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Open AccessFeature PaperArticle

A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii

1
Laboratory of Photosynthesis and Bioenergy, Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy
2
Laboratorio Nacional de Genómica para la Biodiversidad (UGA) Cinvestav, 36821 Irapuato, Guanajuato, Mexico
3
Institute of Genomics for Crop Abiotic Stress Tolerance, Department of Plant and Soil Sciences, Texas Tech University, Box 42122, Lubbock, TX 79409, USA
*
Author to whom correspondence should be addressed.
Plants 2020, 9(4), 473; https://doi.org/10.3390/plants9040473
Received: 4 March 2020 / Revised: 28 March 2020 / Accepted: 31 March 2020 / Published: 8 April 2020
(This article belongs to the Collection Feature Papers in Plant Physiology and Metabolism)
Heterologous expression of the NAD+-dependent phosphite dehydrogenase (PTXD) bacterial enzyme from Pseudomonas stutzerii enables selective growth of transgenic organisms by using phosphite as sole phosphorous source. Combining phosphite fertilization with nuclear expression of the ptxD transgene was shown to be an alternative to herbicides in controlling weeds and contamination of algal cultures. Chloroplast expression of ptxD in Chlamydomonas reinhardtii was proposed as an environmentally friendly alternative to antibiotic resistance genes for plastid transformation. However, PTXD activity in the chloroplast is low, possibly due to the low NAD+/NADP+ ratio, limiting the efficiency of phosphite assimilation. We addressed the intrinsic constraints of the PTXD activity in the chloroplast and improved its catalytic efficiency in vivo via rational mutagenesis of key residues involved in cofactor binding. Transplastomic lines carrying a mutagenized PTXD version promiscuously used NADP+ and NAD+ for converting phosphite into phosphate and grew faster compared to those expressing the wild type protein. The modified PTXD enzyme also enabled faster and reproducible selection of transplastomic colonies by directly plating on phosphite-containing medium. These results allow using phosphite as selective agent for chloroplast transformation and for controlling biological contaminants when expressing heterologous proteins in algal chloroplasts, without compromising on culture performance. View Full-Text
Keywords: phosphite dehydrogenase (PTXD); Chlamydomonas reinhardtii; chloroplast genome engineering; algal cultivation; culture contamination prevention; nicotinamide adenine dinucleotide; site-directed mutagenesis; phosphorous metabolism phosphite dehydrogenase (PTXD); Chlamydomonas reinhardtii; chloroplast genome engineering; algal cultivation; culture contamination prevention; nicotinamide adenine dinucleotide; site-directed mutagenesis; phosphorous metabolism
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MDPI and ACS Style

Cutolo, E.; Tosoni, M.; Barera, S.; Herrera-Estrella, L.; Dall’Osto, L.; Bassi, R. A Phosphite Dehydrogenase Variant with Promiscuous Access to Nicotinamide Cofactor Pools Sustains Fast Phosphite-Dependent Growth of Transplastomic Chlamydomonas reinhardtii. Plants 2020, 9, 473.

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