MATTEI MARIA BENEDETTA (IT)
GIOVANNONI MOIRA (IT)
WO2011140520A2 | 2011-11-10 | |||
WO2012150968A1 | 2012-11-08 |
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CLAIMS 1. A Penicillium sumatraense (P. sumatraense) strain comprising in its genome: a) an RNA polymerase II second largest subunit (RPB2) gene sequence having 100%, 99%, or 98% sequence identity with the nucleic acid of SEQ ID NO: 1; b) β-tubulin (BenA) gene sequence having 100%, 99%, or 98% sequence identity with the nucleic acid of SEQ ID NO: 2; c) Calmodulin (CaM) gene sequence having 100%, 99%, or 98% sequence identity with the nucleic acid of SEQ ID NO: 3; and d) an Internal Transcribed Spacer (ITS) sequence having 100%, 99%, or 98% sequence identity with the nucleic acid of SEQ ID NO: 4 and further comprising in its genome, a gene comprising or consisting of a sequence having 100, 99, 97, 65, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86 or 85% sequence identity with SEQ ID NO:30 and/or wherein said strain produces an enzyme that comprises or consists of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88 or 87% sequence identity with SEQ ID NO:80 or functional fragments comprising the catalytic domain. 2. The P. sumatraense strain according to claim 1 being capable of metabolizing Chlorella vulgaris (C. vulgaris). 3. The P. sumatraense strain according to claim 1 or 2, comprising at least one gene comprising or consisting of a sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86 or 85% sequence identity with one of the sequences selected from the group consisting of: SEQ ID NOs: 5-29, 31-54, preferably comprising in its genome one gene comprising or consisting of a sequence selected from SEQ ID NOs: 5-29, 31-54. 4. The P. sumatraense strain according to claim 3, comprising at least one gene comprising or consisting of a sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86 or 85% sequence identity with one of the sequences selected from the group consisting of: SEQ ID NOs: 7, 8, 9, 12, 13, 25, 28, 39, 46, 55, 17, 36, 37, 38, 51, 53 preferably comprising in its genome one gene comprising or consisting of a sequence selected from SEQ ID NOs: 7, 8, 9, 12, 13, 25, 28, 39, 46, 55, 17, 36, 37, 38, 51, 53, more preferably comprising in its genome SEQ ID Nos: 7, 8, 9, 12, 13, 25, 28, 39, 46, 55 and optionally SEQ ID Nos: 17, 36, 37, 38, 51, 53. 5. The P. sumatraense strain according to claim 4, comprising in its genome sequences having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86 or 85% sequence identity with SEQ ID NOs: 5-54, preferably comprising in its genome SEQ ID NOs: 5-54. 6. Use of the P. sumatraense strain according to any one of previous claims or of a culture filtrate thereof or of a a composition comprising a blend of two or more enzymes secreted by the fungus P. sumatraense strain according to any one of previous claims in pretreatment of a biomass for increasing the extraction efficiency of biological products from said biomass, such as carotenoids and lipids, wherein the biomass is preferably an algal or plant biomass, with the latter preferably rich in xylans such as an agricultural residue preferably straw, cob or raw bran. 7. Use of a Penicillium sumatraense (P. sumatraense) strain comprising in its genome: a) an RNA polymerase II second largest subunit (RPB2) gene sequence having 100%, 99%, or 98% sequence identity with the nucleic acid of SEQ ID NO: 1; b) β-tubulin (BenA) gene sequence having 100%, 99%, or 98% sequence identity with the nucleic acid of SEQ ID NO: 2; c) Calmodulin (CaM) gene sequence having 100%, 99%, or 98% sequence identity with the nucleic acid of SEQ ID NO: 3; and d) an Internal Transcribed Spacer (ITS) sequence having 100%, 99%, or 98% sequence identity with the nucleic acid of SEQ ID NO: 4 said strain being characterized by an alginase activity or of a culture filtrate thereof or of a composition comprising a blend of two or more enzymes secreted by said P. sumatraense strain in pretreatment of a biomass for increasing the extraction efficiency of biological products from said biomass, such as carotenoids and lipids, wherein the biomass is preferably an algal or plant biomass, with the latter preferably rich in xylans such as an agricultural residue preferably straw, cob or raw bran. 8. Use of a composition comprising a blend of two or more isolated or synthetic or recombinant enzymes wherein the enzyme comprises or consists of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88 or 87% sequence identity with SEQ ID NO:80 or functional fragments comprising the catalytic domain or derivatives thereof and optionally the further enzyme comprises or consists of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90 or 85% sequence identity with a sequence selected from SEQ ID NOs:55-79, 81-104, or functional fragments comprising the catalytic domain or derivatives thereof in pretreatment of a biomass for increasing the extraction efficiency of biological products from said biomass, such as carotenoids and lipids, wherein the biomass is preferably an algal or plant biomass, with the latter preferably rich in xylans such as an agricultural residue preferably straw, cob or raw bran. 9. Use of a composition comprising a blend of two or more isolated or synthetic or recombinant enzymes wherein the enzyme comprises or consists of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90 or 89% sequence identity with SEQ ID NO:112 or functional fragments comprising the catalytic domain or derivatives thereof and optionally the further enzyme comprises or consists of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90 or 85% sequence identity with a sequence selected from SEQ ID NOs: 55-79, 81-104, or functional fragments comprising the catalytic domain or derivatives thereof in pretreatment of a biomass for increasing the extraction efficiency of biological products from said biomass, such as carotenoids and lipids, wherein the biomass is preferably an algal or plant biomass, with the latter preferably rich in xylans such as an agricultural residue preferably straw, cob or raw bran. 10. The use of the composition according to any one of claims 6-9, wherein said enzymes are proteases and glycosidases, preferably they are dipeptidyl- and amino-peptidases, and exo- glycosidases including α- and β-glucosidases, β-glucuronidases, α-mannosidases and exo-β-1,3- glucanases, preferably wherein the blend comprises enzymes comprising or consisting of SEQ ID NOs:55-104 or functional fragments comprising the catalytic domain or derivatives thereof. 11. The use of the strain or of the culture filtrate thereof or of the composition according to any one of claims 6-10 wherein the the algae is from a genus selected from the group consisting of Actinocyclus, Bellerochea, Cyclotella, Cryptomonas, Chlorella, Monochrysis Chlorella, Chaetoceros, Dunalliela, Haematococcu, Nannochloropsis, Pleurochrysis, Gracilaria, Sargassum, Dunaliella, Cyclotella, Navicula, Nitzschia, Spirulina, Phaeodactylum, Thalassiosira, Skeletonema, Porphyra and combinations thereof, preferably the algae is C.^vulgaris. 12. An isolated or synthetic or recombinant enzyme comprising or consisting of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88 or 87% sequence identity with SEQ ID NOs:80, or functional fragments comprising the catalytic domain or derivatives thereof, said isolated or synthetic or recombinant enzyme preferably comprising or consisting of SEQ ID NOs:80. 13. An isolated or synthetic or recombinant enzyme comprising or consisting of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90 or 89% sequence identity with SEQ ID NO:112, or functional fragments comprising the catalytic domain or derivatives thereof, said isolated or synthetic or recombinant enzyme preferably comprising or consisting of SEQ ID NO:112. 14. An isolated or synthetic or recombinant enzyme comprising or consisting of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90 or 85% sequence identity with a sequence selected from SEQ ID NOs:55-79, 81-104, or functional fragments comprising the catalytic domain or derivatives thereof, said isolated or synthetic or recombinant enzyme preferably comprising or consisting of a sequence selected from SEQ ID NOs:55-79, 81-104. 