Biology subjects

Mehrshahi, P.

Publications and source records attributed to Mehrshahi, P..

6 recordsLinked to original sources

bioRxiv2026

Cellular basis of B12 uptake and remodelling in microalgae revealed using a novel bioassay

O_LIVitamin B12, an essential micronutrient for many microalgae and humans, is synthesised only by certain prokaryotes. B12 is a complex tetrapyrrole that can exist in many forms (vitamers), some more bioavailable than others. Some microalgae are able to interconvert, or remodel, different B12 vitamers. As microalgae are important primary producers, it is crucial to understand how diverse microalgae acquire, utilise, and remodel this micronutrient. C_LIO_LIThrough the development of a novel algal bioassay for B12 quantification that distinguishes between B12 vitamers with different lower axial ligands, and the generation of targeted knock-out lines, we characterised the role of proteins involved in algal B12 uptake and remodelling. C_LIO_LIWe found that the previously characterised protein CoBalamin-Acquisition protein 1 (CBA1) is also necessary for the acquisition of pseudocobalamin, a less bioavailable form of B12. In addition, we provide the first experimental evidence that COBT is required for Chlamydomonas reinhardtii to remodel B12. C_LIO_LIWe apply the algal B12 bioassay to show that the edible alga Chlorella vulgaris can accumulate pseudocobalamin but is unable to remodel it, highlighting the need for thorough investigation of the metabolic requirements and capabilities of microalgae, especially given the growing interest in microalgae-based food additives. C_LI

microbiology↗

bioRxiv2023

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. One sentence summaryKnockout mutants and physiological studies demonstrate that the CBA1 protein is essential for uptake of vitamin B12 in both Chlamydomonas reinhardtii and the unrelated Phaeodactylum tricornutum.

biochemistry↗

bioRxiv2022

Overexpression of PSR1 in Chlamydomonas reinhardtii induces luxury phosphorus uptake

Remediation using micro-algae offers an attractive solution to environmental phosphate (Pi) pollution. However, for maximum efficiency, pre-conditioning of algae to induce luxury phosphorus (P) uptake is needed. Here we show that natural pre-conditioning can be mimicked through over-expression of a single gene, the global regulator PSR1 (Myb transcription factor: Phosphate Starvation Response 1), raising P levels to 8% dry cell weight from 2% in control. Complete removal of Pi occurred in log phase, unlike the control. This was associated with increases in PolyP granule size and uptake of Mg2+, the principal counterion. Hyper-accumulation of P depended on a feed-forward mechanism, where a small set of Class I genes were activated despite abundant external Pi levels. This drove a reduction in external Pi levels, permitting more genes to be expressed (Class II), leading to more Pi-uptake. These discoveries enable a bio-circular approach of recycling nutrients from wastewater back to agriculture. TeaserManipulating a single gene drove uptake of P and a Mg2+ counter-ion for increased PolyP accumulation.

bioengineering↗

bioRxiv2022

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

O_LIThiamine 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 approximately 20% of global primary production. They have been predicted to contain TPP aptamers in the 3UTR of some thiamine metabolism-related genes, but little is known about their function and regulation. C_LIO_LIWe 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. C_LIO_LIWe found that thiamine-related genes with putative TPP aptamers are not responsive to thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and the targeted mutation of the TPP aptamer in the HMP-P synthase (THIC) does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtSSSP is necessary for thiamine uptake and that PtTHIC is essential for thiamine biosynthesis. C_LIO_LIOur 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. C_LI

molecular biology↗

bioRxiv2021

Exploring the impact of terminators on transgene expression in Chlamydomonas reinhardtii 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.

synthetic biology↗

bioRxiv2019

Physiological and molecular responses of a newly evolved auxotroph of Chlamydomonas to B12 deprivation

The corrinoid B12 is synthesised only by prokaryotes yet is widely required by eukaryotes as an enzyme cofactor. Microalgae have evolved B12 dependence on multiple occasions and we previously demonstrated that experimental evolution of the non-requiring alga Chlamydomonas reinhardtii in media supplemented with B12 generated a B12-dependent mutant (hereafter metE7). This clone provides a unique opportunity to study the physiology of a nascent B12 auxotroph. Our analyses demonstrate that B12 deprivation of metE7 disrupted C1 metabolism, caused an accumulation of starch and triacylglycerides and a decrease in photosynthetic pigments, proteins and free amino acids. B12 deprivation also caused a substantial increase in reactive oxygen species (ROS), which preceded rapid cell death. Surprisingly, survival could be improved without compromising growth by simultaneously depriving the cells of nitrogen, suggesting a type of cross protection. Significantly, we found further improvements in survival under B12 limitation and an increase in B12 use-efficiency after metE7 underwent a further period of experimental evolution, this time in coculture with a B12-producing bacterium. Therefore, although an early B12-dependent alga would likely be poorly adapted to B12 deprivation, association with B12-producers can ensure long-term survival whilst also providing the environment to evolve mechanisms to better tolerate B12 limitation.

biochemistry↗