PRODUCTION OF NEW WAP-8294A CYCLODEPSIPEPTIDES BY THE BIOLOGICAL CONTROL AGENT <i>LYSOBACTER ENZYMOGENES</i> OH11

doi:10.15302/J-FASE-2021410

Front. Agr. Sci. Eng.

2022, Vol. 9

Issue (1) : 120-132 https://doi.org/10.15302/J-FASE-2021410

RESEARCH ARTICLE

PRODUCTION OF NEW WAP-8294A CYCLODEPSIPEPTIDES BY THE BIOLOGICAL CONTROL AGENT LYSOBACTER ENZYMOGENES OH11

Jing ZHU^1,², Yuan CHEN¹, Liangcheng DU¹(

)

¹. Department of Chemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, USA
². State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China

Download: PDF(2261 KB) HTML
Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks

Abstract

● Lysobacter enzymogenes mutants were generated for WAP-8294A biosynthesis.

● Essential and non-essential accessory genes for WAP-8294A biosynthesis were determined.

● Six new WAP-8294A analogs were identified using UHPLC-HR-MS/MS.

● Three deoxy analogs were detected supporting the function of ORF4 in asparagine hydroxylation.

Naturally-occurring environmental microorganisms may provide ‘green’ and effective biocontrol tools for disease management in agricultural crops. Due to the constant threat of resistant pathogens there is a pressing and continual need to search for new biocontrol tools. This study investigated the production of new analogs of WAP-8294A compounds by the biocontrol agent Lysobacter enzymogenes OH11 through biosynthetic engineering. WAP-8294As are a family of natural cyclic lipodepsipeptides with potent activity against Gram-positive bacteria. A series of genetic manipulations was therefore conducted on the accessory genes in the WAP biosynthetic gene cluster. The resulting strains containing a single-point mutation in ORF4, which was predicted to encode a 2-ketoglutarate dependent dioxygenase, produced deoxy-WAP-8294As. This result provides evidence for the function of ORF4 in catalyzing β-hydroxylation of the D-asparagine residue in WAP-8294As. In addition, six new analogs of WAP-8294As were identified by UHPLC-HR-MS/MS. This is the first attempt to produce new WAP-8294As in Lysobacter and shows that the spectrum of the biocontrol compounds may be expanded through the manipulation of biosynthetic genes.

Keywords biocontrol biosynthesis Lysobacter natural products WAP-8294A

Corresponding Author(s): Liangcheng DU

Just Accepted Date: 12 July 2021 Online First Date: 20 August 2021 Issue Date: 17 January 2022

Cite this article:

Jing ZHU,Yuan CHEN,Liangcheng DU. PRODUCTION OF NEW WAP-8294A CYCLODEPSIPEPTIDES BY THE BIOLOGICAL CONTROL AGENT LYSOBACTER ENZYMOGENES OH11[J]. Front. Agr. Sci. Eng. , 2022, 9(1): 120-132.

URL:

https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2021410
https://academic.hep.com.cn/fase/EN/Y2022/V9/I1/120

Fig.1 The biosynthetic gene cluster for WAP-8294A in Lysobacter enzymogenes. ORFs 1–3 are structural genes responsible for the biosynthesis of the peptide scaffold of WAP-8294A compounds, whereas ORFs 4–10 are accessory genes predicted for tailoring, transporting and regulating WAP-8294A compounds.

Tab.1 WAP-8294A compounds detected by the ESI MS analyses

Fig.2 (a) HPLC analysis of metabolites from Lysobacter enzymogenes mutants generated by in-frame deletion of the accessory genes in the WAP gene cluster. (b) Mass spectrometry of the three main WAP-8294A compounds, shown as [M + 2H]²⁺ ions. (c) Chemical structure of WAP-8294A1, A2 and A4. OH11-WT, the wild type; OH11?WAP-ORF6, deletion of ORF6; OH11?WAP-ORF7, deletion of ORF7; OH11?WAP-ORF9, deletion of ORF9; OH11?WAP-ORF10, deletion of ORF10; and OH11?WAP-ORFs 6–10, quint gene deletion of ORF6 through ORF10.

