Lasso peptides from Actinobacteria

236 Pages·2017·11 MB·English

by  Jimmy Mevaere

Lasso peptides from Actinobacteria - Chemical diversity
and ecological role
Jimmy Mevaere
To cite this version:
Jimmy Mevaere. Lasso peptides from Actinobacteria - Chemical diversity and ecological role.
Biochemistry [q-bio.BM]. Université Pierre et Marie Curie - Paris VI, 2016. English. ￿NNT:
2016PA066617￿. ￿tel-01924455￿
HAL Id: tel-01924455
https://theses.hal.science/tel-01924455
Submitted on 16 Nov 2018
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Université Pierre et Marie Curie
ED 227 : Sciences de la Nature et de l’Homme : écologie et évolution
Unité Molécules de communication et Adaptation des Micro-organismes (MCAM) /
Equipe Molécules de défense et de communication dans les écosystèmes microbiens
(MDCEM)
Lasso peptides from Actinobacteria
Chemical diversity and ecological role
Par Jimmy Mevaere

Thèse de doctorat de biochimie et biologie moléculaire

Présentée et soutenue publiquement le 14 novembre 2016

Devant un jury composé de :

Véronique Monnet Directeur de recherche Rapporteur
Jean-Luc Pernodet Directeur de recherche Rapporteur
Jean-Michel Camadro Directeur de recherche Examinateur
Marcelino T. Suzuki Professeur Examinateur
Sergey B. Zotchev Professeur Examinateur
Séverine Zirah Maître de conférences Directrice de thèse
Yanyan Li Chargée de recherche Codirectrice de thèse

Acknowledgements
At first, I would like to thank Séverine Zirah and Yanyan Li, my PhD supervisor, and
Prof. Sylvie Rebuffat for having entrusted me as a candidate for the PhD scholarship with my
little knowledge of molecular microbiology and how to best achieve science. I must
acknowledge them for their support and guidance. Thank you for giving me the opportunity to
work in this unique place which is MNHN and MCAM research lab.
I want to thanks the members and colleagues of the laboratory for the very useful helps
they provided me during these three years and for the discussion: Didier, Bastien, Sebastien,
Stéphane, Soizic, Caroline, Alain P, Carine, Christine, Michel, Isabelle, Arlette, Marie-lise,
Alyssa, Jean, Amandine, Linda, Benjamin M, Alain B, Alexandre and Séverine A. Particular
thanks go to Jean-baptiste Rioux who welcomed me so nicely and who teaches me the basis of
molecular biology at the bench. I must acknowledge the work and the help of Haiyan Ma who
preceded me and started the heterologous expression lasso peptides. I want to thanks Christophe
Goulard who helped me a lot with the cultivation and the purification of those tricky lasso
peptides while I was battling against Streptomyces sviceus morphologies. Many thanks to
Adrienne for support, advice and discussion. Many thanks go to the student who came in
Yanyan’s team, Hortense, Maylis, Saravanane, Andrea, Paul and Victor. I want to acknowledge
the other student and post-doc from the lab, Mehdi, Agathe, Béatrice, Marine, Alison,
Benjamin, Wei, Anaïs, Ambre, Margot, Anne, Frédéric, Mélanie, Johann, Sophie, Natacha and
Clara.
I must acknowledge Manon and Laura for their help in microbiology, Soraya for qPCR
experiments, and Lionel and Arul for mass spectrometry experiments. Many thanks to Nora,
Djena and Brice for their helps in administration matters.
Finaly my thanks go to my friends and my family, in particular Ten, la Mére, le Frére,
la Soeur, Clémence, Yves, Emmanuel, Bastien, Rémi, Erwan, Marion, Morgane, Matthieu, Ly,
Christophe, Raphaël, Dara, Jérôme, Mickael, for their support (Wanna have a beer?), guidance
(Don’t do PhD fool) and encouragement during the past three years, in particular through the
most difficult phases of the project.

