Post on 13-Aug-2020
Supplementary information
Discovery of recombinases enables genome mining of cryptic
biosynthetic gene clusters in Burkholderiales species
Xue Wanga,1, Haibo Zhoua,1, Hanna Chena,b,1, Xiaoshu Jinga, Wentao Zhenga, Ruijuan Lia, Tao
Suna, Jiaqi Liua, Jun Fua, Liujie Huoa, Yue-zhong Lia, Yuemao Shena, Xiaoming Dingc, Rolf
Müllerd, Xiaoying Biana,2, and Youming Zhanga,2
aShandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of
Microbial Technology, School of Life Sciences, Shandong University, 266237 Qingdao,
China;
b Hunan Provincial Key Laboratory for Microbial Molecular Biology-State Key Laboratory
Breeding Base of Microbial Molecular Biology, State key laboratory of freshwater fish
development biology, College of Life Science, Hunan Normal University, Changsha, 410081,
People’s Republic of China;
cCollaborative Innovation Center for Genetics and Development, State Key Laboratory of
Genetic Engineering, Department of Microbiology, School of Life Sciences, Fudan
University, 200433 Shanghai, China;
dDepartment of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research,
Helmholtz Centre for Infection Research and Saarland University, 66123 Saarbrücken,
Germany
1X.W., H.Z. and H.C. contributed equally to this work.
2To whom correspondence should be addressed. Email: bianxiaoying@sdu.edu.cn or
zhangyouming@sdu.edu.cn.
1
Materials and methods
Supplementary Results
Supplementary Tables
Table S1. Selected different recombinase pairs in Burkholderiales species
Table S2. Strains, plasmids and mutants in this work
Table S3. Transcript level (fpkm) of BGC 6A, BGC 7, BGC 11, glb gene clusters and Redαβ7029 of
DSM 7029 in six different conditions
Table S4. Sequences of promoters used in this study
Table S5. Predicted gene function of five activated gene clusters investigated in this study
Table S6. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 1 in MeOD-d4
Table S7. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 1 in DMSO-d6
Table S8. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 2 in DMSO-d6
Table S9. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 3 in DMSO-d6
Table S10. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 4 in DMSO-d6
Table S11. The activity of glidopeptin A and rhizomide A against six plant diseases.
Table S12. Cytotoxic activity of glidopeptin A and rhizomide A.
Table S13. Primers used in this study
Supplementary Figures
Fig. S1. Screening of effective and stringent inducible promoters in DSM 7029
Fig. S2. The recombination efficiency of the LCHR assay mediated by different combinations of Redγ,
Redαβ7029 and Redαβ in E. coli and DSM 7029, respectively
Fig. S3. The recombination efficiency of the LCHR and LLHR mediated by Redγ—Redαβ7029 in E.
coli
Fig. S4. Optimization of work conditions of Redγ-Redαβ7029 in DSM 7029
Fig. S5. Diagram for construction, verification and metabolic analysis of the clean deletion of the
glidobactin biosynthetic gene cluster in DSM 7029
Fig. S6. Diagram for construction, verification, and metabolic analysis of BGC 6A activation and
inactivation in DSM 7029-Δglb
Fig. S7. Diagram for construction, verification, and metabolic analysis of BGC 7 activation and
inactivation in DSM 7029-Δglb
Fig. S8. Diagram for construction, verification, and metabolic analysis of BGC 11 activation and
inactivation in DSM 7029-Δglb.
Fig. S9. Diagram for construction, verification, and metabolic analysis of the deletion of rhizoxin
biosynthetic gene cluster in P. rhizoxinica HKI 454
Fig. S10. Diagram for construction, verification, and metabolic analysis of BGC P1 activation and
inactivation in P. rhizoxinica HKI454-Δrhi.
Fig. S11. Diagram for construction, verification, and metabolic analysis of BGC P7 activation and
inactivation in P. rhizoxinica HKI454-Δrhi.
Fig. S12. Diagram for construction, verification of BGC 2 activation and inactivation in P.
phytofirmans PsJN.
Fig. S13. Marfey’s analysis of the amino acid constituents of glidopeptin A (1)
Fig. S14. Marfey’s analysis of the amino acid constituents of rhizomides A-C (2-4)
Fig. S15. Complete structures and Key COSY and HMBC correlations of (1-4)
Fig. S16. 1H NMR spectrum of glidopeptin A (1) in MeOD- d4
2
Fig. S17. 13C NMR spectrum of glidopeptin A (1) in MeOD-d4
Fig. S18. DEPT spectrum of glidopeptin A (1) in MeOD-d4
Fig. S19. HSQC spectrum of glidopeptin A (1) in MeOD-d4
Fig. S20. 1H-1H COSY spectrum of glidopeptin A (1) in MeOD-d4
Fig. S21. HMBC spectrum of glidopeptin A (1) in MeOD-d4
Fig. S22. 1H NMR spectrum of glidopeptin A (1) in DMSO-d6
Fig. S23. 13C NMR spectrum of glidopeptin A (1) in DMSO-d6
Fig. S24. DEPT spectrum of glidopeptin A (1) in DMSO-d6
Fig. S25. HSQC spectrum of glidopeptin A (1) in DMSO-d6
Fig. S26. 1H-1H COSY spectrum of glidopeptin A (1) in DMSO-d6
Fig. S27. HMBC spectrum of glidopeptin A (1) in DMSO-d6
Fig. S28. 1H NMR spectrum of rhizomide A (2) in DMSO-d6
Fig. S29. 13C NMR spectrum of rhizomide A (2) in DMSO-d6
Fig. S30. DEPT spectrum of rhizomide A (2) in DMSO-d6
Fig. S31. HSQC spectrum of rhizomide A (2) in DMSO-d6
Fig. S32. 1H-1H COSY spectrum of rhizomide A (2) in DMSO-d6
Fig. S33. HMBC spectrum of rhizomide A (2) in DMSO-d6
Fig. S34. 1H NMR spectrum of rhizomide B (3) in DMSO-d6
Fig. S35. 13C NMR spectrum of rhizomide B (3) in DMSO-d6
Fig. S36. DEPT spectrum of rhizomide B (3) in DMSO-d6
Fig. S37. HSQC spectrum of rhizomide B (3) in DMSO-d6
Fig. S38. 1H-1H COSY spectrum of rhizomide B (3) in DMSO-d6
Fig. S39. HMBC spectrum of rhizomide B (3) in DMSO-d6
Fig. S40. 1H NMR spectrum of rhizomide C (4) in DMSO-d6
Fig. S41. 13C NMR spectrum of rhizomide C (4) in DMSO-d6
Fig. S42. DEPT spectrum of rhizomide C (4) in DMSO-d6
Fig. S43. HSQC spectrum of rhizomide C (4) in DMSO-d6
Fig. S44. 1H-1H COSY spectrum of rhizomide C (4) in DMSO-d6
Fig. S45. HMBC spectrum of rhizomide C (4) in DMSO-d6
Fig. S46. IR spectrum of glidopeptin A (1)
Fig. S47. IR spectrum of rhizomide A (2)
Fig. S48. IR spectrum of rhizomide B (3)
Fig. S49. IR spectrum of rhizomide C (4)
Fig. S50. Antibiotic activity Test of glidopeptin A and rhizomide A
3
Materials and methods
Strains, plasmids and reagents
The wild type bacterial strains and the plasmids used in this study are listed in
Table S2. The plasmids were constructed via recombineering either in E. coli GB 2005-
red for linear plus circular homologous recombination (LCHR) or in E. coli GB 2005-
dir for linear plus linear homologous recombination (LLHR) (1). Genes encoding
different recombinases were amplified using polymerase chain reaction (PCR) products
from corresponding genomic DNA or synthesized according to the original sequences
in GenBank by Sangon Biotech in China. All oligonucleotides were synthesized by
Sangon Biotech in China. Restriction enzymes and DNA markers were supplied by
New England Biolabs. The antibiotics were purchased from Invitrogen. E. coli cells
were cultured in Luria-Bertani (LB) broth or on LB agar plates (1.2% agar) with
ampicillin [amp] (100 μg/mL), kanamycin [km] (15 μg/mL), chloramphenicol [cm] (15
μg/mL), or gentamicin [genta] (5 μg/mL) as required. Burkholderiales strain DSM 7029
and P. rhizoxinica HKI 454 were cultured in CYMG (8 g/L Casein peptone, 4 g/L Yeast
extract, 4.06 g/L MgCl2·2H2O, 10 mL/L glycerin) broth or agar plates with apramycin
[apra] (20 μg/mL), kanamycin [km] (20 μg/mL), gentamicin [genta] (15 μg/mL) and
chloramphenicol [cm] (15 μg/mL). P. phytofirmans strain PsJN was cultured in
Trypticase Soy Broth (Oxoid CM129) or agar plates.
General Experimental Procedures
Optical rotations were obtained on a JASCO P-1020 digital polarimeter. UV
spectra were recorded on a Thermo Scientific Dionex Ultimate 3000 DAD detector. IR
spectra were taken on a Nicolet NEXUS 470 spectrophotometer as KBr disks. 1H and 13C NMR, DEPT, and 2D NMR spectra were recorded on an Agilent 500 MHz DD2
using TMS as an internal standard. HRESIMS spectra were measured on a Bruker
Impact HD microTOF Q III mass spectrometer (BrukerDaltonics, Bremen, Germany)
using the standard ESI source. UHPLC-MS was operated using an Thermo Scientific
Dionex Ultimate 3000 system coupled with the Bruker amazon SL Ion Trap mass
spectrometry (Bruker Corporation), controlled by Hystar v3.2 and Chromeleon Xpress
software. A Thermo Scientific™ Acclaim™ C18 column (2.1×100 mm, 2.2 μm) was
used. The mobile phase consisted of H2O containing 0.1% FA and ACN. Semi-
preparative HPLC was performed using an ODS column [Bruker ZORBAX SB-C18,
9.4×250 mm, 5 μm, 3 mL/min]. TLC and column chromatography (CC) were
performed on plates precoated with silica gel GF254 (10-40 μm) and over silica gel
(200-300 mesh, Qingdao Marine Chemical Factory) and Sephadex LH-20 (GE
Healthcare), respectively. Vacuum-liquid chromatography (VLC) was carried out over
silica gel H (Qingdao Marine Chemical Factory).
Bioinformatics analysis
The Redαβ or RecET phage protein homologs were examined in the NCBI non-
redundant protein sequences database using the DELTA-BLAST (Domain Enhanced
Lookup Time Accelerated BLAST) and PSI-BLAST (Position-Specific Iterative
BLAST) (2). Only adjacent Redαβ-like or RecET-like recombinases present in a
bacterial genome were selected in Table S1. The putative biosynthetic gene clusters of
4
strains investigated in this work were predicted using antiSMASH analysis (3, 4) .
Transcriptome Sequencing
The transcriptome sequencing was carried out by Novogene Biotech in China.
DSM 7029 cells were shaken at 250 rpm in the CYMG medium and M9 medium,
respectively. All cultivations were carried out at 30 ℃. Cultures of DSM 7029 in liquid
CYMG and M9 were sampled at three different culture times: 16 h, 24 h and 40 h for
massively parallel RNA sequencing. A total amount of 3 μg RNA per sample was used
as input material for the RNA sample preparations. Samples were sequenced twice to
obtain appropriate deep sequencing results. Differential expression analysis of two
conditions/groups (two biological replicates per condition) was performed using the
DESeqR package. The clustering of the index-coded samples was performed on a cBot
Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia)
according to the manufacturer’s instructions. After cluster generation, library
preparations were sequenced on an Illumina Hiseq platform and paired-end reads were
generated. The RNA libraries were prepared and analyzed by NEBNext® Ultra™
Directional RNA Library Prep Kit for Illumina® (NEB, USA). Gene expression levels
could be reflected by FPKM (expected number of Fragments Per Kilobase of transcript
sequence per Millions base pairs sequenced).
Construction of plasmids with different inducible promoters
The test plasmid was a pBBR1 origin and harbored a firefly luciferase reporter
gene under the control of different promoters. Oligonucleotides used for the
construction are listed in Table S13. The original pBBR1-ccdB-hyg was digested with
ApaLI and the ccdB-cm cassette amplified from pR6K-cm-ccdB (5) with primers 01
cm-ccdB-3/01 cm-ccdB-5 were cotransformed into GB05-dir-gyrA462 (5) to yield
pBBR1-ccdB-cm by LLHR. The linear fragment of pBBR1-km digested (BamHI) from
pBBR1-ccdB-cm was transferred to GB05-Red harboring pSC101-BAD-ccdA-Rha-
gam-tac-beta to obtain pBBR1-tac containing the tac promoter and km resistance gene.
The linear pBBR1-km-tac fragment derived from digestion of pBBR1-tac with NdeI
was mixed with the firefly luciferase reporter gene which was amplified from
pBeloBAC11-firefly using primes 03 Firefly-3/03 Firefly-5, and the mixture was co-
transformed into GB05-dir to yield pBBR1-tac-firefly via triple LLHR. In addition, the
linear pBBR1-km-tac derived from AseI digestion was mixed with three fragments,
which were the Rha promoter amplified from pSC101-Rha-ETgA-tet using primers 04
Rha-3 and 04 Rha-5, the BAD promoter amplified from pSC101-BAD-Red gbaA-amp
using primes 05 BAD-3 and 05 BAD-5, and the tet promoter amplified from pSC101-
tetR-tetO-eGFP-km NEW using primes 06 Ptet-3 and 06 Ptet-5 into GB05-dir,
respectively, to get three plasmids: pBBR1-Rha-firefly, pBBR1-BAD-firefly, pBBR1-
tet-firefly. All plasmids were verified by restriction analysis and sequencing. These four
plasmids were transformed into DSM 7029 by electroporation. Selected promoters
sequences are listed in Table S4.
Promoter screening by quantitative analysis of reporter gene
According to the manual of the reporter gene detection kits (Luciferase Assay
System E1500), the recombinants of DSM 7029 harboring different constructs pBBR1-
promoter-firefly-km (Fig. S1a) were inoculated into 800 µL liquid CYMG medium
5
with kanamycin (15 μg/mL) and cultured overnight at 30 ℃. The culture was
normalized (OD600=0.15), and then 30-40 μL culture was transferred into 1 mL fresh
liquid CYMG with kanamycin (15 μg/mL) and cultivated at 30 ℃, 900 rpm for 4 h.
