Guided microbial remodeling, a platform for the rational improvement of microbial species for agriculture

Abstract

The present disclosure provides guided microbial remodeling (GMR) methods for the rational improvement of plant-associated microbes to perform plant-beneficial functions. The GMR methods described herein allow for non-intergeneric genetic optimization of key regulatory networks within the microbes, which improve plant-beneficial functions over wild-type microbes but don't have the risks associated with transgenic approaches (e.g., unpredictable gene function, public and regulatory concerns, etc.). The present disclosure also provides remodeled microbes and compositions thereof. The utilization of remodeled microbes and compositions thereof will enable farmers to realize more productive and predictable crop yields without the nutrient degradation, leaching, or toxic runoff associated with traditional synthetically derived fertilizers.

1 . A guided microbial remodeling method for the rational improvement of plant-associated microbes to perform plant-beneficial functions, comprising:
 a. providing a plurality of microbial species that are associated with a target plant of interest;   b. assaying the plurality of microbial species for: colonization metrics, and ability to perform a plant-beneficial function of interest;   c. selecting a candidate microbial species from the plurality of assayed microbial species;   d. introducing one or more targeted non-intergeneric genetic variations into the candidate microbial species;   e. confirming integration of the non-intergeneric genetic variation at the target genomic locus and absence of any transgenetic sequence; and   f. repeating steps d) and e) one or more times, until the candidate microbial species has acquired an improved ability to perform the plant-beneficial function of interest.   
     
     
         2 . The guided microbial remodeling method according to  claim 1 , wherein the step b) comprises assaying the plurality of microbial species for transcriptionally active genes under metabolically relevant environmental conditions. 
     
     
         3 . The guided microbial remodeling method according to  claim 1 , comprising a step of sequencing the genomes of the plurality of microbial species obtained in step a) and characterizing one or more genomic pathways, or sets of genes, that are associated with a plant-beneficial function of interest. 
     
     
         4 . The guided microbial remodeling method according to  claim 1 , wherein the step d) of introducing one or more targeted non-intergeneric genetic variations into the candidate microbial species comprises:
 a. transforming the candidate microbial species with a transformation plasmid comprising (i) a selection marker, (ii) a counterselection marker, (iii) a DNA fragment comprising: a non-intergeneric genetic variation to be introduced into the candidate microbial species at a target genomic locus in one or more genomic pathways, or sets of genes, that are associated with a plant-beneficial function of interest, and homology arms to the target genomic locus flanking the non-intergeneric genetic variation, and (iv) plasmid backbone;   b. selecting for a candidate microbial species that has undergone an initial homologous recombination and has the non-intergeneric genetic variation integrated into the target genomic locus based on the presence of the selection marker in the genome; and   c. selecting for a candidate microbial species that has the non-intergeneric genetic variation integrated into the target genomic locus, but has undergone an additional homologous recombination that loops-out the plasmid backbone, based on the absence of the counterselection marker.   
     
     
         5 . The guided microbial remodeling method according to  claim 1 , wherein the step e) of confirming integration of the non-intergeneric genetic variation at the target genomic locus and absence of any transgenetic sequence comprises sequencing the genome of the transformed candidate microbial species. 
     
     
         6 . (canceled) 
     
     
         7 . The guided microbial remodeling method according to  claim 1 , wherein the plant-beneficial function of interest is nitrogen fixation, phosphate solubilizatoin, or microbial colonization. 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . The guided microbial remodeling method according to  claim 3 , wherein the step of sequencing comprises whole genome sequencing. 
     
     
         11 . The guided microbial remodeling method according to  claim 3 , comprising characterizing the nitrogen fixation pathway. 
     
     
         12 . (canceled) 
     
     
         13 . The guided microbial remodeling method according to  claim 3 , comprising characterizing one or more genes involved in a pathway selected from the group consisting of: exopolysaccharide production, endo-polygalaturonase production, trehalose production, and glutamine conversion. 
     
     
         14 . (canceled) 
     
     
         15 . The guided microbial remodeling method according to  claim 3 , comprising characterizing one or more genes selected from the group consisting of: nifA, nifL, ntrB, ntrC, polynucleotide encoding glutamine synthetase, glnA, glnB, glnK, drat, amtB, polynucleotide encoding glutaminase, glnD, glnE, nifJ, nifH, nifD, nifK, nifY, nifE, nifN, nifU, nifS, nifV, nifW, nifZ, nifM, nifF, nifB, nifQ, a gene associated with biosynthesis of a nitrogenase enzyme, bcsii, bcsiii, yjbE, fhaB, pehA, otsB, treZ, glsA2, and combinations thereof. 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . The guided microbial remodeling method according to  claim 1 , wherein step b) comprises assaying the plurality of microbial species for colonization metrics under greenhouse conditions, lab based conditions, and/or under field conditions. 
     
     
         19 . The guided microbial remodeling method according to  claim 1 , wherein a colonization metric assayed in step b) comprises at least one of the following: spatial colonization patterns, temporal colonization dynamics, density of colonization, or combinations thereof. 
     
     
         20 . (canceled) 
     
     
         21 . The guided microbial remodeling method according to  claim 2 , wherein assaying the plurality of microbial species for transcriptionally active genes under metabolically relevant environmental conditions occurs in vitro, under greenhouse, or lab based, or field conditions and comprises measuring the transcriptomic profile of the microbial species. 
     
     
         22 . The guided microbial remodeling method according to  claim 2 , wherein assaying the plurality of microbial species for transcriptionally active genes under metabolically relevant environmental conditions occurs in vitro, under greenhouse, or lab based, or field conditions and comprises measuring the transcriptomic activity of genes associated with the microbial species ability to perform a plant-beneficial function of interest. 
     
