METHOD FOR PREPARATION AND SCREENING OF FUNGAL MUTANT WITH HIGH HYDROLYTIC ACTIVITY AND CATABOLITE DEREPRESSED CHARACTER

Abstract

The present invention relates to a mutant fungal strain of Penicillium funiculosum MRJ-16 characterized by the ability to produce high titer of enzyme mixture comprising FPase, CMCase, Cellobiase, -glucosidase, endoglucanase, -L arabinofuranosidase, -xylosidase, xylanase, pectinase, cellobiohydrase and oxidases and produce enzymes in the presence of a catabolite repressor molecule like glucose and/or xylose. The titer of enzyme mixture produced using mutant fungal strain MRJ-16 is at least two fold higher than naive Penicillium funiculosum strain NCIM 1228, when used in a fermentation process. The mutant strain MRJ-16 with high hydrolytic activity and catabolite derepressed character is having application in the method of degrading or saccharifying biomass to produce valuable products for example-bioethanol.

Claims

1. A mutant fungal strain of Penicillium funiculosum MRJ-16 characterized by the ability to produce high titer of enzyme mixture comprising FPase, CMCase, Cellobiase, -glucosidase, endoglucanase, -L arabinofuranosidase, -xylosidase, xylanase, pectinase, cellobiohydrase and oxidases and is having catabolite derepressed character.

2. The mutant fungal strain as claimed in claim 1, wherein the enzyme mixture comprises cellulase and -glucosidase.

3. The mutant fungal strain as claimed in claim 1, wherein catabolite repressor molecule is glucose and/or xylose.

4. The mutant fungal strain as claimed in claim 1, wherein the titer of enzyme mixture is at least two fold higher than the titer of enzyme mixture produced by naive Penicillium funiculosum NCIM 1228 strain.

5. A method of preparing a mutant fungal strain comprising the steps of: (i) selecting and subjecting Penicillium funiculosum NCIM 1228 to aerobic culture media to prepare spore suspension of about 110.sup.6 spore/mL; (ii) mutating spore suspension of step (i) by method selected from chemical mutagenesis, physical mutagenesis, a combination of both; (iii) screening mutant colonies with cellulolytic activity using screening media comprising amorphous cellulose in a concentration 0.1%-2% (w/w) and glucose in a concentration 0.1-4% (w/w). (iv) obtaining mutant fungal strain Penicillium funiculosum MRJ-16.

6. The method as claimed in claim 5, wherein chemical mutagenesis in step (ii) is carried out with ethyl methanesulfonate (EMS) or diethyl sulfate (DES) in concentration of about 10-50 L/mL spore suspension or with both in 1:1 concentration for 15 hours under dark at room temperature.

7. The method as claimed in claim 5, wherein physical mutagenesis of step (ii) is carried out with UV light of wavelength 254 nm for 2-5 minutes at a distance of 15 cm.

8. The method as claimed in claim 5, wherein chemical mutagenesis is followed by physical mutagenesis.

9. A process for the production of cellulase enzymes using Penicillium funiculosum MRJ 16 strain as claimed in claim 1.

10. A method for degrading or saccharifying biomass using Penicillium funiculosum MRJ 16 strain as claimed in claim 1.

Description

DESCRIPTION OF THE ACCOMPANYING DRAWING

[0028] FIG. 1: Penicillium funiculosum NCIM 1228 (naive strain) and mutant colony on screening media plate

DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention relates to a mutant fungal strain derived from Penicillium funiculosum NCIM 1228, which can produce high titer of enzyme mixture using cheap growth components, having catabolite derepressed character, can produce enzymes in the presence of catabolite repressor molecule like glucose and/or xylose. Hence, present invention provides a commercially viable and sustainable process of enzyme cocktail preparation for the hydrolysis of pretreated lignocellulosic biomass, which is useful for the production of biofuels.