Fig.3 HPLC analysis of metabolites from L. enzymogenes mutants generated by in-frame deletion of ORF5 (a) and by in-frame deletion or single-point mutagenesis of ORF4 in the biosynthetic genes for WAP-8294A (b). OH11-WT, the wild type; OH11?WAP-ORF5, deletion of ORF5; OH11?WAP-ORF4, deletion of ORF4; OH11WAP-ORF4-H104A, histidine residue at position-104 of ORF4 changed to alanine residue; and OH11WAP-ORF4-H298A, histidine residue at position-298 of ORF4 changed to alanine residue.

Fig.4 High-resolution LC-MS of metabolites from the point mutant OH11WAP-ORF4-H104A, with a comparison to the metabolites from the wild type OH11. (a) Total ion chromatogram (TIC) of the wild type OH11-WT and mutant OH11-WAP-ORF4-H104A. The minor metabolite complex is indicated as Ax. (b) MS of deoxy-WAP-8294A1 (expected m/z 766.9012, observed 766.9077), deoxy-WAP-8294A2 (expected m/z 773.9168, observed 773.9156), and deoxy-WAP-8294A4 (expected m/z 780.9247, observed 780.9231) from mutant OH11-WAP-ORF4-H104A. (c) Extracted ion chromatogram (EIC) of deoxy-WAP-8294A1 at 8.7 min. Note that the wild type contains two analogs (AZ1 and AZ2) with the similar mass (m/z 766.9090, see Table 1 and Fig. 6) as deoxy-WAP-8294A1. (d) EIC of deoxy-WAP-8294A2 at 9.1 min. (e) EIC of deoxy-WAP-8294A4 at 9.4 min. The asterisk denoted an unidentified deoxy-WAP at 9.0 min, with m/z in the range of 780.9231. (f–h) Chemical structure of deoxy-WAP-8294A1, deoxy-WAP-8294A2, and deoxy-WAP-8294A4.

Fig.5 Feature-based molecular networking of the WAP metabolites isolated from mutant OH11?WAP-ORF4 and wild type OH11. The metabolites were analyzed by ultra-high-performance liquid chromatography and high-resolution tandem mass spectrometry (UHPLC-HR-MS/MS) (see Fig. S3 and Fig. S4). The node size indicates semiquantitative differences in metabolite concentrations. Nodes are presented as pie charts with different colors, representing the presence of each metabolite in the wild type (blue) and ORF4 mutant (magenta). Metabolites WAP-8294A1, WAP-8294A2 and WAP8-294A4 are indicated as A1, A2 and A4, respectively. Metabolites deoxy-WAP-8294A1, deoxy-WAP-8294A2 and deoxy-WAP-8294A4 as dA1, dA2 and dA4, respectively. AZ1-AZ6 are the WAP-8294A analogs identified in this study (AZ5 is the same as Ax8, see Fig. 6 and Table 1). Note that a new deoxy-WAP-8294Ax (m/z 794.9248, in magenta) was also identified in the networking analysis.

Fig.6 Chemical structure of new WAP-8294A compounds identified in this study (a) and high-resolution MS/MS fragment analysis of an unusual WAP-8294A analog, AZ7 (b). Details of HR-MS/MS fragments of the six compounds are included in Table S2. Note that compound AZ5 is the same as Ax8^[²⁵^], and the structural variations in the new compounds versus WAP-8294A2 are highlighted. In the MS/MS of AZ7, the numbers inside the parentheses indicate the position of the building blocks, the amino acid residues #4-12 of WAP-8294A2.

