PRODUCTION OF ITACONIC ACID AND RELATED MOLECULES FROM AROMATIC COMPOUNDS

• US 20190330665A1
• Filed: 04/29/2019
• Published: 10/31/2019
• Est. Priority Date: 04/30/2018
• Status: Active Grant

• Current Assignee: Alliance For Sustainable Energy LLC, UT-Battelle LLC
• Original Assignee: Alliance For Sustainable Energy LLC, UT-Battelle LLC
• Inventors: Elmore, Joshua R., Huenemann, Jay, Salvachua, Davinia, Beckham, Gregg T., Guss, Adam M.
• Granted Patent: US 10,738,333 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/32 from Bacillus (G)
• CPC Class Codes: C07K 14/375 from Basidiomycetes
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 9/1025 Acyltransferases (2.3)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 7/44 Polycarboxylic acids
• CPC Class Codes: C12P 7/46 Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
• CPC Class Codes: C12P 7/48 Tricarboxylic acids, e.g. citric acid
• CPC Class Codes: C12R 2001/38 Pseudomonas
• CPC Class Codes: C12Y 203/03001 Citrate (Si)-synthase (2.3.3.1)
• CPC Class Codes: C12Y 401/01006 Aconitate decarboxylase (4.1.1.6)
• CPC Class Codes: C12Y 503/03007 Aconitate DELTA-isomerase (5.3.3.7)

Abstract

This disclosure provides a genetically-modified bacterium from the genus Pseudomonas that produces itaconate or trans-aconitate. The disclosure further provides methods for producing itaconate or trans-aconitate using a genetically-modified bacterium from the genus Pseudomonas.

Claims

1. A genetically-modified bacterium from the genus Pseudomonas comprising an exogenous nucleic acid encoding an enzyme that uses cis-aconitate as a substrate.

2. The genetically-modified bacterium of claim 1, wherein the exogenous nucleic acid encodes a cis-aconitate decarboxylase.

3. The genetically-modified bacterium of claim 1, wherein the exogenous nucleic acid encodes an aconitate isomerase.

4. The genetically-modified bacterium of claim 3, wherein the bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate decarboxylase.

5. The genetically-modified bacterium of claim 3, wherein the bacterium does not have an exogenous nucleic acid encoding a trans-aconitate decarboxylase.

6. The genetically-modified bacterium of claim 1, wherein the bacterium comprises an exogenous nucleic acid encoding a cis-aconitate decarboxylase, an exogenous nucleic acid encoding an aconitate isomerase, and an exogenous nucleic acid encoding a trans-aconitate decarboxylase.

7. The genetically-modified bacterium of claim 1, wherein both the endogenous phaC1 gene and the endogenous phaC2 gene are inactivated in the bacterium.

8. The genetically-modified bacterium of claim 1, wherein the bacterium is grown on lignin or a breakdown product of lignin as a carbon source.

9. The genetically-modified bacterium of claim 1, wherein the breakdown product of lignin comprises p-coumaric acid, ferulic acid, or saccharides.

10. The genetically-modified bacterium of claim 1, wherein the bacterium is grown on an organic compound selected from the group consisting of an aromatic compound, a saccharide, an organic acid, and an alcohol.

11. The genetically-modified bacterium of claim 1, wherein the bacterium is grown on an organic compound selected from the group consisting glycerol, a diacid, a fatty acid, and benzoic acid.

12. The genetically-modified bacterium of claim 1, wherein the bacterium further comprises an exogenous nucleic acid encoding a citrate synthase.

13. The genetically-modified bacterium of claim 12, wherein the citrate synthase is a mutant enzyme that is immune to allosteric inhibition.

14. The genetically-modified bacterium of claim 1, wherein the level of isocitrate dehydrogenase in the bacterium is reduced compared to a non-genetically modified bacterium.

15. The genetically-modified bacterium of claim 14, wherein (i) the start codon of the isocitrate dehydrogenase gene is either “

GTG”

or “

TTG,”

(ii) the isocitrate dehydrogenase gene promoter comprises a mutation, (iii) the ribosome binding site of the isocitrate dehydrogenase gene transcript comprises a mutation, or (iv) the isocitrate dehydrogenase encoded by the isocitrate dehydrogenase gene comprises a protease recognition sequence.

16. The genetically-modified bacterium of claim 1, wherein the bacterium is selected from the group consisting of P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borborid, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. asplenii, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. corrugate, P. fragi, P. lundensis, P. taetrolens, P. Antarctica, P. azotoformans, P. blatchfordae, P. brassicacearum, P. brenneri, P. cedrina, P. corrugate, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. putida group, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parafulva P. plecoglossicida, P. putida, P. balearica, P. luteola, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. helianthin, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. protegens, P. psychrophile, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. teessidea, P. thermotolerans, P. toyotomiensis, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P. xanthomarina, P. taiwanensis.

17. The genetically-modified bacterium of claim 1, wherein the bacterium further comprises an exogenous nucleic acid encoding an itaconic acid efflux pump.

18. The genetically-modified bacterium of claim 5, wherein the bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate efflux pump.

