Cristiana Silva Faria Mestrado em Microbiologia Aplicada 2010€¦ · organismos têm sido isolados...
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Universidade de Lisboa Faculdade de Ciências Departamento de Biologia Vegetal Engineering Saccharomyces cerevisiae for the production of mannosylglycerate - a yeast model for assessing its application in protein stabilization Cristiana Silva Faria Mestrado em Microbiologia Aplicada 2010
Transcript of Cristiana Silva Faria Mestrado em Microbiologia Aplicada 2010€¦ · organismos têm sido isolados...
Universidade de Lisboa Faculdade de Ciências
Departamento de Biologia Vegetal
Engineering Saccharomyces cerevisiae for the production of mannosylglycerate - a yeast model for
assessing its application in protein stabilization
Cristiana Silva Faria
Mestrado em Microbiologia Aplicada 2010
Universidade de Lisboa Faculdade de Ciências
Departamento de Biologia Vegetal
Engineering Saccharomyces cerevisiae for the production of mannosylglycerate - a yeast model for assessing its
application in protein stabilization
Dissertação orientada por: Professora Helena Santos (ITQB-UNL) Professora Manuela Carolino (FCUL)
Cristiana Silva Faria
Mestrado em Microbiologia Aplicada 2010
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Acknowledgments
I would like to express my gratitude to the people without whom this thesis would have
not been done.
First of all, I thank my Supervisor, Professor Helena Santos, and my co-Supervisor, Dr.
Nuno Borges, for all the support, guidance, motivation and dedication to science. I am
sincerely grateful for their constant availability to discuss and help me to overcome the
obstacles. I am honored to have had the opportunity to learn from them.
To Dr. Tiago Outeiro (IMM) for his enthusiasm to collaborate in this work and for
valuable suggestions; to Dr. Sandra Tenreiro (IMM) for teaching me how to cultivate
and maintain cultures of S. cerevisiae and for being always ready to help me.
I would also like to thank all my colleagues from the “Cell Physiology & NMR” group,
and “Lactic Acid Bacteria & in vivo NMR” group for their generous assistance, as well
as for making a friendly and warm working environment. I thank all of them for helping
me during all the stages of this work, and especially Dr. Carla Jorge for revising some
of the chapters.
To all my friends!
Aos meus pais, a quem tudo devo.
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Abstract
Mannosylglycerate (MG) is an osmolyte widespread in microorganisms adapted to hot
environments. The effect of MG on the protection of model proteins against
heat-induced denaturation and aggregation has been amply demonstrated in vitro.
These characteristics make MG a valuable compound in biotechnological applications.
However, the practical application of MG is hampered by the high production cost.
In this work, we engineered Saccharomyces cerevisiae for the production of MG. Two
strains (VSY71 and CEN.PK2) and several carbon sources were examined to optimize
the production of MG. Using glucose as carbon source, the strain VSY71 accumulated
90 µmol MG/g of dry mass, an increase of 2-fold compared with strain CEN.PK2. The
effect of several carbon sources (sucrose, mannose, galactose and raffinose) on MG
production by strain VSY71 was also investigated. Synthesis of MG (28.1 µmol/g of dry
mass) was detected with sucrose only. In addition to the biotechnological importance of
being a MG producer, this strain can serve as a platform to assess protein stabilization
by osmolytes in vivo. Therefore, we used it to evaluate the effect of MG on the
aggregating-prone protein, α-synuclein (α-Syn). For that, the S. cerevisiae strain
VSY72, harboring two copies of α-Syn-EGFP integrated in the genome, was
engineered to synthesize MG. As a control, a mutant transformed with the empty
MG-plasmid was also constructed. The accumulation of MG (20 µmol/g of dry mass)
prior to α-Syn over-production clearly reduced the formation of inclusions of this
protein; moreover, the formation of reactive oxygen species induced by α-Syn over-
production decreased in the MG-producing strain. Additionally, when MG accumulation
was induced in cells harboring large inclusions of α-Syn there was an effective
solubilization of these forms. This work clearly shows that MG inhibits the aggregation
of α-Syn and promotes the solubilization of α-Syn inclusions in vivo.
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Resumo
Microrganismos adaptados a proliferar optimamente em ambientes com temperatura
elevada são designados termófilos ou hipertermófilos, consoante a temperatura óptima
de crescimento se situa entre 60ºC e 80ºC ou acima de 80ºC, respectivamente. Estes
organismos têm sido isolados a partir de amostras de zonas geotermais, quer
terrestres quer marinhas, superficiais ou de profundidade, bem como de ambientes
aquecidos artificialmente.
Tal como a maioria dos microrganismos halofílicos ou halotolerantes, os
microrganismos termofílicos e hipertermofílicos provenientes de ambientes marinhos
acumulam compostos orgânicos denominados “solutos compatíveis” para
contrabalançar a pressão osmótica exterior. São moléculas de baixa massa molecular,
com elevada solubilidade em água e que podem atingir concentrações elevadas no
interior das células (da ordem de 1 M) sem interferirem negativamente com as funções
celulares. Para além da sua função na adaptação a flutuações na osmolaridade do
meio exterior, alguns destes solutos são preferencialmente acumulados em resposta a
temperaturas supra-óptimas. Além disso, os solutos típicos de (hiper)termófilos são
quimicamente diferentes dos que geralmente são encontrados em organismos
mesofílicos, nomeadamente no que se refere à carga eléctrica da molécula que é
negativa nos primeiros e neutra nos segundos. Este conjunto de observações levou à
hipótese de que os solutos compatíveis de (hiper)termófilos desempenhariam um
papel relevante na protecção de componentes celulares contra os efeitos
desnaturantes provocados por temperaturas elevadas.
Manosilglicerato é um dos solutos compatíveis mais frequentemente encontrado em
microrganismos adaptados a ambientes quentes. Foi identificado inicialmente em
Rhodothermus marinus, uma bactéria termofílica isolada de uma nascente geotermal
dos Açores, mas posteriormente foi detectado em muitos outros (hiper)termófilos
(trabalho da nossa equipa). A maioria dos organismos estudados acumula este soluto
em resposta a stress osmótico, no entanto acumulação em resposta a stress térmico
tem sido observada em alguns casos. Ensaios in vitro com proteínas modelo
mostraram que manosilglicerato actua como estabilizador, exibindo capacidade
termoprotectora muito superior à da trealose, um soluto característico de organismos
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mesofilos. Manosilglicerato tem-se revelado um excelente protector da estrutura de
proteínas, impedindo a sua desnaturação e subsequente agregação, bem como a
perda de actividade no caso de enzimas. Por estas razões, o manosilglicerato tem
grande potencialidade de aplicação, nomeadamente na indústria cosmética, como
hidratante e protector solar, na conservação de vacinas durante armazenamento e
transporte, como estabilizador de enzimas em “kits” clínicos e analíticos e na
estabilização de proteínas usadas em biosensores. Em particular, manosilglicerato
aumenta o tempo de vida de vectores retrovirais e melhora a sensibilidade em
metodologias com microgrelhas de DNA. Trata-se portanto de um composto valioso
sob o ponto de vista biotecnológico. Contudo, a produção de manosilglicerato é
actualmente um processo altamente dispendioso, uma vez que o seu isolamento é
realizado a partir da biomassa de um produtor natural, Rhodothermus marinus, que
acumula apenas 15 g por kg de biomassa húmida (resultados não publicados). Neste
contexto, torna-se premente o desenvolvimento de um produtor industrial de
manosilglicerato de modo a conseguir custos de produção competitivos.
A estratégia mais promissora envolverá necessariamente a manipulação genética de
uma estirpe industrial (nomeadamente, Escherichia coli, Saccharomyces cerevisiae,
Bacillus subtilis) de modo a dotá-la com a capacidade para produzir manosilglicerato
em larga escala. Esta tarefa é exequível visto que os genes envolvidos na síntese
deste composto são conhecidos.
Um dos objectivos do presente trabalho foi melhorar a produção de manosilglicerato
usando uma organismo industrial. Para tal seleccionou-se a estirpe S. cerevisiae
VSY71 que foi transformada com o plasmídeo (p425::mgsD), contendo o gene que
codifica a enzima bifuncional manosil-3-fosfoglicerato síntase/fosfatase da bactéria
mesofílica Dehalococcoides ethenogenes. Esta enzima sintetiza manosilglicerato a
partir de GDP-manose e 3-fosfoglicerato. Estratégia idêntica tinha sido anteriormente
seguida usando como hospedeiro a estirpe CEN.PK2 (Empadinhas et al., 2004), pelo
que transformámos CEN.PK2 também com p425::mgsD e usámo-la para fins de
comparação de produção.
Para optimização da produção de manosilglicerato usou-se S. cerevisiae estirpe
VSY71, contendo o plasmídeo para a síntese de manosilglicerato, e testaram-se várias
fontes de carbono e várias salinidades no meio de crescimento. Os substratos
rafinose, manose e galactose não conduziram à síntese de manosilglicerato, apesar
de suportarem crescimento celular ainda que com velocidades de crescimento
inferiores às registadas em glucose. Células cultivadas em sucrose acumularam 28
µmol de manosilglicerato por grama de biomassa seca. Produção máxima, 90 µmol de
manosilglicerato por grama de biomassa seca, foi observada usando glucose como
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fonte de carbono. Este valor é cerca de duas vezes superior á produção de
manosilglicerato observada com a estirpe S. cerevisiae CEN.PK2. Os resultados
mostram que a produção de manosilglicerato depende em grande medida da estirpe
utilizada pelo que há todo um trabalho de rastreio de colecções de S. cerevisiae que
será susceptível de conduzir a produções de manosilglicerato consideravelmente
superiores. Mesmo assim, vale a pena destacar que a estratégia seguida neste
trabalho conduziu a um melhoramento considerável da produção específica em
comparação com a estratégia descrita na literatura.
