METHODS AND MICROORGANISMS FOR MAKING 2,3-BUTANEDIOL AND DERIVATIVES THEREOF FROM C1 CARBONS
Genetically modified microorganisms that have the ability to convert carbon substrates into chemical products such as 2,3-BDO are disclosed. For example, genetically modified methanotrophs that are capable of generating 2,3-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 . (canceled)
2 . The microorganism of claim 128 , comprising: (a) a heterologous acetoin reductase; (b) a heterologous alpha-acetolactate decarboxylase; and/or (c) a heterologous acetolactate synthase.
3 - 5 . (canceled)
6 . The microorganism of claim 2 , wherein the heterologous acetolactate synthase is non-constitutively expressed.
7 . The microorganism of claim 2 , wherein: (a) the heterologous acetolactate synthase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 19; (b) the heterologous alpha-acetolactate decarboxylase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7; and/or (c) the heterologous acetoin reductase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9.
8 . (canceled)
9 . The microorganism of claim 2 , wherein the acetoin reductase is NADPH-dependent.
10 - 13 . (canceled)
14 . The microorganism of claim 128 , wherein the microorganism is a methanotroph.
16 - 16 . (canceled)
17 . The microorganism of claim 128 , wherein the microorganism is Methylococcus capsulatus.
18 - 31 . (canceled)
32 . A vector comprising two or more genes encoding an acetoin reductase, an alpha-acetolactate decarboxylase, and/or an acetolactate synthase.
33 - 41 . (canceled)
42 . A method of making the microorganism of claim 128 , the method comprising transforming a microorganism with a heterologous nucleic acid encoding an acetoin reductase, an alpha-acetolactate decarboxylase, and/or an acetolactate synthase.
43 - 62 . (canceled)
63 . A method of making 2,3-BDO, the method comprising:
a) contacting the microorganism of claim 2 with a C1 carbon; and
b) growing the said microorganism to produce 2,3-BDO.
64 - 69 . (canceled)
70 . The method of claim 63 , wherein the C1 carbon is methane.
71 . The method of claim 63 , wherein the microorganism is grown at a temperature between 32° C. and 49° C.
72 - 74 . (canceled)
75 . The method of claim 63 , further comprising contacting the microorganism with media containing at least 1 μM lanthanum before growing the microorganism to produce 2,3-BDO.
76 . The method of claim 75 , further comprising diluting the lanthanum in media before growing the microorganism to produce 2,3-BDO.
77 . The method of claim 63 , wherein the microorganism comprises a heterologous gene encoding an alpha-acetolactate decarboxylase and is grown to produce acetoin.
78 - 88 . (canceled)
89 . The method of claim 63 , further comprising:
contacting the 2,3-BDO with a catalyst to produce butadiene or methyl ethyl ketone.
90 - 127 . (canceled)
128 . A genetically modified microorganism capable of converting a C1 carbon to 2,3-BDO, the microorganism comprising at least one heterologous gene encoding; (a) an acetoin reductase; (b) an alpha-acetolactate decarboxylase; and/or (c) an acetolactate synthase.
129 . (canceled)
130 . The microorganism of claim 128 , wherein the heterologous gene(s) is/are integrated by an integration vector into the genome of said microorganism.
131 . The microorganism of claim 128 , wherein the heterologous gene(s) is/are expressed on an episomal vector.
132 . The microorganism of claim 128 comprising:
(a) a heterologous gene encoding an acetolactate synthase that is 5′ in relation to any other heterologous gene;
(b) a heterologous gene encoding an acetoin reductase that is 3′ in relation to any other heterologous gene; and/or
(c) a heterologous gene encoding an alpha-acetolactate decarboxylase that is neither 5′ or 3′ in relation to any other heterologous gene.
133 - 134 . (canceled)
135 . The microorganism of claim 128 , wherein the heterologous gene(s) is/are under the control of a switch.
136 - 146 . (canceled)