Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii
Author(s)
Young, Rosanna EB
Purton, Saul
Type
Journal Article
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.
Date Issued
2016-05-01
Date Acceptance
2015-09-13
Citation
Plant Biotechnology Journal, 2016, 14 (5), pp.1251-1260
ISSN
1467-7644
Publisher
Wiley Open Access
Start Page
1251
End Page
1260
Journal / Book Title
Plant Biotechnology Journal
Volume
14
Issue
5
Copyright Statement
© 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000374423500008&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Subjects
Science & Technology
Life Sciences & Biomedicine
Biotechnology & Applied Microbiology
Plant Sciences
Chlamydomonas reinhardtii
chloroplast
microalgae
non-sense suppression
transfer RNA
RIBULOSEBISPHOSPHATE CARBOXYLASE OXYGENASE
TRANSFER-RNA HYDROLYSIS
UNNATURAL AMINO-ACIDS
I REACTION-CENTER
ESCHERICHIA-COLI
PHOTOSYSTEM-I
ANTIMICROBIAL PEPTIDES
ORGANELLE GENOMES
MAMMALIAN-CELLS
RELEASE FACTOR
Publication Status
Published
Date Publish Online
2015-10-15