Um segundo objectivo deste trabalho foi avaliar a eficácia de manosilglicerato na
estabilização de proteínas in vivo, ou seja, no meio extremamente viscoso e com
elevada concentração molecular que constitui o citoplasma celular. O esforço investido
neste tópico teve uma dupla finalidade: i) encontrar novas aplicações para
manosilglicerato, eventualmente relacionadas com abordagens para prevenir
agregação proteica induzida por erros no enrolamento de cadeias polipeptídicas; ii) e
obter evidência experimental para apoiar a hipótese de que manosilglicerato exerce de
facto um papel protector da estrutura de proteínas in vivo. Para realizar este objectivo
usou-se um modelo de levedura para a doença de Parkinson, no qual o gene
codificante da α-sinucleína humana (fundido com o gene codificante para uma
proteína super-fluorescente) foi super-expresso em S. cerevisiae VSY72, onde
também foi expresso o gene para a síntese de manosilglicerato. As células
recombinantes produziram α-sinucleína e manosilglicerato, permitindo comparar o
efeito da presença e ausência de manosilglicerato na agregação de α−sinucleína em
ambiente intracelular.
α-Sinucleína é uma proteína encontrada apenas no cérebro de mamíferos. Embora a
sua função fisiológica seja desconhecida, sabe-se que está associada a várias
doenças neurodegenerativas (doença de Parkinson entre outras menos comuns) visto
ser um dos componentes dos agregados proteicos que caracterizam estas patologias.
α−Sinucleína é uma proteína intrinsecamente desenrolada, isto é, a forma nativa não
tem estrutura definida. A proteína tem tendência para auto-associação formando em
muitos casos estruturas fibrilares, designadas amilóides. Quando produzida na
levedura S. cerevisiae, por manipulação genética, a α−sinucleína é transportada para
a membrana plasmática onde se ancora. A super-produção desta proteína leva à
formação, perto da membrana, de pequenas inclusões citoplasmáticas que aumentam
progressivamente de tamanho, originando grandes inclusões, visíveis por microscopia
de fluorescência.
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Neste trabalho, usámos a estirpe VSY72 que contém duas cópias do gene da
α−sinucleína integradas no cromossoma e que foi transformada com o plasmídeo que
contém o gene que codifica para a síntese do manosilglicerato. Como controlo foi
usada a estirpe VSY72 transformada com o plasmídeo vazio. A produção de
manosilglicerato é regulada por um promotor constitutivo, enquanto que a produção de
α−sinucleína é controlada por um promotor sensível à galactose. A acumulação
intracelular de manosilglicerato (20 µmol por grama de biomassa seca) reduziu em 3,5
vezes o número de células com inclusões de α−sinucleína, em comparação com a
estirpe controlo (incapaz de sintetizar manosilglicerato). Estes resultados referem-se a
células examinadas dez horas após indução da produção de α−sinucleína. Tivemos o
cuidado de verificar que as duas estirpes apresentam níveis semelhantes de produção
de α−sinucleína (análises por “western blot”). Em paralelo, monitorizaram-se os níveis
de espécies reactivas de oxigénio induzidas pela produção excessiva de α−sinucleína.
Os resultados mostraram que a presença de manosilglicerato no citoplasma causou
uma diminuição no nível de espécies reactivas de oxigénio em comparação com o
controlo. Por outras palavras, ao prevenir a formação de inclusões de α−sinucleína, o
manosilglicerato diminuiu também o nível de stress oxidativo associado ao processo
de super-expressão da proteína, possivelmente promovendo a estabilização de formas
nativas de α−sinucleína, supostamente com menor toxicidade para a célula.
Tendo-se demonstrado o efeito inibitório de manosilglicerato na formação de inclusões
de α−sinucleína, pôs-se a questão de saber se este composto teria também efeito ao
nível da solubilização de inclusões pré-formadas. Para tal, a produção de α−sinucleína
foi induzida nove horas antes da indução da síntese de manosilglicerato, por permuta
de meio contendo galactose para meio fresco contendo glucose como única fonte de
carbono. Os resultados obtidos mostraram que o manosilglicerato tem uma acção de
chaperona molecular, promovendo a desagregação das inclusões de α−sinucleína:
uma hora após indução da síntese de manosilglicerato o número de inclusões visíveis
por microscopia de fluorescência era 2,8 vezes inferior ao detectado em células
controlo.
O efeito do manosilglicerato na prevenção/solubilização de inclusões de α−sinucleína
pode ser atribuído á activação de mecanismos celulares que conduzem à degradação
de formas anormais de α−sinucleína, ou que promovem o seu enrolamento correcto.
Alternativamente, o efeito benéfico observado pode resultar de uma interacção directa
soluto/proteína que estabilize preferencialmente a estrutura nativa da α−sinucleína,
prevenindo assim a formação de inclusões. Os resultados descritos nesta tese não
permitem tirar conclusões definitivas sobre o tipo de mecanismo envolvido, no entanto
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a observação de efeitos semelhantes em ensaios in vitro monitorizados por
microscopia electrónica (resultados de outros membros da equipa) fundamentam a
hipótese de que interacções directas manosilglicerato/proteína terão uma contribuição
relevante quer in vivo quer in vitro.
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Contents
Acknowledgements5555555555555555555555555.i
Abstract55555555555555555555555555555.5.ii
Resumo555555555555555555555555555555..iii
Contents555555555555555555555555555555.viii
Abbreviations 555555555555555555555555555..ix
Introduction55555555555555555555555555.55.1
Materials and Methods55555555555555555.555.555.10
Results 555555555555555555555555555.555.15
Discussion555555555555555555555555.5.555..25
References555555555555555555555555.55..55.30
Appendix 55555555555555555555555555555..36
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Abbreviations
α-Syn α-Synuclein
MG Mannosylglycerate
EGFP Enhanced Green Fluorescent Protein
SNCA α-Synuclein gene
msgD mannosyl-3-phosphoglycerate synthase/phosphatase gene
NMR Nuclear Magnetic Resonance
DIP Di-myo-inositol phosphate
MDIP Mannosyl-di-myo-inositol phosphate
3-PG 3-Phosphoglycerate
PAGE Polyacrylamide Gel Electrophoresis
SDS Sodium Dodecyl Sulfate
ROS Reactive Oxygen Species
SC Synthetic Complete medium
DHR123 Dihydrorhodamine 123
R. marinus Rhodothermus marinus
S. cerevisiae Saccharomyces cerevisiae
1
Introduction
Compatible solutes from (hyper)thermophiles
In the two last decades of the twentieth century the scientific community was amazed
with the discovery of microbial life in environments with temperatures far outside the
optimal range for most organisms, namely plants and animals. Such scalding habitats
are found in terrestrial as well as in shallow or deep-marine geothermal sites (Stetter
1995).
Microorganisms with optimum growth temperatures between 65ºC and 80ºC are
named thermophiles, those with optimum growth temperature equal or above 80ºC are
designated hyperthermophiles (Blöchl et al., 1995). Most thermophiles and
hyperthermophiles, herein designated (hyper)thermophiles, belong to the domains
Bacteria and Archaea. Actually, the most hyperthermophilic microorganisms known to
date are found exclusively in the domain Archaea.
Aqueous environments are susceptible to fluctuations in the concentrations of salts.
Therefore, microorganism that survive and proliferate in such habitats must adapt to
the environmental osmolarity to avoid cell lysis (due to excessive water uptake) or
dehydratation due to outflow of water molecules. Two strategies are used by
microorganisms to cope with high salt concentration. One of them involves the influx of
inorganic ions, generally KCl, into the cytoplasm and has been designated the “salt-in-
the-cytoplasm” type of adaptation (Galinski, 1995). The second strategy relies on the
accumulation of small organic compounds to balance the external osmotic pressure.
These compounds can accumulate to very high levels (molar concentration) inside the
cells without disturbing cellular functions, and for this reason they are called
“compatible solutes” (Brown, 1976). Besides their major role in osmoadaptation, recent
studies have shown that compatible solutes act as protectors of cells and cell
structures against a variety of stresses, like heat, cold, acid, free-radicals or drought.
(da Costa et al., 1998; Argüelles, 2000; Welsh, 2000; Benaroudj et al., 2001; Santos &
da Costa, 2001; Cardoso et al., 2004; Kandror et al., 2002; Mahmud et al., 2010).
The accumulation of compatible solutes for osmoadaptation is widespread in
organisms, ranging from archaea to bacteria, yeast filamentous fungi, algae, plants and
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animals. Trehalose is widespread in all domains of the Tree of Life; polyols, such as
glycerol, are commonly found in fungi and algae; ectoine or hydroxyectoine seem to be
restricted to bacteria, while glycine betaine and α-glutamate are usually found in
bacteria and archaea (da Costa et al., 1998). It is interesting to note the strong
prevalence of neutral or zwitterionic solutes in mesophilic organisms.
