Hyper-producing trichoderma reesei strain having an enhanced beta-glucosidase activity

Abstract

The present invention relates to a Trichoderma reesei strain which is hyper-producing and which has enhanced -glucosidase activity, as well as the use of said strain.

Claims

1. A Trichoderma reesei strain deposited with Collection Nationale de Cultures de Microorganismes (CNCM) under the Accession Number CNCM I-5221.

2. A method for producing -glucosidase, comprising: culturing the Trichoderma reesei strain of claim 1 in a culture medium comprising a suitable substrate and recovering the -glucosidase from the culture medium.

Description

FIGURES

(1) FIG. 1 represents fed-flask protein production.

(2) FIG. 2 represents the principle of the IFP nergies nouvelles cellulase production process.

(3) FIG. 3A represents the evaluation of the quantity of biomass during fermentation in a DASGIP bioreactor.

(4) FIG. 3B represents the evaluation of the produced quantity of proteins produced during fermentation in a DASGIP bioreactor.

(5) FIG. 4 represents the genealogy of the strains obtained from the strain QM6a. The strains marked with an asterisk were tested by crossing with the strain RuA149 (see example 2). The references of the strains shown in the Figure are as follows:

(6) (1) Mandels, M., Weber, J., & Parizek, R. (1971). Enhanced cellulase production by a mutant of Trichoderma viride. Applied microbiology, 21(1), 152-4. (2) Mandels M. (1975). Microbial sources of cellulase. Biotechnol Bioeng. 5, 81-105. (3) Gruber, F., Visser, J., Kubicek, C. P., & De Graaff, L. H. (1990). The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain. Current genetics, 18(1), 71-6. (4) Durand, H., Clanet, M., & Tiraby, G. (1988). Genetic improvement of Trichoderma reesei for large scale cellulase production. Enzyme and microbial technology, 10 (June), 341-346. (5) Eveleigh D E, Montenecourt B S (1979) Increasing yields of extracellular enzymes. Adv Appl Microbiol 25:57-74. (6) Montenecourt B S, Eveleigh D E (1977). Preparation of Mutants of Trichoderma reesei with Enhanced Cellulase Production. Applied and environmental microbiology, 34(6), 777-782.

EXAMPLES

Example 1: Preparation of the Strain According to the Present Invention (i.e. the Strain as Deposited Under the Number CNCM I-5221) and Determining the Characteristics Thereof

(7) 1. Preparation

(8) The cellulase hyper-producing strain RutC30 (or Rut-C30) (Montenecourt and Eveleigh, 1977) was crossed with the strain A2 according to the protocol described in Seidl et al. The strain RutC30 (deposited at ATCC under the number 56765) was obtained by means of three mutagenesis steps from the strain QM6a and is one of the best producers of cellulases in the public domain (Peterson and Nevalainen, 2012).

(9) After crossing, a plurality of ascospore isolation campaigns were carried out. 295 purified descendants were obtained. The latter were then subject to screening in three steps. These successive screens make it possible to reduce the number of candidates from a few hundred to the individual with the highest performance for cellulase production.

(10) 2. Cellulase ProductionMicroplate Test

(11) The first screening step is based on a method for measuring the production of extracellular proteins using the Bradford method making it possible to rank the candidates and intended to reduce the number thereof drastically so as to only transfer to the second step around ten individuals of the most interest.

(12) The second screen is based on a miniaturisation of the industrial conditions (Jourdier et al., 2012) and makes it possible to select a few individuals for which the performances will be tested in bioreactors.

(13) During the first screen, the descendants are cultured in a microplate in 2 mL of cellulase induction medium (such as Avicel, marketed by MSDS under the reference No.: 9004-34-6).

(14) After seven days of culture, the cellulase production is measured by assaying the quantity of total proteins secreted in the supernatant according to the Bradford method. A correlation between total secreted proteins and cellulases may be made as in T. reesei, the main exoglucanases (CBHI, CBHII) and endoglucanases (EGI, EGII) may represent up to 90% of the total quantity of proteins secreted (Markov et al. 2005).

(15) Only the ten best producers of cellulases, exhibiting a standard deviation less than 10%, were selected.

(16) 3. Cellulase ProductionMiniaturisation of Industrial Conditions

(17) The ten best producers of cellulases were selected to be subjected to a second screen which uses the fed-flask protocol developed by Jourdier et al., 2012 and which is based on the miniaturisation of a fed-batch protocol suitable for maximising the enzyme production (Jeude et al., 2006). This protocol makes it possible to produce cellulases in a flask with a controlled feed with a stoichiometric mixture of carbonated substrate and NH3 base, which makes it possible to stabilise the pH of the culture without a pH control system. An incubator with a rotary stirrer is used to control the stirring and the temperature (Jourdier et al., 2012).

(18) The fed-flask methodology makes it possible to test eight strains in parallel. Each flask is fed by pumps with a flow rate of 0.3 mL/hr with a solution containing 50 g/L of lactose, 0.8 g/L of (NH4)2SO4, and 160 mM NH3. At 96 hrs, the total secreted proteins are assayed. The results of the strains QM6A, A2, RutC30 and RuA-149 are shown in FIG. 1. The fed-flask protein production was only evaluated once for all the strains except for the reference strain RutC30. The percentages represent the rate of protein production with respect to the reference strain RutC30.