I
II
Abbreviations
ABC transporter ATP-binding cassette transporter
ACN Acetonitrile
ACP Acyl carrier protein
ACT Actinorhodin
AT Acyltransferase
ATP Adenosine triphosphate
BGC Biosynthetic gene cluster
bp Base pair
C Cytosine
CDA Calcium-dependent antibiotics
cDNA Complementary cDNA
CoA Coenzyme A
Dha 2,3-didehydroalanine
DHB 2, 5-dihydroxybenzoic acid
Dhb (Z)-2,3-didehydrobutyrine
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
Dnase Deoxyribonuclease
dsDNA Double-stranded DNA
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
ESI Electrospray ionization
FA Formic acid
G Guanine
GBAP Gelatinase biosynthesis-activating pheromone
GlcNAc N-acetylglucosamine
HCCA α-cyano-4-hydroxycinnamic acid
HK Histidine kinase
HPLC High-performance liquid chromatography
HTH helix-turn-helix
KS β-ketoacyl synthase
Lan meso-lanthionine
III
LC-MS Liquid chromatography-mass spectrometry
LP Lasso peptide
MeLan 3-methyllanthionine
MIC Minimal inhibitory concentration
M Molecular weight
w
NGS Next-generation sequencing
NMR Nuclear magnetic resonance
NRP Non-ribosomal peptide
NRPS Non-ribosomal peptide synthase
OCS One-component system
OD Optical density
PCR Polymerase chain reaction
PKS Polyketide synthase
PTM Post-translational modification
qPCR Real-time quantitative polymerase chain reaction
QS Quorum sensing
Q-tof Quadrupole-time of flight
RED Undecylprodigiosin
RiPP Ribosomally synthesized and post-translationally modified peptide
RNA Ribonucleic acid
Rnase Ribonuclease
rpm Revolutions per minute
RR Response regulator
rRNA Ribosomal ribonucleic acid
RT-PCR Reverse transcription polymerase chain reaction
SAM S-adenosylmethionine-dependent methyltransferase
SARP Streptomyces antibiotic regulatory protein
SDS Sodium dodecyl sulfate
SEM Scanning electron microscopy
SET SDS, EDTA, Tris buffer
ssDNA Single-stranded DNA
TCS Two-component system
TE trishydroxyméthylaminométhane-EDTA
Tm Melting temperature
IV
Table of contents
Acknowledgements _________________________________________________________ I
Abbreviations ____________________________________________________________ III
Table of contents __________________________________________________________ V
Figures __________________________________________________________________ IX
Tables ___________________________________________________________________ XI
List of Annexes _________________________________________________________ XIII
Introduction ______________________________________________________________ 15
Literature review __________________________________________________________ 17
I. Microbial natural product discovery in the genomics era _____________________ 17
I.1. Diversity of microbial specialized metabolites _________________________ 17
I.2. From genes to molecules __________________________________________ 23
I.3. Streptomyces, a prolific metabolite producer ___________________________ 24
I.3.a. The life cycle of Streptomyces __________________________________ 26
I.3.b. Regulation of specialized metabolite biosynthesis in Streptomyces ______ 27
I.3.b.i Pathway-specific regulators __________________________________ 29
I.3.b.ii Pleiotropic regulators _______________________________________ 31
II. Ribosomally synthesized and post-translationally modified peptides ___________ 32
II.1. Diversity _______________________________________________________ 32
II.2. Lanthipeptides __________________________________________________ 34
II.2.a. Structure ___________________________________________________ 34
II.2.b. Biosynthesis ________________________________________________ 34
II.2.c. Classification ________________________________________________ 36
II.2.d. Biological activities __________________________________________ 36
II.3. Thiopeptides ____________________________________________________ 37
II.3.a. Structure ___________________________________________________ 37
II.3.b. Biosynthesis ________________________________________________ 37
II.3.c. Classification ________________________________________________ 38
II.3.d. Biological activities __________________________________________ 38
II.4. Lasso peptides __________________________________________________ 40
II.4.a. Diversity ___________________________________________________ 40
II.4.b. Discovery __________________________________________________ 45
II.4.c. Biosynthesis ________________________________________________ 47
II.4.c.i Gene cluster organization ____________________________________ 47
II.4.c.ii Maturation process _________________________________________ 49
II.4.c.iii Additional modifications ____________________________________ 50
II.4.d. Biological activities __________________________________________ 54
II.4.e. Bioengineering using lasso peptides ______________________________ 55
III. Ecological role of RiPPs ______________________________________________ 56
IV. Objectives of the thesis _______________________________________________ 60
Materials and methods _____________________________________________________ 63
I. Chemicals and biological materials _____________________________________ 63
V
II. General DNA and microbiology methods_________________________________ 63
II.1. Purification of plasmids and cosmids ________________________________ 63
II.2. Isolation of genomic DNA _________________________________________ 64
II.3. General Polymerase Chain Reaction methods (PCR) ____________________ 64
II.4. Agarose gel electrophoresis ________________________________________ 65
II.5. General cloning procedure _________________________________________ 65
II.6. Preparation and transformation of competent E. coli cells ________________ 66
II.6.a. Electrocompetent cells ________________________________________ 66
II.6.b. Chemical competent cells ______________________________________ 66