Thermophiles and hyperthermophiles accumulate compatible solutes generally
different from those found in mesophiles (Fig. 1). In respect to their chemical nature,
solutes from (hyper)thermophiles can be divided in two categories: α-hexose derivates
and polyol-phosphodiesters. Among the α-hexose derivatives, mannosylglycerate (MG)
is the most common compound in hyperthermophiles, either bacteria or archaea
(Bouveng et al., 1955). Mannosylglyceramide, an uncharged solute chemically related
to MG, was found only in a few strains of Rhodothermus marinus (Silva et al., 1999).
Mannosyl-glucosylglycerate and glucosyl-glucosylglycerate are two MG-derivatives
found in Petrotoga spp. and in Persephonela marina, respectively (Jorge et al., 2007;
Fernandes et al., 2010). Another related compound is glucosylglycerate, commonly
found in halotolerant, mesophilic bacteria and archaea (Kollman et al., 1979; Robertson
et al., 1992; Goude et al., 2004; Empadinhas & da Costa 2008). Among
(hyper)thermophiles, glucosylglycerate was found only in the thermophilic bacterium
Persephonella marina (Santos et al., 2007a).
FIGURE 1- Compatible solutes highly restricted to (hyper)thermophiles.
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Di-myo-inositol-phosphate (DIP) is a solute restricted to microorganisms with optimal
growth temperatures above 60ºC, hence it is regarded as the most characteristic
member of the polyol-phosphodiester group (Santos et al., 2007a). DIP is also the only
compatible solute accumulated by the most robust hyperthermophile, Pyrolobus fumarii
(Gonçalves et al., 2008; Blöchl et al., 1997). Other examples of the polyol-
phosphodiester group are diglycerol phosphate and glycero-phospho-inositol, which
were found in members of the genus Archaeoglobus; the latter solute has also been
reported in Aquifex spp. (Gonçalves et al., 2003; Lamosa et al., 2006). Two DIP-
derivates, mannosyl-myo-inositol phosphate and di-mannosyl-di-myo-inositol
phosphate, have been found in representatives of the genera Thermotoga and Aquifex
(Rodrigues et al., 2009). Di-N-acetyl-glucosamine phosphate is a minor solute thus far
only found in Rubrobacter xylanophilus (Empadinhas et al., 2007).
Examples of other solutes that do not belong to these two categories are: β-glutamate,
present in methanogenes (Robertson et al., 1989; Robertson et al., 1990) and also in
hyperthermophilic bacteria (Martins et al., 1996; Lamosa et al., 2006; Fernandes et al.,
2010); aspartate that occurs in Palaecoccus and in Thermococcus spp. (Neves et al.,
2005; Lamosa et al., 1998); and 1,3,4,6-tetracarboxy-hexane and
galactosylhydroxylysine, rare solutes found in the thermophilic archaeon,
Methanothermobacter marburgensis and in Thermococcus litoralis, respectively
(Gorkovenko et al., 1994; Lamosa et al., 1998; Martin et al.,1999; Müller et al.,2005).
Role of the compatible solute mannosylglycerate Mannosylglycerate (MG) is accumulated in several thermophilic bacteria like Thermus
thermophilus and Rhodothermus marinus, and also in hyperthermophiles such as
Pyrococcus spp. and in some species of the genus Thermococcus (Santos et al.,
2007a). It is interesting to note that mannosylglycerate was initially found in an
eukaryotic red algae from the order Ceramiales, but its role in these organisms seems
unrelated with osmoregulation (Bouveng et al., 1955).
The synthesis of MG in R. marinus proceeds via two pathways (Fig. 2). In the single-
step pathway, mannosylglycerate synthase catalyzes the condensation of
GDP-mannose and D-glycerate into MG. In the other pathway (two-step pathway), the
mannosyl-3-phosphoglycerate synthase catalyzes the conversion of GDP-mannose
and D-3-phosphoglycerate into mannosyl-3-phosphoglycerate. Dephosphorylation of
mannosyl-3-phosphoglycerate is catalyzed by mannosyl-3-phosphoglycerate
phosphatase, yielding MG (Martins et al., 1999; Borges et al., 2004). To date, R.
marinus is the only organism known to possess the two routes for MG synthesis;
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Pyrococcus spp., Thermus thermophilus and many other organisms synthesize MG via
the two-step pathway (Borges et al., 2004; Empadinhas et al., 2001; Empadinhas et al.,
2003).
An interesting finding was the discovery of a bifuncional mannosyl-3-phosphoglycerate
synthase/phosphatase in the mesophilic bacterium Dehalococcoides ethenogenes that
catalyses the synthesis of MG at moderate temperatures. The encoding gene was
successfully cloned in Saccharomyces cerevisiae and led to the production of MG
(Empadinhas et al., 2004).
FIGURE 2 – Pathways for the synthesis of mannosylglycerate. The single-step pathway using
mannosylglycerate synthase 1 has been characterized in R. marinus only. The two-step
pathway involves mannosyl-3-phosphoglycerate synthase 2 and mannosyl-3-
phosphoglycerate phosphatase 3 . The two-step pathway has been detected in many
organisms adapted to hot environments (Santos et al., 2007a).
The accumulation of MG by microorganisms is generally connected with a response to
osmotic stress. The only exception to this behavior was found in R. marinus in which
MG is the major solute at supra-optimal growth temperatures (Silva et al., 1999). Since
MG is mostly found in organisms adapted to hot environments it is acceptable to
hypothesize that it could have a role in the protection of cellular components under
heat stress. In fact, in vitro assays with model proteins show that MG has a remarkable
ability to improve protein thermostability, namely, in respect to structural stability
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(measured by the melting temperature), to inhibition of aggregation induced by heat,
and to protection against heat inactivation (Ramos et al.,1997, Borges et al., 2004,
Faria et al., 2003, Faria et al., 2008). Therefore, MG is a valuable compound from the
biotechnological point of view, and several patents have been filled by our group and
others (Santos et al., 1996; Santos et al., 1998; da Costa et al., 2003; Lamosa et al.,
2006; Lentzen & Schwarz, 2006). Two of the patents originating in our team have been
licensed to a German company bitop AG, Witten (http://www.bitop.de). Major potential
fields of application are: the cosmetic industry, as moisturizer and skin protector
against UV damage, storage of vaccines and other biomaterials, protein stabilizer in
analytical and clinical kits, and in biosensors. In particular, MG proved to increase the
life-span of retroviral vectors (Cruz et al., 2006) and to improve DNA microarray
experiments (Mascellani et al., 2007).
The major bottleneck towards the industrial utilization of MG is the high production
costs. Although a procedure for the chemical synthesis of MG has been developed
(Lamosa et al., 2006), the yield is very poor. Therefore, the solution for this difficulty
should reside in an optimized fermentation process. Currently, commercial MG is
produced (gram amounts) from fermentation and “milking” of R. marinus, a natural
producer. However, the high production cost prevents a wide utilization of this
compound. In this context, the development of an efficient industrial producer of MG is
mandatory.
Yeast as industrial and model organism Since ancient times the yeast Saccharomyces cerevisiae has been used in the
production of food and alcoholic beverages (Mortimer, 2000). In addition to the
traditional utilization in the food industry, yeast is also an excellent host for the
production of recombinant proteins, offering straightforward genetic tools and highly
optimized cultivation processes. Fast growth rates up to huge cell density make it
probably the most important industrial organism (Goffeau et al., 1996). Moreover,
yeasts are able to perform complex eukaryotic-type post-translational modifications and
are able to produce proteins similar to those of mammalian origin. Since the early
1980s, a number of vaccines, antigens, hormones and other bio-therapeutic
compounds have reached to a commercial production. That is the case of insulin,
interferon and hepatitis A, among others (Hitzeman et al., 1984; Valenzuela et al.,
1982).
In addition to this biotechnological importance, yeast is recognized as a basic
eukaryotic model system (Botstein et al., 1997). More specifically, the baker’s yeast
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(Saccharomyces cerevisiae) has been proven very useful to understanding
fundamental cellular and molecular processes (Botstein et al., 1997). Despite the
evolutionary distance between yeast and higher eukaryotes (including humans), a high
level of conservation is shared in several cellular mechanisms such as, DNA
replication, cell division, protein turnover, vesicular trafficking, aging, and cell death
(Sugiyama et al., 2009).
S. cerevisiae is an attractive organism to work since it is non-pathogenic, very simple
and inexpensive to manipulate and easy to maintain as stable haploids or diploids. The
duplication time of S. cerevisiae is around 90 min in complex medium; moreover, cells
are easily transformable and it is straightforward to integrate foreign genes in the
genome (Sugiyama et al., 2009).
The sequence of the yeast genome was completed 14 years ago (Suter et al., 2006);
presently, around 80% of the 6000 encoding genes have a predicted function (Christie
et al., 2009). A very active and collaborative yeast research community built a series of
biological resources such as, the yeast gene deletion strains (YGDS), where every
protein-coding gene in the genome was deleted (Winzeler et al., 1999); the green
fluorescent protein (GFP)-tagged collection of strains, where each gene was tagged
with GFP (Huh et al., 2003); and a strain collection engineered for protein over-
production (Jones et al., 2008). Another major advantage of the yeast system is the
availability of a rich and easily accessible dataset on genetic interactions, protein-
protein interactions, transcriptional changes and protein localization (Gitler, 2008).
In summary, the powerful genetic resources together with the extensive accumulated
knowledge makes this simple yeast a key-organism for applied as well as for basic
research.
