A high-throughput heterologous expression platform for plant synthetic biology based on Arabidopsis suspension cells.

Efficient heterologous expression platforms are essential for plant synthetic biology, particularly for engineering complex multigene pathways. Here, we establish a high-throughput system for transient and stable transformation of Arabidopsis thaliana suspension cells using plant cell pack infiltration. This method requires no specialized equipment or consumables and is compatible with several cell lines. It enables rapid generation of 100 g of transgenic cells within 2 weeks and allows expression of at least six stacked genes from a single construct. We characterized constitutive promoters for gene expression in Arabidopsis cells and validated plastid-targeting peptides. A library of homologs of the nitrogenase iron-molybdenum cofactor maturase NifB was screened for expression and solubility, and several archaeal variants suitable for plant expression were identified. We further engineered stable cell lines expressing up to six genes, encoding the NifB module components NifU, NifS, FdxN, and NifB, demonstrating that the newly developed platform integrates into an established workflow for nitrogenase engineering. The platform accelerates design-build-test cycles and facilitates the production of unstable and low-abundance proteins that require large amounts of transgenic biomass. It represents a versatile and scalable tool for advancing synthetic biology and for tackling major biotechnological challenges, such as biological nitrogen fixation.