INVITATION
To attend the defence of my PhD
thesis:
“Reducing the genetic complexity
of glycolysis in Saccharomyces
cerevisiae”
on Monday
March 16
th, 2015 at 12:30
in the Aula of TU Delft
Mekelweg 6, Delft
Prior to the defence (12:00)
there will be a presentation
of the thesis for non-experts.
You are also invited to the
reception which follows
in t’Keldertje,
Department of Biotechnology,
Julianalaan 67, Delft
Daniel Solís Escalante
danielsoes@gmail.com
Paranymphs:
Teodoro Solís Escalante
teosoet@gmail.com
Stefan de Kok
stefandekok@gmail.com
Invitation_17022015.pdf 1 17-2-2015 11:00:11
Glycolysis is at the core of the metabolism in
Saccharomyces cerevisiae (baker’s yeast) with several of
its intermediates serving as precursors for a wide range
of valuable compounds. Despite the large amount of
information available about all individual glycolytic
components, the limited understanding on how they
interact and are coordinated has defied all metabolic
engineering attempts to significantly accelerate glycolytic
flux. A particularly poorly understood factor in glycolysis is
its genetic redundancy.
The tendency of S. cerevisiae to produce ethanol under
aerobic conditions (Crabtree effect) and its high glycolytic
capacity were proposed to be the result of a
whole-genome duplication event and resulting duplication
of all glycolytic genes in S. cerevisiae’s ancestor (ca. 100
million years ago). The contribution of the glycolytic
paralogs to the glycolytic flux is unknown and the
simultaneous presence of different isoenzymes
complicates mathematical predictions.
Reduction of the genetic complexity in S. cerevisiae’s
glycolysis could deliver a more predictable and malleable
glycolytic pathway. These characteristics could greatly
benefit the biotechnology industry and our understanding
about glycolysis. Additionally, such a system could serve
as a basis for a more extreme genetic and pathway
engineering strategy aiming at the redesign of glycolysis.
This thesis focuses on the construction and analysis of a
S. cerevisiae strain with a genetically reduced glycolysis.
Reducing the genetic
complexity of glycolysis
in Saccharomyces
cerevisiae
Daniel Solís Escalante
Reducing the genetic complexity of glycolysis in
Saccharomyces cerevisiae
Daniel Solís Escalante
Reducing the genetic complexity of glycolysis in
Saccharomyces cerevisiae.
Proefschrift
ter verkrijging van de graad van doctor aan de Technische Universiteit Delft,
op gezag van de Rector Magnificus prof. ir. K.Ch.A.M. Luyben, voorzitter van het College voor Promoties,
in het openbaar te verdedigen op Maandag 16th 2015 om 12:30 uur
door
Daniel SOLIS ESCALANTE
ingenieur in Life Science and Technology geboren te Mexico City, Mexico.
This dissertation has been approved by the Promotor: Prof. dr. J.T. Pronk
Copromotor:
Dr. ir. P. Daran-Lapujade
Composition of the doctoral committee:
Rector Magnificus chairperson
Prof. dr. J.T. Pronk Technische Universiteit Delft, promotor Dr. ir. P. Daran-Lapujade Technische Universiteit Delft, copromotor Dr. J-M Daran Technische Universiteit Delft
Independent members:
Prof. dr. U. Hanefeld Technische Universiteit Delft Prof. C. Gancedo Universidad Autónoma de Madrid Prof. dr. B. Teusink Vrije Universiteit Amsterdam Dr. P. Kötter Goethe University Frankfurt
The studies presented in this thesis were performed at the Industrial Microbiology section, Department of Biotechnology, Delft University of Technology, the Netherlands and financed by the Ministry of Education, Culture and Science via the Netherlands Organisation for Scientific Research (NOW) through the Technology foundation STW (Vidi Grant 10776).
Table of Contents
Samenvatting / Summary ...1
Chapter 1 ...13
General introduction
Chapter 2 ...35
The genome sequence of the popular hexose-transport-deficient
Saccharomyces cerevisiae strain EBY.VW4000 reveals LoxP/Cre-induced
translocations and gene loss
Chapter 3 ...59
amdSYM, a new dominant recyclable marker cassette for Saccharomyces
cerevisiae
Chapter 4 ...79
Efficient simultaneous excision of multiple selectable marker cassettes using
I-SceI-induced double-strand DNA breaks in Saccharomyces cerevisiae
Chapter 5 ...103
A Saccharomyces cerevisiae strain with a minimal complement of glycolytic
genes reveals strong redundancies in central metabolism
Acknowledgments ...137
Curriculum vitae ...141
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
2
Samenvatting
van het proefschrift
“Vermindering van de genetische complexiteit van glycolyse in
Saccharomyces cerevisiae”
Glycolyse, een biochemische route die glucose oxideert tot pyrodruivenzuur, vormt de kern van de suikerstofwisseling in Saccharomyces cerevisiae (bakkersgist). Glycolyse is niet alleen belangrijk voor de energiehuishouding van de cel, maar levert ook bouwstenen voor groei van nieuwe cellen. Daarnaast zijn verschillende glycolytische intermediairen belangrijke bouwstenen voor de productie van een breed scala aan waardevolle verbindingen. Het meest voor de hand liggende voorbeeld is de productie van ethanol vanuit pyrodruivenzuur. Gezien haar vermogen snel suikers naar ethanol om te zetten (‘fermenteren’), is S. cerevisiae een belangrijke speler in grootschalige productie van biobrandstoffen. Vanwege het belang van glycolyse in cellulaire processen en in de biotechnologie industrie, is deze stofwisselingsroute in S. cerevisiae tot in detail bestudeerd. Ondanks de grote hoeveelheid informatie over alle onderdelen van de glycolyse, heeft ons beperkte begrip van hoe deze onderdelen samenwerken en worden gereguleerd ertoe geleid dat, tot nu toe, alle pogingen om de glycolytische ‘flux’ aanzienlijk te verhogen niet succesvol zijn geweest. Tot dusver zijn de mechanismen die de glycolytische flux bepalen niet volledig bekend. Een bijzonder slecht begrepen factor is de aanwezigheid van meerdere genen per glycolytische stap. Dit fenomeen wordt waargenomen in meerdere organismen, maar vooral S. cerevisiae bezit meerdere isoenzymen en bijbehorende paraloge genen voor de meeste glycolytische reacties. De bijdrage van de verschillende glycolytische paralogen aan de glycolytische flux is onbekend en de gelijktijdige aanwezigheid van verschillende isoenzymen – met mogelijk verschillende kinetische en regulatoire eigenschappen – compliceert het wiskundig modelleren dat nodig is voor een beter begrip van de regulatie van deze industrieel relevante stofwisselingsroute.
De glycolytische stofwisselingsroute van S. cerevisiae bestaat uit tien biochemische reacties. In deze ‘Crabtree-positieve’ gist is glycolyse onder de meeste omstandigheden gekoppeld aan ethanolvorming. Zonder het zeer complexe transport van glucose vanuit de extracellulaire naar de intracellulaire omgeving te beschouwen, omvat glycolyse en ethanolvorming samen 12 biochemische reacties. Deze biochemische reacties worden gekatalyseerd door enzymen die gecodeerd zijn in 27 glycolytische genen, welke kunnen worden verdeeld in acht paraloge families en vier unieke structurele genen.
