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

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 strat...

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Bibliographic Details
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
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1804992115