908 research outputs found
Efficient production of L-lactic acid from xylose by metabolically engineered yeast Pichia stipitis
No full textPichia stipitis, a yeast which ferments naturally xylose, was genetically engineered for L-(+)-lactate production. A P. stipitis strain expressing the L-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter was constructed. Either xylose or glucose was used as the carbon source for lactate production under oxygen restricted conditions. Remarkably, the constructed P. stipitis strain produced a higher lactate concentration and yield on xylose than on glucose. Lactate accumulated as the main product on xylose-containing medium: 58 g/l lactate was produced from 100 g/l xylose. Relatively efficient lactate production was also observed on glucose medium, 41 g/l lactate was produced at a yield of 0.44 g/g glucose. Lactate was produced at the expense of ethanol production which was decreased to approximately 20% of the wild type levels on xylose-containing medium and to 75% on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact
Efficient production of L-lactic acid from xylose by metabolically engineered yeast Pichia stipitis
No full textPichia stipitis, a yeast which ferments naturally xylose, was genetically engineered for L-(+)-lactate production. A P. stipitis strain expressing the L-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter was constructed. Either xylose or glucose was used as the carbon source for lactate production under oxygen restricted conditions. Remarkably, the constructed P. stipitis strain produced a higher lactate concentration and yield on xylose than on glucose. Lactate accumulated as the main product on xylose-containing medium: 58 g/l lactate was produced from 100 g/l xylose. Relatively efficient lactate production was also observed on glucose medium, 41 g/l lactate was produced at a yield of 0.44 g/g glucose. Lactate was produced at the expense of ethanol production which was decreased to approximately 20% of the wild type levels on xylose-containing medium and to 75% on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact
Enhancing the Attendee Co-creation : a Case Study of Hackathon Concept Revamp
Full text linkTutkimus tarkastelee yhteiskehittämiseen pohjautuvia tilaisuuksia, ja sitä kuinka osallistujakokemusta parantamalla voidaan edesauttaa avointen innovaatioiden syntymistä. Tutkimuksen tarkoituksena on arvioida kohdeyrityksen hackathon-tapahtumakonseptin eri osasia, sekä konseptin mahdollisia sudenkuppia.
Kohdeyrityksen nykyisen hackathon-konseptin arvioimisessa sekä uuden, parannellun konseptin luomisessa on hyödynnetty Hackathon-kanvaasia (Duchateau, M, 2016). Tutkimusta tukeva teoreettinen viitekehys pohjautuu innovaatioihin ja yhteiskehittämiseen, erityisesti tapahtuman osallistujien asiakaskokemuksen näkökulmasta tarkasteltuna.
Tutkimus pyrkii ymmärtämään syvällisesti yksittäistä tapausta tapaustutkimuksen menetelmiin nojautuen, ja kvalitatiivisen tutkimuksen periaatteita noudattaen. Toimintatutkimusta on sovellettu tässä työssä kehittämistutkimuksen muodossa niin nykytila-analyysissa, kuin nykyisen hackathon-tapahtumakonseptin puutteiden arvioinnissa, ja edelleen parannellun tapahtumakonseptin luomisessa. Lisäksi palvelumuotoilun menetelmiä hyödynnettiin persoonien sekä asiakaspolkujen luomisessa hackathon-tapahtumien eri osallistujaryhmille.
Tutkimustieto pohjautuu suurimmaksi osaksi puolistrukturoituihin haastatteluihin kohdeyrityksessä hackathonia järjestävien henkilöiden kanssa. Kaikki haastattelut nauhoitettiin, litteroitiin sekä analysoitiin huolellisesti. Lisäksi yhtenä aineistonhankinnan menetelmänä tutkimuksen tekijä osallistui kohdeyrityksen järjestämään hackathon-tapahtumaan, jotta pystytiin muodostamaan mahdollisimman laaja ymmärrys tapahtumien nykytilasta.
