Carbohydrate-enriched recombinant microorganisms

Abstract

The present disclosure relates to recombinant microorganisms engineered for enhanced production of a desired carbohydrate, as well as related biomass, and compositions which are useful, inter alia, as animal feed ingredients. The present disclosure also provides related methods.

What is claimed is: 
     
         1 . A recombinant methanotrophic bacterium, comprising an exogenous nucleic acid that encodes a gluconeogenesis enzyme,
 wherein the encoded gluconeogenesis enzyme is one or more of a pyruvate carboxylase, a phosphoenolpyruvate carboxykinase, an enolase, a phosphoglycerate mutase, a phosphoglycerate kinase, a glyceraldehyde-3-phosphate dehydrogenase, a Type A aldolase, a fructose 1,6-bisphosphatase, a phosphofructokinase, a phosphoglucose isomerase, a hexokinase, and a glucose-6-phosphate,   wherein the methanotrophic bacterium is selected from the group consisting of  Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylocystis, Methylomicrobium, Methanomonas , and  Methylocella , and   wherein the recombinant methanotrophic bacterium grown on a C 1  substrate feedstock is capable of producing glucose at a level that is greater than that produced by the parent methanotrophic bacterium.   
     
     
         2 . The recombinant methanotrophic bacterium of  claim 1 , wherein the C 1  substrate feedstock comprises natural gas or methane. 
     
     
         3 . The recombinant methanotrophic bacterium of  claim 1 , wherein the methanotrophic bacterium is an obligate methanotrophic bacterium. 
     
     
         4 . The recombinant methanotrophic bacterium of  claim 1 , wherein the methanotrophic bacterium is a facultative methanotrophic bacterium. 
     
     
         5 . The recombinant methanotrophic bacterium of  claim 1 , wherein the methanotrophic bacterium is selected from the group consisting of  Methylococcus capsulatus  Bath,  Methylomonas  sp. 16a,  Methylosinus trichosporium  OB3b,  Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus  Y,  Methylomonas flagellata  AJ-3670,  Methylacidiphilum infernorum, Methylacidiphdum fumariolicum, Methylomicrobium alcaliphdum , and  Methyloacida kamchatkensis.    
     
     
         6 . The recombinant methanotrophic bacterium of  claim 1 , wherein the exogenous nucleic acid encoding the gluconeogenesis enzyme is endogenous to an organism selected from the group consisting of a bacterium, a yeast, a fungi, and a plant. 
     
     
         7 . The recombinant methanotrophic bacterium of  claim 1 , wherein the exogenous nucleic acid comprises an expression control sequence that is operably linked to the nucleic acid encoding the gluconeogenesis enzyme. 
     
     
         8 . The recombinant methanotrophic bacterium of  claim 1 , wherein the exogenous nucleic acid encoding the gluconeogenesis enzyme is endogenous to  Escherichia coli  or  Corynebacterium glutamicum.    
     
     
         9 . The recombinant methanotrophic bacterium of  claim 1 , wherein the exogenous nucleic acid encoding the gluconeogenesis enzyme is endogenous to  Saccharomyces cerevisiae.    
     
     
         10 . The recombinant methanotrophic bacterium of  claim 1 , wherein the exogenous nucleic acid encoding the gluconeogenesis enzyme is codon optimized for the methanotrophic bacterium. 
     
     
         11 . The recombinant methanotrophic bacterium of  claim 1 , wherein the carbohydrates of the recombinant methanotrophic bacterium exhibits a δ 13 C that is less than −30‰ or is less than −40‰. 
     
     
         12 . The recombinant methanotrophic bacterium of  claim 1 , wherein the methanotrophic bacterium further comprises an exogenous polynucleotide encoding one or more glycogenesis enzymes selected from the group consisting of a glucose-1-phosphate adenyltransferase, a glycogen synthase, and a 1,4-alpha-glucan-branching protein. 
     
     
         13 . The recombinant methanotrophic bacterium of  claim 12 , wherein the further exogenous polynucleotide encoding one or more glycogenesis enzymes comprises an expression control sequence that is operably linked to the polynucleotide encoding the one or more glycogenesis enzymes, wherein the one or more glycogenesis enzymes are heterologous glycogenesis enzymes, native glycogenesis enzymes, or a combination thereof. 
     
     
         14 . The recombinant methanotrophic bacterium of  claim 12 , wherein the exogenous nucleic acid encoding the one or more glycogenesis enzymes is codon optimized for the methanotrophic bacterium. 
     
     
         15 . A biomass derived from whole and/or lysed cells of the recombinant methanotrophic bacterium of  claim 1 . 
     
     
         16 . A carbohydrate composition, comprising carbohydrates extracted from a biomass derived from the methanotrophic bacterium of  claim 1 , wherein the composition exhibits a δ 13 C that is less than −30‰ or is less than −40‰. 
     
     
         17 . An animal feed, comprising the recombinant methanotrophic bacterium of  claim 1 , a biomass derived from whole and/or lysed cells of the recombinant methanotrophic bacterium of  claim 1 , or a carbohydrate composition comprising carbohydrates extracted from a biomass derived from the recombinant methanotrophic bacterium of  claim 1 . 
     
     
         18 . The animal feed of  claim 17 , further comprising an additive selected from the group consisting of a plant-derived material, an animal-derived material, and a microorganism-derived material. 
     
     
         19 . The animal feed of  claim 18 , wherein the additive is plant-derived material and the plant-derived material is selected from the group consisting of corn, soybean meal, pea protein, or a combination thereof. 
     
     
         20 . The animal feed of  claim 18 , wherein the additive is an animal-derived material and the animal-derived material is fish meal. 
     
     
         21 . A method of producing a desired carbohydrate, the method comprising culturing the recombinant methanotrophic bacterium of  claim 1  in the presence of a C 1  substrate feedstock comprising methane under conditions sufficient to produce the glucose. 
     
     
         22 . The method of  claim 21 , wherein the natural gas-derived carbon feedstock is natural gas. 
     
     
         23 . The method of  claim 21 , wherein the methanotrophic bacterium is selected from the group consisting of  Methylococcus capsulatus  Bath,  Methylomonas  sp. 16a,  Methylosinus trichosporium  OB3b,  Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus  Y,  Methylomonas flagellata  AJ-3670,  Methylacidiphilum infernorum, Methylacidiphilum fumariolicum, Methylomicrobium alcaliphilum , and  Methyloacida kamchatkensis.