Production of an Organic Acid and/or Related Chemicals

• US 20110125118A1
• Filed: 11/22/2010
• Published: 05/26/2011
• Est. Priority Date: 11/20/2009
• Status: Abandoned Application

• Current Assignee: OPX Biotechnologies, Inc. (Cargill Incorporated)
• Original Assignee: OPX Biotechnologies, Inc. (Cargill Incorporated)
• Inventors: Lynch, Michael D.
• Application Number:
  US12/952,149
• Publication Number:
  US 20110125118A1
• Time in Patent Office: Days
• US Class Current: 604/367
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12P 7/40 containing a carboxyl group including Peroxycarboxylic acids
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids

Abstract

The present invention relates to methods, systems and compositions, including genetically modified microorganisms, e.g., recombinant microorganisms, adapted to utilize one or more synthesis gas components in a microbial bio-production of one or more desired biomolecules and products of commercial interest.

Claims

1. A method for producing 3-hydroxypropionic acid (3-HP) comprising:

i) combining hydrogen, a carbon source selected from carbon monoxide and carbon dioxide, and a culture of microorganism cells, wherein

a) said microorganism cells are genetically transformed to introduce or increase one or more enzymatic activities for conversion of the carbon source to malonate semialdehyde, wherein said enzymatic activities are selected from the group consisting of phosphoglucose isomerase, inositol-1-phosphate synthase, inositol deoxy-D-gluconate isomerase, 5-dehydro-2-deoxygluconokinase, and deoxyphophogluconate aldolase; and

b1) said microorganism cells are genetically transformed to introduce or increase enzymatic activity for conversion of malonate semialdehyde to 3-HP, and/or

b2) said microorganism cells are capable of producing 3-HP at a rate of at least 1 g/L/hr in the absence of genetic modification for conversion of malonate semialdehyde to 3-HP; and

ii) maintaining the combined hydrogen, carbon source, and microorganism cells for a suitable time and under conditions sufficient to produce malonate semialdehyde and convert the malonate semialdehyde to 3-HP.

2. A method of producing acrylic acid comprising:

i) producing 3-HP according to the method of claim 1; and

ii) converting said 3-HP to acrylic acid.

3. A method of producing an acrylic acid-based product comprising;

i) producing acrylic acid according to the method of claim 2; and

ii) converting said acrylic acid into an acrylic acid-based product.

4. The method of claim 1, wherein said carbon source has a ratio of carbon-14 to carbon-12 of about 1.0×

10<sup>−

14</sup> or greater.

5. The method of claim 1, wherein said carbon source has a percentage of petroleum origin selected from less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 1%, or essentially free of petroleum origin.

6. The method of claim 1, wherein said carbon source has an amount of glucose, sucrose, fructose, dextrose, lactose, glycerol, and/or combinations thereof that is selected from the group consisting of less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, and less than about 1% by weight.

7. The method of claim 1, wherein said cell culture comprises an inhibitor of fatty acid synthesis or said microorganism is genetically modified for reduced enzymatic activity in one or more of the microorganism'"'"'s fatty acid synthesis pathways.

8. The method of claim 7, wherein said inhibitor of fatty acid synthesis is selected from the group consisting of thiolactomycin, triclosan, cerulenin, thienodiazaborine, isoniazid, and analogs thereof.

9. The method of claim 1, wherein the percentage of carbon source converted to 3-HP is selected from greater than 25%, greater than 35%, greater than 45%, greater than 55%, greater than 65%, greater than 75%, greater than 85%, and greater than 95%.

10. The method of claim 1, wherein the volumetric productivity for 3-HP is selected from at least 1/g/L/hr and at least 2/g/L/hr.

27. The method of claim 1, wherein said genetically modified microorganism is further modified to decrease activity in an enzyme selected from the group consisting of lactate dehydrogenase, phophate acetyltransferase, pyruvate oxidase, pyruvate-formate lyase, and combinations thereof.

28. The method of claim 1, further comprising separating and/or purifying 3-HP from said cell culture by extraction of 3-HP from said culture in the presence of a tertiary amine.

29. The method of claim 1, wherein said 3-HP is produced at a specific productivity of at least 0.005 g/gDCW-hr, 0.05 g/gDCW-hr, 1 g/gDCW-hr, or at least 2 g/gDCW-hr, or at a volumetric productivity of at least 0.50 grams per liter per hour.

30. The method of claim 3, wherein said consumer product is selected from the group consisting of diapers, carpet, paint, and adhesives.

31. The method of claim 30, wherein said consumer product is diapers.

32. Biologically-produced 3-hydroxypropionic acid, wherein said 3-hydroxypropionic acid is produced according to any one of claims 1-31.

