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. A genetically modified microorganism capable of converting a Cl carbon to 2,3-BDO, wherein the microorganism is from the species Methylococcus capsulatus and comprises at least one heterologous gene encoding: i) an acetoin reductase; ii) an alpha-acetolactate decarboxylase; and/or iii) an acetolactate synthase.
2. The microorganism of claim 1 , comprising: (i) a heterologous acetoin reductase; (ii) a heterologous alpha-acetolactate decarboxylase; and/or (iii) a heterologous acetolactate synthase.
3. The microorganism of claim 2 , wherein the acetolactate synthase is non-constitutively expressed.
4. The microorganism of claim 2 , wherein: (i) the acetolactate synthase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 19; (ii) the alpha-acetolactate decarboxylase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7; and/or (iii) the acetoin reductase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 9.
5. The microorganism of claim 2 , wherein the acetoin reductase is NADPH-dependent.
6. The microorganism of claim 1 , wherein the heterologous gene(s) is/are integrated by an integration vector into the genome of said microorganism.
7. The microorganism of claim 1 , wherein the heterologous gene(s) is/are expressed on an episomal vector.
8. The microorganism of claim 1 comprising:
(a) a heterologous gene encoding acetolactate synthase that is 5′ in relation to any other heterologous gene;
(b) a heterologous gene encoding acetoin reductase that is 3′ in relation to any other heterologous gene; and/or
(c) a heterologous gene encoding alpha-acetolactate decarboxylase that is neither 5′ or 3′ in relation to any other heterologous gene.
9. The microorganism of claim 1 , wherein the heterologous gene(s) is/are under the control of a switch.
10. The microorganism of claim 1 , wherein: (i) the acetolactate synthase comprises an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 1, 3, or 19; (ii) the alpha-acetolactate decarboxylase comprises an amino acid sequence at least 90% identical to SEQ ID NO:
5 or 7; and/or (iii) the acetoin reductase comprises an amino acid sequence at least 90% identical to any one of SEQ ID NO: 9, 11, or 13.
11. A method of making the microorganism of claim 1 , the method comprising transforming the microorganism with a heterologous nucleic acid comprising a gene encoding an acetoin reductase, an alpha-acetolactate decarboxylase, and/or an acetolactate synthase.
12. A method of making 2,3-BDO, the method comprising:
a) contacting the microorganism of claim 1 with a Cl carbon; and
b) growing the microorganism to produce 2,3-BDO.
13. The method of claim 12 , wherein the Cl carbon is methane.
14. The method of claim 12 , wherein the microorganism is grown at a temperature between 32° C. and 49° C.
15. The method of claim 12 , further comprising contacting the microorganism with media containing at least 1 μM lanthanum before growing the microorganism to produce 2,3-BDO.
16. The method of claim 15 , further comprising diluting the lanthanum in media before growing the microorganism to produce 2,3-BDO.
17. The method of claim 12 , wherein the microorganism comprises a heterologous gene encoding alpha-acetolactate decarboxylase and is grown to produce acetoin.
18. The method of claim 12 , further comprising contacting the 2,3-BDO with a catalyst to produce butadiene or methyl ethyl ketone.