α-Synuclein as a model to study protein stabilization α-Synuclein (α-Syn) is an intrinsically unfolded protein with 140 amino acids present
only in mammalian brains (Jo et al., 2000). This protein has a tendency to self-
assemble and aggregate, and is associated with several devastating
neurodegenerative diseases, known as synucleinopathies (Cole et al., 2002). α-Syn
comprises an N-terminal region that acts as a membrane binding domain (Fig. 3), a
central hydrophobic domain, essential for oligomerization and fibrilization, and a
C-terminal domain that hinders aggregation for its high solubility (Davidson et al., 1998;
Ulmer et al., 2005; Georgieva et al., 2008).
7
FIGURE 3 – Primary and secondary structure of wild-type α-Syn. Three main domains can be
considered: the N-terminus that confers α-helix structure, a central hydrophobic domain (NAC)
that confers β-sheet and is essential for oligomerization, and a C-terminus that promotes protein
solubilization. Adapted from Vamvaca et al., 2009.
This protein is natively unfolded and when associated with membranes forms two
α-helices in the N-terminal region (Fig. 4). An increment in the concentration of α-Syn
nearby the plasmatic membrane facilitates the formation of dimers on the membrane
surface or in the cytoplasm. Through dimerization, α-Syn adopts β-sheet secondary
structure and can associate with other monomers or dimers leading to oligomer
formation. The oligomers act as nuclei for fibril formation and amyloid deposits
(Reviewed in Auluck et al., 2010).
FIGURE 4 – Model of α-Syn interactions with membranes. α-Syn is natively unfolded and when
associated to membranes forms two α-helices. Increase in concentration of α-Syn favors the
formation of oligomeric forms through association into dimmers and higher oligomers. Adapted
from Auluck et al., 2010.
8
Effects of α-synuclein in yeast physiology In yeast, α-Syn is delivered to the plasma membrane via the classical secretory
pathway: the protein migrates from the endoplasmatic reticulum (ER) to the Golgi
apparatus and is further transported to the plasma membrane in secretory vesicles
(Outeiro & Linquist, 2003; Dixon et al., 2005). ER is the first compartment encountered
by α-Syn, where it impairs the docking and fusion of ER vesicles into the Golgi
apparatus (Cooper et al., 2006). As a consequence, these vesicles accumulate near
the plasma membrane (Soper et al., 2008). The accumulation of α-Syn close to the
plasma membrane leads to the formation of small inclusions that progressively grow in
size towards the cytoplasm. Some of these inclusions stain positive for thioflavin-S
confirming the presence of β-sheet in α-Syn aggregates, while others are α-Syn-
induced aggregates of cytoplasmic vesicles (Outeiro & Linquist, 2003; Zabrocki et al.,
2005).
Another consequence of ER stress is the over-production of reactive oxygen species
(ROS), which indirectly stimulate the production of ROS in the mitochondria and
activates apoptosis (Flower et al., 2005). Moreover, α-Syn over-production induces the
impairment of the proteasome activity as a result of modifications in the proteasome
composition. It is believed that these alterations derive from abnormal protein synthesis
in the ER (Chen et al., 2005). Another effect of ER malfunction is the retardation of
endocytosis, caused by the obstruction in post-vesicle internalization steps and defects
in vacuolar fusion (Outeiro & Linquist, 2003; Zabrocki et al.,2008).
Toxicity of α-Syn in yeast cells also stems from post-translational modifications
catalyzed by enzymes such as, acetyltransferase B complex (acetylation) and casein
kinases I and II (phosphorylation), among others ( Zabrocki et al.,2008). Nevertheless,
α-Syn toxicity is dependent on the genetic background and type of plasmid used. In
fact, certain yeast strains display a high number of α-Syn inclusions without showing
any growth defect; conversely, α-Syn over-production may be highly toxic to cells
without inclusion formation (Sharma et al., 2006; Zabrocki et al., 2005).
Aim of this work
The fermentation of the baker´s yeast, Saccharomyces cerevisiae, to produce
high-value products is a common practice in industry. Since the use of native
producers, such as Rhodothermus marinus, for the production of MG is not
economically feasible, we set out to optimize the production of MG using engineered
strains of S. cerevisiae.
9
A second objective of this work is to assess the efficacy of MG for the stabilization of
proteins in vivo, i.e., in the molecular overcrowded medium that constitutes the
cytoplasm of living cells. This research effort has a dual purpose: i) to find novel
applications for MG, related with its potential ability to suppress protein aggregation
inside the cells; ii) and, to obtain experimental evidence in support of the hypothesis
that MG acts as a protector of protein structures in vivo. To this end, we took
advantage of a yeast model for Parkinson´s disease and investigated the effect of MG
on the aggregation of α-synuclein, by over-producing this aggregating-prone protein
together with the MG-biosynthetic enzymes.
10
Materials and Methods
Optimization of mannosylglycerate production by engineered Saccharomyces
cerevisiae strains
Strains and genetic procedures
S. cerevisiae strain VSY71 (a derivative of W303-1A (MATa, ade2-1, can1-100, ura3-1,
leu2-3,112, his3-11,15, trp1-1) with the plasmids pRS306::URA3 and pRS304::TRP1
integrated in the chromosome)) and strain CEN.PK2 (MATa/MATa, ura3-52/ura3-52,
trp1-289/trp1-289, leu2-3,112/leu2-3,112, his3 D1/his3 D1, MAL2-8C/MAL2-8C) were
transformed with the plasmid p425::msgD. The plasmid p425::msgD, containing the
msgD gene that encodes the bifunctional mannosyl-3-phosphoglycerate
synthase/phosphatase from Dehalococcoides ethenogenes, was kindly provided by
Dr. Milton S. da Costa (University of Coimbra).
S. cerevisiae cells were transformed with the plasmid p425::msgD by using the
standard lithium acetate (LiOAc) procedure (Guthrie & Fink, 1991) with slight
modifications. Briefly, cells were grown overnight in liquid synthetic complete medium
(SC medium) with 2% glucose at 30ºC and 140 rpm. After harvesting by centrifugation
(7000 × g, 10 min, 4ºC), cells were re-suspended in water to a final concentration of 5
× 108 cells/ml. An aliquot (5 ml) was collected, centrifuged, and re-suspended in 360 µl
of a solution containing 33% (w/v) poly(ethylene glycol) 3350, 0.1 M LiOAc, 0.3 mg/ml
boiled salmon sperm DNA, and 1 µg plasmid DNA. The cell suspension was incubated
in a water bath at 42ºC for 1 h, and then cells were washed with water, and plated onto
selective solid medium. The transformants derived from the strain VSY71 were
selected in SC medium without uracil, leucine, and tryptophan; the transformants
derived from the strain CEN.PK2 were isolated in SC medium without leucine.
Growth conditions
S. cerevisiae strains (VSY71 and CEN.PK2) harboring the plasmid p425::msgD, were
grown in appropriate selective medium supplemented with 2% glucose (glucose
medium) at 30ºC and 140 rpm. Cell growth was monitored by measuring the turbidity at
600 nm (OD600). To determine the effect of different carbon sources in the production of
MG in this mutant, cells were grown overnight in SC medium supplemented with 2% of
the following carbon sources: glucose, sucrose, mannose, raffinose, and galactose.
11
Each overnight-grown culture was diluted to a final OD600 of 0.1 with fresh SC medium
supplemented with the respective carbon source to a final volume of 200 ml. The
cultures were incubated at 30ºC and 140 rpm. At the mid-exponential phase (OD600 =
1.6), cells were centrifuged (7000 × g, 10 min, 4ºC), washed with water, and frozen at
-20ºC until further analysis. The effect on growth of the NaCl concentration was also
studied. Cells were grown at 37ºC in medium containing different NaCl concentrations
(0%, 2%, 3%, and 4%, wt/vol). Samples of each culture (200 ml) were removed during
the mid-exponential phase and the pool of compatible solutes was determined in cell
extracts by nuclear magnetic resonance (NMR) analysis.
Extraction and quantification of intracellular organic solutes
Ethanol-chloroform extracts of S. cerevisiae strains were performed as previously
described (Santos et al., 2006). Briefly, a culture of 200 ml was grown until
mid-exponential phase, harvested, and centrifuged (7000 × g, 10 min, 4ºC).
Compatible solutes were extracted from cells by boiling for 10 min with 80% ethanol.
The extraction was repeated twice and the combined extracts evaporated to dryness
under negative pressure; the residue was re-suspended in water:chloroform (1:3, v/v),
and centrifuged to remove solid components. The aqueous extracts were lyophilized
and re-suspended in D2O for nuclear magnetic resonance (NMR) analysis. For
compatible solute quantification purposes, formate was added as a concentration
standard, and the 1H-NMR spectra was acquired with a repetition delay of 60 s on a
Bruker AMX 300 spectrometer. A sample of cell culture (5 ml) was removed, passed
through a 0.22 µm pore filter (Millipore, USA), and the filter was dried overnight at 85ºC
in order to determine dry mass.
