Genetic and metabolic engineering of acetogenic chassis by implementing novel genetic tools and metabolic pathways
Creators: name: Bourgade, Barbara
Publisher: name: Loughborough University
Year: 2022
Subjects: classificationCode: 400499
subject: Chemical engineering not elsewhere classified

description: The modern industry, currently relying on petroleum resources, must reinvent itself to decrease its environmental impacts and face the imminent shortage of fossil fuels. In this challenging context, recent efforts have focused on creating and improving sustainable alternative production processes, such as bioprocesses. Microbial hosts offer a promising opportunity for the efficient production of fuels and chemicals from a range of substrates, such as complex carbohydrates or waste compounds. More recently, acetogenic bacteria have regained interest as they can grow autotrophically, using CO<sub>2</sub>, H<sub>2</sub> or CO, i.e. problematic greenhouse gases, as their sole carbon and energy substrates. During this gas fermentation process, acetogens metabolise C1-gases into acetate, ethanol and species-specific products using the Wood-Ljungdahl pathway for carbon fixation. Although acetogenic metabolic constraints, such as ATP availability, may limit their metabolic flexibility and the engineering strategies applicable, our understanding of the acetogenic metabolism, especially cofactor balancing, has greatly improved in the past decade, allowing us to exploit their abilities for industrial applications in a more efficient manner. In addition, the modern era of genetics has further contributed to improving acetogens through genetic and metabolic engineering. Indeed, recently developed genetic tools have led to the manipulation of metabolic fluxes and production of non-native compounds in several acetogens. Therefore, acetogens stand as key chassis organisms for the sustainable production of fuels and chemicals.Although genetic tools have been adapted for several acetogens, a discrepancy of genetic accessibility persists amongst acetogens. For example, the thermophilic acetogen Moorella thermoacetica has proven challenging to genetically modify as tools available remain limited. M. thermoacetica has previously successfully been transformed but low reported efficiency prevents many genetic applications. These suboptimal results are disappointing as M. thermoacetica thermophilic characteristics are highly beneficial in an industrial context. However, for its wider use in an industrial setting, it must be amenable to genetic manipulations to shape strains to industrial demands and better understand its metabolism. Thus, this insufficient genetic toolkit has motivated this research project to focus on improving transformation efficiency for M. thermoacetica. To do so, a shuttle vector, with a compatible Gram-positive replicon, was created and allowed plasmid maintenance in the population, although further characterisation of the plasmid proved difficult. Nonetheless, the engineered plasmid was later used for other genetic applications, notably introduction of ethanol production in M. thermoacetica. While additional work is still needed to improve these tools, the improvements described here undeniably support other genetic and metabolic engineering strategies to fully unlock M. thermoacetica’s industrial potential.In contrast to M. thermoacetica, the mesophilic acetogen Clostridium autoethanogenum has arguably the most extensive genetic toolkit available amongst acetogens. In fact, it has been manipulated for the production of non-native compounds and even implemented in industrial bioprocesses, promoting C. autoethanogenum as the current workhorse of industrial gas fermentation. Non-native production has relied on expressing naturally occurring metabolic pathways from other microorganisms, such as Clostridium acetobutylicum. However, the rise of computational tools in genetics and metabolomics has led to the creation of novel software for the design and analysis of fully synthetic metabolic pathways, expanding the catalogue of bioproducts to virtually any desired compound, although target numbers might be constrained by host-specific energy requirements, such as limited ATP availability in acetogens during autotrophic growth. Even though experimental implementation comes with many challenges, this strategy has shown success in several model organisms, such as Escherichia coli. This progress has further encouraged to adopt this approach in more complex host organisms, such as acetogens. Thus, the second part of this research project aimed at designing and analysing synthetic pathways for a few value-added chemicals and explore a potential implementation in C. autoethanogenum as described in this thesis. Although several obstacles prevented a straight-forward pathway implementation, a C. autoethanogenum strain was engineered in this work for production of the platform chemical ethylene glycol using a fully synthetic metabolic pathway. However, the other candidate pathway tested in this work could not be successfully expressed, further highlighting the remaining gap between computational analysis and experimental work.