Application of metabolic flux and transcript analyses to understanding the physiology of engineered Geobacillus thermoglucosidasius

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Title: Application of metabolic flux and transcript analyses to understanding the physiology of engineered Geobacillus thermoglucosidasius
Authors: Ward, Charlotte
Item Type: Thesis or dissertation
Abstract: Geobacillus thermoglucosidasius has been identified as an organism capable of producing bioethanol from lignocellulosic biomass based on its ability to ferment both hexose and pentose sugars. Engineering of the wild-type strain DL33 (wt) has produced a single knock out strain DL44 (∆ldh) and a double knock out strain DL66 (∆ldh∆pfl↑pdh), both of which have increased capacity for bioethanol production. The nutritional requirements of the strains under anaerobic conditions are yet to be fully understood. In this study, a systems approach to understanding the metabolism of the wild-type and engineered strains has been taken in order to further understand the changes in metabolism resulting from the mutations introduced. For the first time 13C-metabolic flux analysis has been applied to the comparative study of the wild-type and engineered strains using global isotopomer balancing. This has revealed flux through the anaplerotic reactions has reversed from being in the direction of pyruvate / phosphoenolpyruvate in the wild-type, to being in the direction of oxaloacetate / malate in the engineered strains. Alterations in TCA cycle flux between the strains were also seen. Furthermore alanine was found to be produced as a fermentation product in each strain. Analysis of the genome sequence has revealed an unusual oxidative branch of the pentose phosphate pathway, missing 6-phosphogluconolactonase but with genes encoding the rest of the pathway still present, suggesting that flux through this pathway may still proceed, dependent on the themolability of glucono-1,5-lactone-6-phosphate. It has been found that RNA extracted from G. thermoglucosidasius is prone to rapid degradation which may affect the outcome of analysis of the transcriptome by RNA-seq. Nonetheless, it has been possible to apply RNA-seq to the wild-type organism grown aerobically and use this to identify transcripts for the major pathways of central carbon metabolism and the most highly expressed transcripts of the culture.
Content Version: Open Access
Issue Date: Oct-2014
Date Awarded: May-2015
Supervisor: Leak, David
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (Great Britain)
TMO Renewables Ltd
Department: Life Sciences
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Life Sciences PhD theses

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