S. cerevisiae is geëvolueerd vanuit een voorouder die, ongeveer 100 miljoen jaar
geleden, een duplicatie van het gehele genoom onderging. Het is gesuggereerd dat veel van haar fenotypische kenmerken het resultaat zijn van deze duplicatie en de daaropvolgende herinrichting van het genoom en verlies en verkrijging van genen door evolutie. In S. cerevisiae
Samenvatting/Summary
3
worden niet minder dan acht van de tien enzymatische reacties in de glycolyse gecodeerd door meerdere paraloge genen. Het complete gistgenoom bevat ongeveer 26% paraloge genen; glycolyse bevat dus een sterke oververtegenwoordiging van paraloge genen. Het is gesuggereerd dat de duplicatie van het gehele genoom en de resulterende dubbele set aan glycolytische genen heeft bijgedragen aan de sterke neiging van deze gist ethanol te vormen onder aërobe omstandigheden (‘Crabtree effect’) en aan haar hoge glycolytische capaciteit. Echter, het effect van het verminderen van het aantal glycolytische paralogen op deze en andere fysiologische kenmerken van S. cerevisiae is niet systematisch onderzocht. Alle glycolytische reacties zijn even essentieel voor groei van gist op glucose. Echter, voor alle paraloge genensets in de glycolyse van gist, met de opmerkelijke uitzondering van fosfofructokinase, ondersteunen genexpressie en gendeletiestudies de hypothese van een enkel belangrijk paraloog gen per reactie, met daarnaast één tot vier minder belangrijke paraloge genen. Daarnaast hebben, behalve voor de pseudogenen GPM2 en
GPM3, alle paralogen hun katalytische functie behouden, hoewel hun contextafhankelijke
expressieprofielen verschillen. Tenslotte heeft verwijdering van de minder belangrijke paralogen voor individuele glycolytische reacties een beperkt effect op de enzymactiviteit in celextracten en op de specifieke groeisnelheid onder laboratoriumcondities.
Vermindering van de genetische complexiteit van de glycolyse van S. cerevisiae kan een meer voorspelbare en aanpasbare glycolyse opleveren. Deze kenmerken kunnen veel voordelen opleveren voor de biotechnologie industrie en ons begrip van de glycolyse. Daarom was het doel van dit proefschrift om een giststam met een minimale set aan glycolytische enzymen te construeren en te bestuderen.
Ondanks de verbazingwekkende genetische toegankelijkheid van S. cerevisiae en de uitgebreide genetische gereedschapskist is grootschalige verwijdering van genen, zoals het verwijderen van 13 genen tijdens dit project, een uitdaging, die – aan het begin van dit project – slechts enkele andere studies hadden ondernomen. Ondanks de grote collectie van selecteerbare genetische markers die beschikbaar zijn voor genetische modificatie van
S. cerevisiae was de beschikbaarheid van markergenen een hindernis wanneer tientallen
genen verwijderd moeten worden. Bovendien kan de aanwezigheid van een markergen in het gastheergenoom de fitheid of prestaties van een stam beïnvloeden. Daarom zijn methoden vereist om markergenen te verwijderen uit het genoom.
Verwijdering van alle hexosetransportergenen (HXT’s) in S. cerevisiae heeft, voor meer dan een decennium, model gestaan voor de vermindering van genetische complexiteit in een grote familie van paraloge genen met overlappende functie. Bakkersgist bevat een grote groep strikt gereguleerde transporteiwitten met verschillende eigenschappen voor opname van glucose. Deze familie bestaat uit genen met soortgelijke functie, maar welke tot expressie komen onder verschillende omstandigheden, waardoor deze gist kan groeien onder en kan omgaan met grote en dynamische veranderingen in glucoseconcentratie. Opmerkelijk is
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
4
dat verwijdering van een of meerdere HXT’s de capaciteit om op glucose te groeien niet opheft. In 1999 hebben Eckhard Boles en zijn team een enorme inspanning geleverd door alle transporteiwitten die in staat zijn glucose te importeren in S. cerevisiae te verwijderen, resulterend in de stam EBY.VW4000. Sinds haar constructie is EBY.VW4000 uitgegroeid tot een veelgebruikt platform voor de ontdekking en karakterisering van transporteiwitten uit een breed scala aan organismen, en als platform voor metabolic engineering projecten. Daarnaast kan EBY.VW4000, met haar minder complexe genetica rondom glucose transport, dienen als een prachtig platform voor de constructie van een stam met een minimale glycolyse. Ondanks het uitgebreide gebruik van deze stam, was de genoomsequentie van EBY.VW4000 tot dit project nog niet in detail gekarakteriseerd. In hoofdstuk 2 wordt
ingegaan op deze ontbrekende informatie en wordt de volledige genoomsequentie van EBY. VW4000 gepresenteerd. Om de opname van glucose uit te schakelen in deze stam, zijn 21 genen (inclusief alle HXT’s) verwijderd tijdens 16 opeenvolgende rondes van gendeletie, gebruik makend van het LoxP / Cre-systeem. Met behulp van een combinatie van analyse van de genoomsequentie, karyotypering en moleculaire analyses, hebben we aangetoond dat de constructie van EBY.VW4000 heeft geleid tot verlies van genen en herschikking van chromosomen, in het specifiek veroorzaakt door het LoxP / Cre-systeem. Daarentegen werden slechts 13 puntmutaties geïdentificeerd. Recombinatie tussen LoxP ‘littekens’ heeft geleid tot de vorming van vier ‘neo-chromosomen’, inkorting van twee chromosomen en het verlies van twee telomeerregio’s. Door karyotypering van de voorouders van EBY. VW4000 werd duidelijk dat de huidige inrichting van de chromosomen het resultaat is van vier translocaties die zich tussen de 6e en de 12e ronde van gendeletie en markerrecycling heeft
plaatsgevonden. Daarnaast bleken sporulatie en sporevorming ernstig aangetast in EBY. VW4000. Deze studie toonde ook aan dat, vanwege de enorme genoomveranderingen door het gebruik van het LoxP / Cre-systeem, zowel EBY.VW400 als het LoxP / Cre-systeem niet geschikt zijn voor de constructie van een stam met een minimale glycolyse. Daarom hebben wij klassieke genetische verwijdering gebruikt en zijn nieuwe methoden voor genetische modificatie ontwikkeld en geïmplementeerd.
In hoofdstuk 3 wordt de nieuwe recyclebare dominante markercassette amdSYM
gepresenteerd, welke bestaat uit de Ashbya gossypii TEF2 promoter en terminator en een codon-geoptimaliseerd acetamidase gen (Aspergillus nidulans amdS). Deze module stelt laboratorium, wilde en industriële Saccharomyces stammen in staat om acetamide te gebruiken als enige stikstofbron. Identieke DNA sequenties aan weerszijden van de markercassette zorgen voor een efficiënte verwijdering van de marker door recombinatie. Voor verlies van de cassette kan snel worden geselecteerd door te groeien in de aanwezigheid van fluoracetamide. De admSYM cassette kan in verschillende genetische achtergronden worden gebruikt en vertegenwoordigt de eerste dominante genetische marker voor S. cerevisiae stammen voor wier verlies kan worden geselecteerd. Tenslotte kan, door slim ontwerp van de cassette, de amdSYM cassette worden verwijderd zonder enig litteken of heterologe DNA sequentie achter te laten in het genoom. De amdSYM cassette is beschikbaar voor de
Samenvatting/Summary
5
wetenschappelijke gemeenschap via de Euroscarf collectie.
