Synthetic Biology and Metabolic Engineering in Plants and Microbes Part A: Metabolism in Microbes

382 Pages·2016·13.19 MB·English

by  Sarah E. O'Connor (Eds.)

METHODS IN ENZYMOLOGY
Editors-in-Chief
ANNA MARIE PYLE
Departments of Molecular, Cellular and Developmental
Biology and Department of Chemistry
Investigator, Howard Hughes Medical Institute
Yale University
DAVID W. CHRISTIANSON
Roy and Diana Vagelos Laboratories
Department of Chemistry
University of Pennsylvania
Philadelphia, PA
Founding Editors
SIDNEY P. COLOWICK and NATHAN O. KAPLAN
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CONTRIBUTORS
L.M.Alkhalaf
UniversityofBritishColumbia,Vancouver,BC,Canada
M.C.Andorfer
UniversityofChicago,Chicago,IL,UnitedStates
L.Bourgeois
CentreforStructuralandFunctionalGenomics,ConcordiaUniversity,Montr(cid:1)eal,QC,
Canada
E.Burton
CentreforStructuralandFunctionalGenomics,ConcordiaUniversity,Montr(cid:1)eal,QC,
Canada
L.Chang
UniversityofCalgary,Calgary,AB,Canada
X.Chen
UniversityofCalgary,Calgary,AB,Canada
R.E.Cobb
UniversityofIllinoisatUrbana-Champaign,Urbana,IL,UnitedStates;CarlR.Woese
InstituteforGenomicBiology,UniversityofIllinoisatUrbana-Champaign,Urbana,IL,
UnitedStates
S.D.Colloms
UniversityofGlasgow,Glasgow,UnitedKingdom
M.Dastmalchi
UniversityofCalgary,Calgary,AB,Canada
Y.-L.Du
UniversityofBritishColumbia,Vancouver,BC,Canada
S.Edgar
MassachusettsInstituteofTechnology,Cambridge,MA,UnitedStates
P.J.Facchini
UniversityofCalgary,Calgary,AB,Canada
E.Fossati
CentreforStructuralandFunctionalGenomics,ConcordiaUniversity,Montr(cid:1)eal,QC,
Canada
M.Gajewi
AllgemeineBiochemie,TUDresden,Dresden,Germany
J.M.Gardiner
SchoolofChemistry,ManchesterInstituteofBiotechnology,UniversityofManchester,
Manchester,UnitedKingdom
xi
xii Contributors
N.D.Gold
CentreforStructuralandFunctionalGenomics,ConcordiaUniversity,Montr(cid:1)eal,QC,
Canada
J.M.Hagel
UniversityofCalgary,Calgary,AB,Canada
L.Humphreys
GlaxoSmithKline,MedicinesResearchCentre,Stevenage,UnitedKingdom
A.Jervis
ManchesterInstituteofBiotechnology,SYNBIOCHEM,UniversityofManchester,
Manchester,UnitedKingdom
J.A.Jones
RensselaerPolytechnicInstitute,Troy,NY,UnitedStates;CenterforBiotechnologyand
InterdisciplinarySciences,RensselaerPolytechnicInstitute,Troy,NY,UnitedStates
M.A.G.Koffas
RensselaerPolytechnicInstitute,Troy,NY,UnitedStates;CenterforBiotechnologyand
InterdisciplinarySciences,RensselaerPolytechnicInstitute,Troy,NY,UnitedStates
J.C.Lewis
UniversityofChicago,Chicago,IL,UnitedStates
J.Li
UniversityofCalgary,Calgary,AB,Canada
W.Liu
StateKeyLaboratoryofBioorganicandNaturalProductsChemistry,ShanghaiInstitute
ofOrganicChemistry,ChineseAcademyofSciences,Shanghai;HuzhouCenterof
Bio-SyntheticInnovation,Huzhou,PRChina
V.J.J.Martin
CentreforStructuralandFunctionalGenomics,ConcordiaUniversity,Montr(cid:1)eal,QC,
Canada
D.Medina-Stacey
SchoolofChemistry,ManchesterInstituteofBiotechnology,TheUniversityofManchester,
Manchester,UnitedKingdom
C.A.Merrick
UniversityofEdinburgh,Edinburgh,UnitedKingdom
J.Micklefield
SchoolofChemistry;CentreforSyntheticBiologyofFineandSpecialityChemicals
(SYNBIOCHEM),ManchesterInstituteofBiotechnology,TheUniversityofManchester,
Manchester,UnitedKingdom
D.Milbredt
AllgemeineBiochemie,TUDresden,Dresden,Germany
J.S.Morris
UniversityofCalgary,Calgary,AB,Canada
Contributors xiii
A.N´ıCheallaigh
SchoolofChemistry,ManchesterInstituteofBiotechnology,UniversityofManchester,