Tutkimustiedon analyysi johdatti seuraaviin aihekokonaisuuksiin: kohdeyrityksen sisäisen osaamisen hyödyntäminen yhteiskehittämisessä; tarve tarkemmin fasilitoidulle kehittämisprossille; ja henkilökohtaisemman osallistujakokemuksen luominen ennen ja jälkeen hackathon-tapahtumaa, sekä sen aikana. Tutkimustulosten perusteella rakentui lopullinen suositus uudistetusta hackathon-tapahtumakonseptista, sekä kehitysideoita siitä, kuinka kohdeyritys voisi tehdä jatkossa hedelmällisempää yhteistyötä hackathon-tapahtumien osallistujien kanssa. Jatkotutkimusta tarvitaan, jotta tapahtumakonseptia voidaan kehittää eteenpäin, ja luoda edellytyksiä yhteiskehittämiselle myös virtuaalisesti tapahtuvissa hackathoneissa.The research problem of this study concerns the co-creational events, and how improving the attendee experience could enhance the creation of open innovation. To address this issue, the purpose of this study is to explore the different elements, as well as the possible pitfalls of current the hackathon concept of the case company’s hackathon events.
In evaluation and in concept creation phases the Hackathon Canvas (Duchateau, M. 2016) is used as a tool for creating a robust hackathon event concept. The theoretical framework supporting the study is based on the literature reviews on open innovation and co-creation, especially from the attendee experience point of view. Service design principles and tools are also applied in this study. In the course of the study, provisional personas were recognized and customer journeys were done for selected event attendees.
The goal of the case study is to both guide the qualitative research principles and provide detailed examination of a single case. Additionally, action research method is applied by first analyzing the current state of the case company’s hackathon events; following the evaluation of the current limitations and furthermore in creating an improved and comprehensive concept for hackathons.
The research data is largely based on semi-structured interviews with the organizers of the hackathon events in the case company. All the interviews were recorded, transcribed and analyzed carefully. Additionally, the author participated in one of the case company’s hackathons in order to form wider understanding by observing the course of the hackathon events.
Analysis of the data lead to the following themes: involving the case company’s internal expertise into collaboration; the need for more carefully facilitated design processes, and more personalized attendee experience creation before, during and after the hackathon events. Based on the findings of this study, the initial proposal of the revamped hackathon concept, as well as the development ideas to enhance fruitful cooperation with the attendees of the hackathon events were delivered to the case company. Nevertheless, further research is needed in order to evolve the hackathon concept further – and to explore the new possibilities of collaboration when organizing the online hackathon events
Caracterização fisiológica do fungo Ashbya gossypii e desenvolvimento de estirpes para produção de proteínas recombinantes
No full textTese de doutoramento em Engenharia Quimica e BiológicaAshbya gossypii is a phytopathogenic hemiascomycete belonging to the Saccharomycetaceae
family. This fungus has attracted attention because of its natural ability to produce riboflavin
(vitamin B2) as a detoxifying and protective mechanism being used at an industrial level as a
biotechnological important producer of riboflavin. The genome sequence of this filamentous fungus
revealed remarkable similarities to that of the budding yeast Saccharomyces cerevisiae both at the
level of homology and synteny. A. gossypii is a very promising experimental system because it
has a small genome (the smallest eukaryotic genome known to date) and haploid nuclei.
Moreover, efficient gene targeting due to the high homologous recombination efficiency in A.
gossypii makes it possible to do one-step gene replacement by PCR-based gene targeting. A.
gossypii also allows extrachromosomal free replication of plasmids bearing an autonomous
replicator. Taking advantage of these unique features, the overhall purpose of this project was to
explore the potential of A. gossypii as an alternative cell factory organism. As little information was
available for this organism, the project began with the physiological characterization of different A.
gossypii strains. Also, the information in the literature regarding its secretory ability was inexistent
and thus two heterologous proteins were used to evaluate this feature. In addition, different
strategies were undertaken to improve its secretion ability and a global transcriptome analysis was
conducted to identify the bottlenecks on A. gossypii secretory pathway.
Colony radial growth rates and specific growth rates of three related A. gossypii strains
ATCC10895, IMI31268, MUCL29450 and an unrelated strain, CBS109.26, were measured on
various carbon and nitrogen sources at pH 4.5 and pH 6.5 to elucidate physiological growth
requirements and strain differences. All strains grew on yeast extract or ammonium as nitrogen
sources, but not on nitrate. Substantial growth at pH 4.5 was observed only on complex medium.
D-Glucose, glycerol and starch were utilised as carbon sources. Ethanol was produced during
growth on glycerol. Conversion of xylose into xylitol demonstrates that the xylose reductase is
active. Phenotypic differences between related strains were greater than expected. It was shown
that A. gossypii utilizes ammonium as sole nitrogen source at pH 6.5, facilitating further
physiological studies using chemically defined media in the future.
