WO2023120538A1 - Procédé de production d'acide 4-hydroxybenzoïque - Google Patents

Procédé de production d'acide 4-hydroxybenzoïque Download PDF

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Publication number
WO2023120538A1
WO2023120538A1 PCT/JP2022/046954 JP2022046954W WO2023120538A1 WO 2023120538 A1 WO2023120538 A1 WO 2023120538A1 JP 2022046954 W JP2022046954 W JP 2022046954W WO 2023120538 A1 WO2023120538 A1 WO 2023120538A1
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Prior art keywords
acid
poba
aqueous solution
bacterium
bacteria
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PCT/JP2022/046954
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English (en)
Japanese (ja)
Inventor
篤生 鶴谷
宏征 栗原
和典 園木
雄大 樋口
Original Assignee
東レ株式会社
国立大学法人弘前大学
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Application filed by 東レ株式会社, 国立大学法人弘前大学 filed Critical 東レ株式会社
Priority to JP2023512010A priority Critical patent/JPWO2023120538A1/ja
Publication of WO2023120538A1 publication Critical patent/WO2023120538A1/fr

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/40Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42Hydroxy-carboxylic acids

Definitions

  • the present invention includes the following [1] to [6].
  • a method for producing 4-hydroxybenzoic acid comprising the following steps (1) and (2).
  • Step (1) Preparing an aqueous solution containing coumaric acid and ferulic acid
  • Step (2) Bacteria deficient in PobA enzyme expression are allowed to act on the aqueous solution prepared in Step (1), and the bacteria produce ferulic acid. as a growth carbon source, and converting the coumaric acid to 4-hydroxybenzoic acid by the bacteria
  • the step (1) includes alkali-treating the lignin-containing biomass to contain coumaric acid and ferulic acid.
  • [3] The method for producing 4-hydroxybenzoic acid according to [1] or [2], wherein the aqueous solution prepared in step (1) has a sugar concentration of less than 5 g/L.
  • ferulic acid and coumaric acid are sometimes covalently bound to polysaccharides in the solid raw materials, in order to increase the extraction efficiency, a hydrolysis reaction is performed in a high-temperature alkaline aqueous solution for several hours to obtain ferulic acid. and coumaric acid are preferably eluted in an alkaline aqueous solution.
  • the solid raw material for obtaining the extract is preferably lignin-containing biomass.
  • Lignin-containing biomass refers to biological resources that contain at least lignin components. Specific examples of lignin-containing biomass include herbaceous biomass such as bagasse, corn cob, switchgrass, napier grass, erianthus, corn stover, rice straw, and straw; woody biomass such as trees and waste building materials; biomass derived from aquatic environments such as algae and seaweed; grain husk biomass such as corn husks, wheat husks, soybean husks and rice husks.
  • step (1) is a step of treating lignin-containing biomass with alkali to obtain an extract containing coumaric acid and ferulic acid as the aqueous solution.
  • the aqueous solution prepared in step (1) may contain sugar, and the sugar concentration in that case is preferably less than 5 g/L, more preferably 1 g/L or less.
  • the lower limit of the sugar concentration of the aqueous solution prepared in step (1) is zero.
  • the sugar concentration of the aqueous solution prepared in step (1) is 5 g/L or more, the 4HBA production rate and the 4HBA yield from coumaric acid decrease. This is because the bacterium of the present invention preferentially consumes sugar as a carbon source.
  • a Pseudomonas bacterium in which the PobA enzyme is functionally inactivated is, for example, a Pseudomonas bacterium (for example, a wild-type bacterium) before deficient expression of the PobA enzyme, in which the activity of the PobA enzyme is reduced or eliminated.
  • Known methods for deficient expression of the PobA enzyme in target bacteria include methods of disrupting the pobA gene by homologous recombination.
  • methods such as introduction of mutations into the pobA gene and inactivation of the pobA gene are also known.
  • Genetic manipulations such as phage transduction, homologous recombination, plasmid vector insertion, and the like can be used to abolish PobA enzyme expression in the bacterium of interest.
  • Methods using homologous recombination include gene knockout for gene disruption and gene knockin for mutation introduction.
  • a method of disrupting the pobA gene of a wild-type bacterium by homologous recombination is used as a method of deficient PobA enzyme expression in a target bacterium.
  • Disruption of the pobA gene may be insertion of a disruption cassette into the pobA gene by homologous recombination, replacement of the pobA gene with the disruption cassette by homologous recombination, or pobA gene by homologous recombination.
  • Table 1 shows the component composition of the bagasse alkali extract.
  • Pseudomonas putida (P. putida) strain KT2440 was inoculated into 10 mL of LB liquid medium and cultured with shaking at 30° C. for 16 hours. A portion of the culture was inoculated into 5 mL of fresh LB liquid medium and cultured with shaking at 30° C. until OD 600 reached 0.3-0.5. Cells were collected from the resulting culture medium by centrifugation (6,000 ⁇ g, 5 min, 4° C.) and washed with a cooled 10% Glycerol solution. The washing operation was repeated 3 times. The washed cells were collected by centrifugation and suspended in 0.1 mL of chilled 10% Glycerol solution.
  • Example 3 Preculture of Pseudomonas bacterium (KT2440 strain ⁇ PobA strain) deficient in PobA enzyme expression KT2440 strain ⁇ PobA strain was statically cultured overnight at 30°C on an LB agar medium. A colony formed on the agar medium was inoculated into 10 mL of an LB liquid medium and cultured with shaking at 30° C. for 16 hours. 1 mL of a cell suspension (suspended in physiological saline) having an OD 600 of 5.0 was prepared from the obtained culture medium and used as an inoculum.

