Modified trichoderma fungal strain for the production of an enzymatic cocktail with enhanced cellulolytic activity

Abstract

The present invention relates to a new Trichoderma strain comprising an additional genetic modification to enable the production of an enzymatic cocktail with enhanced cellulolytic activity, allowing an increase in glucose release and in the saccharification efficiency of plant biomasses.

Description

Modified Trichoderma fungal strain for the production of an enzymatic cocktail with enhanced cellulolytic activity. DESCRIPTION FIELD

[0001] The present invention relates to a new strain of Trichoderma comprising additional gene modification to enable the production of an enzymatic cocktail with enhanced cellulolytic activity compared to that described in BR 11 2022 000830 9. The present invention is applicable in the industrial biotechnology sector, in biomass saccharification processes for the production of biofuels and renewable chemicals. FUNDAMENTALS OF DESCRIPTION

[0002] The genus Trichoderma comprises filamentous fungi that are of great importance in biotechnology, especially in the field of industrial enzyme production. This genus is noteworthy for its ability to produce large quantities of cellulase enzymes, which are crucial for breaking down cellulose, one of the main components of plant cell walls, into simpler sugars. These sugars serve industries such as biofuel production and biorenewable chemicals, for example. Furthermore, the enzymes are used in the textile industry for fabric softening and in the food industry to improve the texture of baked goods.

[0003] Over the years, scientists have extensively studied the genetics of fungi of the genus Trichoderma and developed genetically modified strains with enhanced enzyme production capabilities. These efforts have significantly improved the efficiency and cost-effectiveness of industrial processes dependent on cellulases.

[0004] Overall, the genus Trichoderma is a prime example of how microorganisms can be harnessed for industrial purposes, contributing to the development of sustainable and environmentally friendly technologies. Patent BR 11 2022 000830 9, defended on February 7, 2024, protects a A new Trichoderma strain comprising gene modifications that enable enhanced production of an enzymatic cocktail, including at least overexpression of the Xyr1 transcription factor, as defined in SEQ ID NO: 1; disruption of the ACE1 gene as defined in SEQ ID NO: 2; disruption of the SLP1 gene as defined in SEQ ID NO: 3; and expression of the Cel3a gene from Rasamsonia emersonii as defined in SEQ ID NO: 4 (CTBE_R4), which resulted in a beta-glucosidase activity 42 times greater than that of a strain containing only the overexpression of the Xyr1 transcription factor, as defined in SEQ ID NO: 1, and the disruption of the ACE1 gene as defined in SEQ ID NO: 2 (indicated in the text as CTBE_R2). However, the CTBE_R4 strain, protected under BR 11 2022 000830 9, has had its activity further enhanced through an additional gene modification, which will be described below. BRIEF DESCRIPTION OF THE INVENTION

[0005] The present invention relates to a Trichoderma strain comprising, in addition to at least overexpression of the transcription factor Xyr1, as defined in SEQ ID NO: 1; disruption of the ACE1 gene as defined in SEQ ID NO: 2; disruption of the SLP1 gene as defined in SEQ ID NO: 3; and the expression of the Cel3a gene of Rasamsonia emersonii as defined in SEQ ID NO: 4, in accordance with BR 11 2022 000830 9, the expression of a LPMO (Lytic polysaccharide monooxygenase) codon optimized for Trichoderma, as defined in SEQ ID NO: 5. In one embodiment, said LPMO is inserted into the xyn 5 locus, wherein said xyn 5, as defined in SEQ ID NO: 6, is deleted prior to the insertion of said LPMO.

[0006] Among the advantages of the present invention are the fact that the Trichoderma strain according to BR 11 2022 000830 9, supplemented with an optimized LPMO (Lytic polysaccharide monooxygenase) codon for Trichoderma, as per SEQ ID NO: 5, proved viable in terms of survival after modifications, and that the LPMO promoted a synergistic effect on the cocktail produced with the strain claimed in BR 11 2022 000830 9, promoting greater glucose release and greater saccharification efficiency after 72 h of processing when compared to the parental strain of BR 11 2022. 000830 9 and with a commercial enzymatic cocktail. The test was performed on different pre-treated plant biomasses and the results are presented in the example. DETAILED DESCRIPTION OF THE INVENTION

[0007] The present invention relates to a modified Trichoderma strain for the production of an enzymatic cocktail with enhanced cellulolytic activity, said strain comprising, at least, overexpression of the transcription factor Xyr1, as defined in SEQ ID NO: 1; disruption of the ACE1 gene as defined in SEQ ID NO: 2; disruption of the SLP1 gene as defined in SEQ ID NO: 3; and the expression of the Cel3a gene of Rasamsonia emersonii as defined in SEQ ID NO: 4, further characterized by comprising a gene encoding an LPMO (Lytic polysaccharide monooxygenase) codon optimized for Trichoderma, as defined in SEQ ID NO: 5, said gene operationally comprising a promoter and a terminator. In this way, the strain of the present invention comes to express said LPMO.

[0008] Preferably, the modified Trichoderma strain is of the species Trichoderma reesei, especially the Rut C30 strain.

[0009] The aforementioned LPMO codon optimized for Trichoderma originates from Lentinus similis.

[0010] In one embodiment, the so-called LPMO is inserted into the xyn 5 locus of the fungus. To do this, xyn 5 (SEQ ID NO: 6) is deleted and the LPMO is operationally linked to the xyn 5 promoter (SEQ ID NO: 7) and the xyn 5 terminator (SEQ ID NO: 8) by any gene editing techniques known in the state of the art.

