IP Library › Granted Patent US 12,378,580
Granted Patent B2
US 12,378,580 · App. 17/856,863 · Granted Aug 5, 2025

Co-production pathway for 3-HPA and acetyl-CoA derivatives from malonate semialdehyde

Inventors: Beatriz Leite Magalhaes (Campinas, BR); Paulo Moises Raduan Alexandrino (Campinas, BR); Felipe Galzerani (Paulínia, BR)
Assignee: Braskem S.A.
C12P7/42C12N1/16C12N1/185C12R2001/865C12Y101/01C12Y203/01009
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Quick Facts
Patent No.
US 12,378,580
App. No.
17/856,863
Granted
Aug 5, 2025
Kind
B2
Abstract

The present disclosure provides methods for utilizing genetically modified microbes to co-produce 3-hydroxypropionic acid (3-HP) and acetyl-CoA, and derivatives thereof from malonate semialdehyde as a common single intermediate. The disclosure further provides modified microbe that co-produce the 3-HP and acetyl-CoA derivatives from malonate semialdehyde.

Claims (38)

1. A recombinant microorganism capable of producing acetone and/or isopropanol from a feedstock comprising one or more carbon sources, wherein the recombinant microorganism comprises:

(a) at least one endogenous and/or exogenous nucleic acid molecule encoding an aspartate amino transferase that catalyzes the production of aspartate from oxaloacetate;

(b) at least one exogenous nucleic acid molecule encoding an aspartate decarboxylase that catalyzes the production of β-alanine from aspartate from (a);

(c) at least one endogenous and/or exogenous nucleic acid molecule encoding a β-alanine pyruvate amino transferase and/or a β-alanine transaminase that catalyzes the conversion of β-alanine from (b) to malonate semialdehyde; and

(d) a pathway for the conversion of malonate semialdehyde to acetone.

2. The recombinant microorganism of claim 1 , wherein the endogenous and/or exogenous nucleic acid molecule encoding an aspartate amino transferase encodes an amino acid sequence comprising SEQ ID NO: 233.

3. The recombinant microorganism of claim 1 , wherein the exogenous nucleic acid molecule encoding an aspartate decarboxylase encodes an amino acid sequence comprising SEQ ID NO: 234 or SEQ ID NO: 275.

4. The recombinant microorganism of claim 1 , wherein the endogenous and/or exogenous nucleic acid molecule encoding a β-alanine pyruvate amino transferase encodes an amino acid sequence comprising SEQ ID NO: 235.

5. The recombinant microorganism of claim 1 , wherein the endogenous and/or exogenous nucleic acid molecule encoding a β-alanine transaminase encodes an amino acid sequence comprising SEQ ID NO: 236.

6. The recombinant microorganism of claim 1 , wherein the pathway for the conversion of malonate semialdehyde to acetone comprises:

(a) at least one endogenous and/or exogenous nucleic acid molecule encoding

(i) a malonate semialdehyde dehydrogenase that catalyzes the production of acetyl-CoA from malonate semialdehyde, and/or

(ii) a malonyl-CoA reductase and/or 2-keto acid decarboxylase that catalyzes the conversion of malonate semialdehyde into malonyl-CoA, and a malonyl-CoA decarboxylase that catalyzes the production of acetyl-CoA from malonyl-CoA; and

(b) at least one or more endogenous and/or exogenous nucleic acid molecules encoding enzymes capable of catalyzing the conversion of acetyl-CoA to acetone.

7. The recombinant microorganism of claim 6 , wherein the endogenous and/or exogenous nucleic acid molecule encoding a malonate semialdehyde dehydrogenase encodes an amino acid sequence comprising MSD.Pa (SEQ ID NO: 111), MSD.Cal (SEQ ID NO: 112), iolA (SEQ ID NO: 113), iolA (SEQ ID NO: 114), iolA (SEQ ID NO: 115), mmsA (SEQ ID NO: 116), dddC (SEQ ID NO: 117), or iolA (SEQ ID NO: 118).

8. The recombinant microorganism of claim 6 , wherein the endogenous and/or exogenous nucleic acid molecule encoding a malonyl-CoA reductase and/or 2-keto acid decarboxylase encodes an amino acid sequence comprising mcr (SEQ ID NO: 278), matA, MLYCD (SEQ ID NO: 279), kivD (SEQ ID NO: 280), kdcA (SEQ ID NO: 281), or ARO10 (SEQ ID NO: 282).