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  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention a pour but de procurer un procédé de production à haut rendement d'acide 4-hydroxybenzoïque (4HBA) à partir d'un matériau de départ tel qu'une biomasse, et fournit un procédé de production d'acide 4-hydroxybenzoïque comprenant les étapes (1) et (2) suivantes. Étape (1) : préparation d'une solution aqueuse contenant de l'acide coumarique et de l'acide férulique ; et étape (2) : action d'une bactérie présentant un défaut d'expression de l'enzyme PobA sur la solution aqueuse préparée à l'étape (1) pour métaboliser l'acide férulique comme source de carbone de croissance par la bactérie et conversion de l'acide coumarique en acide 4-hydroxybenzoïque par la bactérie.
PCT/JP2022/046954 2021-12-21 2022-12-20 Procédé de production d'acide 4-hydroxybenzoïque WO2023120538A1 (fr)

Priority Applications (1)

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JP2023512010A JPWO2023120538A1 (fr) 2021-12-21 2022-12-20

Applications Claiming Priority (2)

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JP2021-207303 2021-12-21
JP2021207303 2021-12-21

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WO2023120538A1 true WO2023120538A1 (fr) 2023-06-29

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016526896A (ja) * 2013-07-22 2016-09-08 ビーエーエスエフ ソシエタス・ヨーロピアBasf Se フェルラ酸からのバニリンの迅速かつ高収率な製造のためのシュードモナス・プチダkt2440の遺伝子操作
WO2017170549A1 (fr) * 2016-03-29 2017-10-05 東レ株式会社 Procédé de production d'acide hydroxycinnamique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016526896A (ja) * 2013-07-22 2016-09-08 ビーエーエスエフ ソシエタス・ヨーロピアBasf Se フェルラ酸からのバニリンの迅速かつ高収率な製造のためのシュードモナス・プチダkt2440の遺伝子操作
WO2017170549A1 (fr) * 2016-03-29 2017-10-05 東レ株式会社 Procédé de production d'acide hydroxycinnamique

