Engineering precursor supply in
Saccharomyces cerevisiae:
new strategies for cytosolic
acetyl-CoA formation
Saccharomyces cerevisiae:
new strategies for cytosolic
acetyl-CoA formation
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
woensdag 28 oktober 2015 om 10:00 uur
door
Barbara Urszula KOZAK
Magister Inżynier Biotechnologii,
Politechnika Wrocławska,
geboren te Wrocław, Polen
Copromotor:
Dr. ir. A.J.A. van Maris
Composition of the doctoral committee:
Rector Magnificus
voorzitter
Prof. dr. J.T. Pronk
Technische Universiteit Delft
Dr. ir. A.J.A. van Maris
Technische Universiteit Delft
Prof. dr. S. de Vries
Technische Universiteit Delft
Independent members:
Prof. dr. R.A.L. Bovenberg
Rijksuniversiteit Groningen
Prof. dr. ir. J.J. Heijnen
Technische Universiteit Delft
Prof. dr. J. Nielsen
Chalmers University of Technology
Dr. R.A. Weusthuis
Wageningen University
Prof. dr. U. Hanefeld
Technische Universiteit Delft, reservelid
The research presented in this thesis was performed at the Industrial
Microbiology Section, Department of Biotechnology, Delft University of
Technology, The Netherlands and financed by the BE-Basic R&D Program,
which in turn was granted a FES subsidy from the Dutch Ministry of
Economic Affairs, Agriculture and Innovation (EL&I).
Cover:
Fluorescent micrographs of single yeast cells and yeast cells aggregates stained with calcofluor-white to visualize chitin deposition in cell walls.
Summary / Samenvatting 1
1 General introduction 15
2 Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis 43
3 Replacement of the initial steps of ethanol metabolism inSaccharomyces
cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase 81 4 Engineering acetyl coenzyme A supply: functional expression of a bacterial
pyruvate dehydrogenase complex in the cytosol ofSaccharomyces cerevisiae 121
Curriculum vitae 161
List of publications 162
Metabolic engineering – the improvement and addition, by genetic modification, of industrially relevant properties of microorganisms with respect to catalysis, transport and regulatory functions – is a well-established method for development of more cost-effective and ‘green’ industrial processes. Rapid depletion of oil reserves and a growing demand for sustainable, environmentally friendly processes provide incentives for efficient exploitation of new, renewable resources for the production of transport fuels and bulk chemicals. The narrow profit margins that are typical for such commodity products, impose a need to optimize processes in terms of kinetics of product formation and, especially, yield of product on feedstock. Metabolic engineering of microorganisms, with its continuously expanding toolbox, allows researchers to address the challenges involved in the development of biotechnological processes that can compete with petrochemical production.
Due to its robustness in industrial fermentation processes and fast developments in yeast synthetic biology,Saccharomyces cerevisiae (a.k.a. baker’s yeast) has become one of the most popular metabolic engineering platforms in modern biotechnology. As a result, this microorganism, after having been used for ages in the production of alcoholic beverages and bread, is now recognized as multi-purpose microbial ‘workhorse’ with numerous industrial applications.
Production of many natural and heterologous compounds with genetically modified strains of S. cerevisiae is under investigation or has already been implemented in industry. Many of those biochemicals, for example n-butanol, isoprenoids, lipids, flavonoids and 3-hydroxypropionic acid, require acetyl coenzyme A (acetyl-CoA) as a key precursor. The metabolism of this compound inS. cerevisiae cells is compartmentalised. The mitochondrial route, responsible for a substantial flux towards acetyl-CoA during respiratory growth on sugars, involves conversion of mitochondrial pyruvate into acetyl-CoA in the reaction catalysed by the pyruvate dehydrogenase complex. Since, inS. cerevisiae, acetyl-CoA cannot be exported from the mitochondria, another pathway is required to cover biosynthetic requirements for acetyl-CoA in the cytosolic compartment. This so-called ‘pyruvate dehydrogenase bypass’ requires the concerted activity of pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase. The latter two reactions are also required for growth on the C2-compounds – acetate (only acetyl-CoA synthetase) and ethanol. When ethanol is used as a carbon source, it is first converted to acetaldehyde in the reaction catalysed by alcohol dehydrogenases and further to acetyl-CoA, which is used to cover all biosynthetic and energetic requirements of ethanol-grown cells.
Heterologous, acetyl-CoA-dependent product pathways expressed in the cytosol of
S. cerevisiae exclusively depend on the cytosolic route for provision of this precursor.
As a result, several previous metabolic engineering studies have been devoted to improving the synthesis of the cytosolic acetyl-CoA. Although successful in increasing the availability of this compound, by improving the capacity of the native route or by introduction of the heterologous pathways of cytosolic acetyl-CoA synthesis, those
studies primarily focused on the kinetic parameters, while the energetic aspects of cytosolic acetyl-CoA synthesis received little attention.
The maximum yield of biomass or of any other industrially relevant product on a substrate depends on the energy (ATP) cost of the biochemical pathways used for precursor and product formation. The synthesis of cytosolic acetyl-CoA inS. cerevisiae involves hydrolysis of ATP to AMP and pyrophosphate (PPi) in the reaction catalysed by acetyl-CoA synthetase (ACS). The subsequent hydrolysis of PPi to inorganic phosphate (Pi) makes the energetic cost of cytosolic acetyl-CoA synthesis equivalent to hydrolysis of 2 ATP to 2 ADP and 2 Pi. This ATP expenditure has a profound impact on the maximum yields of acetyl-CoA-dependent products that can be achieved in engineered yeast strains and, therefore, negatively influences the economy of the production process. Especially in the case of bulk chemicals and bio-fuels, this single ATP-consuming reaction can become cost-prohibitive. This thesis explores metabolic engineering strategies to address this key challenge in yeast biotechnology.
After general introduction to metabolic engineering andS. cerevisiae, Chapter 1 describes the strategies that have hitherto been explored to increase the availability of cytosolic acetyl-CoA. Moreover, this Chapter discusses other pathways of cytosolic acetyl-CoA synthesis that occur in nature, some of which do not require an input of ATP and could therefore, upon expression inS. cerevisiae, lead to increase of maximum yields of acetyl-CoA-dependent products on substrate in engineered yeast strains.
In S. cerevisiae, cytosolic acetyl-CoA synthesis and growth strictly depend on functional expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Before the research described in this thesis, viableS. cerevisiae strains in which bothACS1 and ACS2 had been deleted, had not been described in the literature. In addition to its anabolic and, under certain conditions, catabolic roles, cytosolic acetyl-CoA also plays key role in cellular regulation via acetylation of proteins, including histones.
Chapter 2 explores the feasibility of replacing the native S. cerevisiae pathway
for cytosolic acetyl-CoA synthesis by two alternative, ATP-independent pathways, and investigates their impact on growth and energetics of the engineered yeast strains. To this end, the native route of cytosolic acetyl-CoA synthesis was replaced by either an acetylating acetaldehyde dehydrogenase (A-ALD) or a pyruvate-formate lyase (PFL). Acetylating acetaldehyde dehydrogenase catalyses direct, ATP-independent oxidation of acetaldehyde to acetyl-CoA, while pyruvate-formate lyase converts cytosolic pyruvate into equimolar amounts of acetyl-CoA and formate. After evaluating the expression of different genes encoding acetylating acetaldehyde dehydrogenase and pyruvate-formate lyase,acs1Δ acs2Δ S. cerevisiae strains were constructed in which A-ALD or PFL functionally replaced ACS. In case of the A-ALD-dependent strains, also all acetaldehyde dehydrogenases were deleted, which resulted in complete replacement of the two-step conversion of acetaldehyde to acetyl-CoA, by a one-step reaction catalysed by A-ALD. The A-ALD-dependent strains showed aerobic growth rates of up to 79% of the reference strain, while anaerobic growth rates of PFL-dependent S. cerevisiae reached up to 73% of the
chemostat cultures. Unexpectedly, the measured biomass yields on glucose of A-ALD-and PFL-dependent strains were 14% A-ALD-and 15% lower than those of the reference strain, respectively. Weak-acid uncoupling by formate, the formation of which was stoichiometrically coupled to growth of PFL-dependent strain, offers a plausible explanation for the reduced biomass yield of this strain. In A-ALD-dependent strain, the reduced biomass yield of the A-ALD-dependent strain was attributed to toxic effects of the acetaldehyde, which was present at higher levels in cultures of the engineered strain than in cultures of the reference strain.