Effect of mannosylglycerate in the stabilization of proteins in vivo using
engineered Saccharomyces cerevisiae strains
Strains and genetic procedures
Multi-copy plasmids for α-Syn expression - S. cerevisiae strain CEN.PK2 was
transformed with the p426GPD::SNCA(wt)EGFP and the respective transformants
were designated “α-Syn mutant”. In addition, strain CEN.PK2 was transformed with a
combination of the following plasmids: (p426GPD::SNCA(wt)EGFP plus pRS425);
(p426GPD::SNCA(wt)EGFP plus p425::msgD); (p426GPD::SNCA(wt)EGFP plus
p425::msgD); (pRS425 plus p426GPD). Transformants were designated
“α-Syn/pRS425 mutant”, “α-Syn/MG mutant”, and “p426GPD/pRS425 mutant”,
respectively. The phenotype of each mutant is described in Table 2. The plasmid
12
p426GPD::SNCA(wt)EGFP, containing the α-Syn gene fused in the N-terminus with an
EGFP gene (enhanced green fluorescent protein), and p426GPD, were kindly provided
by Dr. Tiago Outeiro (IMM, Portugal). The plasmid pRS425 was kindly provided by Dr.
Milton S. da Costa (Coimbra University). The plasmids used are multi-copy plasmids
containing constitutive promoters, and are listed in Table 1. Transformants were
selected in SC medium without uracil and leucine and supplemented with 2% glucose.
TABLE 1. Vectors used in this study
Vector Type
p426GPD multi-copy plasmid
p426GPD::SNCA(wt)EGFP multi-copy plasmid
pRS425 multi-copy plasmid
p425::msgD multi-copy plasmid
p406::SNCA(wt)EGFP Integrative
p404::SNCA(wt)EGFP Integrative
TABLE 2. Mutants constructed in this study
Strain Vector harboring Phenotype
α-Syn mutant p426GPD::SNCA(wt)EGFP α-Syn production
α-Syn/pRS425 mutant P426GPD::SNCA(wt)EGFP plus
pRS425 α-Syn production
α-Syn/MG mutant p426GPD::SNCA(wt)EGFP plus
p425::msgD α-Syn and MG
production
p426GPD/pRS425 mutant pRS425 plus p426GPD -----
Control mutant p406::SNCA(wt)EGFP plus
p404::SNCA(wt)EGFP plus pRS425
α-Syn production
MG mutant p406::SNCA(wt)EGFP plus
p404::SNCA(wt)EGFP plus p425::msgD
α-Syn and MG production
Integrative plasmids for α-Syn production - Strain VSY72 (a derivative of W303-1A
(MATa, ade2-1, can1-100, ura3-1, leu2-3,112, his3-11,15, trp1-1) with plasmids
p406::SNCA(wt)EGFP and p404::SNCA(wt)EGFP integrated in the chromosome)) was
kindly provided by Dr. Tiago Outeiro (IMM, Portugal). This strain has two copies of the
SNCA(wt)EGFP gene integrated in the genome (Fleming et al., 2008). The expression
of α-Syn is controlled by a galactose inducible promoter, allowing synchronizing
expression of α-Syn. This strain was transformed with the plasmids p425::msgD or
pRS425 (Table 1). Selection was carried out in SC medium without uracil, leucine, and
13
tryptophan supplemented with 2% of glucose, leading to the isolation of MG and
Control mutant strains, respectively (Table 2).
Growth conditions
S. cerevisiae mutants with multi-copy plasmids – Initially, the effect of MG on the
inhibition of α-Syn aggregation was investigated using the S. cerevisiae strain
CEN.PK2 producing α-Syn and MG (α-Syn/MG mutant); the respective control mutants
(α-Syn/pRS425 and p426GPD/pRS425 mutants) were used for comparison. Cells were
grown overnight at 30ºC and 140 rpm in glucose medium, and diluted to with fresh
medium to a final volume of 400 ml (OD600 of 0.1). Cell growth was monitored by
measuring the OD600. Culture samples were withdrawn at early, mid-exponential, and
late-exponential phases, and the percentage of cells with α-Syn inclusions was
determined by fluorescence microscopy (details described below).
S. cerevisiae mutants with integrative plasmids – We found that mutants with multi-
copy plasmids were unstable, i.e., the level of α-Syn decreased considerably in
successive growths. Therefore, we resorted to an alternative strategy that used
integrative plasmids. The S. cerevisiae strain VSY72 producing α-Syn and MG (MG
mutant) and the strain VSY72, producing only α-Syn (Control mutant), were grown in
50 ml glucose medium until mid-exponential, centrifuged (7000 × g, 10 min, 4ºC), and
re-suspended in TE buffer (Tris-HCl 10 mM, 1 mM EDTA, pH 7.5). This cell suspension
was used to inoculate 600 ml of SC medium supplemented with galactose (galactose
medium) to a final OD600 of 0.2. Culture samples (200 ml) were removed during the
early-exponential phase (10 and 12 hours), and compatible solutes were extracted and
quantified as described above. The dry mass was calculated using 20 ml of culture.
Culture samples were also collected at 4, 6, 8, 10, 12, and 24 hours after induction and
the percentage of cells with α-Syn inclusions was determined by fluorescence
microscopy (details described below).
To evaluate the ability of MG to promote solubilization of the α-Syn inclusions, MG and
Control mutants were grown in 50 ml of SC medium supplemented with 2% raffinose
(raffinose medium) until mid-exponential phase (around 40 h). At this stage, cells were
harvested, re-suspended in TE buffer, and inoculated in 600 ml of galactose medium to
a final OD600 of 0.2. After allowing for 9 h of growth in this medium, the galactose
medium was replaced by glucose medium. After 1 and 3 hours of growth in glucose
medium, the following sample volumes were collected; 200 ml for solute analysis; 20
ml for dry mass determination; and 20 ml for western blot analysis.
14
Fluorescence microscopy
Fluorescence microscopy analysis was performed with a Zeiss AxioImager (Zeiss,
Oberkochen, Germany) microscope. Cells were observed with a 63x/1.4 (Zeiss Plan-
APO oil) objective and a GFP filter. For each culture sample, a total of 300 cells were
inspected for the presence of α-Syn inclusions. The percentage of cells with α-Syn
inclusions was determined by dividing the number of cells containing at least one
inclusion of α-Syn by the total number of cells counted, and multiplication by 100.
Western blot analysis
S. cerevisiae cells were harvested and washed with water. For each culture sample,
the number of cells per ml was determined with a Neubauer chamber. Samples
containing 6.1 x 107 cells were taken, re-suspended in SDS/PAGE buffer (Laemmli,
1970), and boiled for 10 min. Proteins were then separated in a 10% SDS/PAGE gel
and transferred to a polyvinylidene fluoride (PVDF) membrane (Millippore, USA) for
α-Syn detection. The membranes were treated for 1 h at room temperature with a
blocking solution (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% (w/v) milk, and 0.1 %
(v/v) tween 20). Then, membranes were incubated overnight with monoclonal antibody
against α-Syn from mouse (BD Transduction LaboratoriesTM) in blocking solution using
a dilution of 1:6000. After incubation, membranes were washed 3 times with TBS buffer
(150 mM NaCl, 50 mM Tris-HCl pH 7.5) and 0.1% (v/v) tween 20, and incubated for 1 h
with the secondary antibody anti-mouse IgG (GE healthcare) diluted in 1:1000 in
blocking solution. A chemoluminescence detection kit (Millipore, USA) was used to
visualize α-Syn.
ROS assay
To determine the amount of Reactive Oxygen Species (ROS) in Control and MG
mutants, we used the dihydrorhodamine 123 (DHR 123) staining (Madeo et al., 2002).
The DHR123 is oxidized intracellularly by ROS into rhodamine 123, which emit
fluorescence at 535 nm when excited at 485 nm. To perform this assay, the mutants
were grown in glucose medium until mid-exponential phase and then centrifuged.
Subsequently, the mutants were re-suspended in galactose medium to induce the
α-Syn expression and allowed to grow for 9 h. In addition, as a negative control
experiment, the Control mutant was re-suspended in fresh glucose medium, and
growth was allowed for 9 hours. Aliquots of the three cultures, containing 1 × 106 cells,
were collected and incubated with DHR 123 dye (final concentration 5 µg/ml) for 1 h.
The fluorescence intensity of the samples was measured with a Varian Cary Eclipse
spectrofluorimeter (Varian, USA).
15
Results
Effect of different carbon sources in the production of mannosylglycerate by
recombinant Saccharomyces cerevisiae strains
Currently, mannosylglycerate (MG) is purified from natural producing organisms
(namely Rhodothermus marinus). The extraction and purification yield is very low, and
therefore, the process is not economically feasible. In this work, we used strains of S.
cerevisiae engineered for the synthesis of MG; moreover, several carbon sources were
tested in an attempt to optimize MG production. The MG-engineered yeast strain
VSY71, harboring the plasmid for MG synthesis, was grown in medium supplemented
with glucose. Under these conditions, the amount of MG determined was 90 µmol/g dry
mass (Table 3). Among all tested carbon sources, growth of S. cerevisiae strain VSY71
in glucose-containing medium led to the production of the highest amount of MG. When
sucrose was used as a carbon source, the amount of MG decreased 69% (28.1 µmol/g
dry mass). No MG accumulation was detected when the strain VSY71 was grown in
SC medium supplemented with mannose, galactose, or raffinose (Table 3). For the
purpose of comparison, we engineered strain CEN.PK2 to produce MG, as described
by Empadinhas et al., 2004, and determined the production of MG in glucose medium.
Under these conditions, the MG-engineered strain CEN.PK2 produced 44 µmol of MG
per gram of dry mass (Table 3), which represents only 48% of the amount produced by
strain VSY71.
TABLE 3. Production of mannosylglcerate in recombinant strains of S. cerevisiae using different
carbon sources.
