Engineering C-4 photosynthesis into C-3 chassis in the synthetic biology age

C-4 photosynthetic plants outperform C-3 plants in hot and arid climates. By concentrating carbon dioxide around Rubisco C-4 plants drastically reduce photorespiration. The frequency with which plants evolved C-4 photosynthesis independently challenges researchers to unravel the genetic mechanisms underlying this convergent evolutionary switch. The conversion of C-3 crops, such as rice, towards C-4 photosynthesis is a long-standing goal. Nevertheless, at the present time, in the age of synthetic biology, this still remains a monumental task, partially because the C-4 carbon-concentrating biochemical cycle spans two cell types and thus requires specialized anatomy. Here we review the advances in understanding the molecular basis and the evolution of the C-4 trait, advances in the last decades that were driven by systems biology methods. In this review we emphasise essential genetic engineering tools needed to translate our theoretical knowledge into engineering approaches. With our current molecular understanding of the biochemical C-4 pathway, we propose a simplified rational engineering model exclusively built with known C-4 metabolic components. Moreover, we discuss an alternative approach to the progressing international engineering attempts that would combine targeted mutagenesis and directed evolution