Changes in the synthesis of cytosolic acetyl-CoA might affect histone acetylation as well as central metabolism via acetylation of non-histone proteins and direct participation of this compound in key reactions. However, transcriptome analysis of A-ALD- and PFL-dependent strains revealed only small sets of genes with altered expression levels relative to the reference strain. Combined with their high growth rates, these observations suggested that these strains did not suffer from major limitations in acetyl-CoA provision. This conclusion was further supported by the minor differences in intracellular metabolite levels of an A-ALD-dependent strain relative to the control strain. Intracellular acetyl-CoA concentrations, which reflect the combination of mitochondrial and nucleocytosolic pools, were also not significantly different between A-ALD-dependent strain and the reference strain, which may suggest that intracellular concentrations of acetyl-CoA are subject to strong homeostatic regulations. Higher intracellular lysine concentrations in A-ALD-dependent strain might even be indicative for increased availability of cytosolic acetyl-CoA. The research presented inChapter 2 demonstrated, for the first time, that the native pathway for cytosolic acetyl-CoA synthesis inS. cerevisiae can be entirely replaced by heterologous, ATP-independent pathways.
During respiratory growth of S. cerevisiae on glucose, cytosolic acetyl-CoA is required to cover biosynthetic requirements of the cell, including the biosynthesis of lysine and of lipids. However, these biosynthetic fluxes are relatively small compared to the overall rate of ATP turnover and, therefore, have a relatively small impact on growth energetics (calculated impact on biomass yield in aerobic, glucose-limited cultures < 0.5%). A fundamentally different situation arises when ethanol is used as a carbon source and acetyl-CoA synthetase is the starting point for all biosynthetic and dissimilatory pathways in growing yeast cells. Under these conditions, saving of the 2 ATP required for synthesis of each molecule of cytosolic acetyl-CoA, should theoretically enable an increase of the biomass yield from 0.57 g/g ethanol for the native route up to 0.80 g/g ethanol for an ATP-independent pathway. Such a dramatic increase in the energetic efficiency of ethanol utilization could be highly relevant for industry, as it should result in higher yield on ethanol of any acetyl-CoA-dependent product. Therefore,Chapter 3 focuses on yeast strains in which the native pathway for acetyl-CoA synthesis was replaced by A-ALD. The A-ALD-dependent strains, however, did not show immediate growth on media with ethanol as the sole carbon source.
Prolonged incubation, followed by long-term laboratory evolution experiments, yielded A-ALD-dependent yeast strains that were able to grow on ethanol with specific growth rates up to 0.11 h−1. Reverse engineering studies showed that mutations in ACS1, the gene that encodes one of theS. cerevisiae cytosolic acetyl-CoA synthetases, were essential for growth on ethanol of the A-ALD-dependent strains. Acquired mutations in A-ALD genes improved VMAX/KM for acetaldehyde of the encoded enzymes, but
were not essential for growth on ethanol.
Although the reverse engineered strains grew on ethanol, their growth rates were lower than the growth rates of the evolved strains. Further analysis of the growth of these strains suggested a limited availability of mitochondrial acetyl-CoA during growth of the A-ALD-dependent strains on ethanol. Out of five evolved strains tested in ethanol-limited chemostat cultures, only one evolved strain showed a 5% increase in the biomass yield on ethanol compared to the reference strain, which was far below the 40% theoretical prediction. Increased production of acetaldehyde and other byproducts was identified as possible cause for these suboptimal biomass yields. This study inChapter 3 proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway that has the potential to strongly improve biomass and product yields. Based on metabolic and evolutionary engineering, whole-genome resequencing, reverse engineering and physiological analysis of evolved strains, we identify intracellular acetaldehyde levels and provision of intramitochondrial acetyl-CoA as key targets for further optimization of ethanol conversion by eukaryotic cell factories.
Pyruvate dehydrogenase (PDH) is a huge, multisubunit enzyme complex whose size is comparable to that of a ribosome. The PDH complex catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA which, in many organisms, is the key reaction at the interface of glycolysis and tricarboxylic acid cycle (TCA). In eukaryotes, the PDH complex occurs exclusively in the mitochondrial matrix and, therefore, cannot contribute to provision of cytosolic acetyl-CoA inS. cerevisiae. In Chapter 4, the challenge of the expression of a functional, heterologous PDH complex in the yeast cytosol is taken up. In order to determine if a heterologous PDH complex can replace the native route of cytosolic acetyl-CoA formation inS. cerevisiae, the PDH complex from the bacteriumEnteroccocus faecalis was selected to be expressed in yeast. Three factors contributed to selecting the PDH complex fromE. faecalis for this study: (i) bacterial PDH subunits presumably lack mitochondrial localization sequences; (ii)E. faecalis PDH is relatively insensitive to high NADH/NAD+ ratios in comparison to other
bacterial PDH’s; and (iii)in vitro experiments indicate that purified subunits of the
E. faecalis PDH can self-assemble into a functional complex. All known PDH complexes
require four cofactors for their activity: FAD+, NAD+, thiamine pyrophosphate (TPP) and lipoic acid. InS. cerevisiae, lipoic acid is synthesized and covalently attached to the PDH complex in the mitochondria. Therefore, cytosolic expression of a bacterial PDH complex was likely to require co-expression of proteins that catalyze lipoylation of PDH, as well as addition of lipoate to the media.
combined with deletion ofACS1 and ACS2, enabled growth of an acs1Δ acs2Δ strain ofS. cerevisiae on glucose. The strict dependency of growth on the addition of lipoic acid confirmed the in vivo activity of the heterologous PDH complex. The aerobic growth rate on glucose of the obtained strain (0.36 h−1) was comparable to the growth rate of the reference strain (0.42 h−1) and independent of the concentration of lipoic acid in the range of 20 ng mL−1 to 1000 ng mL−1. The Acs− yeast strain expressing the bacterial PDH complex also showed a near-wild-type growth rate under anaerobic conditions (0.30 h−1 and 0.33 h−1 for engineered and wild-type strain, respectively). Functioning of theE. faecalis PDH complex in anaerobic yeast cultures indicates that it can also be applied in the design and construction of anaerobic product pathways. Enzyme activity assays indicated that, in an engineered strain, PDH fromE. faecalis yielded higher specific activity (53 nmol min−1 mg protein−1) than the activity of the mitochondrial PDH ofS. cerevisiae (12 nmol min−1 mg protein−1).
The cytosolic localization of the heterologously expressedE. faecalis PDH complex in the yeast cytosol was confirmed by subcellular fractionation, combined with enzyme activity assay and mass-spectrometry-based proteomics. While the specific activities of the PDH in the mitochondrial fractions of engineered strain and the wild-type strain were not significantly different, the specific activity of PDH measured in the cytosolic fraction of Acs− strain expressing PDH ofE. faecalis was 32-fold higher than that of the wild-type strain. The cytosolic localization of the four subunits of theE. faecalis PDH complex, as well as of the two lipoate ligases ofE. faecalis in the cytosol of
S. cerevisiae was further confirmed by mass spectrometry-based proteome analysis
of the cytosolic fraction. InE. faecalis, PDH occurs as a protein complex consisting of 210 subunits. Gel filtration of a cytosolic fraction, combined with enzyme activity measurements and proteomics analysis, demonstrated thatE. faecalis PDH was present in the cytosol ofS. cerevisiae as a complex with a size, a specific activity, and a relative abundance of the E1, E2, and E3 subunits comparable to those reported for native
E. faecalis PDH. Chemostat-based physiological characterization in glucose-limited
chemostat cultures showed comparable biomass yields and rates of sugar dissimilation for the Acs− strain expressing PDH and a reference strain. These results, together with the absence of strong differences in the transcriptome of engineered and native strains, indicated that replacement of the cytosolic acetyl-CoA synthesis with PDH complex fromE. faecalis did not lead to significant disturbances of the physiology of
S. cerevisiae.
The research described in this thesis demonstrates that complete replacement of the native route of cytosolic acetyl-CoA formation inS. cerevisiae with heterologous ATP-independent pathways can result in viable yeast strains. Moreover, it delivers proof of concept that implementation of the ATP-independent pathways may result in increased biomass yield on substrate. These strategies appear to be particularly suited for improving the product yield on sugar of any compound, produced inS. cerevisiae, that uses cytosolic acetyl-CoA as a precursor. Therefore, the results presented in this
thesis provide metabolic engineers with new strategies to optimize the performance ofSaccharomyces cerevisiae as a ‘cell factory’ for sustainable production of fuels and chemicals.
