Dataset Information

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.

SUBMITTER: Cutolo E

PROVIDER: S-EPMC7238262 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

Cutolo Edoardo E Tosoni Matteo M Barera Simone S Herrera-Estrella Luis L Dall'Osto Luca L Bassi Roberto R

Plants (Basel, Switzerland) 20200408 4

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 w ...[more]

PMID: 32276527

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Project description:'Marker-free' strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient strain carrying a plastome mutation in a key photosynthesis gene. Selection for transformant colonies is carried out on minimal media, such that only those cells in which the mutated gene has been replaced with a wild-type copy carried on the transgenic DNA are capable of phototrophic growth. However, this approach can suffer from issues of efficiency due to the slow growth of C. reinhardtii on minimal media and the slow die-back of the untransformed lawn of cells when using mutant strains with a limited photosensitivity phenotype. Furthermore, such phototrophic rescue has tended to rely on existing mutants that are not necessarily ideal for transformation and targeted transgene insertion: Mutants carrying point mutations can easily revert, and those with deletions that do not extend to the intended transgene insertion site can give rise to a sub-population of rescued lines that lack the transgene. In order to improve and accelerate the transformation pipeline for C. reinhardtii, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of psaA, which encodes one of the core subunits of photosystem I (PSI). Such PSI mutants are highly light-sensitive allowing faster recovery of transformant colonies by selecting for light-tolerance on acetate-containing media, rather than phototrophic growth on minimal media. The deletion extends to a site upstream of psaA-3 that serves as a neutral locus for transgene insertion, thereby ensuring that all of the recovered colonies are transformants containing the transgene. We demonstrate the application of HNT6 using a luciferase reporter.

Dataset Information

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

Publications

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

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