WO2023002065A2 - Fungus strain and uses thereof - Google Patents
Fungus strain and uses thereof Download PDFInfo
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- WO2023002065A2 WO2023002065A2 PCT/EP2022/070846 EP2022070846W WO2023002065A2 WO 2023002065 A2 WO2023002065 A2 WO 2023002065A2 EP 2022070846 W EP2022070846 W EP 2022070846W WO 2023002065 A2 WO2023002065 A2 WO 2023002065A2
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- biomass
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- sumatraense
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Classifications
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4728—Calcium binding proteins, e.g. calmodulin
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/145—Fungal isolates
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
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- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- sumatraense AQ67100 can be considered a versatile saprophyte (or saprotroph) in accordance with the biological role of this category of microbes.
- the filtrate of P. sumatraense AQ67100 from the algal-supplemented culture, referred to as F-blend was used to treat C. vulgaris cells.
- Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.
- the Plant journal for cell and molecular biology. 2019;100(6).
- 31. Coelho D, Lopes P, Cardoso V, Ponte P, Brás J, Madeira M, et al. Novel combination of feed enzymes to improve the degradation of Chlorella vulgaris recalcitrant cell wall. Scientific reports.2019;9(1). 32. Meng L, Li X, Lv C, Li C, Xu G, Huang C, et al.
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Abstract
The present invention refers to 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, 98, 97, 96, 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.
Description
FUNGUS STRAIN AND USES THEREOF TECHNICAL FIELD The present invention refers to a Penicillium sumatraense (P. sumatraense) strain which is an algal saprophyte (or saprotroph), preferably capable of metabolizing Chlorella vulgaris (C. vulgaris), to the enzymes produced by said strain, and to uses thereof for improving the extraction and yield of biological compounds produced by microalgae. PRIOR ART Microalgae have recently gained increasing interest because of their potential application in a wide range of industrial sectors, from the biofuel to the pharmaceutical field. Microalgae evolved a great biodiversity, making them a precious source of valuable metabolites such as long chain polyunsaturated fatty acids, vitamins, polysaccharides and proteins (1). Moreover, microalgae are efficient producers of lipid-rich biomass, making them a key component in developing a sustainable energy source, alternative to fossil fuels. In this regard, some microalgae species accumulate large amounts of triacylglycerols that, in turn, can be converted to biodiesel through the transesterification process (2). In addition, microalgae are the bio-factory of first choice for the production of carotenoids with the highest anti-oxidant activities (3). Except for few model species used in basic research such as Chlamydomonas reinhardtii (4), the study of microalgal biology is mainly focused on their exploitation. Knowledge on biological processes such as those related to microalga-saprophyte interactions is still scarce and fragmentary. Similarly to what occurred in the plant-microbe interaction field, in which the study of plant cell walls and microbial Cell Wall Degrading Enzymes (CWDEs) paved the way to the enzymatic saccharification of lignocellulosic wastes for biofuel production (5), so the understanding of degrading processes acted by algivorous and saprophytic microbes towards microalgae can be fundamental for maximizing their use in many applied fields (6). Indeed, the recalcitrant cell wall of several microalgal species negatively impacts the extractability of triacylglycerol and carotenoids from the algal cell (7, 8) as well as the poor digestibility of microalgae also affects their dosage in feed and food additives. Moreover, the low extractability of triacylglycerols from microalgae by physical methods negatively impacts the production yield of biodiesel from algal-source at industrial scale (9) whereas lipid extraction by chemical (and polluting) methods clashes with the rationale of using microalgae to produce clean forms of fuel. Compared to chemical and physical methods employed to break algal cells, the biological method is an eco-friendlier option and it is easily extendible to large-scale; moreover, residual biomass from solvent-free extraction techniques can be further valorised by conversion into animal feed or
other forms of fuels (10). To date, the enzymatic treatment of algal cell wall exploited CWDE blends from saprophytic fungi as obtained by fermentation processes in which plant materials are used as feed for such microbes (11-13). However, these enzymatic arsenals were not evolved to hydrolyse the cell walls of microalgae, resulting in fewer effective reactions and low degradation efficiency. In other cases, the enzymatic mixtures consisted of degrading enzymes selected from the most disparate organisms such as chicken, fungi and snails, resulting in highly expensive blends (14, 15) and negatively impacting the production cost of extracted metabolites. Therefore, several factors such as low productivity, expensive harvesting procedures and difficult metabolite extractability limit microalgae full utilization at industrial scale. As the enzymatic arsenals from lignocellulolytic fungi are successfully employed to convert lignocellulose into fermentable sugars for bioethanol production, specific algalytic formulations could be used to improve the extractability of lipids from microalgae to produce biodiesel. WO2011140520 refers to compositions comprising an isolated cellulose degrading fungus, to compositions and bioreactor compositions comprising the cellulose degrading fungus bioprocessing facilities for and methods for producing co-products resulting from one or more bioprocesses of the cellulose degrading fungus. WO2012150968 refers to a system and method for the pelletization of single cell microalgae through co-cultivation with filamentous fungi to enable the low-cost separation of microalgae from liquid medium and to significantly increase the biomass and lipid yield. Currently, the research areas related to algivorous organisms, algal saprophytes and the enzymes responsible for the hydrolysis of algal cell wall are still little explored. Therefore, there is still the need of a microorganism or related enzymes able to efficiently hydrolyze algal cells and produce biological products. SUMMARY OF THE INVENTION All these aspects prompted the inventors to invest their efforts towards the isolation of novel microbes to exploit in the biological treatment of algal biomass. By using an algal trap, inventors captured a filamentous fungus, later identified as a novel Penicillium sumatraense isolate, capable of assimilating Chlorella vulgaris. Penicillium is a heterogenous genus occurring worldwide and its species play important roles as decomposers of organic materials and cause destructive rots in the food industry where they produce a wide range of mycotoxins. Some Penicillium species are common indoor air allergens whereas certain species are considered enzyme factories (16). Here, an integrated approach of enzymatic assays/LC-MS/MS protein analysis revealed the enzymes responsible for the hydrolysis and subsequent assimilation of the oleaginous microalga C. vulgaris
by P. sumatraense, leading to useful insights exploitable in algal bio-refinery processes. As a proof of concept, the algalytic activity of the enzymatic mixture from P. sumatraense was tested for its ability of promoting the release of sugars, chlorophylls and lipids from C. vulgaris and compared to that of other commercial degrading enzymes. DESCRIPTION OF THE INVENTION Authors found out that an algal trap method for capturing actively growing microorganisms was successfully used to isolate a filamentous fungus, that was identified by whole genome sequencing, assembly and annotation as a novel Penicillium sumatraense isolate. The fungus, classified as P. sumatraense AQ67100, was able to assimilate heat-killed Chlorella vulgaris cells by an enzymatic arsenal composed of proteases such as dipeptidyl- and amino-peptidases, and exo-glycosidases including α- and β-glucosidases, β-glucuronidases, α-mannosidases and exo-β-1,3-glucanases. The treatment of C. vulgaris with the filtrate from P. sumatraense AQ67100 improved the release of chlorophylls and lipids from algal cells by 42.6 and 48.9%, respectively. The improved lipid extractability from C. vulgaris biomass treated with the fungal filtrate highlighted the potential of algal saprophytes in the bioprocessing of microalgae posing the basis for the sustainable transformation of algal metabolites into biofuel-related compounds. The blend of enzymes produced and secreted by the fungus of the invention is therefore capable of promoting a higher release of biological products (including metabolites), such as chlorophylls and lipids, upon extraction, e.g. upon ethanolic extraction, from biomasses, in particular from algae. It is therefore an object of the invention a P. sumatraense strain comprising in its genome a RNA polymerase II second largest subunit (RPB2) gene sequence having at least 98% nucleotide sequence identity with the nucleic acid of SEQ ID NO: 1 and/or β-tubulin (BenA)^gene sequence having at least 98% nucleotide sequence identity with the nucleic acid of SEQ ID NO: 2 and/or Calmodulin (CaM) gene sequence having at least 98% nucleotide sequence identity with the nucleic acid of SEQ ID NO: 3 and/or an Internal Transcribed Spacer (ITS) sequence with at least 98% sequence identity to SEQ ID NO: 4. A further object of the invention is 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. An object of the invention is 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, 98, 97, 96, 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. In the present invention a functional fragment comprising the catalytic domain of the 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 is preferably the 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. Preferably the P. sumatraense strain according to the invention is capable of metabolizing Chlorella vulgaris (C. vulgaris). Preferably the P. sumatraense strain according to the invention comprises 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, More preferably it comprises in its genome one gene comprising or consisting of a sequence selected from SEQ ID NOs: 5-29, 31-54. Preferably the P. sumatraense strain according to the invention comprises a 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 SEQ ID NOs: 108. Preferably the P. sumatraense strain comprises 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 it comprises 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 it comprises in its genome SEQ ID Nos: 7, 8, 9, 12, 13, 25, 28, 39, 46 and 55 and optionally SEQ ID Nos: 17, 36, 37, 38, 51 and 53. Preferably the P. sumatraense strain according to the invention comprises 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 it comprises in its genome SEQ ID NOs: 5-54. Preferably the strain of the invention is characterized by an alginase activity. Preferably the strain of the invention produces one or more enzyme that comprise or consist 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:55-104 or functional fragments comprising the catalytic domain. Preferably the strain of the invention produces one or more enzyme that comprise or consist 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:112-156 or functional fragments comprising the catalytic domain. Preferably the strain of the invention produces one or more enzyme that comprise or consist 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:109 or 113 or functional fragments comprising the catalytic domain. Preferably the strain of the invention produces one or more enzyme that comprise or consist 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:55-104.