15. A composition comprising a blend of two or more isolated or synthetic or recombinant enzymes wherein said enzyme comprises or consists of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88 or 87% sequence identity with SEQ ID NO:80 or functional fragments comprising the catalytic domain or derivatives thereof and optionally the further enzyme comprises or consists of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90 or 85% sequence identity with a sequence selected from the group consisting of: SEQ ID NOs:55-104, or functional fragments comprising the catalytic domain or derivatives thereof, preferably wherein said enzymes are proteases and glycosidases, preferably they are dipeptidyl- and amino-peptidases, and exo-glycosidases including α- and β-glucosidases, β-glucuronidases, α-mannosidases and exo-β-1,3-glucanases, preferably wherein the blend comprises enzymes comprising or consisting of SEQ ID NOs:55- 104. 16. An isolated or synthetic or recombinant nucleic acid encoding the enzyme of claim 12 or 13 or 14, preferably said nucleic acid comprising or consisting of a sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90 or 85% sequence identity with a sequence selected from the group consisting of: SEQ ID NOs:5-54, preferably comprising or consisting of a sequence selected from SEQ NOs:5-54. 17. A method of producing a composition comprising a blend of degradative enzymes, comprising the following steps: a) cultivating, preferably for at least 7-10 days, at least one fungus as defined in any one of claims 1-7 with at least one dead algal species in a growth medium in dark condition; b) isolating the growth medium to obtain a first composition comprising a blend of degradative enzymes; and optionally c) centrifugating the composition comprising the degrative enzymes to obtain a supernatant, filtering the supernatant, dialyzing and concentrating the filtered supernatant to obtain a second composition comprising a blend of degradative enzymes. 18. A method of producing a biological product, comprising the following steps: a) cultivating, preferably for at least 7-10 days, at least one fungus as defined in any one of claims 1-7 with at least one dead algal species in a growth medium, said algal species being preferably C.^ vulgaris, in dark condition; b) isolating the growth medium to obtain a first composition comprising a blend of degradative enzymes; and optionally centrifugating the composition comprising the degrative enzymes to obtain a supernatant, filtering the supernatant, dialyzing and concentrating the filtered supernatant to obtain a second composition comprising a blend of degradative enzymes; c) treating a biomass with the first or second composition obtained in step b); and d) extracting from the treated biomass a biological product wherein the biomass is preferably an algal or plant biomass, with the latter preferably rich in xylans such as an agricultural residue preferably straw, cob or raw bran. 19. A method of producing a biological product, comprising the following steps: a) treating a biomass with the composition according to claim 6-8, 9, 10, 11 or 15; and b) extracting from the treated biomass a biological product wherein the biomass is preferably an algal or plant biomass, with the latter preferably rich in xylans, such as an agricultural residue preferably straw, cob or raw bran. 20. The method of claim 17 or 18 or 19 wherein the algae is from a genus selected from the group consisting of Actinocyclus, Bellerochea, Cyclotella, Cryptomonas, Chlorella, Monochrysis Chlorella, Chaetoceros, Dunalliela, Haematococcu, Nannochloropsis, Pleurochrysis, Gracilaria, Sargassum, Dunaliella, Cyclotella, Navicula, Nitzschia, Spirulina, Phaeodactylum, Thalassiosira, Skeletonema, Porphyra and combinations thereof, preferably the algae is C.^vulgaris. 21. A system for the production of a biological product, comprising: - at least one fungus of the P. sumatraense strain as defined in any one of claims 1-7 or a culture filtrate thereof and/or - the composition as defined in any one of claims 6-8, 9, 10, 11 or 15 and/or - at least one of the isolated enzymes or proteins as defined in claim 12 or 13 or 14 and - a biomass; and - a growth medium. |
Table 7. Mature proteins identified in P. sumatraense AQ67100 culture filtrate by LC- MS/MS analysis. Mature proteins are the same proteins listed in Table 6 but devoid of predicted signal peptides. Mature proteins are identified by using the suffix “.nsp” at the end of each Protein ID. Signal peptide prediction was performed by the online tool (https://services.healthtech.dtu.dk/service.php?SignalP-5.0) . Proteins g7401.t1 (SEQ ID NO: 81), g8605.t1 (SEQ ID NO: 89), g4584.t1 (SEQ ID NO: 98) and g9296.t1 (SEQ ID NO: 104) were not predicted to have a signal peptide. See Figure 6, Table 4 and Table 6 for further details on identified proteins.
Amino acid sequences of wild type and recombinant g9376.t1. Protein sequence of the a) putative “1,3-b-exoglucanase” g9376.t1 from P. sumatraense AQ67100 with (up) and without (below) the predicted native signal peptide sequence and b) the corresponding fusion protein as expressed in P. pastoris. In bold amino acids: predicted native signal peptide sequence. Underlined amino acids: α-factor secretion signal. Grey amino acids: c-myc epitope. Italicized amino acids: 6xhis-tag. a) MHFASAITLVSLVSSVHTQLLEIPAVDELVSSALQPLEAWTDYQGPTGIASSALSKSTHA IVANVAVEAADASYWLADISHQGKAAFNPNPSSYKVFRNVKDYGAKGDGVTDDTAAI NSAISDGGRYGPSSRQSSTTTPAIVYFPAGTYLISTPIIDYYFTQLIGNPNSMPVIKATA GFS GLGLIDGDQYQSDGNQGWTSTNVFFRQIRNLKLDLTNIPASSAATGIHWPTGQATSIQN VDIVMSSASGTQHQGIFIENGSGGFLADITITGGLYGANVGNQQFTMRNLVITDAVTAIS QIWDWGWTYQGLTVTNCSTALSVDNGGAGNQLVGSVIVLDSTIQDCSTFVTSAWQAST FSNGSLILENISLENVPVAVKGPSGTVLVGGTMTISAWGQGHKYTPNGPTNFQGTFTAPT RPSSLLASGSSRYYTKSKPQYETLSQSSFVSTRSAGATGDGSTDDTSAIQSALNSAASSG K IVFFDQGTYKVTDTIYVPPGSRITGEAYPVIMASGSAFSSISKPVPVVQVGKSGESGSVE W SDMIVSTQGSTPGAVLIEWNLAANSGSGMWDVHTRIGGFSGSQQQVAQCPTSAAVSAA CEVAYMSMHITDSASGVYLDNVWLWTADHDLDSADNTRISVYSGRGLLIEGQTIWLYG TGVEHHSLYQYQFSGASSVVAGFIQTETPYYQPNPDAANGPYPSNPDLKDPDYSSCLSG NCDSLGLRVLDSSDIVIYGAGLYSFFNNYSTDCSTFPVPENCQSEIFSIEGDTSNLVVYA L STVGTTNMIVKDGTSLAVVSDNLATYAATIAYFTL (SEQ ID NO:80) QLLEIPAVDELVSSALQPLEAWTDYQGPTGIASSALSKSTHAIVANVAVEAADASYWLA DISHQGKAAFNPNPSSYKVFRNVKDYGAKGDGVTDDTAAINSAISDGGRYGPSSRQSST TTPAIVYFPAGTYLISTPIIDYYFTQLIGNPNSMPVIKATAGFSGLGLIDGDQYQSDGNQ G WTSTNVFFRQIRNLKLDLTNIPASSAATGIHWPTGQATSIQNVDIVMSSASGTQHQGIFI E NGSGGFLADITITGGLYGANVGNQQFTMRNLVITDAVTAISQIWDWGWTYQGLTVTNC STALSVDNGGAGNQLVGSVIVLDSTIQDCSTFVTSAWQASTFSNGSLILENISLENVPVA VKGPSGTVLVGGTMTISAWGQGHKYTPNGPTNFQGTFTAPTRPSSLLASGSSRYYTKSK PQYETLSQSSFVSTRSAGATGDGSTDDTSAIQSALNSAASSGKIVFFDQGTYKVTDTIYV P PGSRITGEAYPVIMASGSAFSSISKPVPVVQVGKSGESGSVEWSDMIVSTQGSTPGAVLI E WNLAANSGSGMWDVHTRIGGFSGSQQQVAQCPTSAAVSAACEVAYMSMHITDSASGV YLDNVWLWTADHDLDSADNTRISVYSGRGLLIEGQTIWLYGTGVEHHSLYQYQFSGAS SVVAGFIQTETPYYQPNPDAANGPYPSNPDLKDPDYSSCLSGNCDSLGLRVLDSSDIVIY GAGLYSFFNNYSTDCSTFPVPENCQSEIFSIEGDTSNLVVYALSTVGTTNMIVKDGTSLA VVSDNLATYAATIAYFTL (SEQ ID NO: 112) b) MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTN N GLLFINTTIASIAAKEEGVSLEKREAEAAAQLLEIPAVDELVSSALQPLEAWTDYQGPTG I ASSALSKSTHAIVANVAVEAADASYWLADISHQGKAAFNPNPSSYKVFRNVKDYGAKG DGVTDDTAAINSAISDGGRYGPSSRQSSTTTPAIVYFPAGTYLISTPIIDYYFTQLIGNP NS MPVIKATAGFSGLGLIDGDQYQSDGNQGWTSTNVFFRQIRNLKLDLTNIPASSAATGIH WPTGQATSIQNVDIVMSSASGTQHQGIFIENGSGGFLADITITGGLYGANVGNQQFTMR NLVITDAVTAISQIWDWGWTYQGLTVTNCSTALSVDNGGAGNQLVGSVIVLDSTIQDCS TFVTSAWQASTFSNGSLILENISLENVPVAVKGPSGTVLVGGTMTISAWGQGHKYTPNG PTNFQGTFTAPTRPSSLLASGSSRYYTKSKPQYETLSQSSFVSTRSAGATGDGSTDDTSA I QSALNSAASSGKIVFFDQGTYKVTDTIYVPPGSRITGEAYPVIMASGSAFSSISKPVPVV Q VGKSGESGSVEWSDMIVSTQGSTPGAVLIEWNLAANSGSGMWDVHTRIGGFSGSQQQV AQCPTSAAVSAACEVAYMSMHITDSASGVYLDNVWLWTADHDLDSADNTRISVYSGR GLLIEGQTIWLYGTGVEHHSLYQYQFSGASSVVAGFIQTETPYYQPNPDAANGPYPSNP DLKDPDYSSCLSGNCDSLGLRVLDSSDIVIYGAGLYSFFNNYSTDCSTFPVPENCQSEIF SI EGDTSNLVVYALSTVGTTNMIVKDGTSLAVVSDNLATYAATIAYFTLALEQKLISEEDL NSAVDHHHHHH (SEQ ID NO: 110) The gene g11905 from P. sumatraense AQ67100 encodes a putative alginate lyase. a) Gene, b) CDS and c) amino acid sequences of g11905.t1 with (up) and without (below) the predicted native signal peptide sequence. In bold amino