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

Highlights

● 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.

Abstract

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.

Graphical abstract

Keywords

biocontrol / biosynthesis / Lysobacter / natural products / WAP-8294A

References

[1] Christensen P , Cook F D . Lysobacter, a new genus of non-fruiting, gliding bacteria with a high base ratio. International Journal of Systematic Bacteriology, 1978, 28( 3): 367–393

[2] Xie Y , Wright S , Shen Y , Du L . Bioactive natural products from Lysobacter. Natural Product Reports, 2012, 29( 11): 1277–1287

[3] Giesler L J , Yuen G Y . Evaluation of Stenotrophomonas maltophilia strain C3 for biocontrol of brown patch disease. Crop Protection, 1998, 17( 6): 509–513

[4] Sullivan R F , Holtman M A , Zylstra G J , White J F Jr , Kobayashi D Y . 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

[5] Zhang Z , Yuen G Y . Biological control of Bipolaris sorakiniana on tall fescue by Stenotrophomonas maltophilia strain C3. Phytopathology, 1999, 89( 9): 817–822

[6] Yuen G Y , Steadman J R , Lindgren D T , Schaff D , Jochum C . Bean rust biological control using bacterial agents. Crop Protection, 2001, 20( 5): 395–402

[7] Jochum C C , Osborne L E , Yuen G Y . Fusarium head blight biological control with Lysobacter enzymogenes. Biological Control, 2006, 39( 3): 336–344

[8] Kobayashi D Y , Reedy R M , Palumbo J D , Zhou J M , Yuen G Y . 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

[9] Li S , Du L , Yuen G , Harris S D . Distinct ceramide synthases regulate polarized growth in the filamentous fungus Aspergillus nidulans. Molecular Biology of the Cell, 2006, 17( 3): 1218–1227

[10] Yu F , Zaleta-Rivera K , Zhu X , Huffman J , Millet J C , Harris S D , Yuen G , Li X C , Du L . 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

[11] Li S , Jochum C C , Yu F , Zaleta-Rivera K , Du L , Harris S D , Yuen G Y . An antibiotic complex from Lysobacter enzymogenes strain C3: antimicrobial activity and role in plant disease control. Phytopathology, 2008, 98( 6): 695–701

[12] Lou L , Qian G , Xie Y , Hang J , Chen H , Zaleta-Rivera K , Li Y , Shen Y , Dussault P H , Liu F , Du L . Biosynthesis of HSAF, a tetramic acid-containing macrolactam from Lysobacter enzymogenes. Journal of the American Chemical Society, 2011, 133( 4): 643–645

[13] Li Y , Chen H , Ding Y , Xie Y , Wang H , Cerny R L , Shen Y , Du L . 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

[14] Li Y , Wang H , Liu Y , Jiao Y , Li S , Shen Y , Du L . Biosynthesis of the polycyclic system in the antifungal HSAF and analogues from Lysobacter enzymogenes. Angewandte Chemie International Edition, 2018, 57( 21): 6221–6225

[15] Lee W , Schaefer K , Qiao Y , Srisuknimit V , Steinmetz H , Müller R , Kahne D , Walker S . The mechanism of action of lysobactin. Journal of the American Chemical Society, 2016, 138( 1): 100–103

[16] Hashizume H , Igarashi M , Hattori S , Hori M , Hamada M , Takeuchi T . Tripropeptins, novel antimicrobial agents produced by Lysobacter sp. I. Taxonomy, isolation and biological activities. Journal of Antibiotics, 2001, 54( 12): 1054–1059

[17] Hashizume H , Hirosawa S , Sawa R , Muraoka Y , Ikeda D , Naganawa H , Igarashi M . Tripropeptins, novel antimicrobial agents produced by Lysobacter sp. Journal of Antibiotics, 2004, 57( 1): 52–58

[18] Itoh H , Tokumoto K , Kaji T , Paudel A , Panthee S , Hamamoto H , Sekimizu K , Inoue M . Total synthesis and biological mode of action of WAP-8294A2: a menaquinone-targeting antibiotic. Journal of Organic Chemistry, 2018, 83( 13): 6924–6935

[19] Zhang W , Li Y , Qian G , Wang Y , Chen H , Li Y Z , Liu F , Shen Y , Du L . 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

