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Proc Natl Acad Sci U S A. 2018 Sep 4;115(36):E8509-E8517. doi: 10.1073/pnas.1804992115. Epub 2018 Jul 30.

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

Yang J1,2, Xie X1,2, Xiang N1,2, Tian ZX1,2, Dixon R3, Wang YP4,2.

Author information

1
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, 100871 Beijing, China.
2
School of Advanced Agriculture Sciences, Peking University, 100871 Beijing, China.
3
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom ray.dixon@jic.ac.uk wangyp@pku.edu.cn.
4
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, 100871 Beijing, China; ray.dixon@jic.ac.uk wangyp@pku.edu.cn.

Abstract

Re-engineering of complex biological systems (CBS) is an important goal for applications in synthetic biology. Efforts have been made 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.

KEYWORDS:

nitrogen fixation; polyprotein; synthetic biology

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PMID:
30061389
PMCID:
PMC6130400
DOI:
10.1073/pnas.1804992115
[Indexed for MEDLINE]
Free PMC Article

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