acids: predicted native signal peptide sequence. a)ATGGTATTTCTCGCAAGCGTTTTATATCTTGTCACAGCGACAATTATTCCGATCGTGA CGGCTGGACAATCTGTATCGGCGACCGCCCCGACAACATGGGCTCACCCAGGGGCA ATGATCAGTGAAAACCAGCTAAGCTTTATCCGAAAGAAAGTACAGAATAAAGAGCA GCCGTGGATGGACGCATATGACGCCTTAATAAAAGACGACGGGATCGCAAATCCCA GAGAGCCATCCCCAGTGACCATTGTTGAATGTGGTTCATACTCCATTCCTGATGTCG GATGTCGTGCAGAACGTAACGACTCAATCGCCGCGTACGGAAACGCGTTGGCATGG GCAATAAGCGGCTCGAAGCAATATGCCGAACAAGCGATCAAGATCATGGATGCATA TTCAGCCGTAATTGAGGGCCACAATAATTCAAATGCCCCTTTACAGTCAGGGTGGGT CGGCTCTGTCTGGGCAAGGACAGGGGAGCTCATTCGATATACGGATGCTGGATGGT CTGACGCCAGCATTGAGAGATTCGGGGCAATGTTGAAGAATGTGTATATGCCTCTCA CGGAAAACGGTACAGATCATAATGTCGCGAATTGGGAAttaggtaagttgagaatggacc aactgtga tttttgtgtggtgacttatattgtgctgTTAGTCATGACCGAAGCAGCAATACTTATGGC AGTCTTTCTT GAAGACTCTGAATCGTACAATACTGCTATGTCATGGTTCCTGAAGCGCATTCCTGCT ACTGTATACATGACAACGGATGGGGAATATCCAGTAGCAGCGCGGGGTCAAAGCTC CGATCCTGATGCAATCATAGCATGGTGGTTCAACCAGACAGTGTTTCGCGAAGATGG ACAGGCGCAGGAGACCTGTCGTGACCTTGAGCATACTGGATACTCATTCGCATCCAT GGCGCACGTAGCAGAGACATCTCGCATCCAAGGGACGGATTTGTACAAGACCGACG TGGGGACGCGGCTCAAATATGGATTGGAATTTCACTCGAAGTTTGTAAATGGCGAAT CTGTTCCTTCCTGGCTATGTGGAGGTAAATTGAGTCTCAGTCTTGGCCCAATCACAG AAGTTGGCTTCAATGGGCTTTCATTCAGGATGGACAATGACATGCCAGAGACCGAG AAATTAACCATCAACCAGAGACCGGCGGGTAATAATGGATTGTTCGTGGCATACGA GACATTGACCCACGCGGAGAACAGTACCTAG (SEQ ID NO:108) b)ATGGTATTTCTCGCAAGCGTTTTATATCTTGTCACAGCGACAATTATTCCGATCGTGA CGGCTGGACAATCTGTATCGGCGACCGCCCCGACAACATGGGCTCACCCAGGGGCA ATGATCAGTGAAAACCAGCTAAGCTTTATCCGAAAGAAAGTACAGAATAAAGAGCA GCCGTGGATGGACGCATATGACGCCTTAATAAAAGACGACGGGATCGCAAATCCCA GAGAGCCATCCCCAGTGACCATTGTTGAATGTGGTTCATACTCCATTCCTGATGTCG GATGTCGTGCAGAACGTAACGACTCAATCGCCGCGTACGGAAACGCGTTGGCATGG GCAATAAGCGGCTCGAAGCAATATGCCGAACAAGCGATCAAGATCATGGATGCATA TTCAGCCGTAATTGAGGGCCACAATAATTCAAATGCCCCTTTACAGTCAGGGTGGGT CGGCTCTGTCTGGGCAAGGACAGGGGAGCTCATTCGATATACGGATGCTGGATGGT CTGACGCCAGCATTGAGAGATTCGGGGCAATGTTGAAGAATGTGTATATGCCTCTCA CGGAAAACGGTACAGATCATAATGTCGCGAATTGGGAATTAGTCATGACCGAAGCA GCAATACTTATGGCAGTCTTTCTTGAAGACTCTGAATCGTACAATACTGCTATGTCA TGGTTCCTGAAGCGCATTCCTGCTACTGTATACATGACAACGGATGGGGAATATCCA GTAGCAGCGCGGGGTCAAAGCTCCGATCCTGATGCAATCATAGCATGGTGGTTCAA CCAGACAGTGTTTCGCGAAGATGGACAGGCGCAGGAGACCTGTCGTGACCTTGAGC ATACTGGATACTCATTCGCATCCATGGCGCACGTAGCAGAGACATCTCGCATCCAAG GGACGGATTTGTACAAGACCGACGTGGGGACGCGGCTCAAATATGGATTGGAATTT CACTCGAAGTTTGTAAATGGCGAATCTGTTCCTTCCTGGCTATGTGGAGGTAAATTG AGTCTCAGTCTTGGCCCAATCACAGAAGTTGGCTTCAATGGGCTTTCATTCAGGATG GACAATGACATGCCAGAGACCGAGAAATTAACCATCAACCAGAGACCGGCGGGTA ATAATGGATTGTTCGTGGCATACGAGACATTGACCCACGCGGAGAACAGTACCTAG (SEQ ID NO: 111) c)MVFLASVLYLVTATIIPIVTAGQSVSATAPTTWAHPGAMISENQLSFIRKKVQNKEQP W MDAYDALIKDDGIANPREPSPVTIVECGSYSIPDVGCRAERNDSIAAYGNALAWAISGSK QYAEQAIKIMDAYSAVIEGHNNSNAPLQSGWVGSVWARTGELIRYTDAGWSDASIERF GAMLKNVYMPLTENGTDHNVANWELVMTEAAILMAVFLEDSESYNTAMSWFLKRIPA TVYMTTDGEYPVAARGQSSDPDAIIAWWFNQTVFREDGQAQETCRDLEHTGYSFASMA HVAETSRIQGTDLYKTDVGTRLKYGLEFHSKFVNGESVPSWLCGGKLSLSLGPITEVGF NGLSFRMDNDMPETEKLTINQRPAGNNGLFVAYETLTHAENST (SEQ ID NO: 109) GQSVSATAPTTWAHPGAMISENQLSFIRKKVQNKEQPWMDAYDALIKDDGIANPREPSP VTIVECGSYSIPDVGCRAERNDSIAAYGNALAWAISGSKQYAEQAIKIMDAYSAVIEGHN NSNAPLQSGWVGSVWARTGELIRYTDAGWSDASIERFGAMLKNVYMPLTENGTDHNV ANWELVMTEAAILMAVFLEDSESYNTAMSWFLKRIPATVYMTTDGEYPVAARGQSSDP DAIIAWWFNQTVFREDGQAQETCRDLEHTGYSFASMAHVAETSRIQGTDLYKTDVGTR LKYGLEFHSKFVNGESVPSWLCGGKLSLSLGPITEVGFNGLSFRMDNDMPETEKLTINQ RPAGNNGLFVAYETLTHAENST (SEQ ID NO:113) EXAMPLE 1 Materials and Methods 1. Capture of P. sumatraense by the algal trap. The algal trap consisted of an open flask containing 5 × 10 8 heat-killed C. vulgaris cells suspended in 200 mL of T-Phi medium. T-Phi medium was a modified version of TAP medium, i.e., TAP medium devoid of acetate and supplemented with 0.7 mM (Phi) instead of 1.2 mM (Pi) (54). Phi was purchased from Wanjie Int., China (CAS No. 13977-65-6). The medium was prepared fresh, pH-adjusted (6.9) and autoclaved. Heat-treatment of C. vulgaris was performed by incubating the cells at 70°C for 50 minutes. For each trial, four traps were posed inside a greenhouse on a rotary shaker (150 rpm). After five days, the traps were visual analysed to detect eventual contaminants. Macroscopical fungal contaminants were isolated by transferring different mycelium portions onto solid MEP medium [2% (w/v) Malt-agar, 1% (w/v) Peptone, 1.5% (w/v) micro-agar, 100 µg mL-1 ampicillin] and incubated at 20°C in a dark chamber. The fungal isolate, later identified as P. sumatraense, was maintained at 20°C in a dark chamber on solid MEP or TC medium [1% (w/v) D-glucose, 1.7% (w/v) malt-agar, 0.1% (w/v) asparagine, 0.001% (w/v) thiamin, 0.2% (w/v) KH2PO4, 0.2% (w/v) yeast extract, 0.1% (w/v) MgSO 4 , 1.5% (w/v) micro-agar]. 2. Algal strain and culture conditions. Chlorella vulgaris wild-type strain 211-11p was obtained from the Culture Collection of Algae (Göttingen University, Germany). C. vulgaris was maintained at 26°C, with a 16/8 h light/dark photoperiod, light intensity of 35 µmol m-2 s-1, in solid or liquid TAP medium on a rotary shaker (180 rpm) according to (19). Growth was followed by measuring cell density using an automated cell counter (Countess II FL Cell Counter, ThermoFisher). C. vulgaris cells from the stationary growth phase were used in all the experiments. 3. Morphological analysis. The morphological characteristics of mycelium were investigated by microscopy analysis using a stereo microscope (Leica S8-Apo) equipped with EC3 camera (8X magnification) or a phase contrast ZeissAxio Imager A2 (100X magnification, oil immersion) equipped with a color microscope camera (Leica DFC 320 R2). For optical microscope analysis, the mycelium was harvested from the algal-supplemented medium and fixed using an 85% (w/v) D-Lactic acid solution (Sigma-Aldritch) as mounting fluid. 4. Genomic extraction and NGS analysis. For genomic extraction, 1-2 grams of fresh mycelium were harvested from solid MEP medium and grinded in liquid nitrogen. The grinded sample was transferred into a 50 mL tube containing 6 mL of extraction buffer (7 M Urea, 0.3 M NaCl, 0.02 M EDTA, 0.03 M N-Lauroyl-sarcosine, 0.05 M Tris-HCl pH 8.0). Then, 3 mL of phenol and 3 mL of chloroform: isoamyl-alcohol (24:1) were added to the sample and vortexed. Upon centrifugation (10.000 × g, 10 minutes), the upper phase was transferred into a fresh tube by adding 3 mL of 7.5 M NH4CH3CO2 and 3.6 mL of isopropanol. Upon mixing and centrifugation (10.000 × g, 5 minutes), the sample was dried, and the resulting pellet dissolved in 0.25 mL of ultrapure water. The genomic preparation was subjected to NGS sequencing by Illumina NovaSeq technology at Dante Labs (L'Aquila, Italia; https://dantelabs.it/) and the sequencing results analysed by bioinformatic tools. 5. Genome assembly, annotation and phylogenetic classification. Illumina paired end sequences were trimmed with Trimmomatic v0.36 (23) and the reads were assembled with SPAdes genome assembler v3.11.1 (55) using default parameters. The assembly obtained was quality assessed with QUAST v5.0.2 (24) and BUSCO (Benchmarking Universal Single-Copy Orthologs) v4.1.4 (25) in order to evaluate the completeness. QUAST was run with k-mer-based quality metrics and ribosomal RNA genes prediction options and BUSCO with fungi_odb10 and eurotiales_odb10 lineage options. The whole ribosomal region was reconstructed by using the fragmented ribosomal sequences found in the assembly as probes for the tool GetOrganelle v1.6.4 (https://github.com/Kinggerm/GetOrganelle) and the reconstructed ribosomal and ITS sequences were extracted by the software ITSx v1.1.2 (56). The sequences were used as a query for a BLASTn (57) search against NCBI (National Center for Biotechnology Information) non redundant database. All the matches with query coverage and identity from 99 to 100% belonged to Penicillium genus. For this reason, all Penicillium proteins (444543 sequences in October 14, 2020) were downloaded from NCBI and used as external evidence to annotate the genome with BRAKER2 v2.1.5 pipeline (26). BRAKER2 is an extension of BRAKER1 (58), which allows fully automated training of the gene prediction tools GeneMark- EP+ (59) and AUGUSTUS (60) from RNA-Seq and/or protein homology information, and it integrates the extrinsic evidence into the prediction. Moreover, BRAKER2 (26) reaches high gene prediction accuracy even in the absence of the annotation of very closely related species and in the absence of RNA-Seq data (EP-mode). So, the pipeline was run in EP-mode, fungus option activated and Penicillium sumatraense as species selected, i.e., the ITS best BLASTn hit. The genome in input for BRAKER2 was repeat masked with a de-novo repeat finding program, i.e., RepeatModeler v1.0.11 (http://www.repeatmasker.org/RepeatModeler/). Furthermore, the gtf file generated by BRAKER2 (26) was quality evaluated again with BUSCO (25) and Eval v2.2.8 (28). Predicted proteins were functionally annotated using PANNZER2 (Protein ANNotation with Z- scoRE), a rapid functional annotation server (27). As described by Houbraken and colleagues in a recent study (22), four phylogenetic marker genes were used to classify Aspergillus and Penicillium species, i.e. the genes encoding β-tubulin (BenA), calmodulin (CaM), RNA polymerase II second largest subunit (RPB2) and the Internal Transcribed Spacer (ITS) region. Therefore, in order to identify with more accuracy our isolate Penicillium, a phylogenetic analysis was performed concatenating the nucleotide sequences of these gene into one unique sequence. So, all markers for Aspergillus and Penicillium accepted species described (22), were downloaded from NCBI and concatenated with a custom python script, adding our isolate and Hamigera avellanea sequences, used as outgroup. The analysis was carried out through NGPhylogeny webservice (29) with a personalised workflow (https://ngphylogeny.fr/). The workflow consists in MAFFT(61) for the multiple alignment, BMGE (62) for alignment curation, FastTree (63) with GTR evolutionary model γ-distributed rate and 1000 as bootstrap value (64) for tree Interference and Newick Display (65) for tree rendering. In order to focus on the Citrina section, another tree was constructed with the same method considering the concatenamer constituted of BenA, CaM and ITS sequences and by adding CBS strains of P. sumatraense available in NCBI sharing these genes. RPB2 gene is unavailable for all these strains and it was not considered. Inventors selected only CBS strains because the CBS- KNAW culture collection is the largest one in the world with more 100.000 strains of fungi (including yeasts) and bacteria (https://wi.knaw.nl/page/Collection). 6. Growth of P. sumatraense AQ67100 in liquid medium and determination of glycosyl hydrolyse (GH) activities. P. sumatraense AQ67100 was grown in liquid medium at 20°C on a rotary shaker (120 rpm) posed in a dark chamber. For liquid cultures, A-medium [0.6% (w/v) K 2 HPO 4 , 0.14% (w/v) (NH 4 ) 2 SO 4 , 0.01% (w/v) MgSO4*7H2O, 0.2% (w/v) KH2PO4], and B-medium [0.6% (w/v) K2HPO4, 0.6% (w/v) (NH4)2SO4, 0.1% (w/v) MgSO4*7H2O, 0.6% (w/v) KH2PO4] were used. All the media were prepared fresh, pH-adjusted (6.5) and autoclaved. Three mycelium squares (5 × 5 mm) were cut out of a plate and inoculated in 50 ml of culture medium. After 2 days of growth, cultures were supplemented with 0.2% (w/v) heat treated C. vulgaris biomass and the growth prolonged up to 20 days. Supernatants from 20-days old cultures were used as starting inoculum (1 % v/v) for other cultures. The growth medium from algal-supplemented cultures was centrifuged (4000 × g, 10 minutes) and the supernatant filtered using a sterile Filtropur 0.22 µm. Then the filtered supernatants were dialyzed and concentrated (10X) using a Vivaspin 10,000 MWCO PES. Samples prepared according to this procedure were referred to as “filtrates”. Enzymatic activity was assayed by incubating the filtrate (10% v/v, 100 µl total volume) in 50 mM Na-Acetate buffer pH 5.5 at 28°C by using the following substrates: 1 % (w/v) carboxy-methylcellulose (CMC) to detect endoglucanase activity, 1% (w/v) xyloglucan (XG) to detect xyloglucanase activity, 1% (w/v) arabinoxylan (AX) to determine xylanase activity, 5 mM p-nitrophenyl-β-glucopyranoside (pNPG) and 5 mM p-nitrophenyl-β-galactopyranoside (pNPGal) to determine β-glucosidase and β-galactosidase activity, respectively. All polysaccharides were purchased from Megazyme (Bray, Ireland) whereas pNPG and pNPGal from Sigma-Aldrich (Saint Louis, USA). Enzyme activity was expressed as Enzyme Units (µmol of reducing sugar equivalents released per minute, or µmol of p-nitrophenol released per minute) per kg substrate or Litre (L) culture. Determination of µmol reducing ends released upon hydrolysis was performed according to (66) using different amounts of glucose as calibration curve. Determination of µmol p-nitrophenol released upon hydrolysis was determined using different amounts of p-nitrophenol as calibration curve. Enzyme Units were expressed as mean of the values determined at two different time-points. 7. Preparation of Alcohol Insoluble Solid (AIS) fraction from C. vulgaris. Preparation of Alcohol Insoluble Solid (AIS) fraction from C. vulgaris cells was performed according to (46) with some modifications. In brief, about 50 mg DW of C. vulgaris biomass was frozen in liquid nitrogen and homogenized with mortar and pestle. The resulting powder was washed three times in 70% (v/v) ethanol, vortexed, and pelleted by centrifugation (20,000 × g, 10 minutes). The pellet was washed twice with a chloroform: methanol mixture [1: 1 (v/v)] and centrifuged (20,000 × g, 10 minutes). The pellet was then washed three times with acetone up to complete discolouration and pelleted by centrifugation (20,000 × g, 10 minutes). After evaporation of the solvent, the pellet was solubilized in B-medium and the resulting suspension used for downstream applications. Following this procedure, the AIS yield was about 40 ±10 % of the starting C. vulgaris biomass. 8. Growth in different polysaccharides-supplemented media and enzymatic analysis. For growth experiments in different carbon source-supplemented media, B-medium was supplemented with 0.5% (w/v) XG, 0.5% (w/v) AX, 0.2% (w/v) heat-treated C. vulgaris biomass (C.v.) and 0.2% (w/v) C. vulgaris AIS (C.v. AIS). B-medium without any carbon source (NS) was used as negative control. All polysaccharides-supplemented media were prepared fresh, pH adjusted (6.5) and filter-sterilized. Filtrates from culture media were prepared and assayed according to the procedure previously described. Alternatively, filtrates were separated in 12.5% of Laemmli gel and then stained by silver nitrate. Proteolytic assay was performed by incubating 1 µg BSA with 20 µL of filtrate from C.v.-supplemented culture for 16 hours at 28°C. BSA was purchased from Sigma-Aldritch (St. Louis, USA). The reaction was separated in 10% of Laemmli gel and then stained by silver nitrate. 9. Protein identification by LC-MS/MS analysis. For protein identification analysis, 40 µL of 10X concentrated NS-, AX-, C.v.- and C.v. AIS- filtrates were incubated in Laemmli loading buffer at 100 °C and then loaded on a 10% acrylamide gel for separation by 1D-SDS-PAGE. Each lane was divided into ten different gel slices for in gel trypsin digestion. Peptides were separated on a Pepmap C18 column (150 mm × 0.75 mm) at 300 nL minute-1 with a 90 min multi step gradient of acetonitrile in 0.1% formic acid, using an Ultimate 3000 nano-chromatography pump (Thermo-Fisher Scientific) coupled to an LTQ Orbitrap Discovery mass spectrometer (Thermo-Fisher, Bremen, Germany) operated in a data dependent mode. MS was acquired at 30.000 FWHM resolution in the FTMS (using a target value of 5 × 105 ions) and MS/MS was carried out in the linear ion trap. Five MS/MS scans were obtained per MS cycle. The raw data from the mass spectrometric analysis were processed using the MaxQuant software v. 1.6.17 (67) supported by Andromeda as the database search engine for peptide identifications, using a protein database constructed ad hoc from the annotated genome of P. sumatraense AQ67100. The main peptide identification parameters were the following: trypsin cleavage specificity, variable methionine oxidation and N-term acetylation, cysteine carbamidomethylation as fixed modification and mass tolerance for parent and fragment ions of ± 20 ppm and ± 0.5 Da, respectively. Protein identification was performed in the MaxQuant Identify module using the following parameters: protein and peptide false discovery rate (FDR) < 0.01, posterior error probability based on Mascot score, minimum peptide length of 7. 