Dataset Information

Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii.

ABSTRACT: There is a growing interest in the use of microalgae as low-cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein-coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a useful synthetic biology tool. Here, we report the assignment of the codon to one for tryptophan and show that this can be used as an effective strategy for addressing a key problem in chloroplast engineering: namely, the assembly of expression cassettes in Escherichia coli when the gene product is toxic to the bacterium. This problem arises because the prokaryotic nature of chloroplast promoters and ribosome-binding sites used in such cassettes often results in transgene expression in E. coli, and is a potential issue when cloning genes for metabolic enzymes, antibacterial proteins and integral membrane proteins. We show that replacement of tryptophan codons with the spare codon (UGG?UGA) within a transgene prevents functional expression in E. coli and in the chloroplast, and that co-introduction of a plastidial trnW gene carrying a modified anticodon restores function only in the latter by allowing UGA readthrough. We demonstrate the utility of this system by expressing two genes known to be highly toxic to E. coli and discuss its value in providing an enhanced level of biocontainment for transplastomic microalgae.

SUBMITTER: Young RE

PROVIDER: S-EPMC5102678 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Publications

Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii.

Young Rosanna E B RE Purton Saul S

Plant biotechnology journal 20151015 5

There is a growing interest in the use of microalgae as low-cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein-coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a us ...[more]

PMID: 26471875

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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

Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii.

Publications

Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii.

Similar Datasets

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