II.7. Site-directed mutagenesis _________________________________________ 67
II.8. Modification of cosmids using PCR targeting __________________________ 67
II.9. Conjugation methods for Streptomyces strains _________________________ 68
III. RNA methods ______________________________________________________ 69
III.1. RNA extraction _________________________________________________ 69
III.2. Reverse transcription PCR (RT-PCR) ________________________________ 70
III.3. Real-time quantitative PCR (qPCR) _________________________________ 70
IV. Heterologous expression of lasso peptide gene clusters ______________________ 70
IV.1. Generation of lasso peptide expression system _________________________ 70
IV.1.a. Cosmid libraries _____________________________________________ 70
IV.1.b. Plasmid vectors ______________________________________________ 71
IV.2. Lasso peptide production in heterologous host _________________________ 72
IV.3. Lasso peptide production and extraction ______________________________ 72
V. Liquid chromatography mass spectrometry (LC-MS) _______________________ 73
VI. Generation of deletion mutants in S. sviceus- ______________________________ 73
VI.1. Inactivation using pKGLP2-based suicide plasmids _____________________ 73
VI.2. Inactivation using modified cosmids _________________________________ 74
VI.2.a. Generation of P4H8, a P4H7 based suicide cosmid __________________ 74
VI.2.b. Modification of P4H8 by PCR Targeting __________________________ 74
VI.2.c. Inactivation using P4H8-based cosmids ___________________________ 74
VI.2.d. Gene complementation in the deletion mutants of S. sviceus ___________ 74
VII. Gene reporter assays _________________________________________________ 75
VIII. Oxidative sensibility assay ____________________________________________ 75
IX. Morphology characterization __________________________________________ 76
IX.1. Optic microscopy ________________________________________________ 76
IX.2. Scanning Electronic microscopy (SEM) ______________________________ 76
X. Autoinduction of sviceucin ____________________________________________ 77
X.1. Gene reporter assay ______________________________________________ 77
X.2. qPCR _________________________________________________________ 77
Chapter I: Heterologous expression of lasso peptides from Actinobacteria ___________ 79
I. Introduction ________________________________________________________ 79
I.1. Common strategies to trigger and improve the expression of cryptic or silent
BGCs 79
I.1.a. Optimization of cultivation conditions ____________________________ 79
I.1.b. Heterologous expression _______________________________________ 80
I.1.c. Metabolic engineering ________________________________________ 80
I.1.d. Synthetic biology ____________________________________________ 81
VI
I.2. Heterologous expression of lasso peptides ____________________________ 82
II. Results and discussion _______________________________________________ 83
II.1. Genome mining _________________________________________________ 83
II.1.a. Stackebrandtia nassauensis cluster _______________________________ 88
II.1.b. Nocardiopsis alba cluster ______________________________________ 88
II.1.c. Actinoalloteichus sp. cluster ____________________________________ 89
II.1.d. Streptomyces noursei cluster ____________________________________ 89
II.1.e. Streptomyces venezuelae cluster _________________________________ 89
II.2. Heterologous production based on native gene clusters __________________ 90
II.2.a. Cosmid-based method _________________________________________ 90
II.2.b. Co-expression with the pathway-specific regulator __________________ 91
II.3. Heterologous expression using genetically-engineered clusters ____________ 92
II.3.a. Promoter exchange strategy ____________________________________ 92
II.3.b. Orthogonal two-plasmid expression system ________________________ 93
III. Conclusion and perspectives ___________________________________________ 98
Chapter II: Regulation mechanism and ecological role of sviceucin in Streptomyces
sviceus ___________________________________________________________________ 99
I. Introduction ________________________________________________________ 99
I.1. Sviceucin as a good model to study LP regulation and ecological roles ______ 99
I.2. Overview of growth and development in Streptomyces _________________ 101
II. Results and discussion ______________________________________________ 102
II.1. Determining the operon structure in the sviceucin gene cluster ___________ 102
II.2. Growth dependence of sviceucin production __________________________ 103
II.3. Probing the regulation mechanism of sviceucin biosynthesis _____________ 106
II.3.a. Is sviceucin biosynthesis controlled by SviR1 and SviR2 ? ___________ 106
II.3.b. Attempts to determine the SviR1 and SviR2 regulon in the svi cluster __ 108
II.3.c. Effects of deleting other genes on sviceucin production _____________ 109
II.3.d. Is sviceucin an autoinducing peptide? ___________________________ 110
II.4. Morphological characterization of S. sviceus mutants ___________________ 118
II.5. Sensitivity to oxidative stress of S. sviceus mutants ____________________ 133
III. Conclusions and perspectives _____________________________________ 135
Conclusion and perspectives _______________________________________________ 137
I. Heterologous expression of lasso peptides from Actinobacteria ______________ 137
II. Regulation mechanism and physiological role of sviceucin __________________ 138
Bibliography ____________________________________________________________ 145
Annexes ________________________________________________________________ 168
Publication ______________________________________________________________ 209

VII