S. cerevisiae Carbon source Mannosylglycerate (µmol/g dry mass)
VSY71
Glucose 90.0
Sucrose 28.1
Mannose 0
Galactose 0
Raffinose 0
CEN.PK2 Glucose 44.0
In order to investigate the effect of an osmotic stress in the accumulation of MG, strain
VSY71 was cultivated in glucose medium supplemented with 0%, 2%, 3%, and 4%
16
(w/v) of NaCl. Analysis of ethanol-chloroform extracts derived from these cultures did
not result in an increase in the production of MG in comparison with the same strain
grown in medium without NaCl (data not shown). Instead, we observed the
accumulation of low levels of trehalose (0.9 µmol/g dry mass) and high amounts of
glycerol (134.7 µmol/g dry mass), two compatible solutes known to accumulate in S.
cerevisiae in response to osmotic stress (Albertyn et al., 1994; Hounsa et al., 1998).
Effect of mannosylglycerate on the formation of α-Syn inclusions – use of multi-
copy plasmids to express the gene encoding α-Syn
The inhibitory effect of MG on the aggregation of several model proteins has been
demonstrated in vitro (Santos et al., 2007a). However, it is not known whether MG
maintains this ability in the molecular overcrowded milieu that constitutes the cell
cytoplasm. To achieve this goal, we constructed a S. cerevisiae mutant that
accumulates MG and concomitantly produces α-Syn, an aggregating-prone protein that
self-aggregates without further stimuli. In a first attempt we used the S. cerevisiae
strain CEN.PK2 containing the plasmid for MG synthesis and transformed these cells
with a plasmid encoding α-Syn fused with the EGFP tag. The genes were expressed
constitutively in 2µ plasmids, a type of yeast plasmid with a high transcription rate.
FIGURE 5. Toxicity of α-Syn in S. cerevisiae strain CEN.PK2. A, Growth curves of the wild-type
(WT) and the α-Syn mutant harboring the plasmid for α-Syn expression. B, Fluorescence
microscopy of the α-Syn mutant during the early exponential phase of growth.
17
Growth of the α-Syn mutant was strongly inhibited when compared to the wild-type
strain, indicating that the over-production of α-Syn is toxic to yeast cells (Fig. 5A).
Fluorescence microscopy analysis of the α-Syn mutant revealed the presence of α-Syn
inclusions in the cytoplasm in the majority of the cell population (Fig. 5B).
In order to evaluate the effect of MG in aggregation of α-Syn the following mutants
were constructed: a strain producing only α-Syn (containing also the empty plasmid
pRS425), designated α-Syn/pRS425 mutant; a strain producing α-Syn and MG,
α-Syn/MG mutant; and a strain containing the two empty plasmids used to produce
α-Syn and MG, p426GPD/pRS425 mutant (see Materials and Methods for details).
These mutants were cultivated successively 3 times and stock cultures were frozen at
-80ºC. Surprisingly, no differences in the growth profiles of the four mutants were
observed (Fig. 6A), in contrast with the initial observations (Fig. 5).
FIGURE 6. Successive growths show that yeast was able to overcome α-Syn toxicity. A, No
significant differences in the growth curves of mutants were observed after 3 successive
growths. B, Successive growth curves of α-Syn mutant. The first culture (1) served as pre-
culture for the second culture (2), which served as pre-culture for the third culture (3). C,
Successive growths of the α-Syn/pRS425 mutant as in B. These experiments were repeated 3
times with similar results.
18
The constructions were repeated using the same methodology leading again to similar
growth profiles for all the mutants. To understand why production of α-Syn did not
inhibit growth as it did in the initial experiment, we monitored growth, localization of α-
Syn in the cell, and α-Syn production during the 3 successive growths.
Surprisingly, the successive cultivations of the mutants α-Syn/pRS425 and α-Syn/MG
produced different growth curves. During the successive growths, the lag phase
progressively decreased and the growth rate increased (Fig. 6, panels B and C).
Moreover, fluorescence microscopy analysis of the cells during the mid-exponential
phase, in the successive cultures, showed a marked decrease in the number of cells
exhibiting cytoplasmatic inclusions of α-Syn (Fig. 7A). To rule out the possibility that
these results (less cells with inclusions) were due to a decrease in the level of α-Syn
production, we determined the level of α-Syn by western blot. These analyses showed
a progressive decrease in the expression of the gene coding for α-Syn over the
successive growths, as illustrated in Fig. 7B.
FIGURE 7. The production of α-Syn decreases in successive cultivations of the α-Syn/pRS425
mutant. A, Fluorescence microscopy of each of the three cultures examined during the
mid-exponential phase. B, Level of α-Syn over the successive growths as determined by
western blot analysis in the first culture (1), the second culture (2) and the third culture (3).
Two hypotheses can be put forward to explain the decrease of α-Syn production in the
successive growths: (1) α-Syn production does not lead to a homogeneous population
of cells harboring inclusions, in other words, there are cells presenting inclusions and
cells without them. Therefore, successive growths may favor cells that do not exhibit
α-Syn inclusions as they could have a higher growth rate; (2) Considering that high
19
levels of α-Syn are highly toxic to the yeast cells, there might be a selective pressure to
reduce the number of plasmid copies without compromising auxotrophy. As a
consequence of a lower production of α-Syn, cells would be able to avoid toxicity
(Outeiro & Linquist, 2003; Zabrocki et al., 2005).
To prevent the observed decrease in α-Syn production we constructed a yeast strain
that contains two copies of the α-Syn gene integrated in the genome, thereby
preventing the decrease in the plasmid copy numbers. Moreover, a galactose-inducible
promoter was used to enable a precise control of the timing for α-Syn expression.
Effect of mannosylglycerate on the formation of α-Syn inclusions – use of
integrative plasmids to express α-Syn
The strain VSY72 contains two copies of the α-Syn gene integrated in the genome,
which are under the control of a galactose inducible promoter. This strain was used as
host for the construction of two mutants. In one of them, the plasmid p425::msgD,
containing the gene encoding the activities involved in the synthesis of MG was
introduced in strain VSY72. This mutant was designated as “MG mutant”. In the second
mutant, the empty plasmid (pRS425) was introduced in strain VSY72, and designated
as “Control mutant”. According to established protocols for α-Syn production,
S. cerevisiae cells were cultivated overnight in SC medium supplemented with 2%
raffinose, and then shifted to medium supplemented with 2% galactose (Outeiro &
Linquist, 2003). However, these experimental conditions did not lead to production of
MG in the MG mutant (Table 4a). To overcome this difficulty, different combinations of
carbon sources were tested in order to determine the growth condition, in which the
number of cytoplasmatic α-Syn inclusions and the accumulation of MG were maximal.
The accumulation of MG was determined using the MG mutant, while the formation of
α-Syn inclusions was monitored using the Control mutant (Table 4a and 4b). The
highest MG accumulation (90 µmol/g dry mass) was observed when the MG mutant
was pre-cultivated in glucose, and then cultivated in 2% glucose or in 1% glucose plus
1% galactose (Table 4a). Under these growth conditions, we did not observe any
cytoplasmatic inclusions of α-Syn in the Control mutant (Table 4b). When this mutant
was grown in a mixture of 0.4% glucose plus 1.6% galactose, the number of cells
containing cytoplasmatic inclusions increased to 20%. Under these growth conditions,
the MG mutant accumulated 60 µmol MG/g dry mass. Indeed, high concentrations of
glucose have a repression effect on the galactose metabolism. It is known that glucose
inhibits the expression of the galactose permease, and consequently blocks galactose
uptake (Nehlin et al., 1991).
20
TABLE 4a. Production of mannosylglycerate in the MG mutant for different combinations of
carbon sources.
Pre-culture Culture Mannosylglycerate (µmol/g dry mass)
2% raffinose 2% galactose 0
2% glucose 2% glucose 90
2% glucose 1% glucose / 1% galactose 90
2% glucose 0.4% glucose /1.6% galactose 60
2% glucose 2% galactose 20
TABLE 4b. Percentage of cells with cytoplasmatic α-Syn inclusions in the Control mutant for
different combinations of carbon sources.
Pre-culture Culture α-Syn inclusions1
(%)
2% raffinose 2% galactose n.d.
2% glucose 2% glucose 0
2% glucose 1% glucose / 1% galactose 0
2% glucose 0.4% glucose / 1.6% galactose 20
2% glucose 2% galactose 40
1 The percentage of cells with cytoplasmatic α-Syn inclusions was determined after 10
hours of galactose induction. n.d. – not determined.
When the medium was supplemented with 2% galactose, the MG mutant accumulated
20 µmol MG/g dry mass and around 40% of the cells of the Control mutant exhibited
α-Syn inclusions (Table 4a and 4b). Therefore, these conditions (cells grown overnight
in glucose medium, and then re-suspended in 2% galactose), were selected as the
most appropriate for further experiments. Sample aliquots were removed during the
period of galactose induction, and the cells were examined by fluorescent microscopy.
After 4 hours of galactose induction, α-Syn appeared evenly distributed in the
cytoplasm and localized intensely in the cytoplasmatic membrane. After 8 hours of
induction, α-Syn is mainly localized in the cytoplasmatic membrane. Continuous
production of α-Syn leads to the formation of small inclusions near the cytoplasmatic
membrane. At 10 hours of induction, the size of cytoplasmatic inclusions increased and
they are dispersed through the cytoplasm. This phenotype is transitory, since after 12
hours, the percentage of cells with α-Syn inclusions decreased and at 24 h no cells
with cytoplasmatic α-Syn inclusions were visualized (Fig. 8).