Manchester,UnitedKingdom
L.Narcross
CentreforStructuralandFunctionalGenomics,ConcordiaUniversity,Montr(cid:1)eal,QC,
Canada
J.E.Paget
UniversityofEdinburgh,Edinburgh,UnitedKingdom
B.Pang
StateKeyLaboratoryofBioorganicandNaturalProductsChemistry,ShanghaiInstituteof
OrganicChemistry,ChineseAcademyofSciences,Shanghai,PRChina
E.P.Patallo
AllgemeineBiochemie,TUDresden,Dresden,Germany
J.T.Payne
University of Chicago, Chicago, IL; Stanford University, Stanford, CA,
United States
M.E.Pyne
CentreforStructuralandFunctionalGenomics,ConcordiaUniversity,Montr(cid:1)eal,QC,
Canada
C.J.Robinson
SchoolofChemistry;CentreforSyntheticBiologyofFineandSpecialityChemicals
(SYNBIOCHEM),ManchesterInstituteofBiotechnology,TheUniversityofManchester,
Manchester,UnitedKingdom
S.J.Rosser
UniversityofEdinburgh,Edinburgh,UnitedKingdom
K.S.Ryan
UniversityofBritishColumbia,Vancouver,BC,Canada
D.Sardar
UniversityofUtah,SaltLakeCity,UT,UnitedStates
E.W.Schmidt
UniversityofUtah,SaltLakeCity,UT,UnitedStates
N.S.Scrutton
ManchesterInstituteofBiotechnology,FacultyofLifeSciences,UniversityofManchester,
Manchester,UnitedKingdom
G.Stephanopoulos
MassachusettsInstituteofTechnology,Cambridge,MA,UnitedStates
S.Tait
ManchesterInstituteofBiotechnology,FacultyofLifeSciences,UniversityofManchester,
Manchester,UnitedKingdom
xiv Contributors
E.Takano
ManchesterInstituteofBiotechnology,FacultyofLifeSciences,UniversityofManchester,
Manchester,UnitedKingdom
Z.Tang
StateKeyLaboratoryofBioorganicandNaturalProductsChemistry,ShanghaiInstituteof
OrganicChemistry,ChineseAcademyofSciences,Shanghai,PRChina
M.D.Tianero
UniversityofUtah,SaltLakeCity,UT,UnitedStates
H.S.Toogood
ManchesterInstituteofBiotechnology,FacultyofLifeSciences,UniversityofManchester,
Manchester,UnitedKingdom
J.W.A.vanDijk
SchoolofPharmacy,UniversityofSouthernCalifornia,LosAngeles,CA,UnitedStates
K.-H.vanP(cid:1)ee
AllgemeineBiochemie,TUDresden,Dresden,Germany
H.A.Vincent
SchoolofChemistry,ManchesterInstituteofBiotechnology,TheUniversityofManchester,
Manchester,UnitedKingdom
C.C.C.Wang
SchoolofPharmacy;DornsifeCollegeofLetters,Arts,andSciences,UniversityofSouthern
California,LosAngeles,CA,UnitedStates
Y.Wang
UniversityofIllinoisatUrbana-Champaign,Urbana,IL,UnitedStates
C.Wardrope
UniversityofEdinburgh,Edinburgh,UnitedKingdom
V.Weichold
AllgemeineBiochemie,TUDresden,Dresden,Germany
M.-C.Wu
SchoolofChemistry,ManchesterInstituteofBiotechnology,TheUniversityofManchester,
Manchester,UnitedKingdom
H.Zhao
UniversityofIllinoisatUrbana-Champaign,Urbana,IL,UnitedStates;CarlR.Woese
InstituteforGenomicBiology,UniversityofIllinoisatUrbana-Champaign,Urbana,IL,
UnitedStates
G.Zhong
StateKeyLaboratoryofBioorganicandNaturalProductsChemistry,ShanghaiInstituteof
OrganicChemistry,ChineseAcademyofSciences,Shanghai,PRChina
K.Zhou
NationalUniversityofSingapore,Singapore,Singapore
PREFACE
Advancesinsequencing,bioinformatics,and genomeeditingnowenableus
toaccesstherichchemistryencodedwithinthemetabolicpathwaysofplants
and microbes. A major focus in metabolism is the secondary, or specialized,
pathways that produce small, biologically active molecules with applications
inpharmaceutical,agrochemical,orotherbiotechnologicalsectors.Inrecent
years, metabolic engineering/synthetic biology approaches have shown
remarkable promise for the exploitation of these pathways for human use.
