Thesis (MSc)--Stellenbosch University, 2011. / 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).
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/18073 |
| Date | 12 1900 |
| Creators | Crous, Christiaan |
| Contributors | Rohwer, J. M., Snoep, J. L., Stellenbosch University. Faculty of Science. Dept. of Biochemistry. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Rights | Stellenbosch University |
Page generated in 0.0678 seconds