Supply-demand analysis of anaerobic free-energy metabolism in Zymomonas mobilis

Crous, Christiaan (2011-12)

Thesis (MSc)--Stellenbosch University, 2011.

Thesis

ENGLISH ABSTRACT: Fermentation in Zymomonas mobilis has been described as a catabolic highway, with 50 % of soluble protein comprising glycolytic and fermentative enzymes. In conjunction with one of the fastest observed fermentations, the conversion of glucose to ethanol forms one of the least efficient energy extractions found in nature. The low energy yield of fermentation in Z. mobilis is a result of the usage of the Entner-Doudoroff glycolytic pathway, which has half the energy yield per mol substrate compared to the well known Embden-Meyerhof-Parnas glycolytic pathway. The work presented in this thesis forms part of a larger project to compare glycolytic regulation in different micro-organisms (i.e., Z. mobilis, Escherichia coli, Saccharomyces cerevisiae and Lactococcus lactis). These organisms were chosen based on their usage of different glycolytic mechanisms. By using supply-demand analysis for quantifying glycolytic regulation as well as similar experimental conditions (e.g. using non-growing cell cultures), we can compare the regulatory behaviour of mechanistically distinct freeenergy supplies. The aim of this thesis was to quantify the importance of anaerobic free-energy generation for the regulation of the Entner-Doudoroff glycolytic pathway in Z. mobilis. We used metabolic control analysis (MCA) and supply-demand analysis to realize this goal. The central message of MCA is that when a metabolic parameter (e.g., a conserved metabolic moiety) is deemed important for affecting a particular steady-state variable (i.e., fermentation flux), its effect on the steady state variable should be tested. An extension to MCA, supply-demand analysis, provides a quantitative framework for analyzing the regulatory importance of cellular commodities such as anaerobic free-energy. This is done through comparing the elasticities of anaerobic free-energy supply and demand, which yields the degree to which the respective reaction blocks control the flux through anaerobic free-energy metabolism, as well as determine the cellular free-energy state (ATP/ADP ratio). The regulation of anaerobic free-energy metabolism in Z. mobilis was investigated with an experimental approach. The key features of our experimental setup were the use of NMR spectroscopy for detecting metabolites, as well as employing non-growing conditions for supply-demand experiments. With NMR spectroscopy metabolites could be detected in real time without using invasive sampling techniques; the use of nongrowing conditions further simplified the analysis by enabling us to correlate fermentative behaviour exclusively with the anaerobic free-energy state. Fermentation of glucose was investigated in the wild type Z. mobilis, a recombinant containing a non-expressing plasmid, or expressing plasmids for over-expressing the glucose facilitator (TCDB 2.A.1.1.4) or glucose-6-phosphate dehydrogenase (EC 1.1.1.49). In addition, ATP demand in the non-expressing recombinant and wild type was perturbed by titrating with the uncoupler acetic acid. Our results show that the anaerobic free-energy demand, the glucose facilitator and glucose-6-phospate dehydrogenase all control the flux of ethanol production in Z. mobilis. The Entner-Doudoroff glycolytic supply activity was found to be sensitive to changes in the ratios of ATP/ADP (elasticity varied between –0.31 and –0.49) and NTP/NDP (elasticity varied between –0.31 and – 0.50).