33. The 3-hydroxypropionic acid according to claim 32, wherein said 3-hydroxypropionic acid is essentially free of chemical catalyst.

34. The 3-hydroxypropionic acid according to claim 33, wherein said chemical catalyst is a molybdenum and/or vanadium based catalyst.

35. The 3-hydroxypropionic acid according to claim 32, wherein said 3-hydroxypropionic acid has a ratio of carbon-14 to carbon-12 of about 1.0×

10<sup>−

14</sup> or greater.

36. The 3-hydroxypropionic acid according to claim 32, wherein said 3-hydroxypropionic acid contains less than about 10% carbon derived from petroleum.

37. The 3-hydroxypropionic acid according to claim 32, wherein said 3-hydroxypropionic acid contains a residual amount of organic material related to its method of production.

38. The 3-hydroxypropionic acid according to claim 37, wherein said 3-hydroxypropionic acid contains a residual amount of organic material in an amount between 1 and 1,000 parts per million of said 3-hydroxypropionic acid.

39. Acrylic acid produced from 3-hydroxypropionic acid according to one of claims 32-38.

40. A polymer produced with acrylic acid according to claim 39.

41. A consumer product produced with acrylic acid according to claim 39.

42. The consumer product of claim 41, wherein said consumer product is selected from diapers, carpet, paint, and adhesives.

43. The consumer product of claim 42, wherein said consumer product is diapers.

44. The method of claim 1, additionally comprising converting the 3-HP molecule(s) to a chemical selected from the group consisting of acrylic acid, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, malonic acid, 1,3-propanediol, methyl acrylate, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxyethyl acrylate, isobutyl acrylate, and 2-ethylhexyl acrylate.

45. The method of claim 1, additionally comprising converting the 3-HP molecule(s) to acrylic acid via a dehydration reaction.

46. The method of claim 45, additionally comprising converting the acrylic acid to a polyacrylic acid via a polymerization reaction.

47. The method of claim 1, additionally comprising converting the 3-HP molecule(s) to methyl acrylate via dehydration and esterification.

48. The method of claim 1, additionally comprising converting the 3-HP molecule(s) to acrylamide via dehydration and amidation.

49. The method of claim 1, additionally comprising converting the 3-HP molecule(s) to acrylonitrile.

50. The method of claim 1, additionally comprising converting the 3-HP molecule(s) to propriolactone.

51. The method of claim 1, additionally comprising converting the 3-HP molecule(s) to ethyl 3-HP.

52. The method of claim 1, additionally comprising converting the 3-HP molecule(s) to malonic acid.

53. The method of claim 1, additionally comprising converting the 3-HP molecule(s) to 1,3-propanediol.

54. The method of claim 1, additionally comprising forming a polymerized-3-HP (poly-3HP) polymer.

55. The method of claim 1, additionally comprising forming a polymer from 3-HP (such as poly-3HP), acrylic acid (such as polyacrylic acid), and/or at least one downstream compound of 3-HP.

56. The method of claim 55 wherein at least one downstream compound of 3-HP is selected from the group consisting of acrylic acid, acrylamide, acrylonitrile, propiolactone, ethyl 3-HP, malonic acid, 1,3-propanediol, methyl acrylate, ethyl acrylate, n-butyl acrylate, hydroxypropyl acrylate, hydroxyethyl acrylate, isobutyl acrylate, and 2-ethylhexyl acrylate.

11. A method for producing malonate semialdehyde comprising:

a) combining hydrogen, a carbon source selected from carbon monoxide and carbon dioxide, and a culture of microorganism cells, wherein said microorganism cells comprise at least two genetic modifications to introduce or increase one or more enzymatic activities selected from the group consisting of phosphoglucose isomerase, inositol-1-phosphate synthase, inositol monophosphatase, myo-inositol dehydrogenase, myo-inosose-2-dehydratase, inositol 2-dehydrogenase, deoxy-D-gluconate isomerase, 5-dehydro-2-deoxygluconokinase, and deoxyphophogluconate aldolase;

b) maintaining the combined hydrogen, carbon source, and microorganism cells for a suitable time and under conditions sufficient to convert the carbon source to malonate semialdehyde.

12. A method for producing 3-HP comprising:

i) producing malonate semialdehyde according to the method of claim 11; and

ii) maintaining the microorganism cells for a suitable time and under conditions sufficient to convert the malonate semialdehyde to 3-HP;

wherein the microorganism cells further comprise a protein that converts malonate semialdehyde to 3-HP.

13. The method of claim 12, wherein the protein has 3-hydroxy acid dehydrogenase activity.

14. The method of claim 13, wherein the protein is E. coli YdfG.

15. A method for producing acrylic acid comprising:

i) producing 3-HP according to the method of claim 12; and

ii) dehydrating said 3-HP to produce acrylic acid.

