Genetically Modified Organisms for Increased Microbial Production of 3-Hydroxypropionic Acid Involving an Oxaloacetate alpha-decarboxylase

Abstract

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

What is claimed is: 
     
         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.