WO2015022496A2 - A process for production of methacrylic acid and derivatives thereof - Google Patents

A process for production of methacrylic acid and derivatives thereof Download PDF

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Publication number
WO2015022496A2
WO2015022496A2 PCT/GB2014/052389 GB2014052389W WO2015022496A2 WO 2015022496 A2 WO2015022496 A2 WO 2015022496A2 GB 2014052389 W GB2014052389 W GB 2014052389W WO 2015022496 A2 WO2015022496 A2 WO 2015022496A2
Authority
WO
WIPO (PCT)
Prior art keywords
acid
microorganism
mesaconic
process according
citramalic
Prior art date
Application number
PCT/GB2014/052389
Other languages
English (en)
French (fr)
Other versions
WO2015022496A3 (en
Inventor
Graham Ronald Eastham
David William Johnson
Ian Archer
Reuben Carr
Joe Webb
Gill Stephens
Original Assignee
Lucite International Uk Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB201313904A external-priority patent/GB201313904D0/en
Priority claimed from GB201410029A external-priority patent/GB201410029D0/en
Application filed by Lucite International Uk Limited filed Critical Lucite International Uk Limited
Publication of WO2015022496A2 publication Critical patent/WO2015022496A2/en
Publication of WO2015022496A3 publication Critical patent/WO2015022496A3/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Definitions

  • step (b) is performed outside the microorganism in such a manner as to avoid significant toxic exposure of the microorganism to methacrylic acid and/or derivatives thereof.
  • the intermediate alpha- ketoglutarate within the citric acid cycle can also be converted to mesaconic acid via glutamic acid.
  • Bacteria, such as Corynebacterium glutamicum are known which can produce large quantities of glutamic acid under aerobic conditions which could be used as a starting point for genetically engineering microorganisms capable of producing mesaconic acid, for example, industrial quantities of mesaconic acid using the oxidative cycle.
  • the one or more microorganism/s may be genetically modified to enhance or reduce the activity of said natural or genetically engineered enzymes.
  • the gene/s of the one or more enzymes of the glycolysis pathway which may be modified within the microorganism/s or transformed into the microorganism/s to enhance production of mesaconic acid and/or citraconic acid and/or citramalic acid include those encoding any of the following enzymes: pyruvate kinase; phosphopyruvate hydratase ; phosphoglycerate mutase; phosphoglycerate kinase; glyceraldehyde phosphate dehydrogenase; those phosphate isomerase; fructose bisphosphate aldolase; 6- phosphofructokinase; glucose-6-phosphate isomerase; or hexokinase.
  • vectors for coryneform bacteria can include pAM330 (Japanese Patent Laid-open No. 58-67699), pHM1519 (Japanese Patent Laid-open No. 58-77895), pSFK6 (Japanese Patent Laid-open No. 2000-262288), pVK7 (USP2003-0175912A), pAJ655, pAJ61 1 , pAJ1844 (Japanese Patent Laid-open No. 58-192900), pCG1 (Japanese Patent Laid- open No. 57-134500), pCG2 (Japanese Patent Laid-open No.
  • the present invention may further comprise modifications that decrease or eliminate the activity of an enzyme which metabolises mesaconic acid, metabolises citraconic acid or metabolises citramalic acid or metabolises an intermediate in the above-mentioned biosynthesis pathways.
  • an enzyme which metabolises mesaconic acid, metabolises citraconic acid or metabolises citramalic acid or metabolises an intermediate in the above-mentioned biosynthesis pathways can include one or more of fSJ-2-methylmalate dehydratase (4.2.1 .34) and 3-isopropylmalate dehydrogenase, (1 .1 .1 .85).
  • the mesaconic acid and/or citraconic acid and/or citramalic acid is secreted, preferably into the fermentation medium.
  • the mesaconic acid and/or citraconic acid and/or citramalic acid is accumulated within the microorganisms, and preferably the mesaconic and/or citraconic acid and/or citramalic acid is released, preferably into the fermentation medium.
  • the mesaconic acid and/or citraconic acid and/or citramalic acid may be secreted or accumulated in any suitable form including the acid or the monobasic salt or the dibasic salt.
  • Suitable salts are ammonium or diammonium, sodium or disodium, potassium or dipotassium.
  • the molar level of base catalyst to the said at least one dicarboxylic acid is maintained at a sub-stoichiometric level in relation to the formation of the first acid salt thereof during the extraction process and the amount of dicarboxylic acid added is determined accordingly.
  • Expression plasmid pET21 b fdS-27AA-fdr-his, which carries fdS-27AA-fdr-his, was constructed by cloning a fusion of C.tepidum Fds and C.tepidum Fdr (both synthesised by DNA2.0) (linked by following DNA sequence: 5'- gccggtggaggatccggaggtggaggatccggaggtggaggatccggaggtggaggatccggaggtggaggatccggaggtggaggatccggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggcggc ggc-3' (SEQ ID No. 7)) using the method disclosed in patent WO2010/070295
  • Biotransformation was carried out with whole cells co-expressing GlmE/MutS and Maal.
  • the whole cells loading, monosodium glutamate and cobamamide concentrations were varied to provide a statistical design of experiments with high and low levels of each factor.
  • Samples were taken periodically from each reaction and analysed for glutamate, ⁇ -methylaspartate and mesaconic acid by HPLC (Amino acid analysis: Agilent Technologies series 1 100, GraceSmart RP 18 5 ⁇ column from Grace; Organic acid analysis: Agilent Technologies series 1200, Rezex ROA organic acid column from Phenomenex).
  • Plasmid retention during biotransformation was tested for WCBT 1 .1 , 1 .2 and 1 .3 (using whole cells of IL3.0.3(G2).0). By comparing the growth observed on LB plates to the growth observed on LB/Amp plates between corresponding biotransformation conditions, the plasmid retention can be observed. The plasmid was retained in biotransformation WCBT 1 .2 and 1 .3.
  • E.coli BL21 (DE3) was used as the host strain for the expression of Malyl-CoA Lyase (Mcl).
  • the mcl gene was the WT sequence from Rhodobacter sphaeroides (Alber et al. 2006). The mcl gene was synthesised by DNA2.0. The gene was cloned into pEKEx3 using the method disclosed in patent WO2010/070295.
  • the Mcl DNA sequence is shown in SEQ ID N0.15. Cell pellets were resuspended in HEPES buffer (200 mM, pH 7.5) containing lysozyme (2mg/ml) to a wet cell weight of 300 g/l. These were incubated on ice (30 min).
  • Enzyme assay was carried out using a modified version of the Mcl assay described by Zarzycki & Kerfeld (2013). Mcl assays were carried out on a 300 ⁇ scale. Assay contained acetyl CoA (1 mM), pyruvate (10 mM), MgCI 2 (5 mM), cell free extract (30 ⁇ ) and HEPES buffer (200 mM, pH 7.5, 180 ⁇ ). Assays were incubated (30° C, 30 min) and then placed on ice. Formic acid (15 ⁇ ) was added to stop the reaction. The products of the reaction were analysed by LC-MS.
  • the present invention uses this discovery in that it provides for the use of cells such as E.coli in the process of making methacrylic acid and derivatives thereof by the production of precursor acids without the in vivo production of methacrylic acid. This reduces the wasteful destruction of cells, and allows such processes to become commercial viable continuous processes, whilst still remaining environmentally friendly.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
PCT/GB2014/052389 2013-08-02 2014-08-04 A process for production of methacrylic acid and derivatives thereof WO2015022496A2 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB1313904.3 2013-08-02
GB201313904A GB201313904D0 (en) 2013-08-02 2013-08-02 A process for production of methacrylic acid and derivatives thereof
GB1410029.1 2014-06-05
GB201410029A GB201410029D0 (en) 2014-06-05 2014-06-05 A process for production of methacrylic acid and derivatives thereof