Voor S. cerevisiae zijn slechts vier genetische markers beschikbaar voor wier verlies kan worden geselecteerd, waaronder de amdSYM marker cassette. Uitgebreide genetische modificatie van stammen wordt ernstig belemmerd door deze beperkte beschikbaarheid van markergenen, en door de verminderde stabiliteit van het genoom die optreedt bij herhaaldelijk gebruik van heterologe recombinase-gebaseerde markerverwijderingsmethoden, zoals het LoxP / Cre-systeem. Hoofdstuk 4 introduceert een efficiënte methode om meerdere
markers tegelijkertijd te recyclen in S. cerevisiae, daarmee de tekortkomingen van bestaande technieken omzeilend en het proces van selectie en markerverwijdering aanzienlijk versnellend. Deze methode is gebaseerd op de kunstmatige introductie van een breuk in beide strengen van het DNA rondom de markercassette met behulp van de meganuclease I-SceI en het daaropvolgende herstel van deze breuk door de gist-eigen machinerie voor homologe recombinatie, gestuurd door identieke DNA sequenties aan beide zijden van de markercassette. Gelijktijdige verwijdering van drie markercassettes werd bereikt met hoge efficiëntie (tot 56%). Deze locus- en marker-onafhankelijke werkwijze kan worden gebruikt voor verwijdering van zowel dominante markergenen en markergenen die auxotrofiën opheffen.
Hoofdstuk 5 beschrijft de experimentele verkenning van de genetische redundantie
van de glycolyse van gist door opeenvolgende verwijdering van alle minder belangrijke paraloge genen, en presenteert een nieuw experimenteel platform voor fundamenteel gistonderzoek door de constructie van een giststam met een functionele, minimale glycolyse. De constructie van deze stam werd uitgevoerd met behulp van zowel klassieke genetische modificatietechnieken als de nieuw ontwikkelde methoden gepresenteerd in dit proefschrift. Het project omvatte verwijdering van 13 glycolytische paraloge genen die beschouwd worden weinig bijdrage te leveren aan de glycolytische flux. Na grondige experimentele analyse, gebruik makend van kwantitatieve en systematische benaderingen, onder groeicondities die leiden tot een hoge glycolytische flux, en na semi-kwantitatieve analyse onder een breed scala aan groeicondities, bleek verrassend dat de verwijdering van deze 13 genen geen zichtbare fenotypische effect opleverde. De hoge glycolytische fluxen tijdens anaërobe cultivatie van de ‘minimale glycolyse stam’ en het kleine effect op het transcriptoom suggereren dat het aantal genen of het back-up effect geen redenen zijn om de paralogen te behouden in het genoom. De groeikinetiek van de minimale glycolyse stam onder aërobe en anaërobe condities is bijna gelijk aan die van een wild-type. Dit valt moeilijk te rijmen met de hypothese dat duplicatie van de glycolytische genen tijdens de duplicatie van het complete genoom een belangrijke rol heeft gespeeld in het vergroten van de glycolytische capaciteit, of in het veroorzaken van het Crabtree effect. Deze vermindering van de genetische complexiteit heeft bijgedragen aan het begrijpen van de glycolyse van gist door, voor het eerst, tegelijkertijd het effect van verwijdering van meerdere glycolytische genen te bestuderen. Bovendien heeft deze aanpak de intrinsieke onzekerheid geëlimineerd die wordt veroorzaakt door de gelijktijdige,
context-Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
6
afhankelijke expressie van de verschillende isoenzymen. Dit draagt bij aan de ontwikkeling en validatie van wiskundige modellen die de kinetiek van deze belangrijke stofwisselingsroute beschrijven. Analyse van de fysiologie en fitheid van de minimale glycolyse stam onder kunstmatige en natuurlijke dynamische omstandigheden kan bijdragen aan de lastige, maar spannende taak van het oplossen van de oorsprong, het lot, de evolutie en de rol van glycolytische paraloge genen. Daarnaast zal deze stam dienen als een basis voor projecten waarin het genoom en stofwisselingsroutes meer extreem worden gemodificeerd, aangezien verschillende glycolytische stofwisselingsroutes kunnen worden herontworpen en verwisseld in twee eenvoudige stappen.
Samenvatting/Summary
7
Summary
of the PhD thesis
“Reducing the genetic complexity of glycolysis in Saccharomyces
cerevisiae”
Glycolysis, a biochemical pathway that oxidizes glucose to pyruvate, is at the core of sugar metabolism in Saccharomyces cerevisiae (bakers’ yeast). Glycolysis is not only a catabolic route involved in energy conservation, but also provides building blocks for anabolism. From an applied perspective, several glycolytic intermediates are key precursors for the production of a wide range of highly valuable compounds. The most obvious case is the production of ethanol from pyruvate. Its ability to rapidly ferment sugars to ethanol has made S. cerevisiae the major microbial player in large-scale biofuel production. Because of its importance in cellular processes and in the biotechnology industry, glycolysis in S. cerevisiae has been studied in detail. However, despite the large amount of information generated about all the components in glycolysis, the limited understanding on how these components interact and are co-ordinately regulated to ensure a robust and balanced pathway, has to date defied all metabolic engineering attempts to significantly accelerate glycolytic flux. So far, the mechanisms that govern the glycolytic flux are not fully known. A particularly poorly understood factor in glycolysis is its high genetic redundancy. This phenomenon is observed in many organisms, but is highly pronounced in S. cerevisiae which, for most glycolytic reactions, harbours multiple isoenzymes and corresponding paralogous genes. The contribution of the glycolytic paralogs to the glycolytic flux is unknown and the simultaneous presence of different isoenzymes – with potentially different kinetic and regulatory properties - complicates the mathematical modelling that is required for a deeper understanding of the regulation of this industrially relevant pathway.
Saccharomyces cerevisiae’s glycolytic pathway consist of ten biochemical reactions.
In this Crabtree positive yeast, glycolysis is under most growth conditions linked to ethanol formation. Without considering the very complex transport of glucose from the extracellular environment to the intracellular compartment, glycolysis and ethanol fermentation together encompass 12 biochemical reactions. These biochemical reactions are catalysed by enzymes encoded in 27 glycolytic genes, separated in eight paralog families and four unique structural genes.
S. cerevisiae evolved from an ancestor that, approximately 100 million years ago,
underwent a whole genome duplication (WGD). Many of its hallmark phenotypic characteristics have been proposed to be the result of this duplication event and the subsequent genome rearrangement, gene loss and gains through evolution. In S. cerevisiae, no fewer than eight of the ten enzyme reactions in glycolysis are represented by multiple paralogs genes. This
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
8
incidence of paralogous combinations represents a significant overrepresentation relative to the ca. 26 % of the yeast genome that consists of paralogous combinations. The WGD event and resulting duplication of glycolytic genes has been proposed to have contributed to the strong tendency of this yeast to produce ethanol under aerobic conditions (Crabtree effect) and to its high glycolytic capacity. However, the impact of reducing the number of glycolytic paralogs on these and other physiological characteristics of S. cerevisiae has not been systematically explored. All glycolytic reactions are equally essential for yeast growth on glucose. However, for all paralogs gene sets in yeast glycolysis, with the notable exception of phosphofructokinase, gene expression and gene deletion studies support the definition of a single, major paralog and one to four minor paralogs. Additionally, except for the pseudogenes
GPM2 and GPM3, all paralogs have retained their original catalytic function, although their
context-dependent expression profiles differ. Furthermore, deletion of minor paralogs for individual glycolytic enzymes has minor effects on enzyme activities in cell extracts and on specific growth rate under standard laboratory conditions.
Reduction of the genetic complexity in S. cerevisiae’s glycolysis could deliver a more predictable and malleable glycolytic pathway. These characteristics could greatly benefit the biotechnology industry and our understanding about glycolysis. Thus, the main goal of this thesis was the construction and analysis of a strain with a minimal set of glycolytic enzymes.
