Abstract
<jats:p> Vitamin B <jats:sub>12</jats:sub> , an essential micronutrient for many microalgae and humans, is synthesised only by certain prokaryotes. B <jats:sub>12</jats:sub> 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 B <jats:sub>12</jats:sub> vitamers. As microalgae are important primary producers, it is crucial to understand how diverse microalgae acquire, utilise, and remodel this micronutrient. Through the development of a novel algal bioassay for B <jats:sub>12</jats:sub> quantification that distinguishes between B <jats:sub>12</jats:sub> vitamers with different lower axial ligands, and the generation of targeted knock-out lines, we characterised the role of proteins involved in algal B <jats:sub>12</jats:sub> uptake and remodelling. We found that the previously characterised protein CoBalamin-Acquisition protein 1 (CBA1) is also necessary for the acquisition of pseudocobalamin, a less bioavailable form of B <jats:sub>12</jats:sub> . In addition, we provide the first experimental evidence that COBT is required for <jats:italic>Chlamydomonas reinhardtii</jats:italic> to remodel B <jats:sub>12</jats:sub> . We apply the algal B <jats:sub>12</jats:sub> bioassay to show that the edible alga <jats:italic>Chlorella vulgaris</jats:italic> 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. </jats:p>