Compositions and methods for recovery of stranded gas and oil

Abstract

The present disclosure provides compositions and methods for using recombinant C1 metabolizing microorganisms capable of metabolizing sulfur containing compounds and other contaminants to biologically convert sour or acidic natural gas into high-value molecules, and to allow recovery of stranded oil.

1 .- 125 . (canceled) 
     
     
         126 . A recombinant methanotrophic or methylotrophic bacterium, comprising:
 (a) a first heterologous nucleic acid molecule encoding a polypeptide capable of metabolizing an S substrate selected from:
 (i) a hydrogen sulfide:NADP +  oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, sulfide: quinone oxidoreductase, sulfur dioxygenase, sulfite oxidase, or any combination thereof; 
 (ii) a hydrogen sulfide:NADP +  oxidoreductase, sulfite oxidase, or both; 
 (iii) a hydrogen sulfide:ferredoxin oxidoreductase, sulfite oxidase, or both; 
 (iv) a sulfide:flavocytochrome-c oxidoreductase, sulfite oxidase, or both; 
 (v) a sulfide:quinone oxidoreductase, sulfite oxidase, or both; and 
 (vi) a hydrogen sulfide:NADP +  oxidoreductase, hydrogen sulfide:ferredoxin oxidoreductase, sulfide: flavocytochrome-c oxidoreductase, or sulfide: quinone oxidoreductase, wherein endogenous sulfite oxidase activity of the bacterium is increased; and 
   (b) a second heterologous nucleic acid molecule encoding a cysteine biosynthesis pathway, homocysteine biosynthesis pathway, or a methionine biosynthesis pathway, wherein:
 (i) the cysteine biosynthesis pathway comprises a cysteine synthase; 
 (ii) the homocysteine biosynthesis pathway comprises an O-acetylhomoserine sulfhydrylase; and 
 (iii) the methionine biosynthesis pathway comprises an O-acetylhomoserine sulfhydrylase and a methionine synthase or a homocysteine methyltransferase; 
   wherein the recombinant methanotrophic or methylotrophic bacterium is capable of converting a feedstock comprising the S substrate and a C 1  substrate into the sulfur-containing amino acid and is capable of oxidizing or assimilating an increased amount of the S substrate as compared to wild-type methanotrophic or methylotrophic bacterium.   
     
     
         127 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 126 , wherein the methanotrophic or methylotrophic bacterium is a methanotrophic bacterium. 
     
     
         128 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 127 , wherein the methanotrophic bacterium is an obligate methanotrophic bacterium. 
     
     
         129 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 127 , wherein the methanotrophic bacterium is  Methylococcus capsulatus  Bath,  Methylomonas  16a,  Methylosinus trichosporium  OB3b,  Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus, Methylobacterium organophilum, Methylomonas  sp. AJ-3670,  Methylocella silvestris, Methylocella palustris, Methylocella tundrae, Methylocystis daltona  strain SB2,  Methylocystis bryophila, Methylocapsa aurea  KYG,  Methylacidiphilum infernorum,  or  Methylomicrobium alcaliphilum.    
     
     
         130 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 127 , wherein the methanotrophic bacterium is  Methylococcus capsulatus  Bath,  Methylosinus trichosporium  OB3b,  Methylomonas  sp. 16a,  Methylomicrobium alcaliphilum,  or a high growth variant thereof. 
     
     
         131 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 127 , wherein the recombinant methanotrophic bacterium is in a culture further comprising a heterologous bacterium. 
     
     
         132 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 126 , wherein the methanotrophic or methylotrophic bacterium is a methylotrophic bacterium. 
     
     
         133 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 132 , wherein the methylotrophic bacterium is  Methylobacterium extorquens, Methylobacterium radiotolerans, Methylobacterium populi, Methylobacterium chloromethanicum,  or  Methylobacterium nodulans.    
     
     
         134 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 126 , wherein the polypeptide capable of metabolizing the S substrate:
 (a) is encoded by a nucleic acid comprising the polynucleotide sequence as set forth in any one of SEQ ID NOS.:21-54;   (b) comprises the amino acid sequence as set forth in any one of SEQ ID NOS.:55-88;   (c) is a sulfur oxygenase; or (d) is a sulfur oxygenase having an amino acid sequence that is at least 75% identical to the sequence set forth in Genbank Accession No. AAK58572.1 or ABN04222.1, or a functional fragment thereof.   
     
     
         135 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 126 , wherein the second heterologous nucleic acid molecule encodes the cysteine biosynthesis pathway comprising the cysteine synthase. 
     
     
         136 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 126 , wherein the second heterologous nucleic acid molecule encodes the homocysteine biosynthesis pathway comprising the O-acetylhomoserine sulfhydrylase. 
     
     
         137 . The recombinant methanotrophic or methylotrophic bacterium according to  claim 126 , wherein the second heterologous nucleic acid molecule encodes the methionine biosynthesis pathway comprising the O-acetylhomoserine sulfhydrylase and the methionine synthase or the homocysteine methyltransferase. 
     
     
         138 . A method for producing a sulfur-containing amino acid from a tainted gas, the method comprising culturing a recombinant methanotrophic bacterium or methylotrophic bacterium according to  claim 126  with a tainted gas feedstock,
 wherein the tainted gas feedstock comprises methane and an S substrate; 
 wherein the methanotrophic bacterium or methylotrophic bacterium assimilates and oxidizes the methane and the S substrate, and converts the S substrate into the sulfur-containing amino acid selected from cysteine, homocysteine, or methionine. 
 
     
     
         139 . The method according to  claim 138 , wherein the tainted gas feedstock comprises natural gas, unconventional natural gas, casinghead gas, wellhead condensate, or any combination thereof. 
     
     
         140 . The method according to  claim 138 , wherein the recombinant methanotrophic bacterium or methylotrophic bacterium is a methanotrophic bacterium selected from  Methylococcus capsulatus  Bath,  Methylosinus trichosporium  OB3b,  Methylomonas  sp. 16a,  Methylomicrobium alcaliphilum,  or a high growth variant thereof. 
     
     
         141 . A system for producing a sulfur-containing amino acid from a tainted gas, comprising:
 a source of tainted gas comprising methane and an S substrate;   a bioreactor comprising a recombinant methanotrophic bacterium or methylotrophic bacterium according to  claim 126 ;   wherein the recombinant methanotrophic bacterium or methylotrophic bacterium cultured in the bioreactor converts the methane and the S substrate of the tainted gas into a sulfur-containing amino acid selected from cysteine, homocysteine, or methionine.