Microbial Conversion of Oils and Fatty Acids to High-Value Chemicals
Microorganisms for the production of high-value chemicals from free fatty acids are provided. The microorganisms comprise genetic mutations that alter fatty acid metabolism. The genetic mutations include a mutation or deletion of a fadR gene in which the FadR enzyme activity is partially or substantially eliminated and a mutation in an atoC gene that provides overexpression of the microorganism's ato operon. Methods of using the microorganisms to produce high-value chemicals are also provided. The high-value chemicals include ethanol, methyl acetate, succinate, gamma-butyrolactone, 1,4-butanediol, acetone, iso-propanol, butyrate, butanol, mevalonate, propionate, ethanolamine and 1,2-propanediol.
1 .- 138 . (canceled)
139 . A microorganism comprising:
a) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes; and
iii) a nucleic acid sequence encoding an alcohol dehydrogenase protein that is active under aerobic conditions;
b) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes; and
(1) increased expression of an acetate kinase gene or a phosphate acetyltransferase gene;
(2) increased expression of an ADP-forming acetyl-CoA synthetase activity; or
(3) expression of an acetyl-CoA synthetase activity that functions primarily to produce acetate from acetyl-CoA;
c) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function; and
iv) reduced sdhA or sdhB expression or function;
d) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function;
iv) reduced sdhA or sdhB expression or function;
v) increased expression or function of one or more of succinic semialdehyde dehydrogenase, succinyl-CoA:CoA transferase, or succinate-CoA ligase;
vi) increased gamma-hydroxybutyric acid dehydrogenase expression or function; and
vii) increased lactonase expression or function;
e) 1) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function;
iv) reduced sdhA or sdhB expression or function;
v) increased expression or function of one or more of succinic semialdehyde dehydrogenase, succinyl-CoA:CoA transferase, or succinate-CoA ligase;
vi) increased gamma-hydroxybutyric acid dehydrogenase expression or function;
vii) increased aldehyde dehydrogenase expression or function; and
viii) increased alcohol dehydrogenase expression or function;
f) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function;
iv) reduced sdhA or sdhB expression or function;
v) increased methylmalonyl-CoA mutase expression or function;
vi) increased methylmalonyl-CoA decarboxylase expression or function; and
vii) increased propionyl-CoA:succinate CoA transferase expression or function;
g) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function; and
v) increased acetoacetate decarboxylase expression or function;
h) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function;
v) increased acetoacetate decarboxylase expression or function; and
vi) a gene encoding an acetone monooxygenase;
i) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function;
v) increased acetoacetate decarboxylase expression or function; and
vi) a gene encoding secondary alcohol dehydrogenase.
j) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function;
v) increased acetoacetate decarboxylase expression or function; and
(1) a gene encoding an acetol monooxygenase and a gene encoding a glycerol dehydrogenase; or
(2) a gene encoding an acetol monooxygenase; a gene encoding an acetol kinase; a gene encoding an L-1,2-propanediol-1-phosphate dehydrogenase; and, a gene encoding a glycerol-1-phosphate phosphatase;
k) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased 3-hydroxybutyryl-CoA dehydrogenase expression or function;
v) increased crotonase expression or function;
vi) a gene encoding butyryl-CoA dehydrogenase; and:
(1) increased acetyl-CoA:acetoacetyl-CoA transferase or butyrate-acetoacetate CoA transferase expression or function; or
(2) a gene encoding phosphotransbutyrylase and a gene encoding butyrate kinase;
l) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased 3-hydroxybutyryl-CoA dehydrogenase expression or function;
v) increased crotonase expression or function;
vi) a gene encoding butyryl-CoA dehydrogenase;
vii) a gene encoding butyraldehyde dehydrogenase; and
viii) a gene encoding butanol dehydrogenase or secondary alcohol dehydrogenase;
m) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase; and
v) a gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase; or
n) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetaldehyde dehydrogenase expression or function; and
iv) genes encoding the regulatory and catalytic subunits of ethanolamine ammonia-lyase.
140 . The microorganism of claim 139 , wherein said microorganism is a bacterium.
141 . The microorganism of claim 140 , wherein said microorganism is a member of the genus Escherichia, Lactobacillus, Lactococcus, Bacillus, Paenibacillus, Klebsiella, Citrobacter, Clostridium, or Zymomonas.
