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

Abstract

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.

ptxD/Phi as alternative selectable marker system for genetic transformation for bio-safety concerns: a review

“…Consequently, ptxD/Phi selection can be used as a universal marker to transform C. reinhardtii wild-type microorganisms on medium containing Phi as a major source of P (Changko et al, 2020;Kanda et al, 2014). Combining phosphite fertilization with expression of the nuclear transgene ptxD provided an ideal substitute for herbicides in controlling weeds and pollution of algal cultures (Cutolo et al, 2020).…”

“…Chloroplast expression of ptxD in Chlamydomonas reinhardtii has been proposed as a more environmentally friendly substitute for antibiotic resistance genes in plastid transformation (Day & Goldschmidt-Clermont, 2011). Transplastomic genotypes expressing the mutant PTXD type utilized NADP+ and NAD + to convert Phi to Pi more efficiently and evolved more rapidly than those expressing the wild-type protein (Cutolo et al, 2020). Loera-Quezada et al (2016) reported that the production of genetically engineered microalgae capable of metabolizing Phi is a viable approach for developing a successful scheme for managing biological contaminants in microalgal development that can be combined with established biological and molecular strategies.…”

Combinatorial engineering for photoautotrophic production of recombinant products from the green microalga Chlamydomonas reinhardtii

“…One strategy for reducing contamination of algal cultures is the introduction of metabolic capacity for metabolism of inorganic phosphite as a phosphorous source. This has been shown in numerous organisms, including C. reinhardtii , to reduce contamination and act as a selection agent (López-Arredondo and Herrera-Estrella, 2012; Loera-Quezada et al, 2016; Changko et al, 2020; Cutolo et al, 2020; Dahlin and Guarnieri, 2022). Engineered expression of Pseudomonas stutzeri WM88 phosphite NAD+ oxidoreductase ptxD from either the chloroplast or nuclear genomes of algae has been shown to confer the ability to metabolize phosphite (López-Arredondo and Herrera-Estrella, 2012; Changko et al, 2020; Cutolo et al, 2020; Dahlin and Guarnieri, 2022).…”

Algae Biomass as a Potential Source of Liquid Fuels

“…New technologies are developed such as the use of alternative source of phosphorus in order to control the contamination, for example the use phosphite deydrogenase (PtXD) that catalyzes the conversion of phosphite in phosphate [14]. Many Chlamydomonas strains have been produced expressing nuclear or chloroplastic PtxD [15,16]. There are many works in literature in which new strategies in tuning photosynthesis are developed.…”