Even though filamentous fungi are excellent producers of a broad spectrum of extracellular
enzymes such as amylases, proteases and catalases, little is known about the secretory capacity
of A. gossypii. To explore the potential of A. gossypii as a host for the expression of recombinant
proteins and to assess whether protein secretion would be more similar to the closely related S.
cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI)
from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids
containing the two micron sequences from S. cerevisiae. The native signal sequences of EGI and
CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl--D-lactoside as substrate, the
protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable
and the specific activity being comparable to that produced by a similar EGI producing S.
cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced.
Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation
when compared with the native T. reesei proteins, but the glycosylation was less extensive than on
cellulases expressed in S. cerevisiae.
In order to improve the general secretion ability, A. gossypii was subject to random
mutagenesis with ethyl methane sulfonate (EMS). Selection and screening was carried out in
order to identify secretion mutants with improved protein secretion ability. Secreted EGI, amylase
and beta glucosidase activities of the parental strain and five key mutants were investigated and
used as an indicator for enhanced protein production after the mutagenic treatment. Mutagenesis
improved EGI and amylase activity in the culture supernatant of the mutants S436 and S466 by 2
and 3-fold increase respectively, compared to the initial parental strain. At the same time, the
mutant S436 also revealed 40% improvement in the beta glucosidase activity. Mutant S397
showed a 2 fold increase in beta glucosidase activity. Overall, mutant S436 seems to be the most
promising A. gossypii strain since all the activities tested were enhanced when comparing to the
parental strain. This means that the general secretion capacity of this mutant was enhanced.
Another attempt to improve the secretion capacity of A. gossypii relied on the deletion of GAS1
gene, which codes for a -1,3-glucanosyltransglycosylase involved in cell wall assembly. GAS1
gene is present as a tandem repeat in A. gossypii genome. With this approach, a higher
permeability of the cell wall was expected and hence an increase in the protein secretion capacity.
However two scenarios where observed. The individual deletion of one copy of the gene severely
impaired growth whereas the abolishment of the other copy resulted in similar amounts of EGI
secreted into the extracellular medium when compared to the initial recombinant strain. As a result
this strategy failed to enhance A. gossypii secretory capacity.
In order to understand and determine the limitations encountered along A. gossypii secretory
pathway, a transcriptomic analysis was carried out in the recombinant EGI producing strain and
also under chemical induced stress by dithiotreitol (DTT). Surprisingly, none of the conditions
tested were able to induce unfolded protein response (UPR) in A. gossypii. EGI production can be
expected to have such an effect since the production levels are very low to cause ER stress.
However, the translation machinery was down regulated under EGI producing conditions, which
can explain the low EGI production levels. Neither DTT, a widely used UPR inducer was able to
activate UPR in A. gossypii. Instead endoplasmatic reticulum associated degradation (ERAD) was
highly induced when the mycelium was treated with DTT. The lack of a strong UPR response
when low levels of an heterologous protein is being produced, or during chemically induced stress,
strongly suggests that non-UPR mediated bottlenecks might exist in A. gossypii that hamper
efficient secretion.Ashbya gossypii é uma hemiascomicete pertencente á família Saccharomycetaceae. Este fungo
tem atraído atenção devido à capacidade de produzir riboflavina (vitamina B2) naturalmente como um
mecanismo de destoxificação e de defesa. Sendo considerado um produtor de riboflavina
biotecnologicamente importante e como tal, tem sido usado a nível industrial. A sequência genómica
deste fungo filamentoso revelou semelhanças extraordinárias com a levedura Saccharomyces
cerevisiae quer ao nível da homologia quer da sintenia. Este fungo é um sistema experimental muito
promissor porque possui um genoma pequeno (o genoma eucariota mais pequeno conhecido até á
data) e núcleos haplóides. Para além disso, a elevada eficiência de recombinação permite a deleção
de genes de modo eficiente, sendo possível de ser feito num único passo por técnicas de PCR
dirigidas. A. gossypii também permite a replicação livre extracromossomal de plasmídeos que possuam
um replicador autónomo. Tomando partido destas vantagens únicas, o objetivo geral deste projeto
consistiu na avaliação do potencial de A. gossypii como uma fábrica celular alternativa. Como havia
pouca informação acerca deste organismo, o projeto começou com a caracterização fisiológica de
diferentes estirpes de A. gossypii. A informação relativa à sua capacidade de secreção era inexistente
e portanto, duas proteínas heterólogas foram usadas para avaliar esta capacidade. Para além disso,
várias estratégias foram implementadas com o objetivo de melhorar a sua capacidade de secreção e
uma análise de transcriptoma foi efetuada para identificar os passos limitantes da via de secreção.