21
FIGURE 8. Localization of α-Syn in the Control mutant at different time points after induction
with galactose. Between 4 and 6 hours of galactose induction, the fluorescence is localized in
the cytoplasmatic membrane. At 8 hours, the α-Syn protein is mainly concentrated in the
cytoplasmatic membrane. With the increase in the concentration of α-Syn, small cytoplasmatic
inclusions of α-Syn were observed close to the cytoplasmatic membrane. These inclusions
enlarge and extend through the cell until 10 h post-induction. At 12 h after induction, the
number of cytoplasmatic inclusions starts to decrease and at 24 h cells showed no inclusions.
The effect of MG in the formation of cytoplasmatic α-Syn inclusions was determined at
10 and 12 h after galactose induction by comparing the percentage of cells with
inclusions in the MG mutant and the Control mutant (Fig. 9A and 9B).
Upon 10 h of induction, 38.6 ± 4.9% of cells expressing only α-Syn (Control mutant)
exhibited cytoplasmatic inclusions of α-Syn. This value should be compared with
11.5 ± 5.4% for the mutant expressing α-Syn plus MG (MG mutant). NMR analysis of
the ethanolic/chloroformic extracts of Control and MG mutants showed no MG and 19.6
µmol MG/g dry mass, respectively (Fig. 9C). We also detected the presence of
trehalose, a compatible solute accumulated naturally by S. cerevisiae. It is known that
trehalose acts as a stress protectant of proteins and biological membranes (Singer &
Linquist, 1998). Nevertheless, we verified that there was no significant difference in the
level of trehalose between the two mutants, indicating that the inhibitory effect of Syn
aggregation observed in the MG mutant is related with MG accumulation and not with
trehalose accumulation. Interestingly, besides the pronounced effect of MG in the
number of cells with cytoplasmatic inclusions, the two mutants showed similar growth
rates (data not show). To prove that α-Syn was produced to similar levels in the two
mutants, we performed western blot analysis with cells collected at 10 h after induction
(Fig. 9D).
22
FIGURE 9. Effect of mannosylglycerate on the formation of cytoplasmatic inclusions of α-Syn.
Cells were grown in glucose medium to allow intracellular accumulation of mannosylglycerate
and switched to galactose medium to induce α-Syn production. A, Percentage of cells showing
inclusions without and with accumulation of mannosylglycerate. The MG mutant contains a
bifunctional enzyme that synthesizes mannosylglycerate from GDP-mannose and
3-phosphoglycerate, and Control mutant contains the empty plasmid pRS425. Data shown are
derived from three independent experiments; for each experiment 300 cells were counted. B,
Fluorescence microscopy of Control and MG mutants at 10 and 12 h post-induction. C, Level of
intracellular compatible solutes in the two mutants studied. D, Western blot analysis of α-Syn-
EGFP proteins detected with an anti-α-Syn antibody in cells at 10 h after induction.
We also investigated the ability of MG to promote solubilization of α-Syn inclusions in
vivo. For that, Control and MG mutants were grown in raffinose until mid-exponential to
avoid MG accumulation, then cells were shifted to galactose medium and cultivated for
further 9 h to form cytoplasmatic inclusions of α-Syn; finally, cells were shifted to
glucose medium to allow MG accumulation. Aliquots were removed at 1 and 3 hours
after the switch to glucose medium, and cells were analyzed for α-Syn inclusions
(fluorescence microscopy), MG accumulation (NMR analysis of cell extracts), and
α-Syn content (western blot). Upon the switch to glucose medium, α-Syn production
was repressed and as result the number of cells presenting cytoplasmatic inclusions
decreased. After the shift to glucose medium, there was a significant decrease in the
23
number of cells with cytoplasmatic inclusions in the MG mutant as compared with the
Control mutant (Fig. 10A). After 1 h in glucose medium, 12.7±4.2 % of MG mutant cells
exhibited cytoplasmatic inclusions of α-Syn, while 33.3±6.7 % of Control mutant cells
had cytoplasmatic inclusions of α-Syn. The difference was more pronounced after 3 h
in glucose medium: 15.0±7.0% of Control mutant cells contained cytoplasmatic α-Syn
inclusions, to compare with 4.7±3.5% in the MG mutant. The MG content was 12 and
22 µmol/g dry mass for cultures removed at 1 and 3 hours, respectively (Fig. 10B).
Western blot showed that α-Syn was expressed in Control and MG mutant in similar
amounts (Fig. 10C).
FIGURE 10. Mannosylglycerate promotes solubilization of the cytoplasmatic inclusions of
α-Syn. MG and Control mutants were cultivated in galactose for 9 h, period of time where we
observed the highest amount of cells with inclusions (42%), and then shifted to glucose medium
to allow MG accumulation. Then, aliquots were removed at 1 and 3 hours to calculate: A)
Percentage of cells with cytoplasmatic inclusions of α-Syn; B) Intracellular amount of
mannosylglycerate in the MG mutant; and to analyze C) Levels of α-Syn expression by western
blot in both mutants after 1 hour in glucose medium. The data correspond to three independent
experiments.
Effect of MG on the amount of reactive oxygen species (ROS) induced by α-Syn
over-production
It is well known that α-Syn over-production leads to the formation of ROS and hydrogen
peroxide (Flower et al., 2005). We used DHR 123 staining, a well establish method to
24
detect the presence of ROS. This dye accumulates in the cell and is oxidized by ROS,
thereby emitting a fluorescence signal.
Control mutant was cultured in glucose (no production of α-Syn) and also in galactose
(high production of α-Syn). The MG mutant was only cultured in galactose. As
expected, the production of α-Syn in the Control mutant increased 3-fold the amount of
ROS in comparison with the same cells without α-Syn production (Fig. 11).
MG-producing cells had ROS levels 30% lower than cells producing only α-Syn,
suggesting that MG prevents the accumulation of ROS.
FIGURE 11. ROS levels are reduced by the accumulation of mannosylglycerate. Control mutant
was grown in medium supplemented with glucose (no production of α-Syn) or galactose (high
production of α-Syn), while MG mutant was grown only in the presence of galactose. ROS
accumulation in these mutants was quantified by fluorescence upon 9 h of cultivation using the
DHR 123 staining method. Data represents the average of three independent experiments.
The results presented in Figs. 9 and 11 indicate that MG accumulation in yeast cells
prior to α-Syn production decreases α-Syn aggregation and the accumulation of ROS.
25
Discussion
Microorganisms can accumulate high levels of compatible solutes, making them good
candidates for the large-scale production of these compounds (Lentzen & Schwarz,
2006). However, the applicability of these bioprocesses demands that the relevant
microbial cultures grow fast, to very high cell densities in industrial scale fermentors.
Alternatively, these solutes can be produced in organisms commonly used in industrial
bioprocesses (e.g. E. coli, yeast, Bacillus spp.) after modification of metabolic
pathways or insertion of foreign genes (Lentzen & Schwarz, 2006).
In this work, we optimized the production of mannosylglycerate (MG) using the yeast
Saccharomyces cerevisiae, a robust organism that is well-established as an industrial
producer. Empadinhas et al., (2004) reported the construction of a S. cerevisiae strain
engineered for the production of MG by expression of the gene msgD encoding
mannosy-3-phosphoglycerate synthase/phosphatase, the activities involved in MG
synthesis. This recombinant strain accumulated a maximum of 49 µmol/g dry mass of
MG in medium supplemented with glucose (Empadinhas et al., 2004). In this work we
followed a similar strategy, but used as host the S. cerevisiae strain VSY71. We
observed a maximal intracellular concentration of MG of 90 µmol/g dry mass
(approximately 0.24 µmol/mg protein) in cells grown in glucose as the sole carbon
source.
In order to optimize MG production, the strain VSY71, harboring the msgD gene, was
cultivated in different carbon sources, such as raffinose, galactose, mannose, and
sucrose. Among the carbon sources examined, MG formation was only observed when
cells were grown on sucrose; however, the MG content in sucrose-grown cells
decreased 31% in comparison with cells grown in glucose.
The engineered strain grew on raffinose, galactose or mannose with a growth yield
comparable to that on glucose, although with a clearly lower growth rate (data not
shown). It is known that yeast uses ultimately the glycolytic pathway to metabolize
raffinose, galactose or mannose (Kruckeberg & Dickinson, 2004), thus it is curious that
cells grown on these sugars are unable to produce detectable amounts of MG. It is
conceivable that synthesis of MG in these cells is limited by substrate availability,
namely GDP-mannose and/or 3-phosphoglycerate. The mannose moiety in
GDP-mannose originates from mannose-6-phosphate, which is formed by
26
isomerization of fructose-6-phosphate, a glycolytic intermediate metabolite, like
3-phosphoglycerate. We speculate that lower glycolytic rates on sugars other than
glucose could lead to insufficient accumulation of these metabolites, and therefore the
activity leading to MG synthesis would be very low.
Egorova and co-workers (2007) developed a process to produce high amounts of MG
using the trehalose deficient Thermus thermophilus RQ-1. The Thermus thermophilus
mutant, grown in fed-batch fermentation, accumulated 0.31 µmol/mg protein of MG.