After addition of inducers, the culture was continually incubated at 30 ℃ without
shaking for 45 min. Then 50 µL induced culture mixed with 40 µL overnight culture of
DSM 7029 wild type, and 10 µL of 1 M K2HPO4 (pH 7.8) and 20 mM EDTA was added
into the mixture. After the quick-frozen on dry ice, the mixture was equilibrated to room
temperature. Then, 300 µL freshly prepared lysis mix was added and incubated at room
temperature for 10 minutes. The 100 µL of cell lysate was mixed with 100 µL of
Luciferase Assay Reagent and then the RLU was measured by GloMax 96 microplate
luminometer.
Construction of recombinase expression plasmids
All the recombinase expression plasmids (Table S2) are based on the pBBR1
origin and under the control of rhamnose inducible promoter (Rha). The original
plasmid pBBR1-Rha-Redgba (Redgba: Redγβα) was digested with HindIII and NdeI to
yield linear fragment (5084 bp). The redαβ7029 genes were amplified from genomic
DNA with primers 7029BA-01A-1/7029BA-01A-2. The above two fragments were co-
transformed into induced GB05-dir to get pBBR1-Rha-BA7029-km (BA7029:
Redαβ7029). The digested (HindIII and DraI) fragment (6892 bp) of pBBR1-Rha-
Redgba-km coupled with the Redβα7029 fragment amplified from DSM 7029 genomic
DNA with primers 7029BA-01B-1 and 7029BA-01A-2, were co-transformed into
GB05-dir to build pBBR1-Rha-RedG-BA7029-km. Meanwhile, the digested (HindIII
and NdeI) pBBR1-Rha-Redgba-km fragment was mixed with H7029 and Redαβ7029
DNA fragments which were amplified from DSM 7029 genomic DNA with primers
H7029-1/H7029-2 and 7029BA-01B-1/7029A-2, respectively, and the mixture was co-
transformed into induced GB05-dir to constuct pBBR1-Rha-BA7029-H7029 by triple
LLHR. Digested pBBR1-Rha-pluG-TEpsy-km (EcoNI and BsrGI) mixed the linear
fragment with the Redβα7029 fragment amplified from DSM 7029 genomic DNA with
primers 7029BA-01C-1 and 7029BA-01A-2, and co-transformed into GB05-dir to get
pBBR1-Rha-pluG-BA7029-km. The pBBR1-Rha-ETh_bdu-km was constructed on the
basis of pBBR1-Rha-TEGpsy-km. In this part, the digested pBBR1-Rha-TEGpsy-km
(NdeI and EcoRV) mixed with the synthesized ETh_bdu and co-transferred into
induced GB05-dir to get pBBR1-Rha-ETh_bdu-km. All the recombinants were selected
on LB plates containing 15 μg/mL kanamycin and incubate at 37 ℃. Correct clones
were verified by restriction analysis and sequencing.
Optimization of work conditions of Redγ-Redαβ7029
The aptitude test of Redαβ7029 was made in E. coli, and both LLHR (Fig. S3a)
and LCHR (Fig. S3b) mediated by Redγ-Redαβ7029 were implemented. In the LCHR
assay, 200 ng RK2-apra-cm plasmid and 200 ng kanamycin resistance gene PCR
product flanked with 50 bp homologous arms were transformed to GB2005 harboring
pBBR1-Rha-RedG-BA7029-km. In the LLHR assay, 200 ng linear RK2-cm vector
fragment and 200 ng PCR product were co-transformed to GB2005 harboring pBBR1-
Rha-RedG-BA7029-km.
The effect of homology arm’s length on recombination was explored through
6
using an apramycin resistance gene flanked with varying length homology arms (50 bp,
80 bp, 100 bp) to replace the 21.2 kb glidobactin biosynthetic gene cluster in DSM 7029
harboring pBBR1-Rha-RedG-BA7029-km. (Fig. S2d)
The effect of different preparative methods of DSM 7029 competent cells on the
recombination efficiency was explored according to the previous method (6). Firstly,
the effect of electrocompetent cells preparing temperature on recombination efficiency
was explored by a glb gene cluster replacement assay. An apramycin resistance gene
(Apra) flanked by 80 bp homologous arms was transformed into DSM 7029 harboring
pBBR1-Rha-RedG-BA7029-km to replace the glbB-glbG region of the 21.2 kb glb
gene cluster at room temperature or low temperature condition (0 ℃), respectively (Fig.
S4a). Secondly, the electrocompetent cells of DSM 7029 harboring pBBR1-Rha-RedG-
BA7029-km was prepared with five different washing buffers: double distilled water
(H2O), 10% sucrose (S), 10% sucrose+2 μM HEPES (S+H), 10% glycerol (G) and 10%
glycerol+2 μM HEPES (G+H), and the same glb gene cluster replacement assay was
implemented (Fig. S4b). To determine the optimum temperature for the activities of the
phage proteins, we examined the optimal temperature and time by the same
recombination assays in DSM 7029, and the competent cells were prepared, induced
and revived at different temperatures ranging from 25 ℃ to 37 ℃ (Fig. S4c). The
optimum time for incubation and induction were examined when the OD600 of the
starting culture was 0.1 and rhamnose induction began at 9 h and then at 1 h intervals
up to 15 h (Fig. S4d). After the determination of optimum time for incubation, the
detection area of the induction time was designed to range from 0.5 h to 2 h (Fig. S4e).
Procedure of recombineering
The expression plasmids of various recombinases were electroporated into E. coli
and Burkholderiales species (7). The E. coli electro-competent cells were prepared
according to our established protocol (1, 8). For DSM 7029 and P. rhizoxinica HKI 454,
overnight cultures containing the recombinase expression plasmids were diluted into
1.3 mL CYMG medium supplemented appropriate antibiotics. The OD600 value of
starting culture was around 0.1, and then the culture was incubated at 30 ℃, 950 rpm
for 14 h until the OD600 was around 2.0. After addition of the inducer L-rhamnose to
a final concentration of 2.5 mg/mL, the culture continued to be cultivated for 90 min.
Cells were then centrifuged for 1 min at 9500 rpm at room temperature. The supernatant
was discarded, the cell pellets were suspended in 1 mL room temperature ddH2O, and
the suspension was centrifuged. This step was repeated once more. Cells were finally
suspended in 30 μL ddH2O (room temperature, ~20 ℃) and PCR product (~1 μg) was
added. Electroporation was performed using room temperature cuvettes (1 mm) and an
Eppendorf 2510 electroporator (1300 V). Then CYMG medium (1 mL) was added after
electroporation. The cells were incubated at 30 ℃ for 4 h with shaking (950 rpm) and
then spread on CYMG plates containing appropriate antibiotics. For P. phytofirmans
strain PsJN, overnight cultures containing the recombinase expression plasmids were
diluted into 1.3 mL 129 medium with appropriate antibiotics. The OD600 value of
starting culture was around 0.1, and culture was incubated at 30 ℃, 950 rpm for 3 h
until the OD600 was around 2.0. After addition of the inducer L-rhamnose (2.5 mg/mL),
the cells continued to be cultivated for 90 min. The electroporation procedure was the
7
same as that of DSM 7029. One milliliter 129 medium was added after electroporation.
The cells were incubated at 30 ℃, for 2 h with shaking, and then spread on appropriate
antibiotic plates.
Plasmid modification assay
In the plasmid modifacation assay (Fig. S2a), standard linear plus circular
homologous recombination (LCHR) assays were employed in DSM 7029 and E. coli
using 1 μg of 1 kb linear dsDNA substrate with 50bp homology arms to replace the
recombinase (Redγ-Redαβ7029) of the pBBR1-Rha-RedG-BA7029-km. The
procedure of Recombineering was the same as the above.
Gene replacement or insertion in the chromosome of DSM 7029 and other
Burkholderiales species
The target genes were replaced by an antibiotic selection marker (apramycin
resistance gene) using the Redγ-Redαβ7029 system for biosynthetic gene inactivation.
The BGC activated mutant was constructed by insertion of an antibiotic selection
marker in front of the main biosynthetic gene of corresponding BGC. The antibiotic
resistance genes flanked with homology arms (~50 bp) were generated by polymerase
chain reaction (PCR) amplification using 2×PrimerSTAR Max polymerases (Takara)
according to the manufacture’s manual, and the template for apra and km resistance
genes is plasmid RK2-Apra-km. For the recombineering, purified PCR products of
resistance genes flanked by 50 bp or 80 bp homology arms were transformed into DSM
7029 (Fig. S6-8) and other Burkholderiales species (Fig. S9-11) containing the
recombinase expression plasmid pBBR1-Rha-RedG-BA7029-km by electroporation,
respectively. Recombinants were selected on CYMG plates containing apramycin (20
μg/mL) or kanamycin (20 μg/mL), respectively. Correct recombinants were verified by
colony PCR. A list of recombinants generated in this study is provided as Table S2.
Oligonucleotides used for gene deletions are listed in Table S13.
Single-strand recombination assay
To determine whether dsDNA recombination can be processed through a full-
length ssDNA intermediate in DSM 7029, we employed the one linear dsDNA
substrates (OO) and two linear ssDNA (leading (ld), lagging (lg)) in a recombination
assay in DSM 7029 harboring pBBR1-Rha-RedG-BA7029-km, pBBR1-Rha-RedG-
B7029-km, pBBR1-Rha-RedG-km, pBBR1-Rha-RedGBA-km and DSM 7029 wild
type respectively. The efficiency of the Redαβ7029 promoting recombineering using
single-stranded oligonucleotide-derived substrates was explored by a glb gene cluster
replacement assay (Fig. S2b), single-strand (ss) and double-strands (ds) of apramycin
resistance gene (Apra) flanked by 80 bp homologous arms was transformed to replace
the glbB-glbG region of the 21.2 kb glb gene cluster at the optimal condition.
The dsDNA substrate was prepared carrying either phosphorylated (P) or
hydroxylated (O) in all three combinations (OO, PO, OP). The PCR products were
purified by Tiangen quick PCR purification kit (Tiangen, Shanghai, China). The single-
strand DNA was prepared according to the protocol of a previous study (9). After
PCR, 5 μg purified dsDNA (PO or OP) was incubated with 25 U lambda exonuclease
(New England Biolabs, Frankfurt am Main, Germany) in a total reaction volume of 50
μL in 1×lambda exonuclease reaction buffer at 37 ℃. The reaction was terminated after
8
150 minutes digestion times by 10 minutes incubation at 75 ℃. The phosphorylated
strand was removed by digestion with lambda exonuclease and the ssDNA was
obtained by gel extraction. The same molar quantity of dsDNA and ssDNA were added
to compare the efficiency of the recombination.
Clean deletion of glb gene cluster in DSM 7029
First, 20 μL rhamnose (10 mg/mL) was added to 1.3 mL 14 h culture of DSM 7029
harboring pBBR1-Rha-RedG-BA7029-km to induce the expression of Redγ-
Redαβ7029. Then the Cre-SSRs-apra cassette including an apramycin resistance gene
(Apra), site-specific recombination (SSR) recognition sites (loxP66/71), and a Cre site-
specific recombinase under the control of a BAD promoter flanked by homologous
arms was introduced to replace the target region and the mutants were selected on
CYMG plates containing appropriate antibiotic and verified by PCR. Then, 10 μL
arabinose (100 mg/mL) was introduced to 1.3 ml 14 h culture of the DSM 7029 with
the desired mutant to induce the expression of Cre and accomplishing the remove of
the Cre-SSRs-apra cassette. The excision was screened out by double-streaking on
CYMG plates containing apramycin or kanamycin and confirmed by PCR verification
(Fig. S5).
Fermentation and Extraction of metabolites from Burkholderiales species
Liquid seed cultures of wild type and engineered DSM 7029 and P. rhizoxinica
HKI 454 were inoculated from a plate in 1.4 mL culture tubes. Seed cultures were
incubated at 30 ℃ with 200 rpm shaking until achieving turbidity or high particle
density (typically 1 day). Seed cultures were diluted at the ratio of 1:100 into 50 mL of
CYMG broth in 250 mL baffled flasks, and the flash cultures were incubated at 30 ℃,
200 rpm. After the cultures were incubated for 18-24 hours, the resin Amberlite XAD-
16 (2%) was added, and the mixture was incubated for 48 h continually. The cells and
XAD-16 were harvested at maximum speed in an Eppendorf 5240R centrifuge for 10
min centrifugation, and the crude extracts were extracted with 50 mL methanol. Finally,
the extract was concentrated in vacuo, and redissolved in 1 ml MeOH for further
UHPLC-HRMS analysis.
UHPLC-HRMS analysis of extracts
The UHPLC system was performed using an ODS column (Luna RP-C18,
4.6×250 mm, 5 μm, 0.75 mL/min) with gradient elution. UV spectra was recorded on a
DAD detector with wavelength ranging from 200 to 600 nm. The HRMS was measured
on a Bruker Impact HD microTOF Q III mass spectrometer (BrukerDaltonics, Bremen,
Germany) using the standard ESI source. Mass spectra was acquired in centroid mode
ranging from 100 to 1500 m/z with positive-mode electrospray ionization and auto MS2
fragmentation. HPLC parameters were as follows: solvent A, H2O with 0.2% TFA;
solvent B, 0.1% TFA in acetonitrile (ACN); gradient at a constant flow rate of 0.2
mL/min, 10-5 min, 5% B; 5-45 min, 5%-95 % B; 45-50 min, 95% B; detection by UV
spectroscopy at 200-600 nm.
Purification of glidopeptin (1)
The resin XAD16 was added after two days into the fermention broth (10 L) and
cultivated at 30 ℃, 170 rpm for four days. The crude extract was subjected to a VLC
fractionation in an open column using silica as the solid phase and a gradient solvent
9
system with CH2Cl2-MeOH or MeOH-H2O. For compound from BGC 6A of DSM
7029, gradient elution resulting in 9 fractions. Fr. 8 (MeOH:H2O=9:1) was concentrated
to further purified by repeated semipreparative HPLC (ODS; 5 μm, 250×10mm,
gradient elution 0-5 min 20% ACN, 5 min 20% ACN, 30 min 40% ACN, 30.1min 95%
ACN, 34 min 95% ACN, 34.1 min 20% ACN, 38 min 20% ACN) to afford 1 (10.0 mg)
with retention time at 23.4 min.
