US 12,281,299 B2
Control of nitrogen fixation in rhizobia that associate with cereals
Christopher A. Voigt, Belmont, MA (US); and Min-Hyung Ryu, Cambridge, MA (US)
Assigned to Massachusetts Institute of Technology, Cambridge, MA (US)
Filed by Massachusetts Institute of Technology, Cambridge, MA (US)
Filed on Jan. 17, 2020, as Appl. No. 16/746,215.
Claims priority of provisional application 62/820,765, filed on Mar. 19, 2019.
Prior Publication US 2020/0299637 A1, Sep. 24, 2020
Int. Cl. C12N 1/20 (2006.01); C05F 11/08 (2006.01); C12N 15/87 (2006.01)
CPC C12N 1/20 (2013.01) [C05F 11/08 (2013.01); C12N 15/87 (2013.01); C12N 2510/00 (2013.01)] 19 Claims
 
1. A rhizobia species that can fix nitrogen under aerobic free-living conditions, comprising a symbiotic Rhizobium Sp. IRBG74 species having an exogenous nif cluster, wherein the exogenous nif cluster confers nitrogen fixation capability on the symbiotic Rhizobium Sp. IRBG74 species under aerobic free-living conditions, wherein the exogenous nif cluster is:
(i) is photosynthetic alphaproteobacteria nif cluster; or
(ii) an inducible refactored nif cluster comprising two or more nif genes which are each preceded by a heterologous promoter, and comprise a heterologous terminator, wherein the nif genes located in the inducible refactored nif cluster encode nitrogenase subunits, proteins involved in the biosynthesis of metalloclusters, proteins involved in electron transport, and regulator proteins,
wherein the nitrogenase subunits are encoded by a nifH sequence, a nifD sequence, and/or a nifK sequence, wherein the proteins involved in electron transport are encoded by a nifF sequence and/or a nifJ sequence, wherein the proteins involved in the biosynthesis of metalloclusters and the regulator proteins are encoded by a nifT sequence, a nifY sequence, a nifE sequence, a nifN sequence, a nifX sequence, a nifB sequence, a nifQ sequence, a nifU sequence, a nifS sequence, a nifV sequence, a nifW sequence, and/or a nifZ sequence,
wherein the inducible refactored nif cluster is an inducible refactored free-living diazotroph nif cluster, an inducible refactored symbiotic diazotroph nif cluster, an inducible refactored photosynthetic alphaproteobacteria nif cluster, an inducible refactored gammaproteobacteria nif cluster, an inducible refactored cyanobacteria nif cluster, or an inducible refactored firmicutes nif cluster.