Genetically Modified Organisms for Increased Microbial Production of 3-Hydroxypropionic Acid Involving an Oxaloacetate alpha-decarboxylase
Microorganism compositions are described that comprise combinations of genetic modifications that include a genetic modification to increase oxaloacetate alpha-decarboxylase enzymatic activity. By such genetic modification a 3-hydroxypropionic acid (“3-HP”) production pathway is provided or improved. In various embodiments, comprising other genetic modifications, including selected gene disruptions, 3-HP production is greater than in a control microorganism lacking such combinations of genetic modifications
1 - 43 . (canceled)
44 . A method for producing an acrylic acid-based consumer product comprising
i) combining a carbon source and a microorganism to produce 3-hydroxypropionic acid, wherein said microorganism comprises
a) a heterologous nucleic acid sequence encoding an enzyme selected from the group consisting of: a native or mutated form of a mmsB protein, and a native or mutated form of a ydfG protein;
b) a genetic modification to decrease or eliminate an enzymatic activity selected from the group consisting of: lactate dehydrogenase, phosphate acetyltransferase, and pyruvate-formate lyase;
c) a heterologous nucleic acid sequence encoding an acetyl-CoA carboxylase; and
d) a heterologous nucleic acid sequence encoding a cyanase,
ii) converting said 3-hydroxypropionic acid to acrylic acid; and
iii) processing said acrylic acid into a consumer product.
45 . The method of claim 44 , wherein said carbon source has a ratio of carbon-14 to carbon-12 of about 1.0×10 −14 or greater.
46 . The method of claim 44 , wherein following (i) said method further comprises separating or purifying 3-hydroxypropionic acid from said cell culture by extraction of 3-hydroxypropionic acid from said culture in the presence of a tertiary amine.
47 . The method of claim 44 , wherein said 3-hydroxypropionic acid is produced at a higher level than in a non-genetically modified microorganism.
48 . The method of claim 44 , wherein said consumer product is selected from the group consisting of diapers, carpet, paint, adhesives, and acrylic glass.
49 . The method of claim 48 , wherein said consumer product is diapers.
50 . The method of claim 44 , wherein said carbon source is predominantly glucose, sucrose, fructose, dextrose, lactose, or a combination thereof.
51 . The method of claim 44 , wherein said carbon source is less than 50% glycerol.
52 . The method of claim 44 , wherein said lactate dehydrogenase is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:19 or has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 20.
53 . The method of claim 44 wherein said pyruvate-formate lyase is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NOs: 21 or has an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22.
54 . The method of claim 44 , wherein said genetically modified microorganism further comprises a heterologous nucleic acid sequence encoding an acetyl-CoA carboxylase.
55 . A method for producing an acrylic acid-based consumer product comprising
i) combining a carbon source and a microorganism to produce 3-hydroxypropionic acid, wherein said microorganism comprises
a) a heterologous nucleic acid sequence encoding a 3-hydroxypropionate dehydrogenase;
b) a heterologous nucleic acid sequence encoding a cyanase or a carbonic anhydrase; and
c) a heterologous nucleic acid sequence encoding an NADPH-dependent transhydrogenase or an acetyl-CoA carboxylase;
ii) converting said 3-hydroxypropionic acid to acrylic acid; and
iii) processing said acrylic acid into a consumer product.
56 . The method of claim 55 , wherein said genetically modified microorganism further comprises at least one heterologous nucleic acid sequence encoding an oxaloacetate alpha-decarboxylase.
57 . The method of claim 56 , wherein said oxaloacetate alpha-decarboxylase is encoded by a sequence selected from the group consisting of: SEQ ID NOs: 54, 55, and 56.
58 . The method of claim 56 , wherein said genetically modified microorganism further comprises at least one heterologous nucleic acid sequence encoding a phosphoenolpyruvate carboxykinase or a phosphoenol pyruvate carboxylase.
59 . The method of claims 56 , wherein said genetically modified microorganism further comprises at least one genetic modification to reduce enzymatic activity of a protein selected from the group consisting of: lactate dehydrogenase, pyruvate formate lyase, phosphate acetyltransferase, heat stable, histidyl phosphorylatable protein, phosphoryl transfer protein, polypeptide chain, pyruvate kinase I, and pyruvate kinase II.
60 . The method of claim 55 , wherein said genetically modified microorganism further comprises a modification of a gene to increase the NADPH/NADP + ratio, wherein the modification is selected from the group consisting of: increasing activity of pgi, increasing activity of pntAB, gapA:gapN substitution or replacement, and disrupting sthA.
61 . The method of claim 55 , wherein the 3-hydroxypropionate dehydrogenase is a native or mutated form of a mmsB protein or a native or mutated form of a ydfG protein.
62 . The method of claim 55 , wherein the said genetically modified microorganism further comprises a heterologous nucleic acid sequence encoding an NADPH-dependent transhydrogenase.
63 . The method of claim 55 , wherein said genetically modified microorganism further comprises a heterologous nucleic acid sequence encoding a cyanase or a carbonic anhydrase that increases intracellular bicarbonate levels.
64 . The method of claim 55 , wherein said genetically modified microorganism further comprises a heterologous nucleic acid sequence encoding an acetyl-CoA carboxylase.
65 . The method of claim 55 , wherein said genetically modified microorganism is selected from the group consisting of: Zymomonas, Escherichia, Pseudomonas, Alcaligenes, Salmonella, Shigella, Burkholderia, Oligotropha, and Klebsiella.
66 . The method of claim 55 , wherein said genetically modified microorganism is selected from the group consisting of: Escherichia coli, Cupriavidus necator, Oligotropha carboxidovorans, and Pseudomonas putida.
67 . The method of claim 55 , wherein said genetically modified microorganism is an Escherichia coli.
68 . The method of claim 55 , wherein said genetically modified microorganism is a gram-positive bacterium.
69 . The method of claim 55 , wherein said genetically modified microorganism is selected from the group consisting of: Clostridium, Rhodococcus, Bacillus, Lactobacillus, Enterococcus, Paenibacillus, Arthrobacter, Corynebacterium, and Brevibacterium.
70 . The method of claim 55 , wherein said genetically modified microorganism is selected from the group consisting of: Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, and Bacillus subtilis.
71 . The method of claim 55 , wherein said genetically modified microorganism is a fungus.
72 . The method of claim 55 , wherein said genetically modified microorganism is a yeast.
73 . The method of claim 55 , wherein said yeast is selected from the group consisting of: Pichia, Candida, Hansenula, and Saccharomyces.
74 . A method for producing an acrylic acid-based consumer product comprising
i) combining a carbon source and a microorganism to produce 3-hydroxypropionic acid, wherein said microorganism comprises
a) a heterologous nucleic acid sequence encoding a 3-hydroxypropionate dehydrogenase; and
b) a heterologous nucleic acid sequence encoding a cyanase or a carbonic anhydrase,
ii) converting said 3-hydroxypropionic acid to acrylic acid; and
iii) processing said acrylic acid into a consumer product.
75 . The method of claim 72 , wherein said genetically modified microorganism further comprises a heterologous nucleic acid sequence encoding an acetyl-CoA carboxylase.