Foram determinadas as taxas de crescimento radial de colónia e taxas específicas de
crescimento em várias fontes de carbono e de azoto a pH 4.5 e pH 6.5 de três espécies de A. gossypii
similares, nomeadamente, ATCC10895, IMI31268, MUCL29450 e de uma espécie mais afastada, CBS
109.26, de modo a elucidar os requisitos fisiológicos de crescimento e as diferenças entre as espécies.
Todas as estirpes cresceram em extrato de levedura ou amónio como fonte de azoto, mas não em
nitrato. Foi observado crescimento substancial a pH 4.5 apenas em meio complexo. D-glucose, glicerol
e amido foram utilizados como fonte de carbono. Durante o crescimento em glicerol, detetou-se
produção de etanol. A conversão de xilose em xilitol demonstrou que a xilose redutase está ativa.
Diferenças fenotípicas entre as estirpes mais semelhantes foram maiores do que o esperado. Foi
mostrado que o A. gossypii utiliza amónio como fonte de azoto a pH 6.5, facilitando estudos fisiológicos
que no futuro usem meio definido.
Embora os fungos filamentosos sejam excelentes produtores de uma vasta gama de enzimas
extracelulares, tais como amilase, protease e catalase, pouco se sabe acerca da capacidade secretória
de A. gossypii. Com o objetivo de explorar o potencial de A. gossypii como um organismo usado para a
expressão de proteínas recombinantes e de modo a avaliar se a secreção de proteínas será mais
semelhante à S. cerevisiae ou a outros fungos filamentoso, a endoglucanase I (EGI) e a
celobiohidrolase I (CBHI) do fungo Trichoderma reesei, foram expressas com sucesso em A. gossypii a
partir de plasmídeos que contêm a sequência 2 micra de S. cerevisiae sob o promotor PGK1 de S.
cerevisiae. A sequência sinal nativa da EGI e da CBHI direcionou a secreção da EGI e da CBHI para o meio de cultura em A. gossypii. Embora a atividade da CBHI não tenha sido detetada com o substrato
4- methylumbelliferyl--D-lactoside, a proteína foi detectada por Western blot realizado com anticorpos
monoclonais. A actividade da EGI foi detetada, sendo que a atividade específica é comparável à de
uma estirpe de S. cerevisiae produtora de EGI. Mais EGI foi secretada em comparação com CBHI ou
foi produzida proteína mais ativa. A caracterização parcial da CBHI e da EGI produzidas em A. gossypii
revelou hiperglicosilação quando comparada com as proteínas nativas de T. reesei, mas a glicosilação
foi menos extensa do que nas celulases expressas em S. cerevisiae.
De modo a melhorar a capacidade geral de secreção, A. gossypii foi sujeito a mutagénese aleatória
com etil metano sulfonato (EMS). A seleção e o rastreio foram realizados com a finalidade de identificar
mutantes com uma capacidade melhorada de secretar proteínas. Foram medidas as atividades das
enzimas secretadas, EGI, amilase e beta glucosidaes da estirpe parental e de cinco mutantes chave e
usadas como um indicador de aumento de produção de proteínas após o tratamento mutagénico. A
mutagénese resultou num aumento de 2 e 3 vezes na atividade da EGI e da amilase no sobrenadante
da cultura dos mutantes S436 e S466, comparado com a estirpe parental. Simultaneamente, o mutante
S436 também revelou um aumento na atividade de beta glucosidase. O mutante mutante S397
apresentou um aumento de 2 vezes na atividade da beta glucosidase. No geral, o mutante S436
parece ser a estirpe de A. gossypii mais promissora uma vez que todas as atividades medidas
sofreram um aumento quando comparadas com a estirpe parental. Isto significa que a capacidade
geral de secreção deste mutante foi melhorada.
Uma outra tentativa para melhorar a capacidade de secreção do A. gossypii baseou-se na deleção
do gene GAS1, que codifica para uma -1,3-glucanosyltransglycosylase envolvida na montagem da
parede celular. No genoma de A. gossypii existem duas cópias do gene GAS1. Com esta abordagem
esperava-se um aumento na permeabilidade da parede celular e consequentemente um aumento na
capacidade de secreção de proteínas. Contudo, foram observados dois cenários. A deleção individual
de uma cópia do gene afetou gravemente o crescimento, ao passo que a eliminação da outra cópia
resultou em quantidades semelhantes de EGI secretadas para o meio extracelular, quando comparado
com a estirpe recombinante inicial. Como resultado, esta estratégia falhou no aumento da capacidade
secretora de A. gossypii.