Despite the astonishing genetic accessibility of S. cerevisiae and its broad genetic toolbox, large-scale deletion strategies, like the deletion of 13 genes undertaken in the present thesis, were a challenging endeavour at the outset of this project, that only few previous studies had tackled. Despite the large collection of selectable marker genes for genetic modification available for S. cerevisiae, marker availability still presented a hurdle when dozens of genetic deletions were required. Additionally, the presence of the selectable markers in the host genome can influence the fitness or performance of the strain, thus, different methods for marker removal were required.
Deletion of the hexose transporter genes (HXT’s) in S. cerevisiae has, for over a decade, represented the paradigm for elimination of complexity in a large, redundant paralog family. This yeast harbours a large group of tightly controlled transporters with different characteristics for glucose uptake. This family is composed of genes with similar function but that are expressed under different conditions, thereby allowing yeast to grow and cope with large and dynamic changes in glucose concentration. Interestingly, deletion of single or several HXT’s does not abolish growth on glucose. In 1999, Eckhard Boles and his team took on the enormous endeavour of removing all transporters capable to import glucose in S. cerevisiae, resulting in the strain EBY.VW4000. Ever since its construction, EBY.VW4000 has become a widely used platform for the discovery and characterization of transporters from a wide range of organisms and as a platform strain for metabolic engineering approaches. Additionally, EBY.VW4000, with its reduced genetic redundancy in glucose transport, could serve as an splendid platform
Samenvatting/Summary
9
for the construction of a minimal glycolytic pathway. Despite the extensive usage of this strain, the genome of EBY.VW4000 had hitherto not been characterized in detail. Chapter 2
addresses this information gap and presents the whole-genome sequence of EBY.VW4000. To abolish glucose uptake in this strain, 21 genes (including all HXT’s) had been knocked-out across 16 successive deletion rounds with the LoxP/Cre system. Based on a combination of whole-genome sequencing, karyotyping and molecular confirmation, we demonstrated that the construction of EBY.VW4000 resulted in gene losses and chromosomal rearrangements guided by LoxP/Cre. In contrast, only 13 single nucleotide variations (SNV’s) were identified. Recombinations between LoxP scars led to the assembly of four neo-chromosomes, truncation of two chromosomes and the loss of two telomeric regions. By karyotyping the EBY.VW4000 lineage, it became clear that its current chromosomal architecture has resulted from four translocations events that occurred between the 6th and the 12th rounds of deletion/marker recycling. Additionally, sporulation and spore germination were found to be severely impaired in EBY.VW4000. This work also demonstrated that, due to the massive LoxP/Cre-induced genome modifications observed, neither EBY.VW4000 nor LoxP/Cre were suitable for the construction of a minimal glycolysis strain. Therefore, a combination of classical genetic deletion and novel tools and methodologies were developed and implemented.
In Chapter 3 the new recyclable dominant marker cassette amdSYM, formed by the
Ashbya gossypii TEF2 promoter and terminator and a codon-optimized acetamidase gene
(Aspergillus nidulans amdS) is presented. This module confers laboratory, wild and industrial
Saccharomyces strains the ability to use acetamide as sole nitrogen source. Direct repeats
flanking the marker cassette allow for its efficient recombinative excision. This cassette loss can be rapidly selected for by growth in the presence of fluoroacetamide. The amdSYM cassette can be used in different genetic backgrounds and represents the first counterselectable dominant marker gene cassette for use in Saccharomyces strains. Furthermore, using astute cassette design, amdSYM excision could be performed without leaving a scar or heterologous sequences in the targeted genome. The amdSYM cassette is available for the scientific community via the Euroscarf collection.
Including the amdSYM marker cassette, only four counter-selectable markers for S.
cerevisiae are available. Extensive strain engineering is severely hampered by this limited
marker availability and by the reduced genome stability that occurs upon repeated use of heterologous recombinase-based marker removal methods such as LoxP/Cre system.
Chapter 4 introduces an efficient method to recycle multiple markers in S. cerevisiae
simultaneously, thereby circumventing shortcomings of existing techniques and substantially accelerating the process of selection-excision. This method relies on artificial generation of double strand breaks around the selection marker cassette by the meganuclease I-SceI and the subsequent repair of these breaks by the yeast homologous recombination machinery, guided by direct repeats. Simultaneous removal of up to three marker cassettes was achieved with high efficiencies (up to 56%). This locus- and marker-independent method can be used
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
10
for both dominant and auxotrophy-complementing marker genes.
Chapter 5 describes the experimental exploration of genetic redundancy in yeast
glycolysis by cumulative deletion of minor paralogs and presents a new experimental platform for fundamental yeast research by constructing a yeast strain with a functional minimal glycolysis. The construction of this strain was performed with a combination of classical genetics tools and the newly developed methodologies presented in this thesis. It encompassed the deletion of 13 glycolytic paralogs considered to have a minor contribution to flux. After thorough experimental analysis, using quantitative and systems approaches, under growth conditions leading to a high glycolytic flux and after semi-quantitative analysis under a wide range of growth conditions, the most remarkable feature of the minimal glycolysis strain was the lack of visible phenotypic response to the deletion of 13 genes. The high glycolytic rates in anaerobic cultures of the minimal glycolysis strain and the small effect on the transcriptome argue against gene dosage or back-up effects as means for fixing minor glycolytic paralogs in the yeast genome. The near-wild type growth kinetics of the minimal glycolysis strain in aerobic and anaerobic cultures are difficult to reconcile with the hypothesis that duplication of glycolytic genes during the WGD event played a major role in increasing its glycolytic capacity or in causing the Crabtree effect. This reduction of genetic complexity contributed to the understanding of yeast glycolysis by, for the first time, studying the synergetic effects of multiple deletions of glycolytic genes. Moreover, it eliminated intrinsic uncertainties caused by the simultaneous, context-dependent expression of different isoenzymes, facilitating the formulation and validation of mathematical models that describe the kinetics of this key metabolic pathway. Analysis of the physiology and fitness of the minimal glycolysis strain under dynamic man-made and natural conditions may contribute to the daunting but exciting task of resolving the origin, fate, evolution and role of glycolytic paralogs. Additionally, it will serve as a basis for a more extreme genetic and pathway engineering strategy, in which different glycolytic pathways can be redesigned and interchanged in two simple steps.
General introduction
1
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
14
Saccharomyces cerevisiae: an industrial and model organism.
For millennia, humans have used microbial fermentations for the production of goods. From simple bakery or alcoholic beverages to the production of pharmaceutical compounds and bulk and fine chemicals, the biotechnology industry has been in a continuous expansion (1). Propelled by its fermentative life style, tolerant characteristics and genetic accessibility, the yeast Saccharomyces cerevisiae is one of the main work-horses in modern biotechnology. The natural ability to rapidly consume sugars and convert them to ethanol and carbon dioxide has set S. cerevisiae as the best organism for the production of alcoholic beverages and more recently of biofuels (1; 2). Additionally, S. cerevisiae displays features that are remarkable for industrial purposes such as high tolerance to elevated osmotic pressures and low pH (3; 4).
Recently, integration of metabolic engineering, systems and synthetic biology approaches expanded the number of high-value compounds, endogenous and heterologous, produced by
S. cerevisiae (1; 5; 6; 7; 8; 9; 10; 11). Furthermore, S. cerevisiae’s genetic accessibility is
astounding, this yeast was the first eukaryotic organisms to have its genome sequenced (12) and the toolbox for its genetic manipulation is rapidly expanding. Genetic engineering in S.
cerevisiae is taking unprecedented stages, currently the design and assembly of an entirely
synthetic yeast genome is under development (13; 14; 15).