Preferably the strain of the invention produces the enzymes that comprise or consist of SEQ ID NO:55-104. Preferably the strain of the invention produces one or more enzyme that comprise or consist 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:112-156. Preferably the strain of the invention produces the enzymes that comprise or consist of SEQ ID NO:112-156. Preferably the strain of the invention produces an enzyme that comprise or consist 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:112. Another object of the invention is the use of the P. sumatraense strain as defined herein 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 as defined herein 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. A further object of the invention is the 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. Another object of the invention is the 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. Another object of the invention is 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 with a sequence selected from SEQ ID NOs: 55-104, 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. Another object of the invention is 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: 113-156 and 109, 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. A further object of the invention is the use of the composition as defined herein, 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. The blend may also comprise enzymes comprising or consisting of SEQ ID Nos:112-156 or functional fragments comprising the catalytic domain or derivatives thereof. The blend may also comprise an enzyme comprising or consisting of SEQ ID NO: 109. In the present invention the algae is preferably 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. Another object of the present invention is 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. In the present invention a functional fragment comprising the catalytic domain of the 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 is preferably the 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. Therefore, a further object of the invention is 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. Another object of the invention is 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. Another object of the invention is 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:109 and 112-156, 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: 109 and 112-156. Another object of the invention is 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, 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. The functional fragment comprising the catalytic domain of the 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 may be the isolated or synthetic or recombinant enzyme
comprising or consisting of an amino acid sequence having 100, 99, 98, 97, 95, 96, 94, 93, 92, 91, 90 or 89% sequence identity with SEQ ID NO:112. The functional fragment comprising the catalytic domain of the enzyme comprising or consisting of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85% sequence identity with SEQ ID NOs:55-79 and 81-104 may be the isolated or synthetic or recombinant enzyme comprising or consisting of an amino acid sequence having 100, 99, 98, 97, 95, 96, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85 % sequence identity with SEQ ID NO:114-156. The functional fragment comprising the catalytic domain of the enzyme comprising or consisting of an amino acid sequence having 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85% sequence identity with SEQ ID NOs:109 may be the isolated or synthetic or recombinant enzyme comprising or consisting of an amino acid sequence having 100, 99, 98, 97, 95, 96, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85 % sequence identity with SEQ ID NO:113. Therefore, an object of the invention is 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 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, 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:55-79, 81-104 and SEQ ID NO:112. Another object of the invention is 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 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, 95, 94, 93, 92, 91, 90 or 85% sequence identity with a sequence selected from the group
consisting of: SEQ ID NOs:113-156, 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:112- 156. Preferably the blend comprises enzymes comprising or consisting of SEQ ID NOs:112, 114-156. Preferably the blend also comprises an enzyme comprising or consisting of SEQ ID NOs:113. A further object of the invention is an isolated or synthetic or recombinant nucleic acid encoding the enzyme as defined herein, 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. A further object of the invention is an isolated or synthetic or recombinant nucleic acid encoding the enzyme as defined herein, 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:108 or 111. Another object of the invention is 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 (or strain) as defined herein 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. The composition comprising the blend of degradative enzymes as defined above may be the first or the second above disclosed compositions, or mixtures thereof. Another object of the invention is a method of producing a biological product, comprising the following steps:
a) cultivating, preferably for at least 7-10 days, at least one fungus (or strain) as defined herein 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. Another object of the invention is a method of producing a biological product, comprising the following steps: a) treating a biomass with the composition as defined herein; 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. Preferably the algae (or algal species or biomass) 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, more preferably the algae (or algal species or biomass) is C.^vulgaris. A further object of the invention is a system for the production of a biological product, comprising: - at least one fungus of the P. sumatraense strain as defined herein or a culture filtrate thereof and/or - the composition as defined herein and/or - at least one of the isolated enzymes or proteins as defined herein and - a biomass; and - a growth medium. Preferably the strain as defined herein comprises in its genome an RNA polymerase II second largest subunit (RPB2) gene comprising or consisting of SEQ ID NO: 1, a β-tubulin (BenA)^gene comprising or consisting of SEQ ID NO: 2, a Calmodulin (CaM) gene comprising or consisting of
SEQ ID NO: 3 and an Internal Transcribed Spacer (ITS) sequence comprising or consisting of SEQ ID NO: 4. The P. sumatraense strain as defined herein preferably further comprises in its genome at least one gene comprising or consisting of a sequence having 100, 99, or 98% sequence identity with one of the sequences selected from the group consisting of: SEQ ID NOs: 5-54, more preferably it comprises in its genome one gene comprising or consisting of a sequence selected from SEQ ID NOs: 5-54, even more preferably it comprises in its genome SEQ ID NOs: 5-54. Preferably, the P. sumatraense strain is defined as “algal saprophyte” or “sapro-phyco”, i.e. an organism capable of metabolizing dead algal biomass, preferably capable of metabolizing Chlorella vulgaris (C. vulgaris). Another object of the invention is the use of: - the P. sumatraense strain as defined herein or of a culture filtrate thereof or of - a composition comprising a blend of two or more enzymes secreted the by the fungus P. sumatraense strain as defined herein or 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, 85% sequence identity with a sequence selected from SEQ ID NOs:55-104, or functional fragments comprising the catalytic domain or derivatives thereof or of - at least one 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, 85% sequence identity with a sequence selected from SEQ ID NOs:55-104, 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. Preferably the above composition comprises blend of two or more enzymes comprising or consists of an amino acid sequence having 100, 99, 98, 97, 95, 94, 93, 92, 91, 90, 85% sequence identity with a sequence selected from SEQ ID NOs:55-104, or functional fragments comprising the catalytic domain or derivatives thereof. Preferably 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.