[20] Hamamoto H , Urai M , Ishii K , Yasukawa J , Paudel A , Murai M , Kaji T , Kuranaga T , Hamase K , Katsu T , Su J , Adachi T , Uchida R , Tomoda H , Yamada M , Souma M , Kurihara H , Inoue M , Sekimizu K . Lysocin E is a new antibiotic that targets menaquinone in the bacterial membrane. Nature Chemical Biology, 2015, 11( 2): 127–133

[21] Murai M , Kaji T , Kuranaga T , Hamamoto H , Sekimizu K , Inoue M . 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

[22] Itoh H , Tokumoto K , Kaji T , Paudel A , Panthee S , Hamamoto H , Sekimizu K , Inoue M . Development of a high-throughput strategy for discovery of potent analogues of antibiotic lysocin E. Nature Communications, 2019, 10( 1): 2992

[23] Sang M , Wang H , Shen Y , Rodrigues de Almeida N , Conda-Sheridan M , Li S , Li Y , Du L . Identification of an anti-MRSA cyclic lipodepsipeptide, WBP-29479A1, by genome mining of Lysobacter antibioticus. Organic Letters, 2019, 21( 16): 6432–6436

[24] Kato A , Nakaya S , Ohashi Y , Hirata H , Fujii K , Harada K . WAP-8294A(2), a novel anti-MRSA antibiotic produced by Lysobacter sp. Journal of the American Chemical Society, 1997, 119( 28): 6680–6681

[25] Kato A , Nakaya S , Kokubo N , Aiba Y , Ohashi Y , Hirata H , Fujii K , Harada K . A new anti-MRSA antibiotic complex, WAP-8294A. I. Taxonomy, isolation and biological activities. Journal of Antibiotic, 1998, 51( 10): 929–935

[26] Kato A , Hirata H , Ohashi Y , Fujii K , Mori K , Harada K . 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

[27] Wang Y , Qian G , Liu F , Li Y Z , Shen Y , Du L . 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

[28] Yu L , Su W , Fey P D , Liu F , Du L . 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

[29] Aron A T , Gentry E C , McPhail K L , Nothias L F , Nothias-Esposito M , Bouslimani A , Petras D , Gauglitz J M , Sikora N , Vargas F , van der Hooft J J J , Ernst M , Kang K B , Aceves C M , Caraballo-Rodríguez A M , Koester I , Weldon K C , Bertrand S , Roullier C , Sun K , Tehan R M , Boya P C A , Christian M H , Gutiérrez M , Ulloa A M , Tejeda Mora J A , Mojica-Flores R , Lakey-Beitia J , Vásquez-Chaves V , Zhang Y , Calderón A I , Tayler N , Keyzers R A , Tugizimana F , Ndlovu N , Aksenov A A , Jarmusch A K , Schmid R , Truman A W , Bandeira N , Wang M , Dorrestein P C . Reproducible molecular networking of untargeted mass spectrometry data using GNPS. Nature Protocols, 2020, 15( 6): 1954–1991

[30] Wang M , Carver J J , Phelan V V , Sanchez L M , Garg N , Peng Y , Nguyen D D , Watrous J , Kapono C A , Luzzatto-Knaan T , Porto C , Bouslimani A , Melnik A V , Meehan M J , Liu W T , Crüsemann M , Boudreau P D , Esquenazi E , Sandoval-Calderón M , Kersten R D , Pace L A , Quinn R A , Duncan K R , Hsu C C , Floros D J , Gavilan R G , Kleigrewe K , Northen T , Dutton R J , Parrot D , Carlson E E , Aigle B , Michelsen C F , Jelsbak L , Sohlenkamp C , Pevzner P , Edlund A , McLean J , Piel J , Murphy B T , Gerwick L , Liaw C C , Yang Y L , Humpf H U , Maansson M , Keyzers R A , Sims A C , Johnson A R , Sidebottom A M , Sedio B E , Klitgaard A , Larson C B , Boya P C A , Torres-Mendoza D , Gonzalez D J , Silva D B , Marques L M , Demarque D P , Pociute E , O’Neill E C , Briand E , Helfrich E J N , Granatosky E A , Glukhov E , Ryffel F , Houson H , Mohimani H , Kharbush J J , Zeng Y , Vorholt J A , Kurita K L , Charusanti P , McPhail K L , Nielsen K F , Vuong L , Elfeki M , Traxler M F , Engene N , Koyama N , Vining O B , Baric R , Silva R R , Mascuch S J , Tomasi S , Jenkins S , Macherla V , Hoffman T , Agarwal V , Williams P G , Dai J , Neupane R , Gurr J , Rodríguez A M C , Lamsa A , Zhang C , Dorrestein K , Duggan B M , Almaliti J , Allard P M , Phapale P , Nothias L F , Alexandrov T , Litaudon M , Wolfender J L , Kyle J E , Metz T O , Peryea T , Nguyen D T , VanLeer D , Shinn P , Jadhav A , Müller R , Waters K M , Shi W , Liu X , Zhang L , Knight R , Jensen P R , Palsson B Ø , Pogliano K , Linington R G , Gutiérrez M , Lopes N P , Gerwick W H , Moore B S , Dorrestein P C , Bandeira N . Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nature Biotechnology, 2016, 34( 8): 828–837

