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US-12624372-B2 - Means and methods for producing isobutene from 3-methylcrotonic acid

US12624372B2US 12624372 B2US12624372 B2US 12624372B2US-12624372-B2

Abstract

Described is a method for the production of isobutene from a carbon source characterized in that it comprises: (a) culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing 3-methylcrotonic acid so that it accumulates in the liquid culture medium; and (b) enzymatically converting 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) into isobutene by: (i) incubating a microorganism expressing ap FMN-dependent decarboxylase associated with an FMN prenyl transferase with liquid culture medium containing 3-methylcrotonic acid obtained in step (a); and/or (ii) incubating a FMN-dependent decarboxylase associated with a FMN prenyl transferase with liquid culture medium containing 3-methylcrotonic acid obtained in step (a); thereby producing isobutene; and (c) recovering the produced isobutene.

Inventors

  • Claude Bensoussan
  • Frederic Ollivier
  • Romain Chayot
  • Denis Thibaut
  • Marc Delcourt

Assignees

  • GLOBAL BIOENERGIES

Dates

Publication Date
20260512
Application Date
20211220
Priority Date
20201221

Claims (18)

  1. 1 . A method for the production of isobutene from a carbon source characterized in that it comprises: (a) culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing said 3-methylcrotonic acid so that it accumulates in the liquid culture medium; and (b) enzymatically converting said 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) into isobutene by: (i) incubating a microorganism expressing an FMN-dependent decarboxylase associated with an FMN prenyl transferase with said liquid culture medium containing 3-methylcrotonic acid obtained in step (a); and/or (ii) incubating an FMN-dependent decarboxylase associated with an FMN prenyl transferase with said liquid culture medium containing 3-methylcrotonic acid obtained in step (a); thereby producing said isobutene; and (c) recovering the produced isobutene.
  2. 2 . The method of claim 1 , wherein said incubation of step (b) is carried out in (a) a vessel without gas supply; or (b) a vessel with gas supply at <0.1 vvm (vessel volume per minute) using an inlet gas.
  3. 3 . The method of claim 1 , wherein the liquid culture medium containing said 3-methylcrotonic acid of step (a) is separated from the microorganism prior to step (b).
  4. 4 . The method of claim 1 , wherein said 3-methylcrotonic acid is isolated or purified from said liquid culture medium prior to step (b) of claim 1 .
  5. 5 . The method of claim 1 , wherein said inlet gas is air, inert gas or a mixture of air and inert gas, wherein said inert gas is selected from nitrogen, helium, argon, neon, CO 2 and a mixture of these gases.
  6. 6 . The method of claim 1 , wherein said carbon source is metabolized into acetyl-CoA prior to its enzymatic conversion into 3- methylcrotonic acid.
  7. 7 . The method of claim 6 , wherein said carbon source is selected from the group consisting of glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid and a mixture thereof.
  8. 8 . The method of claim 1 , wherein said microorganism used in claim 1 (b) (i) is pre-cultured in a suitable liquid culture medium under suitable conditions prior to the conversion step (b) (i) of claim 1 .
  9. 9 . The method of claim 1 , further comprising purifying/enriching the recovered isobutene.
  10. 10 . The method of claim 1 , wherein said microorganism is a bacterium, a yeast, a fungus or an algae.
  11. 11 . A method for the production of isobutene from a carbon source characterized in that it comprise: (a) culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing said 3-methylcrotonic acid so that it accumulates in the liquid culture medium; and (b) converting said 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) into isobutene at a temperature between 180° C. and 400° C.; and (c) recovering the produced isobutene.
  12. 12 . The method of claim 11 , wherein the liquid culture medium containing said 3-methylcrotonic acid of step (a) is separated from the microorganism prior to step (b).
  13. 13 . The method of claim 11 , wherein said 3-methylcrotonic acid is isolated or purified from said liquid culture medium prior to step (b).
  14. 14 . The method of claim 11 , wherein said carbon source is metabolized into acetyl-CoA prior to its enzymatic conversion into 3-methylcrotonic acid.
  15. 15 . The method of claim 14 , wherein said carbon source is selected from the group consisting of glucose, fructose, sucrose, xylose, glycerol, starch, ethanol, lactic acid, acetic acid and a mixture thereof.
  16. 16 . The method of claim 11 , wherein said microorganism is pre-cultured in a suitable liquid culture medium under suitable conditions prior to the conversion step (b).
  17. 17 . The method of claim 11 , further comprising purifying/enriching the recovered isobutene.