More preferably the blend comprises enzymes comprising or consisting of SEQ ID NOs:55-104. A further object of the invention is an isolated or synthetic or recombinant enzyme comprising or consisting of an amino acid sequence having 100, 99, 98, 97, 95, 94, 93, 92, 91, 90, 85% sequence identity with SEQ ID NOs:55-104, 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:55-104. Another object of the invention is 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, 95, 94, 93, 92, 91, 90, 85% sequence identity with one of the sequences selected from the group consisting of: SEQ ID NOs:55-104, or functional fragments comprising the catalytic domain or derivatives thereof. Preferably 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 the blend comprises enzymes comprising or consisting of SEQ ID NOs:55-104. A further object of the invention is an isolated or synthetic or recombinant nucleic acid encoding the above enzyme, preferably said nucleic acid comprising or consisting of a sequence having 100, 99, 98, 97, 95, 94, 93, 92, 91, 90, 85% sequence identity with one of the sequences selected from the group consisting of: SEQ ID NOs:5-54, preferably comprising or consisting of a sequence selected from SEQ NOs:5-54. Another object of the invention is 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 above 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, and filtering the supernatant, and dialyzing and concentrating the filtered supernatant to obtain a second composition comprising a blend of degradative enzymes. The composition comprising the blend of degradative enzymes as defined above may be the first or the second above disclosed compositions, or mixtures thereof.
A further object of the invention is 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 above 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, and 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. Another object of the invention is a method of producing a biological product, comprising the following steps: a) treating a biomass with the above composition; 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. Another object of the invention is a method of producing a biological product, comprising the following steps: a) treating a biomass with at least one enzyme as defined above and/or with the culture filtrate as herein defined; 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. Preferably the fungus of the invention produces at least one protein comprising or consisting of a sequence having at least 85% sequence identity with one sequence selected from the group consisting of: SEQ ID Nos:105-107. Preferably the fungus of the invention produces at least one protein comprising or consisting of one sequence selected from the group consisting of: SEQ ID Nos:105-107. Preferably the fungus of the invention produces the proteins having the following sequences: SEQ ID Nos:105-107.
Another object of the invention is a system for the production of a biological product, comprising: - at least one fungus of the P. sumatraense strain as defined above or a culture filtrate thereof and/or - the composition as defined above and/or - at least one of the isolated enzymes or proteins as defined above and - a biomass; and - a growth medium. Another object of the invention is a vector comprising or expressing at least one polynucleotide or protein or enzyme as defined above or a host cell genetically engineered expressing said at least one enzyme or protein or polynucleotide. Preferably the biological product is selected from the group consisting of a biofuel, a biomass, preferably a lipid-rich biomass, a terpenoid compound, a bioplastic, a pigment, an antioxidant, a vitamin, an antibiotic, a surfactant protein, a peptide, a lipid, a long chain polyunsaturated fatty acid, a polysaccharide, a protein, a triacylglycerol, a carotenoid, a therapeutic compound such as a nutraceutical compound or combinations thereof. Preferably the algae (or the algal species or biomass) is from a class selected from the group consisting of Actinochrysophyceae, Bacillariophyceae, Bryopsidophyceae, Bolidophyceae, Chlorarachnea, Chlorophyceae, Chrysophyceae, Cryptophyceae, Cyanophyceae, Diatomophyceae, Dinophyceae, Eustigmatophyccae, Glaucophyceae, Haptophyceae, Noctiluciphyceae, Pedinophyceae, Picophagophyceae, Pleurastrophyceae, Prasinophyceae, Prymnesiophyceae, Raphidophyceae, Synchromophyceae,Synd in iophyceae, Synurophyceae, Trebouxiophyceae, Ulvophyceae, Xanthophyceae, and combinations thereof. More preferably the algae (or the algal species or biomass) 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. Even more preferably the algae (or the algal species or biomass) is C.^vulgaris. In the context of the present invention the fungus “culture filtrate” or “filtrate” is the incubation or growth medium in which the fungus has grown with algae (i.e. a growth medium from algal- supplemented fungus cultures) and which comprises degradative enzymes secreted by the fungus.
Preferably, the growth medium is centrifuged (e.g.4000 × g, 10 minutes) to obtain a supernatant. The supernatant is then filtered, e.g. using a sterile Filtropur 0.22 µm. Then the filtered supernatant is dialyzed and concentrated (e.g.10X) e.g. using a Vivaspin 10,000 MWCO PES. Fungi have indeed an external digestion and secrete degradative enzymes. Therefore, in the context of the present invention, term “produce(s)” in certain embodiment is to be intended as “produce(s) and secrete(s)”. The incubation or growth medium may be liquid or solid. In the case of a solid medium, the latter is rinsed to solubilize secreted enzymes. The incubation or growth medium must comprise salts for an optimal growth of the fungus. In the context of the present invention “capable of metabolizing” means that the biomass has to be completely assimilated by the fungus which grows at the same time, as e.g. shown in figure 4. In the context of the present invention, the enzymes herein defined by their name or sequence include also the corresponding enzymes without the signal peptide. In the context of the present invention, when referring to specific DNA sequences, it is intended that it is comprised within the invention also RNA molecules identical to said polynucleotides, except for the fact that the RNA sequence contains uracil instead of thymine and the backbone of the RNA molecule contains ribose instead of deoxyribose, RNA sequence complementary the sequences therein disclosed, functional fragments, mutants and derivatives thereof, proteins encoded therefrom, functional fragments, mutants and derivatives thereof. The term “complementary” sequence refers to a polynucleotide which is non-identical to the sequence but either has a complementary base sequence to the first sequence or encodes the same amino acid sequence as the first sequence. A complementary sequence may include DNA and RNA polynucleotides. The term “functional” may be understood as capable of maintaining the same activity. “Fragments” are preferably long at least 10 aa., 20 aa., 30 aa., 40 aa., 50 aa., 60 aa., 70 aa., 80 aa., 90 aa., 100 aa., 150 aa., 200 aa., 300 aa., 400 aa., 500 aa., 600 aa., 700 aa., 800 aa., 900 aa., 1000 aa., 1200 aa., 1400 aa., 1600 aa., 1800 aa. or 2000 aa. “Derivatives” may be recombinant or synthetic. The term "derivative" as used herein in relation to a protein means a chemically modified protein or an analogue thereof, wherein at least one substituent is not present in the unmodified protein or an analogue thereof, i.e. a protein which has been covalently modified. Typical modifications are amides, carbohydrates, alkyl groups, acyl groups, esters and the like. As used herein, the term "derivatives" also refers to longer or shorter polynucleotides/proteins and/or
