Additional file 1 of Investigating the initial steps of auricin biosynthesis using synthetic biology
Creators: name: Csolleiova, Dominika
name: Javorova, Rachel
name: Novakova, Renata
name: Feckova, Lubomira
name: Matulova, Maria
name: Opaterny, Filip
name: Rezuchova, Bronislava
name: Sevcikova, Beatrica
name: Kormanec, Jan
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Table S1. Bacterial strains and plasmids used in this study. Table S2. Oligonucleotides used in this study. Cloning sites are underlined. Figure S1. Genetic organization of the aur1 BGC for auricin and its flanking regions. Arrows indicates the position and direction of expression of indivilual genes. Details of the genes and their products are desribed in Genbank Acc. No. KJ396772. Figure S2. Comparison of auricin aur1 BGC core gene organization from S. lavendulae subsp. lavendulae CCM 3239 (Kormanec et al. 2014) with incomplete griseusin BGC from S. griseus K-63 (Yu et al. 1994) and other angucycline BGCs. Homologous genes are indicated by arrows with the same colour. BGC accession numbers are: Streptomyces sp. PGA64 gaudimycin (pga) BGC (AY034378), S. ambofaciens ATCC 23877 kinamycin (alp) BGC (AY338477), S. cyanogenus S136 landomycin (lan) BGC (AF080235), S. fradiae Tu2717 urdamycin (urd) BGC (X87093), S. antibioticus ATCC 11891 oviedomycin (ovm) BGC (AJ632203), S. antibioticus Tu6040 simocyclinone (sim) BGC (AF324838), S. griseoflavus Goe 3592 gilvocarcin (gil) BGC (AY233211), Streptomyces sp. SCC-2136 angucyclines Sch 47554 and Sch 47555 (sch) BGC (AJ628018). The conserved central regions encode oxygenase (OXY), angucycline-specific cyclase (CYC), ketosynthase α (KSα), ketosynthase β (KS β), acyl carrier protein (ACP), ketoreductase (KR), and aromatase (ARO). In three BGCs, this region is interrupted by a gene (aur1B, pgaY, alpJ) encoding a conserved TetR family regulator. Figure S3. Structure of auricin, 3´-O-α-D-forosaminyl-( +)-griseusin A, 4´-dehydro-deacetylgriseusin A (griseusin C), and landomycin E (angucycline group), and medermycin (pyranonaphthoquinone group). Numbering of positions is according to the published data for each compound. Figure S4. a Genetic organization of the aur1 BGC around the aur1DE genes in wild-type S. lavendulae subsp. lavendulae CCM 3239 (Kormanec et al. 2014; Matulova et al. 2019) and the disrupted strain S. lavendulae Δaur1DE::AprR. Coloured arrows indicate individual genes; green correspond to the aur1 BGC and red to the regulatory gene. The yellow box with the blue arrow indicates the AprR aac3(IV) gene with the oriT origin of transfer (red column) from pIJ773 (Gust et al. 2003). The blue bar below the maps indicates the position of the probe 1 used for Southern hybridization analysis. Relevant restriction sites are included. (b) Southern blot hybridization analysis of five S. lavendulae Δaur1DE::AprR clones and wild-type S. lavendulae subsp. lavendulae CCM 3239 (WT) as a control. 1 µg of DNA from the respective strain was digested with the indicated restriction endonucleases and separated by electrophoresis in a 0.8% (w/v) agarose gel. After transfer to a Hybond N membrane, hybridization was performed according to the standard DIG protocol as desribed in materials and methods using DIG-labelled probe 1 covering the aur1B gene. BstEII-digested lambda DNA was used as a size standard. Figure S5. a Genetic organization of the type II PKS BGC aur2 around two translationally-coupled genes aur2A and aur2B encoding KSα and KSβ, in wild-type S. lavendulae subsp. lavendulae CCM 3239 (Kormanec et al. 2014; Matulova et al. 2019) and the disrupted strain S. lavendulae Δaur2AB::AprR. Coloured arrows indicate individual genes; purple correspond to the aur2 BGC and yellow to the pSA3239 replication gene. The yellow box with the blue arrow indicates the AprR aac3(IV) gene with the oriT origin of transfer (red column) from pIJ773 (Gust et al. 2003). The blue bar below the maps indicates the position of the probe 2 used for Southern hybridization analysis. Relevant restriction sites are included. (b) Southern blot hybridization analysis of four S. lavendulae Δaur2AB::AprR clones and wild-type S. lavendulae subsp. lavendulae CCM 3239 (WT) as a control. 1 µg of DNA from the respective strain was digested with the indicated restriction endonucleases and separated by electrophoresis in a 0.8% (w/v) agarose gel. After transfer to a Hybond N membrane, hybridization was performed according to the standard DIG protocol as desribed in materials and methods using DIG-labelled probe 2 covering the aur2BCD gene region. BstEII-digested lambda DNA was used as a size standard. Figure S6. Cloning strategy to create the artificial operon lanABCFDLE. Each individual lan gene was PCR amplified together with a strong RBS site and cloned into pBluescript II SK to obtain plasmids pBS-lanA, pBS-lanB, pBS-lanC, pBS-lanF, pBS-lanD, pBS-lanL, and pBS-lanE. Subsequently, the genes were inserted in the final operon according to the scheme to obtain the final plasmid