Scientists have acquired large amounts of information on the genetics and physiological characteristics of S. cerevisiae, almost all of it freely distributed to the community through a diverse set of databases, Saccharomyces Genome Database (http://www.yeastgenome.org/) as an example (16). This, combined with its ease of cultivation and similarities with higher eukaryotes, has propelled S. cerevisiae as an extraordinary eukaryotic model comparable with traditional models such as Caenorhabditis elegans (nematode), Drosophila melanogaster (fruit fly) and Mus musculus (mouse) (17; 18; 19).
Evolution of S. cerevisiae towards a fermentative life style.
The genus Saccharomyces consist of seven different members (S. cerevisiae, S.
paradoxus, S. mikate, S. kudriavzevii, S. arboricola, S. eubayanus and S. uvarum) and
although they differ largely (their protein divergence is similar to those of human and chicken (20)), they share some interesting physiological characteristics. Among these, the production of carbon dioxide and ethanol, even in the presence of oxygen when glucose is in excess (Crabtree effect), and anaerobic growth are the most valuable for society and the most studied (21; 22). While most of the species in the Saccharomyces genus were isolated from the bakery, brewery and winery industries, their recent identification in oak trees and their surrounding suggest that this environment may be their natural habitat (23). However, understanding
Saccharomyces ecology remains a challenge since many factors are suspected to have
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Approximately 100 million years ago, the ancestor of the Saccharomyces sensu stricto yeasts underwent a duplication of its whole genome (24) (Fig. 1). This whole-genome duplication (WGD) event was followed by massive chromosomal restructuring, loss and gain of several different genes (25). Among the ca. 6000 genes of the S. cerevisiae genome, approximately 1500 are duplicates from which ca. 500 originate from WGD. Remarkably, 295 duplicated genes encode proteins involved in metabolism, thus revealing a strong enrichment of paralogs among metabolic genes (24; 26; 27). With the very well characterized genomes of S. cerevisiae and its close and distant relatives, the Saccharomyces genus has become an extraordinary model for the study of genome evolution and gene duplications. Many hypotheses about the origin, fate and consequences of duplicated genes have been suggested or proven in studies using S. cerevisiae (27; 28; 29; 30; 31; 32; 33; 34; 35). Interestingly, most of the major changes in the S. cerevisiae genome since the WGD seem to be related to the adaptation of this organism to a fermentative life style (22; 25). It has been suggested that after this event, yeast families diverged in their ability to grow in the absence of oxygen and to rapidly convert glucose to ethanol even in the presence of oxygen (Crabtree effect) (22) (Fig 1). Different computational and manual analysis of the genome sequences of large sets of yeast strains have partially confirmed this by the identification of different genes and gene families that contribute to ethanol production and consumption, removal of enzymatic steps that require molecular oxygen and decoupling mitochondrial respiration from biosynthesis (25). It has been suggested that a direct contribution of WGD to the Crabtree effect was the increase of the gene dosage of the glycolytic pathway which later became the potential reason for the high glycolytic flux characteristic of this yeast (36).
Figure 1. Anaerobic growth and Crabtree effect distribution in a phylogenetic tree of the
Saccha-romyces complex, adapted from
(22). Whole-genome duplication that occurred approximately 100 million years ago is indicated with a star. Species capable to grow anaerobically in synthetic media are shown in red, species capable to grow anaerobically in enriched (addition of lysine and glutamic acid or acetoin) synthetic media are shown in blue, species unable to grow anaerobically in synthetic media are shown in black. The level of the Crabtree effect of the different species was evaluated in controlled aerobic batch cultures and quantified as the specific rate of ethanol production (qEtOH,
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
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Saccharomyces cerevisiae’s glycolytic pathway: regulation and genetic
redundancy.
In most organisms, the set of reactions leading to the conversion of glucose to pyruvate is at the core of metabolism. Although different variants and bypasses exist, glycolysis is highly conserved across all levels of organization (37). For instance, the 10 catalytic steps of the Embden-Meyerhof-Parnas route, generally recognized as the “standard” glycolysis, are present in all eukaryotic cells (38), including S. cerevisiae (Fig. 2). Glycolysis, literally the lysis of glucose, is not only part of the catabolism and source of the energetic molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH), but also serves as source of key metabolites for anabolic routes (39). Moreover, many of the metabolites in this set play important roles in an industrial context. Beyond the obvious relevance of ethanol production from pyruvate, 3-phosphoglycerate, phosphoenolpyruvate and pyruvate are precursors for the production of different amino acids and industrially relevant molecules (7; 40). Furthermore, a great variety of biotechnological products share acetyl-CoA as precursor, a molecule located a single biochemical step away from pyruvate (8; 10). Glycolytic intermediates, such glucose-6-phosphate, precursor of trehalose (41), are also important for stress resistance and robustness of S. cerevisiae towards harsh growth conditions encountered in industrial settings.
Due to the central role of this pathway in S. cerevisiae as source of important building blocks for both cellular processes and the biotechnology industry, glycolysis has attracted much attention and is unarguably one of the most investigated pathways. Many studies have attempted to predict and manipulate the glycolytic flux. Although all the individual components of S. cerevisiae‘s glycolysis are known and well-studied, the understanding of the dynamics in this metabolic route remains challenging. Additionally, classical approaches for increasing the glycolytic flux via overexpression of “key” or several steps have failed (42; 43). While this glycolytic robustness to gene dosage may be explained by the fact that the glycolytic enzymes often operate far from saturation, it unveils the current lack of understanding of the regulatory mechanisms that govern the glycolytic flux (44; 45; 46; 47).
This lack of understanding in glycolysis control and flux is not exclusive to yeast. Nearly all metastatic cancer cells exhibit an altered glycolysis where the conversion of glucose to lactic acid even in the presence of oxygen (aerobic glycolysis or Warburg effect, (48)) is common. Despite the extensive study of this effect and the development of few anticancer treatments based on inhibition of glycolysis (49), the mechanisms governing this flux change are not completely elucidated (50).
Glycolysis, which originated approximately 1800 million years ago, is one of the slowest evolving pathways (5% of the residues in glycolytic enzymes are changed every 100 million years) (51). Despite many studies that include enzyme structure and phylogenic analysis, the
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evolutionary mechanisms that shaped the glycolytic pathway have not been unveiled (37; 51). On the other hand, the involvement of gene duplication events in the evolutionary history of this pathway is well recognized (51; 52). In different organisms, several families of glycolytic isoenzymes have been identified and can be classified according to their expression patterns in a tissue-, compartment- and stage of development-specific fashion, additionally, some glycolytic isoenzymes are separated by regulation at transcriptional or post-transcriptional levels, suggesting specificity towards different environmental conditions (37; 51). It is clear that glycolysis is enriched for duplicated genes and that glycolytic isoenzymes are widespread in both prokaryotic and eukaryotic genomes (37). The retention of many paralogs has been attributed in humans to differential tissue-specific expression (51; 52), in plants to different compartmentalization (53), in S. cerevisiae to gene dosage effects (36) and to secondary functions unrelated to their catalytic activities in glycolysis (51; 54; 55).
The existence of many paralogous families represents a challenge for the understanding of glycolysis in general and in S. cerevisiae in particular. While most of these genes and gene families have been individually investigated in S. cerevisiae, their contribution to the glycolytic activity and yeast physiology at large remains unknown. Furthermore the unknown contribution of the different glycolytic isoenzymes to flux is a hurdle for the mathematical modeling of this essential pathway (56).