16. A method for producing an acrylic-acid-based product comprising:

i) producing acrylic acid according to the method of claim 15; and

ii) processing the acrylic acid into an acrylic-acid-based product.

17. A genetically modified microorganism for the production of 3-HP, wherein said microorganism comprises at least one heterologous nucleic acid molecule selected from the groups of nucleic acid molecules encoding

a. phosphoglucose isomerase, inositol-1-phosphate synthase, inositol monophosphatase, myo-inositol dehydrogenase, myo-inosose-2-dehydratase, inositol 2-dehydrogenase, deoxy-D-gluconate isomerase, 5-dehydro-2-deoxygluconate kinase, 5-dehydro-2-deoxyphophogluconate aldolase;

or

b. 3-hydroxy acid dehydrogenase.

18. The genetically modified microorganism of claim 17, wherein the number of genetic modifications is selected from at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, and at least ten enzymatic activities.

19. The genetically modified microorganism of claim 17, wherein said microorganism is selected from the group consisting of chemolithotrophic bacteria.

20. The genetically modified microorganism of claim 17, wherein said microorganism is selected from the group consisting Oligotropha carboxidovorans, Cupriavidus necator, and strain H16 of Cupriavidus necator.

21. The genetically modified microorganism of claim 17, wherein the heterologous nucleic acid molecule is selected from the group:

i) phosphoglucose isomerase encoded by the pgi gene of E. coli;

ii) inositol-1-phosphate synthase encoded by the ino-1 gene of S. cerevisiae;

iii) inositol monophosphatase encoded by the subB gene of E. coli;

iv) myo-inositol dehydrogenase encoded by the iolG gene of B. subtilis;

v) myo-inosose-2-dehydratase encoded by the iolE gene of B. subtilis;

vi) inositol 2-dehydrogenase encoded by the iolD gene of B. subtilis;

vii) deoxy-D-gluconate isomerase encoded by the iolB gene of B. subtilis;

viii) 5-dehydro-2-deoxygluconokinase encoded by the iolC gene of B. subtilis;

ix) 5-dehydro-2-deoxyphophogluconate aldolase encoded by the iolJ gene of B. subtilis;

or

x) 3-hydroxy acid dehydrogenase encoded by the ydfG gene of E. coli.

22. The genetically modified microorganism of claim 17 comprising at least one genetic modification to introduce or increase one or more enzymatic activities provided by amino acid sequences having at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to one or more amino acid sequences selected from the group consisting of SEQ ID NO:

002, SEQ ID NO;

004, SEQ ID NO;

006, SEQ ID NO;

008, SEQ ID NO;

010, SEQ ID NO;

012, SEQ ID NO;

014, SEQ ID NO;

016, SEQ ID NO;

018, SEQ ID NO;

020, and conservatively modified variants thereof.

23. The genetically modified microorganism of claim 17 comprising at least one genetic modification provided by a polynucleotide comprising a nucleic acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to one or more nucleic acid sequences from the group consisting of SEQ ID NO:

001, SEQ ID NO;

003, SEQ ID NO;

005, SEQ ID NO;

007, SEQ ID NO;

009, SEQ ID NO;

011, SEQ ID NO;

013, SEQ ID NO;

015, SEQ ID NO;

017, SEQ ID NO;

019, and conservatively modified variants thereof.

24. A culture system comprising

(i) a population of genetically modified microorganisms of any one of claims 17 to 23;

and (ii) a media comprising nutrients for said population.

25. A method of making a genetically modified microorganism according to claim 17 comprising providing to a microorganism at least one genetic modification to introduce or increase one or more enzymatic activities provided by amino acid sequences having at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to one or more amino acid sequences selected from the group consisting of SEQ ID NO:

002, SEQ ID NO;

004, SEQ ID NO;

006, SEQ ID NO;

008, SEQ ID NO;

010, SEQ ID NO;

012, SEQ ID NO;

014, SEQ ID NO;

016, SEQ ID NO;

018, SEQ ID NO;

020, and conservatively modified variants thereof.

26. A method of making a genetically modified microorganism according to claim 17, comprising providing to a selected microorganism at least one genetic modification comprising through a polynucleotide comprising a nucleic acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% sequence identity to one or more nucleic acid sequences from the group consisting of SEQ ID NO:

001, SEQ ID NO;

003, SEQ ID NO;

005, SEQ ID NO;

007, SEQ ID NO;

009, SEQ ID NO;

011, SEQ ID NO;

013, SEQ ID NO;

015, SEQ ID NO;

017, SEQ ID NO;

019, and conservatively modified variants thereof.