WO2026015965A1 - Cocktail enzymatique, procédé de déconstruction de biomasse lignocellulosique et micro-organisme génétiquement modifié pour la production du cocktail - Google Patents

Cocktail enzymatique, procédé de déconstruction de biomasse lignocellulosique et micro-organisme génétiquement modifié pour la production du cocktail

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Publication number
WO2026015965A1
WO2026015965A1 PCT/BR2025/050317 BR2025050317W WO2026015965A1 WO 2026015965 A1 WO2026015965 A1 WO 2026015965A1 BR 2025050317 W BR2025050317 W BR 2025050317W WO 2026015965 A1 WO2026015965 A1 WO 2026015965A1
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WO
WIPO (PCT)
Prior art keywords
cocktail
seq
microorganism
enzyme
deconstruction
Prior art date
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Pending
Application number
PCT/BR2025/050317
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English (en)
Portuguese (pt)
Inventor
Clelton Aparecido DOS SANTOS
Fernanda MANDELLI
Evandro Antonio DE LIMA
Gabriela Felix PERSINOTI
Mario Tyago MURAKAMI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centro Nacional de Pesquisa em Energia e Materiais CNPEM
Original Assignee
Centro Nacional de Pesquisa em Energia e Materiais CNPEM
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Publication of WO2026015965A1 publication Critical patent/WO2026015965A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/885—Trichoderma