[0030] According to first aspect, the present invention provides a mutant fungal strain of Penicillium funiculosum MRJ-16 (MTCC Accession No. 25142 and date of deposition 12 Jun. 2017) characterized by the ability to produce high titer of enzyme mixture comprising FPase, CMCase, Cellobiase, -glucosidase, endoglucanase, -L arabinofuranosidase, -xylosidase, xylanase, pectinase, cellobiohydrase and oxidases. In an embodiment of the present invention, the mutant fungal strain MRJ-16 produces enzyme mixture comprising -glucosidase 62-64 IU/ml, Filter paper activity 6.2-6.4 FPU/ml, endoglucanase 92-98 IU/ml, -L arabinofuranosidase 0.02 IU/ml, -xylosidase 0.5-0.7 IU/ml, xylanase 212-235 IU/ml, pectinase 82-97 IU/ml and oxidases 4.65-5.04 IU/ml as shown in examples 8 and 9.

[0031] The naive strain of P. funiculosum NCIM 1228 shows catabolite repression on enzyme production i.e. it does not secrete enzymes in the presence of a catabolite repressor molecule like glucose and/or xylose. The present invention overcomes this limitation. According to second aspect, the present invention provides a mutant fungal strain of Penicillium funiculosum MRJ-16 having catabolite derepressed character. In an embodiment of the present invention, the P. funiculosum MRJ-16 cultured on fermentation media of examples 3 and 5 comprising glucose 3% (w/v), after 120 hours of fermentation process produce 1.39 FPU/ml and 21 IU/ml -glucosidase (BGL). Hence, the titre of enzyme mixture is at least two to twenty two times higher as shown in Table 6.

[0032] According to third aspect, the present invention provides a method of preparing a mutant fungal strain comprising the steps of: [0033] (i) selecting and subjecting Penicillium funiculosum NCIM 1228 to aerobic culture media to prepare spore suspension of about 110.sup.6 spore/mL; [0034] (ii) mutating spore suspension of step (i) by method selected from chemical mutagenesis, physical mutagenesis, a combination of both; [0035] (iii) screening mutant colonies with cellulolytic activity using screening media comprising amorphous cellulose in a concentration 0.1%-2% (w/w) and glucose in a concentration 0.1-4% (w/w). [0036] (iv) obtaining mutant fungal strain Penicillium funiculosum

[0037] In an embodiment of the present invention, the spore suspension of naive strain of Penicillium funiculosum NCIM 1228 is prepared in a sterile saline solution containing 0.1% Tween-80 from a 7 day old potato dextrose agar (PDA) culture grown at 30 C.

[0038] In an preferred embodiment of the present invention, the spore suspension of naive strain of Penicillium funiculosum NCIM 1228 is subjected to chemical mutagenesis with ethyl methanesulfonate (EMS) or diethyl sulfate (DES) in a concentration of about 10-50 L/mL spore suspension or with both in 1:1 concentration for 15 hours under dark at room temperature.

[0039] In another embodiment of the present invention, the spore suspension of naive strain of Penicillium funiculosum NCIM 1228 is subjected to physical mutagenesis with UV light and/or NTG (N-methyl-N-nitro-N-nitrosoguanidine) and/or EMS (Ethyl methanesulfonate). In a preferred embodiment, the spore suspension of parent strain NCIM 1228 is treated with UV light (254 nm, Philips TUV-30 W lamp) for 2-5 minutes at a distance of 15 cm in a wooden UV-box. In a preferred embodiment, the method comprises chemical mutagenesis followed by physical mutagenesis.

[0040] Selection of mutual fungal strain is carried out by visually selection on the basis of yellow pigmentation and measuring the amorphous cellulose hydrolyzed zone in the screening media. In a preferred embodiment of present invention, the process of screening mutant colonies with cellulolytic activity is carried out using solid media comprising amorphous cellulose ranging from 0.1% to about 2% (w/w) and a catabolite repressor molecule like glucose and or xylose in a concentration of about 0.1-4% (w/w). In an embodiment, the mutant colonies are selected by measuring the diameter of hydrolyzed zone surrounding the colonies. In a preferred embodiment, MRJ-16 produce wide clear zone with diameter of about 28-32 mm and the colony diameter of about 21-24 mm.