Metabolic engineering – de verbetering en toevoeging, door middel van genetische modificatie, van industrieel relevante eigenschappen van micro-organismen met betrekking tot katalyse, transport en regulatoire functies – is een bewezen methode voor de ontwikkeling van meer kosteneffectieve en groene industriële processen. Snelle uitputting van oliereserves en een groeiende vraag naar duurzame en milieuvriendelijke processen voorzien in een stimulans voor efficiënte exploitatie van nieuwe, hernieuwbare bronnen voor de productie van transportbrandstoffen en bulkchemicaliën. De smalle winstmarges die typerend zijn voor dergelijke bulkgoederen maken het noodzakelijk om de processen te optimaliseren met betrekking tot de kinetiek van productvorming en, in het bijzonder, de opbrengst van het product per hoeveelheid grondstof. Metabolic engineering van micro-organismen, met haar continu uitbreidende ‘gereedschapskist’, stelt onderzoekers in staat het hoofd te bieden aan de uitdagingen van de ontwikkeling van biotechnologische processen die kunnen wedijveren met de petrochemische productie.
Door haar robuustheid in industriële fermentatieprocessen en snelle ontwikkelingen in gistsynthetische biologie, is Saccharomyces cerevisiae (alias bakkersgist) een van de meest populaire metabolic engineering platforms geworden in de moderne biotechnologie. Als gevolg hiervan is dit micro-organisme, na vele eeuwen te zijn gebruikt voor productie van alcoholische dranken en brood, nu erkend als een multipurpose microbieel werkpaard met veel industriële toepassingen.
De productie van veel natuurlijke en heterologe stoffen met genetisch gemodificeerde stammen van S. cerevisiae wordt nu onderzocht of is reeds geïmplementeerd in de industrie. Veel van deze biochemicaliën, zoals bijvoorbeeld
n-butanol, isoprenoïden, vetten, flavonoïden en 3-hydroxypropionzuur, vereisen acetyl
coenzyme A (acetyl-CoA) als belangrijke bouwsteen. Het metabolisme van dit molecuul in S. cerevisiae is gecompartimenteerd. De mitochondriële route, verantwoordelijk voor een substantiële flux richting acetyl-CoA tijdens respiratoire groei op suikers, omvat de omzetting van mitochondrieel pyruvaat in acetyl-CoA in de reactie gekatalyseerd door het pyruvaatdehydrogenase-complex. Omdat, in
S. cerevisiae, acetyl-CoA niet uit het mitochondrion getransporteerd kan worden, is
er een andere route nodig om te voorzien in de acetyl-CoA-vraag voor de biosynthese in het cytosol. Deze zogenaamde ‘pyruvaatdehydrogenase-omleiding’ vereist de gecoördineerde activiteit van pyruvaatdecarboxylase, aceetaldehyde-dehydrogenase en acetyl-CoA-synthase. De twee laatstgenoemde reacties zijn ook noodzakelijk voor groei op C2-verbindingen, acetaat (alleen acetyl-CoA-synthase) en ethanol. Wanneer ethanol wordt gebruikt als koolstofbron, wordt dit eerst omgezet in aceetaldehyde in de reactie gekatalyseerd door alcohol-dehydrogenase en vervolgens tot acetyl-CoA, die gebruikt wordt om te voorzien in alle biosynthetische en energetische behoefte van cellen die groeien op ethanol.
Heterologe, acetyl-CoA-afhankelijke productroutes die tot expressie worden gebracht in het cytosol van S. cerevisiae zijn exclusief afhankelijk van cytosolische
aanvoerroutes in het cytosol van deze verbinding. Als gevolg hiervan zijn er diverse studies uitgevoerd met als doel de synthese van acetyl-CoA in het cytosol te verbeteren. Ondanks het succesvol verhogen van de beschikbaarheid van deze verbinding, door verbetering van de capaciteit van de natuurlijke route of door inzet van heterologe routes voor cytosolische acetyl-CoA-synthese, concentreerden deze studies zich voornamelijk op de kinetische parameters, terwijl de energetische aspecten van de acetyl-CoA synthese in het cytosol onderbelicht blijven.
De maximale opbrengst van biomassa of enig ander voor de industrie relevant product gevormd op substraat hangt af van de energiekosten (ATP) van de biochemische route die wordt gebruikt voor bouwsteen- en productvorming. De synthese van acetyl-CoA in het cytosol vanS. cerevisiae omvat de hydrolyse van ATP naar AMP en pyrofosfaat (PPi) in de reactie gekatalyseerd door acetyl-CoA synthase (ACS). De hieropvolgende hydrolyse van PPi tot anorganisch fosfaat (Pi) stelt de energetische kosten van de acetyl-CoA-synthese in het cytosol gelijk aan de hydrolyse van 2 ATP tot 2 ADP en 2 Pi. Deze ATP-uitgave heeft diepgaande gevolgen voor de maximale opbrengst van acetyl-CoA-afhankelijke producten die bereikt kan worden in gemodificeerde giststammen en heeft dientengevolge een negatieve invloed op de economische balans van het productieproces. In het bijzonder in het geval van bulkchemicaliën en biobrandstoffen kan deze ATP-consumerende reactie bepalend zijn voor de economische haalbaarheid. Dit proefschrift beschrijft het onderzoek van ‘metabolic engineering’ strategieën om deze cruciale uitdagingen in de
gistbiotechnologie het hoofd te bieden.
Hoofdstuk 1 beschrijft, na een algemene introductie in ‘metabolic engineering’ en
S. cerevisiae’, de strategieën die tot op heden zijn onderzocht om de beschikbaarheid
van cytosolisch acetyl-CoA te verhogen. Daarnaast beschrijft dit hoofdstuk andere routes voor cytosolische acetyl-CoA-synthese die in de natuur voorkomen, waarvan sommige geen ATP kosten en daarom, na expressie inS. cerevisiae, zouden kunnen leiden tot een verhoging van de maximumopbrengst van acetyl-CoA-afhankelijke producten in gemodificeerde gist stammen.
InS. cerevisiae hangen groei en cytosolische acetyl-CoA synthese volledig af van functionele expressie van óf het Acs1 óf het Acs2 als iso-enzym van acetyl-CoA-synthase (ACS). Vóór het verschijnen van het onderzoek in dit proefschrift, waren er geen literatuurvermeldingen van levensvatbareS. cerevisiae stammen waarin zowel ACS1 alsACS2 was verwijderd. Naast haar anabole en, onder bepaalde condities, katabole rollen, speelt acetyl-CoA ook een sleutelrol in cellulaire regulatie via acetylering van eiwitten, inclusief histonen.