Definitions

  • the present invention relates to an enzymatic cocktail, a method for the deconstruction of lignocellulosic biomass using said cocktail, and a genetically modified microorganism to produce heterologous enzyme.
  • the objective of the present invention is to make the biomass fermentable into biorenewable products.
  • the present invention is in the field of industrial biotechnology.
  • Cellulose is the most abundant renewable polymer on Earth, being a notable source of carbon for biotransformation into fuels, chemicals and other materials via microbial routes. However, because it is formed by glucose chains with some amorphous regions and others highly organized in a crystalline structure, the degradation of cellulose requires a set of enzymes with different specializations acting in synergy.
  • the depolymerization of cellulose through biological degradation represents a major challenge for the large-scale use of lignocellulosic materials in biorefineries: notably, the conversion of cellulose into glucose can represent up to 50% of the total cost of bioproducts such as bioethanol, organic acids, and hydrocarbons, highlighting the importance of this process for the transition to a circular economy based on bioenergy and biorenewables. [0003] In nature, the degradation of cellulose occurs very slowly. Thus, the development of an effective industrial bioprocess involving the depolymerization of lignocellulosic materials requires the composition of effective enzymatic cocktails and the development of suitable conditions for their action, in order to make it technically and economically viable.
  • LPMOs in situ generation of hydrogen peroxide (H2O2) is a limiting factor for their activity.
  • H2O2 hydrogen peroxide
  • LPMOs are orders of magnitude more effective in the presence of hydrogen peroxide than oxygen and require a source or supply of peroxide for their activity. The peroxide, then, acts as a co-substrate, allowing the LPMO to perform its catalytic function.
  • One of the objectives of the present invention is to disclose an enzymatic cocktail comprising the enzyme defined as SEQ ID No: 1 and cellulolytic enzymes.
  • the enzyme SEQ ID No: 1 has been demonstrably able to increase the decomposition of lignocellulose both in vitro (provided exogenously, supplementing a cellulase-rich cocktail) and in vivo (co-expressed in fungi of the genus Trichoderma), showing synergy with endocellulases under industrially relevant conditions.
  • SEQ ID No: 1 has shown activity under anaerobic conditions when exogenous hydrogen peroxide was provided, exhibiting lytic cellulosic peroxygenase activity. Comparative analysis of the time course of oxidized product formation in the presence of oxygen or hydrogen peroxide revealed similar catalytic rates between them, which differs from what happens with LPMOs, which are orders of magnitude more efficient in the presence of hydrogen peroxide compared to oxygen. In this sense, in situ peroxide generation is not a limiting factor for the cocktail of the present invention, circumventing this well-established problem for cocktails comprising LPMOs.
  • the enzymatic cocktail of the present invention increased the saccharification efficiency by 24% and 16.5%, respectively, for pretreated eucalyptus and sugarcane bagasse materials, two industrially relevant biomasses, under high solids concentrations in the saccharification reactions. High solids concentrations are understood to be those greater than 15%.
  • the present invention aims to disclose a method for deconstructing lignocellulosic biomass using the cocktail of the present invention and a microorganism genetically modified to express SEQ ID No: 1.
  • FIGURE 1 shows the relative production of hydrogen peroxide from SEQ ID No: 1 in the presence of increasing amounts of Avicel.
  • FIGURE 2 shows the synergism of SEQ ID No: 1 with endocellulases and genetic engineering of the T. reesei strain.
  • (C) Saccharification efficiency of the enzyme cocktail produced by the modified strain (Br_TrR04 co-expressing SEQ ID No: 1) under relevant industrial conditions, using pre-treated eucalyptus and sugarcane bagasse. The results of panels A and C are expressed as mean ⁇ standard deviation (sd) of three independent experiments (n 3).
  • FIGURE 3 shows the production of extracellular proteins in a bioreactor by the modified Trichoderma reesei strain co-expressing SEQ ID No: 1 (Br_TrR04), comparing the Br_TrR03 (parental) strain and the modified Br_TrR04 strain in bioreactor cultures with inducer-rich medium.
  • FIGURE 4 shows the characterization of the cocktails Enzymatic hydrolysates produced in bioreactors by the modified and parental strains, showing specific activity levels of xylanase (XYN), filter paper activity (FPase), carboxymethyl cellulase (CMCase), ⁇ -xylosidase (XYL), and ⁇ -glucosidase (BGL).
  • XYN xylanase
  • FPase filter paper activity
  • CMCase carboxymethyl cellulase
  • XYL ⁇ -xylosidase
  • BGL ⁇ -glucosidase
  • FIGURE 5 shows the production of cellobionic acid in enzymatic hydrolysates of sugarcane bagasse and eucalyptus pretreated with the secretome of the parental (Br_TrR03) and modified (Br_TrR04) T. reesei strains.
  • Cellobionic acid was quantified by HPAEC-PAD analysis using a standard curve.
  • the present invention relates to an enzymatic cocktail comprising the enzyme defined as SEQ ID No. 1 and cellulolytic enzymes.
  • SEQ ID No. 1 is a copper-dependent metalloenzyme and exhibits a mechanism involved in cellulose oxidation. Elucidation of its structure confirms an exo-action mechanism and a dual catalytic model, in which dimerization allows in situ peroxide generation while interacting with the substrate. It was observed that increasing cellulose concentrations did not cease the peroxide-generating capacity of SEQ ID No.
  • SEQ ID No. 1 (Fig. 1), unlike what happens with LPMOs. In this sense, SEQ ID No. 1 exhibits a sophisticated mechanism to overcome the typical limiting factor for the peroxygenase activity of LPMOs. [0019] Surprisingly, SEQ ID No. 1 was able to increase the activity of endoglucanases in the breakdown of lignocellulose, acting synergistically with them in the process. As already indicated, the cocktail composed of cellulolytic enzymes and SEQ ID No. 1 increased the breakdown efficiency by 24% and 16.5%, respectively, for pre-treated eucalyptus and sugarcane bagasse materials. Therefore, in one embodiment of the present invention, the cellulases in the cocktail are of the endoglucanase type.
  • T. reesei in particular, is known for its ability to produce cellulases that are currently used in various industries, including biofuel production, paper and pulp processing, the textile industry, and the food industry.
  • T. reesei is capable of secreting a cocktail of cellulolytic enzymes that includes endoglucanases, cellobiohydrolases and ⁇ -glucosidases and, therefore, another embodiment of the present invention is a cocktail comprising SEQ ID No.
  • the present invention also relates to a method for the deconstruction of lignocellulosic biomass.
  • the method is characterized by comprising: a) contacting a cocktail of the present invention with a lignocellulosic biomass, and b) obtaining a hydrolyzed material. wherein the contact of the cocktail with the biomass occurs at pH 4 to 6 and at a temperature of 37° to 50°, under agitation.
  • the concentration of total solids in the reaction medium of step (a) is at least 15%.
  • the concentration of enzymatic cocktail in the same reaction medium is at least 1 mg/g of biomass.
  • the hydrolyzed material obtained in step (b) of the method of the present invention can be biotransformed into products such as biofuels, organic acids, hydrocarbons and other chemicals according to the microorganism and process employed.
  • products such as biofuels, organic acids, hydrocarbons and other chemicals according to the microorganism and process employed.
  • a person skilled in the art will know how to choose the best microorganism and process for the final product he intends to obtain.
  • the present invention relates to a microorganism capable of producing the enzyme as defined as SEQ ID No: 1.
  • Said microorganism is characterized by comprising the gene as defined as SEQ ID No 2 operationally linked to a promoter and a terminator.
  • SEQ ID No 2 is inserted in the genome of the microorganism.
  • SEQ ID No 2 is inserted in the plasmid of the microorganism.
  • SEQ ID No: 2 can be a codon optimized to match the gene expression of the microorganism, as known to those skilled in the art, without its product losing activity and characteristics.
  • the gene can be inserted into a vector.
  • the insertion of SEQ ID No 2 into the genome or plasmid of the microorganism can be done by various techniques known in the state of the art, such as techniques related to CRISPR/Cas9 systems, however without limitation to these.
  • the microorganism is a fungus, preferably of the genus Trichoderma. More preferably, the fungus is T. reesei.
  • SEQ ID No. 2 can be located between the promoter and the terminator of the xylanase IV locus (xyn4). Although this is not the only locus, it is understood that by placing SEQ ID No. 2 in this position, there will be concomitant expression with the other genes that encode for cellulolytic enzymes, forming an enzymatic cocktail with improved activity compared to the basal cocktail of the fungus.
  • Example 1 Based on its ability to bind to crystalline cellulose and its distinct mode of action that exclusively releases cellobionic acid, possible synergies with other cellulose-active CAZymes such as those of the GH5 and GH45 families (Fig. 2A) were evaluated.
  • SEQ ID No: 1 is also an exo-acting enzyme and its catalytic activity does not produce new reducing terminals in the cellulose fiber corroborates the lack of synergism with Cel7A. This is consistent with the fact that Cel7A activity is negatively affected by cellulose oxidation by some LPMOs. Thus, SEQ ID No: 1 does not favor synergy with exo-acting enzymes, while it is beneficial for endoglucanases.
  • the sequence SEQ ID No: 2 which codes for the enzyme as defined herein as SEQ ID No: 1, was inserted into the xyn4 locus of the Trichoderma reesei Br_TrR03 strain developed for lignocellulose biorefineries using a customized CRISPR/Cas9 approach (Fig. 2B).
  • the secretome produced by this engineered strain under industrially relevant conditions significantly increased saccharification efficiency by 24% and 16.5% for pretreated eucalyptus and sugarcane bagasse materials, respectively (Fig. 2C, and Figs. 3 and 4, and Table 1).