Inthesetwovolumes,wehighlightsome ofthemost important approaches
that have been used to harness microbial and plant metabolic pathways.
In Volume 1, we focus on advances that have been made in microbial-
basedsystems.Thediscoverysomethreedecadesagothatbacterialspecial-
izedmetabolicpathwaysareclusteredonthegenomehasgreatlyfacilitated
the identification and characterization of these pathway genes. Coupled
withthefactthatbacterialgenomescannowbesequencedrapidlyandinex-
pensively,thelastdecadehasseenastaggeringincreaseinourknowledgeof
bacterial specialized metabolism. Additionally, it is now known that fungal
specializedpathwaysalsoclusteronthegenome.Whilefungalgenomesare
larger than those from bacteria, these genomes can still can be easily
sequenced, and substantial advances in elucidating fungal metabolism have
beenmade.Consequently,awealthofnewopportunitiesinmetabolicengi-
neeringhavebeenopened.Inthisvolume,wehighlighthowbetterproduc-
tion of these compounds can be achieved, and how these biosynthetic
enzymescanbeengineeredtogeneratenewbiocatalystsandnewproducts.
Wealsodiscusshowmicrobialspeciescanbemanipulatedtoserveasahost
for reconstitution of plant pathways. The volume concludes with several
representative examples of new tools that allow us to rapidly manipulate
the genetic material of the microbial host.
Volume2focusesonthemetabolismofplants.Historically,elucidating
plant metabolism has been challenging due to the lack of tightly genome-
clustered pathways that are observed in microbial systems, along with the
large size of plant genomes and transcriptomes. The first set of articles in
this volume describe a variety of strategies to elucidate plant-specialized
metabolism. Notably, the specialized metabolism of plants is controlled
bycomplexregulatoryprocesses.Furthermore,plantbiosyntheticprocesses
are also complicated by the fact that the metabolic reactions occur in a
xv
xvi Preface
variety of different cell types and subcellular compartments. Therefore,
several articles in thisvolume alsodescribe effortsto control the regulatory
networksthatmaintainthelevelsofmetabolismproductioninplants,along
withmethodstounderstandthemechanismsoftransportandlocalizationof
specialized metabolic intermediates. Finally, we highlight emerging tools
to harness plant metabolism: new plant-based expression platforms and
expression tools for production of metabolites are discussed.
Metabolic engineering has progressed rapidly in the last several years.
The advent of genome editing, the ability to sequence complex genomes
quickly and inexpensively, and the successful manipulation of plant and
microbial hosts for moreeffectivepathway reconstitutionhave collectively
demonstrated that metabolic engineering holds substantial promise for
improving our access to the end products of specialized metabolism.
Inotethatthesetwovolumesscratchthesurfaceofthisfield,providingonly
asurveyofsomeoftheeffortsbeingmadeinthisarea.Iamdeeplyindebted
toallofthecontributorstothisvolumewhograciouslyprovidedtheirtime
and effort to make a contribution to this work.
