Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella

Abstract

The production of recombinant proteins for health applications accounts for a large share of the biopharmaceutical market. While many drugs are produced in microbial and mammalian systems, plants gain more attention as expression hosts to produce eukaryotic proteins. In particular the GMP-compliant moss Physcomitrella (Physcomitrium patens) has outstanding features such as excellent genetic amenability, reproducible bioreactor cultivation, and humanized protein glycosylation patterns. In this study, we selected and characterized new terminators for their effects on heterologous gene expression. The Physcomitrella genome contains 53,346 unique 3 prime UTRs (untranslated regions) of which 7,964 transcripts contain at least one intron. Over 91% of 3 prime UTRs exhibit more than one polyadenylation site, indicating the prevalence of alternative polyadenylation in Physcomitrella. Out of all 3 prime UTRs, 14 terminator candidates were selected and characterized via transient Dual Luciferase assays, yielding a collection of endogenous terminators performing equally high as established heterologous terminators CaMV35S, AtHSP90, and NOS. High performing candidates were selected for testing as double terminators which impact reporter levels, dependent on terminator identity and positioning. Testing of 3 prime UTRs among the different promoters NOS, CaMV35S, and PpActin5 showed an increase of more than 1,000-fold between promoters PpActin5 and NOS, whereas terminators increased reporter levels by less than 10-fold, demonstrating the stronger effect promoters play as compared to terminators. The number of polyadenylation sites as well as polyadenylation signals were found to be major determinants of terminator performance. Our results improve the biotechnology platform Physcomitrella and further our understanding of how terminators influence gene expression in plants in general.

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

“…While most production hosts are vascular plants, such as Nicotiana benthamiana (Nicotiana) and Daucus carota (carrot), the non-vascular moss Physcomitrella has a proven track-record for the production of pharmaceuticals and bioactive ingredients (Decker and Reski, 2020;Verdú-Navarro et al, 2023;Munoz et al, 2024). In our attempts to constantly optimize this moss for molecular farming approaches (Reski et al, 2015;Ruiz-Molina et al, 2023), we concentrate on gene expression (e.g., Top et al, 2021;Niederau et al, 2024), bioproduction (e.g., Ruiz-Molina et al, 2022), and glycoengineering (e.g., Decker and Reski, 2012;Bohlender et al, 2022). In the latter field, plant-typical glyco-structures have been abolished (Parsons et al, 2012), and stable in-vivo protein sialylation has been achieved (Bohlender et al, 2020).…”

Mobius Assembly for Plant Systems highlights promoter-coding sequences-terminator interaction in gene regulation

“…This involves combining these parts and quantifying the output reporter gene expression. While promoters are generally more well characterized, recently, attention has been also directed towards terminators; diverse arrays of terminators have been characterized in bacteria 16 , yeast 17 , and moss 18 . However, for plant systems the number of available terminator parts was limited for a long time, and their characterization was performed with the Nicotiana benthamiana leaf infiltration system 19,20 .…”