[31] Chambers M C , Maclean B , Burke R , Amodei D , Ruderman D L , Neumann S , Gatto L , Fischer B , Pratt B , Egertson J , Hoff K , Kessner D , Tasman N , Shulman N , Frewen B , Baker T A , Brusniak M Y , Paulse C , Creasy D , Flashner L , Kani K , Moulding C , Seymour S L , Nuwaysir L M , Lefebvre B , Kuhlmann F , Roark J , Rainer P , Detlev S , Hemenway T , Huhmer A , Langridge J , Connolly B , Chadick T , Holly K , Eckels J , Deutsch E W , Moritz R L , Katz J E , Agus D B , MacCoss M , Tabb D L , Mallick P . A cross-platform toolkit for mass spectrometry and proteomics. Nature Biotechnology, 2012, 30( 10): 918–920

[32] Pluskal T , Castillo S , Villar-Briones A , Oresic M . MZmine 2: modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics, 2010, 11( 1): 395

[33] Shannon P , Markiel A , Ozier O , Baliga N S , Wang J T , Ramage D , Amin N , Schwikowski B , Ideker T . Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Research, 2003, 13( 11): 2498–2504

[34] Elkins J M , Ryle M J , Clifton I J , Dunning Hotopp J C , Lloyd J S , Burzlaff N I , Baldwin J E , Hausinger R P , Roach P L . X-ray crystal structure of Escherichia coli taurine/alpha-ketoglutarate dioxygenase complexed to ferrous iron and substrates. Biochemistry, 2002, 41( 16): 5185–5192

[35] Chen H , Olson A S , Su W , Dussault P H , Du L . Fatty acyl incorporation in the biosynthesis of WAP-8294A, a group of potent anti-MRSA cyclic lipodepsipeptides. RSC Advances, 2015, 5( 128): 105753–105759

[36] Strieker M , Kopp F , Mahlert C , Essen L O , Marahiel M A . Mechanistic and structural basis of stereospecific Cbeta-hydroxylation in calcium-dependent antibiotic, a daptomycin-type lipopeptide. ACS Chemical Biology, 2007, 2( 3): 187–196

[37] Chen H , Hubbard B K , O’Connor S E , Walsh C T . Formation of beta-hydroxy histidine in the biosynthesis of nikkomycin antibiotics. Chemistry & Biology, 2002, 9( 1): 103–112

[38] Yu L , Du F , Chen X , Zheng Y , Morton M , Liu F , Du L . 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

[39] Li S , Wu X , Zhang L , Shen Y , Du L . 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

Supplementary materials

The online version of this article at https://doi.org/10.15302/J-FASE-2021410 contains supplementary materials (Tables S1−S2, Figs. S1−S4).

Acknowledgements

This study was supported in part by University of Nebraska Collaboration Initiative Seed Grant. Jing Zhu and Yuan Chen were recipients of a China Scholarship Council fellowship.

Compliance with ethics guidelines

Jing Zhu, Yuan Chen, and Liangcheng Du declare that they have no conflicts of interest or financial conflicts to disclose. This article does not contain any study with human or animal subjects performed by any of the authors.

RIGHTS & PERMISSIONS

The Author(s) 2021. Published by Higher Education Press. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0)