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dc.contributor.authorHolzer, Andre
dc.date.accessioned2022-02-01T01:09:22Z
dc.date.available2022-02-01T01:09:22Z
dc.date.submitted2021-10
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/333485
dc.description.abstractAlgae, a globally widespread group of photosynthetic eukaryotes, are the primary producers in the aquatic world. Their photoautotrophic lifestyle together with rapid growth rates and diverse metabolic capabilities make them sustainable, renewable and environmentally friendly production platforms with great potential for biotechnological applications including the production of biofuels, medicinal products or food supplements. Better understanding of algal biology including their predominately vitamin B12 dependency is however desperately needed. While de novo biosynthesis of nature’s most complex vitamin is limited to prokaryotes, a vast number of eukaryotic species, comprising all mammals and more than half of all algal species, require B12 as an enzyme cofactor. The phylogenetic distribution of B12 auxotrophy across the eukaryotic tree of life suggests that this trait has evolved multiple times in the algal kingdom, indicating that it has a selective advantage of some sort. Indeed, previous artificial evolution experiments alongside physiological studies from our group resulted in mutant strains of the unicellular green model alga Chlamydomonas reinhardtii that now require B12 for growth, providing evidence for how this might have arisen in the environment. Here, I exploit the unique opportunity these early B12 auxotroph strains of C. reinhardtii, together with their B12-independent progenitors, provide to study the molecular, cellular and evolutionary mechanisms underlying B12 auxotrophy by applying an interdisciplinary research strategy, coupling multi-omics with targeted metabolite analysis, computational genomics and classical molecular biology approaches. First, I present the global transcriptomic and one- carbon cycle related metabolite alterations in response to vitamin B12 scarcity in a B12 auxotroph and its B12-independent progenitor of C. reinhardtii and characterise common strategies eukaryotic cells use to cope with low B12. Findings highlight that the “B12-specific response” includes reduced B12 usage, fine-tuning of methionine cycle genes, accumulation of folates and upregulation of uptake, salvage and remodelling capacity for B12. At the same time, a much broader “starvation response” characterised by the downregulation of flagella, microtubule, circadian clock, histone, photosystem, carotenoid biosynthesis as well as starch and sucrose metabolism genes alongside strong upregulation of one-carbon metabolism, heme binding, purine metabolism, protein degradation, deoxyribonucleic acid (DNA) and histone methyltransferase genes and accumulation of homocysteine, is induced in B12- dependent cells. Accumulation of S-adenosylhomocysteine further results in a significantly reduced methylation potential that likely promotes DNA hypomethylation and suggests that B12 starvation results in a strong alteration of the epigenetic landscape, influencing transcriptional regulation and genetic stability. With several lines of evidence for the involvement of mobile genetic elements, or transposable elements (TE) in the evolution of B12 dependency in algae, a comparative genomic analysis was performed, characterising the nuclear genome architecture of three assembled C. reinhardtii lab strains. Besides revealing numerous improvements in the new, near complete version 6 C. reinhardtii genome assemblies, the updated repeat maps presented here highlight that consistently ~10.5% of the nuclear C. reinhardtii genome is comprised of TEs. Approximately half of all structural variants were found to be associated with TEs, representing a comprehensive list of active TE families in C. reinhardtii. Further, a custom developed multi-tool approach that overcomes common challenges in accurate mapping of DNA transposons is presented and applied to the example of mapping, classifying, characterising and tracking the C. reinhardtii specific DNA transposon family Gulliver. Finally, I present an experimental model system to study TEs in C. reinhardtii and propose a methodology to validate connections between environmental B12 signalling and (epi-)genetic changes. Using short-read whole-genome shotgun sequencing, I map nucleotide and structural variants in the various C. reinhardtii lines derived from the artificial evolution experiment and track TE translocations between strains as a result of vitamin B12 deprivation stress. This study provides key genomic resources for the algal community and expands our understanding of B12 auxotrophy in single-cell eukaryotes, linking environmental B12 signalling to metabolic and transcriptomic status as well as TE activity. The results further improve our understanding regarding the biological function of transposons in algae and their role in adaptation to environmental stress.
dc.description.sponsorshipGates Cambridge Trust (Graduate Student Fellowship) & Bill and Melinda Gates Foundation (OPP1144) & Frank Smart Studentship 2018 (Department of Plant Sciences)
dc.rightsAll Rights Reserved
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.subjectSystems Biology
dc.subjectMicroalgae
dc.subjectChlamydomonas reinhardtii
dc.subjectVitamin B12
dc.subjectCobalamin
dc.subjectMulti-omics
dc.subjectTranscriptomics
dc.subjectOne-carbon metabolism
dc.subjectTransposable elements
dc.subjectGenomics
dc.subjectAuxotrophy
dc.subjectAdaptation
dc.titleInvestigating the genomic, transcriptomic and metabolic landscape of Chlamydomonas reinhardtii in the context of vitamin B12 availability
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.date.updated2022-01-31T13:30:24Z
dc.identifier.doi10.17863/CAM.80905
rioxxterms.licenseref.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.contributor.orcidHolzer, Andre [0000-0003-2439-6364]
rioxxterms.typeThesis
dc.publisher.collegeChrists
cam.supervisorSmith, Alison
cam.supervisorHenderson, Ian
cam.supervisor.orcidSmith, Alison [0000-0001-6511-5704]
cam.depositDate2022-01-31
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement
rioxxterms.freetoread.startdate2023-02-01


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