLYSY-27 / cosNL31C with the ECO-orf27deleted and ampicillin resistance gene replaced by apramycin resistance gene aac(3)IV and the oriT of plasmid RP4 / This work
pLYZL102 / pUC19 containing the apramycin resistance gene aac(3)IV with the oriT and SceI cutting site / This work
pULVK2A / a derivative of pULVK2 containing the aac3(IV) geneconferring resistance to apramycin / Ding et al. 2003
pLYZL102-27 / pLYZL102containing ECO-orf27 flankedwith 3kb upstream and downstream of ketoreductase located in vancomycin gene cluster / This work
pLYZL102-TE I / pLYZL102containing ECO-TE I flanked with 3kb upstream and downstream of ketoreductase located in vancomycin gene cluster / This work
pLYZL102-pikAV / pLYZL102containing pikAV flanked with 3kb upstream and downstream of ketoreductase located in vancomycin gene cluster / This work
pULVK2A-27 / pULVK2A containing ECO-orf27 / This work

Supplementary Table 1Bacterial strains and plasmids used in this study

Supplementary Table 2 Primers used in this study. The recognition sites for restriction enzymes are underlined.

Primer / Nucleotide sequence(5’-3’) / Purpose / Gene name
orf27aadA-F
orf27aadA-R / gtgggatcgattctgatgtgcaccacgga
agggctccttATTCCGGGGATCCGTCGACC
ACGGGCGATCCGCCCGTCGCCGACGCAGG
AGGTGGCCGGTGTAGGCTGGAGCTGCTTC / Construction of ECO-orf27
mutant strains / ECO-orf27
cosaac(3)IV-F
cosaac(3)IV-R / gtaactgtcagaccaagtttactcatatat
actttagatattccggggatccgtcgacc
tgtaggctggagctgcttcactcttccttt
ttcaatattattgaagcatttatcaggg / Change resistance of SupercosI / bla
orf27up-F
orf27down-R / GCTGATCTTGACCCCGGCCTGGT
GTCGCCGGGTCTTCGACCTGA / PCR analysis ECO-orf27
mutant strains / upstream and downstream of ECO-orf27
orf27probe-F
orf27probe-R / TCACTGGGTGATATGGAGGTCG
GCTATTTCTTCCCGGTCTCACG / Southern blot analysis
ECO-orf27
mutant strains / ECO-orf27 fragment
orf27R-F
orf27R-R
orf27I-F
orf27I-R
TE I-F
TE I-R
pikAV-F
pikAV-R
KRup-F
KRup-R
KRdown-F
KRdown-R / AAGCTTTCACTGGGTGATATGGAGGTCGG
AAGCTTGTGGCGGACAACGACGAGGG
TCTAGATCACTGGGTGATATGGAGGTCGG
TCTAGAGTGGCGGACAACGACGAGGG
TCTAGACTGCAACCCGTCCGGCTCGC
TCTAGAGGGAGCCTTCAGCCCACGACC
TCTAGAGTGACCGACAGACCTCTGAACG
TCTAGATCACCGTGGGTTCTGCCATC
AAGCTTggcccggcggctgggcacgaaggtctc
TCTAGAgcgcagccacggggcggtgcctga
TCTAGAtgaccacgcgcgtatgggactac
GAGCTCTTCACGCTCGTCCTCGGCTCGATC / Over expression
Complementation / ECO-orf27
ECO-TE I
pikAV
upstream of Van-ketoreductase
downstream of Van-ketoreductase
Apr-F
Apr-R / CGGCATCGCATTCTTCGCAT
TGACACATTCTGGCGCCTGC / Identification of apramycin resistant gene / aac(3)IV

SupplementaryFig.1 Southernblot analysis of ΔECO-orf27mutant.

Southern blot analysis of BamHI-digested genomic DNA from A.orientalis dA9 and A.orientalisdA9 ΔECO-orf27. A 500bp internal ECO-orf27 fragment obtained by PCR was labeled with DIG and used as a probe.Lane1: A.orientalisdA9; lane2: A.orientalisdA9 ΔECO-orf27.

Supplementary Fig.2HPLC analysis of ECO-0501 production in A.orientalis

a:A. orientalis dA9; b: ECO-orf27 disrupted mutant; c: ECO-orf27 disrupted mutant complemented with intact ECO-orf27; d: ECO-orf27 disrupted mutant complemented with ECO-TE I; e: ECO-orf27 disrupted mutant complemented with pikAV; f:A. orientalis dA9/pULVK2A-27.

SupplementaryFig.3 LC-MS analysis of ECO-0501 and N-demethyl ECO-0501 inA.orientalis dA9