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
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  • Organic Chemistry (AREA)
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  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne un cocktail enzymatique, un procédé de déconstruction de biomasse lignocellulosique utilisant ledit cocktail et le micro-organisme génétiquement modifié pour produire une enzyme hétérologue visant à transformer la biomasse fermentable en produits biorenouvelables, tels que des biocarburants, des acides organiques et des hydrocarbures.
PCT/BR2025/050317 2024-07-19 2025-07-18 Cocktail enzymatique, procédé de déconstruction de biomasse lignocellulosique et micro-organisme génétiquement modifié pour la production du cocktail Pending WO2026015965A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
BR1020240148363 2024-07-19
BR102024014836 2024-07-19

Publications (1)

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WO2026015965A1 true WO2026015965A1 (fr) 2026-01-22

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2807702A1 (fr) * 2010-08-20 2012-02-22 Codexis, Inc. Utilisation de proteines de la famille 61 des glycoside hydrolases dans le traitement de la cellulose
WO2015187935A1 (fr) * 2014-06-06 2015-12-10 Novozymes A/S Compositions enzymatiques et leurs utilisations
WO2017070219A1 (fr) * 2015-10-20 2017-04-27 Novozymes A/S Variants de polysaccharide mono-oxygénase lytique (lpmo) et polynucléotides codant pour ceux-ci

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2807702A1 (fr) * 2010-08-20 2012-02-22 Codexis, Inc. Utilisation de proteines de la famille 61 des glycoside hydrolases dans le traitement de la cellulose
WO2015187935A1 (fr) * 2014-06-06 2015-12-10 Novozymes A/S Compositions enzymatiques et leurs utilisations
WO2017070219A1 (fr) * 2015-10-20 2017-04-27 Novozymes A/S Variants de polysaccharide mono-oxygénase lytique (lpmo) et polynucléotides codant pour ceux-ci

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AMANDA HILDEBRAND, TAKAO KASUGA, ZHILIANG FAN: "Production of Cellobionate from Cellulose Using an Engineered Neurospora crassa Strain with Laccase and Redox Mediator Addition", PLOS ONE, vol. 10, no. 4, US, pages e0123006, XP055235006, DOI: 10.1371/journal.pone.0123006 *
ANGELTVEIT CAMILLA F., VÁRNAI ANIKÓ, EIJSINK VINCENT G. H., HORN SVEIN J.: "Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities", BIOTECHNOLOGY FOR BIOFUELS AND BIOPRODUCTS, vol. 17, no. 1, 9 March 2024 (2024-03-09), pages 1 - 14, XP093392344, ISSN: 2731-3654, DOI: 10.1186/s13068-024-02485-6 *
KONT RIIN, BISSARO BASTIEN, EIJSINK VINCENT G. H., VÄLJAMÄE PRIIT: "Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)", NATURE COMMUNICATIONS, vol. 11, no. 1, 13 November 2020 (2020-11-13), UK, pages 1 - 10, XP093392342, ISSN: 2041-1723, DOI: 10.1038/s41467-020-19561-8 *

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