having e.g. a percentage of identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, more preferably of at least 99% with the sequences herein disclosed. In the present invention “at least 70 % identity” means that the identity may be at least 70%, or 75%, or 80%, or 85 % or 90% or 95% or 100% sequence identity to referred sequences. This applies to all the mentioned % of identity. Preferably, the % of identity relates to the full length of the referred sequence. The derivative of the invention also includes “functional mutants” of the polypeptides, which are polypeptides that may be generated by mutating one or more amino acids in their sequences and that maintain their activity. Indeed, the polypeptide of the invention, if required, can be modified in vitro and/or in vivo, for example by glycosylation, myristoylation, amidation, carboxylation or phosphorylation, and may be obtained, for example, by synthetic or recombinant techniques known in the art. In the present invention “functional” is intended for example as “maintaining their activity” e.g. degrading activity. Also within the scope of the subject invention are polynucleotides which have the same nucleotide sequences of a polynucleotide exemplified herein except for nucleotide substitutions, additions, or deletions within the sequence of the polynucleotide, as long as these variant polynucleotides retain substantially the same relevant functional activity as the polynucleotides specifically exemplified herein (e.g., they encode a protein having the same amino acid sequence or the same functional activity as encoded by the exemplified polynucleotide). Thus, the polynucleotides disclosed herein should be understood to include mutants, derivatives, variants and fragments, as discussed above, of the specifically exemplified sequences. The subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the polynucleotide sequences of the invention so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis, T. et al, 1982). Polynucleotides described herein can also be defined in terms of more particular identity and/or similarity ranges with those exemplified herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% or greater as compared to a sequence exemplified herein. Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into
the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See NCBI/N1H website. In the context of the present invention, with the term “algal saprophyte” or “sapro-phyco” it is intended that the fungus feeds on of dead algae. The amount of fungus inoculation is preferably in the range of 0.1 to 10% by volume, the temperature during the cultivation is in the range of 4 to 40°C, preferably of 25°C and the incubation time for the cultivation is preferably 7-10 days. Algae are typically administered at 0.1-0.2% in the culture medium. Preferably the culture medium is a basal salt medium composed of K2HPO4, (NH4)2SO4, MgSO4 and KH2PO4. In the context of the present invention, treating a biomass means incubating the biomass with the agent of the invention, e.g. with the composition or with the fungus filtrate of the invention. The treatment of the biomass with the composition of the invention preferably ranges from 6 to 18 hours and/or is carried out in a culture medium. Any known method of extraction of biological products may be used. Included in the present invention are also nucleic acid or amino acid sequences derived from the sequences herein shown, e.g. functional fragments, mutants, derivatives, analogues, and sequences having a % of identity of at least 70% with the herein disclosed sequences. The term “isolated or synthetic or recombinant nucleic acid encoding the enzyme” includes the gene and the related coding sequence (CDS), e.g. the gene without the herein indicated introns. In the present context, the term "comprising" has the broad common meaning "which includes", "which covers", "which contains" or "which consists of" or “which have”. It includes the element or elements explicitly recited and allows, but does not require, the presence of another or other elements not recited. In addition to this broad meaning, in the present context, the term "comprising" also covers the limiting meaning of "consisting of", according to which only the explicitly recited element or elements are present and no other. Furthermore, the term "comprising" or “consisting of” or “having” also includes the meaning of "essentially consisting of", which
means that one or more additional elements may be present in addition to those explicitly stated, provided that the additional element or elements present do not alter the effect technical obtained from the element or elements explicitly recited. ABBREVIATIONS AIS: Alcohol Insoluble Solid AX: arabinoxylan C.v.: Chlorella vulgaris CWDE: Cell Wall Degrading Enzyme GH: glycosyl hydrolase Phi: Phosphite Pi: Phosphate pNPG: p-nitrophenyl-β-glucopyranoside pNPGal: p-nitrophenyl-β-galactopyranoside XG: xyloglucan The present invention will be described by means of non-limiting examples, referring to the following figures: Figure 1. Capture of an unknown fungus by the algal trap. a) Capture of the fungus by the algal trap. Trapping experiments were repeated twice with similar results. b) Isolation of different fungal mycelium portions from the algal trap. Highlighted numbers indicate five different mycelium portions as grown on solid MEP medium. c) Phenotype of the fungus as grown on solid MEP and TC medium. Figure 2. Phylogenetic tree with all Penicillium and Aspergillus accepted species. The tree shows the phylogenetic proximity of our fungal isolate, i.e., P. sumatraense AQ67100, with P. sumatraense (CBS 281.36) (red area). Aspergillus and Penicillium species are highlighted in cyan and green, respectively, whereas Citrina section is in dark green. Figure 3. Phylogenetic tree with all Citrina Penicillium section. The phylogenetic tree shows the clustering of P. sumatraense AQ67100 with P. sumatraense CBS strains from Citrina section (orange area). Figure 4. Growth of P. sumatraense AQ67100 in algal-supplemented medium. Left, Representative images of fungal cultures as grown in B-medium supplemented with 0.2 % (w/v) heat-treated C. vulgaris biomass at a) 7 and b) 18 days from algal administration. Middle-Right,
Optical microscope images of the fungus from the same cultures shown in (a, b). Scale bars are also indicated [C.v., C. vulgaris cells; co, conidia]. Figure 5. GH activities from P. sumatraense AQ67100 cultures upon growth in different cell wall polysaccharides-supplemented media. a) Analysis of GH activities in filtrates from different cell wall polysaccharides-supplemented cultures upon 10 days of growth. B-medium was supplemented with 0.5% (w/v) arabinoxylan (AX), 0.5% (w/v) xyloglucan (XG), 0.2% (w/v) C. vulgaris AIS (C.v. AIS) and 0.2% (w/v) heat-treated C. vulgaris biomass (C.v.). B-medium without any carbon source (NS) was used as negative control. The corresponding cultures are shown below the graph. Units are expressed as µmol reducing ends (for glucanase, xyloglucanase and xylanase activities) and µmol pnitrophenol (for β-glucosidase and β-galactosidase activities) released per minute. Experiments were performed in triplicate with consistent results. b) SDS- PAGE analysis carried out on the same filtrates assayed in (a). AX- and NS-filtrates were analysed as positive and negative control of CWDE-production, respectively. Figure 6. Proteins identified in AX-, C.v.- and C.v. AIS-filtrates as determined by LC- MS/MS analysis. The protein intensity (sum of all identified peptide intensities) and the predicted protein function is reported for each identified protein. Proteins are listed from left to right in decreasing order of sequence coverage. Only proteins identified with the highest score (323.31) are reported. Further details on the identified proteins are reported in Table 4, Figure 7. Algal treatment using P. sumatraense AQ67100 filtrate improves the release of metabolites from C. vulgaris. a) Sugars released in the incubation medium from enzymatically treated C. vulgaris cells. b, c) Amount of b) chlorophylls and c) lipids in ethanolic extracts from enzymatically treated C. vulgaris cells. [N-T: no treatment; C-T: treatment with cellulase blend; LCS-T: treatment with lysozyme-chitinase-sulfatase blend; F-T: treatment with P. sumatraense blend; N-T/DMSO: no treatment + DMSO-extraction; RB: total lipids from raw algal biomass]. Data are expressed as mean ± SD (N≥5). Asterisks indicate statistically significant difference against control (N-T) according to Student’s t test (*, p ≤ 0.005; **, p ≤ 0.0005; ***, p ≤0.0001). Figure 8. Biological treatment of algal biomass by enzymatic blends from algal saprophytes. Schematic representation of the workflow for the design of algalytic formulations using the algal trap approach. Figure 9. Morphological characteristics of the unknown fungal isolate. a) Conidial head of the fungus as grown on solid MEP medium by stereo microscope analysis (8X magnification). b) Conidiophore and conidia of the fungus as grown on solid MEP medium by optical microscope
