METHODS AND MICROORGANISMS FOR MAKING 1,4-BUTANEDIOL AND DERIVATIVES THEREOF FROM C1 CARBONS
Genetically modified microorganisms that have the ability to convert carbon substrates into chemical products such as 1,4-BDO are disclosed. For example, genetically modified methanotrophs that are capable of generating 1,4-BDO at high titers from a methane source are disclosed. Methods of making these genetically modified microorganisms and methods of using them are also disclosed.
1 - 141 . (canceled)
142 . A method of making succinate comprising:
(a) contacting a C1 carbon with a genetically-modified microorganism that comprises a heterologous fumarate hydratase and/or a heterologous fumarate reductase; and
(b) growing the genetically microorganism to produce succinate.
143 . The method of claim 142 , wherein the genetically microorganism comprises a pyruvate dehydrogenase (aceEF), citrate synthase (gltA), aconitate hydratase 1 (acnA), and/or isocitrate dehydrogenase (icdA).
144 . The method of claim 142 , wherein the succinate from (b) is isolated.
145 . The method of claim 142 , wherein the succinate is converted into 1,4-BDO.
146 . The method of claim 142 , wherein the genetically-modified microorganism is grown for at least 96 hours.
147 . The method of claim 142 , wherein the fumarate hydratase comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 45, 47, and 49.
148 . The method of claim 142 , wherein the fumarate hydratase comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NOs: 45, 47, and 49.
149 . The method of claim 142 , wherein the fumarate hydratase comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NOs: 45, 47, and 49.
150 . The method of claim 142 , wherein the fumarate hydratase comprises the amino acid sequence of any one of SEQ ID NOs: 45, 47, and 49.
151 . The method of claim 142 , wherein the fumarate reductase is from a bacteria.
152 . The method of claim 142 , wherein the fumarate reductase comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NOs: 51, 53, 55, and 57.
153 . The method of claim 142 , wherein the fumarate reductase comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NOs: 51, 53, 55, and 57.
154 . The method of claim 142 , wherein the fumarate reductase comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NOs: 51, 53, 55, and 57.
155 . The method of claim 142 , wherein the fumarate reductase comprises the amino acid sequence of any one of SEQ ID NOs: 51, 53, 55, and 57.
156 . The method of claim 142 , wherein the genetically-modified microorganism is a methanotroph.
157 . The method of claim 142 , wherein the genetically-modified microorganism is from the genus Methylococcus.
158 . The method of claim 142 , wherein the genetically modified microorganism is an M. capsulatus.
159 . The method of claim 142 , wherein the genetically-modified microorganism comprises a heterologous fumarate reductase gene under the control of a pMxaF, J23111, or J23100 promoter.
160 . The method of claim 142 , wherein the genetically-modified microorganism comprises a heterologous fumarate reductase gene under the control of a J23111 or J23100 promoter.
161 . The method of claim 142 , wherein the genetically modified microorganism comprises a heterologous fumarate reductase gene under the control of a J23111 promoter.