1	P Christensen , F D Cook . Lysobacter, a new genus of non-fruiting, gliding bacteria with a high base ratio. International Journal of Systematic Bacteriology, 1978, 28( 3): 367–393 https://doi.org/10.1099/00207713-28-3-367
2	Y Xie , S Wright , Y Shen , L Du . Bioactive natural products from Lysobacter. Natural Product Reports, 2012, 29( 11): 1277–1287 https://doi.org/10.1039/c2np20064c
3	L J Giesler , G Y Yuen . Evaluation of Stenotrophomonas maltophilia strain C3 for biocontrol of brown patch disease. Crop Protection, 1998, 17( 6): 509–513 https://doi.org/10.1016/S0261-2194(98)00049-0
4	R F Sullivan , M A Holtman , G J Zylstra , J F Jr White , D Y Kobayashi . Taxonomic positioning of two biological control agents for plant diseases as Lysobacter enzymogenes based on phylogenetic analysis of 16S rDNA, fatty acid composition and phenotypic characteristics. Journal of Applied Microbiology, 2003, 94( 6): 1079–1086 https://doi.org/10.1046/j.1365-2672.2003.01932.x
5	Z Zhang , G Y Yuen . Biological control of Bipolaris sorakiniana on tall fescue by Stenotrophomonas maltophilia strain C3. Phytopathology, 1999, 89( 9): 817–822 https://doi.org/10.1094/PHYTO.1999.89.9.817
6	G Y Yuen , J R Steadman , D T Lindgren , D Schaff , C Jochum . Bean rust biological control using bacterial agents. Crop Protection, 2001, 20( 5): 395–402 https://doi.org/10.1016/S0261-2194(00)00154-X
7	C C Jochum , L E Osborne , G Y Yuen . Fusarium head blight biological control with Lysobacter enzymogenes. Biological Control, 2006, 39( 3): 336–344 https://doi.org/10.1016/j.biocontrol.2006.05.004
8	D Y Kobayashi , R M Reedy , J D Palumbo , J M Zhou , G Y Yuen . A clp gene homologue belonging to the Crp gene family globally regulates lytic enzyme production, antimicrobial activity, and biological control activity expressed by Lysobacter enzymogenes strain C3. Applied and Environmental Microbiology, 2005, 71( 1): 261–269 https://doi.org/10.1128/AEM.71.1.261-269.2005
9	S Li , L Du , G Yuen , S D Harris . Distinct ceramide synthases regulate polarized growth in the filamentous fungus Aspergillus nidulans. Molecular Biology of the Cell, 2006, 17( 3): 1218–1227 https://doi.org/10.1091/mbc.e05-06-0533
10	F Yu , K Zaleta-Rivera , X Zhu , J Huffman , J C Millet , S D Harris , G Yuen , X C Li , L Du . Structure and biosynthesis of heat-stable antifungal factor (HSAF), a broad-spectrum antimycotic with a novel mode of action. Antimicrobial Agents and Chemotherapy, 2007, 51( 1): 64–72 https://doi.org/10.1128/AAC.00931-06
11	S Li , C C Jochum , F Yu , K Zaleta-Rivera , L Du , S D Harris , G Y Yuen . An antibiotic complex from Lysobacter enzymogenes strain C3: antimicrobial activity and role in plant disease control. Phytopathology, 2008, 98( 6): 695–701 https://doi.org/10.1094/PHYTO-98-6-0695
12	L Lou , G Qian , Y Xie , J Hang , H Chen , K Zaleta-Rivera , Y Li , Y Shen , P H Dussault , F Liu , L Du . Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes. Journal of the American Chemical Society, 2011, 133( 4): 643–645 https://doi.org/10.1021/ja105732c
13	Y Li , H Chen , Y Ding , Y Xie , H Wang , R L Cerny , Y Shen , L Du . Iterative assembly of two separate polyketide chains by the same single-module bacterial polyketide synthase in the biosynthesis of HSAF. Angewandte Chemie International Edition, 2014, 53( 29): 7524–7530 https://doi.org/10.1002/anie.201403500