analysis (100X magnification). Black arrows point to three different metulae. Scale bars are also indicated. Figure 10. Extraction of genomic DNA from the unknown fungal isolate. a) Agarose gel electrophoresis of fungal gDNA. Molecular weight marker (1kb+) is also shown. Figure 11. Growth of P. sumatraense AQ67100 in different algal-supplemented media. Fungal cultures as grown in A- and B-medium supplemented with 0.2 % (w/v) heat-treated C. vulgaris biomass at a) 2, b) 4 and c) 7 days from algal administration. Figure 12. Time-course analysis of GH activities from P. sumatraense AQ67100 cultures as grown in algal-supplemented media. GH activities were evaluated in the filtrates from algal- supplemented cultures upon different days of growth. B-medium was supplemented with 0.2% (w/v) heat-treated C. vulgaris biomass upon two days of growth (grey arrow). Units are expressed as µmol reducing ends (for glucanase activity) and µmol pnitrophenol (for β-glucosidase and β- galactosidase activities) released per minute. Xylanolytic and xyloglucanolytic activities were not significantly detected. Experiments were repeated twice with consistent results. Figure 13. Analysis of GH activities from P. sumatraense AQ67100 cultures upon growth in different cell wall polysaccharides-supplemented media. a) Analysis of GH activities in filtrates from different cell wall polysaccharides-supplemented cultures upon 10 days of growth. B- medium was supplemented with 0.5% (w/v) arabinoxylan (AX), 0.5% (w/v) xyloglucan (XG), 0.2% (w/v) C.vulgaris AIS (C.v. AIS) and 0.2% (w/v) heat-treated C. vulgaris biomass (C.v.). Units are expressed as µmol reducing ends (for glucanase, xyloglucanase, xylanase activities) and µmol pnitrophenol (for β-glucosidase and β-galactosidase activities) released per minute. Experiments were repeated twice with consistent results. Figure 14. Evaluation of protease activity in C.v.-filtrate. BSA was incubated with C.v.-filtrate for 16 hours at 28°C (BSA + C.v.-filtrate) and the reaction analyzed by SDS-PAGE. An equivalent amount of 200 ng of BSA and 4 µL of C.v.-filtrate was analyzed [BSA, bovine serum albumin; NI, not incubated]. Fig. 15. Purification of recombinant g9376.t1 from P. pastoris. SDS-PAGE/Comassie blue staining analysis of g9376.t1 after elution from the IMAC chromatography. Two different protein amounts (µg) were evaluated. Molecular weight marker (MM) is also reported. Fig. 16. Enzymatic characterization of exo-1,3-β-glucanase g9376.t1. a) pH- and b) temperature-dependent activity of exo-1,3-β-glucanase g9376.t1 using laminarin (0.2% w/v) as
substrate. c) Residual activity of exo-1,3-β-glucanase g9376.t1 at pH 5.0 towards (0.2% w/v) laminarin after 1 hour-incubation at different temperatures. d) Specific activity expressed as Units (µmol reducing end/D-Glucose min-1) per mg of exo-1,3-β-glucanase g9376.t1 at pH 5.0 towards 1,3-β-D-LAM5P. [1,3-β-D-LAM5P, 1,3-β-D-Laminaripentaitol borohydride]. Fig. 17. Analysis of degradation products released from different 1,3-β-glucan oligomers upon incubation with exo-1,3-β-glucanase g9376.t1. Chromatographic analysis of equal amounts of five different 1,3-β-glucan oligomers (upper panel) alone and (lower panel) upon 1-h incubation with exo-1,3-β-glucanase g9376.t1. In the upper panel, glucose is also analysed. [GLC, D-Glucose; LAM2, Laminaribiose; LAM3, Laminaritriose; LAM4, Laminaritetraose; LAM5, Laminaripentaose; LAM6, Laminarihexaose]. Fig. 18. Analysis of degradation products released from two different 1,3-β-glucan pentamers upon incubation with exo-1,3-β-glucanase g9376.t1. Chromatographic analysis of (upper panel) equal amounts of five different 1,3-β-glucan oligomers alone and of (lower panel) enzymatic products obtained from 1,3-β-glucan pentamer (LAM5) and 1,3-β-D-Laminaripentaitol borohydride (1,3-β-D-LAM5P) upon 3-h incubation with exo-1,3-β-glucanase g9376.t1. * indicates an intermediate-product detected during the reaction. In the upper panel, glucose was also analysed. [GLC, D-Glucose; LAM2, Laminaribiose; LAM3, Laminaritriose; LAM4, Laminaritetraose; LAM5, Laminaripentaose; 1,3-β-D-LAM5P, 1,3-β-D-Laminaripentaitol borohydride]. Fig.19. Growth of P. sumatraense AQ67100 in Ulva lactuca-supplemented medium. (a-c, Left) 10-day old cultures of P. sumatraense grown in B-medium supplemented with a) 0.2% (w/v) Ulva lactuca biomass and P. sumatraense AQ67100 inoculum, b) P. sumatraense AQ67100 inoculum and c) 0.2% (w/v) Ulva lactuca biomass. a,c) Middle-right, Optical stereo microscope images of the same cultures shown in (a,c, Left) (12,5X and 50X magnification, respectively). Scale bars are also indicated [P.s., P. sumatraense AQ67100; U.l., Ulva lactuca biomass]. Fig.20. Growth of P. sumatraense AQ67100 in an alginate-supplemented medium. a) 10-days old cultures of P. sumatraense grown in B-medium supplemented with only 0.2 % (w/v) alginate (sample A), with only fungal inoculum (sample B) and with 0.2% (w/v) alginate and fungal inoculum (sample C). +/- indicates presence/absence of the substrate (Alginate) or fungus (P.s.). [P.s., P. sumatraense AQ67100]. b) Alginase activity detected in the culture filtrates from the corresponding cultures shown in a). Units are expressed as µmol reducing ends released per minute.
Table 1- QUAST results. Genome assembly statistics calculated using QUAST tool. All statistics are based on contigs of size >= 500 bp, unless otherwise noted [e.g., "# contigs (>= 0 bp)" and "#Total length (>= 0 bp)" include all contigs].
Table 2. BUSCO results. The completeness of genomic, transcriptomic or annotated gene datasets in terms of the expected gene content based on evolutionary principles was quantified by BUSCO (Benchmarking Universal Single-Copy Orthologs) tool. Table displays the completeness of genome assembly and predicted proteins by BRAKER2 on the basis of two datasets (fungi_odb10 and eurotiales_odb10) [No Sp: Number of Species contained in the reference database].
Table 3- Eval statistics. General statistics on the gene prediction obtained by BRAKER2 using Eval software.
Table 4. Proteins identified in AX-, C.v.- and C.v. AIS-filtrates by LC-MS/MS analysis. The sequence coverage (percentage of the protein sequence covered by identified peptides), unique peptides (number of peptide sequences that are unique to a protein), the protein ID, the expected molecular weight and the predicted protein function are reported for each identified protein. Proteins are listed in decreasing order of sequence coverage. Only proteins identified with the highest score (323.31) are listed. Identification was performed by using the annotated genome of P. sumatraense AQ67100 as reference database. Proteins specifically identified in AX-filtrate are highlighted in bold. All the remaining proteins were identified in AX- and at least in 1 out of 2 filtrates from algal-supplemented cultures. See Figure 6 for further details on identified proteins.
