Carbon fixation is a prerequisite for accumulating biomass and storing energy in most of the living world. Improvements to the central enzyme performing CO2 fixation in the biosphere, RuBisCO, can transform agriculture and open new routes towards economically sustainable production of biofuels. While the on-going quest towards a “super-charged” RuBisCO is a highly significant challenge in the fields of both plant science and metabolic engineering, so far only a tiny subset of the ~8000 sequenced RuBisCO genes have been kinetically characterized using in vitro biochemical assays. All of our knowledge and assumptions regarding the kinetic limits of RuBisCO are based on this small fraction covering less than 1% of currently known sequences. By leveraging breakthroughs in gene synthesis capabilities and metagenomic data, as well as a new robust screening system we developed, hemiautotrophic E. coli, we are in the process of exploring the properties of natural RuBisCO variants more comprehensively than ever before. We are exploring the sequence diversity of RuBisCO by synthesizing hundreds of phylogenetically distributed variants. Enzymes with promising kinetic properties are rapidly identified after they are expressed in our hemiautotrophic E. coli strain. Once identified, kinetically interesting variants are being tested in vitro for activity and specificity, with the goal of improving our understanding of the structure-function relationship in this critical protein. We employ a novel approach to a long-lasting challenge by harnessing two recent biotechnological revolutions. High-throughput sequencing has yielded an unprecedented wealth of RuBisCO metagenomic data from across the biosphere and DNA synthesis enables the production of hundreds of gene variants, effectively covering the phylogenetic space. We intend to make strides toward the holy grail of improving crop yields and CO2 conversion to biofuels, or at the very least clarify the constraints on doing so.

Integrating <i>in vivo</i> and <i>in vitro</i> approaches for metagenomic RuBisCO sequence mining to improve carbon fixation

254th National Meeting (2017)

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Integrating in vivo and in vitro approaches for metagenomic RuBisCO sequence mining to improve carbon fixation