Publications (2)

Publication Number Publication Date
WO2015022496A2 true WO2015022496A2 (en) 2015-02-19
WO2015022496A3 WO2015022496A3 (en) 2015-06-25

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2014/052389 WO2015022496A2 (en) 2013-08-02 2014-08-04 A process for production of methacrylic acid and derivatives thereof

Country Status (3)

Country Link
TW (1) TW201508064A (es)
UY (1) UY35692A (es)
WO (1) WO2015022496A2 (es)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018203076A1 (en) 2017-05-03 2018-11-08 Gradley Michelle Modified microorganisms and methods for production of branched c5 carbon compounds
US20190284587A1 (en) * 2016-11-23 2019-09-19 Lucite International Uk Limited Process for the production of methyl methacrylate
US10731185B2 (en) * 2016-03-22 2020-08-04 University Of Georgia Research Foundation, Inc. Genetically engineered microbes and methods for producing citramalate

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3173176B2 (ja) * 1992-10-09 2001-06-04 三菱化学株式会社 Ｓ（＋）−シトラマル酸の製造方法
AU2011333557A1 (en) * 2010-11-24 2013-06-27 Lucite International Uk Limited A process for the production of methacrylic acid and its derivatives and polymers produced therefrom
EP2694663A4 (en) * 2011-04-01 2015-04-29 Genomatica Inc MICRO-ORGANISMS FOR THE PREPARATION OF METHACRYLIC ACID AND METHACRYLATE ESTERS AND METHODS RELATED THEREWITH

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10731185B2 (en) * 2016-03-22 2020-08-04 University Of Georgia Research Foundation, Inc. Genetically engineered microbes and methods for producing citramalate
US20190284587A1 (en) * 2016-11-23 2019-09-19 Lucite International Uk Limited Process for the production of methyl methacrylate
CN110520537A (zh) * 2016-11-23 2019-11-29 卢塞特英国国际有限公司 用于产生甲基丙烯酸甲酯的方法
US11981951B2 (en) * 2016-11-23 2024-05-14 Mitsubishi Chemical UK Limited Process for the production of methyl methacrylate
WO2018203076A1 (en) 2017-05-03 2018-11-08 Gradley Michelle Modified microorganisms and methods for production of branched c5 carbon compounds

Also Published As

Publication number Publication date
WO2015022496A3 (en) 2015-06-25
TW201508064A (zh) 2015-03-01
UY35692A (es) 2015-02-27

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