Hoofdstuk 2 onderzoekt de haalbaarheid van het vervangen van de natuurlijke
route voor cytosolische acetyl-CoA-synthese door twee alternatieve, ATP-onafhankelijke routes. Ook wordt in dit hoofdstuk de impact op de groei en de energetica van de gemodificeerde giststammen onderzocht. Met dit doel werd de oorspronkelijke route voor acetyl-CoA synthese vervangen door acetylerende aceetaldehyde-dehydrogenase (A-ALD) of pyruvate-formiaatlyase (PFL). Acetylerend aceetaldehyde-dehydrogenase katalyseert de directe, ATP-onafhankelijke oxidatie van
verschillende bacteriële genen voor acetylerend aceetaldehyde-dehydrogenase en pyruvate-formiaatlyase, werden eracs1Δ acs2Δ S. cerevisiae stammen geconstrueerd waarin ACS functioneel vervangen werd door A-ALD of PFL. In het geval van A-ALD-afhankelijke stammen, werden ook alle aceetaldehyde-dehydrogenases verwijderd, wat resulteerde in een volledige vervanging van de twee-stapsomzetting van aceetaldehyde naar acetyl-CoA, één enkele reactie gekatalyseerd door A-ALD. De aerobe groeisnelheden van A-ALD-afhankelijke stammen waren 79% t.o.v. de referentiestam, terwijl de anaerobe groeisnelheden van de PFL-afhankelijke
S. cerevisiae 73% van de groeisnelheid van de referentiestam haalden. Fysiologische
karakterisering van de snelst groeiende A-ALD- en PFL-afhankelijke stammen werd gedaan in glucose-gelimiteerde chemostaatcultures. De gemeten biomassa-opbrengsten op glucose van de A-ALD- en PFL-afhankelijke stammen waren onverwacht respectievelijk 14% en 15% lager dan die van de referentiestam. Ontkoppeling door de aanwezigheid van formiaat, een zwak zuur waarvan de vorming stoichiometrisch gekoppeld is aan de groei van de PFL-afhankelijke stam, levert een plausibele verklaring voor verlaagde biomassa-opbrengst van deze stam. In de A-ALD-afhankelijke stam werd de verlaagde biomassa-opbrengst geweten aan de toxische effecten van aceetaldehyde, dat in culturen van de gemodificeerde stam in hogere concentraties aanwezig was dan in die van de referentiestam.
Veranderingen in de cytosolische acetyl-CoA-synthese kunnen effect hebben op zowel acetylering van histonen als ook op het centrale metabolisme via acetylering van non-histon-eiwitten en de directe betrokkenheid van dit molecuul bij sleutelreacties in het metabolisme. Echter, analyse van het transcriptoom van A-ALD- en PFL-afhankelijke stammen liet slechts een kleine set genen met een veranderde expressie t.o.v. de referentiestam zien. In combinatie met hun hoge groeisnelheden, suggereerden deze waarnemingen dat deze stammen geen majeure beperkingen hadden in de acetyl-CoA-voorziening. Deze conclusie werd verder ondersteund door de kleine verschillen in intracellulaire metabolietniveaus van een A-ALD-afhankelijke stam ten opzichte van de referentiestam. Intracellulaire acetyl-CoA-concentraties, die een combinatie van mitochondriële en nucleo-cytosolische verzamelingen weergeven, waren ook niet significant verschillend tussen de A-ALD-afhankelijke stam en de controlestam, wat erop zou kunnen duiden dat intracellulaire acetyl-CoA-concentraties onderhevig zijn aan sterk homeostatische regulatie. Hogere intracellulaire concentraties van lysine in de A-ALD-afhankelijke stam kunnen wellicht zelfs duiden op een verhoogde beschikbaarheid van cytosolisch acetyl-CoA. Het onderzoek inHoofdstuk 2 heeft voor het eerst laten zien dat de oorspronkelijke route voor cytosolische acetyl-CoA-synthese inS. cerevisiae volledig vervangen kan worden door heterologe, ATP-onafhankelijke routes.
Tijdens respiratoire groei vanS. cerevisiae op glucose, is cytosolisch acetyl-CoA nodig om aan de biosynthetische behoefte van de cel, inclusief de biosynthese van lysine en vetten, te voldoen. Echter, deze biosynthetische fluxen zijn relatief klein in
vergelijking met de totale turnoversnelheid van ATP en hebben daarom een relatief kleine impact op de groei-energetica (berekende impact op biomassa-opbrengst in aerobe glucose-gelimiteerde cultures < 0,5%). Een fundamenteel andere situatie ontstaat wanneer ethanol wordt gebruikt als koolstofbron en acetyl-CoA-synthase het startpunt is voor alle biosynthetische en dissimilatoire routes in groeiende gistcellen. Besparing van de 2 ATP nodig voor synthese van elk cytosolisch acetyl-CoA-molecuul zou, onder deze condities, theoretisch een toename in de biomassa-opbrengst van 0,57 g/g ethanol voor de natuurlijke route tot 0,80 g/g ethanol voor de ATP-onafhankelijke route mogelijk moeten maken. Een dergelijke dramatische toename in de energetische efficiëntie van ethanolgebruik zou hoogst relevant voor de industrie kunnen zijn, omdat het zou moeten resulteren in een hogere acetyl-CoA-afhankelijke productopbrengst op ethanol. Hoofdstuk 3 focust om die reden op giststammen waarin de oorspronkelijke route voor acetyl-CoA-synthese is vervangen door A-ALD. De A-ALD-afhankelijke stammen echter, lieten geen onmiddellijke groei zien op media met ethanol als enige koolstofbron. Verlengde incubatie, gevolgd door langetermijnlaboratoriumevolutie-experimenten leverde A-ALD-afhankelijke stammen op die in staat waren te groeien op ethanol met specifieke groeisnelheden tot 0,11 uur−1. Reverse engineering studies laten zien dat mutaties in ACS1, het gen dat codeert voor een van de cytosolische acetyl-CoA synthases, essentieel waren voor groei op ethanol van de A-ALD-afhankelijke stammen. Verkregen mutaties in A-ALD-genen verbeterden de VMAX/KM voor aceetaldehyde van de gecodeerde enzymen, maar
waren niet essentieel voor groei op ethanol.
Alhoewel de reverse engineered stammen groeiden op ethanol, waren hun groeisnelheden lager dan de groeisnelheden van de geëvolueerde stammen. Verdere analyse van de groei van deze stammen suggereerde een beperkte beschikbaarheid van mitochondrieel acetyl-CoA tijdens de groei van de A-ALD-afhankelijke stammen op ethanol. Van de vijf geëvolueerde stammen die getest zijn in ethanol gelimiteerde chemostaatcultures, liet slechts een geëvolueerde stam 5% toename in de biomassaopbrengst op ethanol zien ten opzichte van de referentiestam, wat beneden de theoretische 30-40% voorspelling was. Een toename in de productie van aceetaldehyde en ander bijproducten was geïdentificeerd als mogelijke oorzaak voor deze suboptimale biomassa opbrengsten. Dit onderzoek inHoofdstuk 3 bewijst dat de natieve gistroute voor omzetting van ethanol naar acetyl-CoA vervangen kan worden door een ge-engineerde route die in potentie biomassa- en productopbrengsten sterk kan verbeteren. Gebaseerd op metabolic en evolutionary engineering, resequencing van het hele genoom, reverse engineering en fysiologische analyse van de geëvolueerde stammen, identificeren wij intracellulaire aceetaldehyde niveaus en voorziening van intra-mitochondrieel acetyl-CoA als primaire doelen voor verder optimalisatie van de omzetting van ethanol door eukaryotische cell factories.
Pyruvaatdehydrogenase (PDH) is een groot, multisubunit enzymcomplex waarvan de grootte vergelijkbaar is met dat van een ribosoom. Het PDH complex katalyseert de oxidatieve decarboxylatie van pyruvaat naar acetyl-CoA wat, in veel organismen, de sleutelreactie is op het snijvlak van glycolyse en de citroenzuurcyclus (TCA). In
in S. cerevisiae. In Hoofdstuk 4, is de uitdaging opgenomen om een functioneel, heteroloog PDH-complex in het cytosol van gist tot expressie te brengen. Om te bepalen of een heteroloog PDH-complex de oorspronkelijke route van cytosolisch acetyl-CoA-vorming inS. cerevisiae kan vervangen, werd het PDH-complex van de bacterieEnteroccoccus faecalis gekozen om tot expressie te worden gebracht in gist. Drie factoren droegen bij aan de selectie van het PDH-complex vanE. faecalis voor deze studie: (i) bacteriële PDH-subunits hebben vermoedelijk geen mitochondriële targeting sequenties. (ii) het PDH van E. faecalis is relatief ongevoelig voor hoge NADH/NAD+ ratioos in vergelijking met andere bacteriële PDH’s; en (iii)in vitro experimenten wijzen erop dat gezuiverde subunits van hetE. faecalis PDH autonoom een functioneel complex vormen. Alle bekende PDH-complexen hebben vier cofactoren nodig voor hun activiteit: FAD+, NAD+, thiamine pyrofosfaat (TPP) en lipoaat. In
S. cerevisiae wordt lipoaat gesynthetiseerd en covalent gebonden aan het PDH-complex
in de mitochondria. Om deze reden was het waarschijnlijk dat cytosolische expressie van een bacterieel PDH-complex een co-expressie van eiwitten nodig zou hebben die de lipoaat van PDH katalyseren en toevoeging van lipoaat aan het medium.