[0041] In an embodiment, the present invention relates to a process for the production of cellulase enzymes using the mutant strain of Penicillium funiculosum MRJ-16. The process comprises culturing Penicillium funiculosum MRJ-16 strain in a culture medium comprising carbon source selected from the group consisting of rice straw, wheat straw, baggase or a mixture thereof. This is followed by collecting the cellulase enzymes from the culture medium. In a preferred embodiment, MRJ 16 mutant strain comprises cellulase enzymes comprising -glucosidase 62-64 IU/ml, Filter paper activity 6.2-6.4 FPU/ml.

[0042] In an embodiment of the present invention, the enzymes produced by Penicillium funiculosum MRJ-16 mutant strain are used without any downstream process. Hydrolysis of 20% pre-treated lignocellulosic biomass such as acid pre-treated rice at pH 4-5, 50 mM citrate buffer, temperature 50 C. at enzyme loadings of 6 FPU/g of dry biomass leads to 60% glucan conversion in 48 hours. In a preferred embodiment, an enzyme loading of 6 FPU/g of dry biomass is either clear enzyme broth or enzyme broth without any downstream processing, as exemplified in Examples 10 and 11.

[0043] Following non-limiting examples are given by way of illustration for specific embodiments thereof and therefore should not be construed to limit the scope of the invention.

EXAMPLES

Example 1

Mutagenesis Procedure for Preparing Mutant Fungal Strain Penicillium Funiculosum MRJ-16

[0044] Spore suspension (approx. 110.sup.6 Spore/mL) of naive strain was prepared in sterile saline containing 0.1% Tween-80 from a 7 day old Potato dextrose agar (PDA) culture grown at 30 C. Spore suspension was subjected to chemical mutagenesis by treating them with ethyl methanesulfonate (EMS) or diethyl sulfate (DES) of concentration about 10-50 L/mL spore suspension or both at 1:1 concentration for 15 hours under dark at room temperature. This was followed by physical mutagenesis of suspension with UV light (254 nm, Philips TUV-30 W lamp) for 2-5 min at a distance of 15 cm in a wooden UV-box. The spore suspension after combined mutagenesis was spread (100 L) onto screening media containing 0.3% -0.5% wt. amorphous cellulose and 0.5-3% glucose. The plates were incubated under dark at 30 C. for 4-6 days and colonies with cellulolytic activity were visually detected by observing a clear transparent halo surrounding the colonies. The obtained mutant colonies were counted and potential mutant colonies were identified on the basis of clear halo zone.

Example 2

Screening of an Efficient Mutant for Enzyme Production

[0045] The naive strain of Penicillium funiculosum NCIM 1228 was used to create mutants for enzyme production. Mutations were done using UV or NTG or EMS mutagens alone or all together. The mutant strains were obtained after repeated multi-stage mutagenesis process. Mutants were selected sequentially on specially designed media containing amorphous cellulose and glucose at different concentration from 1-4% (w/w) (screening media composition in table 1). Naive strain didn't hydrolyze amorphous cellulose in the presence of glucose. Mutant strain that hydrolyzes the amorphous cellulose in the presence of 3% glucose was selected after visualizing and measuring the hydrolyzed zone. Stability of mutant stain for enzyme production capability was tested for multiple cycles and then used for further study.

TABLE-US-00001 TABLE 1 Screening media composition S. No. Chemical Components Quantity (g/L) 1 Ammonium Sulphate 1.4 2 KH.sub.2PO.sub.4 2.0 3 MgSO.sub.47H.sub.2O 0.3 4 CaCl.sub.23H.sub.2O 0.3 5 Urea 0.3 6 Tween-80 0.1 7 Peptone 0.25 8 Yeast Extract 0.10 9 ZnSO.sub.47H.sub.2O 0.0014 10 FeSO.sub.47H.sub.2O 0.005 11 MnSO.sub.4H.sub.2O 0.0016 12 CoCl.sub.26H.sub.2O 0.002 13 Glucose 15 14 Amorphous cellulose 1.5 15 Agar 20 16 pH 5.0

[0046] The visual observation of the Penicillium funiculosum NCIM 1228 (naive strain) and mutant (MRJ-16) cultured on screening media plate showed that in the presence of 3% glucose, naive stain do not produce clear zone and mycelia appear white till 10-13 days of incubation, while MRJ-16 started turning yellow before the onset of spores as shown in FIG. 1. More wrinkled surface was observed in case of MRJ-16 than naive strain. MRJ-16 produce wide clear zone with diameter 28 mm, the colony diameter of naive and MRJ-16 mutant strain was 25 mm and 21 mm respectively.

