Enzyme cocktail, method for the deconstruction of lignocellulosic biomass, and genetically modified microorganism for producing the cocktail

Abstract

The present invention relates to an enzyme cocktail, a method for the deconstruction of lignocellulosic biomass using said cocktail, and a genetically modified microorganism for producing heterologous enzyme aimed at making biomass fermentable into biorenewable products such as biofuels, organic acids, and hydrocarbon.

Description

Enzymatic cocktail, method for the deconstruction of lignocellulosic biomass and genetically modified microorganism for the production of the cocktail. Field of the invention.

[0001] The present invention relates to an enzymatic cocktail, a method for deconstructing lignocellulosic biomass using said cocktail, and a genetically modified microorganism to produce heterologous enzymes. 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. BACKGROUND OF THE INVENTION

[0002] Cellulose is the most abundant renewable polymer on Earth, and a remarkable 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 to 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, cellulose degradation occurs very slowly. Therefore, 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. In this area, extensive research on filamentous fungi and bacteria has culminated in a canonical model involving at least three main hydrolytic activities: endo-glucanases, cellobiohydrolases, and β-glucosidases. In this model, endo-glucanases and cellobiohydrolases attack cellulose, releasing cellooligosaccharides that are then converted into glucose by β-glucosidases.

[0004] Despite its effectiveness, this canonical triad still has room for improvement. With the inclusion of oxidative enzymes called lytic polysaccharide monooxygenases (LPMOs), it has been shown to be possible to act on crystalline areas of cellulose. Thus, the action of LPMOs in these recalcitrant stretches improves the efficiency of depolymerization when acting together with the hydrolytic enzymatic cascade. An important point regarding LPMOs, however, is that the in situ generation of hydrogen peroxide (H2O2) is a limiting factor for their activity. 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.

[0005] Enzymatic depolymerization of cellulose remains challenging, and new enzymes are constantly being sought, developed, and improved. Documents such as JP4767534, EP2824177, and EP2744898 are examples that claim new modified microorganisms, polynucleotides, and cellulases capable of cleaving both crystalline and amorphous cellulose, but new enzymes and their combinations that increase the efficiency of this process are welcome for the advancement of the production of biorenewable products from lignocellulosic materials. Given the high cost of this step, it is essential that the catalytic efficiency of the cocktail be increasingly maximized, especially in processes where the goal is to cleave cellulose in a medium containing a high concentration of solids. BRIEF DESCRIPTION OF THE INVENTION

[0006] 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.

[0007] The enzyme SEQ ID No: 1 was demonstrably able to increase lignocellulose breakdown 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.

[0008] Furthermore, regarding the co-substrate, SEQ ID No: 1 showed activity under anaerobic conditions when exogenous hydrogen peroxide was supplied, 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.

[0009] The enzymatic cocktail of the present invention increased the saccharification efficiency by 24% and 16.5%, respectively, for pre-treated eucalyptus and sugarcane bagasse materials, two industrially relevant biomasses, under high solids concentrations in the saccharification reactions. High solids concentrations are defined as those greater than 15%.

[0010] In addition to the cocktail, the objectives of the present invention are 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. In microorganisms of the genus Trichoderma, an industrially relevant genus, genetic modification for the co-expression of SEQ ID No: 1 together with its enzymatic cocktail results in an increase in the activity of endocellulases naturally secreted by this fungus. BRIEF DESCRIPTION OF THE FIGURES

[0011] The present invention is illustrated in the embodiments shown in the figures, as briefly described below.

[0012] Figure 1 shows the relative hydrogen peroxide production of SEQ ID No: 1 in the presence of increasing amounts of Avicel. Increases in Avicel concentrations resulted in a decrease in hydrogen peroxide production for TtLPMO9J, an LPMO used for comparison, and for the monomeric mutant of SEQ ID No: 1 (CellOx^2-5), suggesting a peroxide generation mechanism by SEQ ID No: 1 similar to that of TtLPMO9J. The original arrangement of SEQ ID No: 1 (CellOx WT) did not show substrate concentration dependence for hydrogen peroxide generation. Results are expressed as mean ± standard deviation (sd) of three independent experiments (n = 3).

