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

[摘要] ENGLISH ABSTRACT: Fermentation in Zymomonas mobilis has been described as a catabolic highway, with50 % of soluble protein comprising glycolytic and fermentative enzymes. In conjunctionwith one of the fastest observed fermentations, the conversion of glucose to ethanol formsone of the least efficient energy extractions found in nature. The low energy yield offermentation in Z. mobilis is a result of the usage of the Entner-Doudoroff glycolyticpathway, which has half the energy yield per mol substrate compared to the well knownEmbden-Meyerhof-Parnas glycolytic pathway.The work presented in this thesis forms part of a larger project to compare glycolyticregulation in different micro-organisms (i.e., Z. mobilis, Escherichia coli, Saccharomycescerevisiae and Lactococcus lactis). These organisms were chosen based on their usage ofdifferent glycolytic mechanisms. By using supply-demand analysis for quantifyingglycolytic regulation as well as similar experimental conditions (e.g. using non-growingcell cultures), we can compare the regulatory behaviour of mechanistically distinct freeenergysupplies.The aim of this thesis was to quantify the importance of anaerobic free-energy generationfor the regulation of the Entner-Doudoroff glycolytic pathway in Z. mobilis. We usedmetabolic control analysis (MCA) and supply-demand analysis to realize this goal. Thecentral message of MCA is that when a metabolic parameter (e.g., a conserved metabolicmoiety) 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 toMCA, supply-demand analysis, provides a quantitative framework for analyzing theregulatory importance of cellular commodities such as anaerobic free-energy. This isdone through comparing the elasticities of anaerobic free-energy supply and demand,which yields the degree to which the respective reaction blocks control the flux throughanaerobic 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 withan experimental approach. The key features of our experimental setup were the use ofNMR spectroscopy for detecting metabolites, as well as employing non-growingconditions for supply-demand experiments. With NMR spectroscopy metabolites couldbe detected in real time without using invasive sampling techniques; the use of nongrowingconditions further simplified the analysis by enabling us to correlatefermentative behaviour exclusively with the anaerobic free-energy state.Fermentation of glucose was investigated in the wild type Z. mobilis, a recombinantcontaining a non-expressing plasmid, or expressing plasmids for over-expressing theglucose facilitator (TCDB 2.A.1.1.4) or glucose-6-phosphate dehydrogenase (EC1.1.1.49). In addition, ATP demand in the non-expressing recombinant and wild type wasperturbed by titrating with the uncoupler acetic acid. Our results show that the anaerobicfree-energy demand, the glucose facilitator and glucose-6-phospate dehydrogenase allcontrol the flux of ethanol production in Z. mobilis. The Entner-Doudoroff glycolyticsupply activity was found to be sensitive to changes in the ratios of ATP/ADP (elasticityvaried between –0.31 and –0.49) and NTP/NDP (elasticity varied between –0.31 and –0.50).

[发布机构] Stellenbosch University