14	Y Li , H Wang , Y Liu , Y Jiao , S Li , Y Shen , L Du . Biosynthesis of the polycyclic system in the antifungal HSAF and analogues from Lysobacter enzymogenes. Angewandte Chemie International Edition, 2018, 57( 21): 6221–6225 https://doi.org/10.1002/anie.201802488
15	W Lee , K Schaefer , Y Qiao , V Srisuknimit , H Steinmetz , R Müller , D Kahne , S Walker . The mechanism of action of lysobactin. Journal of the American Chemical Society, 2016, 138( 1): 100–103 https://doi.org/10.1021/jacs.5b11807
16	H Hashizume , M Igarashi , S Hattori , M Hori , M Hamada , T Takeuchi . Tripropeptins, novel antimicrobial agents produced by Lysobacter sp. I. Taxonomy, isolation and biological activities. Journal of Antibiotics, 2001, 54( 12): 1054–1059 https://doi.org/10.7164/antibiotics.54.1054
17	H Hashizume , S Hirosawa , R Sawa , Y Muraoka , D Ikeda , H Naganawa , M Igarashi . Tripropeptins, novel antimicrobial agents produced by Lysobacter sp. Journal of Antibiotics, 2004, 57( 1): 52–58 https://doi.org/10.7164/antibiotics.57.52
18	H Itoh , K Tokumoto , T Kaji , A Paudel , S Panthee , H Hamamoto , K Sekimizu , M Inoue . Total synthesis and biological mode of action of WAP-8294A2: a menaquinone-targeting antibiotic. Journal of Organic Chemistry, 2018, 83( 13): 6924–6935 https://doi.org/10.1021/acs.joc.7b02318
19	W Zhang , Y Li , G Qian , Y Wang , H Chen , Y Z Li , F Liu , Y Shen , L Du . Identification and characterization of the anti-methicillin-resistant Staphylococcus aureus WAP-8294A2 biosynthetic gene cluster from Lysobacter enzymogenes OH11. Antimicrobial Agents and Chemotherapy, 2011, 55( 12): 5581–5589 https://doi.org/10.1128/AAC.05370-11
20	H Hamamoto , M Urai , K Ishii , J Yasukawa , A Paudel , M Murai , T Kaji , T Kuranaga , K Hamase , T Katsu , J Su , T Adachi , R Uchida , H Tomoda , M Yamada , M Souma , H Kurihara , M Inoue , K Sekimizu . Lysocin E is a new antibiotic that targets menaquinone in the bacterial membrane. Nature Chemical Biology, 2015, 11( 2): 127–133 https://doi.org/10.1038/nchembio.1710
21	M Murai , T Kaji , T Kuranaga , H Hamamoto , K Sekimizu , M Inoue . Total synthesis and biological evaluation of the antibiotic lysocin E and its enantiomeric, epimeric, and N-demethylated analogues. Angewandte Chemie International Edition, 2015, 54( 5): 1556–1560 https://doi.org/10.1002/anie.201410270
22	H Itoh , K Tokumoto , T Kaji , A Paudel , S Panthee , H Hamamoto , K Sekimizu , M Inoue . Development of a high-throughput strategy for discovery of potent analogues of antibiotic lysocin E. Nature Communications, 2019, 10( 1): 2992 https://doi.org/10.1038/s41467-019-10754-4
23	M Sang , H Wang , Y Shen , N Rodrigues de Almeida , M Conda-Sheridan , S Li , Y Li , L Du . Identification of an anti-MRSA cyclic lipodepsipeptide, WBP-29479A1, by genome mining of Lysobacter antibioticus. Organic Letters, 2019, 21( 16): 6432–6436 https://doi.org/10.1021/acs.orglett.9b02333
24	A Kato , S Nakaya , Y Ohashi , H Hirata , K Fujii , K Harada . WAP-8294A(2), a novel anti-MRSA antibiotic produced by Lysobacter sp. Journal of the American Chemical Society, 1997, 119( 28): 6680–6681 https://doi.org/10.1021/ja970895o