[0013] FIGURE 2 shows the synergism of SEQ ID No: 1 with endocellulases and genetic engineering of the T. reesei strain. (A) Synergism of SEQ ID No: 1 with (A) Endo- and exo-acting cellulases in Avicel, measuring the release of reducing sugars (note that SEQ ID No: 1 does not release reducing sugars). (B) Schematic representation of the genetic engineering strategy used to integrate the SEQ ID No: 2 sequence into the T. reesei genome. (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).

[0014] 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. The data consist of three independent experiments and are expressed as mean ± standard deviation (sd) (n = 3).

[0015] Figure 4 presents the characterization of the enzymatic cocktails produced in bioreactors by the modified and parental strain, showing specific activity levels of xylanase (XYN), filter paper activity (FPase), carboxymethyl cellulase (CMCase), β-xylosidase (XYL), and β-glucosidase (BGL). The data consist of three independent experiments and are expressed as mean ± standard deviation (SD) (n = 3). Tukey's post-hoc tests were performed (P < 0.05), and bars with an asterisk indicate statistically significant differences.

[0016] Figure 5 shows the production of cellobionic acid in enzymatic hydrolysates of sugarcane bagasse and eucalyptus pretreated with the secretome of parental (Br_TrR03) and modified (Br_TrR04) T. reesei strains. Cellobionic acid was quantified by HPAEC-PAD analysis using a standard curve. An increased release of cellobionic acid was observed in saccharification assays performed with the secretome of the strain co-expressing SEQ ID No: 1. Data were recorded after 72 hours of hydrolysis. The data consist of three independent experiments and are expressed as mean ± standard deviation (SD) (n = 3). Tukey's post hoc tests were performed (P < 0.05) and bars with an asterisk indicate statistically significant differences. DETAILED DESCRIPTION OF THE INVENTION

[0017] In one embodiment, the present invention relates to an enzymatic cocktail comprising the enzyme defined as SEQ ID No. 1 and cellulolytic enzymes.

[0018] The enzyme 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 for 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. 1 (Fig. 1), unlike what occurs 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 pretreated eucalyptus and sugarcane bagasse materials. Therefore, in one embodiment of the present invention, the cellulases in the cocktail are of the endoglucanase type.

[0020] At this point, it is worth highlighting that microorganisms of the genus Trichoderma are filamentous fungi widely studied and used in biotechnology, especially for their ability to produce large quantities of cellulolytic enzymes. 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 β-glucosidases, and therefore, another embodiment of the present invention is a cocktail comprising SEQ ID No. 1 and cellulolytic enzymes from Trichoderma, preferably from T. reesei.

[0021] 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 lignocellulosic biomass, and b) obtaining a hydrolyzed material. in which the cocktail comes into contact with the biomass at a pH of 4 to 6 and a temperature of 37° to 50°, under agitation.

[0022] In one embodiment of the method of the present invention, the concentration of total solids in the reaction medium of step (a) is at least 15%. Furthermore, the concentration of enzymatic cocktail in the same reaction medium is at least 1 mg / g of biomass.

[0023] It should be noted that, before applying the method of the present invention, it may be necessary to carry out a pretreatment step of the lignocellulosic biomass. Known pretreatments are divided into physical pretreatments, which include processes such as grinding and extrusion that reduce particle size and increase surface area; chemical pretreatments, among which are acidic, alkaline, organosoluble and oxidative pretreatments; and physicochemical pretreatments, such as steam explosion or supercritical CO₂. A person skilled in the art will know how to choose the best pretreatment for the lignocellulosic biomass with which they intend to work.

[0024] 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 depending on 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 they wish to obtain.

[0025] Finally, 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. In one embodiment, SEQ ID No: 2 is inserted into the microorganism's genome. Alternatively, SEQ ID No: 2 is inserted into the microorganism's plasmid. SEQ ID No: 2 can be codon-optimized to suit the microorganism's gene expression, as known to those skilled in the art, without its product losing activity and characteristics.

[0026] To transform a microorganism with the gene defined by SEQ ID No. 2, this gene can be inserted into a vector. The insertion of SEQ ID No. 2 into the microorganism's genome or plasmid can be achieved using various known state-of-the-art techniques, such as those related to CRISPR / Cas9 systems; however... without limitation to these.

