Chemical Engineering Seminar - Thinking outside the flask - how to do something useful with algal metabolism

There is enormous potential to use microalgae as feedstocks for everything from recombinant proteins and high value chemicals to biofuels, but to implement this technology in a sustainable and economic manner, it will be necessary to optimize many parameters, and metabolic engineering strategies will be essential. However, in comparison with the well-developed molecular biology tools available for manipulation of bacteria, yeast, and even land plants, those for algae are limited, even for the well-studied Chlamydomonas reinhardtii. But this can also be an opportunity to think outside the box and develop novel approaches. Vitamins, in particular the water-soluble B-vitamins, are a class of metabolites that offer several unique aspects that could be exploited in this context. Thiamine pyrophosphate (TPP) riboswitches are regions in messenger RNA that bind to TPP directly without the involvement of protein factors. They are present ubiquitously in bacteria, and are the only riboswitches found in eukaryotes. In these latter organisms, binding of the ligand results in alternative splicing of the transcript, which then leads to changes in expression of the mRNA. We have identified and characterised riboswitches in the green alga Chlamydomonas reinhardtii. Key nucleotides in the mechanism of action of these sequences have been established using a combination of site-directed mutagenesis, error-prone PCR and suppression mutagenesis, and as a result we demonstrated that the riboswitches are modular and versatile, responding to nM levels of the ligand, and not just TPP but also analogues and thiamine biosynthetic intermediates.  At the same time, we have developed synthetic genetic circuits using a suite of engineered TPP riboswitches that can ‘tune’ expression of one or more transgenes, both in the nucleus and the chloroplast, offering the means for sophisticated metabolic engineering strategies.