Multifactorial analysis of terminator performance on heterologous gene expression in Physcomitrella

Area of Science:

• Plant biotechnology
• Molecular biology
• Biopharmaceutical production

Background:

• Plants are emerging as efficient hosts for eukaryotic protein production, particularly the moss Physcomitrella (Physcomitrium patens), known for its GMP compliance and humanized glycosylation.
• Understanding gene expression regulation, including the role of 3' untranslated regions (3'UTRs) and terminators, is crucial for optimizing protein yields in plant-based systems.

Purpose of the Study:

• To characterize novel endogenous terminators from Physcomitrella (Physcomitrium patens) for use in molecular pharming.
• To evaluate the impact of these terminators, individually and in combination, on heterologous gene expression in Physcomitrella.

Main Methods:

• Genome-wide analysis of Physcomitrella 3'UTRs to identify potential terminator candidates, noting the prevalence of alternative polyadenylation.
• Transient Dual-Luciferase assays were employed to characterize 14 selected terminator candidates across different promoters (NOS, CaMV35S, PpActin5).
• High-performing terminators were tested as double terminators to assess their combined effects on reporter gene expression.

Main Results:

• Identified endogenous Physcomitrella terminators that perform comparably to established heterologous terminators (CaMV35S, AtHSP90, NOS).
• Demonstrated that promoters have a significantly larger impact (over 1000-fold) on gene expression than terminators (less than 10-fold).
• Found that the number of polyadenylation sites and polyadenylation signals within 3'UTRs are key factors influencing terminator performance.

Conclusions:

• Developed a collection of endogenous single and double terminators from Physcomitrella for use in molecular pharming applications.
• Enhanced the Physcomitrella biotechnology platform and contributed to a broader understanding of terminator function in plant gene expression.
• These findings support the use of Physcomitrella as a robust system for producing recombinant proteins for health applications.