[0027] In one embodiment of the present invention, the microorganism is a fungus, preferably of the genus Trichoderma. More preferably, the fungus is T. reesei. In the case of 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. In this sense, the xyn4 locus presents itself as a viable locus possibility, but the invention is not limited to this, so that a person skilled in the art may choose other loci for the insertion of SEQ ID No. 2. EXAMPLES OF EMBODIMENTS OF THE INVENTION

[0028] What follows presents exemplary, non-restrictive examples of the object described herein, illustrating the results and advantages achieved.

[0029] 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 from the GH5 and GH45 families (Fig. 2A) were evaluated. A notable degree of synergism was observed with endo-acting cellulases. However, it was not observed with exo-acting cellulases, such as the cellobiohydrolase GH7 from Trichoderma reesei (Cel7A, Fig. 2A). The fact that 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 endo-glucanases.

[0030] In the current state of the art, all available data on the synergism of CAZymes with oxidative enzymes such as LPMOs are exclusively based on in vitro supplementation assays, preventing the prediction of the tangible impact of oxidative enzymes in these enzyme cocktails when co-expressed by the same strain. For example, there is no record of any Trichoderma strain engineered with oxidative enzymes to date, although most commercially available enzyme cocktails contain them. Oxidative enzymes to enhance lignocellulose degradation. Therefore, aiming to fill this gap, 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). The presence of the SEQ ID No: 1 enzyme in the secretome was confirmed by proteomics (Table 1), and increased cellobionic acid release was observed in saccharification assays performed with the secretome of the strain co-expressing SEQ ID No: 1 (Fig. 5).Furthermore, an increase in CMCase activity was observed in the enzyme cocktail containing SEQ ID No: 1, corroborating the synergism assays (Fig. 2A). The observed improvement in saccharification is notably relevant, since the secretome of the parental strain is already highly effective and competitive for lignocellulose deconstruction. Taken together, these results demonstrate that SEQ ID No: 1 can enhance lignocellulose degradation both in vitro (provided exogenously, supplementing a cellulosic enzyme cocktail) and in vivo (co-expressed in Trichoderma), under industrially relevant conditions, exhibiting synergism with endocellulases.

[0031] Table 1. Secretome of the Br_TrR04 strain co-expressing SEQ ID No:1. Accession FUN_999995 Unique peptides 13 Confidence score 84.2 Description NMFMLLOJ_02003_CellOx OS=Trichoderma reesei (strain L11) OX=51453 GN=CellOxWT PE=4 SV=1 Abundance 1 4940 2 5346 3 4891 Fmol 1 5.6 2 6.0 3 5.4 Percentage 1 0 relative 2 0.1 3 0.1 Average 0.1 SD 0.00 CV (%) 3 Fmol Average 5.7 SD 0.31 CV (%) 5.4

Claims

CLAIMS 1. Enzymatic cocktail characterized by comprising the enzyme defined as SEQ ID No. 1 and cellulolytic enzymes.

2. Enzymatic cocktail, according to claim 1, characterized in that the cellulolytic enzymes are of the endoglucanase type.

3. Enzymatic cocktail, according to claim 1, characterized in that the cellulolytic enzymes are enzymes from Trichoderma, preferably from T. reesei.

4. Method for the deconstruction of lignocellulosic biomass characterized by comprising: a) contacting a cocktail as defined in claims 1 to 3 with a lignocellulosic biomass, and b) obtaining a hydrolyzed material in which the contact of the cocktail with the biomass occurs at pH 4 to 6 and at a temperature of 37° to 50°, under agitation.

5. Method according to claim 4, characterized in that the concentration of total solids in the reaction medium of step (a) is at least 15%. 6.Method according to claim 4, characterized in that the concentration of enzymatic cocktail in the reaction medium of step (a) is at least 1 mg / g biomass.

7. Genetically modified microorganism characterized in comprising the gene as defined as SEQ ID No. 2 operationally linked to a promoter and a terminator in its genome or plasmid.

8. Microorganism according to claim 7, characterized in that said microorganism is a fungus, preferably of the genus Trichoderma.

9. Microorganism according to claim 8, characterized in that said microorganism is T. reesei.

10. Microorganism, according to claims 7 or 8, characterized in that SEQ ID No. 2 is located between the promoter and the terminator of the xylanase IV locus (xyn4).

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