Development and Application of Synthetic Biology Tools to Modulate Decarboxylation in Arabidopsis

We recently described an advanced system, named TNT-cloning, for joining DNA fragments from a universal library that automatically keeps open reading frames (ORFs) and does not require linkers, adaptors, sequence homology, amplification or mutation of fragments containing restriction enzyme sites in order to work properly. This all-inclusive method, which is enhanced by a unique buffer formulation, increases the current capabilities of testing and sharing multi-gene circuitry assembled from various DNA segments. Here, we present an improvement of the method for higher cloning efficiency and its expansion for use in multiple model systems. Also, we applied such genetic circuitry to create multi-gene loss-of-function lines and a proof-of-concept for manipulation of decarboxylation in Arabidopsis. We targeted all the four malic enzymes (NADP-ME1-4) in Arabidopsis, which are responsible for malate breakdown into pyruvate and CO₂ in plant cells. Diel regulation of the malic enzymes, and therefore the decarboxylation step, are key steps acquired by evolution for the adaptation of CAM (Crassulacean Acid Metabolism) plants to water-limited environments. Therefore, the capability of manipulating diel carboxylation/decarboxylation in plants is critical for CAM-into-C₃ engineering to increase water use efficiency (WUE) and drought tolerance in C₃ plants. NADP-ME loss-of-function lines showed increased photosynthetic rate, higher net CO₂ fixation rates, higher biomass yield and improved WUE with higher drought tolerance relative to the wild-type Arabidopsis plants. Furthermore, diel metabolomics analysis showed the accumulation of malate is temporal and becomes readily converted into sugars/carbohydrates along with precursors of cell wall biogenesis. To demonstrate the feasibility of creating an inducible and reprogrammable metabolic state for malate decarboxylation, we complemented the NADP-ME loss-of-function lines with a dark-induced promoter and monitored gas exchange during a 24-hour period. Our results demonstrate the robustness of a controller, comprised of YES (1), AND (2) and XOR (2) gates, to effectively link 3 inputs, which includes an ON-OFF switch, and generate two distinct outputs for diel control of intracellular CO₂ in leaves. Taken together, our research leveraged synthetic biology tools for bioengineering of complex traits using genetic circuitry, particularly in plants.