Phylogenetic markers used for the classification of Penicillium sumatraense AQ67100. The genes encoding the RNA polymerase II second largest subunit (RPB2, gene ID: g10996), β- tubulin (BenA, gene ID: g5792) and calmodulin (CaM, gene ID: g2696), together with the Internal Transcribed Spacer (ITS) region were used as genetic markers for the phylogenetic analysis shown in Figure 2. Introns are reported in lowercase. RPB2 ATGGCCGACTATGACGAAGCTTATGAGGATGAGTTTTACGACGACGCGGATGAGGG TATCACCTCTGAGGACTGCTGGTCCGTGATCTCTTCCTTCTTCGATACCAAGGGTCTT GTGTCACAGCAACTCGACTCTTTCGATGAATTCATCTCTTCGACGATGCAGGAACTT GTGGAGGAACAAGGACAAGTGGTTCTCGACCAGACACTTCCGCCCGCCGAAGACGA AATCGACCCGGTCGTCGTTCGCCGTTATGAGCTCAAATTCGGAACCATCATGCTTTC GCGACCATCCGTGACGGAAGGCGATGGCGCTACGACAATCATGTTGCCGCAAGAAG CTCGCCTCCGAAACTTGACTTACGCCAGTCCGTTGTACCTGAACGTCTCAAAGAAGA TTATGGAGGGCCGAGAGCGCATGGTGGGTGATGGCGATGAGGATGAGGGCGAGGC GGATGAAGACCGCAAGAACCGTGGAACATACCTGCAATGGGAGACAAAGGAACTA CCAGAGCAGCAAAGGAAAGAGGATACAGTTTTCATTGGAAAGATGCCCATCATGCT GAAATCCAAGTACTGTATCTTGAAGGACTTGAACGAGCAGGCTCTTTACGCTTGGAA CGAGTGTCCCTACGATTCCGGTGGCTACTTTATCATCAACGGAAGTGAAAAGGTTCT GATCGCACAGGAGCGCAGTGCTGGTAACATTGTGCAAGTCTTCAAGAAAGCGCCCC CGAGTCCCACACCCTACGTTGCCGAAATCCGAAGTGCCGTCGAGAAGGGATCCCGA CTCCTGTCGCAACTTGCCCTCAAGCTATTCGCCAAGGGTGACAGTGCCAAGGGCGGA TTCGGTCCGACTATTCGCTCCACCCTTCCCTACGTCAAGGCCGATATTCCCATTGTCG TTGTTTTCCGAGCCCTCGGCGTTGTCTCCGATGAAGATATCCTTAACCACATTTGCTA CGACCGCAATGATACCCCTATGCTAGAGATGCTCAAGCCGTGTATTGAGGAAGGAT TCGTCATTCAAGACCGAGAAGTCGCTCTGGATTTCATCGCCAAGCGTGGCTCTTCCC AGTCCAACCTCAACCACGAGCGTCGTGTGCGCTACGCCCGGGAGATTATGCAGAAG GAACTGCTGCCTCACATCTCGCAGAGCGAGGGCAGTGAAACCCGTAAGGCTTTCTTC TTGGGTTACATGGTTCACCGTCTTCTCCAGTGTGCCCTTGGCCGCCGTGACGTCGAC
GACCGTGATCACTTCGGAAAGAAGCGTCTTGATCTCGGTGGTCCTCTTCTTGCCAAC CTTTTCCGTGTCCTTTTCACTCGTGTCACTCGCGATCTGCAGCGTTATGTCCAACGGT GTGTTGAGACCAACCGTGAGATCTACTTGAACATTGGTCTCAAGGCCGCTACCTTGA CCGGTGGTTTGAAGTATGCCCTTGCTACAGGTAACTGGGGTGAACAGAAGAAGGCA GCCAGCGCTAAGGCCGGTGTGTCCCAAGTGCTGAGTCGCTACACATTCGCTTCTTCG CTGTCGCATTTGCGCCGTACCAACACGCCTATTGGTCGTGATGGTAAGATTGCCAAA CCGCGTCAACTTCATAATACCCACTGGGGTCTGGTGTGTCCCGCAGAAACACCCGAA GGACAGGCTTGTGGTCTTGTCAAGAACTTGGCACTTATGTGCTATATCACTGTTGGT ACTCCTAGTGAGCCCATCATCGATTTCATGATTCAGCGAAATATGGAAGTCTTGGAA GAGTTTGAACCTCAAGTCACGCCGAATGCCACAAAGGTCTTCGTTAATGGTGTCTGG GTTGGTATCCACCGTGATCCTTCTCACCTGGTTAACACTATGCAGTCCCTGCGTCGAC GCAACATGATTTCTCACGAAGTCAGTTTGATTCGGGATATTCGTGAGCGAGAGTTCA AGATTTTCACCGATACCGGCCGTGTCTGCCGCCCCTTGTTCGTTGTCGACAATGATCC CAAGAGCGAAAATGCGGGATCTTTGATTCTGAATAAGGAGCACATTCACAAGCTGG AACAGGACAAGGACCTGCCTCTAGATATGGATGTTGAAGAGCGACGAGAGCGCTAC TTCGGATGGGATGGTCTGGTTCGATCAGGAGCTGTTGAGTATGTCGATGCGGAAGA AGAAGAGACTATAATGATTGTGATGACGCCTGAAGATTTGGAGATTTCCAAGCAGC TCCAGGCCGGCTACGCATTGCCCGAGGAAGAGTCCAGTGACCCGAACAAGCGAGTC CGCTCGATTCTCAGCCAGCGGGCGCACACCTGGACACACTGTGAGATTCATCCCAGT ATGATTCTCGGTGTTTGTGCCAGTATTATTCCTTTCCCGGATCACAACCAGTCTCCTC GTAACACTTACCAGTCTGCCATGGGTAAGCAGGCCATGGGTGTTTTCCTGACAAACT TTGCCCAGCGCATGGAGACCATGGCCAATATTCTCTACTACCCCCAGAAGCCTCTGG CCACAACTCGATCAATGGAGTTCTTGCGCTTCCGCGAGCTTCCTGCTGGACAGAACG CCATTGTCGCCATTGCCACTTACTCCGGTTACAACCAAGAAGATTCCGTTATTATGA ACCAGAGCAGTATCGATCGTGGACTGTTCCGCAGTTTGTTCTACCGTACATACACCG ATTCCGAGAAGATGGTTGGTTTGACAGTTGTCGAGCGATTCGAGAAGCCCATGCGCT CCGACACAATTGGTATGCGCAAGGGTACCTACGACAAGTTGGACGAGGATGGTATT ATCGCCCCTGGTGTTCGTGTTTCCGGAGAAGATATCATCATCGGAAAGACCGCACCT TTGGCAGCCGATGCAGAGGAGCTTGGTCAACGTACCAAGGCACACACCAAGATCGA TGTGTCGACGCCACTGCGAAGTACCGAGAACGGTATTGTGGATCAGGTCTTGATCTC TACTGGCAATGACGATCTCAAATTCGTCAAGGTCCGTATGCGTACCACAAAGGTTCC CCAGATTGGTGACAAGTTCGCGTCTCGTCACGGTCAAAAGGGTACCATTGGTATCAC CTACCGACAGGAGGATATGCCTTTCACTCGCGAGGGTGTTGTCCCCGATCTGATTAT