This amount was raised up to 0.61 µmol/mg protein when the mutant was cultivated
under osmotic stress (Egorova et al., 2007). Therefore, the MG production by the
Thermus thermophilus mutant is 2.5-fold higher in comparison with S. cerevisiae
engineered in our work. Despite this lower production, the strategy using recombinant
S. cerevisiae has several advantages, such as: i) large-scale fermentation of yeast is
well-established and thoroughly optimized; ii) S. cerevisiae grows at 30ºC while the
growth temperature of Thermus thermophilus is 77ºC, which implies extra cost with
energy consumption; iii) genetic manipulation of S. cerevisiae is straightforward and
inexpensive, which makes this organism more suitable for further rounds of
optimization.
In view of the 2-fold increase in the production of MG when comparing strains VSY71
and CEN.PK2 it would be worth screening a strain collection for other strains of S.
cerevisiae that might be even better MG producers. Further strategies to improve MG
production could involve the knock-out of pathways consuming GDP-mannose in an
attempt to increase the pool of this substrate. Moreover, more effort should be invested
in medium manipulation that is likely to result in considerable improvement in
productivity.
Compatible solutes are potentially useful in a broad range of applications, from
biotechnology to medicine (Lentzen & Schwarz, 2006). In biotechnology their
applicability goes from stabilization of proteins, like enzymes or vaccines, to the
prevention of the aggregation of heterologous proteins when produced to high levels
(Carrió et al., 2005; Schrödel & Marco, 2005).
One of the applications derives from the protective effect of compatible solutes against
protein misfolding and aggregation (Ross & Poirier, 2004). These characteristics make
compatible solutes good candidates for lead compounds for the treatment of diseases
associated with the unfolding of proteins, such as prion and amyloid-related illnesses
(Ross & Poirier, 2004). In fact, several studies were conducted to evaluate the effect of
these solutes in the formation of aggregated proteins. Ryu and co-workers (2008)
studied the effect of MG, mannosylglyceramide, and glycerol on the aggregation of
β-amyloid protein in mammalian cells. MG had a significant inhibitory effect on
27
β-amyloid formation and reduced the toxicity of amyloid aggregates in human
neuroblastoma cells, while being innocuous to cells. In contrast, the other compounds
showed no effect in the prevention of β-amyloid formation.
In another report, the role of four compatible solutes (MG, mannosylglyceramide,
ectoine and hydroxyectoine) on the aggregation of the prion peptide was explored. In
this case, MG and hydroxyectoine showed no effect in the prevention of the prion
peptide aggregation. On the other hand, mannosylglyceramide and ectoine were able
to inhibit the aggregation of this peptide and to decrease the prion peptide-induced
toxicity in human neuroblastoma cells (Kanapathipillai et al., 2008). The authors
suggested that the ability of solutes to prevent prion aggregation depends highly on
their structure and it was inferred that the preferential binding properties of each solute
could play a key role in the inhibition of aggregation.
In the present work we propose the use of the MG-accumulating yeast as a model to
study protein stabilization in living cells. For that, the S. cerevisiae strain VSY72,
harboring two copies of α-Syn-EGFP integrated in the genome, was engineered to
synthesize MG (MG mutant). Remarkably, the accumulation of MG in the MG mutant
led to a 3.5-fold decrease in the number of cells with α-Syn inclusions in comparison
with the Control mutant. Moreover, microscopy analysis of MG mutant cells showed
bright fluorescence around the plasma membrane, suggesting that α-Syn was re-
localized to the membrane.
Moreover, we confirmed that α-Syn over-production was accompanied by a 3-fold
increase in the level of ROS; most importantly, this increase was clearly attenuated by
the accumulation of MG in the cells. In healthy cells, the formation of unfolded and
misfolded proteins is controlled by protein control systems that promote the refolding
and repair of these proteins. When refold or repair is not possible, these aberrant
proteins are marked and eliminated either by the ubiquitin proteasome system or by the
autophagy-lysosome pathway (Haliwell, 2006). The over-production of α-Syn seems to
overload the capacity of the protein control systems, which raises the magnitude of the
oxidative stress imposed to cells, consequently leading to accumulation of ROS. The
observation that the level of ROS decreased in the MG mutant supports the view that
MG alleviates the oxidative burden associated with α-Syn over-production, most
probably by shifting the equilibrium towards α-Syn forms that are less toxic than
oligomers or even mature aggregates.
It is important to note that there is a controversy about whether α-Syn production in
yeast cells results in the formation of fibrils. It has been reported that α-Syn aggregates
stained positive with thioflavin-S, proving that they had an amyloid-like structure
(Zabrocki et al., 2005). More recently, on the basis of electron microscopy studies, it
28
was argued that α-Syn accumulations do not contain fibril structures, but solely clusters
of vesicles (Soper et al., 2008). In this work, we did not characterize the type of
structures within the bright spots, but since we used the same strain as Zabrocki et al.,
(2005), it is reasonable to assume that at least some of the aggregates are amyloid-like
structures.
Besides the ability of MG to prevent the formation of α-Syn inclusions, we also
demonstrated that MG was able to promote the solubilization of these inclusions. This
unexpected result revealed a new role for this compatible solute beyond protein
stabilization. In fact, our work indicates that MG acts as a molecular chaperone since it
effectively disrupts α-Syn inclusions, probably promoting the release of native forms of
α-Syn. The role of MG in the prevention / disruption of α-Syn inclusions can derive from
three different modes of action: 1) MG may induce cell mechanisms namely,
proteasome and autophagy, which actively degrade misfolded α-Syn; 2) MG may
induce or activate chaperones that promote the correct folding of α-Syn; 3) MG may
directly stabilize the structure of α-Syn preventing the formation of aggregates, similarly
to what happens in in vitro assays of protein stabilization.
Since western analysis showed that both mutants (Control and MG mutant) had similar
levels of α-Syn, probably the MG effect is not due to the induction of cell mechanisms
that degraded α-Syn. To check the second hypothesis, it would be interesting to
compare the transcriptional profiles of the MG mutant and the Control mutant to have a
global picture of the impact of MG at the transcriptional level. For example, if MG
induces chaperone expression it is possible that the effect could be noted by the DNA-
microarray methodology. The data available do not allow us to draw a final conclusion,
but it is possible that the mode of action of MG involves a direct effect, either by binding
to specific regions of the protein or protein-oligomers, or indirectly by changes in the
structure/dynamics of the protein hydration shell.
Concluding remarks
We constructed a strain of S. cerevisiae that produced 2-fold higher amounts of MG
than the strain earlier described. Moreover, the specific production was similar to that
of the best natural producer, Rhodothermus marinus. This is an encouraging result,
especially if we take into account that, unlike R. marinus, S. cerevisiae can be
fermented to very high cell densities. Although much work needs to be done to achieve
the final goal of producing MG at a competitive cost, this is already an important
achievement that should be followed in further optimization rounds.
We designed an elegant strategy to demonstrate the efficacy of MG in the protection of
proteins in living cells. MG was able to prevent the aggregation of α-Syn and thereby
29
decrease its toxicity (reduction of ROS level). Moreover, it was shown, for the first time,
that MG acts as a molecular chaperone by effectively accelerating the disruption of
α-Syn inclusions. The yeast model developed in this work can be easily extended to
test the effect of MG in the protection of other proteins, thereby verifying whether MG
can be used as a general protein protector.
30
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Appendix
We were interested in assessing the effect of mannosyl-di-myo-inositol phosphate
(MDIP) on the protection of model proteins against heat-induced denaturation,
aggregation and inactivation. So far, MDIP has only been found in hyperthermophilic
bacteria of the genera Thermotoga and Aquifex (Santos et al., 2007): in Aquifex spp.,
MDIP accumulates in response to osmotic stress, whereas it contributes to the heat
stress response in Thermotoga spp.
In order to determine the growth conditions that lead to maximum accumulation of
MDIP in T. maritima, this hyperthermophile was grown at different temperatures (80ºC
and 85ºC) and the cultures were harvested at different growth phases (mid- and late-
exponential phases). For each growth condition, ethanolic-chloroformic extractions
were performed as described in Material and Methods.
FIGURE 12 - Effect of the growth temperature on the accumulation of di-myo-inositol phosphate
(DIP) and mannosyl-di-myo-inositol phosphate (MDIP) in Thermotoga maritima at mid- and
late-exponential growth phases.
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The pool of compatible solutes (DIP and MDIP) was quantified by 31P-NMR. The
highest MDIP accumulation was observed when cells were grown at a temperature of
85ºC and harvested during the mid-exponential phase (Fig. 12).
Subsequently, the T. maritima was grown in a 300 L fermentor at 85ºC, and the
compatible solutes were extracted from the biomass by using ethanol-chloroform
extraction. The extract was loaded onto a QAE-Sephadex A25 column previously
equilibrated with 5.0 mM sodium bicarbonate, pH 9.8. The elution was performed with
one bed volume of the same buffer, followed by a linear gradient of 5.0 mM to 1 M
sodium bicarbonate at a flow rate of 4 ml.min-1. The fractions eluted were analyzed by
31P-NMR to monitor the presence of MDIP. Two fractions were obtained: the first
fraction contained pure MDIP was eluted at 100 mM sodium bicarbonate, while the
second fraction was eluted at 200 mM sodium bicarbonate and contained a mixture of
DIP plus MDIP. Unfortunately, the amount of pure MDIP obtained after the purification
process was not sufficient to examine the effect of MDIP on the thermal stabilization of
model proteins.
