Microbiology - Doctoral Theses

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    Marine bacteria as a source of polyester-degrading enzymes with biocatalytic potential
    (University College Cork, 2023) Carr, Clodagh M.; Dobson, Alan; Clarke, David J.; Science Foundation Ireland; Synthesis and Solid State Pharmaceutical Centre
    The issue of plastic waste continues to surge, as does our collective awareness of this global problem and interest in finding sustainable solutions for its control. Bacteria, which are among the earliest known forms of life, have evolved over millions of years to degrade organic matter found in the environment by producing enzymes that can catalyse the breakdown of various compounds for energy and nutrient gain. With biotechnological advances in recent decades, bacterial enzymes have emerged as a tool for the catalysis of chemical reactions, where they can aid in the development of safer, more efficient, and more environmentally friendly processes. The conventional recycling of plastic waste has typically consisted of a thermo-mechanical process, where waste is ground down, melted, and reformed into new, but lower-quality products that are less likely to be recycled multiple times. While alternative chemical methods can improve recyclability by facilitating recovery of the raw materials used to make plastic, enzyme-based treatments enable the same process to be conducted under milder, less energy-intensive reaction conditions without the use of hazardous reagents and solvents. Enzymes that are specialized to break apart ester bond-containing compounds in nature can be employed for the degradation of polyester-based plastics, allowing for the sustainable recycling of these materials after use. Polyester-degrading enzymes (termed polyesterases) have been studied with a particular focus on the recycling of polyethylene terephthalate (PET), a synthetic polyester which is mass-produced for use in food and beverage packaging or as a fiber in the textile industry. By an enzymatic hydrolysis mechanism, the PET polymer may be degraded into its short-chain oligomeric intermediates MHET and BHET and/or its constituent monomers ethylene glycol (EG) and terephthalic acid (TPA) which can subsequently be recycled into PET or upcycled into value-added products. In chapter 1, a literature review was conducted on microbial PET hydrolase enzymes to assess existing knowledge in the field, identify key challenges, and determine important areas for future research. Thermophilic bacteria from the phylum Actinomycetota such as Thermobifida fusca emerged as a major source of PET-hydrolyzing enzymes as well as those with fungal and metagenomic origins, while Ideonella sakaiensis, a mesophilic bacterium isolated from a PET-contaminated site served as a model system featuring both PET and MHET hydrolases predicted to work in tandem. Relatively few PET-hydrolyzing enzymes were reported from marine environments, an aspect which we hoped to expand upon. In chapter 2, activity screening and genome mining of marine bacterial isolates led to the identification of a polyesterase, BgP, from a deep-sea, marine sponge-derived Brachybacterium sp. isolate. BgP was explored as a structural homolog of cutinase-like enzymes, such as Cut190, LCC, and TfCut2, which had previously been reported for efficient hydrolysis of PET and its hydrolytic activity was confirmed on the PET model substrate polycaprolactone (PCL). In chapter 3, PET-hydrolyzing activity was confirmed for SM14est, a marine sponge-derived polyesterase from Streptomyces sp. SM14 with this enzyme exhibiting a preference for high-salt conditions and moderate temperatures (up to 45°C). In chapter 4, MarCE, a marine carboxylesterase was found encoded in the genome of a Maribacter sp. isolated from a sea sponge sample collected at Lough Hyne. MarCE was shown to hydrolyze polycaprolactone diol and putative binding of PET oligomers was demonstrated by molecular docking analysis. The work presented on BgP, SM14est, and MarCE makes a case for the continued exploration of marine-derived bacteria, in particular those found within the unique marine sponge ecosystem, as a source of potentially novel polyesterases with relevance for the biological degradation of synthetic polyesters among other biocatalytic applications.