De expressie van genen coderend voor de vier subunits van het E. faecalis PDH-complex, samen met twee lipoaat-ligases van E. faecalis in het cytosol van
S. cerevisiae, gecombineerd met de deletie van ACS1 en ACS2, maakte groei van een acs1Δ acs2Δ stram van S. cerevisiae op glucose mogelijk. De strikte afhankelijkheid
voor groei van de toevoeging van lipoaat, bevestigde de in vivo activiteit van het heterologe PDH-complex. De aerobe groeisnelheid op glucose van de verkregen stam (0,36 uur−1) was vergelijkbaar met de groei van de referentiestam (0,42 uur−1) en onafhankelijk van de concentratie van lipoaat in de range van 20 ng mL−1 tot 1000 ng mL−1. De Acs− giststam met het bacterieel PDH-complex had ook een groeisnelheid die bijna gelijk was aan het wildtype onder anaerobe condities (respectievelijk 0,30 uur−1 en 0,33 uur−1 voor de engineered en de wildtypestam). Het functioneren van hetE. faecalis PDH-complex in anaerobe gistcultures laat zien dat dit ook toegepast kan worden in het ontwerp en de constructie van anaerobe productroutes. Enzymactiviteitmetingen laten zien dat in een aangepaste stam, PDH vanE. faecalis, een hogere specifieke activiteit (53 nmol min−1 [mg eiwit]−1) bereikte dan de activiteit van een mitochondrieel PDH uitS. cerevisiae (12 nmol min−1 [mg eiwit]−1).
De cytosolische lokalisatie van het heteroloog tot expressie gebrachteE. faecalis PDH-complex in het gistcytosol, werd bevestigd door subcellulaire fractionering in combinatie met enzymactiviteitmetingen en proteomics op basis van massaspectrometrie. Terwijl de specifieke activiteit van het PDH in de mitochondriële fracties van de aangepaste stam en de wildtypestam niet wezenlijk verschilde, was de activiteit van het PDH gemeten in de cytosolische fracties van de Acs− stam met het
E. faecalis PDH 32-maal hoger dan die van de wildtypestam. De cytosolische
lokalisatie van de vier subunits van hetE. faecalis PDH-complex, zowel als van de twee lipoaatligases van E. faecalis in het cytosol van S. cerevisiae werden verder
bevestigd door proteoomanalyse op basis van massaspectrometrie van de cytosolische fractie. In E. faecalis komt PDH voor als een eiwitcomplex bestaande uit 210 subunits. Gelfiltratie van een cytosolische fractie, gecombineerd met enzymactiviteitmetingen en een proteoomanalyse, toonden aan datE. faecalis PDH aanwezig was in het cytosol vanS. cerevisiae als een complex met een grootte, een specifieke activiteit en een relatieve overmaat van de E1-, E2- en E3-subunits vergelijkbaar met dat wat gerapporteerd is voor het natieve E. faecalis PDH. Fysiologische karakterisering op basis van glucose-gelimiteerde chemostaat cultures toonde vergelijkbare biomassa-opbrengst en suikerdissimilatiesnelheden voor de Acs−-stam met PDH-expressie en de referentiestam. In combinatie met de afwezigheid van grote verschillen in het transcriptoom van de aangepaste en de oorspronkelijke stammen, toonden deze resultaten aan dat de vervanging van de cytosolische acetyl-CoA-synthese met het PDH-complex vanE. faecalis niet leidde tot significante verstoringen van de fysiologie van S. cerevisiae.
Het onderzoek in hoofdstuk 2-4 van dit proefschrift laat zien dat het volledig vervangen van de oorspronkelijke route voor cytosolische acetyl-CoA synthese in
S. cerevisiae door heterologe ATP-onafhankelijke routes kan resulteren in
levensvatbare giststammen. Bovendien levert het conceptueel bewijs dat implementatie van ATP-onafhankelijke routes kan resulteren in toename van de biomassa-opbrengst op substraat. Deze strategieën lijken in het bijzonder geschikt voor het verbeteren van de productopbrengst op suiker van willekeurig welke verbinding geproduceerd inS. cerevisiae, die cytosolisch acetyl-CoA nodig heeft als bouwsteen. Om deze reden voorzien de resultaten gepresenteerd in dit proefschrift de metabolic engineers van nieuwe strategieën om de prestaties vanS. cerevisiae als ‘cell factory’ te optimaliseren voor duurzame productie van brandstoffen en chemicaliën.
CHAPTER
1
General introduction
Introduction to metabolic engineering
The origins of metabolic engineering date back to 1973, when Herbert Boyer and Stanley N. Cohen first introduced a recombinant DNA molecule intoEscherichia coli. Soon after, genetic engineering was applied for the development and optimization of new biotechnological processes, such as production of penicillin V byAspergillus niger [1] and human insulin byE. coli [2]. These developments led to two papers which, in 1991, introduced metabolic engineering as a new field of research [3, 4]. Bailey defined metabolic engineering as ‘the improvement of enzymatic, transport and regulatory functions of the cell with use of the recombinant DNA technology’ [3]. As predicted by Bailey and Stephanopolous, metabolic engineering developed into a fast growing field of science, as reflected by the increasing number of papers devoted to the topic. The number of published papers on metabolic engineering increased from 3730 in 1991 to 13100 in 2000 and 33700 in the year 2014 (number of scientific publications containing the term ‘metabolic engineering’ obtained by a Google Scholar search).
Nowadays, metabolic engineering of microorganisms is recognized as an established strategy for development of more cost-effective and green industrial processes. Increasing concerns about decreasing oil reserves, as well as a growing ambition to move towards sustainable and environmental friendly processes have boosted interest in production of fuels and commodity chemicals from renewable resources such as corn starch, cane sugar and cellulosic biomass. In order to compete with petrochemical based production, those processes need to be cost-effective, which often requires optimization of titers, yields and productivities. Metabolic engineering facilitates improvement of the native microbial product pathways in terms of product formation kinetics and yields, in order to assure profitable production of (bio)fuels and chemicals. It also enables design and engineering of novel metabolic pathways to implement microbial synthesis of chemical compounds that so far were derived from fossil fuels.
The variety of chemicals produced by metabolically engineered microorganisms, as well as the engineering strategies applied to generate them, were extensively reviewed in two recent papers by Lee et al. and Rabinovitch-Deere et al. [5, 6]. Lee et al. divided chemical compounds produced by microorganisms in four categories: natural–inherent – endogenous chemicals produced by non-engineered microorganisms (e.g. amino acids, lactate and 2,3-butanediol); natural–noninherent – chemicals that are found in nature but are produced by microorganisms as a result of introduction of heterologous pathway(s) (e.g. isoprene, taxadiene and lycopene); non-natural– noninherent – chemicals that have not yet been found in nature but can be produced by microorganisms by establishing heterologous pathways and enzymes often originating from different natural sources (e.g. 6-aminocaproic acid); non-natural–created – chemicals that have not yet been found in nature and are produced by synthetically designed or evolved enzymes with new catalytic properties (e.g. 1-homoalanine and 5-methyl-1-heptanol) [5]. The large variety of chemical compounds whose production by microorganisms was enabled by metabolic engineering, illustrates the innumerable possibilities of this field of research.
In addition to the list of synthesized products, metabolic engineering also extended the range of substrates that can be used for microbial production. The sustainability attributed to utilization of renewable carbon sources is one of the main reasons to engineer microorganisms for production of biofuels and chemicals from biomass. Among others, lignocellulosic plant biomass, due to its abundance and low cost, is a very attractive raw material for synthesis of low-value added products. Lignocellulosic biomass consists of cellulose, hemicellulose and lignin, and the efficient use of this feedstock requires utilization of most of those components. While glucose obtained from hydrolysis of cellulose is a common substrate for many microorganisms, pentose sugars (xylose, arabinose) that originate from hemicellulose hydrolysates and which contribute up to 26% of plant material [7], cannot be used as a carbon source by many popular industrial microorganisms. Huge efforts have therefore been made to extend the substrate range of those organisms by introduction of xylose and/or arabinose utilization pathways [7].
