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

Saul Purton,Rosanna E B Young

doi:10.1111/pbi.12490

Abstract

SummaryThere 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.

Highlights

The microalgal chloroplast has many advantages as a production platform for recombinant proteins and small molecules including low culturing costs, lack of toxins and ease of genetic manipulation
The psaA promoter/ 50 untranslated region (50 UTR) is often used to drive robust expression of foreign genes in the C. reinhardtii chloroplast (Michelet et al, 2011; Specht and Mayfield, 2013; Young and Purton, 2014), but it cannot be used for proteins that are detrimental to Escherichia coli as they will be expressed during cloning in this host and will prevent successful production of the plasmid vector for subsequent transfer to the microalga
For example the PanDaTox database lists over 40 000 microbial genes that are predicted to be toxic to E. coli based on their failure to be propagated during genome sequencing projects (Amitai and Sorek, 2012)

Summary

Introduction

The microalgal chloroplast has many advantages as a production platform for recombinant proteins and small molecules including low culturing costs, lack of toxins and ease of genetic manipulation. The transformation of C. reinhardtii W2 with plasmid pWUCA1 was found to elicit the accumulation of full-length CrCD protein (Figure 2a, cell lines W2A and W2B), demonstrating that the synthetic trnWUCA gene is expressed and recognizes the UGA codon. This confirms that the 100 bp flanking sequences included around trnWUCA were sufficient for its transcription and any subsequent 50 and 30 processing by RNaseP and other RNases. There was no observable difference in CrCD protein yield between W2A and a control C. reinhardtii cell line that had been transformed with pCD (Figure S2)

Results

Discussion

Conclusion