S.E. O’CONNOR
The John Innes Centre
Department of Biological Chemistry
CHAPTER ONE
Directing Biosynthesis: Practical
Supply of Natural and Unnatural
Cyanobactins
D. Sardar, M.D. Tianero, E.W. Schmidt1
UniversityofUtah,SaltLakeCity,UT,UnitedStates
1Correspondingauthor:e-mailaddress:[email protected]
Contents
1. Introduction 1
2. DiscoveryofCyanobactinPathways 2
3. ElucidatingNaturalRulesofEngineeringinCyanobactinPathways 3
4. HeterologousExpressionofCyanobactinPathwaysinE.coli 5
5. OptimizationforIncreasedYieldofCyanobactinsinE.coli 9
6. SynthesisofCyanobactinsInVitro 11
7. Conclusions 16
8. Outlook 18
Acknowledgments 18
References 18
Abstract
Theincreasinglyrapidaccumulationofgenomicinformationisrevolutionizingnatural
products discovery. However, the translation of sequence data to chemical products
remainsachallenge.Here,wedetailmethodsusedtocircumventthesupplyproblem
ofcyanobactinnaturalproducts,bothbyengineeredsynthesisinEscherichiacoliandby
usingpurifiedenzymesinvitro.Suchmethodologiesexploitnature'sstrategiesofcom-
binatorialchemistryinthecyanobactinclassofRiPPnaturalproducts.Asaresult,itis
possibletosynthesizeawidevarietyofnaturalandunnaturalcompounds.
1. INTRODUCTION
Naturalproductsremainamajorcomponentofdrugdiscoveryefforts
(Newman&Cragg,2012),butsucheffortsarestillhinderedbytheneedto
synthesizeasufficientsupplyandtocreateanalogs.Mimickingbiosynthesis
has emerged as a powerful tool to overcome these problems, providing a
tractable alternative to traditional chemical synthesis. The key advance
MethodsinEnzymology,Volume575 #2016ElsevierInc. 1
ISSN0076-6879 Allrightsreserved.
http://dx.doi.org/10.1016/bs.mie.2016.02.012
2 D.Sardaretal.
thathasaidedthisistheexplosioningenomicdata,whichhasallowedcon-
nectingnaturalproductstotheircorrespondinggenes(Walsh&Fischbach,
2010).Inturn,rapidgeneidentificationenablesheterologousexpressionof
enzymestoperformtotalsynthesisinvivoorinvitro.Connectinggenesto
molecules has thus aided discovery based on sequencing, overcoming
limitations of conventional natural products discovery, which relies on
actual physical isolation of compounds, often from far-reaching locations
of the earth. In addition, assigning genes to specific chemical modifica-
tions allows exploitation of enzyme function to modify nature’s arsenal
of chemistry far beyond, by technologies such as directed evolution
(Renata, Wang, & Arnold, 2015).
Thischapterfocusesondirectingbiosynthesisusingthecyanobactinbio-
synthetic machinery to engineer synthesis. Cyanobactins are natural prod-
ucts found in marine animals or in relatively slow-growing cyanobacteria.
Duetotherareandvariabledistributionoftheproducingorganisms,supply
isanissuehinderingcyanobactindevelopment.Anadvantageisthatseveral
cyanobactin pathways are exceptionally broad-substrate tolerant, which
allows cyanobactin enzymes and pathways to be used in the synthesis of
thousands of derivatives. Recent advances enable such synthesis at scale in
Escherichia coli and in vitro using purified enzymes. Later, we describe
methods enabling the practical synthesis and engineering of cyanobactin
pathways.
2. DISCOVERY OF CYANOBACTIN PATHWAYS
ThecyanobactinsbelongtoRiPP(ribosomallysynthesizedandpost-
translationallymodifiedpeptides)classofnaturalproductsandarepresentin
about 30% of all cyanobacteria (Arnison et al., 2013; Sivonen, Leikoski,
Fewer, & Jokela, 2010). The first cyanobactins, patellamides, were isolated
from marine ascidian animals (Ireland & Scheuer, 1980), and related com-
poundshavealsobeenfoundinbothascidiansandfree-livingcyanobacteria.
Subsequently,biosyntheticgenesforcyanobactinswerediscovered.Asmall
sample (several grams) of the ascidian Lissoclinum patella was obtained near
breaking surf on the reef flat above Blue Corner, Palau. Metagenome
sequencingofthemarineanimalanditssymbioticbacterialedtoidentifica-
tionofaRiPPpathway,patinsymbioticcyanobacteria,Prochloron.patcar-
riedageneencodingtheaminoacidsequenceofpatellamide.Heterologous
expressionofpatinE.coliconfirmedthesymbiontProchloronasthesourceof