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    Shotgun metagenomics-based exploration of kefir microbiomes
    (University College Cork, 2023) Walsh, Liam; Cotter, Paul; O'Toole, Paul W.; Teagasc
    Fermentation is among the world’s oldest forms of food processing, having been applied for millennia to preserve or enhance foods and still accounts for a significant component in the human diet. One such fermented food is milk kefir, which is regularly consumed in eastern European countries and is becoming increasingly popular in western society as a functional food, with numerous scientific studies and reviews associating health benefits with its consumption. Water kefir represents another fermented food of considerable interest, which is increasingly being perceived as a non-dairy alternative to milk kefir. In this thesis, we demonstrate that metagenomic analysis is a valuable tool to (i) expand our, and the general public’s understanding on the microbiology of kefir fermentations, (ii) determine the potential functionality of specific microbes therein and (iii) investigate the impact of milk kefir on the host gut microbiome. In chapter 1, we critically analyse the tools and pipelines that have been used, or that could be applied, to the analysis of metagenomic and metatranscriptomic data relating to fermented foods. In addition, we critically analyse a number of studies of fermented foods in which these tools have previously been applied to highlight the insights that these approaches can provide. Chapter 2 is focused on the shotgun metagenomics-based analysis of 256 kefir milk samples produced from milk kefir grains sourced from various parts of the world. This chapter provides considerable insight into the heterogeneity of these populations, while also uncovering conserved features such as the presence of Lactococcus lactis and Lactococcus cremoris, which may help to define the minimal components required for a fermented milk product to be considered a milk kefir. In chapter 3, we show that shotgun metagenomics, when used alongside metabolomics, can provide evidence that milk kefir modulates the gut microbiome. We show that daily consumption of kefir in a healthy cohort has a subtle impact on the urinal metabolome and gut microbiome. The principle change to the gut microbiome was the detection of Lactococcus raffinolactis post kefir consumption. The detection of Lactococcus raffinolactis is particularly notable given its low relative abundance across kefir metagenomes generated in chapter 2. In chapter 4, we describe Kefir4All, a citizen science project designed to provide the general public with an opportunity to expand their awareness, knowledge and practical skills relating to microbiology, introduced from the perspective of producing a fermented food, i.e., milk kefir or water kefir. In chapter 5, we highlight how research relating to the milk kefir and water kefir microbiome was greatly extended through the efforts of the Kefir4All citizen scientists through the investigation of compositional, functional and evolutionary change in milk and water kefir microbial communities over 21 weeks of repeat regular fermentation by citizen scientists. Overall, this thesis highlights that bioinformatic analysis of high throughput sequencing datasets can expand our knowledge of microbial communities associated with fermented foods and in the host following consumption, while also highlighting the merits of employing fermented food-related studies to raise awareness, knowledge and interest in microbiology and fermentation.
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    Molecular characterisation of a conjugative Bifidobacterium megaplasmid
    (University College Cork, 2023) Dineen, Rebecca L.; van Sinderen, Douwe; O'Connell Motherway, Mary; Science Foundation Ireland
    Bifidobacterium species are highly abundant autochthonous bacteria of the human gut microbiota, particularly during host infancy. Various members of the Bifidobacterium genus have been associated with a plethora of health-promoting attributes, among which maintaining gut homeostasis, limiting pathogenic bacterial invasion as well as their purported ability to modulate host immune responses are notable examples. Due to their positive association with human health, this genus has received substantial scientific attention and commercial interest. Plasmids were once considered an atypical feature of Bifidobacterium and those identified within this genus were primarily small cryptic plasmids which were presumed to replicate by a so-called rolling circle mechanism. However, the recent availability of long-read single-molecule sequencing technologies precipitated the resolution of the first reported bifidobacterial megaplasmid isolated from the common and abundant inhabitant of the human gut, Bifidobacterium breve JCM7017. The discovery of this >190 kb conjugative megaplasmid, denoted pMP7017, and the subsequent identification of pMP7017 homologs in several B. longum subsp. longum strains, highlights the prevalence of this megaplasmid family within this genus, representing an unexplored feature. Conjugative plasmids such as pMP7017 play a central role in bacterial evolution and have the potential to significantly influence the activity of the microbiome community and, by extension, impact human health and physiology. The research described in this thesis covers the replication functions of megaplasmid pMP7017 and exploits these functions for the development of important molecular tools that facilitate the genetic engineering of these genetically recalcitrant bacteria. As pMP7017 represents the first and, thus far, only experimentally validated conjugative plasmid of bifidobacterial origin, research performed within the context of this thesis also concerned examination of the conjugative functions of this megaplasmid. While the molecular characterisation of pMP7017 represents an opportunity for the development of much needed molecular tools and provides a starting point in the understanding of bifidobacterial DNA transfer systems, an integrated approach of comparative analyses and metagenomic data mining has generated highly relevant and insightful information concerning the biology and distribution of pMP7017 and related megaplasmids.
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    Development of synthetic biology tools for Kluyveromyces marxianus and their application to produce aromatic molecules in this yeast
    (University College Cork, 2023) Akinola, Joel Abidemi; Morrissey, John P.; Horizon 2020; Eli Lilly and Company
    The experimental work in this thesis was carried out as part of the European Union-funded Horizon 2020 project CHASSY (https://www.ucc.ie/en/eri/projects/chassy.html). 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.