19. A method for converting an organic compound to itaconic acid or trans-aconitate, the method comprising inoculating an aqueous solution containing said organic compound with a genetically-modified bacterium from the genus Pseudomonas, wherein the bacterium comprises an exogenous nucleic acid encoding an enzyme that uses cis-aconitate as a substrate.

20. The method of claim 19, wherein the organic compound is selected from aromatic compounds, saccharides, organic acids, and alcohols.

21. The method of claim 19, wherein the organic compound is a breakdown product of lignin produced during a lignin depolymerization process.

22. The method of claim 19, wherein the organic compound is selected from the group consisting of aromatic compounds, glycerol, diacids, fatty acids, and benzoic acid.

23. The method of claim 19, wherein the aqueous solution is a lignin depolymerization stream or derived from a lignin depolymerization stream.

24. The method of claim 19, wherein the lignin depolymerization stream contains p-coumaric acid, ferulic acid, and saccharides.

25. The method of claim 19, wherein the bacterium produces itaconate, and wherein the exogenous nucleic acid encodes a cis-aconitate decarboxylase.

26. The method of claim 19, wherein the exogenous nucleic acid encodes an aconitate isomerase.

27. The method of claim 26, wherein the bacterium produces itaconate, and wherein the bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate decarboxylase.

28. The method of claim 26, wherein the bacterium produces trans-itaconate, and wherein the bacterium does not have an exogenous nucleic acid encoding a trans-aconitate decarboxylase.

29. The method of claim 19, wherein both the endogenous phaC1 gene and the endogenous phaC2 gene are inactivated in the bacterium.

30. The method of claim 19, wherein the bacterium further comprises an exogenous nucleic acid encoding a citrate synthase.

31. The method of claim 30, wherein the citrate synthase is a mutant enzyme that is immune to allosteric inhibition.

32. The method of claim 19, wherein the level of isocitrate dehydrogenase in the bacterium is reduced compared to a non-genetically modified bacterium.

33. The genetically-modified bacterium of claim 32, wherein (i) the start codon of the isocitrate dehydrogenase gene is either “

GTG”

or “

TTG,”

(ii) the isocitrate dehydrogenase gene promoter comprises a mutation, (iii) the ribosome binding site of the isocitrate dehydrogenase gene transcript comprises a mutation, or (iv) the isocitrate dehydrogenase encoded by the isocitrate dehydrogenase gene comprises a protease recognition sequence.

34. The method of claim 19, wherein the bacterium is selected from the group consisting of P. aeruginosa, P. alcaligenes, P. anguilliseptica, P. argentinensis, P. borborid, P. citronellolis, P. flavescens, P. mendocina, P. nitroreducens, P. oleovorans, P. pseudoalcaligenes, P. resinovorans, P. straminea, P. asplenii, P. aurantiaca, P. aureofaciens, P. chlororaphis, P. corrugate, P. fragi, P. lundensis, P. taetrolens, P. Antarctica, P. azotoformans, P. blatchfordae, P. brassicacearum, P. brenneri, P. cedrina, P. corrugate, P. fluorescens, P. gessardii, P. libanensis, P. mandelii, P. marginalis, P. mediterranea, P. meridiana, P. migulae, P. mucidolens, P. orientalis, P. panacis, P. protegens, P. proteolytica, P. rhodesiae, P. synxantha, P. thivervalensis, P. tolaasii, P. veronii, P. denitrificans, P. pertucinogena, P. putida group, P. cremoricolorata, P. entomophila, P. fulva, P. monteilii, P. mosselii, P. oryzihabitans, P. parajidva, P. plecoglossicida, P. putida, P. balearica, P. luteola, P. stutzeri, P. amygdali, P. avellanae, P. caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P. helianthin, P. meliae, P. savastanoi, P. syringae, P. tomato, P. viridiflava, P. abietaniphila, P. acidophila, P. agarici, P. alcaliphila, P. alkanolytica, P. amyloderamosa, P. asplenii, P. azotifigens, P. cannabina, P. coenobios, P. congelans, P. costantinii, P. cruciviae, P. delhiensis, P. excibis, P. extremorientalis, P. frederiksbergensis, P. fuscovaginae, P. gelidicola, P. grimontii, P. indica, P. jessenii, P. jinjuensis, P. kilonensis, P. knackmussii, P. koreensis, P. lini, P. lutea, P. moraviensis, P. otitidis, P. pachastrellae, P. palleroniana, P. papaveris, P. peli, P. perolens, P. poae, P. pohangensis, P. protegens, P. psychrophile, P. psychrotolerans, P. rathonis, P. reptilivora, P. resiniphila, P. rhizosphaerae, P. rubescens, P. salomonii, P. segitis, P. septica, P. simiae, P. suis, P. teessidea, P. thermotolerans, P. toyotomiensis, P. tremae, P. trivialis, P. turbinellae, P. tuticorinensis, P. umsongensis, P. vancouverensis, P. vranovensis, P. xanthomarina, P. taiwanensis.

35. The method of claim 19, wherein the bacterium further comprises an exogenous nucleic acid encoding an itaconic acid efflux pump.

36. The method of claim 28, wherein the bacterium further comprises an exogenous nucleic acid encoding a trans-aconitate efflux pump.