Supplementary information Discovery of recombinases enables … · 2018-04-14 · Supplementary...

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: [email protected] or

[email protected].

mailto:[email protected]

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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 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


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

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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

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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

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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

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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

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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



This study



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


BGC 7


upstream of the core biosynthetic region (3215613) of BGC 7 in DSM

7029 Δglb

This study


BGC 11


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


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


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


This study

P. phytofirmans PsJN

ΔBGC 2

The region (90392-94838) of BGC 2 was completely replaced by an


This study

P. phytofirmans PsJN

Papra- BGC 2


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



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



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



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



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


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


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


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


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)

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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