Example 3

Screening of an Efficient Mutant for Enzyme Production

[0047] The naive strain of Penicillium funiculosum NCIM 1228 was used to create mutants for enzyme production. Mutations were done using UV or NTG or EMS mutagens alone or all together. The mutant strains were obtained after repeated multi-stage mutagenesis process. Mutants were selected sequentially on specially designed media containing amorphous cellulose and glucose at different concentration from 1-4% (w/w) (screening media composition in table 2). Naive strain didn't hydrolyze amorphous cellulose in the presence of glucose. Mutant strain that hydrolyzes the amorphous cellulose in the presence of 4% glucose was selected after visualizing and measuring the hydrolyzed zone. Stability of mutant stain for enzyme production capability was tested for multiple cycles and then used for further study.

TABLE-US-00002 TABLE 2 Screening media composition S. No. Chemical Components Quantity (g/L) 1 Ammonium Sulphate 2.4 2 KH.sub.2PO.sub.4 3.0 3 MgSO.sub.47H.sub.2O 0.1 4 CaCl.sub.23H.sub.2O 0.1 5 Urea 0.2 6 Tween-80 1.0 7 Peptone 0.5 8 Yeast Extract 1.0 9 ZnSO.sub.47H.sub.2O 0.005 10 FeSO.sub.47H.sub.2O 0.005 11 MnSO.sub.4H.sub.2O 0.002 12 CoCl.sub.26H.sub.2O 0.002 13 Glucose 25 14 Amorphous cellulose 2.5 15 Agar 20 16 pH 5.5

[0048] The visual observation of the Penicillium funiculosum NCIM 1228 (naive strain) and mutant (MRJ-16) cultured on screening media plate showed that in the presence of 3% glucose, naive stain do not produce clear zone and mycelia appear white till 10-13 days of incubation, while MRJ-16 started turning yellow before the onset of spores. More wrinkled surface was observed in case of MRJ-16 than naive strain. MRJ-16 produce wide clear zone with diameter 32 mm, the colony diameter of naive and MRJ-16 mutant strain was 28 mm and 24 mm respectively.

Example 4

SNPs Analysis of Mutant Fungal Strain MRJ-16 and Naive Strain

[0049] The filtered paired end fastq files of sample M (mutant strain MRJ-16) were aligned to the sample V (naive strain) assembly using bowtie2 aligner. The mapping had an overall alignment rate of 95.38%. The alignment sequence map file was further sorted and indexed before the SNPs were predicted. The SNPs prediction was facilitated by samtools mpileup and bcftools view functions. VarFilter tool was used to filter the SNP sites with QUAL value greater than or equal to 100. There were a total of 1655 SNP sites observed in the sample M (mutant strain MRJ-16) when compared to sample V (naive strain). Sample V (naive strain) protein annotations from previous step were used to annotate the SNP sites in the sample M (mutant strain MRJ-16) using the SnpEff software. The results of SNPs analysis is shown in table 3.