Utilization of renewable carbon sources often also requires engineering of cellular physiology for adaptation to process conditions. For example, acetic acid and furfural, which are produced during pretreatment of lignocellulosic biomass, are inhibitory or even toxic for production strains. Engineering of the tolerance of industrial microorganisms to these compounds is therefore a key element in the development of robust fermentation processes based on lignocellulosic hydrolysates [8]. Also the product itself can become toxic for a producing organism as it accumulates in the culture broth. In such a case, improved tolerance towards the product may be a prerequisite to reach economically feasible production titers of biofuels or commodity chemicals [9].
Development of pathways leading to new products, elimination or reduction of byproduct formation, enhancement of yields and productivities, extension of substrate range and improved cellular physiology of microorganisms were identified as important metabolic engineering targets in microbial strain improvement [10]. Another application of metabolic engineering is development of natural pathways for degradation of xenobiotics, as well as introduction of new pathways in strains already established as a good platform for bioremediation, which is a promising technology for treatment of environmental pollutions [11, 12]. Strain engineering for heterologous protein expression is another great example of fast and extensive applicability of metabolic engineering. Initiated by production of human insulin inE. coli in 1979, nowadays production of proteins in heterologous hosts includes production of pharmaceuticals (hormones, antibodies, vaccines) as well as various enzymes for use in agriculture, nutrition, detergents production, substrate pretreatment and for other applications. In production of many of those proteins, prokaryotic systems are used. Additionally, the need for post-translational modifications (e.g. proper folding, glycosylation or phosphorylation) in case of many eukaryotic proteins resulted in a wide variety of different eukaryotic host systems for protein production including yeast, insect cells, mammalian cells, plants and molds [13–17].
Irrespective of the goal, modern metabolic engineering involves use of many sophisticated tools that help scientists on every step of strain construction and analysis. Although genetic engineering and molecular biology are indispensable components of metabolic engineering, efficient design, construction and optimization of a pathway requires additional expertise in areas such as biochemistry, thermodynamics and kinetic analysis of different pathway configurations, flux analysis and modeling, bioinformatics, inverse metabolic engineering and others [18]. Fast development and integration of these disciplines, contributes to continual expansion of the available metabolic engineering tools and of the complexity of the challenges that metabolic engineering can successfully address.
One of the first questions that has to be answered after a product of interest is identified, is which metabolic pathways lead to the desired compound. Additionally, steps which may result in byproduct formation should be identified in order to avoid suboptimal product yields. Several computational pathway prediction algorithms have been generated to assist scientist in those analytical steps. Those algorithms can be divided into two types of models. Algorithms such as OptKnock, OptGene and OptForce allow scientists to add changes to existing pathways or metabolic networks by in silico evaluation of gene knockouts or amplification [19–21]. Some of these algorithms can also be used to determine the group of reactions that lead to desired product or to optimize the set of existing reactions to achieve better performance of a strain whose genome was sequenced. For example, once a genome sequence of a microorganism is available, it can be combined with literature data (such as functional annotation of genome, identification of the associated reactions and determination of their stoichiometry, etc.) to build genome-scale metabolic models. Such models can be used to predict the growth supporting fluxes through a linear optimization approach [22, 23] and to determine the distribution of fluxes that supports an optimal rate or yield of the product formation. In contrast to the aforementioned algorithms, which use known, existing pathways as their input, Biochemical Network Integrated Computational Explorer (BNICE) utilizes information about chemical structures of the substrates and products and a set of biotransformation rules based on the Enzyme Classification (EC) system. This approach enables thein silico design of new, thermodynamically feasible pathways that lead from the substrate to the product [24]. This algorithm is especially useful in building possible synthesis pathways for production of non-natural–noninherent and non-natural–created chemicals.
Once identified, a heterologous pathway has to be introduced and optimized in a suitable microbial host strain. Introduction of heterologous genes, as well as deletion of those encoding unwanted steps that can lead to byproduct(s) formation and result in lower yield, requires availability of efficient transformation systems. Initially, powerful, well developed genetic engineering tools were available for only a limited number of industrial microorganisms, includingSaccharomyces cerevisiae andE. coli [25–27]. Efficient methods for chromosomal integration, the possibility of gene expression from high copy-number plasmids and the availability of many auxotrophic and dominant marker genes, madeS. cerevisiae and E. coli two of the
most often used strains in academic and industrial research. Nevertheless, the need for use of other (micro)organisms, better adapted to certain production conditions (e.g. high temperature, low pH) or already expressing desired product pathways drives a sustained, but slower, development of molecular biology techniques for other host strains [27, 28]. A promising tool that enables gene deletion and integration in many organisms, including non-conventional industrial microorganisms is the CRISPR-Cas system [29, 30]. This bacterial adaptive immune defense system, which was already successfully applied in genetic engineering of many eukaryotes, has dramatically changed the ability to edit genomes of diverse organisms [29–34].
Optimal performance of an engineered pathway depends on expression of the introduced genes andin vivo activities of the resulting enzymes. Those factors can be optimized at the transcriptional, post-transcriptional and post-translational levels [35]. For example, to assure proper expression of genes, a number of promoter libraries has been developed for prokaryotic and eukaryotic organisms. Such libraries include both naturally occurring promoters as well as promoters that contain synthetic regulatory sequences [36–39]. Alternative approaches for transcriptional regulation include engineering of the global transcription machinery [40, 41] and the design of new transcription factors [42]. These approaches allow for reprogramming gene networks and metabolism, by simultaneously affecting the regulation of multiple genes. Post-transcriptional regulation can be modified by engineering the structural elements present around the translation initiation regions of mRNA. This approach was first demonstrated by Pfleger et al. [43], who generated libraries of tunable intergenic regions for optimization of the expression of multiple genes within an operon. Additional modification can be introduced at the level of the gene sequence and result in a more optimal translation process. Optimization of protein coding sequence for expression in a given host, including tuning of the codon-pair fitness, may lead to significant improvement of expression of heterologous proteins [44]. Finally, post-translational modifications, such as the expression of protein scaffolds, can result in higher titers by increasing the probability of the efficient collisions between enzymes and substrates. Dueber et al. used eukaryotic protein-protein interaction domains to optimize the stoichiometry of the three enzymes involved in the biosynthesis of mevalonate and combine them in a synthetic protein scaffold, which resulted in a 77-fold increase in titer [45].
Analysis of the modified strain and comparison of its performance to the parental strain or to another strain(s), in which different engineering strategies were applied, is important to evaluate the engineered pathway and to identify possible bottlenecks. Advances in transcriptomics, proteomics (including post-translational modifications), metabolomics and flux determination combined with computer-aided modeling and high-throughput systems facilitate such analyses and greatly contribute to development of any metabolic engineering strategy. The development of rapid sampling and 13C-labelling for identification of metabolic network structures and
levels of metabolites, supported by advancements in many analytical techniques such as GC-MS, LC-MS or13C-NMR and modeling are important to quantify the fluxes
through the different branches of the network [46–49]. Determination of the fluxome under different (dynamic) conditions and their deviations from the controlled (steady) state can be used in a platform of metabolic flux-analysis to determine kinetics of the metabolic network and identify the limiting step. Such information can be used for further design of the next metabolic engineering step in the process of performance optimization in terms of yield, productivity and titer.
Progress in ‘omics’ technologies and in high-throughput technologies for strain construction and characterization also support other, more random methods of strains and enzyme optimization. Once the rate-limiting step has been identified, the properties of the enzyme (e.g. KM or VMAX) can be optimized, e.g. by error-prone
PCR and high-throughput screening for improved enzyme kinetics. Tolerance of microbial cells to high concentrations of substrate, product or any organic solvents present in industrial processes often involves complex and therefore difficult to predict physiological responses. Evolutionary engineering, which imitates and sometimes accelerates natural evolution, followed by whole genome and/or plasmid sequencing and strain re-engineering, has proven to be a powerful approach to obtain robust phenotypes that would have been difficult to obtain by targeted methods for genetic modification [50]. Random mutagenesis (e.g. by UV-radiation), combined with appropriate selection strategies, is another method that can deliver strains with new, better properties without a need for detailed knowledge on the molecular basis for improved performance [51].