25	A Kato , S Nakaya , N Kokubo , Y Aiba , Y Ohashi , H Hirata , K Fujii , K Harada . A new anti-MRSA antibiotic complex, WAP-8294A. I. Taxonomy, isolation and biological activities. Journal of Antibiotic, 1998, 51( 10): 929–935 https://doi.org/10.7164/antibiotics.51.929
26	A Kato , H Hirata , Y Ohashi , K Fujii , K Mori , K Harada . A new anti-MRSA antibiotic complex, WAP-8294A II. Structure characterization of minor components by ESI LCMS and MS/MS. Journal of Antibiotics, 2011, 64( 5): 373–379 https://doi.org/10.1038/ja.2011.9
27	Y Wang , G Qian , F Liu , Y Z Li , Y Shen , L Du . Facile method for site-specific gene integration in Lysobacter enzymogenes for yield improvement of the anti-MRSA antibiotics WAP-8294A and the antifungal antibiotic HSAF. ACS Synthetic Biology, 2013, 2( 11): 670–678 https://doi.org/10.1021/sb4000806
28	L Yu , W Su , P D Fey , F Liu , L Du . Yield improvement of the anti-MRSA antibiotics WAP-8294A by CRISPR/dCas9 combined with refactoring self-protection genes in Lysobacter enzymogenes OH11. ACS Synthetic Biology, 2018, 7( 1): 258–266 https://doi.org/10.1021/acssynbio.7b00293
29	A T Aron , E C Gentry , K L McPhail , L F Nothias , M Nothias-Esposito , A Bouslimani , D Petras , J M Gauglitz , N Sikora , F Vargas , der Hooft J J J van , M Ernst , K B Kang , C M Aceves , A M Caraballo-Rodríguez , I Koester , K C Weldon , S Bertrand , C Roullier , K Sun , R M Tehan , P C A Boya , M H Christian , M Gutiérrez , A M Ulloa , Mora J A Tejeda , R Mojica-Flores , J Lakey-Beitia , V Vásquez-Chaves , Y Zhang , A I Calderón , N Tayler , R A Keyzers , F Tugizimana , N Ndlovu , A A Aksenov , A K Jarmusch , R Schmid , A W Truman , N Bandeira , M Wang , P C Dorrestein . Reproducible molecular networking of untargeted mass spectrometry data using GNPS. Nature Protocols, 2020, 15( 6): 1954–1991 https://doi.org/10.1038/s41596-020-0317-5
30	M Wang , J J Carver , V V Phelan , L M Sanchez , N Garg , Y Peng , D D Nguyen , J Watrous , C A Kapono , T Luzzatto-Knaan , C Porto , A Bouslimani , A V Melnik , M J Meehan , W T Liu , M Crüsemann , P D Boudreau , E Esquenazi , M Sandoval-Calderón , R D Kersten , L A Pace , R A Quinn , K R Duncan , C C Hsu , D J Floros , R G Gavilan , K Kleigrewe , T Northen , R J Dutton , D Parrot , E E Carlson , B Aigle , C F Michelsen , L Jelsbak , C Sohlenkamp , P Pevzner , A Edlund , J McLean , J Piel , B T Murphy , L Gerwick , C C Liaw , Y L Yang , H U Humpf , M Maansson , R A Keyzers , A C Sims , A R Johnson , A M Sidebottom , B E Sedio , A Klitgaard , C B Larson , P C A Boya , D Torres-Mendoza , D J Gonzalez , D B Silva , L M Marques , D P Demarque , E Pociute , E C O’Neill , E Briand , E J N Helfrich , E A Granatosky , E Glukhov , F Ryffel , H Houson , H Mohimani , J J Kharbush , Y Zeng , J A Vorholt , K L Kurita , P Charusanti , K L McPhail , K F Nielsen , L Vuong , M Elfeki , M F Traxler , N Engene , N Koyama , O B Vining , R Baric , R R Silva , S J Mascuch , S Tomasi , S Jenkins , V Macherla , T Hoffman , V Agarwal , P G Williams , J Dai , R Neupane , J Gurr , A M C Rodríguez , A Lamsa , C Zhang , K Dorrestein , B M Duggan , J Almaliti , P M Allard , P Phapale , L F Nothias , T Alexandrov , M Litaudon , J L Wolfender , J E Kyle , T O Metz , T Peryea , D T Nguyen , D VanLeer , P Shinn , A Jadhav , R Müller , K M Waters , W Shi , X Liu , L Zhang , R Knight , P R Jensen , B Ø Palsson , K Pogliano , R G Linington , M Gutiérrez , N P Lopes , W H Gerwick , B S Moore , P C Dorrestein , N Bandeira . Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nature Biotechnology, 2016, 34( 8): 828–837 https://doi.org/10.1038/nbt.3597