9. The recombinant microorganism of claim 6 , wherein the at least one or more endogenous and/or exogenous nucleic acid molecules encoding enzymes capable of catalyzing the conversion of acetyl-CoA to acetone comprise:

(a) at least one endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase that catalyzes the production of acetoacetyl-CoA from acetyl-CoA;

(b) at least one endogenous and/or exogenous nucleic acid molecule encoding

(i) an acetoacetyl-CoA transferase that catalyzes the production of acetoacetate from acetoacetyl-CoA; and/or

(ii) at least one endogenous and/or exogenous nucleic acid molecule encoding

(1) a hydroxymethylglutaryl-CoA synthase that catalyzes the production of HMG-CoA from acetoacetyl-CoA, and

(2) a hydroxymethylglutaryl-CoA lyase that catalyzes the production of acetoacetate from HMG-COA; and

(c) at least one endogenous and/or exogenous nucleic acid molecule encoding an acetoacetate decarboxylase that catalyzes the production of acetone from acetoacetate.

10. The recombinant microorganism of claim 9 , wherein the endogenous and/or exogenous nucleic acid molecule encoding a thiolase or an acetyl-CoA acetyltransferase encodes an amino acid sequence comprising ERG10 (SEQ ID NO: 209), thIA (SEQ ID NO: 210), atoB (SEQ ID NO: 211), H16_B0759 (SEQ ID NO: 212), Msed_0656 (SEQ ID NO: 213), or AAT1 (SEQ ID NO: 214).

11. The recombinant microorganism of claim 9 , wherein the endogenous and/or exogenous nucleic acid molecule encoding an acetoacetyl-CoA transferase encodes an amino acid sequence comprising atoA/atoD (SEQ ID NO: 215 and 216), ctfA/ctfB (SEQ ID NO: 219 and 220), ctfA/ctfB (SEQ ID NO: 221 and 222), or ctfA/ctfB (SEQ ID NO: 223 and 224).

12. The recombinant microorganism of claim 9 , wherein the endogenous and/or exogenous nucleic acid molecule encoding a hydroxymethylglutaryl-CoA synthase and a hydroxymethylglutaryl-CoA lyase encode an amino acid sequence comprising ERG13 (SEQ ID NO: 283) and yngG (SEQ ID NO: 284).

13. The recombinant microorganism of claim 9 , wherein the endogenous and/or exogenous nucleic acid molecule encoding an acetoacetate decarboxylase encodes an amino acid sequence comprising Adc.Ca (SEQ ID NO: 225), Adc.Cbe (SEQ ID NO: 226), Adc (SEQ ID NO: 227), Adc (SEQ ID NO: 228), Adc (SEQ ID NO: 229) or Adc.Pp (SEQ ID NO: 230).

14. The recombinant microorganism of claim 1 , wherein the recombinant microorganism produces isopropanol.

15. The recombinant microorganism of claim 14 , further comprising a 2-propanol dehydrogenase.

16. The recombinant microorganism of claim 1 , wherein the recombinant microorganism co-produces 3-hydroxypropionic acid (3-HP), and/or derivatives of 3-HP, and acetone, and/or isopropanol, wherein the recombinant microorganism comprises at least one endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionic acid dehydrogenase that catalyzes the production of 3-HP from malonate semialdehyde;

wherein the derivatives of 3-HP are selected from acrylic acid, 1-propanol, propene, polypropylene.

17. The recombinant microorganism of claim 16 , wherein at least a portion of excess NAD (P) H generated in the production of acetone and/or isopropanol is utilized as a source of reducing equivalents in the production of 3-HP and/or derivatives of 3-HP.

18. The recombinant microorganism of claim 16 , wherein the endogenous and/or exogenous nucleic acid molecule encoding a 3-hydroxypropionic acid dehydrogenase encodes an amino acid sequence comprising MCR-Nterm.Cau (SEQ ID NO: 105), ADH.Ae (SEQ ID NO: 106), MMSB.Bce (SEQ ID NO: 107), YDFG-0.Ec (SEQ ID NO: 108), YMR226C (YDF1) (SEQ ID NO: 109), or HPD1 (SEQ ID NO: 110).

19. The recombinant microorganism of claim 1 , wherein the microorganism is selected from a bacterium, a fungus, or a yeast.

20. A method of producing acetone and/or isopropanol by contacting the recombinant microorganism of claim 1 with a fermentable carbon source under conditions and for a sufficient period of time to produce acetone and/or isopropanol.

21. The method of claim 20 , wherein the carbon source is selected from sugars, glycerol, alcohols, organic acids, alkanes, fatty acids, lignocellulose, proteins, carbon dioxide, and carbon monoxide.

22. The method of claim 20 , wherein the recombinant microorganism produces acetone and/or isopropanol in an aerobic, microanaerobic or anaerobic production process.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 30, 2022
From: ALEXANDRINO, PAULO MOISES RADUAN; GALZERANI, FELIPE
To: BRASKEM S.A.
Reel/Frame 060678/0479 →
Continuity (3)
Continuation 16719833 · Dec 18, 2019
Provisional Application 62781511 · Dec 18, 2018
Related Publication 20230037707A1 · Feb 9, 2023
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