Μελετήθηκε ο μεταβολισμός της γλυκερόλης στη ζύμη Yarrowia lipolytica ACA–DC 50109 με έμφαση στη μετατροπή της σε λιπίδια και κιτρικό οξύ, μεταβολικά προϊόντα που παρουσιάζουν ιδιαίτερο ενδιαφέρον για τη βιοτεχνολογία. Σε καλλιέργειες που πραγματοποιήθηκαν σε βιοαντιδραστήρα διαλείποντος έργου, επί πολλαπλώς περιοριστικού μέσου, διαπιστώθηκε η ύπαρξη τριών διακριτών φάσεων αύξησης που χαρακτηρίζονται από ιδιαίτερα μορφολογικά και βιοχημικά χαρακτηριστικά: η φάση βιοσύνθεσης κυτταρικής μάζας (κατά την οποία συντέθηκαν 4–4,5 g/l βιομάζας), η ελαιογόνος φάση (κατά την οποία πραγματοποιήθηκε συσσώρευση λιπιδίων 20–22% wt/wt επί ξηρής βιομάζας, 90% wt/wt των οποίων ήταν ουδέτερα) και η φάση παραγωγής κιτρικού οξέος (κατά την οποία εκκρίθηκαν στο περιβάλλον της αύξησης 14–30 g/l κιτρικού οξέος). Κατά τη διάρκεια των ανωτέρω φάσεων η ζύμη διήλθε από διάφορα μορφολογικά στάδια: μικρού μήκους αληθή μυκήλια και ψευδομυκήλια που κυριάρχησαν των κυττάρων ζύμης κατά τη φάση βιοσύνθεσης κυτταρικής μάζας, ευμεγέθη κύτταρα κατά τη φάση της ελαιογένεσης και μικρού μεγέθους κύτταρα ζύμης κατά τη φάση παραγωγής κιτρικού οξέος. Η γλυκερόλη διαπερνά την κυτταροπλασματική μεμβράνη με διευκολυνόμενη διάχυση και καταβολίζεται μέσω των αντιδράσεων της κινάσης της γλυκερόλης – GK και της NAD+ εξαρτώμενης αφυδρογονάσης της 3–P–γλυκερόλης. Την υψηλή ενεργότητα της NAD+ εξαρτώμενης ισοκιτρικής αφυδρογονάσης (NAD+–ICDH) κατά τη διάρκεια της φάσης βιοσύνθεσης κυτταρικής μάζας διαδέχθηκε σημαντική πτώση της ενεργότητάς της, επάγοντας τη λιπογένεση. Απρόσμενη αποδόμηση των αποθεματικών (ουδέτερων) λιπιδίων και σημαντική βιοσύνθεση γλυκολιπιδίων, σφιγγολιπιδίων και φωσφολιπιδίων – Ρ παρατηρήθηκε κατά τη διάρκεια της φάσης παραγωγής κιτρικού οξέος, φάση κατά την οποία η ενεργότητα της GK είχε μειωθεί σημαντικά ενώ η ενεργότητα της NAD+–ICDH είχε σχεδόν μηδενιστεί. Το ελαϊκό οξύ ήταν το κυριότερο λιπαρό οξύ ενώ η φωσφατιδυλχολίνη – PC το κύριο Ρ. Σε συνεχές σύστημα καλλιέργειας επί θρεπτικού υλικού περιοριστικού σε άζωτο, βιοσυντέθηκαν περιορισμένες μόνο ποσότητες λιπιδίων (~10% wt/wt, επί της ξηρής βιομάζας), γεγονός που μπορεί αποδοθεί στο ότι δεν υπήρχε μια περιοχή του ειδικού ρυθμού αραίωσης (D, h–1) στην οποία τα ένζυμα – κλειδιά που εμπλέκονται στη λιπογένεση (όπως η ΑΤΡ:κιτρική λυάση – ATP:CL και το μηλικό ένζυμο – ME) να παρουσιάζουν συγχρόνως υψηλές ενεργότητες, ενώ η ενεργότητα της NAD+–ICDH μειώθηκε, όχι όμως σημαντικά, στους χαμηλούς D. Η ενεργότητα της ATP:CL χαρακτηρίστηκε από υψηλές τιμές (60–300 Units/mg DW) σε D 0,033 h–1 ενώ οι μέγιστες τιμές ενεργότητας του ME (650 Units/mg DW) εμφανίστηκαν σε D=0,104 h–1. Τα λιπίδια της ζύμης ήταν περισσότερο ακόρεστα σε ενδιάμεσες τιμές D. Σε όλους τους D η φωσφατιδυλαιθανολαμίνη – PE, η φωσφατιδυλινοσιτόλη – PI και η PC αντιπροσωπεύουν τις κυριότερες κλάσεις των Ρ. Όσον αφορά τη μορφολογία της ζύμης, βρέθηκε ότι σε D<0,055 h–1 επικρατούσαν αληθή μυκήλια και ψευδομυκήλια ενώ σε D 0,055 h–1 παρατηρήθηκαν μόνο κύτταρα ζύμης. Σε πειράματα που πραγματοποιήθηκαν επί θρεπτικού υλικού περιοριστικού σε άζωτο, σε D=0,026 h–1, σε διαφορετικές συγκεντρώσεις διαλυμένου οξυγόνου – DO παρατηρήθηκε αυξημένο ποσοστό του κλάσματος των Ρ επί των ολικών λιπιδίων στις ακραίες σε τιμές DO ( 70% και 7%). Ανεξάρτητα των τιμών DO η PC ήταν η κλάση με το μεγαλύτερο ποσοστό, ακολουθούμενη από την PI και PE. Ειδικότερα το ποσοστό της ΡΕ παρουσιάστηκε ιδιαίτερα αυξημένο σε ενδιάμεσες τιμές DO (20% και 30%). Σε DΟ 50% επικρατούσαν αληθή μυκήλια και ψευδομυκήλια ενώ σε DΟ 50% εμφανίστηκαν στην καλλιέργεια περισσότερα κύτταρα ζύμης. Σε πειράματα που πραγματοποιήθηκαν σε D=0,026 h–1 βρέθηκε ότι ο περιορισμός της αύξησης από ιχνοστοιχεία όπως το μαγνήσιο και το ασβέστιο τα οποία εμπλέκονται σε πολλαπλές κυτταρικές