Construction of DNA Tools for Hyperexpression in Marchantia Chloroplasts.

Sze Wai Tse,Yang Liu,Harald Schneider,Jim Haseloff,Kasey Markel,Susanna Sauret-Gueto,Artemis Perraki,Eftychios Frangedakis,Fernando Guzman-Chavez,Bernard Goffinet,Marius Rebmann,Ying Yu,Jenna Rever

doi:10.1021/acssynbio.0c00637

Abstract

Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated. However, plastid transformation is a slow process and currently limited to a few plant species. The liverwort Marchantia polymorpha is a simple model plant that allows rapid transformation studies; however, its potential for protein hyperexpression has not been fully exploited. This is partially due to the fact that chloroplast post-transcriptional regulation is poorly characterized in this plant. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 bryophyte species and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilization. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplastic transformed plants. We have produced novel DNA tools for protein hyperexpression in this facile plant system that is a test-bed for chloroplast engineering.

Highlights

Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated
Foreign proteins have been produced in chloroplasts at high levels, sometimes reaching a major proportion of the total soluble proteins in transformed plants.[1−3] previous attempts to harness this capacity for routine hyperexpression (>1% soluble protein) have been irregular and sporadic
Plastid RNA transcripts are subject to a series of complex processing steps that are primarily mediated by nucleus-encoded factors, including pentatricopeptide repeat (PPR) containing proteins

Summary

Introduction

Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated. The liverwort Marchantia polymorpha is a simple model plant that allows rapid transformation studies; its potential for protein hyperexpression has not been fully exploited This is partially due to the fact that chloroplast post-transcriptional regulation is poorly characterized in this plant. The PPR proteins are a large family of RNA-binding proteins that have undergone a substantial expansion in plants[11] and are required for stabilization of mRNAs by protection from exonuclease activity in the plastid.[9,12] The sequence-specific RNA-binding properties and defined target sites for these proteins make them excellent candidates as artificial regulators of RNA degradation, in addition to being used as highly effective tools for enhancing gene expression in chloroplasts.[9,10] Post-transcriptional regulation of chloroplast mRNAs in Marchantia is relatively simple compared to vascular plants. No evidence of PPR protein-mediated base editing has been found in Marchantia chloroplast transcripts.[15]

Methods

Results

Conclusion