Glidopeptin A (1): colorless oil; [α]20D +6 (c 0.15, MeOH); UV (MeOH) λmax (log
ε) 220 (3.70) nm; IR (KBr) vmax 3256, 2929, 1672, 1524, 1206, 1140, 802, 724 cm-1; 1H and 13C NMR, Tables S6-S7; HRESIMS m/z 676.3833 [M + 2H]2+ (calculated for
C60H103N16O19 676.3826).
Purification of rhizomides A-C (2-4)
For compounds produced by BGC P1 of P. rhizoxinica, gradient elution resulting
in 11 fractions. Fr. 4 (CH2Cl2:MeOH 12:1) was further purified by repeated
semipreparative HPLC (ODS; 5 μm, 250×10 mm, gradient elution 0-3 min 40% ACN,
3-10 min 55% ACN, 10-23 min 85% ACN, 23.1 min 95% ACN, 23.1-27 min 95%
ACN, 27.1 min 40% ACN, 27.1-31 min 40% ACN) to afford 2 (~100 mg) and 3 (3 mg)
at retention time 22.8 min and 21.4 min, respectively. Fr.6 (CH2Cl2:MeOH 8:1) was
further purified by repeated semipreparative HPLC (ODS; 5 μm, 250 ×10 mm, gradient
elution 0-3 min, 5% ACN; 3-25 min, 5%-46% ACN; 25.1 min 95% ACN; 25.1-29 min,
95% ACN; 29.1 min, 5% ACN; 29.1-33 min, 5% ACN) to yield compound 4 (2.5 mg)
at retention time 21.6 min.
Rhizomide A (2): white solid; [α]20D -9 (c 0.15, MeOH); UV (MeOH) λmax (log ε)
224 (3.20), 276 (1.20) nm; IR (KBr) vmax 3292, 2962, 1656, 1517, 1450, 1374, 1238,
1062 cm-1; 1H and 13C NMR, Table S8; HRESIMS m/z 732.3930 [M + H]+ (calculated
for C35H54N7O10 732.3927).
Rhizomide B (3): white solid; [α]20D -6 (c 0.15, MeOH); UV (MeOH) λmax (log ε)
224 (3.30) nm; IR (KBr) vmax 3275, 2962, 1736, 1657, 1517, 1450, 1374, 1238, 1063
cm-1; 1H and 13C NMR, Table S9; HRESIMS m/z 748.3873 [M + H]+ (calculated for
C35H54N7O11 748.3876).
Rhizomide C (4): white solid; [α]20D -5 (c 0.15, MeOH); UV (MeOH) λmax (log ε)
223 (3.50) nm; IR (KBr) vmax 3292, 2961, 1737, 1657, 1517, 1450, 1374, 1238, 1063
669 cm-1; 1H and 13C NMR, Table S10; HRESIMS m/z 748.3873 [M + H]+ (calculated
for C35H54N7O11 748.3876).
Marfey’s analysis of the amino acid constituents of compounds
Each compound was hydrolyzed in 6N HCl at 60 ℃ for 24 hours. The acid
hydrolysates of 1-4 were redissolved in H2O (50 μL), and then 0.25 μM L-FDAA in
100 μL of acetone was added, followed by 1 N NaHCO3 (25 μL). The mixtures were
heated for 1 h at 40 ℃. After cooling to room temperature, the reaction was quenched
by the addition 2 N HCl (25 μL). Finally the resulting solution was filtered through a
small 2.5 μm filter and analyzed by LC-MS using AcclaimTM RSLC 120 C18 column
(2.1×100 mm 2.2 μm) with a linear gradient of ACN and 0.1% aqueous formic acid
with different elution conditions (5%-95% ACN in 15 min for 2, 30 min for 1, 40 min
for Asn, or 45 min for 3 and 4) at a flow rate of 0.3 mL/min and UV detection at 330
nm. For the analysis of L-Thr and L-allo-Thr, the elution condition is a linear gradient
of ACN 5%-55% in 45 min. Amino acid standards were derivatized with L-FDAA in a
similar manner. Each chromatographic peak was identified by comparing its retention
10
times and molecular weight for the L-FDAA derivatives of the L- and D-amino acid
standards (10, 11).
Bioactivity assay
The biocontrol protective activity of glidopeptin A and rhizomide A against
Pseudoperonospora cubenis (CDM), Colletotrichum lagenarium (CA), Blumeria
graminis (WPM), Puccinia sorghi Schw (CSR) on the 2-leaf stagies of cucumber
seedlings, wheet seedlings and maize seedlings, respectively, under greenhouse
conditions were tested by Shenyang Sinochem Agrochemicals R&D Co.Ltd. The
results were based on the "A Manual of Assessment Keys for Plant Diseases" written
by the American Phytopathological Society (APS), with 100~0 to indicate the grade of
disease while 100 presents disease-free and 0 marks a dire threat.
The cytotoxic and antimicrobial activities of the new compound were assessed
using sulforhodamine B (SRB) and agar disk diffusion assays, respectively.
Aliquot samples of Human gastric cancer cell line MGC-803, Human breast
cancer cell lines MCF-7, Human hepatocellular carcinoma cell line Huh-7, Human
hepatocellular carcinoma cell line HepG-2, Human cervical carcinoma cell line Hela,
Human gastric cancer cell line SGC-7901, Human lung adenocarcinoma cell line NCI-
H1975 and normal human hepatic cell line LO2 were transferred to 96-well plates and
incubated overnight at 37 ℃ in 5 % CO2/air. Test compounds were added to the plates
in DMSO and serially diluted (from 160 μM to 0.3125 μM ). The plates were then
further incubated for another 72 h, and at the end of this period, a CellTiter 96 Aqueous
non-radioactive cell proliferation assay (Promega) was used to assess cell viability. This
method relies on the property of SRB, which binds stoichiometrically to proteins under
mild acidic conditions and then can be extracted using basic conditions; thus, the
amount of bound dye can be used as a proxy for cell mass, which can then be
extrapolated to measure cell proliferation.
The antimicrobial activities of glidopeptin A and rhizomide A were evaluated
using Kirby-Bauer disk diffusion method (12). The tested microorganisms included
Gram-positive bacteria Staphylococcus aureus ATCC 29213 (Sa) and Bacillus subtilis
ATCC 6633 (Bc), Gram-negative bacteria Escherichia coli ATCC 35218 (E. coli) and
Pseudomonas aeruginosa ATCC 27853 (PAOI). They were obtained from the China
General Microbiological Culture Collection Center, CGMCC. The overnight culture of
different micro-organisms was mixed with the warm Muller Hinton agar medium to
make sure each plate contains 4*107 CFU of different micro-organisms respectivly.
Sterile filter paper disks (6 mm diameter) were impregnated with 5 μL of the tested
compound in methanol that was prepared in four different concentrations (500 μM, 50
μM, 5 μM, 500 nM) and applied on the inoculated plates. The plates were incubated at
37 ℃ for 24 h. The control disks impregnated with methanol were used to determine
the solvent activity.
11
Supplementary Results
The optimal work conditions of Redγ-Redαβ7029 in DSM 7029 strain
Cultivation of DSM 7029 cells at 30 ℃ for 14 h to OD600 reaching 2.0, addition
of inducer rhamnose and continued cultivation at 30 ℃ for 1.5 h, and competent cell
preparation using ddH2O at room temperature (~20 ℃).
Structural elucidation of glidopeptin A (1)
Glidopeptin A (1) was isolated as a colorless oil. Its molecular formula was
established as C60H102N16O19 by the HRESIMS ion at m/z 676.3833 [M+2H]2+
(calculated for 676.3826). Analysis of the 1D and 2D NMR data established the
structures of seven proteinogenic amino acid residues including single Glu, Lys, Leu,
Asn, Gly and two Ser, and five nonproteinogenic amino acid residues, two Dab, two
2,3-dehydrobutyric acids (Dhb) and one α,β-dehydrovaline (Dhv) (Table S6-S7). The
remaining hydrogen and carbon signals were assigned as decanoic acid based on a
series of contiguous COSY and HMBC correlations (Fig. S15). Starting from the fatty
acid residue, HMBC correlations from a Ser (Ser1) H-2 to the fatty acid C-1, Glu H-2
to Ser C-1, Dab (Dab1) H-2 to Glu C-1, and Lys H-2 to Dab C-1 established a partial
sequence of (N-Acyl)-Ser1-Glu-Dab-Lys (Fig. S15). HMBC correlations from a Leu
H-2 to Dhb (Dhb1) C-1, Dab (Dab2) H-2 to Leu C-1, and Ser (Ser2) H-2 to Dab
(Dab2) C-1 established a partial sequence of Dhb-Leu-Dab-Ser. HMBC correlations
from a Asn H-2 to Dhb (Dhb2) C-1, Gly H-2 to Asn C-1, and Dhv H-4 to Gly C-1
established a partial sequence of Dhb-Asn-Gly-Dhv. The three parts were connected
to the complete structure by HMBC correlations from Dhb1 NH to Lys C-1 and from
Dhb2 NH to Ser2 C-1 (Observed in DMSO-d6 solvent, Table S7 and Fig. S22-S27).
Configurations of the amino acid residues was assigned as D-Ser, L-Glu, D-Dab, L-
Lys, L-Leu, D-Dab, L-Ser, L-Asn using Marfey’s method combined with the presence
of dual condensation/epimerization domains (Fig. 5 and Fig. S13).
Structural elucidation of rhizomides A-C (2-4)
Compounds 2-4 were purified as white amorphous powders from methanol extracts
by preparative reverse-phase HPLC. The molecular formula of 2 was determined to be
C35H53N7O10 according to the protonated HRESIMS peak at m/z 732.3930 [M+H]+
(calculated for 732.3927). The chemical shifts of 1D NMR spectra indicated the
presence of 7 amino acids, which was further confirmed by the HMBC correlations
(Table S8, Fig. S15). HMBC correlations between amide protons and adjacent carbonyl
groups defined the order of the 7 amino acids. Intramolecular cyclization through the
Thr side chain is supported by an HMBC correlation from Thr H-3 (δH 5.19) to Val C-
1 (δC 169.4). An HMBC correlation between the Leu amide proton (δH 8.33) and the
carbonyl (δC‑1 170.0) confirms attachment of acetyl group to the N-terminus of the Leu.
The absolute configurations of amino acids were also confirmed by Marfey’s method
and bioinformatics analysis of C domains (Fig. 6c, Fig. S14). Minor products 3 and 4
differs from 2 by an Oxygen atom, based on the same MS-predicted formula (m/z
[M+H]+ calculated for C35H53N7O11, observed, 748.3873; expected 748.3876). The 1H
and 13C NMR spectra of 3 (Table S9) were very similar to those of compound 2, with
the exception of the presence of an extra hydroxy group and absence of a methyl group.
12
Further analysis of the 2D NMR correlations of 3 indicated that a Ser instead of the
third alanine (Fig. S15). Carefully compared these differences between 3 and 4
suggested that the position of Ser in compound 4 was changed which was determined
by 2D NMR analysis (Fig. S15). The HMBC correlations from Ser H-2 to Ala1 C-1
and from Ala2 NH to Ser C-1 together with the COSY correlations between Ser H-2
and Ser H-3 located the Ser between the two Ala. Therefore, the third and second Ala
in compound 2 was instead of Ser in compound 3 and 4, respectively, they should be
derivatives of 2, which was also consist with its absolute configurations determination.
The Marfey’s analysis of compound 3 and 4 demonstrated the presence of D- and L-
Ser in 3 and 4 (Fig. S14), respectively, supporting the proposed structures.
13
Supplementary Tables
Table S1 Selected recombinase pairs in Burkholderiales species
Strains locus tag Size
(aa)
protein id identity annotation
Burkholderiales
strain DSM 7029
AAW51_RS103
65
220 WP_0471945
57.1
48/201 (24%)
identical to Redα
exonuclease
AAW51_RS103
70
291 WP_0530134
64.1
53/172 (31%)
identical to Redβ
phage recombination
protein Bet
Burkholderia
ubonensis MSMB1802WG
S
WJ91_RS11250 226 WP_0599901
48.1
57/199 (29%)
identical to Redα
YqaJ-like viral
recombinase
WJ91_RS11255 289 WP_0885061
37.1
43/157 (27%)
identical to Rec T
recombinase RecT
Burkholderia
multivorans
MSMB612WGS
WL98_RS24830 217 WP_0601462
15.1
63/213 (30%)
identical to Redα
exonuclease
WL98_RS24835 288 WP_0601462
75.1
4/7 (57%)
identical to Redβ
single-stranded DNA-
binding protein
Paraburkholderi
a dilworthii
WSM3556
F759_RS010159
5
215 WP_0277982
89.1
60/206 (29%)
identical to Redα
exonuclease
F759_RS010160
0
142 WP_0277982
90.1
no single-stranded DNA-
binding protein
Burkholderia
pseudomultivora
ns
MSMB368WGS
WT56_RS13745 217 WP_0602418
28.1
61/213 (29%)
identical to Redα
exonuclease
WT56_RS13750 288 WP_0602421
98.1
4/7 (57%)
identical to Redβ
single-stranded DNA-
binding protein
Burkholderia
territorii
MSMB1918WG
S
WT41_RS28880 217 WP_0601766
23.1
62/209 (30%)
identical to Redα
exonuclease
WT41_RS28885 288 WP_0601766
24.1
no single-stranded DNA-
binding protein
Burkholderia sp.
KK1
A9R05_RS0745
5
210 WP_0771569
80.1
48/192 (25%)
identical to Redα
exonuclease
A9R05_RS0746
0
334 WP_0771569
81.1
6/21(29%)
identical to Redβ
DNA recombinase
Burkholderia sp.
