Supply-demand analysis of anaerobic free-energy metabolism in Saccharomyces cerevisiae

Kroukamp, Marthinus (2003-12)

Thesis (MSc)--Stellenbosch University, 2003.

Thesis

ENGLISH ABSTRACT: Scientists and biochemical engineers alike are very interested in the control and regulation of free-energy metabolism in micro-organisms, whether the findings purely satisfy scientific curiosity or translate into the meeting of biotechnology company deadlines. We used a rather fundamental approach to investigate experimentally the control and regulation of yeast free-energy metabolism in anaerobic chemostat cultures using supply-demand analysis. This conceptually simple, quantitative framework, however, may lead to insight into the control properties of various metabolic pathways to be used in biotechnological applications. Supply-demand analysis is based on the theoretical framework of metabolic control analysis (MCA).Sections (of arbitrary size) of a metabolic pathway are grouped together around a linking metabolite. Those steps that produce the intermediate are combined into the supply block while the reactions that remove/consume the intermediate are grouped together as the demand. The elasticity coefficients of the supply and demand blocks (with regard to the linking metabolite concentration) can be used to determine the flux and concentration control coefficients by using the traditional MCAsummation and connectivity theorems. Supply and demand rate characteristics are a powerful visual approach to determine and display the control structure of the pathway under consideration and sets supply-demand analysis apart from traditional top-down analysis. Our first tool of analysis was a structured kinetic model of yeast growing in a chemos tat, constructed by using methods developed in our research group for modelling systems with variable volumes. Independent perturbations of the linking metabolite concentration resulted in a control profile where the control resided mainly in the demand (flux control coefficient of 0.92), as a result of a large negative supply elasticity. This elasticity, however, varied greatly under different conditions, leading to increased flux control by the supply in some cases. We extended our research to an experimental setup of Saccharomyces cerevisiae growing in a glucose-limited chemos tat supplemented with yeast extract as a source of carbon intermediates. This allowed glucose to act solely as the freeenergy source, as confirmed by balancing the glucose flux with the fluxes towards the fermentation products, ethanol and carbon dioxide. We obtained the supply rate characteristic by perturbing the ATPdemand through the addition of benzoate, which uncouples the proton gradient across the cell membrane. The demand rate characteristic was obtained by perturbing the ATP supply through changes in the dilution rate and thus the residual glucose concentration in the fermentor. The concentrations of ATPand ADPwere measured using a luciferase bioluminescence assay, while the fermentation products were measured with HPLCand C02 with an acoustic off-gas analyser. For our experimental conditions the flux-control of energy metabolism resided predominantly in the supply with respect to the linking metabolite [ATP]/[ADP](chosen as an indication of the free-energy state of the cell), i.e. a flux control coefficient of 0.90. Further, the [ATP]/[ADP]was under strong homeostatic control, as evidenced by the low [ATP]/[ADP]control coefficients of ± 0.12. We adjusted the structured kinetic model by varying strategic parameters, so that the results resembled the experimental observations more closely. However, the kinetics of our core model seem to be too simplistic to capture fully the extent of regulation displayed by the experimental system. The model did, however, reveal the regulatory importance of glucose transport into the cell. We conclude that the control and regulation of free energy metabolism in yeast strongly depend on the culturing conditions and on the steady state being analysed.