10. Enzymatic treatment of C. vulgaris cells and determination of metabolites. For the enzymatic treatment, 0.33 mg of C. vulgaris biomass was washed, heat-treated (70°C, 50 minutes) and incubated with 0.3 mL of different enzymatic mixtures. The enzymatic mixtures consisted of (i) a recombinant cellulase from Aspergillus niger (0.24 U mL-1), (ii) a blend composed of lysozyme from chicken hen egg white (100.000 U mL-1), chitinase from Streptomyces griseus (0.15 U mL-1) and sulfatase H1 from Helix pomatia (10 U mL-1) according to (15) and (iii) a filtrate (10X) of P. sumatraense AQ67100 as obtained from 10-days old C. vulgaris supplemented-culture (0.13 ± 0.03 U mL-1 of β-glucosidase activity). Lysozyme, chitinase and sulfatase were purchased from Sigma-Aldritch (St. Louis, USA) whereas the cellulase was purchased from Megazyme (Bray, Ireland). All the enzyme mixtures were prepared fresh, filter- sterilized and dialyzed using B-medium as buffer exchange. The reactions were incubated at 28°C for 16 hours. Following the incubation, the reaction mixtures were centrifuged (14000 × g, 10 minutes) and the amount of sugars, chlorophylls and lipids spectrophotometrically determined in the supernatants. Determination of reducing and total sugars was performed in accordance with (66) and (68), respectively, using different amounts of glucose to build a calibration curve. Determination of chlorophylls was performed in accordance with (69). Determination of lipids was performed by the sulfo-phospho-vanillin (SPV) assay according to Mishra et al., 2014 (70). Extraction of metabolites from enzymatically treated C. vulgaris cells was performed by using 1 mL of 60% (v/v) ethanol or 1.5 mL of [DMSO: acetone: H2O] (10: 80: 10, v: v: v). The chlorophyll yield obtained by using [DMSO: acetone: H 2 O] was reported as reference. Determination of lipids in both ethanolic extracts and raw C. vulgaris biomass was performed by SPV assay. The lipid yield obtained from raw C. vulgaris biomass was reported as reference. All the spectrophotometric determinations were performed by using an Infinite® M Nano200 spectrophotometer (Tecan AG, Männedorf, Switzerland). RESULTS Capture of the unknown fungal isolate by the algal trap. An algal trap was employed to capture potential algivorous and algal saprophytic microbes. The algal trap consisted of an open flask containing heat-killed cells of C. vulgaris suspended in T-Phi medium, i.e., a TAP medium devoid of acetate in which Phi replaced Pi as Phosphorous source. The algal trap employs C. vulgaris as potential substrate and Phi as selective agent to isolate actively growing microorganisms. Phi is only metabolizable by chemolithotrophic bacteria carrying the Phosphite Dehydrogenase enzyme (PTXD) such as Pseudomonas stutzeri (17) and therefore, an eventual microbial growth in such medium could only be sustained at the expense of intracellular phosphate stored in the algal cells. Moreover, Phi is employed as antimicrobial agent to restrict contamination in transgenic algal cultures over-expressing PTXD (18-20), making the selection in the algal trap highly stringent. The algal traps were posed inside a greenhouse, an environment expected to contain plant parasites, saprophytes, endophytic microbes and, at lower extent, phytopathogens. During such attempts, our attention was attracted by a fungus that in two independent trials grew in the traps at the expense of microalgae (Figure 1a). The fungus was able to grow in T-Phi medium exploiting C. vulgaris as both carbon and phosphorous source, thereby suggesting its capability of metabolizing dead algal biomass. The fungus grew in the form of small mycelium balls capable of adsorbing the microalgae that, in turn, conferred to the mycelium a slight green colour (Figure 1a); for the isolation, different mycelium portions were harvested from the algal traps and plated onto solid MEP medium. All the mycelium portions expanded on the solid medium displaying a similar growth phenotype, thereby suggesting that they could be ascribed to the same organism (Figure 1b). The mycelium phenotype was similar to that displayed by fungi belonging to Penicillium and Aspergillus genus (21, 22) appearing as a greenish or whitish colony based on the type of medium used for growth (Figure 1c). Microscopy analysis was used to investigate the morphological characteristics of colony and conidiophores of the unknown fungal isolate (Figure 9a, b). The conidiophores were biverticillate as those found in several Penicillium species, i.e., whorl of three or more metulae between the end of the stipe and the phialides (Figure 9b) (16), supporting the identification of the fungus as a Penicillium isolate. Identification of the unknown fungal isolate by genomic sequencing. The unknown fungal isolate was subjected to whole-genome sequencing for identification. Genomic DNA was extracted from the mycelium and then subjected to NGS sequencing. By following the procedure described in Methods 4, inventors obtained a highly pure RNA-free gDNA preparation without adding RNAse to the sample, suggesting that such procedure did not disable fungal RNAses (Figure 10). A total of more than 290 million reads 2x150bp were obtained. The de-novo assembly of the Penicillium isolate, obtained by SPAdes (23), was a high-quality draft genome consisting of 129 scaffolds and a size of 35.196.323 bp (scaffolds size >= 1000 bp). The GC content was 46.56% whereas N50 value was 1455188 bp in accordance with QUAST statistics (24) (Table 1). It resulted complete and in single copy at 99.3% and 98.2% for fungi and Eurotiales, respectively (Table 2). BUSCO tool (25) quantified the completeness of genomic, transcriptomic or annotated gene datasets in terms of the expected gene content based on evolutionary principles and its metric was complementary to technical metrics like N50 (25). Regarding on gene prediction and annotation, a total of 14204 genes were predicted by BRAKER2 pipeline (26) trained with protein sequences belonging to Penicillium species downloaded from GenBank; data of predicted protein library and the corresponding gene annotations are partially reported in tables 5 and 6. PANNZER2 webserver (27) annotations output reported 10481 proteins with a description and 9854 with at least one Gene Ontology (GO) term. Meanwhile, running BUSCO on predicted proteins, resulted in 100% and 99.3% prediction with fungi and Eurotiales datasets, respectively (Table 2). Other statistics on gene prediction calculated by Eval tool (28) are displayed in Table 3. The predicted genes, CDSs and proteins were listed according to incremental numerical identifiers, i.e., gene-, CDS- and protein-IDs, as automatically generated by the BRAKER pipeline; here, each gene and the corresponding CDS and encoded protein are identified by the same number, whereas the suffix “.t” is referred to the specific CDS and protein isoform. A recently published work (22) about the phylogeny of Eurotiales defined the accepted species and strains of Penicillium genus, allowing us to construct a phylogeny tree based on the concatenation of four marker genes. According to the phylogenetic analysis performed by NGPhylogeny (29) with “à la carte” workflow, our isolate was placed near Penicillium sumatraense (i.e., Penicillium sumatraense CBS 281.36) and therefore classified as P. sumatraense AQ67100 on the basis of such result (Figure 2). A further study on the other P. sumatraense strains was necessary to understand how they clustered with our isolate. In accordance with previous analysis, P. sumatraense AQ67100 clustered with the other strains belonging to this species (Figure 3). Penicillium sumatraense belongs to series Sumatraensia subgen. Aspergilloides, Citrina section and is phylogenetically related to series Copticolarum (22). Regarding morphology and physiology, colonies are characterized by moderated or fast growth. Conidial colour ranges from blue green to dull and dark green, and the conidiophores are predominantly biverticillate in accordance with the morphological analysis