TABLE-US-00003 TABLE 3 SNPs analysis of mutant strain MRJ-16 and naive strain A B C D E #CHROM POS REF ALT QUAL F G H I scaffold1 size1605473 737632C T 221.999 missense_variant MODERATE PCH03293.1 Chromatin-remodelling complex, RSC scaffold1 size1605473 1001984C A 221.999 upstream_gene_variant MODIFIER PCG95362.1 Amidase scaffold2 size1468793 191909C T 221.999 upstream_gene_variant MODIFIER PCG91910.1 Succinate dehydrogenase, flavoprotein scaffold2 size1468793 554756T A 221.999 upstream_gene_variant MODIFIER PCH06707.1 Heat shock protein DnaJ scaffold2 size1468793 1199260G A 221.999 synonymous_variant LOW PCH07343.1 Hypothetical protein PENO1_012430 scaffold2 size1468793 1199261A T 221.999 missense_variant MODERATE PCH07343.1 Hypothetical protein PENO1_012430 scaffold2 size1468793 1298011A T 156.012 upstream_gene_variant MODIFIER PCG90448.1 Hypothetical protein PENO1_099160 scaffold2 size1468793 1463248A G 221.999 upstream_gene_variant MODIFIER PCG93125.1 Phytanoyl-CoA dioxygenase A. Scaffold1_size1605473: Reference scaffold position in sample V. B. POS: position at which the snp is present on the scaffold. C. REF: allele present in reference sequence of sample V. D. ALT: alternative allele present in the sample M. E. QUAL: quality value of the SNP position. F. Type of the SNP: missense_variant, upstream_gene_variant, to name a few. G. Effect of the SNP: Moderate, Modifier, High, Low. H. GenBank Protein Id. I. Putative Protein Annotation. indicates data missing or illegible when filed

Example 5

Enzyme Production Efficiency of Mutant Strain MRJ-16 and Naive Strain

[0050] Fermentation process was carried out in aerated stirred tank bioreactor of 2 L glass jacketed vessel, with 1.8 L working volume. The media components of fermentation media used were ammonium sulphate 5 g/L, KH.sub.2PO.sub.4 6 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, CaCO.sub.3 5 g/L, Glycerol 2.5 g/L, Corn steep solids 27 g/L, cellulose 30 g/L and Tween-80 2 ml/L. The fermenter containing 1.5 L medium was sterilized at 120 C. for 20 min. After cooling, the temperature was kept at 30 C., pH adjusted to 5.5 and inoculated with 10% active liquid seed (seed media composition in table 4) of Penicillium mutant strain. After 96 hours of fermentation, the enzyme broth was collected, centrifuged and analysis of clear enzyme broth was done.

TABLE-US-00004 TABLE 4 Seed/Inoculum Media composition S. No. Chemical Components Quantity (g/L) 1 Ammonium Sulphate 4 2 KH.sub.2PO.sub.4 3 3 MgSO.sub.47H.sub.2O 0.1 4 CaCO.sub.3 2 5 Sucrose 5 6 Corn Steep Liquor 15 7 Cellulose 10 8 Tween-80 2 9 pH 5.5

[0051] The results obtained after 96 hours of incubation were that parent strain produces 8.5 g/L of protein, 22 IU/ml of -glucosidase and 3.8 FPU/ml of filter paper activity, while MRJ-16 produces 15 g/L of protein, 62 IU/ml of -glucosidase and 6.4 FPU/ml of filter paper activity. Hence, the mutant strain MRJ 16 possesses significantly enhanced enzyme production in comparison to parent strain.

Example 6

Enzyme Production Efficiency of Mutant Strain MRJ-16 and Naive Strain

[0052] Fermentation process was carried out in aerated stirred tank bioreactor of 2 L glass jacketed vessel, with 1.8 L working volume. The media components of fermentation media used were ammonium sulphate 3.5 g/L, KH.sub.2PO.sub.4 4 g/L, MgSO.sub.4.7H.sub.2O 0.5 g/L, CaCO.sub.3 2.5 g/L, Glycerol 2.5 g/L, Corn steep solids 20 g/L, cellulose 20 g/L and Tween-80 2 ml/L. The fermenter containing 1.5 L medium was sterilized at 120 C. for 20 min. After cooling, the temperature was kept at 30 C., pH adjusted to 5.5 and inoculated with 10% active liquid seed (seed media composition in table 5) of Penicillium MRJ-16 mutant strain. After 96 hours of fermentation, the enzyme broth was collected, centrifuged and analysis of clear enzyme broth was done.

