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.

Keywords: Chlamydomonas reinhardtii; chloroplast; microalgae; non-sense suppression; transfer RNA.

Figure 1

Strategy to clone genes into a chloroplast expression vector whilst preventing their expression in Escherichia coli. The gene of interest ( GOI ) is redesigned such that one or more tryptophan (TGG) codons are altered to TGA, indicated by asterisks. These changes can be integrated into the codon‐optimized gene design prior to ordering the synthetic gene and integrating it into the Chlamydomonas reinhardtii chloroplast genome. A tRNA gene based on the C. reinhardtii plastidial trnW, but with the anticodon sequence altered to recognize UGA, is also introduced (trn W _UCA ). This enables readthrough of the GOI in C. reinhardtii. Flanking regions amplified from chloroplast DNA allow targeted integration of the constructs into the chloroplast genome by homologous recombination; the target site is a neutral region downstream of either psbH or psaA exon 3, depending on the construct. The psbH gene can be used for selection in a psbH mutant recipient strain.

Figure 2

Introduction of a synthetic trn W _UCA gene into the Chlamydomonas reinhardtii chloroplast genome allows the expression of full‐length, active CrCD protein from the crCD** gene. (a) Western analysis. Equalized cell lysates were subjected to SDS‐PAGE and two identical blots were probed with antibodies as indicated on the left. (b) Growth tests demonstrating that cell lines A1, W2A and W2B contain active CrCD protein that leads to cell death on media containing 5‐fluorocytosine (5‐FC). C. reinhardtii cultures were adjusted to equal optical densities and spotted onto TAP agar containing no drug (left panel) or 5‐FC (right panel), with serial fivefold dilutions left to right. Plates were incubated under 50 μE/m²/s light for 10 days.

Figure 3

In Escherichia coli, the synthetic Chlamydomonas reinhardtii chloroplast trn W _UCA gene does not permit full length CrCD protein expression from the crCD** gene. (a) Western analysis using equalized lysates of E. coli DH5α carrying four different plasmids. The blot was probed with an αHA antibody to detect HA‐tagged CrCD protein. (b) Growth curve of E. coli DH5α carrying the four different plasmids.

Figure 4

The mutation of a tryptophan codon that is essential for PsaA function in Chlamydomonas reinhardtii can be complemented by trnW_UCA . (a) PCR on C. reinhardtii cell lines showing the homoplasmic integration of aadA downstream of psaA in the chloroplast genome, with the concurrent mutation of the psaA W693 codon to TGA (confirmed by sequencing). (b) PCR showing the homoplasmic integration of pWUCA2 into the C. reinhardtii pPsaA* cell line. Selection was directly for trn W _UCA to restore phototrophic growth by allowing the translation of psaA*. (c) Growth properties of the cell lines on media containing acetate (TAP) or minimal medium (HSM). Chlamydomonas reinhardtii cultures were adjusted to equal optical densities, spotted onto agar and incubated at 25 °C in the presence of oxygen.

Figure 5

Use of the trn W _UCA system to clone and express genes in C. reinhardtii whose products are toxic to Escherichia coli. (a) PCR demonstrating homoplasmic integration of the transgenes into C. reinhardtii cell line TN72. (b) Western blot with anti‐HA antibody, demonstrating accumulation of the foreign proteins.