CAACCCACACGCCATTCCCTCTCGTATGACTATTGCTCACTTGATCGAGTGTCAATTG AGTAAGGTCTCAGCTCTCCGTGGTTTCGAAGGTGATGCCACTCCATTCACCGATGTC ACTGTCGACTCCATCTCGCGTCTACTGCGCGAGCACGGTTACCAATCTCGTGGTTTC GAAGTTATGTTCAACGGCCATACCGGTCGCAAGCTTGTTGCACAGGTCTTCCTGGGT CCAACCTACTACCAGCGTCTTCGTCACATGGTAGACGACAAGATTCACGCTCGTGCT CGTGGCCCTACCCAAATTCTCACCCGTCAACCCGTCGAAGGTCGTGCACGTGATGGT GGTCTGCGTTTCGGAGAGATGGAACGCGATTGCATGATTGCCCATGGTGCCAGTGCC TTCTTGAAGGAGCGTCTCTTCGATGTTTCCGATCCTTTCCGCGTTCACATTTGCGACG ACTGCGGTCTGATGACCCCTATTGCgtaagttctacatccctatctcagctgcatctctgcatctcttcttggtataccga catcttactaacacatcaacagAAAACTGAAGAAGGGTCTCTTCGAGTGCCGTCTCTGCAATAAC AAACACCGCATCTCACAGGTCCACATCCCCTACGCCGCCAAGCTTCTGTTCCAAGAG CTGGCCTCGATGAACATTGCCGCTCGGATGTTTACCGACCGGTCGGGTGTATCGGTG CGATAA (SEQ ID NO:1) BenA ATGCGTGAGATCgtacgtcctcttgtcccaagttcaacgcgtctttgttgacctacccctgaacggacccccacttgttcatcctgct aacctgagctttttttccccaacgtatagGTTCACCTTCAGACCGGCCAGTGTgtaagtgcacattcgaagaatccaat gccctcatgatcgggcaaaaagacacaactatatgagtatgatggttcgaatgaatgctaaatgatatgtggggacatacagGGTAAC CAAATTGGTGCCGCTTTCTGgtacgtgctgcaatcccaaaacaatcaattgttgaatgcatgaagcaataaactaatcaatt caacagGCAAACCATTGCTGGCGAGCACGGCCTTGATGGCGATGGACAgtgagtgatttcgacca ggttttgattttcgagaatggcggtctgatatttttgggcagCTACAACGGTACCTCTGACCTCCAGCTGGAGC GCATGAACGTTTACTTCACCCATgtaagcgacaacaatcccatcaatacaattagtcttgattctaacggcttgtttttctg tttacaatagGCTTCCGGTGACAAGTATGTTCCCCGTGCCGTTCTGGTTGATCTGGAGCCCG GTACCATGGACGCTGTCCGTGCCGGTCCCTTCGGCAAGCTCTTCCGCCCCGACAACT TCGTCTTCGGTCAGTCTGGTGCTGGTAACAACTGGGCCAAGGGTCACTACACTGAGG GTGCCGAGCTCGTTGACCAGGTCGTCGATGTCGTCCGCCGTGAGGCCGAGGCTTGCG ACTGCCTCCAGGGTTTCCAGATCACCCACTCCCTGGGTGGTGGTACCGGTGCCGGTA TGGGTACACTCCTGATCTCCAAGATCCGTGAGGAGTTCCCCGACCGTATGATGGCCA CCTTCTCCGTTGTTCCCTCCCCCAAGGTCTCGGATACCGTTGTCGAGCCTTACAACGC TACCCTGTCCGTTCACCAGCTGGTTGAGCACTCCGACGAGACTTTCTGTATCGATAA CGAGgtatggatagtggcctatgaatgccctacgaaaacagccaattgactaatttcatgatagGCTCTGTACGACATCT GCATGCGCACCCTTAAGCTGTCTCAGCCCTCCTACGGTGACCTGAACCACCTGGTCT CTGCCGTCATGTCCGGTGTCACCACCTCGCTCCGTTTCCCCGGTCAGCTCAACTCCGA TCTCCGCAAGCTGGCTGTCAACATGGTTCCTTTCCCTCGTCTCCACTTCTTCATGGTT
GGATTCGCTCCCCTGACCAGCCGTAACGCCAATGCCTACCGCCAGGTCAGCGTTCCC GAGCTGACCCAGCAGATGTTCGACCCCAAGAACATGATGGCTGCTTCTGACTTCCGT AACGGCCGTTACCTCACCTGCTCCGCTCTGTTCCGCGGTAAGGTCTCCATGAAGGAG GTCGAGGACCAGATGCGCAGCATCCAGACCAAGAACCAGAGCTACTTCGTCGAGTG GATTCCCAACAATGTCCAGACCGCCCTGTGCTCCGTTCCTCCCCGCGGCCTGCGCAT GTCCTCCACCTTCGTCGGTAACAGCACCTCTATCCAGGAGCTGTTCAAGCGTATCGG TGACCAGTTCACTGCCATGTTCCGCCGCAAGGCTTTCTTGCACTGGTACACTGGTGA GGGTATGGACGAGATGGAGTTCACTGAGGCTGAGAGCAACATGAACGACCTGGTCT CTGAATACCAGCAATACCAGGATGCCTCCATCTCCGAGGGTGAGGAGGAGTACCTC GCTGAGGAGGCTGCTCTCGAGGATGAGGTCTAA (SEQ ID NO:2) CaM ATGgtatgtctcttacttgacacctctttggcctcactactaccccctgttatgtatctcggggtgcaggacctcccatcccgagacacttcgc actgcggcataccccattgttgtcccgaatgttgttgactaactgcgccatcactgctaaatagGCCGATTCTCTGACTGAA GAGCAAGTTTCCGAGTACAAGGAGGCGTTCTCCCTCTTTgtgagtaattcgatccaggaattgaaattgt gaacgtgtgatcgattttaggctgacggggggttatcttgtgatcgacagGACAAGGATGGTGATGgtgagttctgtcgtttgt gaacgacccgtgtcctttctacgggcggtgtttctcttgcgattcgtttccatcaaaattcactcgaaatatactgattgatcgatgataaatagG ACAAATCACCACCAAGGAGCTCGGCACTGTCATGCGCTCCCTCGGCCAGAACCCCTC CGAGTCCGAGCTACAGGACATGATCAATGAGGTCGATGCCGATAACAATGGCACCA TTGATTTCCCTGgtacgaatcccagagtccatattcctatgactccctctcttcacctacattatgccccatcgtcgcatctcgatacg aaagaaataaatattgacatgcgcccacagAGTTCCTGACCATGATGGCTCGTAAGATGAAGGATACC GACTCCGAGGAGGAGATCCGCGAGGCTTTCAAGGTGTTTGATCGCGATAACAACGG ATTCATCTCTGCCGCTGAGCTGCGCCACGTCATGACCTCCATCGGCGAGAAGCTGAC CGACGATGAGGTCGATGAGATGATCCGTGAGGCCGATCAGGACGGCGACGGCCGTA TCGACTgtacatatcccccgctcccgcatcctggaatttctcgagaggggacatgctaacaattctcttttttagACAACGAGTT CGTCCAGCTCATGATGCAAAAATAA (SEQ ID NO:3) ITS CTGAGTGAGGGCCCCTCGGGGTCCAACCTCCCACCCGTGTTTAACGAACCTTTGTTG CTTCGGCGGGCCCGCCTCACGGCCGCCGGGGGGCTCCTGCCCCCGGGCCCGCGCCC GCCGAAGCCCCCCCTTGAACGCTGTCTGAAGTTTGCAGTCTGAGAAACTAGCTAAAT TAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCG AAATGCGATAACTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGC ACATTGCGCCCTCTGGTATTCCGGAGGGCATGCCTGTCCGAGCGTCATTGCTGCCCT
CAAGCACGGCTTGTGTGTTGGGCCCCCGTCCCCCCCTCTGCCGGGGGGACGGGCCCG AAAGGCAGCGGCGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTCGTCACCCGC TCTTGTAGGCCCGGCCGGCGCCAGCCGACCCCAACCCTAAATTTTTTTCAG (SEQ ID NO:4) The protein encoded by the above RNA polymerase II second largest subunit (RPB2, protein ID: g10996.t1) has the following sequence: MADYDEAYEDEFYDDADEGITSEDCWSVISSFFDTKGLVSQQLDSFDEFISSTMQELVEE QGQVVLDQTLPPAEDEIDPVVVRRYELKFGTIMLSRPSVTEGDGATTIMLPQEARLRNLT YASPLYLNVSKKIMEGRERMVGDGDEDEGEADEDRKNRGTYLQWETKELPEQQRKED TVFIGKMPIMLKSKYCILKDLNEQALYAWNECPYDSGGYFIINGSEKVLIAQERSAGNIV QVFKKAPPSPTPYVAEIRSAVEKGSRLLSQLALKLFAKGDSAKGGFGPTIRSTLPYVKAD IPIVVVFRALGVVSDEDILNHICYDRNDTPMLEMLKPCIEEGFVIQDREVALDFIAKRGSS