Application of metabolic flux and transcript analyses to understanding the physiology of engineered Geobacillus thermoglucosidasius
File(s)
Author(s)
Ward, Charlotte
Type
Thesis
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.
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.
Version
Open Access
Date Issued
2014-10
Date Awarded
2015-05
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Leak, David
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
TMO Renewables Ltd
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)