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
AKUTSU, MIHO; KAMIMURA, NAOHUMI; MASAI, EIJI; SONOKI, TOMONORI: "2A16a13 Application of Pseudomonas sp. NGC7 to the production of 4-hydroxybenzoic acid from lignin", ANNUAL MEETING OF JAPAN SOCIETY FOR BIOSCIENCE, BIOTECHNOLOGY, AND AGROCHEMISTRY (JSBBA2020); MARCH 25 - 28, 2020, vol. 2020, 1 January 2020 (2020-01-01) - 28 March 2020 (2020-03-28), pages 272, XP009547239 *
CHRISTOPHER W. JOHNSON, ET AL.: "Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity", METABOLIC ENGINEERING COMMUNICATIONS, ELSEVIER, NL, vol. 3, 1 December 2016 (2016-12-01), NL , pages 111 - 119, XP055537131, ISSN: 2214-0301, DOI: 10.1016/j.meteno.2016.04.002 *
KARLEN STEVEN D., FASAHATI PEYMAN, MAZAHERI MONA, SERATE JOSE, SMITH REBECCA A., SIROBHUSHANAM SIRISHA, CHEN MINGJIE, TYMOKHIN VIT: "Assessing the Viability of Recovery of Hydroxycinnamic Acids from Lignocellulosic Biorefinery Alkaline Pretreatment Waste Streams", CHEMSUSCHEM, WILEY-VCH, DE, vol. 13, no. 8, 21 April 2020 (2020-04-21), DE , pages 2012 - 2024, XP093074000, ISSN: 1864-5631, DOI: 10.1002/cssc.201903345 *
NONAKA KAZUHIRO, OHTA HIROYUKI, SATO YOSHINORI, HOSOKAWA KEIICHI: "Utilization of Phenylpropanoids by Pseudomonas putida Soil Isolates and Its Probable Taxonomic Significance", MICROBES AND ENVIRONMENTS, vol. 23, no. 4, 1 January 2008 (2008-01-01), pages 360 - 364, XP093073993, ISSN: 1342-6311, DOI: 10.1264/jsme2.ME08545 *
VERHOEF, S. RUIJSSENAARS, H.J. DE BONT, J.A.M. WERY, J.: "Bioproduction of p-hydroxybenzoate from renewable feedstock by solvent-tolerant Pseudomonas putida S12", JOURNAL OF BIOTECHNOLOGY, ELSEVIER, AMSTERDAM NL, vol. 132, no. 1, 5 October 2007 (2007-10-05), Amsterdam NL , pages 49 - 56, XP022285747, ISSN: 0168-1656, DOI: 10.1016/j.jbiotec.2007.08.031 *
WADA AYUMU, PRATES ÉRICA T., HIRANO RYO, WERNER ALLISON Z., KAMIMURA NAOFUMI, JACOBSON DANIEL A., BECKHAM GREGG T., MASAI EIJI: "Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics", METABOLIC ENGINEERING, ACADEMIC PRESS, AMSTERDAM, NL, vol. 64, 1 March 2021 (2021-03-01), AMSTERDAM, NL, pages 167 - 179, XP093073986, ISSN: 1096-7176, DOI: 10.1016/j.ymben.2021.01.013 *
WANG WEI, CHEN XIAOWEN, KATAHIRA RUI, TUCKER MELVIN: "Characterization and Deconstruction of Oligosaccharides in Black Liquor From Deacetylation Process of Corn Stover", FRONTIERS IN ENERGY RESEARCH, vol. 7, XP093073997, DOI: 10.3389/fenrg.2019.00054 *
XU, F. SUN, R.C. SUN, J.X. LIU, C.F. HE, B.H. FAN, J.S.: "Determination of cell wall ferulic and p-coumaric acids in sugarcane bagasse", ANALYTICA CHIMICA ACTA, ELSEVIER, AMSTERDAM, NL, vol. 552, no. 1-2, 3 November 2005 (2005-11-03), AMSTERDAM, NL , pages 207 - 217, XP005116955, ISSN: 0003-2670, DOI: 10.1016/j.aca.2005.07.037 *

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