λειτουργίες, είχαν δυσμενή επίδραση στη φυσιολογία της ζύμης, ωστόσο η σύσταση των λιπιδίων σε λιπαρά οξέα δεν επηρεάστηκε από τη φύση του περιοριστικού για την αύξηση παράγοντα. Η παρούσα διδακτορική διατριβή φιλοδοξεί να συμβάλει στη μελέτη της φυσιολογίας των ελαιογόνων μικροοργανισμών και στη χρήση της γλυκερόλης ως υποστρώματος σε μελλοντικές βιοτεχνολογικές εφαρμογές. / In this thesis the metabolism of glycerol in Yarrowia lipolytica ACA–DC 50109, with emphasis on glycerol conversion into value–added biotechnological products, such as single cell oils and citric acid, was studied. The growth of Y. lipolytica was studied in bioreactor batch cultures in multiple limited medium and three distinct phases were identified during growth cycle. In each phase, yeast cells were characterized by specific morphological and biochemical features: biomass formation phase (in which 4–4.5 g/l of biomass were synthesized), lipogenic phase (in which 20–22% lipids wt/wt in dry weight were accumulated in biomass, containing 90% wt/wt neutral lipids) and citric acid production phase (in which 14–30 g/l of citric acid were secreted in the growth environment). Distinct cellular forms of Y. lipolytica were developed during the above phases: in biomass formation phase short true mycelia and pseudo–mycelia were predominant while a few yeast–like cells were observed, in lipogenic phase large obese cells were predominant and in citric acid production phase cells size was diminished. Glycerol passes into the microbial cell by facilitated diffusion. Y. lipolytica successfully converts glycerol via phosphorylation pathway, in which glycerol kinase (GK) and glycerol–3–P–dehydrogenase are implicated. Though high activity of NAD+ dependent isocitric dehydrogenase (NAD+–ICDH) was detected during biomass formation phase, this activity was significantly decreased afterwards inducing lipogenesis. Surprisingly, storage (neutral) lipid turnover and synthesis of glycolipids, sphingolipids and phospholipids – Ρ simultaneously occurred with citric acid production, and happened when GK activity was considerably reduced and NAD+–ICDH activity was minimised. Oleic acid was the major fatty acid in all lipid fractions and phosphatidylcholine – PC was the main Ρ. In continuous culture in nitrogen limited medium Y. lipolytica accumulated low quantities of lipids (~10% w/w, in dry weight), maybe due to the fact that there was not a region of specific dilution rate (D, h–1) in which the key–enzymes that are implicated in lipogenesis (i.e. ΑΤΡ:citrate lyase – ATP:CL and malic enzyme – ME) presented simultaneously high activity while NAD+–ICDH activity was insignificantly decreased in low D. ATP:CL presented high activity (60–300 Units/mg DW) in D 0,033 h–1 while ME presented maximum activity (650 Units/mg DW) in D=0,104 h–1. Lipids were more unsaturated in intermediate D values while phosphatidylethanolamine – PE, phosphatidylinositol – PI and PC are the main Ρ classes. As far as the morphology is concerned, in D<0,055 h–1 short true mycelia and pseudo–mycelia were predominant in culture medium while in D 0,055 h–1 only yeast cells were observed. In experiments performed in nitrogen limited medium in D=0,026 h–1 in different dissolved oxygen – DO concentrations, it was found that in extreme DO values ( 70% and 7%) the percentage of P was increased. Independently the DO concentration PC was the main class followed by PI and PE. The morphology of Y. lipolytica was influenced by the different concentration of DO and it was observed that in DΟ 50% short true mycelia and pseudo–mycelia were predominant in culture medium while in DΟ 50% more yeast cells were appeared. In experiments performed in D=0,026 h–1, it was found that the absence of micronutrients from the growth medium, i.e. magnesium and calcium that are implicated in multiple cellular functions, had severe effects in yeast physiology, while the fatty acid composition of cellular lipids was not affected by the nature of the growth limiting factor. The present thesis aspires to contribute in the study of oleaginous microorganisms’ physiology and in use of glycerol as substrate in future biotechnological applications.