shown in Figure 9). The sexual morphology is unknown and sclerotia are not observed in culture (22). Moreover, this species produces curvularins such as curvularin, dehydrocurvularin, sumalactone A-D, sumalarins and citridones E-G and it was considered as the causative agent of blue mould rot in Vitis vinifera and Sparassis crispa (22). Growth of the fungus in algal-supplemented media. In order to confirm the saprophytic nature of P. sumatraense AQ67100 towards algal biomass, different mycelium portions were inoculated in two different basal salt media, named as A- and B-medium, whose salt compositions resembled those used for culturing fungi belonging to Penicillium and Aspergillus genus. Upon 2 days of culturing, heat-treated C. vulgaris biomass (0.2% w/v) was added to each medium. The use of heat-killed microalgae avoided undesired algal responses that, in turn, could complicate the comprehension of the interaction between the fungus and its feed. Between the two different media, B-medium was the most suitable for supporting the growth of P. sumatraense AQ67100 in the presence of heat-killed C. vulgaris cells as substrate (Figure 11) and therefore it was selected for all the subsequent growth experiments. Four days after algal supplementation, the mycelium started to expand whereas the green colour of the medium, indicating the presence of microalgae, turned to pale green (Figure 11b). This effect was clearly visible 7 days after algal supplementation (Figure 11c); here, the optical microscope analysis revealed that algal cells were homogenously adsorbed to the fungal hyphae, indicating a direct contact between the mycelium and dead algal biomass (Figure 4a). Eighteen days after algal supplementation, the fungus had metabolized most of the microalgae whereas the amount of mycelium biomass and conidia had increased (Figure 4b). All these results taken together indicated that the fungal growth was sustained by the algal biomass since the medium was devoid of any other carbon source except of microalgae, thus demonstrating the saprophytic nature of P. sumatraense AQ67100 towards algal biomass. Determination of GH activities in algal-supplemented culture. C. vulgaris is an oleaginous microalga capable of growing in wastewaters, thus allowing to combine the production of triacylglycerols with waste-water remediation (19). However, the cell wall of C. vulgaris is composed of highly recalcitrant chitin- and glucan-like polysaccharides (14) that limit its large-scale exploitation (5). To overcome this limitation, inventors decided to investigate the GH activities secreted by P. sumatraense AQ67100 to assimilate C. vulgaris. In a time-course analysis, the supernatant from the algal-supplemented culture was collected at different days of growth and processed according to the procedure described in Methods 6. The extracellular enzyme profiles of each culture filtrate, abbreviated as filtrate, were evaluated using different substrates in order to identify the different CWDE activities secreted by the fungus. The time-course analysis revealed that GH activities reached the highest value at 8 days from algal supplementation (i.e., 10-days old culture) and remained stable up to 2 days later (i.e., 12-day old cultures). The filtrates from 10-days old cultures were therefore chosen for all the subsequent analysis. Among the different activities tested, the highest activity was ascribed to β-glucosidase and, at lower extent, to endoglucanase and β-galactosidase whereas xylanolytic and xyloglucanolytic activities were almost absent (Figure 12). Analysis of degrading activities of P. sumatraense AQ67100 from different polysaccharides- supplemented media. The degrading arsenal of P. sumatraense AQ67100 was further investigated by growing the fungus in different polysaccharide-supplemented media. Different substrates were employed in order to stimulate the production of different categories of CWDEs, since fungi are highly sensitive to the carbon source used for their growth (5). Cultures were performed in B-medium supplemented with different carbon sources such as heat-treated C. vulgaris biomass (C.v.), C. vulgaris cell walls (C.v. AIS), arabinoxylan (AX) and xyloglucan (XG). The fungal culture obtained by inoculating the fungus in basal salt B-medium, i.e., without any carbon source, was used as negative control (NS). After 10-days of incubation, the mycelium displayed different growth phenotypes depending on the carbon source used whereas, as expected, the fungus did not grow on NS medium (Figure 5a). The supernatants from each culture were collected and the CWDE activities secreted by the fungus were evaluated using different substrates. The enzyme activities were expressed as Enzyme Units both per kg substrate (Figure 5a) and litre culture (Figure 13). Notably, P. sumatraense AQ67100 secreted a high level of xylanolytic activities when the growth was performed using arabinoxylan as carbon source, reaching up to 3.2 × 105 Units kg arabinoxylan-1 (Figure 5a). The use of xyloglucan-supplemented media induced the expression of different GH activities in accordance with the heterogenous nature of xyloglucan, a β-glucan decorated with D-xylose units and at lower extent, with D-galactose, L-arabinose and D-glucuronic acid residues (Figure 5a). Instead, when the fungus was grown using the cell wall of C. vulgaris or the whole cells as substrates, cellulolytic and hemicellulolytic activities were significantly lower. Interestingly, β- glucosidase activity was the most prominent activity in both algal-supplemented media; this activity tripled from C.v.- (5.7× 10 3 Units kg substrate-1) to C.v. AIS-filtrate (17.9 × 103 Units kg substrate-1) suggesting a key role for β-glucosidase in cell wall metabolism of C. vulgaris (Figure 5a). Equal volumes of C.v.- and C.v. AIS-filtrates were then analysed by SDS-PAGE including the AX- and NS-filtrates, here used as positive and negative control of enzyme production, respectively (Figure 5b). The amount of secreted proteins was lower in C.v.- filtrate than in AX- filtrate whereas a higher abundance of proteins was present in C.v. AIS-filtrate in accordance with the level of enzyme activities detected in the same filtrates (Figure 5a, b). Identification of enzymes in the filtrates by LC-MS/MS analysis. Although the enzymatic assays allowed to detect and quantify several enzymatic activities, such approach was limited by the number of substrates tested. In order to reveal the whole enzymatic arsenal secreted by P. sumatraense AQ67100, C.v.-, C.v. AIS- and AX-filtrates were also subjected to LC-MS/MS analysis. About 50 proteins were identified at the highest confidence score using a protein database constructed ad hoc from the annotated genome of P. sumatraense AQ67100 (Table 4). In parallel, protein identification using a database constructed on C. vulgaris genome (30) failed in identifying putative algal proteins in both C.v.- and C.v. AIS-filtrates (data not shown). Interestingly, the majority of the proteins identified in C.v.- and C.v. AIS-filtrates were related to two main enzymatic classes: proteases and exo-glycosidases (Table 4, Figure 6). A wide array of proteases was detected in both C.v.- and C.v. AIS-filtrate, suggesting that protein hydrolysis is fundamental to achieve the digestion of C. vulgaris biomass. Among the different proteases identified, a secreted isoform of dipeptidyl-peptidase 5 (g4775.t1, Table 4) and aminopeptidase (g1317.t1, Table 4) were the most prominent in both filtrates (Figure 6). In order to confirm the presence of proteolytic activities in the filtrates from algal-supplemented cultures, C. v.-filtrate was incubated with BSA; upon 16 hours of incubation, BSA resulted almost completely degraded, thus confirming the presence of high proteolytic activity in C.v.-filtrate (Figure 14). Other highly represented CWDEs in algal filtrates were exo-glycosidases such as α-glucosidase (g10014.t1, Table 4), β-glucosidases (g4340.t1, g10362.t1, g484.t1, Table 4), α-1,2-mannosidase (g1651.t1, Table 4) and β-glucuronidase (g4150.t1, Table 4). Interestingly, four enzymes related to the metabolism of β-1,3-glucan were also identified (g9376.t1, g5736.t1, g7048.t1, g7605.t1, Table 4) with an exo-β-1,3-glucanase (g9376.t1, Table 4, Figure 6) being the most prominent in both C.v.