Abstract
Phosphorus (P) is a nonrenewable resource, which is one of the major challenges for sustainable agriculture. Although phosphite (Phi) can be absorbed by the plant cells through the Pi transporters, it cannot be metabolized by plant and unable to use as P fertilizers for crops. However, transgenic plants that overexpressed phosphite dehydrogenase (PtxD) from bacteria can utilize phosphite as the sole P source. In this study, we aimed to improve the catalytic efficiency of PtxD from Ralstonia sp.4506 (PtxDR4506), by directed evolution. Five mutations were generated by saturation mutagenesis at the 139th site of PtxD R4506 and showed higher catalytic efficiency than native PtxDR4506. The PtxDQ showed the highest catalytic efficiency (5.83-fold as compared to PtxDR4506) contributed by the 41.1% decrease in the K m and 2.5-fold increase in the k cat values. Overexpression of PtxDQ in Arabidopsis and rice showed increased efficiency of phosphite utilization and excellent development when phosphite was used as the primary source of P. High-efficiency PtxD transgenic plant is an essential prerequisite for future agricultural production using phosphite as P fertilizers.
Highlights
Phosphorus (P) is one of the essential major macronutrients required by all living organisms
The exact concentration of Phi in the cytosol of plant cells is still not measured, the plants can only uptake limited Phi at relatively high concentrations of Pi in the environment (Danova-Alt et al, 2008; Thao and Yamakawa, 2009). This could explain why the overexpression of ptxDPS only showed a significant effect for Phi utilizing efficiency under no or low Pi (LP) conditions (López-Arredondo and Herrera-Estrella, 2012; Manna et al, 2016; Heuer et al, 2017)
For protein PtxD expression in E. coli, the synthesized ptxDR4506 gene was cloned into the pGEX-6p-1 expression plasmid
Summary
Phosphorus (P) is one of the essential major macronutrients required by all living organisms. It is a limiting nutrient that controls growth in many ecosystems (Du et al, 2020). The formation and breakdown of phosphate esters under the control of kinases and phosphatases regulates the temporal protein activity and is responsible for the generation, distribution, and utilization of free energy throughout the cell in a number of metabolic pathways (Deng et al, 2020). Most P exists in its completely oxidized state (valence of +5) as phosphate anion (PO43-, Pi), phosphate-containing minerals, and organic phosphate esters. Phosphonates, which contain a carbon-phosphorus (C-P) bond and are characterized by phosphite ester, are found in a wide range of organisms (Andrea and William, 2007)
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