BDU8
WS71_RS13965 321 WP_0664897
06.1
53/184 (28.8 %)
identical to RecE
hypothetical protein
WS71_RS13960 320 WP_0664897
03.1
77/216 (36 %)
identical to RecT
recombinase RecT
WS71_RS13955 210 WP_0664896
94.1
26/210 (13.1%))
identical to Redγ
hypothetical protein
Burkholderia
pseudomallei RNS3Bp1
AHE88_RS1969
5
271 WP_0580367
54.1
104/275(38%)
identical to RecE
exonuclease VIII
AHE88_RS1969
0
350 WP_0580367
53.1
111/237(47%)
identical to RecT
recombinase RecT
Burkholderia
multivorans 800_BMUL
1240
ADJ22_RS2587
5
270 WP_0489962
31.1
99/277(36%)
identical to RecE
exonuclease VIII
ADJ22_RS2587
0
350 WP_0489962
29.1
111/245(45%)
identical to RecT
recombinase RecT
Burkholderia
vietnamiensis G4
Bcep1808_4506 271 ABO57470.1 99/275 (36%)
identical to RecE
exonuclease VIII, 5' -> 3'
specific dsDNA
exonuclease
Bcep1808_4505 350 ABO57469.1 125/287 (46%)
identical to RecT
RecT protein
Burkholderia
multivorans
LMG 29310
UA21_RS09045 271 WP_0889301
83.1
98/276 (36%)
identical to RecE
exonuclease VIII
UA21_RS09050 350 WP_0889301
84.1
109/237 (46%)
identical to RecT
recombinase RecT
Burkholderia sp.
CCA53
BCR55_RS1641
0
270 WP_0654939
92.1
99/277 (36%)
identical to RecE
exonuclease VIII
BCR55_RS1641
5
350 WP_0654939
93.1
108/236 (46%)
identical to RecT
recombinase RecT
Burkholderia sp.
LMG 28154
BSIN_5371 272 SMG01703.1 99/275 (36%)
identical to RecE
Exodeoxyribonuclease
VIII
BSIN_5370 350 SMG01702.1 111/244 (45%) Recombinational DNA
14
identical to RecT repair protein RecT
Burkholderia sp.
TSV86
WS68_RS21005 273 WP_0595725
95.1
98/273 (36%)
identical to RecE
exonuclease VIII
WS68_RS20995 350 WP_0595725
92.1
112/245 (46%)
identical to RecT
recombinase RecT
Burkholderia
singularis LMG
28154
BSIN_RS17770 271 WP_0893414
65.1
99/275 (36%)
identical to RecE
exonuclease VIII
BSIN_RS17765 350 WP_0893414
64.1
111/244 (45%)
identical to RecT
recombinase RecT
Burkholderia sp.
YI23
BYI23_RS1728
0
279 WP_0417316
15.1
100/284 (35%)
identical to RecE
exonuclease VIII
BYI23_RS1727
0
349 WP_0142506
99.1
108/236 (46%)
identical to RecT
recombinase RecT
15
Table S2 Strains, plasmids and mutants in this work
strains Description Source
E. coli GB2005 (HS996, ∆recET, ∆ybcC). The endogenous recET locus and the DLP12
prophage ybcC, which encodes a putative exonuclease similar to the
Redα, were deleted
(13, 14)
E. coli GB05-dir (GB2005, araC-BAD-ETgA) recE, recT, redγ and recA under BAD
promoter were inserted at the ybcC locus
(1)
E. coli GB05-red (GB2005, araC-BAD-γβαA) redγβα and recA under BAD promoter
were inserted at the ybcC locus
(13)
Burkholderiales strain
DSM 7029
[Polyangium] brachysporum DSM 7029 (K481-B101; ATCC 53080) (7)
Paraburkholderia
phytofirmans PsJN
Plant Growth-Promoting Endophyte P. phytofirmans Strain PsJN,
DSM 17436
DSMZ
Paraburkholderia
rhizoxinica HKI 454
An Endosymbiont of Rhizopus microspores, DSM 19002 DSMZ
plasmid Characteristics Source
pBBR1-ccdB-hyg pBBR1 replicon. PCR templates to amplify ccdB-hyg cassette (plasmid
DNA digested with BseRI)
(5)
pBBR1-ccdB-cm pBBR1 replicon. PCR templates to amplify ccdB-cm cassette (plasmid
DNA digested with BseRI)
this work
pSC101-BAD-ccdA-
Rha-gam-tac-beta
pSC101 replicon. ccdA under the control of BAD promoter, redγ under
the control of Rha promoter and redβ under the control of tac promoter
our lab
pBBR1-tac-km pBBR1 replicon, kmR.. PCR templates to amplify tac promoter this work
pSC101-Rha-ETgA-tet pSC101 replicon, tetR, recETγA under the control of Rha promoter (15)
pSC101-BAD-gbaA-
amp
pSC101 replicon, ampR, redγβαA under the control of BAD promoter (1)
pSC101-tetR-tetO-
eGFP-km
pSC101 replicon, kmR, eGFP under the control of tetO promoter. (16)
pBBR1-tac- firefly-km pBBR1 replicon, kmR, firefly luciferase reporter genes under tac
promoter
this work
pAD123-xyl-gfp E. coli-B. subtilis shuttle plasmid, eGFP reporter gene under xyl
promoter
our lab
pBBR1-Rha-firefly pBBR1 replicon, kmR, firefly luciferase under the control of Rha
promoter
our lab
pBBR1-BAD-firefly pBBR1 replicon, kmR, firefly luciferase under BAD promoter our lab
pBBR1-tetO-firefly pBBR1 replicon, kmR, firefly luciferase under tetO promoter our lab
pBBR1-Rha-gba pBBR1 replicon, kmR, redγβα under the control of Rha promoter our lab
pBBR1-Rha-BA_7029-
km
pBBR1 replicon, kmR, redβα7029 under the control of Rha promoter this work
pBBR1-Rha-B_7029-
km
pBBR1 replicon, kmR, redβ7029 under the control of Rha promoter this work
pBBR1-Rha-RedG-
B_7029-km
pBBR1 replicon, kmR, redγ and redβ7029 under the control of Rha
promoter
this work
pBBR1-Rha-RedG-
BA_7029
pBBR1 replicon, kmR, redγ and redβα7029 under the control of Rha
promoter
this work
pBBR1-Rha-pluG-
TEpsy-km
pBBR1 replicon, kmR, pluγ and TEpsy under the control of Rha
promoter
our lab
pBBR1-Rha-pluG-
BA_7029-km
pBBR1 replicon, kmR, pluγ and redβα7029 under the control of Rha
promoter
this work
pBBR1-Rha-TEpsy-km pBBR1 replicon, kmR. TEpsy system from Pseudomonas syringae pv.
tomato DC3000 under the control of Rha promoter
this work
pBBR1-Rha-ETh_bdu-
km
pBBR1 replicon, kmR, ETh-bdu under the control of Rha promoter this work
pBBR1-Rha-BA_ 7029-
H7029-km
pBBR1 replicon, kmR, redβα7029 and H7029 under the control of Rha
promoter
this work
pBBR1-Rha-RedG-
BA_7029-H7029-km
pBBR1 replicon, kmR, redγ, redβα7029 and H7029 under the control of
Rha promoter
this work
Mutants Characteristics source
DSM 7029 Δglb the glidobactin biosynthetic gene cluster (2675183—2696188) was
replaced by an apramycin resistance gene, and the apraR was then
removed by Cre
This study
16
DSM 7029 Δ50 kb The deletion of 50 kb (3054675—3111708) region on DSM 7029
chromosome was replaced by apramycin resistance gene
This study
DSM 7029 Δ100 kb The deletion of 100 kb (3054675—3154840) region on DSM 7029
chromosome was replaced by apramycin resistance gene
This study
DSM 7029 Δ200 kb The deletion of 200 kb (3054675—3254511) region on DSM 7029
chromosome was replaced by apramycin resistance gene
This study
DSM 7029 Δglb ΔBGC
6A The region (3068779—3112764) of BGC 6A was replaced by
apramycin resistance gene in DSM 7029 Δglb
This study
DSM 7029 Δglb
ΔBGC7 The (3207610—3208985) region of BGC 7 of DSM 7029 was replaced
by apramycin resistance gene
This study
DSM 7029 Δglb ΔBGC
11 The region (4012149—4013625) of BGC 11 was replaced by
apramycin resistance gene in DSM 7029 Δglb
This study
DSM 7029 Δglb Papra-
BGC 6A
The Papra promoter and apramycin resistance gene was inserted
upstream of core biosynthetic region (3067766) of BGC 6A in DSM
7029 Δglb
This study
DSM 7029 Δglb Papra-
BGC 7
The Papra promoter and apramycin resistance gene was inserted
upstream of the core biosynthetic region (3215613) of BGC 7 in DSM
7029 Δglb
This study
DSM 7029 Δglb Papra-
BGC 11
The Papra promoter and apramycin resistance gene was inserted
upstream of the core biosynthetic region (4013625) of BGC 11 in DSM
7029 Δglb
This study
HKI 454 Δrhi The rhi gene cluster (1646382-1647728) was replaced by apramycin
resistance gene in HKI 454
This study
HKI 454 Δrhi Papra-
BGC P1
The Papra promoter and apramycin resistance gene was inserted
upstream of BGC P1 (19923-20,001) located on pBRH01 plasmid of
HKI 454Δrhi.
This study
HKI 454 Δrhi ΔBGC -
P1
Part of BGC P1 (21439-22629) of HKI 454 Δrhi was replaced by an
apramycin resistance gene.
This study
HKI 454 Δrhi Papra-
BGC P7
The Papra promoter and apramycin resistance gene was inserted
upstream of BGC P7 (528842-530137) of HKI 454Δrhi
This study
HKI 454 Δrhi ΔBGC
P7
Part of BGC P7 (530186-531534) of HKI 454 Δrhi was replaced by an
apramycin resistance gene.
This study
P. phytofirmans PsJN
ΔBGC 2
The region (90392-94838) of BGC 2 was completely replaced by an
apramycin resistance gene.
This study
P. phytofirmans PsJN
Papra- BGC 2
The Papra promoter and apramycin resistance gene was inserted
upstream of the core biosynthetic region of BGC 2 (90314) of P.
phytofirmans PsJN
This study
17
Table S3 Transcript level (fpkm) of BGC 6A, BGC 7, BGC 11, glb gene clusters and Redαβ7029 of
DSM 7029 in different conditions
Gene_id transcription level(fpkm value)
CYMG
16h
CYMG 26h CYMG 40h M9 16h M9 26h M9 40h
BGC 6A
AAW51_RS13550 (glpA) 6.28 0.93 0.77 1.05 1.39 97.27
AAW51_RS13555 (glpB) 13.36 0 0 1.57 0.26 59.95
AAW51_RS13560 (glpC) 10.30 0.64 0.17 2.41 1.54 26.21
AAW51_RS13565 (glpD) 12.32 1.07 0.47 2.59 1.54 41.40
AAW51_RS13570 (glpE) 12.47 1.92 0.54 2.50 1.79 34.99
AAW51_RS13575 (glpF) 14.19 12.51 4.54 9.34 10.52 68.40
AAW51_RS13580 (glpG) 22.02 40.47 16.85 7.95 13.42 64.11
AAW51_RS13585 (glpH) 22.15 9.89 5.19 18.80 13.08 36.38
BGC 7
AAW51_RS13870 (A) 19.75 50.48 52.1 39.96 603.90 402.33
AAW51_RS13865 (B) 19.46 31.03 32.23 30.76 353.69 226.39
AAW51_RS13860 (C) 38.95 79.96 104.61 112.61 947.69 409.82
AAW51_RS13855 (D) 15.63 12.18 29.81 27.19 333.98 296.67
AAW51_RS13850 (E) 14.52 17.27 25.94 23.42 318.36 290.65
AAW51_RS13845 (F) 12.544 11.51 11.47 13.51 198.63 170.10
AAW51_RS13840 (G) 20.30 27.27 25.28 21.48 320.19 359.79
AAW51_RS13835 (H) 20.65 37.52 21.23 14.79 288.70 403.02
AAW51_RS13830 (I) 17.78 63.88 23.89 14.36 256.41 326.18
AAW51_RS13825 (J) 23.29 68.13 23.89 44.54 219.19 253.41
AAW51_RS13825 (K) 23.48 40.87 14.47 22.09 124.84 208.99
BGC 11
AAW51_RS16830 (A) 21.36 5.99 1.98 27.66 5.78 88.90
AAW51_RS28270 (B) 8.56 2.14 0.70 6.91 2.53 62.07
AAW51_RS16815 (C) 16.38 4.43 2.16 11.05 6.37 62.34
AAW51_RS16810 (D) 18.34 8.51 4.35 14.19 6.27 94.27
glb
AAW51_RS11945(glbA) 47.31 73.36 17.74 6.95 2.21 288.97
AAW51_RS11940 (glbB) 776.84 1005.50 254.99 28.37 4.53 275.12
AAW51_RS11935(glbC) 921.75 1744.78 323.91 33.72 2.02 356.12
AAW51_RS11930(glbD) 368.71 856.62 163.72 16.96 0.60 217.04
AAW51_RS11925(glbE) 514.00 592.74 115.33 15.24 0 210.94
AAW51_RS11920(glbF) 1418.16 2830.92 454.46 43.18 3.30 367.13
AAW51_RS11915(glbG) 1356.04 2506.48 397.06 37.76 5.10 216.60
AAW51_RS11910(glbH) 926.31 1816.63 298.89 32.60 4.24 283.60
Redαβ7029
AAW51_RS10365(Redα7029) 2.93 0.47 0.52 0 0.56 25.66
AAW51_RS10370(Redβ7029) 4.44 0.35 0 0.96 1.28 34.22
18
Table S4 Sequences of promoters used in this study.