31	M C Chambers , B Maclean , R Burke , D Amodei , D L Ruderman , S Neumann , L Gatto , B Fischer , B Pratt , J Egertson , K Hoff , D Kessner , N Tasman , N Shulman , B Frewen , T A Baker , M Y Brusniak , C Paulse , D Creasy , L Flashner , K Kani , C Moulding , S L Seymour , L M Nuwaysir , B Lefebvre , F Kuhlmann , J Roark , P Rainer , S Detlev , T Hemenway , A Huhmer , J Langridge , B Connolly , T Chadick , K Holly , J Eckels , E W Deutsch , R L Moritz , J E Katz , D B Agus , M MacCoss , D L Tabb , P Mallick . A cross-platform toolkit for mass spectrometry and proteomics. Nature Biotechnology, 2012, 30( 10): 918–920 https://doi.org/10.1038/nbt.2377
32	T Pluskal , S Castillo , A Villar-Briones , M Oresic . MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics, 2010, 11( 1): 395 https://doi.org/10.1186/1471-2105-11-395
33	P Shannon , A Markiel , O Ozier , N S Baliga , J T Wang , D Ramage , N Amin , B Schwikowski , T Ideker . Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Research, 2003, 13( 11): 2498–2504 https://doi.org/10.1101/gr.1239303
34	J M Elkins , M J Ryle , I J Clifton , J C Dunning Hotopp , J S Lloyd , N I Burzlaff , J E Baldwin , R P Hausinger , P L Roach . X-ray crystal structure of Escherichia coli taurine/alpha-ketoglutarate dioxygenase complexed to ferrous iron and substrates. Biochemistry, 2002, 41( 16): 5185–5192 https://doi.org/10.1021/bi016014e
35	H Chen , A S Olson , W Su , P H Dussault , L Du . Fatty acyl incorporation in the biosynthesis of WAP-8294A, a group of potent anti-MRSA cyclic lipodepsipeptides. RSC Advances, 2015, 5( 128): 105753–105759 https://doi.org/10.1039/C5RA20784C
36	M Strieker , F Kopp , C Mahlert , L O Essen , M A Marahiel . Mechanistic and structural basis of stereospecific Cbeta-hydroxylation in calcium-dependent antibiotic, a daptomycin-type lipopeptide. ACS Chemical Biology, 2007, 2( 3): 187–196 https://doi.org/10.1021/cb700012y
37	H Chen , B K Hubbard , S E O’Connor , C T Walsh . Formation of beta-hydroxy histidine in the biosynthesis of nikkomycin antibiotics. Chemistry & Biology, 2002, 9( 1): 103–112 https://doi.org/10.1016/S1074-5521(02)00090-X
38	L Yu , F Du , X Chen , Y Zheng , M Morton , F Liu , L Du . Identification of the biosynthetic gene cluster for the anti-MRSA lysocins through gene cluster activation using strong promoters of housekeeping genes and production of new analogs in Lysobacter sp. 3655. ACS Synthetic Biology, 2020, 9( 8): 1989–1997 https://doi.org/10.1021/acssynbio.0c00067
39	S Li , X Wu , L Zhang , Y Shen , L Du . Activation of a cryptic gene cluster in Lysobacter enzymogenes reveals a module/domain portable mechanism of nonribosomal peptide synthetases in the biosynthesis of pyrrolopyrazines. Organic Letters, 2017, 19( 19): 5010–5013 https://doi.org/10.1021/acs.orglett.7b01611

[1]

FASE-21410-OF-ZJ_suppl_1

Download

Viewed

Full text

Abstract

Cited

Shared

Discussed