Even in the presence of oxygen, S. cerevisiae has a preference for a fermentative life-style. Fermentation, the two-step biochemical conversion of pyruvate to ethanol, enables the reoxidation of NADH produced in glycolysis, thereby maintaining the cellular redox balance (21). In most growth environments, S. cerevisiae‘s glycolysis is therefore inseparable from fermentation. In the present work, glycolysis refers to the combination of glycolysis and fermentation in S. cerevisiae, and consists of 12 steps (Fig. 2). In S. cerevisiae, these 12 glycolytic reactions are catalyzed by 27 isoenzymes encoded by 27 distinct glycolytic genes. These 27 genes are clustered in eight paralog families, originated by WGD (Fig. 2, blue circles) and other small scale duplications (SSD) (Fig. 2, black circles) (22; 25; 36), and four singletons. From these 27 genes, only seven are classified as essential (57; 58; 59), suggesting a high level of redundancy in the pathway. Some of the most interesting features of the members of the glycolytic paralogs families are summarized below.
Hexokinase (HXK). Three proteins can catalyze the phosphorylation of glucose, Hxk1,
Hxk2 and Glk1. HXK1 and GLK1 present 77 % and 48 % sequence similarity to HXK2 respectively and the proteins that they encode 77 % and 34 %. The genes HXK1 and GLK1 are glucose-repressed whereas HXK2 is the main glucose phosphorylation enzyme in glucose excess conditions (60). Additionally, HXK2 is involved in glucose repression and has a direct impact on the expression of HXK1 and GLK1 (61; 62; 63; 64; 65). Accordingly, Hxk2 is localized in both cytoplasm and nucleus, and has been shown to be part of the Mig1 repressor complex (64). Interestingly, the regulatory and catalytic functions of Hxk2 can be dissected
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
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Figure 2. Saccharomyces
cerevi-siae glycolytic and fermentative
pathways. Glycolysis and ethanol
formation in S. cerevisiae consist of 12 biochemical reactions. There are 27 glycolytic genes distributed in eight paralogs families and four singletons. The paralogs originat-ing from small scale duplication are indicated by a black dot while paralogs generated by whole-ge-nome duplication are indicated by blue dot, next to the gene name. Gluc = glucose, Gluc-6-P = glucose 6-phosphate, Fruc-6-P = fructose 6-phosphate, Fruc-1,6-bP = fruc-tose 1,6-bisphosphate, DHAP = dihydroxyacetone phosphate, GAP = glyceraldehyde 3-phosphate, 1,3-bP-G = 1,3-bisphosphoglycer-ate, 3PG = 3-phosphoglycer1,3-bisphosphoglycer-ate, 2PG = 2-phosphoglycerate, PEP = phosphoenolpyruvate, Pyr = py-ruvate, AcH = acetaldehyde and EtOH = ethanol.
Chapter 1
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by the change in few amino acids (64). hxk1, hxk2 and glk1 individual mutants are viable but
hxk2 mutants showed a steep decrease in enzymatic activity. Individual expression of HXK1, HXK2 and GLK1 under the control of their native promoters is sufficient to support growth on
glucose (60).
Phosphofructokinase (PFK). PFK1 and PFK2, generated by SSD events, encode
the α and β subunits of the heterooctomeric phosphofructokinase (four subunits α and four subunits β) (66). Pfk is allosterically inactivated by ATP and activated by AMP and fructose-2,6-bisphosphate, both subunits possess regulatory and catalytic functions (67). Deletion of each subunit leads to decreased growth on glucose and reduced ethanol production (68).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). TDH1, TDH2 and TDH3
encode for the three glyceraldehyde-3-phosphate dehydrogenases in S. cerevisiae. TDH2 and TDH3 are expressed in exponential growing cells and they can individually support growth on glucose (69; 70). Despite the fact that all isoforms have catalytic activity, tdh2 tdh3 double mutants present deleterious phenotypes in media containing glucose as carbon source (70). Although in most conditions TDH1 is weakly expressed, it is up-regulated under specific conditions such as osmotic and reductive stress and stationary phase, which suggested a potential role of TDH1 other than glycolysis (70; 71). Additionally, it has been proposed that
TDH3 and TDH2 may be involved in oxidative stress resistance (72).
Phosphoglycerate mutase (GPM). The reversible conversion of 3-phosphoglycerate
to 2-phosphoglycerate is carried out in S. cerevisiae by Gpm1. Although two GPM1 paralogs exist, GPM2 and GPM3, their encoded proteins cannot complement gpm1 mutants when gene expression is guided by their native weak promoters (73). However, when GPM2 or
GPM3 are under the control of strong promoters, they can individually restore growth on
glucose although enzyme activity is not detected (73). Interestingly, despite the fact that
GPM2 and GPM3 are paralogs that arose from WGD and have been maintained in the yeast
genome for several million years, no functionality has been attributed to these genes and they are considered non-functional copies of GPM1.
Enolase (ENO). ENO1 and ENO2 encode the two enolases in S. cerevisiae. The
abundance of Eno2 in yeast cells during logarithmic growth on glucose is approximately 20 times higher than that of Eno1, whereas the levels of both isoenzymes are similar when the cultivation is performed in non-fermentable carbon sources (74). ENO2 is an essential gene for growth on glucose, while eno1 mutants are viable under this condition. Both Eno1 and Eno2 participate in vacuole formation, a function not directly related to glycolysis (58). Deletion of ENO1, reduction of ENO2 expression, or a combination of both, result in vacuolar fragmentation.
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
20
to be the main pyruvate kinase (57). pyk1 mutants cannot grow on glucose as carbon source and only overexpression of PYK2 can restore growth. Pyk1 is activated by fructose-1,6-bisphosphate and glucose induces expression of PYK1 whereas PYK2 is glucose-repressed and Pyk2 is insensitive to fructose-1,6-bisphosphate. Based on these characteristics, it was suggest that Pyk2 is used under conditions where the glycolytic flux is low such as starvation (57) and during the diauxic shift (75; 76).
Pyruvate decarboxylase (PDC). The paralogs PDC5 and PDC6 were gained via SSD
after the WGD from the ancestor PDC1 (25). PDC5 is induced upon deletion of PDC1 or in thiamine limiting conditions and can support growth on glucose in pdc1 mutants (77; 78). Pyruvate decarboxylase activity is not detected in pdc1 and pdc5 mutants (79). PDC6 expression is induced under sulfur limitation, presumably to salvage sulfur as Pdc6 contains less methionine and cysteine than Pdc1 and Pdc5 (80; 81; 82).
Alcohol dehydrogenase (ADH). Five functional alcohol dehydrogenases are present in
S. cerevisiae, Adh1 to Adh5. Although each individual enzyme is capable to support growth
on glucose and produce ethanol, only Adh1 produces this compound in large quantities and is considered as the main alcohol dehydrogenase in S. cerevisiae (83). Based on the higher affinity of Adh2 for ethanol as compared to Adh1 and the repression of ADH2 by glucose, Adh2 has been proposed to be involved in ethanol consumption rather than production (84). However, ethanol consumption is only marginally reduced in adh2 mutants containing functional ADH1 (83). Comparative genomic studies suggest that ADH2 has been acquired after WGD and has contributed to S. cerevisiae’s evolution towards a make-accumulate-consume lifestyle (22; 25). Adh3 is a mitochondrial alcohol dehydrogenase involved in the ethanol-acetaldehyde redox shuttle, while deletion of ADH3 does not have a significant impact on aerobic growth, under anaerobic conditions the growth rate is significantly lowered (85). The amino acid sequence of Adh4 is more related to bacterial dehydrogenases than the eukaryotic versions and different from the other S. cerevisiae’s alcohol dehydrogenases. Adh4 is activated by zinc ions instead of ferrous ions (84) and ADH4 expression seems to be induced by glucose (86) and zinc limitation (87). A large-scale protein localization study suggested a mitochondrial localization for Adh4 (88). Adh5 activity has only been detected in
adh1 adh3 double mutants (89).
