Minimal Cells: Design, Construction, Biotechnological Applications

233 Pages·2020·5.654 MB·English

by  Alvaro R. Lara, Guillermo Gosset

Alvaro R. Lara
Guillermo Gosset  Editors
Minimal
Cells: Design,
Construction,
Biotechnological
Applications
Minimal Cells: Design, Construction,
Biotechnological Applications
(cid:129)
Alvaro R. Lara Guillermo Gosset
Editors
Minimal Cells: Design,
Construction,
Biotechnological
Applications
Editors
AlvaroR.Lara GuillermoGosset
DepartamentodeProcesosyTecnología DepartamentodeIngenieríaCelular
UniversidadAutónoma yBiocatálisis
Metropolitana-Cuajimalpa InstitutodeBiotecnología,Universidad
CiudaddeMexico,Mexico NacionalAutónomadeMéxico
Cuernavaca,Morelos,Mexico
ISBN978-3-030-31896-3 ISBN978-3-030-31897-0 (eBook)
https://doi.org/10.1007/978-3-030-31897-0
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Preface
The goal of industrial or white biotechnology is the generation of products and
servicesbyemployinglivingorganismsortheircomponents.Theaimisthedevel-
opment of commercially viable processes for the transformation of renewable raw
materials into useful products, thus replacing technologies based on the use of
nonrenewable fossil feedstocks whose processing and utilization generates toxic
by-products.Whilemostorganismscanbeemployedinbiotechnologicalprocesses,
microbes are widely used since they can be grown and genetically modified with
relativeease.Thedevelopmentandoptimizationofabiotechnologicalprocessentail
diverse goals; one of them is to improve the performance of a microbial cell as a
factory.Thisobjectiveismainlyachievedbyemployingawidearrayoftechniques,
collectively known as genetic engineering, which enable the modification of the
information in the genome of the organism. The capacity for genetic modification
startedasmodestchangesinspecificgeneregionsandthetransferandexpressionof
genesamongspecies.Overthelastfewyears,geneticengineeringtechniqueshave
improvedconsiderably,allowingpreciseandextensivegeneticmodifications.These
methodologies have been recently complemented by the emergence of synthetic
biology. This new field applies engineering principles to biology and is based on
mathematical modeling to design and create novel biological parts, devices, and
systems.
Thegenomeofeachorganismspecifiesallthecellularfunctionsrequiredforits
growthandsurvival.Afractionofthegenesinthegenomeisrequiredforessential
functions,whilemanyothergenesbecomeactiveonlyunderspecificconditionsin
the continuously changing natural environment. The gene set required for self-
replicating life isconsidered the minimalgenome. Approaches such asthe genera-
tionandanalysisofmutants,thecomparisonofsequencedgenomes,thegeneration
andanalysisofgenome-scalemetabolicmodels,andthestudyofbacteriawithsmall
genomes have enabled the estimation of the minimal number of genes that could
sustainlife.Thesemethodsplacethenumberofessentialgenesaroundtwotothree
hundred. In addition to its value for basic science, the minimal cell concept has
implications in biotechnology. In contrast to a natural environment, in industrial
v
vi Preface
productionprocesses,physicochemicalconditionsarehighlycontrolledtoremainat
specificvalues.Inthiscontext,manyofthegenesinthegenomearenotrequiredfor
production performance. The replication and expression of genes required for
survivalinthenaturalenvironmentcouldrepresentawasteofenergyinanindustrial
bioreactor.Inaddition,thepresenceofrepeatedandself-replicatinggeneticelements
inthegenomecanresultinproductionstraininstability.Therefore,itisexpectedthat
the elimination of genome regions that are not required for cell replication and the
synthesis of the desired product could result in minimal cells with improved pro-
ductionperformance.Acellwithareducedorminimalgenomecanbeconsideredas
a chassis where natural or synthetic functions could be added to generate a cell
specialized for the synthesis of a specific biotechnological product. Moreover, a
reducedgenomemaydecreasetherateofinteractionsbetweenthesyntheticgenetic
program and the chassis genome (e.g., insertions of genes of the genome into an
expressionvectorplasmid).