[0011] Additionally, the modified Trichoderma strain comprises a disruption of the PEP1 gene as per SEQ ID NO: 9, a disruption that should be understood as a point mutation, insertion, inversion, or deletion of one or more base pairs of DNA, among other constants of the state of the art.

[0012] It is also optional that the modified Trichoderma strain express the SucA gene from Aspergillus niger according to SEQ ID NO: 10.

[0013] It should be noted that this locus is one option for the insertion site of LPMO SEQ ID NO: 5 in the Trichoderma genome, but this is not the only location, as a person skilled in the art will know. Other analogous location options can be used and are equally contemplated in the present invention. Therefore, the description should be interpreted not as limiting, but merely as examples of particular embodiments that embody the inventive concept presented herein. EXAMPLE

[0014] The strain of the present invention was constructed from the protected strain in BR 11 2022 000830 9, which is a Trichoderma reesei RUTC30 containing the overexpression of the transcription factor Xyr1, as defined in SEQ ID NO: 1; disruption of the ACE1 gene as defined in SEQ ID NO: 2; disruption of the SLP1 gene as defined in SEQ ID NO: 3; and the expression of the Cel3a gene of Rasamsonia emersonii as defined in SEQ ID NO: 4, as well as containing the disruption of the PEP1 gene and the expression of the SucA gene of Aspergillus niger (Original strain).

[0015] In the original lineage, the xyn 5 gene (SEQ ID NO: 6) was deleted and the Lentinus similis codon-optimized LPMO (Lytic polysaccharide monooxygenase) for Trichoderma, as per SEQ ID NO: 5, was operationally linked to the xyn 5 promoter (SEQ ID NO: 7) and the xyn 5 terminator (SEQ ID NO: 8) (Present invention).

[0016] For the production of enzymatic cocktails, the original fungal strain and the strain of the present invention were cultivated in 3 L BioFIo / CelliGen 115 benchtop bioreactors containing 1 liter of industrial medium based on sugarcane molasses (20 gL- 1 of yeast cells, 20 gL- 1 of ammonium sulfate and 50 gL- 1 of total reducing sugars (TRS), pH 4.5). The reactors were inoculated with 10% (v / v) inoculum, which was prepared by cultivating spores at a concentration of 10 6 spores / mL in Erlenmeyer flasks containing the industrial medium at 200 rpm, for 4 days at 28 °C. Aeration was The mixture was maintained at a constant rate of 0.7 slpm of compressed air, and a cascade agitation system (400-1000 rpm) was employed to ensure that the dissolved oxygen concentration remained above 20%. The pH during cultivation was maintained at 4.5% ± 0.5 by adding 2M phosphoric acid and 10% (v / v) ammonium hydroxide. After 25 hours, when the glucose concentration reached approximately 5 g / L... 1 Continuous feeding was initiated with a solution of cane molasses (450 g / l) 1 of ART) containing 1 gL 1 of antifoaming agent. The molasses feed rate was adjusted to 1.3 gART.I_- 1 .h- 1 in relation to the instantaneous mass in the bioreactor, generating a non-linear feed profile. After fermentation, all the broth was centrifuged (6,000 rpm, 30 min, 4 °C) and the supernatant was recovered and stored at 4 °C.

[0017] The cocktails were used for the saccharification, for 72 hours, of hydrothermally pretreated sugarcane bagasse, sugarcane bagasse pretreated with diluted acid, pretreated corn straw, and pretreated eucalyptus.

[0018] Under these process conditions, it was possible to observe an increase in released glucose of approximately 4 g / L compared to the original strain and 3 to 6 g / L compared to the commercial cocktail, considering these biomasses and their respective pretreatments. This translates to a 6 to 10 percentage point increase in saccharification efficiency compared to the original strain and 6 to 16 points compared to the commercial cocktail. *The superscript letters next to the mean values ​​refer to the ANOVA statistical analysis of the results. Different letters indicate a significant difference between the groups according to Tukey's test (p < 0.05). * If the result of glucose release with the enzymatic cocktail of the original BR 11 strain 2022 000830 9 for considered as 100%.

Claims

CLAIMS 1. A modified Trichoderma fungal strain for the production of an enzymatic cocktail with enhanced cellulolytic activity comprising at least overexpression of the Xyr1 transcription factor, as defined in SEQ ID NO: 1; disruption of the ACE1 gene as defined in SEQ ID NO: 2; disruption of the SLP1 gene as defined in SEQ ID NO: 3; and expression of the Cel3a gene from Rasamsonia emersonii as defined in SEQ ID NO: 4, further characterized by comprising the LPMO (Lytic polysaccharide monooxygenase) gene as defined in SEQ ID NO: 5, operationally linked to a promoter and a terminator.

2. A strain according to claim 1, characterized in that the genetically modified fungus is preferentially Trichoderma reesei.

3. A strain according to claim 2, characterized in that the genetically modified fungus is preferably Trichoderma reesei of the Rut C30 strain.

4. A lineage according to claim 4, characterized in that the LPMO is inserted at locus xyn 5, operationally linked to the xyn 5 promoter (SEQ ID NO: 7) and the xyn 5 terminator (SEQ ID NO: 8).

5. A strain according to claim 1, characterized in that it comprises disruption of the PEP1 gene as per SEQ ID NO:

9.

6. Strain, according to claim 1, characterized in that it comprises expression of the SucA gene of Aspergillus niger according to SEQ ID NO: 10.

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