Suga[R]evolution
Synthetic biology aims at understanding by building. To do so, various tools and methodologies were developed and allow design of metabolic pathways on demand. In particular, microorganisms can be used as cell factories which paves the way to a world based on a renewable chemistry. The main source of energy of these cells is sugar. However, industrial use of these heterotrophic cells requires massive production of sugar with canes and beets which has dramatic ecological and environmental impacts. To bring a solution to this problem, we want to engineer Chlamydomonas Reinhardtti, a green microalgae adapted to live in marine water, to produce trehalose (a disaccharide of glucose) in large amounts. This way, sugar production should not compete for arable lands which is increasingly demanded for feeding the world. Using microalgae as a photosynthetic cellular chassis is a promising alternative to heterotrophic microorganisms such as bacterium or yeast (the current favorite chassis). In order to spread the use of Chlamydomonas reinhardtii as a green cell factory, more genetic tools to engineer it are still required. To do so, we will enrich the recently developed Modular Cloning (MoClo) toolkit for C. reinhardtti (Pierre Crozet, Stephane Lemaire and their collaborators, publication submitted) with a synthetic retrotransposon to generate in vivo continuous directed evolution. It will be the first time that such genetic tool is applied to non-baring plasmids cells such as plants or microalgae. This approach allows to either generate new proteins with tailor-made functional properties or to optimize biological systems. To adapt this system, a transposable element which relies on a reverse-transcription event will be used. During the transposition, the target sequence (CARGO) inserted within the transposable element undergoes an important mutagenesis linked to the error rate of the reverse transcriptase. This strategy allows us to quickly generate a large library of sequences in vivo speeding up screening for phenotype. Indeed, in classical directed evolution, the library is generated in vitro and then transformed into the organism. As transforming alga could be a limiting factor in the design/build/test cycles, the proposed methodology should greatly increase the speed of this cycles and thus make C. reinhardtii a better chassis. The proof-of-concept of this designed retrotransposon will be made on a gene conferring resistance to paromomycine, and then applied to the trehalose production. We identified in the literature a fluorescent biosensor for trehalose that will allow us to efficiently screen best producing clones.
