Heterologous expression of a cryptic gene cluster from Streptomyces leeuwenhoekii C34T yields a novel lasso peptide, leepeptin

Juan Pablo Gomez-Escribano, Jean Franco Castro, Valeria Razmilic, Scott A Jarmusch, Gerhard Saalbach, Rainer Ebel, Marcel Jaspars, Barbara Andrews, Juan A Asenjo, Mervyn J Bibb*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor. The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides.

IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.
Original languageEnglish
Article numbere01752-19
Number of pages13
JournalApplied and Environmental Microbiology
Volume85
Issue number23
Early online date14 Nov 2019
DOIs
Publication statusPublished - 1 Dec 2019

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Streptomyces
Multigene Family
multigene family
peptide
peptides
Peptides
gene
Streptomyces coelicolor
Biological Products
genome
alachlor
Genome
environmental cue
bioinformatics
Actinobacteria
new family
Computational Biology
Cues
Sequence Analysis
nuclear magnetic resonance

Keywords

  • Streptomyces
  • natural products
  • RiPP
  • Lasso peptides
  • Heterologous expression
  • COELICOLOR
  • DESERT
  • heterologous expression
  • RESTRICTION
  • BIOSYNTHESIS
  • CHAXAMYCINS
  • SEQUENCE
  • RESISTANCE
  • lasso peptide
  • BINDING
  • TOOL

Cite this

Heterologous expression of a cryptic gene cluster from Streptomyces leeuwenhoekii C34T yields a novel lasso peptide, leepeptin. / Gomez-Escribano, Juan Pablo; Castro, Jean Franco; Razmilic, Valeria; Jarmusch, Scott A; Saalbach, Gerhard; Ebel, Rainer; Jaspars, Marcel; Andrews, Barbara; Asenjo, Juan A; Bibb, Mervyn J.

In: Applied and Environmental Microbiology, Vol. 85, No. 23, e01752-19, 01.12.2019.

Research output: Contribution to journalArticle

Gomez-Escribano, Juan Pablo ; Castro, Jean Franco ; Razmilic, Valeria ; Jarmusch, Scott A ; Saalbach, Gerhard ; Ebel, Rainer ; Jaspars, Marcel ; Andrews, Barbara ; Asenjo, Juan A ; Bibb, Mervyn J. / Heterologous expression of a cryptic gene cluster from Streptomyces leeuwenhoekii C34T yields a novel lasso peptide, leepeptin. In: Applied and Environmental Microbiology. 2019 ; Vol. 85, No. 23.
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abstract = "Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor. The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides.IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.",
keywords = "Streptomyces, natural products, RiPP, Lasso peptides, Heterologous expression, COELICOLOR, DESERT, heterologous expression, RESTRICTION, BIOSYNTHESIS, CHAXAMYCINS, SEQUENCE, RESISTANCE, lasso peptide, BINDING, TOOL",
author = "Gomez-Escribano, {Juan Pablo} and Castro, {Jean Franco} and Valeria Razmilic and Jarmusch, {Scott A} and Gerhard Saalbach and Rainer Ebel and Marcel Jaspars and Barbara Andrews and Asenjo, {Juan A} and Bibb, {Mervyn J}",
note = "ACKNOWLEDGEMENTS. We are grateful to Michael Goodfellow and Alan Bull for providing S. leeuwenhoekii C34T , and to Michael Fischbach and Jan Claesen for S. viridochromogenes and S. pristinaspiralis, Matthias Mach for S. davawensis, and Kristian Apel on October 31, 2019 at University of Aberdeen http://aem.asm.org/ Downloaded from 17 for S. roseochromogenes. We thank Govind Chandra for advice on blastP analyses of the lasso peptide data sets, Sol{\`e}ne Rollet for technical support in the isolation of leepeptin and Andrew Truman for his comments on the manuscript. J.F.C. and V.R. received National PhD Scholarships (#21110356 and #21110384, respectively) and Visiting Student Scholarships (Becas Chile, 2013–2014) from the National Commission for Scientific and Technological Research (CONICYT). S.A.J. thanks the University of Aberdeen for an Elphinstone Scholarship. This work was supported financially by the Biotechnological and Biological Sciences Research Council (BBSRC, United Kingdom) Institute Strategic Programme Grant “Understanding and Exploiting Plant and Microbial Secondary Metabolism” (BB/J004561/1), the Basal Programme of CONICYT (Chile) for funding of the Centre for Biotechnology and Bioengineering, CeBiB (project FB0001) and the UK Newton Project for UK–Chile collaboration (grant JIC CA586).",
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TY - JOUR

T1 - Heterologous expression of a cryptic gene cluster from Streptomyces leeuwenhoekii C34T yields a novel lasso peptide, leepeptin

AU - Gomez-Escribano, Juan Pablo

AU - Castro, Jean Franco

AU - Razmilic, Valeria

AU - Jarmusch, Scott A

AU - Saalbach, Gerhard

AU - Ebel, Rainer

AU - Jaspars, Marcel

AU - Andrews, Barbara

AU - Asenjo, Juan A

AU - Bibb, Mervyn J

N1 - ACKNOWLEDGEMENTS. We are grateful to Michael Goodfellow and Alan Bull for providing S. leeuwenhoekii C34T , and to Michael Fischbach and Jan Claesen for S. viridochromogenes and S. pristinaspiralis, Matthias Mach for S. davawensis, and Kristian Apel on October 31, 2019 at University of Aberdeen http://aem.asm.org/ Downloaded from 17 for S. roseochromogenes. We thank Govind Chandra for advice on blastP analyses of the lasso peptide data sets, Solène Rollet for technical support in the isolation of leepeptin and Andrew Truman for his comments on the manuscript. J.F.C. and V.R. received National PhD Scholarships (#21110356 and #21110384, respectively) and Visiting Student Scholarships (Becas Chile, 2013–2014) from the National Commission for Scientific and Technological Research (CONICYT). S.A.J. thanks the University of Aberdeen for an Elphinstone Scholarship. This work was supported financially by the Biotechnological and Biological Sciences Research Council (BBSRC, United Kingdom) Institute Strategic Programme Grant “Understanding and Exploiting Plant and Microbial Secondary Metabolism” (BB/J004561/1), the Basal Programme of CONICYT (Chile) for funding of the Centre for Biotechnology and Bioengineering, CeBiB (project FB0001) and the UK Newton Project for UK–Chile collaboration (grant JIC CA586).

PY - 2019/12/1

Y1 - 2019/12/1

N2 - Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor. The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides.IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

AB - Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor. The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides.IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

KW - Streptomyces

KW - natural products

KW - RiPP

KW - Lasso peptides

KW - Heterologous expression

KW - COELICOLOR

KW - DESERT

KW - heterologous expression

KW - RESTRICTION

KW - BIOSYNTHESIS

KW - CHAXAMYCINS

KW - SEQUENCE

KW - RESISTANCE

KW - lasso peptide

KW - BINDING

KW - TOOL

U2 - 10.1128/AEM.01752-19

DO - 10.1128/AEM.01752-19

M3 - Article

C2 - 31562169

VL - 85

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 23

M1 - e01752-19

ER -