High-throughput methods for isolating and characterizing ammonium-excreting mutant libraries generated by chemical mutagenesis

Inventors

Ryu, Min-Hyung • ESKIYENENTURK, Bilge Ozaydin • Tamsir, Alvin

Assignees

Pivot Bio Inc

Abstract

The present disclosure provides high-throughput methods for rapidly mutagenizing, screening, and targeting candidate microbes that are capable of fixing atmospheric nitrogen in the presence of exogenous nitrogen. The methods utilize a microbial biosensor capable of detecting the presence/absence of ammonium and/or glutamine in a composition and signaling with a fluorescent reporter. The present disclosure further utilizes rapid visual detection assays capable of processing thousands of candidate microbes. The disclosed methods and biosensor can be used to identify mutant bacteria with improved nitrogen fixing capabilities. Mutant bacteria with improved nitrogen fixing capabilities are also disclosed, as well as methods of utilizing these novel bacteria to provide fixed nitrogen to a plant.

Core Innovation

The disclosure relates to mutant nitrogen-fixation regulatory targets in bacteria, particularly mutant glnA and mutant GlnA, and identifies a nitrogen-fixing bacterium as Klebsiella variicola. In particular examples, the mutant GlnA protein includes an N339D amino acid substitution at amino acid position 339 relative to a Klebsiella GlnA protein, and the corresponding mutant glnA gene includes a nucleotide substitution at nucleotide position 1015 relative to a Klebsiella glnA gene.

The disclosure uses Nif regulon control to relate nitrogen fixation state to a fluorescent biosensor circuit and to support selection and screening of nitrogen-fixing, ammonium-excreting microbial mutants. A fluorescent reporter under control of the nifH promoter is described as being turned off or on depending on the presence or absence of free ammonium or glutamine, in conjunction with NifL/NifA nitrogen fixation regulation, and the selection and screening strategy is stated as high-throughput.

Biosensor assays and readouts are used to identify mutants with enhanced atmospheric nitrogen fixation and ammonium excretion, and genome sequencing findings report SNPs primarily in glnA and also in glnE. The disclosure further describes nitrogen-fixing bacterial strains and microbial compositions intended to improve plant traits and to provide fixed nitrogen to plants using a microbial composition that can include an agriculturally acceptable carrier.

Claims Coverage

The claim set covers five inventive features across nitrogen-fixing bacteria, microbial compositions, and plant application. The main inventive features are the defined mutant glnA/GlnA substitutions, the specification of Klebsiella variicola and a deposited strain identity, and the use of a microbial composition, optionally with a carrier, for plant fixed-nitrogen provision.

The claim set centers on nitrogen-fixing Klebsiella variicola bacteria defined by specific mutant glnA/GlnA substitutions, including N339D at amino acid position 339 and a nucleotide substitution at nucleotide position 1015. It also covers a deposited strain identity, microbial compositions comprising one or more isolated deposited bacterial strains, and application of a microbial composition to a plant or its growth locus to provide fixed nitrogen.

Stated Advantages

Documented Applications