Promoter Sequnce
Papra CGCTCAGTGGAACGAGGTTCATGTGCAGCTCCATCAGCAAAAGGGGATGATAAGTTTATC
ACCACCGACTATTTGCAACAGTGCCGTTGATCGTGCTATGATCGACTGATGTCATCAGCGG
TGGAGTGCAATGTC
Prha ACTGGCCTCCTGATGTCGTCAACACGGCGAAATAGTAATCACGAGGTCAGGTTCTTACCTT
AAATTTTCGACGGAAAACCACGTAAAAAACGTCGATTTTTCAAGATACAGCGTGAATTTT
CAGGAAATGCGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAACATCATCACGTT
CATCTTTCCCTGGTTGCCAATGGCCCATTTTCCTGTCAGTAACGAGAAGGTCGCGAATTCA
GGCGCTTTTTAGACTGGTCGTAATGAACAATTCTTAAGAAGGAGATATACAT
PBAD TTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTC
GGGCTGGCCCCGGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCA
ACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGG
CTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGA
TGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATT
GCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGT
GGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTA
TCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACA
GGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATA
TTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGT
GATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACA
GCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACC
GCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCG
AGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTAT
CCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAG
AAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTT
CTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAA
AGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGA
TTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATC
CTACCTG
19
Table S5 Predicted gene function of five BGCs investigated in this study
Gene Orf Predicted Protein Function
DSM 7029 BGC 6A (glp) AAW51_RS13550 glpA 2,4-diaminobutyrate 4-aminotransferase
AAW51_RS13555 glpB MFS transporter
AAW51_RS13560 glpC NRPS(Acyl-Ser-Glu-Dab)
AAW51_RS13565 glpD NRPS(Lys-Thr-Leu-Dab)
AAW51_RS13570 glpE NRPS(Ser-Thr-Asn-Gly-Val)
AAW51_RS13575 glpF MBL fold metallo-hydrolase
AAW51_RS13580 glpG histidinol-phosphatase
AAW51_RS13585 glpH membrane protein
DSM 7029 BGC 7
AAW51_RS13870 A 2,4-diaminobutyrate 4-aminotransferase
AAW51_RS13865 B thioesterase
AAW51_RS13860 C mbtH-like_protein
AAW51_RS13855 D Dioxygenase_TauD/TfdA
AAW51_RS13850 E ACL-PCP-C
AAW51_RS13845 F taurine catabolism dioxygenase TauD
AAW51_RS13840 G NRPS (Ser-Arg-Dab)
AAW51_RS13835 H NRPS (Asp-Thr)
AAW51_RS13830 I NRPS (His-Dab)
AAW51_RS13825 J TonB-dependent siderophore receptor
AAW51_RS13820 K ABC transporter
DSM 7029 BGC 11 AAW51_RS16830 A NRPS (Acyl-Leu-Asn-Pro)
AAW51_RS28270 B NRPS (Nrp-Phe-Pro-Trp/Ser-Val/Ile-Val-Ala-Ala-Ser-Ala)
AAW51_RS16815 C ABC transporter
AAW51_RS16810 D Hypothetical Protein
HKI 454 BGC P1 (rzm) RBRH_RS12370 rzmA NRPS (Acyl-Leu-Thr-Tyr-Ala-Ala-Ala-Val)
HKI 454 BGC P7 RBRH_RS16800 A NRPS (Val-Phe-Gly-Ile/Val-Ala- Ile/Val)
20
Table S6. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 1 in MeOD-d4
No. δC δH Mult. (J in Hz)
Decanoic Acid 1 177.2, C
2 37.0, CH2 2.34 ma
3 27.0, CH2 1.62 ma
4 33.2, CH2 1.30 ma
5
6
7
8
9
10
30.8, CH2
30.7, CH2
30.6, CH2
30.6, CH2
23.9, CH2
23.6, CH3
1.30
1.30
1.30
1.30
1.51
0.95
ma
ma
ma
ma
ma
t (5.8)
Ser1 1 174.1, C
2 57.9, CH 4.30 ma
3a
3b
63.0, CH2 3.95
3.88
ma
ma
Glu 1 175.0, C
2 55.7, CH 4.22 dd (4.5, 9.4)
3a
3b
27.7, CH2 2.17
1.99
ma
ma
4
5
32.6, CH2
177.8, C
2.34 ma
Dab1 1 173.7, C
2 52.8, CH 4.41 dd (4.8, 9.3)
3 30.1, CH2 2.14 ma
4 38.1, CH2 3.06 ma
Lys 1 174.5, C
2 55.8, CH 4.35 ma
3a
3b
4
5
31.7, CH2
24.1, CH2
28.2, CH2
1.99
1.88
1.30
1.70
ma
m
ma
ma
6 40.6, CH2 2.95 ma
Dhb1 1 168.1, C
2
3
131.2, C
131.1, CH
6.46
q (7.0)
4 13.5, CH3 1.76 d (7.0)
Leu 1 175.9, C
2 54.6, CH 4.30 ma
3
4
5
6
40.9, CH2
26.2, CH
22.0, CH3
14.6, CH3
1.70
1.62
0.89
0.91
ma
ma
d (7.2)
d (7.2)
Dab2 1 173.8, C
21
2 52.8, CH 4.51 dd (5.2, 8.5)
3
4
30.4, CH2
38.1, CH2
2.26
3.06
ma
ma
Ser2 1 173.0, C
2 58.3, CH 4.35 ma
3a
3b
62.6, CH2 3.88
3.81
ma
ma
Dhb2 1 167.1, C
2
3
130.8, C
133.1, CH
6.62
q (7.1)
4 13.6, CH3 1.78 d (7.1)
Asn 1
2
3a
3b
4
173.9, C
52.1, CH
36.7, CH2
174.4, C
4.78
2.95
2.86
dd (6.5, 7.7)
ma
dd (7.7, 17.0)
Gly
1
2a
2b
171.1, C
43.8, CH2
3.97
3.94
d (16.8)
d (16.8)
Dehydrovaline
1
2
3
4
5
168.4, C
123.1, C
146.2, C
22.6, CH3
21.5, CH3
1.84
2.14
s
s
aoverlapped
22
Table S7. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 1 in DMSO-d6
No. δC δH Mult. (J in Hz)
Decanoic Acid 1 172.9, C
2 35.1, CH2 2.15 ma
3 25.2, CH2 1.47 ma
4 31.3, CH2 1.23 ma
5
6
7
8
9
10
28.9, CH2
28.8, CH2
28.7, CH2
28.7, CH2
22.1, CH2
21.7, CH3
1.23
1.23
1.23
1.23
0.85
0.87
ma
ma
ma
ma
ma
t (6.4)
Ser1 1 170.7, C
2 55.4, CH 4.44 m
3a
3b
NH
OH
61.6, CH2 3.75
3.60
8.33
5.37
ma
ma
d (7.6)
brs
Glu 1 171.9, C
2 52.7, CH 4.19 ma
3a
3b
26.6, CH2 1.47
1.33
ma
ma
4
5
NH
COOH
30.9, CH2
173.8, C
2.09
7.80a
12,33
ma
brs
Dab1 1 169.6, C
2 50.1, CH 4.29 ma
3 27.4, CH2 1.54 ma
4
NH
NH2
36.1, CH2 2.77
8.11
7.80a
ma
d (8.1)
Lys 1 170.9, C
2 52.8, CH 4.37 ma
3a
3b
4
5
29.8, CH2
22.8, CH2
26.6, CH2
1.85
1.76
0.87
1.47
ma
m
ma
ma
6
NH
NH2
38.7, CH2 2.77
8.16a
7.80a
ma
Dhb1 1 158.2, C
2 130.0, C
23
3 128.9, CH 6.41 q (7.0)
4
NH
13.0, CH3 1.61
8.84
d (7.0)
s
Leu 1 172.4, C
2 52.1, CH 4.29 ma
3
4
5
6
NH
40.2, CH2
24.2, CH
21.4, CH3
14.0, CH3
1.54
1.33
0.87
0.87
8.16a
ma
ma
ma
ma
Dab2 1 170.6, C
2 50.5, CH 4.60 dd (7.1, 14.1)
3
4
NH
NH2
27.4, CH2
36.1, CH2
1.46
2.77
7.97
7.80a
ma
ma
d (8.2)
Ser2 1 167.6, C
2 55.7, CH 4.19 ma
3
NH
OH
61.6, CH2 3.60
7.95
5.04
ma
d (7.2)
brs
Dhb2 1 157.9, C
2
3
127.6, C
130.3, CH
6.29
q (7.1)
4
NH
13.1, CH3 1.64
7.26
d (7.1)
s
Asn 1
2
3
4
NH
NH2
170.7, C
49.9, CH
31.3, CH2
170.8, C
4.19
2.77
8.21
9.30, 9.16
ma
ma
d (7.4)
s
Gly
1
2
NH
164.8, C
42.1, CH2
3.75
8.05
ma
t (5.9)
Dehydrovaline
1
2
3
4
5
NH
164.1, C
122.6, C
140.5, C
21.7, CH3
20.6, CH3
1.71
2.00
6.79
s
s
s
aoverlapped
24
Table S8. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 2 in DMSO-d6
No. δC δH Mult. (J in Hz)
HAc 1 170.0, C
2 22.3, CH3 1.86 s
Leu 1 172.7, C
2 50.9, CH 4.48 m
3a
3b
39.5, CH2 1.53
1.42
m
m
4 24.0, CH 1.61 m
5 23.1, CH3 0.89 d (6.5)
6 21.2, CH3 0.83 d (6.5)
NH 8.33 d (8.1)
Thr 1 168.1, C
2 55.2, CH 4.39 dd (2.3, 8.9)
3 70.1, CH 5.20 dq (2.3, 6.5)
4 15.4, CH3 1.00 d (6.5)
NH 7.76 d (8.9)
Tyr 1 170.8, C
2 54.6, CH 4.38 ddd (4.8, 8.0, 9.0)
3a
3b
36.0, CH2 2.98
2.83
dd (4.8, 14.4),
dd (9.0, 14.4)
4 127.0, C
5/9 130.0, CH 7.02 d (8.4)
6/8 115.0, CH 6.63 d (8.4)
7 155.9, C
NH 7.30 d (8.0)
OH 8.26 s
Ala1 1 172.5, C
2 49.0, CH 4.02 dq (4.8, 6.9)
3 17.1, CH3 1.29 d (6.9)
NH 8.17 d (4.8)
Ala2 1 171.8, C
2 48.6, CH 4.29 m
3 16.2, CH3 1.22 d (7.6)
NH 8.60 d (7.2)
Ala3 1 171.6, C
2 49.4, CH 4.17 m
3 17.5, CH3 1.23 d (7.6)
NH 7.35 d (9.0)
Val 1 169.4, C
2 57.5, CH 4.19 dd (7.4, 14.5)
3 30.3, CH 1.85 m
4 18.8, CH3 0.78 d (6.7)
5 17.9, CH3 0.72 d (6.7)
NH 8.05 d (7.4)
25
Table S9. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 3 in DMSO-d6
No. δC δH Mult. (J in Hz)
HAc 1 170.4, C
2 22.4, CH3 1.87 s
Leu 1 173.0, C
2 51.6, CH 4.33 m
3a
3b
39.7, CH2 1.55
1.42
m
m
4 24.1, CH 1.65 m
5 23.0, CH3 0.92 d (6.5)
6 21.1, CH3 0.85 d (6.5)
NH 8.36 d (7.1)
Thr 1 168.2, C
2 55.0, CH 4.46 dd (1.8, 9.2)
3 70.3, CH 5.18 dq (1.8, 6.4)
4 15.5, CH3 0.99 d (6.4)
NH 7.81 d (9.2)
Tyr 1 171.6, C
2 54.4, CH 4.42 m
3a
3b
36.4, CH2 3.02
2.78
dd (3.6, 14.2),
dd (10.5, 14.2)
4 127.3, C
5/9 129.9, CH 7.02 d (8.2)
6/8 115.0, CH 6.62 d (8.2)
7 155.9, C
NH 7.30 d (8.1)
Ala1 1 172.2, C
2 49.4, CH 4.00 dq (3.8, 6.9)
3 16.8, CH3 1.30 d (6.9)
NH 8.43 d (3.8)
Ala2 1 171.9, C
2 48.6, CH 4.33 m
3 16.2, CH3 1.25 d (7.2)
NH 8.62 d (6.9)
Ser 1 169.8, C
2 56.6, CH 4.20 m
3a
3b
61.5, CH2 3.76
3.68
dd (5.3, 11.3)
dd (4.4, 11.3)
NH 7.99 d (7.7)
Val 1 169.6, C
2 57.0, CH 4.23 dd (6.4, 9.2)
3 29.9, CH 1.95 m
4 19.0, CH3 0.78 d (6.7)
5 17.7, CH3 0.71 d (6.7)
NH 7.28 d (9.2)
26
Table S10. The 1H (500 MHz) and 13C NMR (125 MHz) Data of 4 in DMSO-d6
No. δC δH Mult. (J in Hz)
HAc 1 170.0, C
2 22.4, CH3 1.86 s
Leu 1 172.8, C
2 51.1, CH 4.39 m
3a
3b
39.7, CH2 1.53
1.43
m
m
4 24.1, CH 1.61 m
5 23.1, CH3 0.89 d (6.7)
6 21.4, CH3 0.83 d (6.7)
NH 8.37 d (7.8)
Thr 1 168.0, C
2 55.2, CH 4.45 dd (2.3, 8.8)
3 70.2, CH 5.17 dq (2.3, 6.5)
4 15.4, CH3 1.01 d (6.5)
NH 7.81 d (8.8)
Tyr 1 171.0, C
2 54.6, CH 4.43 m
3a
3b
36.2, CH2 2.97
2.81
dd (4.7, 14.3),
dd (8.8, 14.3)
4 127.0, C
5/9 130.0, CH 7.01 d (8.5)
6/8 115.0, CH 6.62 d (8.5)
7 156.0, C
NH
OH
7.39
8.49
d (8.4)
brs
Ala1 1 172.7, C
2 49.3, CH 4.08 dq (3.8, 6.9)
3 16.9, CH3 1.27 d (6.9)
NH 8.37 d (3.8)
Ser 1 169.8, C
2 55.8, CH 4.18 m
3a 60.3, CH2 3.66 d (6.3)
NH 7.39 d (8.4)
Ala2 1 171.9, C
2 49.2, CH 4.23 m
3 17.6, CH3 1.23 d (7.2)
NH 8.62 d (7.1)
Val 1 169.5, C
2 57.6, CH 4.16 dd (7.1, 8.7)
3 30.2, CH 1.90 m
4 18.9, CH3 0.78 d (6.7)
5 17.9, CH3 0.73 d (6.7)
NH 8.14 d (7.5)
27
Table S11. The activity (%) of glidopeptin A and rhizomide A against 6 plant diseases.
Bio-No. 100 mg/L 6.25 mg/L
CDM WPM CSR CA RB CGM
Glidopeptin A 30% 0 0 0 0 0
Rhizomide A 20% 0 0 0 0 0
Cyazofamid 100% / / / / /
Azoxystrobin / 100% 100% 100% / /
SYP-Z048 / / / / 100% 100%
methyl alcohol 0 0 0 0 0 0
Pseudoperonospora cubenis (CDM), Colletotrichum lagenarium (CA), Blumeria graminis (WPM),
Puccinia sorghi Schw (CSR) Pyricularia oryzae (RB), Botrytis cinerea(CGM). 100~0 indicate the grade
of disease while 100 present disease-free and 0 marks a dire threat.
