Molecular biology of B vitamin metabolism genes and their regulation in Phaeodactylum tricornutum

Thiamine (Vitamin B1) in its diphosphate form is an essential cofactor for virtually all organisms. Thiamine biosynthesis is an expensive metabolic process involving suicidal and low-turnover enzymes, and many organisms have lost the ability to synthesise thiamine and depend on an exogenous source. Those prototrophs that synthesise thiamine, including species of bacteria, fungi, green algae and plants, have been shown to tightly regulate their thiamine-related genes in response to exogenous thiamine. In many cases this is via riboswitches, sequences in mRNA that fold into a tertiary structure (aptamer) able to specifically recognise a ligand and mediate a change of genetic expression in response. In diatoms, marine algae responsible for ~20 % of net primary productivity, THIC, which encodes one of the first enzymes in the thiamine biosynthetic pathway, has been predicted to have a thiamine pyrophosphate (TPP) riboswitch in the 3’UTR. Additionally, THIC has been previously observed to be downregulated by cobalamin (vitamin B12) supplementation in diatoms reflecting the interconnectedness and co-regulation of different B vitamin metabolisms. The aim of this thesis is to investigate different aspects of the regulation of thiamine and cobalamin-related genes and the transport and metabolism of thiamine and cobalamin in diatoms. Homology-based bioinformatic tools confirmed that all available diatom genomes contain homologues of THIC encoding the first enzyme in the pyrimidine branch of the thiamine biosynthesis pathway in bacteria, and/or NMT1, which catalyses the equivalent step in fungi. Additionally, it was found that many of these genes, and those coding for SSSP, a putative thiamine transporter, had predicted TPP aptamers in their 3’UTRs. A conserved polyadenylation site was found overlapping the diatom TPP aptamer sequences, which might be involved in a hypothetical mechanism of action. However, experiments using RT-qPCR and 3’-RACE showed that the THIC and SSSP genes in Phaeodactylum tricornutum did not respond to thiamine supplementation at the transcriptional or post-transcriptional level, even though thiamine is taken up by the cells. Furthermore, unlike in the green alga Chlamydomonas reinhardtii, which has experimentally characterised TPP riboswitches, the diatoms P. tricornutum and Thalassiosira pseudonana were insensitive to pyrithiamine, a thiamine antimetabolite that primarily inhibits growth by binding TPP riboswitches and downregulating thiamine biosynthesis genes. Reporter constructs confirmed that the PtTHIC regulatory sequences (promoter, 5’UTR, 3’UTR) could not regulate heterologous genes in response to thiamine supplementation. Nor did the PtTHIC aptamer respond in C. reinhardtii chimeric constructs. Finally, site-directed mutagenesis of the PtTHIC aptamer did not alter endogenous thiamine levels in P. tricornutum, in contrast to equivalent mutations in other organisms with confirmed riboswitches. Together, these results demonstrate that the predicted TPP aptamer in the PtTHIC gene does not act as a riboswitch. RT-qPCR experiments suggested that PtTHIC is downregulated by cobalamin supplementation similarly to cobalamin-independent methionine synthase (PtMETE). A motif-prediction algorithm revealed a conserved 14 bp motif in the promoters of THIC, METE and other cobalamin-downregulated genes in diatoms that could indicate the co-regulation of cobalamin and thiamine metabolism. Reporter construct experiments confirmed that the PtTHIC promoter could downregulate a reporter in response to cobalamin and that the PtMETE promoter with a mutation in the motif did not drive the expression of a reporter, demonstrating that the conserved motif is necessary for gene expression. Finally, a CRISPR/Cas9 method was developed in collaboration with other members of the group to generate knock-out mutants of several genes in P. tricornutum. The THIC knock-out did not require thiamine for growth in f/2 minimal media, suggesting P. tricornutum can obtain thiamine or its pyrimidine moiety from an alternative source or pathway. A knock-out of SSSP demonstrated that this gene is required for thiamine uptake. The METE knock-out was auxotrophic for cobalamin and the Cobalamin Acquisition Protein 1 (CBA1) knock-out confirmed it is necessary for cobalamin uptake. Taken together, the results in this thesis provide new insights into thiamine and cobalamin metabolism, transport and genetic regulation in diatoms. Unexpected results such as the unaltered growth of the THIC knock-out and the lack of function of the P. tricornutum predicted TPP riboswitches stand in contrast with existing knowledge of thiamine metabolism in other organisms. This raises important questions to understand the role of thiamine in the physiology, ecology and evolution of an algal group with global ecological relevance

The political ecology of oil and gas corporations : TotalEnergies and post-colonial exploitation to concentrate energy in industrial economies

Altres ajuts: acords transformatius de la UABUnidad de excelencia María de Maeztu CEX2019-000940-MIndustrial economies require a steady supply of energy to reproduce and grow. Oil and gas companies fulfil that socio-economic function by constantly finding, extracting and transporting energy sources. The steady extraction and concentration of fossil resources in industrialised centres requires the constant expansion of extraction frontiers and the exploitation of the environment and local communities in unindustrialised areas. This leads to conflicts where local environmental justice organisations fight for the preservation of their lives, livelihood and culture, while companies defend their profits. Thus, oil companies become vectors of an oppression that links the societies enjoying the benefits of lavish energy with those that suffer the impacts of extraction. In this work, based on the Environmental Justice Atlas database, we systematically analyse 50 environmental conflicts related to one of such companies - the French oil major TotalEnergies. Our research reveals the social and environmental cost of the energy resources that power industrial economies. We find that, despite a recent narrative focused on the company's 'greening', TotalEnergies' extraction and concentration functions remain inextricably linked to fossil fuels. Furthermore, the interests and operations of TotalEnergies and the French State are inextricably intertwined and reproduce colonial relationships of power. Our findings support theories of change based on the abandonment of colonial and extractive State models, rather than pursuing fiscal and regulatory measures alone

Development of Novel Riboswitches for Synthetic Biology in the Green Alga Chlamydomonas.

Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression. Because of their modular composition, where a ligand-sensing aptamer domain is combined with an expression platform, riboswitches offer unique tools for synthetic biology applications. Here we took a mutational approach to determine functionally important nucleotide residues in the thiamine pyrophosphate (TPP) riboswitch in the THI4 gene of the model alga Chlamydomonas reinhardtii, allowing us to carry out aptamer swap using THIC aptamers from Chlamydomonas and Arabidopsis thaliana. These chimeric riboswitches displayed a distinct specificity and dynamic range of responses to different ligands. Our studies demonstrate ease of assembly as 5'UTR DNA parts, predictability of output, and utility for controlled production of a high-value compound in Chlamydomonas. The simplicity of riboswitch incorporation in current design platforms will facilitate the generation of genetic circuits to advance synthetic biology and metabolic engineering of microalgae.BBSR

Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualised by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities

Engineered light controlled cell development for enhanced hydrogen production in Nostoc punctiforme ATCC 29133

The aim of this thesis is to enhance heterocyst-based hydrogen production inNostoc punctiforme ATCC 29133. We envision to do so by finely regulatingthe ratio of heterocyst in order to optimize the filament energy balance. Wehereby report the development of an optogenetic synthetic switch basedon the native PcpeC promoter. The optogenetic switch featured a 24-folddynamic range when measuring reporter sfGFP fluorescence. Such a geneticgate was conceived to artificially drive the expression of hetR, the masterregulator of heterocyst development. We achieved to induce enhancedheterocyst differentiation in the presence of ammonia only by changing thechromatic properties of the light source. Thus, the natural cell developmentregulation was substituted by effectively introducing a full person-drivencontrol over the process

Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualized by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities

Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches.

Funder: Cambridge TrustsThiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for c. 20% of global primary production. They have been predicted to contain TPP aptamers in the 3'UTR of some thiamine metabolism-related genes, but little information is known about their function and regulation. We used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. We found that thiamine-related genes with putative TPP aptamers are not responsive to supplementation with thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and targeted mutation of the TPP aptamer in the THIC gene encoding HMP-P synthase does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtTHIC is essential for thiamine biosynthesis and another gene, PtSSSP, is necessary for thiamine uptake. Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance

The conserved protein CBA1 is required for vitamin B12 uptake in different algal lineages

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling, but despite their phototrophic lifestyle, over half of algal species depend on a supply of the corrinoid vitamin B12 (cobalamin) for growth. This essential organic micronutrient is produced only by a subset of prokaryotic organisms, which implies that for algal species to use this compound, they must first acquire it from external sources. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about how it is taken up in algae. Here, we demonstrate the essential role of a protein, CBA1 (for cobalamin acquisition protein 1), in B12 uptake in Phaeodactylum tricornutum, using CRISPR-Cas9 to generate targeted knockouts, and in Chlamydomonas reinhardtii, by insertional mutagenesis. In both cases, CBA1 knockout lines are no longer able to take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wildtype CBA1 gene restores B12 uptake, and regulation of CBA1 expression via a riboswitch element can be used to control the phenotype. When visualised by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 shows association with membranes. A bioinformatics analysis found that CBA1-like sequences are present in all the major eukaryotic phyla. Its presence is correlated with B12-dependent enzymes in many, although not all, taxa, suggesting CBA1 has a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities.University Broodbank Fund, Gates Cambridge Trus

Conserved cobalamin acquisition protein 1 is essential for vitamin B12 uptake in both Chlamydomonas and Phaeodactylum.

Microalgae play an essential role in global net primary productivity and global biogeochemical cycling. Despite their phototrophic lifestyle, over half of algal species depend for growth on acquiring an external supply of the corrinoid vitamin B12 (cobalamin), a micronutrient produced only by a subset of prokaryotic organisms. Previous studies have identified protein components involved in vitamin B12 uptake in bacterial species and humans. However, little is known about its uptake in algae. Here, we demonstrate the essential role of a protein, cobalamin acquisition protein 1 (CBA1), in B12 uptake in Phaeodactylum tricornutum using CRISPR-Cas9 to generate targeted knockouts and in Chlamydomonas reinhardtii by insertional mutagenesis. In both cases, CBA1 knockout lines could not take up exogenous vitamin B12. Complementation of the C. reinhardtii mutants with the wild-type CBA1 gene restored B12 uptake, and regulation of CBA1 expression via a riboswitch element enabled control of the phenotype. When visualised by confocal microscopy, a YFP-fusion with C. reinhardtii CBA1 showed association with membranes. Bioinformatics analysis found that CBA1-like sequences are present in all major eukaryotic phyla. In algal taxa, the majority that encoded CBA1 also had genes for B12-dependent enzymes, suggesting CBA1 plays a conserved role. Our results thus provide insight into the molecular basis of algal B12 acquisition, a process that likely underpins many interactions in aquatic microbial communities