Exploring the Impact of Terminators on Transgene Expression in <i>Chlamydomonas reinhardtii</i> with a Synthetic Biology Approach.

Chlamydomonas reinhardtii has many attractive features for use as a model organism for both fundamental studies and as a biotechnological platform. Nonetheless, despite the many molecular tools and resources that have been developed, there are challenges for its successful engineering, in particular to obtain reproducible and high levels of transgene expression. Here we describe a synthetic biology approach to screen several hundred independent transformants using standardised parts to explore different parameters that might affect transgene expression. We focused on terminators and, using a standardised workflow and quantitative outputs, tested 9 different elements representing three different size classes of native terminators to determine their ability to support high level expression of a GFP reporter gene. We found that the optimal size reflected the median size of element found in the C. reinhardtii genome. The behaviour of the terminator parts was similar with different promoters, in different host strains and with different transgenes. This approach is applicable to the systematic testing of other genetic elements, facilitating comparison to determine optimal transgene design

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

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology

Characterising the functional importance of folate polyglytamylation in plants

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

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

Naked Clams to open a new sector in sustainable nutritious food production

Acknowledgements: D.F.W. was funded by a Henslow Fellowship at Murray Edwards College, University of Cambridge, and also the British Ecological Society (SR21/1376) and Fishmongers’ Company. J.R.S was funded by the National Science Foundation award (NSF-IOS ID1940165). Both D.F.W and J.R.S were supported by the Seale Hayne Educational Trust. D.C.A. was funded by a Dawson’s Fellowship at St Catherine’s College, University of Cambridge. KPP, AGS, LA, PM were supported by funding from Royal Society International Collaboration Awards 2019 ICA\R1\191196, and Research England GCRF QR 2018-19. We thank Dr Samuel Furse for his guidance on the nutritional analyses, and Professor Barry Goodell (University of Massachusetts, Amherst) and the Goodell laboratory for the provision of Teredo navalis, and Francesca Willcocks (University of Plymouth) for help with the Energy Dispersive Spectroscopy sample preparation and imaging. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Funder: Henslow Fellowship, Murray Edwards CollegeFunder: Fishmongers' CompanyFunder: Seale Hayne TrustFunder: Dawson’s Fellowship, St Catherine’s CollegeThe global population urgently requires alternative food sources that provide the micronutrient-rich profile of meat and fish but with lower environmental cost. We present a solution in the form of ‘Naked Clams’ (teredinids/shipworms) - a seldom researched group of bivalves, that feature tiny shells and live in and feed on wood, turning it into protein and essential nutrients. We report the first pilot system for Naked Clam aquaculture, the first nutritional profile and feeding efficacy assessment, and demonstrate value offered by microencapsulated feeds in fortifying Naked Clams. Naked Clams were rich in nutrients including vitamin B12 and monounsaturated fatty acids, and shared the high protein content of conventional bivalves such as blue mussels (Mytilus edulis). Microencapsulated algal feeds enriched the Naked Clams with essential PUFAs including EPA and DHA, with potential for further tailoring. Additional work is required, but this study represents a gateway to a new form of sustainable food production.D.F.W. was funded by a Henslow Fellowship at Murray Edwards College, University of Cambridge, and also the British Ecological Society (SR21/1376) and Fishmongers’ Company. J.R.S was funded by the National Science Foundation award (NSF-IOS ID1940165). Both D.F.W and J.R.S were supported by the Seale Hayne Educational Trust. D.C.A. was funded by a Dawson’s Fellowship at St Catherine's College, University of Cambridge. KPP, AGS, LA, PM were supported by funding from Royal Society International Collaboration Awards 2019 ICA\R1\191196, and Research England GCRF QR 2018-19

Naked Clams to open a new sector in sustainable nutritious food production

The global population urgently requires alternative food sources that provide the micronutrient-rich profile of meat and fish but with lower environmental cost. We present a solution in the form of ‘Naked Clams’ (teredinids/shipworms) - a seldom researched group of bivalves, that feature tiny shells and live in and feed on wood, turning it into protein and essential nutrients. We report the first pilot system for Naked Clam aquaculture, the first nutritional profile and feeding efficacy assessment, and demonstrate value offered by microencapsulated feeds in fortifying Naked Clams. Naked Clams were rich in nutrients including vitamin B12 and monounsaturated fatty acids, and shared the high protein content of conventional bivalves such as blue mussels (Mytilus edulis). Microencapsulated algal feeds enriched the Naked Clams with essential PUFAs including EPA and DHA, with potential for further tailoring. Additional work is required, but this study represents a gateway to a new form of sustainable food production.D.F.W. was funded by a Henslow Fellowship at Murray Edwards College, University of Cambridge, and also the British Ecological Society (SR21/1376) and Fishmongers’ Company. J.R.S was funded by the National Science Foundation award (NSF-IOS ID1940165). Both D.F.W and J.R.S were supported by the Seale Hayne Educational Trust. D.C.A. was funded by a Dawson’s Fellowship at St Catherine's College, University of Cambridge. KPP, AGS, LA, PM were supported by funding from Royal Society International Collaboration Awards 2019 ICA\R1\191196, and Research England GCRF QR 2018-19