Biological production of multi-carbon compounds from methane
Multi-carbon compounds such as ethanol, n-butanol, sec-butanol, isobutanol, tert-butanol, fatty (or aliphatic long chain) alcohols, fatty acid methyl esters, 2,3-butanediol and the like, are important industrial commodity chemicals with a variety of applications. The present invention provides metabolically engineered host microorganisms which metabolize methane (CH 4 ) as their sole carbon source to produce multi-carbon compounds for use in fuels (e.g., bio-fuel, bio-diesel) and bio-based chemicals. Furthermore, use of the metabolically engineered host microorganisms of the invention (which utilize methane as the sole carbon source) mitigate current industry practices and methods of producing multi-carbon compounds from petroleum or petroleum-derived feedstocks, and ameliorate much of the ongoing depletion of arable food source “farmland” currently being diverted to grow bio-fuel feedstocks, and as such, improve the environmental footprint of future bio-fuel, bio-diesel and bio-based chemical compositions.
1. A genetically modified methanotroph comprising a heterologous polynucleotide encoding for an alcohol dehydrogenase (ADH), wherein the alcohol dehydrogenase can catalyze the conversion of isobutyraldehyde to isobutanol and comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 10, and wherein said methanotroph is capable of converting formaldehyde to pyruvate through a type I RuMP pathway or a type II serine pathway.
2. The methanotroph of claim 1 , wherein the methanotroph further comprises a heterologous polynucleotide encoding a ketoacid decarboxylase (KDC), wherein the ketoacid decarboxylase can catalyze the conversion of ketoisovalerate to isobutryaldehyde.
3. The methanotroph of claim 2 , wherein the methanotroph further comprises a heterologous polynucleotide encoding an acetolactate synthase (ALS), a heterologous polynucleotide encoding a ketol-acid reductoisomerase (KARI), and a heterologous polynucleotide encoding a dihydroxy-acid dehydratase (DHAD); wherein the acetolactate synthase can catalyze the conversion of pyruvate to acetolactate, wherein the ketol-acid reductoisomerase can catalyze the conversion of acetolactate to 2,3-dihydroxyisovalerate, and wherein the dihydroxy-acid dehydratase can catalyze the conversion of 2,3-dihydroxyisovalerate to ketoisovalerate.
4. The methanotroph of claim 3 , wherein the methanotroph further comprises a heterologous polynucleotide encoding a ketoacid decarboxylase (KDC), wherein the ketoacid decarboxylase can catalyze the conversion of ketoisovalerate to isobutryaldehyde.
5. The methanotroph of claim 4 , wherein the ketoacid decarboxylase (KDC) comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 8.
6. The methanotroph of claim 1 , wherein the methanotroph further comprises a heterologous polynucleotide encoding acetolactate synthase (ALS), a heterologous polynucleotide encoding a ketol-acid reductoisomerase (KARI), heterologous polynucleotide encoding a dihydroxy-acid dehydratase (DHAD), and a heterologous polynucleotide encoding a ketoacid decarboxylase (KDC); wherein the acetolactate synthase can catalyze the conversion of pyruvate to acetolactate and comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 2, wherein the ketol-acid reductoisomerase can catalyze the conversion of acetolactate to 2,3-dihydroxyisovalerate and comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 4, wherein the dihydroxy-acid dehydratase can catalyze the conversion of 2,3-dihydroxyisovalerate to ketoisovalerate and comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 6, and wherein the ketoacid decarboxylase can catalyze the conversion of ketoisovalerate to isobutryaldehyde and comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 8.
7. The methanotroph of claim 1 , wherein the methanotroph further comprises a polynucleotide sequence encoding for the alcohol dehydrogenase (ADH) and a promoter, wherein said promoter can direct the expression of the alcohol dehydrogenase in the methanotroph.
8. The methanotroph of claim 2 , wherein the methanotroph further comprises a polynucleotide sequence encoding for the ketoacid decarboxylase (KDC), the alcohol dehydrogenase (ADH), and a promoter, wherein said promoter can direct the expression of the ketoacid decarboxylase and the alcohol dehydrogenase (ADH) in the methanotroph.
9. The methanotroph of claim 3 , wherein the methanotroph further comprises a polynucleotide sequence encoding for the acetolactate synthase (ALS), the ketol-acid reductoisomerase (KARI), the dihydroxy-acid dehydratase (DHAD), the ketoacid decarboxylase (KDC), the alcohol dehydrogenase (ADH) and a promoter, wherein said promoter can direct the expression of the acetolactate synthase (ALS), the ketol-acid reductoisomerase (KARI), the dihydroxy-acid dehydratase (DHAD), the ketoacid decarboxylase (KDC), and the alcohol dehydrogenase (ADH) in a methanotroph.
10. The methanotroph of claim 9 , wherein said promoter is constitutive.
11. The methanotroph of claim 9 , wherein said promoter is inducible.
12. The methanotroph of claim 1 , wherein said methanotroph is from the genus Methylobacter, Methylomicrobium, Methylomonas, Methylocaldum, Methylococcus, Methylosoma, Methylosarcina, Methylothermus, Methylohalobius, Methylogaea, Methylovulum, Crenothrix , Clonothrix, Methylosphaera, Methylocapsa, Methylocella, Methylosinus, Methylocystis , or Methyloacidophilum.
13. The methanotroph of claim 1 , wherein said methanotroph is from the genus Methylococcus.
14. The methanotroph of claim 1 , wherein said methanotroph is from the species Methylococcus capsulatus.
15. The methanotroph of claim 1 , wherein said methanotroph is from the strain Methylococcus capsulatus strain Bath.
16. A method of making a multi-carbon compound comprising:
(a) contacting a genetically modified methanotroph with a multi-carbon product precursor comprising a heterologous polynucleotide encoding for an alcohol dehydrogenase (ADH), wherein the ADH can catalyze the conversion of isobutyraldehyde to isobutanol and comprises an amino acid sequence having at least 90% sequence homology to SEQ ID NO: 10, and wherein said methanotroph is capable of converting formaldehyde to pyruvate through a type I RuMP pathway or a type II serine pathway; and
(b) growing said methanotroph in conditions to produce a multi-carbon compound.
17. The method of claim 15 wherein said precursor is methane.
18. The method of claim 16 wherein said multi-carbon compound is isobutanol.
19. The method of claim 16 wherein said multi-carbon product is 1-butanol.