Theobjectiveofthisbookistoprovidereviewsonthecurrentknowledgerelated
tothedesign,characterization,anduseofminimalcells.Leadingexpertswrotethe
bookchaptersandincludedup-to-dateinformationaswellasthein-depthanalysisof
current issues and challenges on this topic. This book aims to become a source of
referenceforresearchersandstudentsworkinginthisfieldinacademiaandindustry.
Chaptersinthisbookdescribethespecificapproachesemployedforthegeneration
of minimal cells of the microbes Escherichia coli, Pseudomonas species, Bacillus
subtilis, Corynebacterium glutamicum, Lactococcus lactis, Streptomyces species,
Schizosaccharomyces pombe, and Saccharomyces cerevisiae. These organisms are
employed as production strains in industry and as models in basic biological
research.Genomereductionintheseorganismshasresultedinstrainswithimproved
productivityandgeneticstability.Specificchaptersalsoaddressthevariousmethods
employedtodefinetheminimalgenesetrequiredforthegenerationofminimalcells.
Genome-scalemetabolicmodelscanbeemployedtopredictgeneessentialitybased
on computational simulations and also to determine the growth phenotypes
expressed from the minimal genomes. Specific chapters also address the concepts
of gene persistence based on metabolic functions and optimal cellular resource
allocationasalternativestodefinetheminimalgenesetforthegenerationofchassis
strains.
Support from the Universidad Autónoma Metropolitana and Universidad
NacionalAutónomadeMéxicoisgratefullyacknowledgedbytheeditors.
CiudaddeMexico,Mexico AlvaroR.Lara
Cuernavaca,Mexico GuillermoGosset
July1,2019
Contents
ReducedandMinimalCellFactoriesinBioprocesses:Towards
aStreamlinedChassis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
MartinZieglerandRalfTakors
ConstructionofMinimalGenomesandSyntheticCells. . . . . . . . . . . . . 45
DonghuiChoe,SunChangKim,BernhardO.Palsson,
andByung-KwanCho
EngineeringReduced-GenomeStrainsofPseudomonasputida
forProductValorization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
NicolasT.WirthandPabloI.Nikel
Genome-ReducedCorynebacteriumglutamicumFitforBiotechnological
Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
VolkerF.Wendisch
ReductionoftheSaccharomycescerevisiaeGenome:Challenges
andPerspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
LuisCaspetaandPrisciluisCaheriSalasNavarrete
TheUseofInSilicoGenome-ScaleModelsfortheRationalDesign
ofMinimalCells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Jean-ChristopheLachance,SébastienRodrigue,andBernhardO.Palsson
FromMinimaltoMinimizedGenomes:FunctionalDesign
ofMicrobialCellFactories. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
PaulLubrano,AntoineDanchin,andCarlosG.Acevedo-Rocha
ResourceAllocationPrinciplesandMinimalCellDesign. . . . . . . . . . . . 211
DavidHidalgoandJoséUtrilla
vii
Reduced and Minimal Cell Factories
in Bioprocesses: Towards a Streamlined
Chassis
MartinZieglerandRalfTakors
Abstract The rapid advances in molecular genome engineering, systems biology
and synthetic biology over the past decade have laid the ground for extensive
engineering of bacterial and fungal genomes and the rational setup of synthetic
biological systems. In order to optimize the production host for biotechnical pro-
cesses, the genomes of many industrial workhorse microorganisms such as
Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Streptomyces
species, Pseudomonas species, Saccharomyces cerevisiae, and Lactococcus lactis
have been successfully reduced. Here, we evaluate this progress in the context of
biotechnicalapplicationfortheproductionofindustriallyattractiveproducts.Based
ontheviewofmicrobialcellsasfactories,wediscusstheconceptofrelevantgenes.