De modo a compreender e a determinar as limitações encontradas ao longo da via de secreção de
A. gossypii, foi realizada uma análise de transcriptoma na estirpe recombinante produtora de EGI e
também sob stress quimicamente induzido por ditiotreitol (DTT). Surpreendentemente, nenhuma das
condições testadas foi capaz de induzir a “Unfolded protein response” (UPR) em A. gossypii. Seria de
esperar tal efeito por parte da EGI, tendo em conta os baixos níveis de expressão. Contudo, a
maquinaria de tradução foi reprimida nas condições de produção de EGI, o que por sua vez ajuda a
explicar os baixos níveis de produção de EGI. Nem mesmo o DTT, um indutor de UPR amplamente
utilizado, foi capaz de ativar a UPR em A. gossypii. Em vez disso, a degradação associada ao reticulo
endoplasmático (ERAD) foi altamente induzida quando o micélio foi tratado com DTT. A ausência de
uma resposta UPR forte quando uma proteína heteróloga está a ser produzida a baixos níveis ou
durante stress quimicamente induzido, sugere que limitações não relacionadas com a UPR possam
existir em A. gossypii e que estejam a condicionar uma secreção eficiente
Understanding the biotechnological potential of Ashbya gossypii
Full text linkTese de doutoramento em BioengenhariaAshbya gossypii (syn. Eremothecium gossypii) is a filamentous Saccharomycete which has long been known in the scientific and industrial communities, first as a cotton pathogen and subsequently as a riboflavin overproducer. This fungus has the smallest free living eukaryotic genome known, which shares a high degree of gene homology and gene order conservation with that of Saccharomyces cerevisiae. It has haploid nuclei and is prone to genetic manipulation, allowing the use of simple PCR-based gene targeting strategies and free propagation of plasmids containing replicons from S. cerevisiae. Moreover, it grows well in a variety of defined, complex and waste-based media, and has a long history of safe use in the industrial production of riboflavin (vitamin B2). These unique features led to expanded interest in A. gossypii as a “minimal” host for the production of, yet unexploited, valuable compounds other than riboflavin, namely heterologous proteins. However, although two heterologous proteins have already been successfully secreted by A. gossypii, little is still known about the protein secretion ability of this fungus.
To further understand the biotechnological potential of A. gossypii as a cell factory organism, this thesis primarily focused on the characterization of the A. gossypii protein secretory pathway at the genomic, transcriptomic and proteomic levels. Based on experimental observations and on the data from the genomic and transcriptomic analyses, a hydrolytic enzyme, invertase, was deduced to be natively secreted by A. gossypii and molecularly characterized. To further address A. gossypii as a heterologous protein producer, the β-galactosidase from Aspergillus niger was expressed in this fungus under the regulation of different native and heterologous promoters. In addition, a new molecular tool for use in A. gossypii was developed to generate mutant strains free of exogenous selection markers, allowing the creation of improved A. gossypii strains suitable for industrial applications.
The results presented in this thesis demonstrate that the amount and variety of proteins natively secreted by A. gossypii to the culture medium is rather low, being more similar to that of yeast than to that of other filamentous fungi. Similarly, the N-glycosylation patterns produced by A. gossypii are generally more similar to those produced by yeast than to those produced by other filamentous fungi. However, extensive hyperglycosylation only occurs in certain culture conditions. Like other filamentous fungi, A. gossypii also seems to be able to trim its N-glycans.
A conventional unfolded protein response (UPR) was not activate in A. gossypii in response to heterologous protein secretion nor to dithiothreitol (DTT)-induced secretion stress, as generally observed in other fungi. However, the transcriptional responses of A. gossypii to DTT-induced stress indicate that alternative mechanisms exist in this fungus to cope with protein secretion stress.
The A. gossypii invertase was demonstrated to be encoded by the AFR529W (AgSUC2) gene, which is functionally complemented by the S. cerevisiae SUC2 (ScSUC2) gene. The signal sequences of both AgSuc2p and ScSuc2p were able to direct the secretion of invertase into the culture medium in A. gossypii. Similarly to the invertases of other fungi, the expression of the A. gossypii invertase is regulated by the sugars present in the medium. These results expanded our knowledge about the A. gossypii native secretion capacities, being invertase the second hydrolytic enzyme natively secreted by this fungus to be experimental characterized.