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    Exploring early-life microbiome transfer and therapeutic applications in bovines and humans
    (University College Cork, 2023) Linehan, Kevin; Stanton, Catherine; Ross, R. Paul; APC Microbiome Institute; Science Foundation Ireland
    The microbiome consists of intricate microbial communities, including bacteria, archaea, eukarya, viruses, bacteriophages, and their associated products. These dynamic entities establish symbiotic relationships with their bovine and human hosts, exerting direct or indirect influences on physiology throughout life, impacting both health and disease outcomes. The early-life microbiome exerts a profound impact on developmental trajectories and long-term health. The extent to which different maternal microbial sources and perinatal factors contribute and shape the initial colonisation, development, and functionality of the neonatal microbiome is a topic of ongoing research. Understanding these factors is crucial for comprehending the early establishment of the microbiome. Given the current antibiotic resistance crisis, there is significant importance in leveraging host-microbiome interactions to develop microbiome-based therapeutics. This thesis explores a number of research foci with a view to gain a better understanding of (1) the influence of different maternal microbial sources and perinatal factors on the initial establishment of the human infant gut microbiome, (2) harnessing the bioactive composition of bovine colostrum for bovine and human health applications, (3) characterising the virome of bovine colostrum and the influence of perinatal factors on its composition, and (4) the potential of microbiome-based therapeutics for disease treatment in bovines and humans. Chapter 1 discusses the impact of perinatal factors, including maternal nutrition, antibiotic use, gestational age, and mode of delivery, on the initial colonisation, development, and function of the human neonatal gut microbiome. The elucidation of the precise extent to which these factors influence gut microbiome establishment and identification of those with the most decisive effects on colonisation are essential for improving infant health. In Chapter 2, the diverse array of bioactive components in bovine colostrum suitable for the development of functional foods, nutraceuticals, and pharmaceuticals with veterinary and human health applications are discussed. The processing techniques used to produce high-value colostrum-based products, and recent studies utilizing bovine colostrum for veterinary and human health are also outlined. In Chapter 3, using a cohort of 18 healthy mother-infant dyads, the microbial composition of three potential maternal sources of microbial transmission (oral, vaginal and placental) to the microbiota of their new-born infant (oral and meconium microbiota) were characterised. This allowed investigation of the contribution of numerous transmission routes and the impact of various perinatal factors on the initial establishment of the infant gut and oral microbiome. The results of this study consolidate and corroborate recent findings surrounding the existence of a meconium microbiome and the absence of a placental microbiome. Furthermore, the study shows that significant vertical transfer, primarily from the maternal oral cavity to the infant oral cavity occurs in early life. In Chapter 4, a reproducible, low cost and high-throughput virome extraction method was developed for bovine colostrum. Shotgun sequencing and viral specific metagenomics bioinformatics were performed on samples from 72 dairy cows, given dry cow therapy (n=48) or naturally dried off (n=24). The impact of farm level variables (location and parity) were also assessed. Phages carrying multidrug resistance genes (smeS, lfrA, kdpE and baeS) were identified. Antibiotic treatments significantly impacted virome composition and the presence of resistance genes specific to the administered antibiotic. This study provides novel insights into disease development and transmission in animals and humans, and the contribution of viruses to the spread of global antimicrobial resistance. In Chapter 5, two novel Staphylococcus aureus bacteriophage species from the genus Phietavirus were isolated. Phages were lytic against several human and bovine mastitis causing strains of Staphylococcus aureus (including MRSA). Phages displayed excellent characteristics for in vivo experiments, with no resistance genes present, stability to variations in pH (4 to 9), temperature (up to 60 °C), chloroform resistant and capable of replicating in mastitic milk. Finally, in Chapter 6, a field trial was undertaken to investigate the efficacy of emulsion based postbiotic and live-biotherapeutic formulations of Lactococcus lactis DPC3147, producer of the bacteriocin lacticin 3147, as alternative therapeutics for bovine mastitis. Twenty eight cows with chronic mastitis were treated with emulsion-based formulations containing either viable L. lactis DPC3147 cells (n=15) or heat-killed L. lactis DPC3147 cells (n=13). The efficacies of the two formulations in stimulating a localised immune response (measuring interleukin-8 concentrations in milk) and cure rates (somatic cell counts reductions and pathogen absence) were evaluated. This study demonstrated that the presence of heat-inactivated bacteria (a postbiotic) was as effective as the live bio-therapeutic in eliciting a localised immune response in cows with chronic mastitis. The results outlined in this thesis provide valuable insights into the intricate dynamics of early-life microbiome transfer and outline novel microbiome-based therapeutics for applications in bovines and humans.