Made available in DSpace on 2016-06-02T19:56:48Z (GMT). No. of bitstreams: 1 4595.pdf: 1437433 bytes, checksum: f83e0ea8c49064050b3f382c7d942d28 (MD5) Previous issue date: 2012-08-27 / Financiadora de Estudos e Projetos / Diseases caused by Streptococcus pneumoniae are one of the main problems of public health in the world. The pneumococcal surface protein A(PspA) is a potential canditate as carrier in a conjugate vaccine against this bacteria. Considering the inherent high losses of the purification and conjugation steps, it is fundamental to adopt a strategy of cultivation and expression that allows the obtainance of large quantities of protein. Thus, the use of Escherichia coli as expression system as well as its cultivation in complex medium constitutes promising alternatives for reducing the cost and increasing the productivity of the process. The goal of this work was to study the influence of the temperature and cultivation medium composition over the production of a PspA belonging to clade 4 protein fragment (PspA4Pro) during rE coli cultivations, aiming at to evaluate the possibility of employing vegetable-based nitrogen sources (soybean protein hydrolisates) instead of the Triptona, an animal-derived nitrogen source. The experiments were carried out in both shakers and benchscale bioreactor, using a complex medium which contained glucose and glycerol as carbon sources, lactose as inducer and Soytone, Phytone or Triptone as nitrogen sources, besides yeast extract. Samples were collected during the experiments to follow the cell growth (measurements of absorbance, dry cell weight and permittivity signal from biomass sensors), the carbon sources consumption and the production of organic acids by HPLC analysis. The stability of the plasmid (agar plates with or without kanamycin) and the production of recombinant protein (Bradford and SDS-PAGE electrophoresis followed by densitometry) were also evaluated. Preliminary experiments were performed in shake flasks, incubated at 300rpm and 37ºC, employing both complex and defined media. The highest productivity was achieved in complex medium, with a 42% superior protein production. Subsequently, nine complementary experiments were conducted in shake flasks with complex medium, under the agitation of 300rpm and temperatures of 37ºC (growth phase) and 25, 31 or 37ºC (induction phase). The largest specific production of soluble PspA4Pro was verified at 25ºC, reaching, respectively, 209±6, 192±5mg/g dry cell mass for Phytone and Triptone, with final absorbance values (after 12h of induction) of 9.0±0.4 and 8.5±0.4. The best protein production for Soytone (124±4mg/g dry cell weight) was observed at 31ºC, yielding a final absorbance 8.0±0.4. From the results obtained in the preliminary tests, the nitrogen source Phytone was selected for experiments in bioreactor. Four batch cultures were conducted in bench-scale bioreactor (5L), containing a modified auto-induction complex medium (10g/L glucose, 60g/L glycerol and 20g/L lactose), being three of them with Phytone and one with Triptone, for comparison. The best results in terms of protein production (245±7mg of PspA4Pro soluble/g dry mass) were obtained in the presence of Phytone, corresponding to an increase of 16% towards the maximum value achieved in the cultivation with Triptone. These results demonstrate the