(19) This production method makes it possible to rank the strains according to their production of secreted proteins which correspond in T. reesei to cellulases (Markov et al., 2005). After a fed-batch process in lactose for 96 hrs, the assay of the proteins produced measured according to the Lowry method (FIG. 1) confirms that the strain A2 produces more proteins than the strain QM6a and two times less than the hyper-producing strain RutC30.

(20) By means of this second screen, the strain RuA-149 was selected to be tested in bioreactors mimicking industrial conditions on a small scale. The bioreactor culture is carried out according to the IFP nergies nouvelles production protocol (Pourquie et al., 1988) which distinguishes two phases (FIG. 2): a batch phase of approximately 30 hours which corresponds to a growth phase of T. reesei which produces biomass until total glucose consumption; a fed-batch phase of approximately 200 hours during which cellulase production is induced by adding lactose at a constant rate so as to maximise and keep constant the production rate.

(21) The production of biomass (FIG. 3A) and proteins (FIG. 3B) (assays with the Lowry method) were evaluated throughout the fermentation in DASGIP bioreactors. The specific Filter Paper (FP) (endoglucanase and exoglucanase activity) and -glucosidase activities are assayed only for the final samples (after 200 hours). These experiments were repeated twice and the results obtained are similar.

(22) The results are shown in Table 1 hereinafter.

(23) TABLE-US-00001 TABLE 1 Specific Filter Paper (FP) and -glucosidase activities of parent strains (A2, RutC30 and of progeny RuA-149). FP activity -glucosidase activity Strain (IU/mg) (IU/mg) RutC30 0.77 0.11 0.45 0.07 A2 0.41 0.01 0.72 0.03 RuA-149 0.8 0.13 1.33 0.09

(24) The strain RuA-149 progressively produces approximately 5 g/L of biomass until total glucose consumption which occurs after 34 hrs. During the fed-batch phase, the biomass is stabilised around 15 g/L (FIG. 3A).

(25) The production of proteins by the strain RuA-149 starts once induction has been carried out but it slows down from 100 hrs. The protein production of the strain RuA-149 is of 34 g/L (FIG. 3B).

(26) Effective cellulose hydrolysis requires the synergistic action of endoglucanases, exoglucanases and -glucosidases (Kubicek et al., 2009). Endoglucanases and exoglucanases may represent up to 90% of the total quantity of secreted proteins whereas -glucosidase represents less than 1% (Lynd et al., 2002; Herpoel-Gimbert et al., 2008). The main -glucosidase (encoded by the gene bgl1 or cel3a in T. reesei) is involved for hydrolysing cellobiose (dimer) during the final step prior to obtaining the glucose monomer (Chauve et al., 2010). The small proportion thereof in the cellulolytic cocktail of T. reesei induces an accumulation of cellobiose the inhibitory effect whereof on exoglucanases induces a significant slowdown of hydrolysis (Chauve et al., 2010). An increase in the -glucosidase activity in the enzyme cocktail is essential for more effective hydrolysis during second-generation bioethanol production.

(27) At the end of fermentation, the quality of the enzyme cocktail produced is evaluated by measuring the enzyme activities (FIGS. 3A and 3B). The endoglucanase and exoglucanase activity is assayed by means of the FP (filter-paper) test which gives an indication of the overall effectiveness of the cellulase cocktail (Ghose, 1987). The limiting activity of -glucosidase is evaluated separately by measuring the hydrolysis of p-nitrophenyl--Dglucopyranoside (pNPG).

(28) The FP assay demonstrates that the strain A2 has a low specific cellulolytic activity with respect to the reference strain RutC30 but the -glucosidase activity thereof is almost two times greater than that of RutC30. The strain according to the invention, RuA-149, has a similar overall FP activity to that of the reference strain RutC30 but it has an approximately three times greater -glucosidase activity.

(29) These results demonstrate that the cross between the strain A2 and the hyper-producing strain RutC30 made it possible to generate an enhanced hyper-producing strain for the -glucosidase activity.

Example 2: Preparation of Fertile Cross Between the Strain According to the Present Invention (i.e. the Strain as Deposited Under the Number CNCM I-5221) with Sterile Female MAT1-2 Strains

(30) The strain RuA-149 was crossed with MAT1-2 sterile female Trichoderma reesei MAT1-2 derived from the isolate QM6a. The MAT1-1 IDC1 strains A2 and QM6a (Linke et al., 2015) were used as a positive control (fertile cross). The crosses are produced by confrontation: the strains were inoculated in mutually facing rows, with approximately 1.5 cm between the two strains. Each row contains four to six inoculums of the same strain from either a plate of conidia or a cryotube. A cross is fertile when ascospores are expelled from the perithecia contained in the stromata. The expulsion of ascospores is observed by the presence of a yellowish substance present in the ostioles of the perithecia. The crosses were produced with the strains RutC30, C1847, QM9414 and Tu-6. The results are given in Table 2 hereinafter. The genealogy of the strains tested is shown in FIG. 4.

(31) TABLE-US-00002 TABLE 2 Cross between the strain according to the invention and sterile female MAT1-2 strains Strains tested (sterile Cross with A2 Cross with strain female MAT1-2) (positive control) RuA-149 RutC30 Fertile Fertile Cl847 Fertile Fertile QM9414 Fertile Fertile Tu-6 Fertile Fertile

(32) These results confirm that the strain according to the invention may be crossed with fertile female strains, and thereby generate new strains.

REFERENCES

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