AFRIKAANSE OPSOMMING: Fermentasie in Zymomonas mobilis word beskryf as ‘n kataboliese snelweg, waar glikolitiese en fermentatiewe ensieme 50% van totale oplosbare proteïene in die sel uitmaak. Hoewel dié fermentasie een van die vinnigstes is wat tot op hede waargeneem is, is die omskakeling van glukose na etanol een van die mees ondoeltreffende energieekstraksies in die natuur. Dié lae energie-opbrengs, soos waarneembaar in fermentasie in Zymomonas mobilis, kan toegeskryf word aan die Entner-Doudoroff metaboliese pad. Hierdie metaboliese pad lewer slegs die helfte van die energie-opbrengs per mol substraat vergeleke met die meer bekende Embden-Meyerhof-Parnas glikolitiese pad. Die navorsing in hierdie tesis is deel van ‘n omvattende projek wat poog om die regulering van glikolise in verskillende mikro-organismes (Z. mobilis, Escherichia coli, Saccharomyces cerevisiae en Lactococcus lactis) te vergelyk. Dié organismes is gekies op grond van die uiteenlopende glikolitiese meganismes waarvan hulle gebruik maak. Ten einde die reguleringsgedrag van meganisties verskillende vry-energie produksieweë m.b.v. vraag-aanbod analise te vergelyk, moet glikolitiese regulering eers onder eenderse eksperimentele kondisies (b.v. nie-groeiende selkulture) gekwantifiseer kan word. Die hoofdoel van hierdie tesis was om die belang van anaerobiese vry-energie produksie vir die regulering van die Entner-Doudoroff glikolitiese pad in Z. mobilis te kwantifiseer. Hiervoor is van Metaboliese kontrole-analise (MKA) en vraag-aanbodanalise (‘n uitbreiding van MKA) gebruik gemaak. MKA is ‘n tegniek waarmee die effek wat ‘n metaboliese parameter (soos metaboliese deel-konservering) op ‘n spesifieke bestendige toestand-veranderlike (soos fermentasiefluksie) het, gekwantifiseer kan word. Vraagaanbodanalise daarenteen, bied ‘n kwantitatiewe raamwerk waardeur die regulatoriese belang van sellulêre kommoditeite (byvoorbeeld anaerobiese vry-energie) geanaliseer kan word. Tydens laasgenoemde proses word die elastisiteit van die anaerobiese vry-energie aanbod en die elastisiteit van die vraag vergelyk. Op hierdie manier kan die mate van beheer wat die onderskeie reaksieblokkie oor die fluksie deur anaerobiese vry-energie metaboliese paaie, sowel as oor die sellulêre vry-energie toestand (ATP/ADP verhouding), bepaal word. In hierdie werk is die regulering van anaerobiese vry-energie metabolisme in Z. mobilis ondersoek deur van ‘n eksperimentele benadering gebruik te maak. Die sleuteleienskappe van dié benadering was om kernmagnetiese-resonansiespektroskopie (KMR spektroskopie) te gebruik om metabolietkonsentrasies te meet, en om van niegroeiende kondisies gebruik te maak vir die vraag-aanbod eksperimente. Metabolietkonsenstrasies kon aaneenlopend bepaal word sonder die gebruik van monsternemingstegnieke wat die reaksie sou kon beïnvloed. Eksterne invloede op die fermentasiegedrag kon ook uitgesluit word deur van nie-groeiende kondisies gebruik te maak, sodat die waargenome fermentasiegedrag uitsluitelik aan die anaerobiese vryenergie toestand toegeskryf kan word. Glukose fermentasie was ondersoek in wilde tipe Z. mobilis, en in drie rekombinante wat onderskeidelik ‘n glukose fasiliteerder ooruitdrukkingsplasmied (TCDB 2.A.1.1.4), ‘n glukose-6-fosfaat dehidrogenase ooruitdrukkingsplasmied (EC 1.1.1.49), en ‘n nieuitdrukkingsplasmied bevat het. Die ATP vraag in die wilde tipe en die nieuitdrukkingsrekombinant is geperturbeer deur titrasies met asynsuur as ontkoppelaar. Die resultate toon dan die anaerobiese vry-energievraag, sowel as die glukose fasiliteerder en glukose-6-fosfaat dehidrogenase, die fluksie van etanolproduksie in Z. mobilis beheer. Die Entner-Doudoroff glikolitiese produksie-aktiwiteit was sensitief vir veranderinge in die ATP/ADP verhouding (elastisiteite was tussen -0.31 en -0.49) en die NTP/NDP verhouding (elastisiteite was tussen -0.31 en -0.50).

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