Development of synthetic biology tools for Kluyveromyces marxianus and their application to produce aromatic molecules in this yeast

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Akinola, Joel Abidemi
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University College Cork
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The experimental work in this thesis was carried out as part of the European Union-funded Horizon 2020 project CHASSY ( CHASSY was a collaborative research project made up of industry and academic partners who set out to unlock the potential of yeasts as microbial cell factories for producing valuable compounds. While Saccharomyces cerevisiae is the most studied yeast for which there are many genetic engineering tools available and is thus commonly used for synthetic biology (SynBio) applications, it has certain limitations for specific applications. This has in recent times led to the exploration of the industrially attractive non-saccharomyces yeast, Kluyveromyces marxianus for various synthetic biology applications. This yeast has a QPS/GRAS status that drives its application in the food industry, it is thermotolerant, able to metabolise and grow on several feedstocks and SynBio tools for achieving heterologous gene expression and genome engineering by CRISPR technologies have been developed for it. Those recent advancements have contributed to research on this yeast in regard to improving our understanding of its physiology and for SynBio applications. To further improve genome editing tools for the yeast, a counter-selectable amidase (amdS) gene from bacteria was developed in this thesis for marker-dependent integration. Close to 100% of the transformants obtained had an amdS marker cassette integrated when the cassette was transformed. A CRISPR-based marker-free system was also developed and optimised for simultaneous editing of more than one locus in the yeast’s genome. This system is highly useful because it eliminates genetic engineering restrictions that arise as a result of the need for markers, and it was made possible thanks to the yeast’s homologous recombination machinery which allows homology directed repair (HDR). Thus, the system was optimised by improving HDR through the inactivation of non-homologous end-joining, the expression of recombinases and chemical synchronisation of yeast cells into the S/G2 phase of cell cycle. The optimisation allowed efficient marker-free deletion of specific genes, integration at specific loci and multi-loci integration at repeat sequences that are distributed around the yeast’s genome. The developed tools in this work are entirely based on the well-established yeast toolkit standard which uses Golden Gate cloning technique for DNA assembly, making it possible to use them in combination with pre-existing tools and they are transferable between SynBio applications. Therefore, the tools were used for unprecedented engineering of K. marxianus for heterologous production of a group of plant aromatic compounds called phenylpropanoids. The biosynthesis of these compounds takes the aromatic amino acids (AAA), phenylalanine, and tyrosine as precursors. Therefore, the key enzymes of the shikimate pathway which is responsible for AAA biosynthesis and the glycolytic and pentose phosphate pathways which supply phosphoenolpyruvate and erythrose 4-phosphate as precursors of the shikimate pathway were overexpressed in order to increase AAA production. The phenylpropanoid pathway begins with the conversion of tyrosine to coumaric acid by means of a single step reaction catalysed by tyrosine ammonia lyase or phenylalanine in two reaction steps where phenylalanine ammonia lyase first converts phenylalanine to cinnamic acid and then cinnamic acid 4-hydroxylase and cytochrome P450 reductase convert cinnamic acid into coumaric acid. Given that coumaric acid is an important phenylpropanoid from which many other phenylpropanoids are produced, its production was first engineered into K. marxianus in with the expectation that it will be possible to further engineer coumaric acid producers to produce more complex phenylpropanoids. This work resulted in the production of coumaric acid at a yield of over 40mg/g glucose in 5L batch bioreactors with 2.5L working volumes of minimal glucose medium. Subsequently, plant genes that catalyse the derivatisation of coumaric acid into an important flavonoid called naringenin (coumaric acid:coenzyme A ligase, chalcone synthase and chalcone isomerase) were expressed in a coumaric acid producer. This however, only produced very low amounts of naringenin with minor decrease in coumaric acid yield, giving rise to a study in which the depletion of coumaric acid was the objective. Since the derivatisation requires coenzyme A and malonyl-CoA, increased biosynthesis of both metabolites was engineered by overexpressing acetyl-CoA carboxylase, acetyl-CoA synthase, and the entire coenzyme A biosynthetic pathway, leading to naringenin yield of 1231µg/g glucose. Due to the fact that freely available intracellular AAAs are catabolised by the Ehrlich pathway in yeasts, a study was conducted to identify the genes that contribute K. marxianus phenylpyruvate decarboxylase activity, a key player that is responsible for driving flux through the pathway. That study revealed that ARO10 contributed most of the enzyme activity with some coming from PDC1 and provided information that enabled the reduction of the activity so that AAAs are available to enter the heterologous phenylpropanoid pathway. Together, this work demonstrates the potential of K. marxianus as a cell factory for producing phenylpropanoids, bringing us closer to the production of these compounds from diverse feedstocks.
Synthetic biology , Aromatics , Yeasts , Kluyveromyces marxianus , Metabolic engineering , CRISPR , Genome editing , Genetic engineering
Akinola, J. A. 2023. Development of synthetic biology tools for Kluyveromyces marxianus and their application to produce aromatic molecules in this yeast. PhD Thesis, University College Cork.
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