28
Table S12. Cytotoxic activity (IC50 value) of glidopeptin A and rhizomide A. rhizomide A(uM) glidopeptin A
(uM)
Paclitaxel(uM)
MGC-803 90.74 >100 5.98
MCF-7 77.33 34.47 67.04 (nM)
Huh-7 >100 >100 42.10
HepG-2 >100 >100 43.80
Hela >100 >100 1.95
SGC-7901 >100 37.73 26.49
NCI-H1975 96.13 >100 1.50
LO2 >100 >100 22.1
Human gastric cancer cell line MGC-803, Human breast cancer cell lines MCF-7, Human
hepatocellular carcinoma cell line Huh-7, Human hepatocellular carcinoma cell line HepG-2,
Human cervical carcinoma cell line Hela, Human gastric cancer cell line SGC-7901, Human lung
adenocarcinoma cell line NCI-H1975, Human hepatic cell line LO2
Table S13. Primers used in this study
primers primer sequences (5'-3') application
01 cm-ccdB-3 cttccggtagtcaataaaccggtaagcCATATGGCTAGCCATATGAATTCCTCCTGT
GTGAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGGAT
CCACAGGAACACTTAACGGCTGACA
pBBR1-
ccdB-Cm
01 cm-ccdB-5 gcttaatgaattacaacagtttttatgcaGATATCaattaatgagcgcctgatgcggtattttctccttacgcat
ctgtgcggtatttcacaGGATCCACGTACTATCAACAGGTTGAACT
02 GFP-5 tgtgtggaattgtgagcggataacaatttcacacaggaggaattcatatgACCATGATTACGCATC
ATC
pBBR1-tac-
GFP-firefly-
km 02 GFP-3 agcggatagaatggcgccgggcctttctttatgtttttggcgtcttccatATTCGAACCTCCTTTAT
TACTTGTACAGCTCGTCCATG
03 Firefly-3 cggtcacactgcttccggtagtcaataaaccggtaagcCATATGGCTAGCTTACAATTTGG
ACTTTCCGCC
03 Firefly-5 ATGGAAGACGCCAAAAACATA
04 Rha-3 tcgcccttgctggatccatgatgatgatgatgatgcgtaatcatggtcatATGTATATCTCCTTCTT
AAGAATTG
pBBR1-Rha
-firefly
04 Rha-5 ggcttccatgtcggcagaatgcttaatgaattacaacagtttttatgcaGATATCAATTAATCTTTC
TGCGAATTGAG
05 BAD-3 ctcgcccttgctggatccatgatgatgatgatgatgcgtaatcatggtCATATGAATTCCTCCTG
CTAGCCCAAAAAAAC
pBBR1-
BAD- firefly
05 BAD-5 gcttccatgtcggcagaatgcttaatgaattacaacagtttttatgcagatatcAATTAATTTATGAC
AACTTGACGGCTAC
06 Ptet-3 caacaagaattgggacaactccagtgaaaagttcttctcctttactcatATGAATtcTCTCTATCAC
TGATAGGGAG
pBBR1-tetO
-firefly
06 Ptet-5 gcttccatgtcggcagaatgcttaatgaattacaacagtttttatgcagatatcAATTAATTTAAGAC
CCACTTTCACATTT
07 Pxyl-3 tcgcccttgctggatccatgatgatgatgatgatgcgtaatcatggtcatActagTTcctCCTTTGATT
TAAGTGAACAAGT
pBBR1-xyl -
firefly
07 Pxyl-5 gcttccatgtcggcagaatgcttaatgaattacaacagtttttatgcaatttaaatCTAACTTATAGGG
GTAACACTT
09 Pm-3 gcccttgctggatccatgatgatgatgatgatgcgtaatcatggtCATGTTCATGACTCCATTA
TTATTG
09 Pm-5 gcttccatgtcggcagaatgcttaatgaattacaacagtttttatgcaGATATCTCAAGCCACTTC
CTTTTTGCA
7029BA-01A-1 tagactggtcgtaatgaacaattcttaagaaggagatagtatacATGACCAACGCCCTCACGA pBBR1-Rha-
BA_7029-
km 7029BA-01A-2 ttgagaagcacacggtcacactgcttccggtagtcaataaaccggtaagcGGATCCTCATGCTG
CCTCCTTCCGCAGTTC
29
7029BA-01B-1 attgctcaccaccaggttgatattgattcagaggtataaaacgagaggagggtatacATGACCAACG
CCCTCACGAAAC
pBBR1-Rha-
RedG-
BA_7029-
km
7029BA-01C-1 ctcttttgtatgaactaagcttaatgaaagtaaaaatggaggatatatgaGGATCCAGGAGGATAC
CATATGACCAACGCCCTCACGAAACAAGAG
pBBR1-Rha-
pluG-
BA_7029-
km
7029BA-01D-2 gatgatttggcactgttgcgcaggatggcgctcagctcggtagtcaTCATATGTACACCTCCTT
CATGCTGCCTCCTTCCGCAGTTC
pBBR1-Rha-
BA_7029-
psyG-km
H7029-1 tcgctcagttcgagaaggaactgcggaaggaggcagcatgaATGATGCCGCGCACCTTCA
TCG
pBBR1-Rha-
BA_7029-
H7029-km H7029-2 atttgagaagcacacggtcacactgcttccggtagtcaatgtaTACTCACGATTTGTTGTCGA
AGTCG
100-apra-glb-1 ccttgcagcaacgcggatgggccaggttcgacgcgaccgacatgcaagtcgcggtcgacgaagcggctga
cttgcagcgcttgacggaatacgcccgaagcACGCTCAGTGGAACGAGGTTC
DSM 7029
Δglb using
100 bp
homology 100-apra-glb-2 gcgagggtcggctttcgcgccgctgctgaaactcggcgtgtgctgcgaccagctcgagaaccggcactggc
tgaggcaaccgatgcagttcttctggtcgcgTCAGCCAATCGACTGGCGAG
80-apra-glb-1 atgggccaggttcgacgcgaccgacatgcaagtcgcggtcgacgaagcggctgacttgcagcgcttgacg
gaatacgcccgAAGCACGCTCAGTGGAACGAGGTTC
DSM 7029
Δglb using
80 bp
homology 80-apra-glb-2 gcgccgctgctgaaactcggcgtgtgctgcgaccagctcgagaaccggcactggctgaggcaaccgatgc
agttcttctggTCGCGTCAGCCAATCGACTGGCGAG
50-apra-glb-1 aagtcgcggtcgacgaagcggctgacttgcagcgcttgacggaatacgcccgAAGCACGCTCA
GTGGAACGAGGTTC
DSM 7029
Δglb using
50 bp
homology 50-apra-glb-2 gaccagctcgagaaccggcactggctgaggcaaccgatgcagttcttctggTCGCGTCAGCCAA
TCGACTGGCGAG
glb-delet -cre-
apra-1
aagtcgcggtcgacgaagcggctgacttgcagcgcttgacggaatacgcccgaagcATTACATTCC
CAACCGCGTGGCACAACAACTGGCGGGC
DSM 7029
Δglb by
SSRs-apra
cassette
glb-delet- cre-
apra-2
accagctcgagaaccggcactggctgaggcaaccgatgcagttcttctggtcgcgCATACCGTTCG
TATAGCATACATTATACGAAGTTATTCGGCTTGAACGAATTG
glb-delet-check-1 TGACAGGACAGGAATGGGCTG DSM 7029
Δglb check glb-delet-check-
2.
AGCAGCAGGGCTGCGAGGCT
C7-delet-apra- 1 gtctcgacagcgtgcccctgttgcacatgctgatcgagcggcaggcccggACGCTCAGTGGAA
CGAGGTT
DSM 7029
ΔBGC 7
C7-delet-apra -2 cagcaaatgcgtgcgcaaggtctcggcggcgggcgccggcgggcgggcgaaaTCTGTACCTCC
TTAAGTCAG
C7-delet-check-1 TCGCTCGCGACGCTGCTGCAG DSM 7029
ΔBGC 7
check C7-delet-check-2 ATGCCCAGGCCGCTCAAGGC
inside apra-1 AGTCCAAGTGGCCCATCTTC
inside apra-2 CAGGTGGCTCAAGGAGAAGA
inside apra-3 ATAGCACGATCAACGGCACTGTTGC DSM 7029
Δglb by
SSRs-apra
cassette
check
inside Cre-2 TTAGCACCGCAGGTGTAGAGAAG
100-100kb-delet-
1
aggcactgaaggctgagttgcaggcgctgaaggccttggtcgcgggcaaggtcaccgcggcggccggca
ccgcagcaccccagcccgaggccgcggtgcACGCTCAGTGGAACGAGGTTC
DSM 7029
Δ50kb using
100bp
homology 100-50kb-delet-2 taacgtgctgcacggccgatgcgatgtaccatcccgcccggcaactgtttgctgacaacaaggtgataggac
aacatggccaagaagatcgatcgtgcTCTGTACCTCCTTAAGTCAG
80-100kb-delet-1 tgcaggcgctgaaggccttggtcgcgggcaaggtcaccgcggcggccggcaccgcagcaccccagccc
gaggccgcggtgcACGCTCAGTGGAACGAGGTTC
DSM 7029
Δ50kb using
80bp
homology 80-50kb-delet-2 gcgatgtaccatcccgcccggcaactgtttgctgacaacaaggtgataggacaacatggccaagaagatcga
tcgtgcTCTGTACCTCCTTAAGTCAG
30
50-100kb-delet-1 aggtcaccgcggcggccggcaccgcagcaccccagcccgaggccgcggtgcACGCTCAGTGG
AACGAGGTTC
DSM 7029
Δ50kb using
50bp
homology 50-50kb-delet-2 aaggtcaccgcggcggccggcaccgcagcaccccagcccgaggccgcggtgcACGCTCAGTG
GAACGAGGTTC
100kb-delet-
check-1
TCGATGAGAGCCGCCAGAGAG DSM 7029
Δ50kb check
50kb-delet-check-
2
ACCCGCTCGGCGAACTGACGC
100-100kb-delet-
1
aggcactgaaggctgagttgcaggcgctgaaggccttggtcgcgggcaaggtcaccgcggcggccggca
ccgcagcaccccagcccgaggccgcggtgcACGCTCAGTGGAACGAGGTTC
DSM 7029
Δ100kb
using 100 bp
homology 100-100kb-delet-
2
acgaggaagatggcccacagcaacctgtcgccgccgacgttcttggtcgtcaacttgtaggcgctcatcgcg
atgatgccgatcaccgcggcgcccacgcTCAGCCAATCGACTGGCGAGC
80-100kb-delet-1 tgcaggcgctgaaggccttggtcgcgggcaaggtcaccgcggcggccggcaccgcagcaccccagccc
gaggccgcggtgcACGCTCAGTGGAACGAGGTTC
DSM 7029
Δ100kb
using 80 bp
homology 80-100kb-delet-2 aacctgtcgccgccgacgttcttggtcgtcaacttgtaggcgctcatcgcgatgatgccgatcaccgcggcgc
ccacgctCAGCCAATCGACTGGCGAGC
50-100kb-delet-1 aggtcaccgcggcggccggcaccgcagcaccccagcccgaggccgcggtgcACGCTCAGTGG
AACGAGGTTC
DSM 7029
Δ100kb
using 50 bp
homology
50-100kb-delet-2 tcaacttgtaggcgctcatcgcgatgatgccgatcaccgcggcgcccacgctCAGCCAATCGACT
GGCGAG
100kb-delet-
check-1
TCGATGAGAGCCGCCAGAGAG DSM 7029
Δ100kb
check 100kb-delet-
check-2
AGTGTGTGCGGTGTGGAGCG
100-200kb-delet-
2
agtgccgtcagccggtgcccctgcagggcctcgtccagcagcgcggcgaaatcgtggtggtggtcgcgca
ccggcaggcgaaagcgcaccgcggacggtcAGCCAATCGACTGGCGAGC
DSM 7029
Δ200kb
using 100
bp, 80 bp
and 50 bp
homology
80-200kb-delet-2 tgcagggcctcgtccagcagcgcggcgaaatcgtggtggtggtcgcgcaccggcaggcgaaagcgcacc
gcggacggtcAGCCAATCGACTGGCGAGC
50-200k-delet-2 aaatcgtggtggtggtcgcgcaccggcaggcgaaagcgcaccgcggacggtCAGCCAATCGAC
TGGCGAGC
200kb-delet-
check-1
TCAGAGACAGACAGGACCGAC DSM 7029
Δ200kb
check
200kb-delet-
check-2
TTGCGCGGCCCGGGGCGTTG
6A-delet-1 acggtgtgcatttcgcgccttatccctatcgctaccgctgtcccttcggcaccgacggcagcgccaccgaccg
cctgtcgatcgactacctgcgcaaccTGGAAGGCACGAACCCAGTTGAC
DSM 7029
ΔBGC 6A
6A-delet-2 gaaggtgatgctttcgtactcgccgagctcgatcacccgctcgaagccgcaatgctgcagcaagtgcttcaac
gagatgtcctgcaggctgccgccggcgTTAGGTGGCGGTACTTGGG
B-mutant-check 1 GGCCACTTCGATGATGACACCACT DSM 7029
ΔBGC 6A
check B-mutant-check 4 AGGTAGATGTCCATCGGGTACTCT
Papra-ABCDE
check 3
GGCAATGGATCAGAGATGATCT DSM 7029
Papra-BGC
6A check Papra-ABCDE
check 2
GGCAGAGCAGATCATCTCTGAT
C11-delet-apra-1 tcgtcaaataagtattgcctggcagcaccccattcgctatatctccggcgcattttgttgccattaatgaACGC
TCAGTGGAACGAGGTT
DSM 7029
ΔBGC 11
C11-delet-apra-2 acatcgaacaagcgtgagcggcggtcctgccccagccccaactgccgcttgatctcggcgaccgggcaac
gttggtggcggtagcagcgTCTGTACCTCCTTAAGTCAG
C11-delet-check-
1
TGAGTACAGATCCGGTCAGC DSM 7029
ΔBGC 11
check C11-delet-check-
2
AAGCACCGTCCTCAACACAT
6A- Papra-1 ccttgggcgtggtgccagtcgaccccttggccgccttccccgaaagccaaaacgctcacggctttcatcaagc
tttcacgCTCAGTGGAACGAGGTT
DSM 7029
PApra-BGC
6A 6A-Papra-2 atggtgatgaccgagcggcaacgcgcccgcacaagacaaacagtcgatgatgtcccgcccggcctggtcg
cgaatcatAATCTGTACCTCCTTAAGTC
Papra-ABCDE
check 1
CGACGTGTATCAGGCAGTAGAT DSM 7029
PApra-BGC
6A check Papra-ABCDE
check 4
CGGCTTCATTGCGCATGA
C7-Papra-1 atgtacatgcgatgaaacggggcgaggcaccacagccgcgccatgggcatcacgaccagggagatcgatc
atcgtcgcACGCTCAGTGGAACGAGGTT
DSM 7029
PApra-BGC 7
31
C7-Papra-2 tcgtgcagctgctctgcgacgagcgcttgcaccgcctgccacgccggcgcgccgggtgcagggggcggc
acgagcacAATCTGTACCTCCTTAAGTCAG
C11-Papra-1 tcgtcaaataagtattgcctggcagcaccccattcgctatatctccggcgcattttgttgccattaatgaACGC
TCAGTGGAACGAGGTT
DSM 7029
PApra-BGC
11 C11-Papra-2 cgcgacgggcgcttctcggcatccgccgattcgactgcgtgggtgtacataaTCTGTACCTCCTT
AAGTCAG
C11-delet-check-
1
TGAGTACAGATCCGGTCAGC DSM 7029
PApra-BGC
11 check C11-Papra-check-2 ATTTCCAGCGAGCGTTCC
C7-Papra-check-1 TGGGTCCTTTCTTGACAGAG DSM 7029
PApra-BGC 7
check C7-Papra-check-2 CTTGCAGCTGGGATTGAGCT
HKI 454-Δrhi-
apra-1