The β-galactosidase from A. niger was successfully expressed in A. gossypii under the regulation of different promoters. The native TEF promoter revealed to be the best promoter for overexpressing heterologous β-galactosidase in A. gossypii, inducing 2-fold higher secreted activity than the A. gossypii GPD promoter and 7-fold higher than the S. cerevisiae PGK1 and ADH1 promoters. The levels of active β-galactosidase secreted by a S. cerevisiae laboratorial strain transformed with the same plasmids were up to 37 times lower than those obtained in A. gossypii. The secretion of active β-galactosidase by A. gossypii was approximately 1.5-fold higher in glycerol- than in glucose-containing medium. These results highlight the potential of A. gossypii as a heterologous protein producer and open new opportunities to further optimize its secretion capacities using this enzyme as a model. As its activity is easy to detect, the screening for improved secretion will be facilitated.
The Cre-loxP recombination system, which has been widely used in other organisms, was successfully adapted for use in A. gossypii, allowing the removal and reuse of selection marker genes in targeted engineering of this fungus. The set of disruption cassettes and plasmids constructed greatly expand the possibilities for genetically engineer A. gossypii, being these suitable for use in both laboratorial and industrial strains, as they do not required any predetermined genetic background. In the future, targeted improvement of A. gossypii strains for industrial applications will benefit from this molecular tool.O Ashbya gossypii (sin. Eremothecium gossypii) é um Saccharomycete filamentoso há muito conhecido nas comunidades científica e industrial, primeiro como patogénio do algodão e subsequentemente como super-produtor de riboflavina. Este fungo tem o mais pequeno genoma eucariótico não parasitário conhecido, o qual partilha um elevado grau de homologia e conservação de ordem génica com o genoma da Saccharomyces cerevisiae. Ele tem núcleos haplóides e é de fácil manipulação genética, permitindo o uso de estratégias de manipulação genética direcionada simples e a livre propagação de plasmídeos contendo sequências de replicação de S. cerevisiae. Além do mais, cresce bem numa variedade de meios definidos, complexos e baseados em resíduos, e tem um longo historial de utilização segura na produção industrial de riboflavina (vitamina B2). Estas características únicas levaram a um interesse alargado em A. gossypii como hospedeiro “mínimo” para a produção de outros compostos de interesse, ainda inexplorados, para além da riboflavina, nomeadamente proteínas heterólogas. No entanto, apesar de duas proteínas heterólogas já terem sido secretadas por A. gossypii com sucesso, pouco ainda se conhece acerca da capacidade de secreção de proteínas deste fungo.
Para melhor compreender o potencial biotecnológico de A. gossypii como fábrica celular, esta tese focou-se primariamente na caracterização da via de secreção de proteínas do A. gossypii aos níveis genómico, transcriptómico e proteómico. Com base em observações experimentais e nos dados das análises genómicas e transcriptómicas, uma enzima hidrolítica, invertase, foi prevista ser nativamente secretada por A. gossypii e molecularmente caracterizada. Para melhor avaliar o A. gossypii como produtor de proteínas heterólogas, a β-galactosidase de Aspergillus niger foi expressa neste fungo sob a regulação de diferentes promotores nativos e heterólogos. Adicionalmente, uma nova ferramenta molecular para uso em A. gossypii foi desenvolvida para gerar estirpes mutantes livres de marcadores de selecção exógenos, permitindo a criação de estirpes de A. gossypii melhoradas para aplicações industriais.
Os resultados apresentados nesta tese demonstram que a quantidade e variedade de proteínas nativamente secretadas por A. gossypii para o meio de cultura é relativamente baixa, sendo mais comparável à das leveduras do que à de outros fungos filamentosos. De modo semelhante, os padrões de N-glicosilação produzidos por A. gossypii são genericamente mais semelhantes aos produzidos pelas leveduras do que aos produzidos por outros fungos filamentosos. No entanto, hiperglicosilação extensa só ocorre em determinadas condições de cultura. Tal como outros fungos filamentosos, o A. gossypii parece ter também a capacidade de produzir N-glicanos truncados.
Uma unfolded protein response (UPR) convencional não foi ativada em A. gossypii em resposta à secreção de proteínas heterólogas nem ao stress de secreção induzido por ditiotreitol (DTT), como geralmente observado noutros fungos. No entanto, as respostas transcricionais do A. gossypii ao stress induzido por DTT indicam que existem mecanismos alternativos neste fungo para lidar com o stress de secreção proteica.