Weattempttoestimatethetheoreticalbenefitsofgenomereductionwhichformthe
basisoftargetselection.Subsequently,wecomprehensivelydiscussexistingstudies
on genome-reduced strains. The current limits of beneficial genome reduction and
potential future developments in both prokaryotic and eukaryotic systems are
considered.
Keywords Genomereduction·Minimumgenome·Microbialcellfactory·
Chassis·Essentialgenes·Relevantgenes
1 Introduction
In1982,thecompletesequencingofthegenomeofBacteriophageLambdamarked
the beginning of a new era in Biosciences (Sanger et al. 1982). The extensive
availability of sequence information promised to revolutionize our understanding
oflife.Technicalprogresssuchasshotgunsequencingtechnologyenabledsequenc-
ing of much larger genomes in the following decade with the Haemophilus
influenzae genome being the first published genome of a free-living organism
M.Ziegler·R.Takors(*)
InstituteofBiochemicalEngineering,UniversityofStuttgart,Stuttgart,Germany
e-mail:[email protected];[email protected]
©SpringerNatureSwitzerlandAG2020 1
A.R.Lara,G.Gosset(eds.),MinimalCells:Design,Construction,
BiotechnologicalApplications,https://doi.org/10.1007/978-3-030-31897-0_1
2 M.ZieglerandR.Takors
(Fleischmann et al. 1995). The complete sequence of Saccharomyces cerevisiae
assembledin1996wasnotonlythefirsteukaryoticsequencebutalsothefirstofan
economically relevant organism (Goffeau et al. 1996). Only 1 year later, the
sequences of model organisms Escherichia coli K-12 and Bacillus subtilis were
reported (Blattner et al. 1997; Kunst et al. 1997), and in 2001 sequencing of the
humangenomewasfinished(Venteretal.2001).Sincethenthousandsofgenomes
havebeensequencedandarepubliclyavailablethroughresourcessuchasGenBank
(Bensonetal.2013).
At the same time, the scientific community initiated first projects yielding cells
withareducedgenome.Theaimwasthegenerationofdeletionsuptothepointofa
minimalset(Koobetal.1994).Alreadythenamajormotivationwastosimplifythe
model organism Escherichia coli for research purposes and to improve its accessi-
bilityandpredictabilityasanindustrialworkhorseorganism.Sincethen,advancesin
molecularbiologyhaveenabledgenomemodificationsandfunctionalgenomicsina
vast variety of microorganisms and eukaryotic cell lines. With the availability of
CRISPR-Castechnology,thisprocesshasonceagainaccelerated(Fordetal.2018;
SalsmanandDellaire2017).Complementaryresearchhastargetedourunderstand-
ing and annotation of genomic information as well as the application of this
knowledge for the purpose of metabolic engineering (Stephanopoulos 1999; Tao
etal.1999).
Thiscontributionaimstoprovideanoverviewofthecurrentprogressingenome
reductionbothfromtheviewpointofthemolecularbiologistandwiththeeyesofthe
metabolicengineer.Ourconsiderationswillalwaysbedirectedtowardstheutiliza-
tionofmicrobesinanindustrialproductionscenario.Thetargetconditionsassumed
are thus controlled cultivations in bioreactor systems. We will begin with a short
presentation of the cell factory concept, followed by an in-depth look at the theo-
retical gains expected from genome reduction. Modern methods from the field of
molecular biology for the generation of genome-reduced organisms will be
addressed only superficially as they have been extensively reviewed elsewhere
(Adli 2018; Freed et al. 2018; Guha et al. 2017; Nakashima and Miyazaki 2014;
Oesterle et al. 2017). Instead we thoroughly summarize and examine published
studies on genome-reduced strains. We critically consider their potential benefits
forindustrialproduction.
2 The Concept of Microbial Cell Factories
In the early 2000s, the first generation of genome reduction studies focusing on
Escherichia coli were published (Kolisnychenko et al. 2002; Yu et al. 2002).