142 . The microorganism of claim 139 , wherein said microorganism is a fungus.
143 . The microorganism of claim 142 , wherein said microorganism is a member of the genus Saccharomyces, Pichia, Schizosaccharomyces, Aspergillus, or Neurospora.
144 . The microorganism of claim 139 , wherein said microorganism comprised reduced fadR expression.
145 . The microorganism of claim 139 , wherein said microorganism comprises reduced fadR function.
146 . The microorganism of claim 139 , wherein said microorganism comprises increased expression of the fad regulon.
147 . The microorganism of claim 139 , wherein said microorganism comprises increased expression of the ato operon.
148 . A method of converting free fatty acids to ethanol, acetate, succinate, gamma-butyrolactone, 1,4 butanediol, propionate, acetone, methyl acetate, isopropanol, 1,2 propanediol, butyrate, butanol, mevalonate, or ethanolamine, comprising culturing a microorganism comprising:
a) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes; and
iii) a nucleic acid sequence encoding an alcohol dehydrogenase protein that is active under aerobic conditions,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to ethanol;
b) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes; and
(1) increased expression of an acetate kinase gene or a phosphate acetyltransferase gene;
(2) increased expression of an ADP-forming acetyl-CoA synthetase activity; or
(3) expression of an acetyl-CoA synthetase activity that functions primarily to produce acetate from acetyl-CoA,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to acetate;
c) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function; and
iv) reduced sdhA or sdhB expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to succinate;
d) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function;
iv) reduced sdhA or sdhB expression or function;
v) increased expression or function of one or more of succinic semialdehyde dehydrogenase, succinyl-CoA:CoA transferase, or succinate-CoA ligase;
vi) increased gamma-hydroxybutyric acid dehydrogenase expression or function; and
vii) increased lactonase expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to gamma-butyrolactone;
e) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function;
iv) reduced sdhA or sdhB expression or function;
v) increased expression or function of one or more of succinic semialdehyde dehydrogenase, succinyl-CoA:CoA transferase, or succinate-CoA ligase;
vi) increased gamma-hydroxybutyric acid dehydrogenase expression or function;
vii) increased aldehyde dehydrogenase expression or function; and
viii) increased alcohol dehydrogenase expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to 1,4-butanediol;
f) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function;
iv) reduced sdhA or sdhB expression or function;
v) increased methylmalonyl-CoA mutase expression or function;
vi) increased methylmalonyl-CoA decarboxylase expression or function; and
vii) increased propionyl-CoA:succinate CoA transferase expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to propionate;
g) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function; and
v) increased acetoacetate decarboxylase expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to acetone;
h) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function;
v) increased acetoacetate decarboxylase expression or function; and
vi) a gene encoding an acetone monooxygenase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to methyl acetate;
i) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function;
v) increased acetoacetate decarboxylase expression or function; and
vi) a gene encoding a secondary alcohol dehydrogenase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to isopropanol;
j) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function;
v) increased acetoacetate decarboxylase expression or function; and
(1) a gene encoding an acetol monooxygenase and a gene encoding a glycerol dehydrogenase; or
(2) a gene encoding an acetol monooxygenase; a gene encoding an acetol kinase; a gene encoding an L-1,2-propanediol-1-phosphate dehydrogenase; and, a gene encoding a glycerol-1-phosphate phosphatase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to 1,2-propanediol;
k) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased 3-hydroxybutyryl-CoA dehydrogenase expression or function;
v) increased crotonase expression or function;
vi) a gene encoding butyryl-CoA dehydrogenase; and:
(1) increased acetyl-CoA:acetoacetyl-CoA transferase or butyrate-acetoacetate CoA transferase expression or function; or
(2) a gene encoding phosphotransbutyrylase and a gene encoding butyrate kinase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to butyrate;
l) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased 3-hydroxybutyryl-CoA dehydrogenase expression or function;
v) increased crotonase expression or function;
vi) a gene encoding butyryl-CoA dehydrogenase;
vii) a gene encoding butyraldehyde dehydrogenase; and
viii) a gene encoding butanol dehydrogenase or secondary alcohol dehydrogenase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to butanol;
m) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase; and
v) a gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to mevalonate; or
n) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetaldehyde dehydrogenase expression or function; and
iv) genes encoding the regulatory and catalytic subunits of ethanolamine ammonia-lyase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby converting said free fatty acids to ethanolamine.