TABLE-US-00005 TABLE 5 Seed/Inoculum Media composition S. No. Chemical Components Quantity (g/L) 1 Ammonium Sulphate 5 2 KH.sub.2PO.sub.4 6 3 MgSO.sub.47H.sub.2O 1 4 CaCO.sub.3 2.5 5 Sucrose 10 6 Corn Steep Liquor 10 7 Cellulose 20 8 Tween-80 2 9 pH 5.5

[0053] The results obtained after 96 hours of incubation were naive strain produces 7.8 g/L of protein, 21 IU/ml of -glucosidase and 3.4 FPU/ml of filter paper activity, while MRJ-16 produces 16.2 g/L of protein, 64 IU/ml of -glucosidase and 6.2 FPU/ml of filter paper activity. Hence, the titer of enzyme produced using mutant strain MRJ 16 is at least two fold higher than enzyme produced using naive strain in fermentation process.

Example 7

Enzyme Production in the Presence of Glucose

[0054] In order to demonstrate the glucose repression on enzyme production, cellulose used as carbon source was replaced with glucose. Enzyme production from Penicillium funiculosum MRJ-16 mutant strain was carried out under the conditions and media composition as described in Examples 3 and 5 except cellulose. Concentrated solution of glucose was autoclaved separately and added into media at 3% w/v concentration. Fermentation was lasted approximately for about 120 hours, enzyme harvested and results analyzed are shown in below mentioned Table 6.

TABLE-US-00006 TABLE 6 Enzyme production from MRJ-16 and parent strain in the presence of glucose Carbon Source FPU/ BGL Fungal strain Concentration (% w/v) ml (IU/ml) Penicillium funiculosum NCIM Cellulose 3% 3.5 22.1 1228 (Parent strain) Glucose 3% 0.01 0 Penicillium funiculosum Cellulose 3% 6.47 62 MRJ-16 mutant Glucose 3% 1.39 21

Example 8

Diversity of Enzyme Secreted

[0055] Cellulases enzymes production using Penicillium funiculosum MRJ-16 mutant strain was performed according to Example 5. The secretome analyses were done it is comprise enzyme activities of -glucosidase 62 IU/ml, Filter paper activity 6.4 FPU/ml, endoglucanase 98 IU/ml, -L arabinofuranosidase 0.02 IU/ml, -xylosidase 0.5 IU/ml, xylanase 212 IU/ml, pectinase 97 IU/ml and oxidases 5.04 IU/ml analyzed using respective substrates.

Example 9

Diversity of Enzyme Secreted

[0056] Cellulases enzymes production using Penicillium funiculosum MRJ-16 mutant strain was performed according to example no. 6. The secretome analyses were done it is comprise enzyme activities of -glucosidase 64 IU/ml, Filter paper activity 6.2 FPU/ml, endoglucanase 92 IU/ml, -L arabinofuranosidase 0.02 IU/ml, -xylosidase 0.7 IU/ml, xylanase 235 IU/ml, pectinase 82 IU/ml and oxidases 4.65 IU/ml analyzed using respective substrates.

Example 10

Hydrolysis of Pre-Treated Lignocellulosic Biomass Using Concentrated Enzyme

[0057] The efficiency of enzyme produced was determined by its ability to hydrolyze lignocellulosic biomass such as acid pretreated rice straw and produce sugars. Enzyme was first separated from fungal mycelia by centrifugation and clear broth was concentrated and used. Hydrolysis was performed at high substrate loading of biomass i.e. 20% at pH 4-5, 50 mM citrate buffer, temperature 50 C. at enzyme loadings of 6 FPU/g of dry biomass. Sugars released were determined at regular interval of time by HPLC. Enzyme cocktail worked efficiently and leads to 60% glucan conversion in 48 hours.

Example 11

Hydrolysis of Pre-Treated Lignocellulosic Biomass Using Enzyme as Such

[0058] The efficiency of enzyme produced was determined by its ability to hydrolyze lignocellulosic biomass such as acid pretreated rice straw and to produce sugars. Enzyme broth was used as such without any downstream processing. Hydrolysis was performed at high substrate loading of biomass i.e. 20% at pH 4-5, 50 mM citrate buffer, temperature 50 C. at enzyme loadings of 6 FPU/g of dry biomass. Sugars released were determined at regular interval of time by HPLC. Enzyme cocktail worked efficiently and leads to 60% glucan conversion in 48 hours.