  18. 18 . The method of claim 11 , wherein said microorganism is a bacterium, a yeast, a fungus or an algae.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a § 371 National Stage Application of PCT/EP2021/086687 filed on Dec. 20, 2021, which claims priority to EP 20215872.1 filed on Dec. 21, 2020, both of which are hereby incorporated by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 7, 2023, and is named GB-35-US_Sequence_Listing.txt and is 14,488 bytes in size. The present invention relates to a method for the production of isobutene from a carbon source characterized in that it comprises (a) culturing a microorganism capable of producing 3-methylcrotonic acid from a carbon source in a liquid culture medium, thereby producing said 3-methylcrotonic acid so that it accumulates in the liquid culture medium; and (b) enzymatically converting said 3-methylcrotonic acid contained in the liquid culture medium obtained in step (a) into isobutene by: (i) incubating a microorganism expressing an FMN-dependent decarboxylase associated with an FMN prenyl transferase with said liquid culture medium containing 3-methylcrotonic acid obtained in step (a); and/or (ii) incubating an FMN-dependent decarboxylase associated with an FMN prenyl transferase with said liquid culture medium containing 3-methylcrotonic acid obtained in step (a); thereby producing said isobutene; and (c) recovering the produced isobutene. A large number of chemical compounds are currently derived from petrochemicals. Alkenes (such as ethylene, propylene, the different butenes, or else the pentenes, for example) are used in the plastics industry, for example for producing polypropylene or polyethylene, and in other areas of the chemical industry and that of fuels. Butylene exists in four forms, one of which, isobutene (also referred to as isobutylene), enters into the composition of methyl-tert-butyl-ether (MTBE), an anti-knock additive for automobile fuel. Isobutene can also be used to produce isooctene, which in turn can be reduced to isooctane (2,2,4-trimethylpentane); the very high octane rating of isooctane makes it the best fuel for so-called “gasoline” engines. Alkenes such as isobutene are currently produced by catalytic cracking of petroleum products (or by a derivative of the Fischer-Tropsch process in the case of hexene, from coal or gas). The production costs are therefore tightly linked to the price of oil. Moreover, catalytic cracking is sometimes associated with considerable technical difficulties which increase process complexity and production costs. The production by a biological pathway of alkenes such as isobutene is called for in the context of a sustainable industrial operation in harmony with geochemical cycles. The first generation of biofuels consisted in the fermentative production of ethanol, as fermentation and distillation processes already existed in the food processing industry. The production of second generation biofuels is in an exploratory phase, encompassing in particular the production of long chain alcohols (butanol and pentanol), terpenes, linear alkanes and fatty acids. Two recent reviews provide a general overview of research in this field: Ladygina et al. (Process Biochemistry 41 (2006), 1001) and Wackett (Current Opinions in Chemical Biology 21 (2008), 187). The conversion of isovalerate to isobutene by the yeast Rhodotorula minuta has been described (Fujii et al. (Appl. Environ. Microbiol. 54 (1988), 583)). Gogerty et al. (Appl. Environm. Microbiol. 76 (2010), 8004-8010) and van Leeuwen et al. (Appl. Microbiol. Biotechnol. 93 (2012), 1377-1387) describe the production of isobutene from acetoacetyl-CoA by enzymatic conversions wherein the last step of the proposed pathway is the conversion of 3-hydroxy-3-methylbutyric acid (also referred to as 3-hydroxyisovalerate (HIV)) by making use of a mevalonate diphosphate decarboxylase. This reaction for the production of isobutene from 3-hydroxy-3-methylbutyric acid is also described in WO2010/001078 which, in general terms, describes methods for generating alkenes through a biological process, in particular methods for producing terminal alkenes (in particular propylene, ethylene, 1-butylene, isobutylene or isoamylene) from molecules of the 3-hydroxyalkanoate type. WO2012/052427 also describes a method for generating alkenes through a biological process while, in particular, a method for producing alkenes (for example propylene, ethylene, 1-butylene, isobutylene or isoamylene) from molecules of the 3-hydroxyalkanoate type is described. In this context, the reaction for the production of isobutene from 3-hydroxy-3-methylbutyric acid is also described in WO2012/052427. WO 2016/042012 describes methods for producing said 3-hydroxy-3-methylbutyric acid. In particular, WO 2016/042012 describes methods for producing 3-hydroxy-3-methylbutyric acid comprising the step of enzymat