Remarkably, in S. cerevisiae most glycolytic reactions encoded by paralog families rely on a single, main isoenzyme. Still, most minor isoenzymes, while seemingly not significantly contributing to the glycolytic flux, have retained their glycolytic enzyme activity. As mentioned above, it has been proposed that the different glycolytic paralogs contributed to increase the glycolytic flux in the ancestor strain soon after the WGD, thereby shaping S. cerevisiae’s current lifestyle (36). The paralogs clearly do not fulfill this function in modern S. cerevisiae and the role of the glycolytic paralogs and the mechanisms responsible for their retention remain obscure.
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Maintenance of redundant genes.
Gene duplication events, at small or whole-genome scale (SSD and WGD respectively), are relatively frequent. It was estimated that the frequency of birth of duplicated genes is approximately 1 per gene per million years (90), and have been confi rmed in several diffe rent organisms, ranging from microbes to plants and vertebrates (24; 91; 92; 93). Interestingly, a large percentage of modern genomes are composed by duplicated genes (Table 1 and (28)).
Table 1. Genome size and percentage of duplicated genes for different organisms
Immediately after gene duplication, the resulting paralog pairs are usually identical and, therefore, functionally redundant. Unless duplicati on confers a selective advantage, either via g ene-dosage effects (9 4) or via mutational acquisition of modifi ed (95) or new functions (96), duplicated genes will eventually be pseudogenized and/or lost from the genome (28; 97). It has been suggested that in the human genome there is at least one pseudogene every two genes (98).
Conversely, by analyzing mutant libraries of different model organisms and humans, it was observed that a high proportion of null mutations do not cause signifi cant effects on the fi tness of the mutants analyzed and that duplicated ge nes, rather than singletons, are prone to have compensatory functions (33; 34; 99; 100; 101). Interestingly, despite the apparent lack of evolutionary force to support their retention, these studies suggested that genetic redundant paralogs seem to be retained for long periods of time (reviewed in (102)).
Retention of duplicated genes solely for their compensatory function seems to contradict evolutionary theories and remains debatable. It has recently been proposed that functional complementation might only be present for certain period of time, where organisms can explore new environmental niches, until the paralogs diverge to give rise to new functions (35). On the other hand, redundancy might be just a sub-product of evolution where the structure or even the function of the protein is required for a new function (piggyback hypothesis (103)).
Although it is clear that gene duplications have undoubtedly played an important role in evolution of all organisms and play roles in speciation, development of co mplex structures and behavior, and adaptation to changing environments (25; 92; 104), in many cases the
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
22
exact mechanisms responsible for the retention of the duplicates, as discussed in the previous section regarding yeast glycolysis, are far from elucidated.
Synthetic and minimal genomes.
“As simple as possible but not simpler”, a modified version of Einstein’s quote, perfectly describes the scientists long pursuit of the minimal set of genes that are required for a free-living organism. This minimal set, also called minimal genome, should contain a minimal set of protein-coding DNA sequences required for survival and to support growth under nutrient-rich, stress-free conditions and independent of any other living organism (105; 106). Different scientific milestones are driving the efforts to obtain a minimal genome. On the one hand, knowing the absolute essential genes for survival and growth can have large implications in health care and the fight against pathogenic organisms (107). On the other hand, genome reduction and minimization may improve metabolic efficiency and decrease redundancy, ideally minimal genomes can deliver engineered systems that are functionally robust and predictable (106; 108).
In the pursuit of minimal genomes, two synthetic biology approaches have been taken: bottom-up and top-down. The former approach attempts to construct a self-replicating system composed of purified essential elements (genes and macromolecules required for generating gene products and self-replication) encapsulated in a synthetic membrane (106). Although this approach has attracted a large amount of attention, a full version of a synthetic cell following this approach is still far from complete. Conversely, top-down approaches focus on nature’s smallest genomes and attempt to reduce them (reduced genomes) in order to identify a set of universally essential genes. Large deletions or inactivation programs based on transposon mutagenesis, interference by antisense RNA or targeted gene deletion (see below) have helped to determine the percentages of essential genes in diverse genomes (59; 109; 110). Additionally, comparisons between small and reduced genomes have served to prove that essentiality can be condition dependent and that there is not a universal set of essential genes but rather a set of essential functions (111). Top-down approaches have already delivered different minimal genomes, however, they differ largely and are composed of as few as 150 genes to as large as ca. 500 genes (105; 111; 112; 113). Additionally, a complete functional reduced genome has been chemically synthesized and transplanted to a host cell (114).
The search for the minimal genome has focused almost exclusively on small prokaryotic organisms. However, recently the eukaryotic model S. cerevisiae has been the center of attention for synthetic biology approaches to reduce its genome. This reduction allowed simplification in the construction of synthetic versions of it and its design-based manipulation (13; 14; 15). Genome reduction requires a large number of genetic modifications for which the development of efficient genetic tools is crucial.
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Tools for genetic reduction in Saccharomyces
cerevisiae.
Propelled by its pivotal role as model eukaryote and industrial workhorse, S. cerevisiae has become a preferred host for the development of an extensive genetic toolbox and, more recently, for synthetic biology advances. This role has unarguably been boosted by S. cerevisiae’s simple cultivation requirements, robustness, ability to incorporate DNA and its preference for homologous recombination for DNA repair (1; 115; 116). Various means for gene expression are available and range from plasmids with high and low copy number, integrative plasmids and integrative cassettes (117; 118; 119; 120). Additionally, different (heterologous) marker genes are available (121) and the genome sequence of S. cerevisiae is known since 1996 (12).
A feature that allowed S. cerevisiae’s easy genetic accessibility is the highly efficient homologous recombination (HR) machinery naturally present in this yeast (Fig. 3). In S. cerevisiae, HR instead of non-homologous end joining is the predominant DNA repair mechanism (115). Additionally, recombination can be achieved with relatively short homologous sequences below 60 bp as compared to the ca. 500 bp required for mammalian cells or filamentous fungi (117; 122; 123). While current knowledge has been largely established using S. cerevisiae as model, the mechanisms of HR are not completely elucidated yet (115; 124). Nonetheless, decades ago molecular biologists have harnessed this high fidelity DNA repair apparatus to perform targeted integrations, remove genes and assemble in vivo plasmids (117; 120; 125; 126) (Fig. 3).
Figure 3. Mechanism of homologous recombination (HR) in Saccharomyces cerevisiae. A) S. cerevisiae HR
steps. 1 - double strand break generation, 2 – Single stranded DNA (ssDNA) resection by unidentified nucleases, followed by covering of ssDNA by the replication protein A (RPA), 3 – filament formation by Rad51, Rad52 and the complex Rad55-Rad57, 4 – homology search by an unknown mechanism, 5 – strand invasion in the homologous chromosome/section and DNA synthesis by a DNA polymerase (DNA pol) and 6 – re-annealing and ligation of the newly synthesized DNA. The power of S. cerevisiae HR has been harnessed by scientist to perform targeted gene deletions (B) and in vivo assembly of plasmids (C).