AFRIKAANSE OPSOMMING: Wetenskaplikes sowel as biochemiese ingenieurs is dikwels geïnteresseerd in die beheer en regulering van vry-energie metabolisme in mikro-organismes, hetsy die bevindinge suiwer wetenskaplike nuuskierigheid bevredig of die haalbaarheid van biotegnologie-maatskappy-mikpunte beteken. Ons het 'n redelik fundamentele benadering gevolg om die beheer en regulering van vry-energie metabolisme in gis eksperimenteel te bepaal in anaerobiese chemostaatkulture met behulp van aanbod- aanvraag analise. Dit is 'n konseptueel eenvoudige, kwantitatiewe raamwerk met die potensiaal om insig te gee in die beheereienskappe van verskeie metaboliese paaie wat nuttig kan wees in biotegnologiese toepassings. Aanbod-aanvraag analise is gebaseer op die teoretiese onderbou van metaboliese kontrole-analise (MKA).Dele (van arbitrêre grootte) van 'n metaboliese pad word gegroepeer rondom 'n verbindingsmetaboliet. Die stappe wat die intermediaat produseer word gekombineer as die aanbod terwyl die reaksies wat die intermediaat verbruik, saamgegroepeer word as die aanvraag. Die elastisiteitskoëffisiënte van die aanbod en aanvraag blokke (met betrekking tot die verbindingsmetabolietkonsentrasie) kan gebruik word om die fluksie en konsentrasie kontrolekoëffisiënte te bereken met behulp van die sommasie en konnektwiteit teoremas van MKA.Aanbod en aanvraag snelheidskenmerkgrafieke is 'n treffende visuele benadering om die kontroleprofiel van die betrokke metaboliese pad te bepaal en te vertoon. Hierdie kenmerk onderskei aanbod-aanvraag analise van bo-na-onder analise. Die eerste deel van ons ondersoek het behels 'n gestruktureerde kinetiese model (van gis wat groei in 'n chemostaat) met behulp van metodes wat in ons groep ontwikkel is om sisteme met variërende volumes te modelleer. Onafhanklike perturbasies van die verbindingsmetaboliet konsentrasie het gelei tot 'n kontroleprofiel waar die kontrole hoofsaaklik in die aanvraag gesetel was (fluksie kontrolekoëffisiënt van 0.92), as gevolg van 'n groot negatiewe aanbod-elastisiteit. Hierdie elastisiteit kan egter grootliks varieer tydens verskillende kondisies, wat lei tot 'n toenemende fluksle-beheer deur die aanbod in sommige gevalle. Ons het ons navorsing uitgebrei na 'n eksperimentele opstelling van Saccharomyces cerevisiae wat groei in 'n glukose-gelimiteerde chemostaat, aangevul met gisekstrak as 'n bron van koolstof-Intermediate. Dit bring mee dat glukose slegs as energiebron dien; dit is wel bevestig deur balanse op te stel van die koolstoffluksie vanaf glukose na koolstofdioksied en etanol as die fermentasieprodukte. Die aanbod snelheidskenmerkgrafiek is gegenereer deur die aanvraag van ATP te manipuleer deur middel van toevoeging van bensoaat, wat die protongradiënt oor die selmembraan ontkoppel. Die snelheidskenmerkgrafiek Vir die aanvraag is gegenereer deur die aanbod van ATP te manipuleer deur middel van 'n variasie in die verdunningstempo en sodoende die residuele glukose konsentrasie in die fermentor. Die konsentrasies van ATPen ADPis bepaal deur middel van 'n lusiferase bioluminessensie-essai, terwyl die fermentasieprodukte met 'n HPLCen CO2 met 'n akoestiese aflaatgasanaliseerder gemeet is. Vir die betrokke eksperimentele toestande was die flukste-kontrole van energiemetabolisme oorwegend in die aanbod met betrekking tot die verbindingsmetaboliet, [ATP]/[ADP](gekies as aanduiding van die vrye-energiestatus van die sel), naamlik 'n fluksie kontrolekoëffisiënt van 0.90. Verder was die [ATP]/[ADP]onder sterk homeostatiese beheer soos duidelik blyk uit die lae [ATP]j[ADP] kontrolekoëffisiënte van ± 0.12. Ons het die gestruktureerde kinetiese model aangepas deur strategiese parameters te verander om sodoende die eksperimentele gedrag te probeer naboots. Die kinetika van ons kernmodel blyk egter te simplisties te wees om die volle omvang van die regulering van die eksperimentele sisteem te vertoon. Die model het egter die belang van glukose transport oor die selmembraan aan die lig gebring. Ons kom tot die gevolgtrekking dat die beheer en regulering van vrye-energie metabolisme in gis sterk afhang van die groeitoestande sowel as die spesifieke bestendige toestand wat ondersoek word.

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