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

Abstract Efficient heterologous expression platforms are essential for plant synthetic biology, particularly for engineering complex multigene pathways. Here, we establish a high-throughput system for both 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 two weeks and allows expression of at least 6 stacked genes from a single construct. We characterized constitutive promoters for gene expression in Arabidopsis cells and validated plastid targeting peptides. A library of NifB homologs 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 delicate proteins that require large amounts of transgenic biomass. It thus represents a versatile and scalable tool for advancing synthetic biology and for tackling major biotechnological challenges, such as biological nitrogen fixation. Highlight We developed a fast and scalable expression platform in Arabidopsis suspension cells, enabling transient and stable multigene expression for applications in plant synthetic biology such as nitrogenase engineering. Competing Interest Statement The authors have declared no competing interest. Footnotes Other e-mail addresses: Germán Alonso-Tolo (german.atolo{at}upm.es), Zeltia Ferreiro-Eiras (zeltiaferreiroeiras{at}gmail.com), Xi Jiang (xi.jiang{at}upm.es), and Stefan Burén (stefan.buren{at}upm.es). Supplemental files added. Supplemental Figures S1-S6. Supplemental Tables S1-S4. Abbreviations - 2,4-D - 2,4-dichlorophenoxyacetic acid - BNF - biological nitrogen fixation - CTP - chloroplast targeting peptide - MS - Murashige and Skoog - NAA - 1-naphthaleneacetic acid - PCP - plant cell pack - TS - Twin-Strep