In the literature, metabolic engineering is often described as a cycle, consisting of three steps [10]:
– construction of a recombinant strain with modifications aiming at improvement of its properties, including modification of an existing pathway, or introduction of new one;
– analysis of the modified strain and comparison of its performance to that of the parental strain, in order to determine the influence of introduced changes on the strain performance;
– design of the next target for genetic engineering based on the achieved results or in relation to further strain development.
The success of this cyclic metabolic engineering strategy may require numerous iterations, as well as an integrative approach that engages many of the available tools and techniques, in order to achieve optimal strain performance. Many challenges remain to be solved in order to further accelerate the metabolic engineering cycle. These include the fast and rational design of new enzymes and pathways for production of non-natural compounds and increasing the accessibility of alternative hosts that can be applied under non-standard, industrially relevant conditions (e.g. high temperatures, low/high pH, high concentrations of solvents, etc.).
Selection of a suitable production host is one of the most crucial elements in every metabolic engineering strategy. Factors that influence the final choice include the product of interest, presence of pathways that lead to synthesis of product or
precursors and favored/required process conditions, including carbon source utilization. Many commodity chemicals and biofuels are already naturally produced by microorganisms. However, those producing organisms are rarely adapted to required industrial process conditions or able to use the preferred substrate. The choice of a suitable microorganism is thus often primarily based on availability of a well-developed genetic engineering toolbox, since introduction of genetic changes may be a difficult and time consuming process. Therefore, in most of the metabolic engineering strategies one applies microorganisms for which genetic engineering and analytical tools, as well as broad knowledge of genome and metabolism are available. Those traits enable relatively fast engineering of those microorganisms, often designated as metabolic engineering platforms, for the production of native and heterologous compounds, utilization of new carbon sources and for adaptation to new, often toxic conditions in order to obtain production process with economically feasible titer, yield and productivity.
Saccharomyces cerevisiae – an industrial workhorse with
numerous applications
Robustness and tolerance towards industrial conditions, such as osmotic stress, low pH, high sugar and ethanol concentrations, together with its insensitivity to phage infection makeS. cerevisiae very well suited microorganism for large-scale industrial application. These traits, together with the extensive fundamental knowledge of the physiology of this organism and the fast development of yeast molecular biology tools, facilitated by whole genome sequencing [52], resulted in many different examples of metabolic engineering ofS. cerevisiae for production of biofuels and various chemicals (Figure 1.1).
One of the oldest and best known applications of S. cerevisiae is production of ethanol, both as an ingredient of fermented beverages and as a biofuel. Limited byproduct formation and high tolerance to ethanol make this yeast an excellent host for industrial production of this compound. To further improve the yield of the ethanol on sugar,S. cerevisiae was engineered to minimize or completely abolish the formation of glycerol. Glycerol is the main byproduct synthesized during anaerobic alcoholic fermentation, produced by the cell in order to achieve redox cofactor balance. Deletion of both genes encoding NADH-dependent glycerol-3-phosphate dehydrogenases [53], altering cellular redox metabolism to reduce NADH formation [54] or reducing growth by lowering the ATP production [55] are only a few examples of metabolic engineering strategies that brought the ethanol yield of S. cerevisiae close to the theoretical maximum yield of 0.51 g ethanol / g glucose. Currently, the bioethanol production exceeds 75 billion liters annually, with the majority being produced with corn starch as the feedstock [56]. The use of corn as the main substrate has raised concerns about the cost of the production process, as well as possible competition for resources between food and fuel production.
S. cerevisiae lacks a natural ability to efficiently metabolize the pentoses (arabinose
glucose glucose-6-P ribulose-5 -P pyruvate lactate phosphoenolpyru vate phenylalanine + tyrosine isobutanol acetaldehyde ethanol acetyl-CoA acetoacetyl-CoA dimeth ylallyl-5-PP malonyl-CoA fatty acids fatty acid ethyl ester s lipids polyketides farnesene taxadiene bisabolone β-carotene squalene acetyl-CoA butanol ribo fl avin sugar alcohols vanillin coumaric acid naringeni n resveratrol malate succin ate citrate Glycolysis P entose phosp hate pathway Shikimate pathway Tricarb oxylic acid cycle Mevalonate pathway TCA Figure 1.1: Sc hematic represen tation of the metab olism of Sac char omyc es cer evisiae ,indicating key precursors and examples of nativ e and heterologous pro ducts of industrial in terest, including (from top left, highligh ted) sugar deriv ativ es, aromatic comp ounds, acids, alcohols, fatt y acid deriv ativ es and isoprenoids.
convert a number of other industrially relevant carbohydrates (e.g. cellulose, starch, xylan, cellobiose and melibiose). Additionally, other sugars present in the biomass feedstock, such as galactose, can be metabolized byS. cerevisiae but their uptake rate is much lower than that of glucose. These features negatively influence the productivity and, consequently, the feasibility of the processes in which lignocellulosic feedstocks are used. Therefore, many metabolic engineering studies were focused on extending the substrate range ofS. cerevisiae. Introduction of heterologous xylose isomerase and arabinose isomerase pathways, in combination with over-expression of the non-oxidative part of the pentose phosphate pathway has resulted in strains that are able to ferment pentoses to ethanol with yields similar to those on glucose [57, 58]. Furthermore, application of evolutionary engineering resulted in mutant strains able to co-consume mixtures of glucose, xylose and arabinose [59].S. cerevisiae was also engineered for fermentation of amorphous cellulose [60, 61], starch [62], xylan [63], melibiose [64], for improved fermentation of galactose [65] and for co-fermentation of xylose and cellobiose by simultaneous expression of cellodextrin transporter, β-galactosidase and enzymes allowing xylose metabolism [66].
Efficient consumption of sugars derived from biomass also requires increased tolerance to numerous compounds that are released during pretreatment and hydrolysis of lignocellulosic feedstocks, such as acetic acid, galacturonic acid, furfural and hydroxymethylfurfural. Evolutionary engineering of S. cerevisiae resulted in significant improvement of yeast tolerance towards acetic acid [67]. An alternative approach was proposed by Guadalupeet al. [68], who engineered yeast to use acetic acid as an external electron acceptor. In this manner, yeast strains unable to produce glycerol, were able to achieve redox cofactor balance in an anaerobic fermentation process, by reducing acetic acid to ethanol [68]. Several studies were also dedicated to galacturonic acid focusing on increasing tolerance to this compound and/or enabling its consumption by yeast [69–71]. Despite functional expression of heterologous enzymes for galacturonate uptake and metabolism, fermentation of galacturonic acid has not been achieved yet.
In recent years, other compounds started to gain more and more attention as potential biofuels, due to their advantages over ethanol or possible applications in diesel engines. Higher energy density, lower hygroscopicity and the possibility to be blended up to 85% with gasoline, render butanol an attractive biofuel [72]. There are four different isomers of butanol, of which isobutanol has the highest octane number [73] and is naturally produced inS. cerevisiae through the Ehrlich pathway, although at a very low rate [74, 75]. The first example of metabolic engineering of yeast for isobutanol production was reported by Chenet al. [76], who overexpressed the endogenous genes of the mitochondrial pathway for valine biosynthesis. Further efforts focused on partial or total relocalization of this pathway to the yeast cytosol, pathway improvement by expression of heterologous genes for isobutanol synthesis and alteration of native yeast metabolism for improved availability of pyruvate. These modifications led to an isobutanol yield of 0.33 g/g of glucose, which is more than 80% of the theoretical yield, and an isobutanol titer of 18.6 g/L [77–80]. The first yeast strain engineered for
synthesis ofn-butanol was reported by Steen et al. [81]. Overexpression of a number of genes originating from different microorganisms yielding a synthesis pathway utilizing acetyl-CoA as a precursor, led ton-butanol production of 2.5 g/L. A similar approach was proposed by researchers from Gevo Inc., who proposed to incorporate the butanol synthesis pathway fromClostridium acetobutylicum and increase the supply of cytosolic acetyl-CoA by expression of the pyruvate dehydrogenase complex fromE. coli, which converts pyruvate to acetyl-CoA [82]. Although S. cerevisiae is able to grow at relatively high concentrations of butanol [83], further improvement of its tolerance to this compound is required for feasible, high-titer production processes. Several mutations and underlying molecular processes were identified by means of evolutionary engineering and reverse engineering, which resulted in improved butanol tolerance [84–86].