pBS-lanABCFDLE. Sequences upstream of individual genes are shown with RBS from pMU1s* (Craney et al. 2007) in green and the ATG codon underlined. The relevant restriction sites are indicated. Figure S7. a Schemes of the luxCDABE reporter PhiBT1 phage integration plasmid pMU1s* (Craney et al. 2007) and recombinant plasmids containing the ermEp* or kasOp* promoter. Arrows indicate the position and orientation of individual genes, and bent arrows indicate the position of promoters. The plasmid backbone contains the AprR aac3(IV) gene (blue arrow), the oriT origin of transfer (red bar), the phiBT1int integrase gene (green arrow) together with the attP site (green bar), the fdT terminator (dark grey box), and the E. coli origin of replication ColE1 (light grey box) from plasmid pMU1s* (Craney et al. 2007). Only relevant restriction nuclease sites are shown. b The luciferase activity of the luxCDABE operon after fusion with the corresponding promoters. The plasmids were introduced into S. coelicolor M1146 by conjugation and the luminescence of eight clones from each construct was determined in relative luminescence units (RLU) after growth and differentiation on solid Bennet medium in 96-well plates at the indicated time points. Each time point represents the mean, and the error bar indicates the standard deviation from the mean. Black arrows above the graph indicate developmental stages. Figure S8. High resolution ESI MS spectrum in positive mode for the rabelomycin peak (m/z = 339.08641 [M = H] + , calculated mass is 339.08631). Figure S9. Phylogenetic trees of selected ketosynthase α (KSα) and ketosynthase β (KSβ), which represent basic groups of aromatic polyketides. The Neighbour Joining method (Saiton and Nei 1987) was used to construct these trees based on the comparison of KSα (Aur1D, Aur2A) and KSβ (Aur1E, Aur2B) from S. lavendulae subsp. lavendulae CCM 3239 with the selected KSα and KSβ from the basic groups of aromatic polyketides. Protein alignments and their desriptions are shown in supplementary figures S8 and S9 below. Figure S10. Comparison of the amino acid sequence of KSα (Aur1D, Aur2A) from S. lavendulae subsp. lavendulae CCM 3239 with selected KSα proteins from the basic groups of aromatic polyketides. Protein sequences (and corresponding accession numbers) are as follows: auricin Aur2A (AIE41912), mithramycin MtmP (CAA61989), granaticin Gra-ORF1 (CAA09653), qinimycin Qin-ORF19 (WP_058047374), medermycin Med-ORF1 (BAC79044), actinorhodin ActI-ORF1 (CAC44200), alnumycin AlnL (ACI88861), frenolicin FrnL (AAC18107), kinamycin AlpA (AAR30152), jadomycin JadA (AAB36562), landomycin LanA (AAD13536), gaudimycin PgaA (AAK57525), simocyclinone SimA1 (AAK06784), urdamycin UrdA (CAA60569), auricin Aur1D (AAX57191), griseusin Gris-ORF1 (CAA54860), elloramycin ElmK (CAP12600), tetracenomycin TcmK (AAA67515), pradimicin PdmA (ABM21747), daunorubicin Dau-ORFA (AAA87618), doxorubicin DpsA (AAA65206), oxytetracycline OxyA (AAZ78325). Figure S11. Comparison of the amino acid sequence of KSβ (Aur1E, Aur2B) from S. lavendulae subsp. lavendulae CCM 3239 with selected KSβ proteins from the basic groups of aromatic polyketides. Protein sequences (and corresponding accession numbers) are as follows: elloramycin ElmL (CAP12601), tetracenomycin TcmL (AAA67516), pradimicin PdmB (ABM21748), jadomycin JadB (AAB36563), urdamycin UrdB (CAA60570), kinamycin AlpB (AAR30151), auricin Aur1E (AAX57192), griseusin Gris-ORF2 (CAA54859), landomycin LanB (AAD13537), gaudimycin PgaB (AAK57526), simocyclinone SimA2 (AAK06785), daunorubicin Dau-ORFB (AAA87619), doxorubicin DpsB (AAA65207), actinorhodin ActI-ORF2 (CAC44201), qinimycin Qin-ORF18 (WP_058047373), granaticin Gra-ORF2 (CAA09654), medermycin Med-ORF2 (BAC79045), alnumycin AlnM (ACI88862), frenolicin FrnM (AAC18108), auricin Aur2B (AIE41911), mithramycin MtmK (CAA61990), oxytetracycline OxyB (AAZ78326). Figure S12. Comparison of the amino acid sequence of Aur1D from S. lavendulae subsp. lavendulae CCM 3239 with the corresponding KSα proteins from the angucycline BGCs. Protein sequences (and corresponding accession numbers) are as follows: PgaA (AAK57525), LanA (AAD13536), AlpA (CAJ87874), JadA (AAB36562), UrdA (caa60569), SimA1 (AAK06784, Aur1D (AAX57191). Amino acid residues highlighted in green correspond to the Cys-His-His catalytic triad (Keatinge-Clay et al. 2004), highlighted in yellow different residues in Aur1D that were conserved in all other KSs. Figure S13. Comparison of the amino acid sequence of Aur1E from S. lavendulae subsp. lavendulae CCM 3239 with the corresponding KSβ proteins from the angucycline BGCs. Protein sequences (and corresponding accession numbers) are as follows: AlpB (CAJ87873), JadB (AAB36563), UrdB (CAA60570), SimA2 (AAK06785), PgaB (AAK57526), Aur1E (AAX57192), LanB (AAD13537). Amino acid residues highlighted in green correspond to the altered catalytic triad in CLF (Keatinge-Clay et al. 2004), highlighted in yellow different residues in Aur1E that were conserved in all other KSs.