Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology

• Published in: Proceedings of the National Academy of Sciences - PNAS 2018-09, Vol.115 (36), p.E8509-E8517
• Main Authors: Yang, Jianguo, Xie, Xiaqing, Xiang, Nan, Tian, Zhe-Xian, Dixon, Ray, Wang, Yi-Ping
• Format: Article
• Language: English
• Subjects: Assembly; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Biological Sciences; Biology; Biosynthesis; E coli; Endopeptidases - genetics; Endopeptidases - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Gene expression; Genes; Klebsiella; Klebsiella oxytoca - genetics; Microorganisms, Genetically-Modified - genetics; Microorganisms, Genetically-Modified - metabolism; Nitrogen Fixation; Nitrogenase; Operon; PNAS Plus; Polyproteins; Polyproteins - biosynthesis; Polyproteins - genetics; Protein expression; Proteins; Reengineering; Ribonucleic acid; RNA; RNA viruses; SEE COMMENTARY; Splicing; Stoichiometry; Synthetic biology; Tobacco; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1804992115

Re-engineering of complex biological systems (CBS) is an important goal for applications in synthetic biology. Efforts have beenmade to simplify CBS by refactoring a large number of genes with rearranged polycistrons and synthetic regulatory circuits. Here, a posttranslational protein-splicing strategy derived from RNA viruses was exploited to minimize gene numbers of the classic nitrogenase system, where the expression stoichiometry is particularly important. Operon-based nif genes from Klebsiella oxytoca were regrouped into giant genes either by fusing genes together or by expressing polyproteins that are subsequently cleaved with Tobacco Etch Virus protease. After several rounds of selection based on protein expression levels and tolerance toward a remnant C-terminal ENLYFQ-tail, a system with only five giant genes showed optimal nitrogenase activity and supported diazotrophic growth of Escherichia coli. This study provides an approach for efficient translation from an operon-based system into a polyprotein-based assembly that has the potential for portable and stoichiometric expression of the complex nitrogenase system in eukaryotic organisms.