caccacaaaggccacgagtttaggatctgctgaaccaggatccttaaccaAATCTGTACCTCCTT
AAGTCAG
HKI 454
Δrhi
HKI 454-Δrhi-
apra-2
ccactggacaagtgcttacagcagttgatcgaagagcaggttgagcgcagaCGCTCAGTGGAAC
GAGGTT
HKI 454-Δrhi -
check-1
GCGCCTTAATGAAATCCCGT HKI 454
Δrhi check
HKI 454-Δrhi -
check-2
AGACACGGACTCATAGCCAG
HKI 454- Δrhi
ΔC1-apra-1
ttgagcgcacgcccaaggctacggcgttggtctatgaagatcaaacactgAATCTGTACCTCCTT
AAGTC
HKI 454
Δrhi ΔBGC
P1 HKI 454- Δrhi
ΔC1-apra-2
ttggccaccacatacgctaccagacgtttatcctggctttcacctgttgcACGCTCAGTGGAACG
AGGTT
HKI 454- Δrhi-
C1-Papra-1
gcaagcgacgtgtcatcaaagccgtagtggttcctgaagagcaacgatagACGCTCAGTGGAA
CGAGGT
HKI 454
Δrhi PApra-
BGC P1 HKI 454- Δrhi-
C1-Papra-2
gtttgggcagcggataatgcatacgtagtggacatgacgctagcgtccatAATCTGTACCTCCTT
AAGTC
HKI 454- Δrhi
ΔC1-check-1
GCACATCAGCGTCGGTTTAT HKI 454
Δrhi ΔBGC
P1 check HKI 454- Δrhi
ΔC1-check-2
CGTTCGACACCCAATAGCTC
HKI 454- Δrhi-
C1-Papra-check-1
CTGGCTTGCCCTGTTCTTTT HKI 454
Δrhi PApra-
BGC P1
check
HKI 454- Δrhi-
C1-Papra-check-2
AGATTGACCGGCTGCTGATA
HKI 454-Δrhi
ΔC7-apra-1
gatttattcgatttcaagggcgtgagtccacctatcccctcacgcgtcacAATCTGTACCTCCTTA
AGTCA
HKI 454
Δrhi ΔBGC
P7 HKI 454-Δrhi
ΔC7-apra-2
atggaacgcgacgcaacaggactatccggcgcaccaatgtattcaccagcAATCTGTACCTCCT
TAAGTC
HKI 454-Δrhi -
C7-Papra-1
gatttattcgatttcaagggcgtgagtccacctatcccctcacgcgtcacAATCTGTACCTCCTTA
AGTCA
HKI 454
Δrhi PApra-
BGC P7 HKI 454-Δrhi-
C7-Papra-2
gcaggccgcacaaacacggcctttgcgatatccacaccaactgctgacgtACGCTCAGTGGAA
CGAGGT
HKI 454-Δrhi
ΔC7-check-1
TTGCCGTGCCAATAACCAAA HKI 454
Δrhi ΔBGC
P7 check HKI 454-Δrhi
ΔC7-check-2
AGGATTTGATGCTTGCGGTC
HKI 454-Δrhi-
C7-Papra-check-1
ACCGGCTCTTCATAATCGGT HKI 454
Δrhi PApra-
BGC P7
check
HKI 454-Δrhi-
C7-Papra-check-2
CGGAAGTTCGCCTATGTTGG
17436-ΔC2-apra-
1
gcaccaccagatccacgcgatggcgcgcatcgatcccgatgcgcccgcgctagcgtcttttacgccacatac
cgtGGCTCAGTGGAACGAGGTTCA
PsJNΔBGC
2
17436-ΔC2-apra-
2
agcgggcgtcgctcgccaaggcgacgaacgaggccagacgcaagcgcccatcgtgctctacggccagcg
tttcggcccagTCAGCCAATCGACTGGCGAG
17436-C2-Papra-1 agcgcgcgcggtaaagatttgcgattgcggatcgtcacaccaatgaagcggcccattgcggcctgcgacttg
gcctatgcggccagttctaacggatttcatgcccCGCTCAGTGGAACGAGGTTC
PsJN PApra-
BGC2
17436-C2-Papra-2 aagacgctagcgcgggcgcatcgggatcgatgcgcgccatcgcgtggatctggtggtgcaacgcaatcgg
gaagctcgtcataatctgtacctccttaagtcagTCAGCCAATCGACTGG
17436-BGC2KO-
check-1
ATGCGCCCGCGCTAGCGTCT PsJN ΔBGC
2 check
17436-BGC2KO-
check-2
CAAGGCGACGAACGAGGCC
17436-C2-papra- TCATCTGCCACGTCAAAAAGATG PsJN PApra-
32
The homology arms are in lower case.
check1 BGC2 check
17436-C2-papra-
check2
CTTCACCGTGCGCGGCAAGC
33
Supplementary Figures
Fig. S1. Screening of effective and stringent inducible promoters in DSM 7029
a. Map of the inducible promoters contained pBBR1 plamsids for screening in DSM 7029
b.The strength (RLU) of different promoters with all inducers in DSM 7029. Tet: tetracycline-regulated
promoter, BAD: arabinose-regulated promoter, tac: IPTG-regulated promoter, Rha: rhamnose-regulated
promoter. ara: arabinose inducer, rha: rhamnose inducer, AHT: anhydrotetracycline inducer, IPTG:
isopropyl-β-d-thiogalactoside. Error bars, SD. n = 3.
34
Fig. S2. The recombination efficiency of the LCHR assay mediated by different combinations of Redγ,
Redαβ7029 and Redαβ, in E. coli and DSM 7029, respectively.
a. Diagram of plasmid modification (linear plus circlar homologous recombination, LCHR) in E. coli
and DSM 7029. PCR products carrying the apramycin resistance gene (Apra) flanked by 50-bp homology
arms (red) was integrated into the expression plasmid in place of recombinase genes.
b. Diagram of genome modification of DSM 7029. The 21.2 kb of glidobactin biosynthetic genes (glbB-
glbG) on chromosome was replaced by an apramycin resistance gene (Apra) flanked by 80 bp homology
arms (red) in DSM 7029, p1: primer glb-delete-check-1, p2: primer glb-delete-check-2.
c. Results of glidobactin biosynthetic gene cluster replacement assay in DSM 7029. BA_7029:
Redαβ7029, RedG_BA_7029: Redγ from E. coli combined with Redαβ7029, RedG_BA_7029_H7029:
Redγ from E. coli combined with Redαβ7029 and H7029. BA_7029_H7029: Redαβ7029 and H7029 .
Colonies were selected on apramycin plates and counted. Error bars, SD. n = 3.
d. Recombination efficiency comparison of genome modification (Fig. S2b) on chromosome using
apramycin resistance gene (Apra) flanked by various length homology arms (50 bp, 80 bp, 100 bp) in
DSM 7029 wild type and DSM 7029 carrying different recombinases. RedG: Redγ of E. coli, RedGBA-
E. coli: Redγ/Redα/Redβ of E. coli. Error bars, SD. n = 3.
e. The map of the expression plasmid that carries the optimal recombinase combination Redγ-Redαβ7029
under the control of Rha promoter for genetic engineering in DSM 7029.
f. PCR verification of the deletion of glidobactin biosynthetic gene cluster. p1: glb-delet-check 1, p2:
glb-delet-check 2, p3: inside apra-1, p4: inside apra-2, Left: size of PCR product: 545 bp (mutants, p2/p4),
Right: size of PCR product: 427 bp (mutants, p1/p3), M: DL5000 DNA ladder, ck: DSM 7029 WT.
35
Fig. S3. The recombination efficiency of the LCHR and LLHR mediated by Redγ-Redαβ7029 in E. coli.
a. Diagram of the LCHR (linear-circle homologous recombination) assay mediated by Redγ-Redαβ7029
in E. coli. Homologous arms (red), Apra: apramycin resistance gene, cm: chloramphenicol resistance
gene, Km: kanamycin resistance gene. RK2: origin of replication.
b. Diagram of the LLHR assay mediated by Redγ-Redαβ7029 in E. coli.
c. Results from LCHR and LLHR assays in E. coli mediated by Redγ-Redαβ7029. Colonies were
selected on LB plate (kanamycin 15 μg /mL) and counted. Error bars, SD; n = 3.
36
Fig. S4. Optimization of the work conditions of Redγ-Redαβ7029 in DSM 7029 for genome modification.
a. Recombination efficiency comparison of genome modification (Fig. S2b) in DSM 7029 on
chromosome mediated by Redγ-Redαβ7029 under different electroporation temperature conditions. RT:
room temperature electroporation conditions, OI: ice-cold temperature electroporation conditions. Error
bars, SD. n = 3.
b. Recombination efficiency comparison of the genome modification assay in DSM 7029 mediated by
Redγ-Redαβ7029. The competent cells were treated with different washing buffer. H2O: double distilled
water, S:10% sucrose, S+H: 10% sucrose+2 μM HEPES, G: 10% glycerol ,G+H: 10% glycerol+2 μM
HEPES. Error bars, SD. n = 3.
c. Recombination efficiency comparison of the genome modification assay in DSM 7029 mediated by
Redγ-Redαβ7029 under different induction temperatures (25 ℃, 30 ℃, 32 ℃, 37 ℃). Error bars, SD. n
= 3.
d. Recombination efficiency comparison of the genome modification assay in DSM 7029 mediated by
Redγ-Redαβ7029 under different growth condition. Error bars, SD. n = 3.
37
e. Recombination efficiency comparison of the genome modification assay in DSM 7029 mediated by
Redγ-Redαβ7029 under different induction time (0.5 h, 1 h, 1.5 h, 2h). Error bars, SD. n = 3.
f. PCR verification of replacement of large genome sequences (50-200 kb) in DSM 7029 under optimized
conditions. p1: 100k-delet-check 1, p2: 100k-delet-check 2, p3: 200 k-delet-check 1, p4: 200 k-delet-
check 2, p5: inside-apra 3, size of PCR product: 2104 bp (mutants, p1/p2 and p3/p4), 801 bp (mutants,
p1/p5), M: DL5000 DNA ladder, ck: DSM 7029 WT.
g. Samples are separated on a 3 % TBE gel and stained with ethidium bromide (EB) nucleic acid stain.
Lane (1) PO and (2) OP dsDNA without lambda exonuclease digestion, (3) lagging strand, dsDNA (PO)
after 2.5 h lambda exonuclease digestion, (4) leading strand, dsDNA (OP) after 2.5 h lambda exonuclease
digestion.
38
Fig. S5. Diagram for constuction, verification and metabolic analysis of the clean deletion of glidobactin
biosynthetic gene cluster in DSM 7029.
a. Diagram for construction of the clean deletion of the glidobactin biosynthetic genes glbB-glbG using
Redγ-Redαβ7029 recombinases combined with site-specific recombinase (Cre/loxP) in DSM 7029. Cre:
site-specific recombinase, lox66 and lox71: the recognition sites of Cre.
b. PCR verification of the glidobactin BGC replacement and excision of the selectable marker in DSM
7029 by Cre. 1/3/5/7: DSM 7029 Δglb-apra with an Apra marker; 2/4/6/8: DSM 7029 Δglb after the
expression of Cre to remove Apra marker; p1: primer glb-delet-check-1, p2: primer inside-cre-check-1,
p3:inside-apra-check-2, p4: glb-delet-check-2; M: DL5000 DNA ladder (TaKaRa, Kuasatsu, Shiga 525-
0058, Japan), size of PCR product: 1747 bp (mutants, p2/p4), 554 bp (mutants, p1/p4), 868 bp (mutants,
p2/p3).
c. HPLC-HRMS analysis (BPC+All MS) of DSM 7029 WT and DSM7029-Δglb showed the abolishment
of glidobactins (RT: 22-42 min) in the mutant.
39
Fig. S6. Diagram for construction, verification and metabolic analysis of BGC 6A activation and
inactivation in DSM 7029-Δglb.
a. Diagram for construction of BGC 6A activation (PApra-BGC6A) and inactivation (ΔBGC6A) in DSM
7029-Δglb using Redγ-Redαβ7029 recombinases.
b. PCR verification of the activation of BGC 6A (PApra-BGC6A). p1: Papra-ABCDE check 1, p2: Papra-
ABCDE check 2, p3: Papra-ABCDE check 4; M: DL 5000 DNA ladder, ck: DSM 7029-Δglb, size of
PCR product: 677 bp (mutants, p1/p2) and 1303 bp (mutants, p1/p3), 898 bp (ck, p1/p3) .
