Geobacillus stearothermophilus NUB3621 as a vector for metabolic engineering Open Access

Blanchard, Kristen (2016)

Permanent URL: https://etd.library.emory.edu/concern/etds/1z40kt564?locale=en
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Abstract

Microbial cells can be coopted to produce desired compounds. Utilizing cells to synthesize products avoids creating racemic mixtures, which can be undesirable when a desired compound behaves differently than its enantiomer. Cellular product synthesis generally requires metabolic engineering of the host strain, or manipulating the cells' metabolic environment to improve production of the target molecule.

Metabolic engineering requires well-studied host systems, and a variety of proteins and pathways from heterologous sources. Although there are many candidates for host strains for metabolic engineering, the genus Geobacillus is especially attractive due to its unique temperature range (39-75 degrees Celsius.) This unique range makes Geobacillus species capable of expressing both mesophilic and thermophilic proteins. Among Geobacillus species, Geobacillus stearothermophilus NUB3621, GsNUB3621, is especially attractive because it is more transformable than other Geobacillus species.

In this work, we seek to improve upon the utility of GsNUB3621 as a host strain for metabolic engineering. To do this, we have sequenced its genome, which should provide insight into GsNUB3621's metabolic network. We have also developed two expression constructs, one inducible and one constitutive, that can be used to express foreign proteins.

These tools help improve the utility of GsNUB3621 as a strain for metabolic engineering. Other Geobacillus strains have already shown use as a vector for ethanol production, and these tools may allow GsNUB3621 to fulfill the same purpose. Because of GsNUB3621's higher transformation efficiency, it may be able to be used for other metabolic engineering purposes less feasible in other Geobacillus strains.

Table of Contents

Table of contents

Chapter 1 General introduction 1

References 8

Chapter 2 Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering 12

Abstract 13

Introduction 14

Materials and methods 16

Results 22

Discussion 27

References 37

Chapter 3

Discussion 43

References 49

Appendix 1 E. coli chromosome evolution 50

References 60

Appendix 2

Catalytic modularity of glutamine synthetase 61

References 68

Figures and tables

Chapter 2 Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering 12 Table 1 Primers used in this study 30 Table 2 Plasmids used in this study 31 Table 3 Contigs in GsNUB3621 draft genome 32 Figure 1 Features identified in GsNUB3621 33 Figure 2 Phylogenetic tree of Geobacillus species 34 Figure 3 Regulatory region of the sucrose utilization operon 35 Figure 4 Alpha galactosidase and sfGFP expression in GsNUB3621 36 Appendix 1 Evolution of the E. coli chromosome 50 Table 1 Mutagenesis results 57 Table 2 Comparison of results with Patrick et al 2007 59 Appendix 2 Catalytic modularity of glutamine synthetase 61 Figure 1 Reactions catalyzed by glutamine synthetase and γ-glutamyl kinase/ glutamate-5-semialdehyde dehydrogenase 66 Figure 2 The E327A mutation rescues ΔproB 66 Figure 3 Growth of ΔproB rescues in low ammonia media 67

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