     
         23 . The guided microbial remodeling method according to  claim 2 , wherein assaying the plurality of microbial species for transcriptionally active genes under metabolically relevant environmental conditions occurs under greenhouse or lab based, or field conditions and comprises at least one of:
 measuring the transcriptomic activity of regulatory gene sequences,   measuring the transcriptomic activity of promotor sequences;   measuring the transcriptomic activity of promotor sequences in the presence of exogenous nitrogen, and   measuring the transcriptomic activity of promoter sequences in the presence of exogenous nitrogen, wherein said transcriptomic activity of the promoter sequences is measured by quantifying the expression of a regulated gene.   
     
     
         24 .- 36 . (canceled) 
     
     
         37 . The guided microbial remodeling method according to  claim 2 , wherein assaying the plurality of microbial species for transcriptionally active genes under metabolically relevant environmental conditions occurs in vitro and comprises at least one of:
 measuring the transcriptomic activity of regulatory gene sequences,   measuring the transcriptomic activity of promotor sequences;   measuring the transcriptomic activity of promotor sequences in N-depleted and N-replete conditions, and   measuring the transcriptomic activity of promoter sequences in N-depleted and N-replete conditions, wherein said transcriptomic activity of the promoter sequences is measured by quantifying the expression of a regulated gene.   
     
     
         38 .- 41 . (canceled) 
     
     
         42 . The guided microbial remodeling method according to  claim 1 , wherein assaying the plurality of microbial species for colonization metrics, comprises: growing said plurality of microbial species in intimate association with a target plant under greenhouse conditions, or lab based conditions, and/or field conditions. 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . The guided microbial remodeling method according to  claim 2 , wherein assaying the plurality of microbial species for transcriptionally active genes under metabolically relevant environmental conditions, comprises: growing said plurality of microbial species in intimate association with a target plant under greenhouse conditions, lab based conditions, or field conditions. 
     
     
         46 . (canceled) 
     
     
         47 . (canceled) 
     
     
         48 . The guided microbial remodeling method according to  claim 1 , wherein step b) comprises assaying the plurality of microbial species for ability to perform a plant-beneficial function of interest under greenhouse or lab based conditions. 
     
     
         49 . The guided microbial remodeling method according to  claim 1 , wherein step b) comprises assaying the plurality of microbial species for nitrogen fixation activity. 
     
     
         50 . The guided microbial remodeling method according to  claim 1 , wherein step b) comprises assaying the plurality of microbial species for nitrogen fixation activity in an acetylene reduction assay or ammonium excretion assay. 
     
     
         51 . (canceled) 
     
     
         52 . The guided microbial remodeling method according to  claim 5 , wherein the sequencing comprises whole genome sequencing. 
     
     
         53 . (canceled) 
     
     
         54 . (canceled) 
     
     
         55 . The guided microbial remodeling method according to  claim 1 , wherein in step d), said non-intergeneric genetic variations are selected from the group consisting of: full gene deletions, partial gene deletions, promoter insertions, single base pair changes, and combinations thereof. 
     
     
         56 . (canceled) 
     
     
         57 . (canceled) 
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . (canceled) 
     
     
         61 . A computationally guided microbial remodeling method for the rational improvement of plant-associated microbes to perform plant-beneficial functions, comprising:
 a. accessing a plurality of microbial whole genome sequences;   b. identifying a plurality of regulatory gene sequences that actively regulate the transcription of a gene under a metabolically relevant environmental condition;   c. identifying a plurality of genes associated with a plant-beneficial function;   d. selecting a regulatory gene sequence and a gene associated with a plant-beneficial function from said pluralities; wherein steps a)-d) occur in silico; and   e. manufacturing, in vivo, a remodeled microbial cell that has the selected regulatory gene sequence operably linked to the selected gene associated with a plant-beneficial function, thereby improving the expression of the gene associated with a plant-beneficial function.   
     
     
         62 . A computationally guided microbial remodeling system for the rational improvement of plant-associated microbes to perform plant-beneficial functions, comprising:
 a. one or more processors; and   b. one or more memories operatively coupled to the one or more processors and having instructions stored thereon, that when executed by the one or more processors, cause the system to:
 i. access a plurality of microbial whole genome sequences; 
 ii. identify a plurality of regulatory gene sequences that actively regulate the transcription of a gene under a metabolically relevant environmental condition; 
 iii. identify a plurality of genes associated with a plant-beneficial function; and 
 iv. select a regulatory gene sequence and a gene associated with a plant-beneficial function from said pluralities. 
   
     
     
         63 . A computationally guided microbial remodeling method for the rational improvement of plant-associated microbes to perform plant-beneficial functions, comprising:
 a. activating a computer system having: one or more processors and one or more memories operatively coupled to the one or more processors and having instructions stored thereon, thereby causing the one or more processors to execute the instructions, and cause the system to:
 i. access a plurality of microbial whole genome sequences; 
 ii. identify a plurality of regulatory gene sequences that actively regulate the transcription of a gene under a metabolically relevant environmental condition; 
 iii. identify a plurality of genes associated with a plant-beneficial function; 
 iv. select a regulatory gene sequence and a gene associated with a plant-beneficial function from said pluralities; and 
   b. manufacturing, in vivo, a remodeled microbial cell that has the selected regulatory gene sequence operably linked to the selected gene associated with a plant-beneficial function, thereby improving the expression of the gene associated with a plant-beneficial function.