149 . A method of producing ethanol, acetate, succinate, gamma-butyrolactone, 1,4 butanediol, propionate, acetone, methyl acetate, isopropanol, 1,2 propanediol, butyrate, butanol, mevalonate, or ethanolamine, comprising culturing a microorganism comprising:
a) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes; and
iii) a nucleic acid sequence encoding an alcohol dehydrogenase protein that is active under aerobic conditions,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing ethanol;
b) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes; and
(1) increased expression of an acetate kinase gene or a phosphate acetyltransferase gene;
(2) increased expression of an ADP-forming acetyl-CoA synthetase activity; or
(3) expression of an acetyl-CoA synthetase activity that functions primarily to produce acetate from acetyl-CoA,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing acetate;
c) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function; and
iv) reduced sdhA or sdhB expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing succinate;
d) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function;
iv) reduced sdhA or sdhB expression or function;
v) increased expression or function of one or more of succinic semialdehyde dehydrogenase, succinyl-CoA:CoA transferase, or succinate-CoA ligase;
vi) increased gamma-hydroxybutyric acid dehydrogenase expression or function; and
vii) increased lactonase expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing gamma-butyrolactone;
e) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function;
iv) reduced sdhA or sdhB expression or function;
v) increased expression or function of one or more of succinic semialdehyde dehydrogenase, succinyl-CoA:CoA transferase, or succinate-CoA ligase;
vi) increased gamma-hydroxybutyric acid dehydrogenase expression or function;
vii) increased aldehyde dehydrogenase expression or function; and
viii) increased alcohol dehydrogenase expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing 1,4-butanediol;
f) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) reduced sucA or sucB expression or function;
iv) reduced sdhA or sdhB expression or function;
v) increased methylmalonyl-CoA mutase expression or function;
vi) increased methylmalonyl-CoA decarboxylase expression or function; and
vii) increased propionyl-CoA:succinate CoA transferase expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing propionate;
g) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function; and
v) increased acetoacetate decarboxylase expression or function,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing acetone;
h) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function;
v) increased acetoacetate decarboxylase expression or function; and
vi) a gene encoding an acetone monooxygenase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing methyl acetate;
i) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function;
v) increased acetoacetate decarboxylase expression or function; and
vi) a gene encoding a secondary alcohol dehydrogenase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing isopropanol;
j) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased acetoacetyl-CoA transferase expression or function;
v) increased acetoacetate decarboxylase expression or function; and
(1) a gene encoding an acetol monooxygenase and a gene encoding a glycerol dehydrogenase; or
(2) a gene encoding an acetol monooxygenase; a gene encoding an acetol kinase; a gene encoding an L-1,2-propanediol-1-phosphate dehydrogenase; and, a gene encoding a glycerol-1-phosphate phosphatase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing 1,2-propanediol;
k) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased 3-hydroxybutyryl-CoA dehydrogenase expression or function;
v) increased crotonase expression or function;
vi) a gene encoding butyryl-CoA dehydrogenase; and:
(1) increased acetyl-CoA:acetoacetyl-CoA transferase or butyrate-acetoacetate CoA transferase expression or function; or
(2) a gene encoding phosphotransbutyrylase and a gene encoding butyrate kinase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing butyrate;
l) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) increased 3-hydroxybutyryl-CoA dehydrogenase expression or function;
v) increased crotonase expression or function;
vi) a gene encoding butyryl-CoA dehydrogenase;
vii) a gene encoding butyraldehyde dehydrogenase; and
viii) a gene encoding butanol dehydrogenase or secondary alcohol dehydrogenase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing butanol;
m) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetyl-CoA acetyltransferase expression or function;
iv) a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase; and
v) a gene encoding 3-hydroxy-3-methylglutaryl-CoA reductase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing mevalonate; or
n) i) reduced fadR expression or function, or increased expression of the fad regulon;
ii) increased expression of the atoDAEB genes;
iii) increased acetaldehyde dehydrogenase expression or function; and
iv) genes encoding the regulatory and catalytic subunits of ethanolamine ammonia-lyase,
in a culture medium comprising free fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, or a combination thereof, thereby producing ethanolamine.