Remarkably, although a significant portion of the E. coli genome was deleted the
resultingstrainsdidnotshowgrowthdefects.Itwasproposedthatthecombination
of these approaches with complete essentiality libraries like the KEIO collection
might even form the basis for accurate modeling of cellular responses to real-time
changes in its environment (Baba et al. 2006; Smalley et al. 2003). Following this
ReducedandMinimalCellFactoriesinBioprocesses:Towardsa... 3
concepttheideaofcreatingcellswithaminimalgenomeresultedinvariousprojects
to reduce the genomes of several industrially relevant microorganisms with the
purpose of improving their basic production parameters (Ara et al. 2007; Giga-
Hama et al. 2007; Mizoguchi et al. 2007). On the contrary, molecular biology and
synthetic biology showed interest in finding the essential set of genes needed to
sustainlifeandtoartificiallycreateit(Burgardetal.2001;Fraseretal.1995).
Bothapproacheshaveincommonthatthecellisregardedasacomplexbutdefined
self-replicating factory. Similar to a macroscopic industrial facility, there are core
componentsthatcannotbereplacedandauxiliarycomponentsforspecialsituations.
Onamolecularlevel,thescientificcommunitygenerallyacceptstheinterpretationof
complex multi-protein structures as biological machines (Alberts and Miake-Lye
1992). A cell is consequently an assembly line consisting of such biological
machines. However, given that the number of some reaction partners in a cell is
verylow—intheextremecaseofgenomicinformationitcanequalone—metabolism
is also inherently stochastic (Kiviet et al. 2014; Kurakin 2005). The inherent
stochasticityofgrowth,enzymaticreactions,andgeneexpressionleadingtopheno-
typicvariabilityofsinglecellsinaclonalpopulationcanbeassessedbasedonsingle-
celldataandcomputationalsimulation(Kivietetal.2014;Thomasetal.2018).Under
the conditions of an industrial large-scale reactor another layer of stochasticity is
added: variations in the local environment of cells due to imperfect mixing. The
extracellularstimulifromfluctuatingsubstrategradientsenhanceexistingphenotypic
variability through their interaction with cellular regulation (Delvigne et al. 2009).
The concept of regarding cells as microbial factories holds though—as long as we
considercellularindividualityandplasticity.
Fromthesimplestpointofview,cellsconvertGibbsfreeenergyofsubstratesinto
biomass and products, thereby linking Gibbs free negative catabolic reactions with
Gibbsfreepositiveanabolism.ItshouldbenotedthatnetGibbsfreeenergyisnegative
which usually coincides with an increase in systemic entropy. Thus, the cell works
verysimilartoachemicalrefinery—withthebonusofbeingabletoduplicateitselfby
self-assembly.Fromachemicalengineer’sperspectivesuchsystemsarecalledauto-
catalytic:Oneofthereactionproducts(biomass)isacatalystforthereaction(substrate
turnover).Forasimpleconversionprocessobservedfrommaximumdistancewecan
describe this behavior as if an entire population of cells was working as a single
catalyticunit.Thisblack-boxapproachleadstoanunstructured,unsegregatedmodel
(Weuster-Botz and Takors 2018). The empiric description of the resulting process
kineticsresultsinthewell-knownMonodmodel(Monod1949).
Looking closerinto what happens inside thecellrevealsthecomplexmetabolic
network and its regulation behind this behavior. The flow of carbon through the
centralmetabolismaloneinvolvesdozensofconversions,forks,andjoints.Models
integratingthisinformationarecalledstructured.Inanalogytoarefinery,enzymes
canbeseenastheworkforce,informationcarrierproteinsserveasmanagersandthe
genome represents the administrative headquarter. If we now consider that not all
cells in a bioreactor are exactly identical, we reach a segregated model including
microbial individuality (Weuster-Botz and Takors 2018). Population heterogeneity
canariseonvariouslevels:Bioreactorpopulationsareclonalexpansionsfromsingle