A invertase de A. gossypii foi demonstrada ser codificada pelo gene AFR529W (AgSUC2), que é funcionalmente complementado pelo gene SUC2 de S. cerevisiae (ScSUC2). As sequências sinal da AgSuc2p e ScSuc2p conseguiram direcionar em A. gossypii a secreção de invertase para o meio de cultura. Similarmente às invertases de outros fungos, a expressão da invertase de A. gossypii é regulada pelos açúcares presentes no meio. Estes resultados expandiram o nosso conhecimento acerca das capacidades de secreção nativas do A. gossypii, uma vez que a invertase é apenas a segunda enzima hidrolítica nativamente secretada por este fungo a ser experimentalmente caracterizada.
A β-galactosidase de A. niger foi espressa com sucesso em A. gossypii sob a regulação e diferentes promotores. O promoter nativo TEF revelou-se o melhor promotor para sobre-expressar β-galactosidase heteróloga em A. gossypii, induzindo 2 vezes mais atividade secretada do que o promotor GPD de A. gossypii e 7 vezes mais do que os promotores PGK1 e ADH1 de S. cerevisiae. Os níveis de β-galactosidase ativa secretada por uma estirpe laboratorial de S. cerevisiae transformada com os mesmos plasmídeos foram até 37 vezes mais baixos do que os obtidos em A. gossypii. A secreção de β-galactosidase ativa por A. gossypii foi cerca de 1.5 vezes mais alta em meio contendo glicerol em vez de glucose. Estes resultados evidenciam o potencial do A. gossypii como produtor de proteínas heterólogas e abrem novas oportunidades para optimizar as suas capacidades de secreção usando esta enzima como modelo. Como ela é de fácil deteção, o rastreio de melhorias na secreção será facilitado.
O sistema de recombinação Cre-loxP, que tem sido amplamente usado em diferentes organismos, foi adaptado com sucesso para utilização em A. gossypii, permitindo a remoção e reuso de marcadores de selecção em engenharia genética direcionada neste fungo. O conjunto de cassetes de deleção e plasmídeos construídos expandem as possibilidades para manipular geneticamente o A. gossypii, sendo adequados quer para uso em estirpes laboratoriais, quer em estirpes industriais, pois não requerem a existência de nenhum background genético predeterminado. No futuro, o melhoramento direcionado de estirpes de A. gossypii para aplicações industriais irá beneficiar desta ferramenta molecular
Protein production in filamentous fungi:cellular responses and strain improvement
No full textIn recent years our understanding of cellular responses in Trichoderma and Aspergillus towards secretion load has increased significantly. Production of large amounts of native proteins or production of difficult foreign proteins provokes secretion stress that is believed in particular to be caused by folding limitations within the ER. Impairnment of folding causes the so called unfolded protein response (UPR), which is mediated by the regulatory protein HAC that causes modification of expression of a number of genes involved in various cellular processes related to protein secretion. In addition to the classical UPR, also other cell responses have been discovered using genome-wide approaches. Furthermore, a novel mechanism (RESS) has been identified that down-regulates expression of genes encoding native secreted proteins such as cellulases and glycoamylase under secretion stress conditions. These results show that the function of the secretory pathway is interlinked with other cellular processes, and that several feed-back mechanisms may exist that serve to balance between expression, folding, glycosylation and secretion of proteins. It is possible to alter these cellular mechanisms and create improved production strains. For instance, overexpression of HACA increases production of calf chymosin and laccase in A.niger. The EC-funded Eurofung consortium has studied protein secretion in filamentous fungi and some of the results obtained will also be presented. References: Pakula, T.M., Laxell, M., Huuskonen, A., Uusitalo, J., Saloheimo, M. & Penttilä, M. 2003. The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells. J. Biol. Chem. 278, 45011-45020. Saloheimo, M., Valkonen, M. & Penttilä, M. 2003. Activation mechanisms of the HACI-mediated unfolded protein response in filamentous fungi. Mol. Microbiol. 47, 1149 1161. Valkonen, M., Penttilä, M. & Saloheimo, M. 2003. Effects of inactivation and constitutive expression of the unfolded-protein response pathway on protein production in the yeast Saccharomyces cerevisiae. Appl. Env. Microb. 69, 2065-2072. Valkonen, M., Ward, M., Wang, H., Penttilä, M. & Saloheimo, M. 2003. Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response. Appl. Env. Microb. 69, 6979-6986. Mulder, H. J., Saloheimo, M., Penttilä, M. & Madrid, S.M. The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and upregulates the transcription of its own gene. Mol. Gen. Genet., in press