potential of vegetable-based nutrients as alternatives to animal-derived nitrogen sources in complex media, contributing to adequate these media formulations to the current guidelines of good manufacturing practices. / Doenças causadas por Streptococcus pneumoniae constituem um dos principais problemas de saúde pública mundial. A proteína A de superfície de pneumococo (PspA) é candidata em potencial a ser carreadora em vacina conjugada contra essa bactéria. Considerando as altas perdas inerentes às etapas de purificação e conjugação da proteína, é fundamental adotar uma estratégia de cultivo e expressão que permita obter grandes quantidades de proteína. Nesse sentido, o emprego da bactéria Escherichia coli como sistema de expressão e o cultivo da mesma em meio complexo se apresentam como alternativas promissoras para redução do custo e aumento da produtividade do processo. O objetivo do presente trabalho foi estudar a influência da temperatura e da composição do meio de cultivo sobre a produção do fragmento da proteína PspA do clado 4 (PspA4Pro) em cultivos de rE. coli, visando avaliar a viabilidade de utilização de fontes de nitrogênio de origem vegetal (hidrolisados protéicos de soja) em substituição à Triptona, de origem animal. Os experimentos foram realizados em câmara incubadora e em biorreatores de bancada, utilizando meio complexo contendo glicose e glicerol e lactose como fontes de carbono, lactose como indutor e Soytone, Phytone ou Triptona como fontes de nitrogênio, além de extrato de levedura. Amostras foram coletadas ao longo dos experimentos para acompanhamento do crescimento celular (medida de absorbância, massa seca e permissividade por sensor de biomassa), do consumo das fontes de carbono e da produção de ácidos orgânicos por análises em cromatografia líquida de alto desempenho. A estabilidade do plasmídeo (plaqueamento em meio contendo ou não canamicina) e a produção de proteína recombinante (Bradford e eletroforese SDS-PAGE seguida por densitometria) também foram avaliadas. Experimentos preliminares foram realizados em frascos agitados e incubados a 300rpm e 37oC, empregando tanto o meio complexo como o definido. A maior produtividade foi obtida em meio complexo, a qual foi 42% superior a alcançada com meio definido. Em seguida, nove experimentos complementares foram conduzidos em frascos agitados em meio complexo sob agitação de 300rpm e à temperatura de 37ºC (fase de crescimento) e de 25, 31 ou 37ºC (fase de indução). Verificou-se que a temperatura de 25ºC proporcionou a maior produção específica de PspA4Pro solúvel, alcançando-se, respectivamente, 209±6, 192±5mg/g massa seca para o Phytone e para a Triptona, com absorbâncias finais (após 12h de indução) de 9,0±0,4 e 8,5±0,4. Já para o Soytone, a melhor produção de proteína (124±4mg/g massa seca) foi observada à temperatura de 31ºC, obtendo-se uma absorbância de final de 8,0±0,4. A partir dos resultados obtidos nos ensaios preliminares, a fonte de nitrogênio de origem vegetal Phytone foi selecionada para experimentos em biorreator. Quatro cultivos em batelada foram conduzidos em biorreator de bancada (5L), contendo meio complexo de autoindução modificado (10g/L glicose, 60g/L glicerol e 20g/L lactose), sendo 3 com Phytone e um com Triptona, para comparação. Os melhores resultados em termos de produção de proteína (245±7mg de PspA4Pro solúvel/g massa seca) foram obtidos na presença de Phytone, correspondendo a um aumento de 16% em relação ao valor máximo alcançado no cultivo com Triptona. Esses resultados comprovam o potencial dos nutrientes de origem vegetal como alternativa às fontes de nitrogênio de origem animal em meios complexos, contribuindo para adequar as formulações desses meios às atuais diretrizes de boas práticas de fabricação.