40
c. PCR verification of the inactivation of BGC 6A (ΔBGC6A). p2: Papra-ABCDE check 2, p4: B-mutant-
check 1, p5: Papra-ABCDE check 3, p6: B-mutant-check 4; M: DL 5000 DNA ladder, ck: DSM 7029-
Δglb, size of PCR product: 1227 bp (mutants, p2/p4) and 650 bp (mutants, p5/p6).
d. HPLC-HRMS analysis (BPC 650.00-700.00 +All MS) of DSM 7029-Δglb and its BGC6A inactivated
and activated mutants showed the activation of glidopeptins (RT: 17-22min).
e. HRMS spectra of products of glidopeptins (1: RT=19.1min, m/z=676 [M+2H]2+; RT=17.1min,
m/z=662 [M+2H]2+; RT=18.1min, m/z=662 [M+2H]2+;RT=19.9min, m/z=676 [M+2H]2+; RT=21.2min,
m/z=690 [M+2H]2+; RT=21.9min, m/z=690 [M+2H]2+).
41
Fig. S7. Diagram for construction, verification and metabolic analysis of BGC 7 activation and
inactivation in DSM 7029-Δglb.
a. Diagram for construction of BGC7 activation (PApra-BGC7) and inactivation (ΔBGC7) in DSM 7029-
Δglb using Redγ-Redαβ7029 recombinases.
b. PCR verification of the inactivation of BGC7 in DSM 7029-Δglb. p3: primer C7-delet-check-1, p4:
primer inside apra-1, p5: primer inside apra-2, p6: primer C7-delet-check-2, M: DL5000 DNA ladder,
ck: DSM 7029-Δglb. size of PCR product: 493 bp (mutants, p3/p5), 416 bp (mutants, p4/p6).
c. PCR verification of the activation of BGC7 in DSM 7029-Δglb. p1: primer C7-Papra-check-1, p2:
primer C7-Papra-check-2, M: DL5000 DNA ladder, ck: DSM 7029-Δglb. size of PCR product: 700 bp (ck,
p1/p2), 1308 bp (mutants, p1/p2).
d. HPLC-HRMS analysis (BPC 450.00-500.00 +All MS) of DSM7029-Δglb and its BGC7 inactivated
and activated mutants showed the products of BGC7 (RT: 14-20min).
e. HRMS and HRMS/MS of a product of BGC7 (RT=13.7min, m/z=478 [M+2H]2+, 955[M+H]+).
42
Fig. S8. Diagram for construction, verification and metabolic analysis of BGC 11 activation and
inactivation in DSM 7029-Δglb.
a. Diagram for construction of BGC11 activation (PApra-BGC11) and inactivation (ΔBGC11) in DSM
7029-Δglb using Redγ-Redαβ7029 recombinases.
b. PCR verification of the activation of BGC 11 in DSM 7029-Δglb. p1: C11-delet-check-1, p2: C11-
Papra-check-2, p3: inside apra-1, p4: inside apra-2; M: DL5000 DNA ladder, ck: DSM 7029-Δglb. size of
PCR product: 486 bp (mutants, p1/p4), and 416 bp (mutants, p2/p3).
c. PCR verification of the inactivation of cluster 11 in DSM 7029-Δglb. p1: C11-delet-check-1, p3: inside
apra-1, p4: inside apra-2, p5: C11-delet-check-2; M: DL5000 DNA ladder, ck:DSM 7029-Δglb, size of
PCR product: 486 bp (mutants, p1/p4), and 1465 bp (mutants, p3/p5).
d. HPLC-HRMS analysis (BPC 750.00-800.00+All MS) of DSM7029-Δglb and its BGC11 inactivated
and activated mutants showed the activated products (RT: 31-36min).
e. HRMS spectra of products of BGC11 (RT=32.5min, m/z=762 [M+2H]2+; RT=33.8min, m/z=785
[M+2H]2+; RT=34.8min, m/z=776 [M+2H]2+).
43
Fig. S9. Diagram for construction, verification and metabolic analysis of the deletion of rhizoxin
biosynthetic gene cluster in P. rhizoxinica HKI 454
a. Diagram for construction of the inactivation of the rhizoxin biosynthetic gene cluster via apramycin
gene in lieu of A domain of gene rhiB in HKI 454 using Redγ-Redαβ7029 recombinases
b. PCR verification of recombinants of inactivation of the rhizoxin biosynthetic gene cluster. p1: primer
HKI 454-ΔRhi-check-1, p2: HKI 454-ΔRhi-check-2; M: DL 5000 DNA ladder, ck: HKI 454 WT, size of
PCR product: 1620 bp (ck, p1/p2), 1304 bp (mutants, p1/p2).
c. HPLC-HRMS analysis (BPC+All MS) of HKI 454 WT and HKI 454-Δrhi showed the abolishemnt of
rhizoxins (RT: 28-42 min) in the mutant.
44
Fig. S10. Diagram for construction, verification and metabolic analysis of BGC P1 activation and
inactivation in P. rhizoxinica HKI454-Δrhi.
a. Diagram for construction of BGC P1 activation (PApra-BGC P1) and inactivation (ΔBGC P1) in
HKI454-Δrhi using Redγ-Redαβ7029 recombinases.
b. PCR verification of the activation and inactivation of BGC P1 in HKI454-Δrhi. Left: inactivation of
BGC P1 of HKI 454, p3: primer HKI 454-ΔrhiΔC1-check-1, p4: primer HKI 454-ΔrhiΔC1-check-2,
size of PCR product: 1712 bp (ck, p3/p4), 1452 bp (mutants, p3/p4); Right: PCR verification of
constructive promoter insertion up stream of BGC P1 of HKI 454, p1: primer HKI 454-Δrhi-C1-Papra-
check-1, p2: primer HKI 454-Δrhi-C1-Papra-check-2, size of PCR product: 678 bp (ck, p1/p2), 1530 bp
(mutants, p1/p2); M: DL5000 DNA ladder, ck: HKI 454-Δrhi.
c. HPLC-HRMS analysis (BPC 700.00-800.00+All MS) of HKI454-Δrhi and its BGC P1 inactivated and
45
activated mutants showed their improved products rhizomides (RT: 22-26min).
d. HRMS spectra of three products of rhizomides A-C (2-4) (2: RT=24.4min, m/z=732 [M+H]+; 3:
RT=24.1min, m/z=748 [M+H]+; 4: RT=23.2min, m/z=748 [M+H]+).
46
Fig. S11. Diagram for construction, verification and metabolic analysis of BGC P7 activation and
inactivation in P. rhizoxinica HKI 454-Δrhi.
a. Diagram for construction of BGC P1 activation (PApra-BGC P7) and inactivation (ΔBGC P7) in
HKI454-Δrhi using Redγ-Redαβ7029 recombinases.
b. PCR verification of the activation and inactivation of BGC P7 in P. rhizoxinica HKI 454-Δrhi. Left:
inactivation of BGC P7 of HKI 454-Δrhi, p3: primer HKI 454-ΔrhiΔC7-check-1, p4: primer HKI 454-
ΔrhiΔC7-check-2, size of PCR product: 2862 bp (ck, p3/p4), 2499 bp (mutants, p3/p4); Right: PCR
verification of constructive promoter insertion up stream of BGC P7 of HKI 454-Δrhi, p1: primer HKI
454-Δrhi-C7-Papra-check-1, p2: primer HKI 454-Δrhi-C7-Papra-check-2, size of PCR product: 1052 bp
(ck, p1/p2), 1629 bp (mutants, p1/p2); M: DL5000 DNA ladder, ck: HKI454-Δrhi.
c. HPLC-HRMS analysis (BPC 750.00-850.00+All MS) of HKI454-Δrhi and its BGC P7 inactivated and
activated mutants showed their improved products (RT: 31-37 min).
d. HRMS spectra of products of BGC P7 (RT=31.9min, m/z=833 [M+H]+; RT=32.2min, m/z=789
[M+H]+; RT=34.7min, m/z=783 [M+H]+ ; RT=35.6min, m/z=817 [M+H]+).
47
Fig. S12. Diagram for construction, verification of BGC2 activation and inactivation in P. phytofirmans
PsJN.
a. Diagram for construction of BGC2 activation (PApra-BGC2) and inactivation (ΔBGC2) in PsJN using
Redγ-Redαβ7029 recombinases.
b. PCR verification of the activation and inactivation of BGC 2 in PsJN. Left: inactivation of BGC2 in
P. phytofirmans PsJN, p3: 17436-BGC2KO-check-1, p4: 17436-BGC2KO-check-2 size of PCR product:
1020 bp. Right: activation of BGC2 in P. phytofirmans PsJN, p1: 17436-BGC2-Papra-check-1, p2:
17436-BGC2-Papra-check-2, 610 bp (ck, p1/p2), 1629 bp (mutants, p1/p2),; M: DL5000 DNA ladder,
ck: PsJN.
48
Fig. S13. Marfey’s analysis of the amino acid constituents of glidopeptin A (1) by LC-MS. The
extracted ion [M + H]+ chromatograph of the L-FDAA-derivatized authentic standards are Glu 400.1,
Dab 371.1, Lys 399.1, Leu 384.1, Ser 358.1, Asn 385.1.
49
Fig. S14. Marfey’s analysis of the amino acid constituents of rhizomides A-C (2-4) by LC-MS with
different elution conditions (see Method). (a) Marfey’s analysis of the amino acids in rhizomide A (2);
(b) Marfey’s analysis of the amino acids in rhizomides B-C (3-4); (c) Marfey’s analysis of L-Thr and
L-allo-Thr in rhizomides A-C (2-4). The ratio of L- and D- Ala (approximately 1:2) of rhizomide A (2)
is in perfect agreement with the predicted configuration of three Ala (Fig. 5c), which indicated that the
1st and 3rd Ala are D configuration while the 2nd is L configuration. In rhizomde B (3), Marfey’s
analysis showed equal number of L- and D- Ala, and the 3rd D-Ala was instead of a D-Ser in 3. In
rhizomide C (4), Marfey’s analysis only showed presence of D-Ala, and also an L- Ser, indicating the
L-Ala (2nd) was instead of an L-Ser in 4. This analysis is consistent with NMR data (Tables S8-S10,
Fig. S15). The extracted ion [M+H]+ chromatograph of the L-FDAA-derivatized authentic standards
are Leu 384.1, Thr 372.1, Tyr 434.1, Ala 342.1, Val 370.1, Ser 358.1.
50
Fig. S15. Complete structures with carbon number (a), key COSY and HMBC correlations of compounds
1-4 (b).
51
Fig. S16. 1H NMR spectrum (500 MHz) of glidopeptin A (1) in MeOD-d4
Fig. S17. 13C NMR spectrum (125 MHz) of glidopeptin A (1) in MeOD-d4
52
Fig. S18. DEPT spectrum (125 MHz) of glidopeptin A (1) in MeOD-d4
Fig. S19. HSQC spectrum of glidopeptin A (1) in MeOD-d4
53
Fig. S20. 1H-1H COSY spectrum of glidopeptin A (1) in MeOD-d4
Fig. S21. HMBC spectrum of glidopeptin A (1) in MeOD-d4
54
Fig. S22. 1H NMR spectrum (500 MHz) of glidopeptin A (1) in DMSO-d6
Fig. S23. 13C NMR spectrum (125 MHz) of glidopeptin A (1) in DMSO-d6
55
Fig. S24. DEPT spectrum (125 MHz) of glidopeptin A (1) in DMSO-d6
Fig. S25. HSQC spectrum of glidopeptin A (1) in DMSO-d6
56
Fig. S26. 1H-1H COSY spectrum of glidopeptin A (1) in DMSO-d6
Fig. S27. HMBC spectrum of glidopeptin A (1) in DMSO-d6
57
Fig. S28. 1H NMR spectrum (125 MHz) of rhizomide A (2) in DMSO-d6
Fig. S29. 13C NMR spectrum (125 MHz) of rhizomide A (2) in DMSO-d6
58
Fig. S30. DEPT spectrum (125 MHz) of rhizomide A (2) in DMSO-d6
Fig. S31.HSQC spectrum of rhizomide A (2) in DMSO-d6
59
Fig. S32. 1H-1H COSY spectrum of rhizomide A (2) in DMSO-d6
Fig. S33. HMBC spectrum of rhizomide A (2) in DMSO-d6
60
Fig. S34. 1H NMR spectrum (500 MHz) of rhizomide B (3) in DMSO-d6
Fig. S35. 13C NMR spectrum (125 MHz) of rhizomide B (3) in DMSO-d6
61
Fig. S36. DEPT spectrum (125 MHz) of rhizomide B (3) in DMSO-d6
Fig. S37. HSQC spectrum of rhizomide B (3) in DMSO-d6
62
Fig. S38. 1H-1H COSY spectrum of rhizomide B (3) in DMSO-d6
Fig. S39. HMBC spectrum of rhizomide B (3) in DMSO-d6
63
Fig. S40. 1H NMR spectrum (125 MHz) of rhizomide C (4) in DMSO-d6
Fig. S41. 13C NMR spectrum (125 MHz) of rhizomide C (4) in DMSO-d6
64
Fig. S42. DEPT spectrum (125 MHz) of rhizomide C (4) in DMSO-d6
Fig. S43. HSQC spectrum of rhizomide C (4) in DMSO-d6
65
Fig. S44. 1H-1H COSY spectrum of rhizomide C (4) in DMSO-d6
Fig. S45. HMBC spectrum of rhizomide C (4) in DMSO-d6
66
Fig. S46. IR spectrum of glidopeptin A (1)
Fig. S47. IR spectrum of rhizomide A (2)
67
Fig. S48. IR spectrum of rhizomide B (3)
Fig. S49. IR spectrum of rhizomide C (4)
68
69
Fig. S50 Antibiotic activity Test of glidopeptin A, rhizomide A performed by Using the Kirby-Bauer
Disk Diffusion Method on Muller-Hinton Agar. 1: glidopeptin A, 2: rhizomide A, 500: 500 μM, 50: 50
μM, 5: 5 μM, 0.5: 0.5 μM, control: 5 μl Methanol. Staphylococcus aureus ATCC 29213 (Sa) and
Bacillus subtilis ATCC 6633 (Bc), Gram-negative bacteria Escherichia coli ATCC 35218 (E. coli) and
Pseudomonas aeruginosa ATCC 27853 (PAO1).
70
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