Protein production in filamentous fungi:cellular responses and strain improvement
No full textIn recent years our understanding of cellular responses in Trichoderma and Aspergillus towards secretion load has increased significantly. Production of large amounts of native proteins or production of difficult foreign proteins provokes secretion stress that is believed in particular to be caused by folding limitations within the ER. Impairnment of folding causes the so called unfolded protein response (UPR), which is mediated by the regulatory protein HAC that causes modification of expression of a number of genes involved in various cellular processes related to protein secretion. In addition to the classical UPR, also other cell responses have been discovered using genome-wide approaches. Furthermore, a novel mechanism (RESS) has been identified that down-regulates expression of genes encoding native secreted proteins such as cellulases and glycoamylase under secretion stress conditions. These results show that the function of the secretory pathway is interlinked with other cellular processes, and that several feed-back mechanisms may exist that serve to balance between expression, folding, glycosylation and secretion of proteins. It is possible to alter these cellular mechanisms and create improved production strains. For instance, overexpression of HACA increases production of calf chymosin and laccase in A.niger. The EC-funded Eurofung consortium has studied protein secretion in filamentous fungi and some of the results obtained will also be presented. References: Pakula, T.M., Laxell, M., Huuskonen, A., Uusitalo, J., Saloheimo, M. & Penttilä, M. 2003. The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells. J. Biol. Chem. 278, 45011-45020. Saloheimo, M., Valkonen, M. & Penttilä, M. 2003. Activation mechanisms of the HACI-mediated unfolded protein response in filamentous fungi. Mol. Microbiol. 47, 1149 1161. Valkonen, M., Penttilä, M. & Saloheimo, M. 2003. Effects of inactivation and constitutive expression of the unfolded-protein response pathway on protein production in the yeast Saccharomyces cerevisiae. Appl. Env. Microb. 69, 2065-2072. Valkonen, M., Ward, M., Wang, H., Penttilä, M. & Saloheimo, M. 2003. Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response. Appl. Env. Microb. 69, 6979-6986. Mulder, H. J., Saloheimo, M., Penttilä, M. & Madrid, S.M. The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and upregulates the transcription of its own gene. Mol. Gen. Genet., in press
Integrative approaches for studying pentose metabolism in <i>Saccharomyces cerevisiae</i>
No full textWe are interested in broadening the substrate utilization range of the yeast Saccharomyces cerevisiae and in connection to this to study nutritional responses and signalling at a global level. S.cerevisiae does not naturally utilize pentose sugars unlike most other fungi, thus recombinant S.cerevisiae strains have been constructed that contain the xylose utilization pathway from other yeasts. The recombinant xylose pathway is believed to create a redox imbalance in the cells, due to different cofactor requirements of the enzymes of the pathway, and limit xylose fermentation. In order to understand redox and nutrient regulation in general, and the physiology of xylose utilising S.cerevisiae, we carried out chemostat and batch cultures at different oxygenation levels on xylose as a carbon source, and compared those with glucose cultures. Transcriptional profiling, total proteomics and metabolite analyses were carried out. New responses towards xylose as a carbon source were discovered such as the upregulation of pathways for alternative carbon source utilisation and responses for nutritional control and starvation. The physiology of the recombinant yeast appears to be neither fully repressed (fermentative) nor derepressed (gluconeogenic). References [1] H. Maaheimo, J. Fiaux, Z. P. Cakar, J. E. Bailey, U. Sauer, T. Szyperski. (2001). Eur. J. Biochem. 268, 2464-2479. [2] M. H. Toivari, A. Aristidou, L. Ruohonen, M. Penttilä. (2001). Metab. Eng. 3, 236-249. [3] J.-P. Pitkänen, A. Aristidou, L. Salusjärvi, L. Ruohonen, M. Penttilä. (2003). Metab. Eng. 5, 16-31. [4] L. Salusjärvi, M. Poutanen, J.-P. Pitkänen, H. Koivistoinen, A. Aristidou, N. Kalkkinen, L. Ruohonen, M. Penttilä. (2003). Yeast 20, 295-314. [5] L. Salusjärvi, J.-P. Pitkänen, A. Aristidou, L. Ruohonen, M. Penttilä. (2004). (submitted). [6] J.-P. Pitkänen, E. Rintala, L. Salusjärvi, A. Aristidou, L. Ruohonen, M. Penttilä. (2004). submitted)
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