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
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A major hurdle in large strain construction programs is the requirement, for each genetic modification, of the concomitant transformation of a marker gene, such as the dominant markers KanMX (120), ble (127) and hph (128) and the auxotrophic markers HIS3 (129) and
LEU2 (130), to select for transformants carrying
the desired genetic modification. The presence and accumulation of these DNA materials can cause undesired deleterious effects in S.
cerevisiae (131; 132; 133). Thus, methods
enabling the removal of marker genes have been explored. Already 20 years ago it was observed that, using the power of yeast HR, marker genes could be removed (frequency of 10-4 – 10-3 if flanked by direct repeats and
by cultivating the mutants in the absence of selection pressure (120)). The efficiency of this process was improved by the development of counter-selection, where the presence (selection) or absence (negative selection or counter-selection) of a marker can be screened for. The most widely used example is the URA3 marker which can be selected for in media without uracil and counter-selected in media containing 5-fluorooritic acid (134). Marker excision efficiency was further enhanced by the generation in vivo DNA double strand breaks (DSB) using endonucleases, which promotes HR repair (115; 124; 135; 136; 137).
Alternatively, heterologous recombinase-based marker removal methods (HeR) were developed (138; 139). HeR methods rely on the presence of specific sites flanking the
Figure 4. Mechanism and applications of the LoxP/Cre system. A) Gene deletion and marker removal using the
LoxP/Cre system occurs according to the following steps: 1 - Gene deletion using an integrative cassette containing
a marker gene (green) flanked by LoxP sites (black triangles) and regions homologous to the targeted deletion site. 2 - Cre recombinase (blue circles) expression. Dimers of Cre recognize and attach to LoxP sequences. 3 – Tetramers of Cre catalyse the recombination of two LoxP sites. 4 – Marker gene is removed from the genome and a LoxP sequence is left in place. Depending on the direction and location of LoxP sites in the genome, and upon expression of Cre, inversions (B) or translocations (C) can be obtained.
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marker gene, which will be recognized and used for recombination by the heterologous recombinase, resulting in the excision of the marker gene. HeR methodologies are highly efficient and standard protocols and tools for their use were developed, with the LoxP/Cre system (Fig. 4) being the most popular (139; 140; 141). Implementation of the LoxP/Cre system has opened the door to so-far impossible large deletion projects, such as the construction of a S. cerevisiae strain deficient for hexose transport. In S. cerevisiae from the CEN.PK background, the deletion of 21 genes was required to completely abolish glucose uptake (142) and thereby to construct EBY.VW4000, a powerful strain used for the characterization and discovery of hexose transporters (143; 144; 145). To avoid the deleterious effects of marker genes accumulation in EBY.VW4000, every deletion was accompanied by a marker removal step using the LoxP/Cre system. Although the LoxP/Cre system accelerated the genetic engineering of S. cerevisiae and allowed the fast construction of multiple-gene mutants (140; 142; 146), it presents drawbacks that have only recently been investigated (this thesis), limiting its use for large strain construction programs.
Clearly novel tools and methodologies for large scale genetic reduction and genome engineering of S. cerevisiae are necessary. Developments in molecular and genetic tools for S. cerevisiae are expanding rapidly. The use of HR and the combination of HR and DSB are now being used more often in novel methodologies for in vivo plasmid assembly and in combination with genome integration (147; 148). New gene targeting methodologies that accelerate the marker removal process (this thesis) or that completely abolish the need for selection, such as the CRISPR-Cas9 system (149) are being rapidly developed. The in vitro and in vivo assembly of DNA fragments have reached sizes and accuracy previously not imagined (150). The current accelerated molecular and genetics tool development share remarkable resemblance with Moore’s law (151).
Reducing genetic redundancy in S. cerevisiae is expected to improve predictability in this yeast and its metabolism which might lead to great advances in the biotechnology industry and it can also be used as a path to address fundamental questions about eukaryotic evolution and genetic robustness and redundancy.
Scope: towards a synthetic glycolysis, the minimal glycolysis.
It is clear that a predictable and malleable glycolytic pathway could greatly benefit the biotechnology industry. In order to achieve these characteristics in S. cerevisiae, the construction and implementation of a “synthetic glycolysis” was proposed in which the set of endogenous glycolytic genes can, in a few simple steps, be replaced by redesigned sets of (heterologous) glycolytic genes. To achieve this ‘pathway swapping’ strategy, three milestones are required. Firstly, methodologies for fast construction, assembly and integration of new sets of glycolytic genes have to be established (PhD thesis, Niels Kuijpers). Secondly, a genetic reduction of the native S. cerevisiae glycolytic pathway is required to trim down to a strict
Reducing the genetic complexity of glycolysis in Saccharomyces cerevisiae
26
minimum the number of glycolytic isoenzymes. Finally, expression of a synthetic glycolysis in this “minimal glycolysis” strain (MG), and removal of the remaining endogenous genes will result in a platform strain in which the simplified and relocated glycolytic pathway can be easily swapped. This ambitious project was divided in two PhD theses, among which the present one focuses on the reduction of the genetic complexity in S. cerevisiae‘s glycolysis. This genetic reduction would not only provide a platform strain for the construction of synthetic glycolysis but also enable the exploration of the role of genetic redundancy in yeast glycolysis.
Genetic reduction of yeast glycolysis implies the deletion of over a dozen of genes. Despite the extensive toolbox available for S. cerevisiae‘s genetic engineering, this task remains daunting and requires the development of novel tools for multiple and sequential gene modifications. Every genetic modification requires a marker for selection of correct transformants. Not only is the number of available markers too small, but accumulation of a dozen of selection markers would be detrimental for the constructed strains. Marker removal is therefore a prerequisite for the successful construction of a minimal glycolysis (MG) strain. While the LoxP/Cre system offers a powerful tool for marker removal, Chapter 2 demonstrates
its limitations for sequential gene deletion. In 1999, Boles and his team removed all genes encoding functional glucose transporters (HXT genes) in S. cerevisiae’s. Removal of HXT genes was performed with 16 consecutive rounds of deletion and marker removal, using the LoxP/Cre system resulting in the strain EBY.VW4000 (142), a splendid and intensively used platform for characterization and discovery of transporters (152; 153; 154; 155; 156; 157; 158). In Chapter 2, the characterization of EBY.VW4000 revealed massive LoxP/Cre-induced chromosomal rearrangements and gene loss. The severe genetic damage caused by repeated utilization of the LoxP/Cre system precluded its use for the construction of the MG strain and revealed the need for alternative systems for marker removal.
The only alternative to the LoxP/Cre system for marker removal lies in counter-selectable markers. S. cerevisiae’s toolbox holds a very limited number of such markers, among which the most intensively used and most robust is the URA3/5-FOA system. To increase the number of selectable markers available for S. cerevisiae with the added benefit of negative selection for recycling, and to expand the strategies for scarless marker removal, the development and applications of the new marker module amdSYM is presented in Chapter 3. Still, each
gene deletion is typically followed by marker removal, a laborious and time-consuming step. In order to reduce the time required for multiple markerless genetic modifications, Chapter 4
describes a method to enable simultaneous multi-marker removal while minimizing the risk of chromosomal translocations.
Finally, combining classical genetics with the newly developed tools and methods developed in Chapters 3 and 4, Chapter 5 describes the construction of a “minimal glycolysis”
(MG) strain with a genetically reduced glycolysis consisting of a single glycolytic isoenzyme per reaction. To evaluate the impact of the cumulated deletions of 13 glycolytic paralogs on