- and C.v. AIS-filtrates (Figure 6). Other enzymes that are usually required for the efficient hydrolysis of plant cell walls, such as endoglucanases, endoxylanases and endopolygalacturonases, were hardly detected (Table 4, Figure 6). In AX-filtrate, in addition to exo-glycosidases and proteases (Figure 6), several xylanolytic enzymes such as endo-xylanases (g11633.t1, g9323.t1, Table 4, Figure 5), β-1,4-xylosidases (g3950.t1, Table 4, Figure 6) and α-L-arabinofuranosidases (g1388.t1, g411.t1, Table 4, Figure 6) were specifically identified (Table 4). Unexpectedly, chitinases were more prominent in AX- filtrate than in C.v.- and C.v. AIS-filtrates, although this class of enzymes was found to be effective in degrading the cell wall of C. vulgaris (14, 15). In our analyses, the ubiquitous presence of chitinases (g9356.t1, g11244.t1, Table 4, Figure 6) suggested their possible involvement in endogenous processes such as the remodelling of fungal hypha rather than towards algal chitin- like polysaccharides. The lack of degrading enzymes exclusively induced by algal supplementation together with the high propensity in the production of xylanolytic activities suggested that arabinoxylan is a preferred substrate for P. sumatraense AQ67100 compared to algal biomass. However, P. sumatraense AQ67100 can be considered a versatile saprophyte (or saprotroph) in accordance with the biological role of this category of microbes. Enzymatic treatments of C. vulgaris using the filtrate of P. sumatraense AQ67100. In order to evaluate the degrading potential of P. sumatraense towards algal biomass, the filtrate of P. sumatraense AQ67100 from the algal-supplemented culture, referred to as F-blend, was used to treat C. vulgaris cells. Other degrading enzymes such as a pure cellulase, referred to as C-blend, and an enzyme mixture composed of lysozyme, chitinase and sulfatase, referred to as LCS-blend, were used in control experiments. Indeed, peptidoglycan-degrading enzymes such as lysozyme have been recently shown to be highly effective towards the cell wall of C. vulgaris (31), and the LCS-blend was also employed to obtain C. vulgaris protoplasts (15). The amount of sugars, chlorophylls and lipids released from the enzymatically treated cells was determined in the incubation medium upon 16h of treatment. Differently from sugars, that may also have a cell wall origin, an increased release of chlorophylls and lipids in the supernatants can be considered as a direct proof of cell lysis, given the intracellular compartmentalization of these metabolites. In parallel, the amount of chlorophylls and lipids was also evaluated in the ethanolic extracts from the same cells. It is worth noting that in normal conditions (i.e., untreated cells), metabolite extraction by 60% ethanol is not efficient for C. vulgaris. Although at different extent, all the enzyme blends promoted the release of sugars (Figure 7a) whereas none of the treatments was able to promote the release of chlorophylls and lipids in the incubation medium, indicating that the released sugars are likely degradation products of cell wall origin rather than of endogenous nature. Among the different blends, the highest release of reducing and total sugars (4.8 ± 0.5 mg g DW-1, 40.1 ± 4.9 mg g DW-1) was promoted by F-blend, followed by LCS-blend (3.7 ± 1.1 mg g DW-1, 12.9 ± 6 mg g DW-1), whereas C-blend had very little effect (Figure 7a). Compared to the control experiment (N-T), F-blend was the only mixture capable of promoting an increased release of both chlorophylls (2.5 ± 0.2 mg g DW-1) and lipids (130.7 ± 10.9 mg g DW-1) from algal biomass upon ethanolic extraction, thus demonstrating the effectiveness of P. sumatraense AQ67100 filtrate in increasing the permeability of C. vulgaris cells (Figure 7b, c). On the other hand, the treatment with LCS-blend lowered the extraction yield of both chlorophylls and lipids with respect to that obtained from untreated cells (Figure 7b, c), suggesting the presence of unexpected side-reactions between LCS blend and the cells of C. vulgaris. The latter result highlighted the importance of selecting the proper enzyme blend for algal bio-refinery processes, that must combine effectiveness (towards algal cell wall) to compatibility with all downstream reactions. EXAMPLE 2 To better characterize the enzymatic activities of P. sumatraense AQ67100, the gene g9376.t1 encoding a putative exo-1,3-β-glucanase (Table 6) was expressed as his-tagged protein in Pichia pastoris (Fig.15). Inventors selected the gene g9376.t1 because the putative exo-1,3-β-glucanase was the most abundant glycoside hydrolase produced by P. sumatraense AQ67100 in the presence of Chlorella vulgaris as determined by mass spectrometry analysis (Fig.6). Among the different substrates tested (Table 7), the recombinant enzyme g9376.t1 was active on laminarin from Laminaria digitata (a 1,3-/1-6-β-mixed glucan of brown algae) and 1,3-β-D- Laminaripentaitol borohydride (1,3-β-D-LAM5P), a 1,3-β-glucan pentamer with a blocked C1- end, demonstrating that g9376.t1 is effectively a 1,3-β-glucan acting enzyme. Table 7. Specific activity of exo-b-1,3-glucanase g9376.t1 towards different substrates. Enzyme activity, expressed as Units (µmol min-1) per mg of enzyme, was evaluated at 25°C and pH 5. Data are expressed as mean ± SD, n = 3. --, no activity detected. [pNPGlc, p-nitrophenyl-β- D-glucopyranoside; pNPGal, p-nitrophenyl-β-D-galactopyranoside; pNPLAM2, p-nitrophenyl-β- D-laminaribioside; CMC, Carboxy-Methyl-Cellulose; PGA, polygalacturonic acid; 1,3-β-D- LAM5P, 1,3-β-D-Laminaripentaitol borohydride]. Once determined the preferred substrate(s) of g9376.t1, inventors proceeded to deeper characterize its activity by using laminarin as reference substrate. The analysis of pH-dependent activity revealed that the enzyme was active in a pH range between 4 and 6, with a temperature optimum around 50°C (Fig. 16a-b). The enzymatic assays revealed that g9376.t1 is characterized by a marked thermostability since its activity was significantly reduced only upon incubation at temperatures higher than 60-70°C (Fig. 16c). Moreover, g9376.t1 acted as an exo-glucanase by releasing D-glucose as main product since its activity generated comparable amounts of reducing ends and D-glucose over reaction time (Fig.16d). HPLC chromatography was employed to analyze the degradation products released by g9376.t1 from 1,3-β-glucan oligomers with different degree of polymerization. The enzyme was active from laminaritriose upwards and released two end-products, i.e., glucose and 1,3-β-laminaribiose (Fig. 17), indicating that the enzyme was unable to operate the catalysis on 1,3-β-glucan oligosaccharides shorter than three residues. The ratio [glucose:laminaribiose] as released from same amounts of 1,3-β-oligomers with different lengths increased in proportional manner to their degree of polymerization, further confirming that the enzyme acted through an exo-mode of action, i.e., by releasing glucose from the end of each oligomer (Fig.17). The analysis of degradation products released from 1,3-β-D-LAM5P demonstrated that glucose was released from the non-reducing end of the oligomer since it accumulated together with a 1,3- β-glucan oligomer characterized by a non-conventional retention time, i.e., plausibly a dimer with a borohydride-blocked reducing end, whereas laminaribiose (LAM2) was not detected neither as intermediate- or end-product (Fig.18). These results clearly revealed the mode of action of g9376.t1, i.e., a glucose-releasing enzyme acting on the non-reducing end of 1,3-β-glucan oligosaccharides with a minimum size of three units [(Glc1–3βGlc1–3βGlc)-n]. g9376.t1 is the first exo-1,3-β-glucanase from Penicillium species so far characterized. EXAMPLE 3 The P. sumatraense isolate AQ67100 shows additional interesting features respect to the strains described in Park M. S. et al., The J. of Microbiol. (2016), 54(10):646-654, in particular: a) P. sumatraense AQ67100 efficiently grows on Ulva lactuga biomass (green macro-alga) (Fig. 19); b) P. sumatraense AQ67100 grows in 0.2% (w/v) alginate (Fig.20a) by displaying a weak alginase activity (about 0.7-0.8 U/L) (Fig.20b). The alginase activity is also supported by the presence of g11905.t1 a protein predicted as a putative alginase lyase (Table 6). DISCUSSION In the present research, a filamentous fungus capable of metabolizing C. vulgaris was captured by an algal trap (Figure 1) and classified as P. sumatraense AQ67100 by genomic analysis (Figure 2, Figure 3). Although different Penicillium species have been widely investigated, the current available information on P. sumatraense is still scarce. The first isolate was obtained from the rhizosphere of the mangrove Lumnitzera racemose (32); subsequently, another isolate from deep- sea sediments was demonstrated to produce sumalactones, a new class of curvularin-type macrolides (33). In 2017, P. sumatraense was identified as one of the fungi responsible for blue mold disease in V. vinifera (34). Nowadays, some research groups are proposing P. sumatraense as a bioreactor for lipase production (35). To our knowledge, the genome of P. sumatraense AQ67100 is the first fully annotated genome available for this fungal species. Although P. sumatraense AQ67100 showed a high propensity in the production of xylanolytic activities in accordance with other fungal species belonging to the same genus (36, 37), our isolate metabolized C. vulgaris by a combined action of different exo-glycosydases and proteases (Figure 5, 6, Table 4-6). The array of enzyme activities secreted by P. sumatraense AQ67100 towards the cell walls and whole cells of C. vulgaris did not resemble the conventional degradative activities required for the degradation of plant cell wall polysaccharides. Amongst the different CWDEs secreted in the algal-supplemented media, exo-glycosidases were the most represented GHs (Figure 6, Table 4). Exo-glycosidases are highly versatile enzymes, employed in several industrial sectors due to their broad substrate specificity. Presumably, P. sumatraense AQ67100 could exploit the versatility of these enzymes towards the heterogenous cell wall of C. vulgaris. Proteases were identified both in C.v.- and C.v. AIS-filtrates, indicating that their degrading activity is directed not only towards proteins released from disrupted cells, but also towards structural proteins of the algal cell wall. Notably, glycoproteins are structural cell wall components in several microalgae species (38, 39) and different researches demonstrated that the treatment of C. vulgaris biomass with commercial proteases improved its assimilation by methanogenic bacteria, resulting in higher biogas yield (40-42). Our results showed that β-1,3-glucan hydrolases may also play a role in the degradation of C. vulgaris biomass (Figure 6). It is worth noting that β-1,3-glucan is a major cell wall component of brown seaweed (43) and of several microalgal species including C. vulgaris (31, 44, 45), and in higher plants it is also known as callose, i.e. a defence-induced polysaccharide highly recalcitrant to CWDE hydrolysis (46). However, the characteristics of polysaccharide accessibility in the C. vulgaris cell wall cannot be inferred based only on the enzymes used by P. sumatraense AQ67100. Other algal saprophytes may employ different enzymes since the arsenal of CWDEs also reflects host preference (47), pointing to the necessity of integrating different microbial secretomes for a more comprehensive scenario. Moreover, ultra- structural, NMR and monosaccharide composition analyses will be fundamental to accurately decipher the complex structure of C. vulgaris cell wall. During the first days of incubation with the algal biomass, the fungal hyphae attracted the dead algal cells to their surface (Figure 4a). For certain aspects, such interaction could resemble a fungal-assisted flocculation event, a phenomenon observed in different fungi-microalgae interactions and proposed as a potential eco-friendly harvesting process (48-50). However, at longer incubation times, P. sumatraense AQ67100 clearly grew at expense of C. vulgaris (Figure 4b, Figure 11). In this regard, the adhesion of fungal hyphae to Chlorella cells could also evoke a epibiotic or endobiotic style of predation (51) similar to that displayed by Vampirovibrio chlorellavorus (52). One aspect that must be taken into account is the tendency of fungi to adhere to organic substrates as well as to inert surfaces (53). In our experiments, the use of dead cells instead of living microalgae focused the analysis on the saprophytic nature of the fungus rather than on its potential predator and pathogenic nature; therefore, further studies will be required to investigate the interaction between P. sumatraense AQ67100 and living cells of C. vulgaris. Although the treatment with two out of three enzymatic blends promoted the release of sugars from the cells of C. vulgaris (Figure 7a), the filtrate from P. sumatraense was the only blend capable of promoting a higher release of chlorophylls and lipids upon ethanolic extraction (Figure 7b, c). The lipid-rich biomass of Chlorella species has attracted considerable interest, due to its application potential as a renewable and sustainable source of biofuels. However, biological constraints still pose significant challenges to the development of economically viable large-scale production of algal biofuel. In this regard, the low efficiency of metabolite extraction is one of the main drawbacks that still limit a sustainable use of microalgae in the biofuel sector. Compared to the untreated algal biomass, the treatment with the fungal filtrate improved the release of chlorophylls and lipids by 42.6 and 48.9%, respectively. Based on our results (Figure 7), at least ~4.7 litres of fungal culture are required to efficiently extract all the lipids from one gram of C. vulgaris biomass. Except for xylanolytic activities (3.2 × 105 Units kg arabinoxylan-1), the level of other CWDE activities secreted by P. sumatraense AQ67100 was significantly lower (Figure 5a), highlighting the importance of optimizing growth conditions to exploit this fungus in industrial processes. Importantly, the activity displayed by the fungal filtrate towards C. vulgaris seemed more permeabilizing than algalytic, indicating that the cell wall of C. vulgaris was degraded only in part. In this regard, the enzymatic hydrolysis of C. vulgaris will require further optimization, e.g., by testing different pH values, temperature conditions, reaction times, substrate concentrations and enzyme/substrate loadings. However, our analyses revealed several degrading enzymes exploitable in algal processing that can be heterologously expressed at high level for further characterization and large-scale exploitation, e.g., as supplements in specific algalytic blends (Table 4-6) (15, 31). Based on the characterization of the enzymatic profiles in P. sumatraense AQ67100 secretome, this fungus cannot be considered as a specialized algal saprophyte, but rather as an opportunistic saprophyte capable of assimilating microalgae in case of necessity, i.e., when the microalgae are the unique available carbon source in the medium (Figure 5). The enzymes used to assimilate C. vulgaris were not exclusively induced by algal biomass (Table 4), in contrast to activities specifically secreted by P. sumatraense AQ67100 for arabinoxylan degradation. However, our results indicate how the fungus remodulated its enzymatic arsenal in relation to C. vulgaris, pointing to exo-glycosidases and proteases as the enzymes responsible for algal assimilation. Probably, the isolation of a facultative algal saprophyte using the algal trap depended on the location of the trap, i.e., a greenhouse usually hosting land-plants such as Nicotiana tabacum, Lycopersicon esculentum and V. vinifera. A further improvement of this approach will consist in positioning the algal trap in proximity of algal open-ponds and marshy areas in order to increase the probability of capturing algivorous organisms and specialized algal saprophytes. CONCLUSIONS The approach here described allowed to unveil the enzymatic arsenal exploited by a novel P. sumatraense isolate to assimilate C. vulgaris. 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