S. cerevisiae was also engineered for production of other compounds with potential
applications as jet fuel or diesel, such as fatty acid ethyl esters (FAEEs) and isoprenoids. In order to enable FAEEs production in yeast, Shiet al. combined overexpression of heterologous wax ester synthase with up-regulation of acetyl-CoA carboxylase, leading to a biodiesel titer of 8.2 mg/L [87]. Production of isoprenoid diesel substitutes by engineered S. cerevisiae was reported by Peralta – Yahya et al., who tested different bisabolene synthases inS. cerevisiae and reached titers of 0.99 g/L [88]. The recent studies by Ozaydin et al. report even higher titers of bisabolene of 5.2 g/L [89]. Farnesene, which can be blended with petroleum diesel up to 35%, is another isoprenoid whose production inS. cerevisiae is intensively studied by Amyris Inc. [90]. Isoprenoids form a chemically diverse group of compounds with a broad range of applications as medicines, fragrances, dyes, food additives and fine chemical intermediates. These compounds were originally extracted only from natural sources, mostly plants, which resulted in their very limited and strongly fluctuating availability. Therefore, production of isoprenoids by genetically modified microorganisms became one of the most intensively studied subjects in microbial biotechnology [91]. For the production of farnesene, Amyris Inc. used knowledge and technology gained during development ofS. cerevisiae strain for production of artemisinic acid [92]. Artemisinic acid is a precursor of the antimalarial drug artemisinin, and, similar to farnesene, it is derived from the precursor farnesyl pyrophosphate (FPP) [93, 94]. Strain engineering for improving FPP synthesis involved overexpression of a substantial number of genes encoding key enzymes of the mevalonate pathway, which is the route that converts acetyl-CoA into isopentenyl pyrophosphate (IPP) and dimethylalallyl pyrophosphate (DMAPP) – two main precursors of all isoprenoids. Additionally, a number of genes that led to byproduct formation were deleted or their expression was reduced. Finally, the availability of acetyl-CoA was increased by overexpression of native genes as well as introduction of a heterologous acetyl-CoA synthetase from Salmonella enterica [93–95]. The diversity of isoprenoids is enormous. So far, S. cerevisiae has been successfully engineered for production of, among others, squalene [96], geranylgeraniol [97], patchoulol [98], β-carotene [99] and taxadiene, an isoprenoid with medical application as a precursor of taxol (drug used in cancer chemotherapy) [100].
Another group of complex molecules that can be produced by S. cerevisiae consists of compounds originating from the shikimate pathway. The shikimate pathway converts erythrose-4-phosphate, an intermediate of the pentose phosphate pathway, and phosphoenolpyruvate, a glycolytic intermediate, into the aromatic amino acids tyrosine and phenylalanine. Aromatic rings are common structural elements of a wide range of compounds that originate from the shikimate pathway. Several studies on engineering ofS. cerevisiae for production of such compounds were reported in the literature. Overexpression of heterologous genes originating from plants resulted in a yeast strain able to produce resveratrol, a compound with potential antioxidant properties, produced in yeast from coumaric acid [101–104]; naringenin, a flavonoid which is considered to have many positive effects on human health and also serves as precursor for other flavonoids [105, 106] and vanillin, with potential application as a flavoring agent [107].
High tolerance ofS. cerevisiae to low pH also makes this yeast a promising host for production of organic acids such as citric acid, succinic acid, malic acid, pyruvic acid and L-lactic acid, many of which are intermediates of the citric acid cycle. Production of those acids at low pH avoids formation of gypsum in the product recovery step, which positively influences economy and sustainability of the process. There are several examples of metabolic engineering ofS. cerevisiae for the synthesis of organic acids. Relocalization of malate dehydrogenase to the cytosol, overexpression of the native pyruvate carboxylase and expression of heterologous exporters of malic acid resulted in malate titers of up to 59 g/L [108]. Deletion of all three genes encoding pyruvate decarboxylases (PDC1, PDC5, PDC6 ), followed by evolutionary engineering to overcome C2-dependency of the Pdc− strain resulted in a mutant strain able to produce pyruvic acid at the yield of 0.54 g/g glucose [109]. Finally, introduction of heterologous genes coding for L-lactic acid dehydrogenases, combined with knocking out pyruvate decarboxylase (PDC1 ) and alcohol dehydrogenase (ADH1 ), led to strain overproducing lactate with a yield of 0.75 g/g glucose [110].
S. cerevisiae was also engineered for production of many other fine and bulk
chemicals (Figure 1.1). All these examples show the broad application ofS. cerevisiae as a production strain. Therefore, metabolic engineering of this microorganism as a cell platform, with optimized supply of central metabolites that in many cases serve as precursors for other chemicals of interest (Figure 1.1), is an important focal point in research aimed at optimizing the production of biofuels and biochemicals from renewable feedstocks.
Acetyl-CoA – physiological functions and synthesis
Acetyl coenzyme A (acetyl-CoA) is a ubiquitous metabolite that can be found in all domains of life. In this compound, an acetyl group is connected to coenzyme A through an ‘energy-rich’ thioester bond, which facilitates transfer of the acetyl moiety to a variety of acceptor molecules. As shown in Figure 1.2, inSaccharomyces cerevisiae there are two major pathways of acetyl-CoA synthesis [111]. When this yeast is grown on glucose, pyruvate generated in glycolysis enters the mitochondria, where it is converted
glucose pyruvate formate acetyl-CoA acetyl-CoA acetate ethanol TCA acetaldehyde pyruvate lipids lysine PDHmit A-ALD ADH ALD PFL ACS PDHcyt PDC CoA CoA NADH NADH CoA NADH NADH CO2 CoA CO2 CO2 NADH AMP+PPi CoA ATP Mitochondrion 2 NADH 2 ATP
Figure 1.2: Synthesis of acetyl-CoA in Saccharomyces cerevisiae via native (black) routes and
heterologous pathways catalyzed by: acetylating acetaldehyde dehydrogenase (blue); pyruvate dehydrogenase complex (green) and pyruvate-formate lyase (yellow). Abbreviations: ACS – acetyl-CoA synthetases; ADH – alcohol dehydrogenases, ALD – acetaldehyde dehydrogenases; A-ALD – acetylating acetaldehyde dehydrogenase; PDC – pyruvate decarboxylases; PDHcyt
– cytosolic pyruvate dehydrogenase; PDHmit – mitochondrial pyruvate dehydrogenase; PFL –
pyruvate-formate lyase.
to acetyl-CoA and CO2in a reaction catalyzed by the pyruvate dehydrogenase (PDH) complex. Acetyl-CoA generated this way cannot leave mitochondria to satisfy the requirements for acetyl-CoA in the cytosol, which is needed for biosynthesis of cellular components such as lipids and the amino acid lysine [112, 113]. S. cerevisiae lacks ATP-citrate lyase, that could convert citrate originating from mitochondria into cytosolic acetyl-CoA [114]. Moreover, althoughS. cerevisiae contains structural genes for the proteins of the carnitine shuttle, it is unable to synthesize carnitine. Therefore, unless carnitine is added to growth media, the carnitine shuttle cannot transfer acetyl-units across the mitochondrial inner membrane [115]. To synthesize cytosolic acetyl-CoA, pyruvate is converted to acetyl-CoA through a pathway known as the ‘pyruvate dehydrogenase bypass’. This pathway involves the combined activity of pyruvate decarboxylases (PDC), acetaldehyde dehydrogenases (ALD) and acetyl-CoA synthetases (ACS) [111]. The latter two enzymes are also required for growth on C2-compounds, such as acetate or ethanol. When the latter serves as a carbon source, it is first converted to acetaldehyde in the reaction catalyzed by alcohol dehydrogenases (ADH) and further to acetyl-CoA (Figure 1.2). Under those conditions, cytosolic acetyl-CoA is also used to fuel tricarboxylic acid cycle (TCA) for generation of energy and TCA intermediates in mitochondria. The mechanism by which the cytosolic acetyl-CoA is fed to the mitochondrial TCA cycle, has not yet been fully resolved.