QSNLNHERRVRYAREIMQKELLPHISQSEGSETRKAFFLGYMVHRLLQCALGRRDVDD RDHFGKKRLDLGGPLLANLFRVLFTRVTRDLQRYVQRCVETNREIYLNIGLKAATLTGG LKYALATGNWGEQKKAASAKAGVSQVLSRYTFASSLSHLRRTNTPIGRDGKIAKPRQL HNTHWGLVCPAETPEGQACGLVKNLALMCYITVGTPSEPIIDFMIQRNMEVLEEFEPQV TPNATKVFVNGVWVGIHRDPSHLVNTMQSLRRRNMISHEVSLIRDIREREFKIFTDTGRV CRPLFVVDNDPKSENAGSLILNKEHIHKLEQDKDLPLDMDVEERRERYFGWDGLVRSG AVEYVDAEEEETIMIVMTPEDLEISKQLQAGYALPEEESSDPNKRVRSILSQRAHTWTHC EIHPSMILGVCASIIPFPDHNQSPRNTYQSAMGKQAMGVFLTNFAQRMETMANILYYPQ KPLATTRSMEFLRFRELPAGQNAIVAIATYSGYNQEDSVIMNQSSIDRGLFRSLFYRTYT DSEKMVGLTVVERFEKPMRSDTIGMRKGTYDKLDEDGIIAPGVRVSGEDIIIGKTAPLAA DAEELGQRTKAHTKIDVSTPLRSTENGIVDQVLISTGNDDLKFVKVRMRTTKVPQIGDK FASRHGQKGTIGITYRQEDMPFTREGVVPDLIINPHAIPSRMTIAHLIECQLSKVSALRGFE GDATPFTDVTVDSISRLLREHGYQSRGFEVMFNGHTGRKLVAQVFLGPTYYQRLRHMV DDKIHARARGPTQILTRQPVEGRARDGGLRFGEMERDCMIAHGASAFLKERLFDVSDPF
RVHICDDCGLMTPIAKLKKGLFECRLCNNKHRISQVHIPYAAKLLFQELASMNIAARMF TDRSGVSVR* (SEQ ID NO: 105) The protein encoded by the above β-tubulin (BenA, protein ID: g5792.t1) has the following sequence: MREIVHLQTGQCGNQIGAAFWQTIAGEHGLDGDGHYNGTSDLQLERMNVYFTHASGD KYVPRAVLVDLEPGTMDAVRAGPFGKLFRPDNFVFGQSGAGNNWAKGHYTEGAELVD QVVDVVRREAEACDCLQGFQITHSLGGGTGAGMGTLLISKIREEFPDRMMATFSVVPSP KVSDTVVEPYNATLSVHQLVEHSDETFCIDNEALYDICMRTLKLSQPSYGDLNHLVSAV MSGVTTSLRFPGQLNSDLRKLAVNMVPFPRLHFFMVGFAPLTSRNANAYRQVSVPELT QQMFDPKNMMAASDFRNGRYLTCSALFRGKVSMKEVEDQMRSIQTKNQSYFVEWIPN NVQTALCSVPPRGLRMSSTFVGNSTSIQELFKRIGDQFTAMFRRKAFLHWYTGEGMDE MEFTEAESNMNDLVSEYQQYQDASISEGEEEYLAEEAALEDEV* (SEQ ID NO: 106) The protein encoded by the above calmodulin (CaM, protein ID: g2696.t1) has the following sequence: MADSLTEEQVSEYKEAFSLFDKDGDGQITTKELGTVMRSLGQNPSESELQDMINEVDAD NNGTIDFPEFLTMMARKMKDTDSEEEIREAFKVFDRDNNGFISAAELRHVMTSIGEKLT DDEVDEMIREADQDGDGRIDYNEFVQLMMQK* (SEQ ID NO: 107) Table 5. Proteins identified in P. sumatraense AQ67100 culture filtrate by LC-MS/MS analysis. The gene sequence, the gene ID and the predicted protein function are reported for each identified protein. Introns are in lowercase. Proteins are listed in decreasing order of sequence coverage according to Table 4. Identification was performed by using the annotated genome of P. sumatraense AQ67100 as reference database. See Figure 6 and Table 4 for further details on identified proteins.
Table 6. Proteins identified in P. sumatraense AQ67100 culture filtrate by LC-MS/MS analysis. The amino acid sequence, the protein ID and the predicted protein function are reported for each identified protein. Proteins are listed in decreasing order of sequence coverage according to Table 4. Identification was performed by using the annotated genome of P. sumatraense AQ67100 as reference database. See Figure 6 and Table 4 for further details on identified proteins.
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 NSAISDGGRYGPSSRQSSTTTPAIVYFPAGTYLISTPIIDYYFTQLIGNPNSMPVIKATAGFS GLGLIDGDQYQSDGNQGWTSTNVFFRQIRNLKLDLTNIPASSAATGIHWPTGQATSIQN VDIVMSSASGTQHQGIFIENGSGGFLADITITGGLYGANVGNQQFTMRNLVITDAVTAIS QIWDWGWTYQGLTVTNCSTALSVDNGGAGNQLVGSVIVLDSTIQDCSTFVTSAWQAST FSNGSLILENISLENVPVAVKGPSGTVLVGGTMTISAWGQGHKYTPNGPTNFQGTFTAPT RPSSLLASGSSRYYTKSKPQYETLSQSSFVSTRSAGATGDGSTDDTSAIQSALNSAASSGK IVFFDQGTYKVTDTIYVPPGSRITGEAYPVIMASGSAFSSISKPVPVVQVGKSGESGSVEW SDMIVSTQGSTPGAVLIEWNLAANSGSGMWDVHTRIGGFSGSQQQVAQCPTSAAVSAA CEVAYMSMHITDSASGVYLDNVWLWTADHDLDSADNTRISVYSGRGLLIEGQTIWLYG TGVEHHSLYQYQFSGASSVVAGFIQTETPYYQPNPDAANGPYPSNPDLKDPDYSSCLSG NCDSLGLRVLDSSDIVIYGAGLYSFFNNYSTDCSTFPVPENCQSEIFSIEGDTSNLVVYAL STVGTTNMIVKDGTSLAVVSDNLATYAATIAYFTL (SEQ ID NO:80) QLLEIPAVDELVSSALQPLEAWTDYQGPTGIASSALSKSTHAIVANVAVEAADASYWLA DISHQGKAAFNPNPSSYKVFRNVKDYGAKGDGVTDDTAAINSAISDGGRYGPSSRQSST TTPAIVYFPAGTYLISTPIIDYYFTQLIGNPNSMPVIKATAGFSGLGLIDGDQYQSDGNQG WTSTNVFFRQIRNLKLDLTNIPASSAATGIHWPTGQATSIQNVDIVMSSASGTQHQGIFIE NGSGGFLADITITGGLYGANVGNQQFTMRNLVITDAVTAISQIWDWGWTYQGLTVTNC STALSVDNGGAGNQLVGSVIVLDSTIQDCSTFVTSAWQASTFSNGSLILENISLENVPVA VKGPSGTVLVGGTMTISAWGQGHKYTPNGPTNFQGTFTAPTRPSSLLASGSSRYYTKSK PQYETLSQSSFVSTRSAGATGDGSTDDTSAIQSALNSAASSGKIVFFDQGTYKVTDTIYVP PGSRITGEAYPVIMASGSAFSSISKPVPVVQVGKSGESGSVEWSDMIVSTQGSTPGAVLIE WNLAANSGSGMWDVHTRIGGFSGSQQQVAQCPTSAAVSAACEVAYMSMHITDSASGV YLDNVWLWTADHDLDSADNTRISVYSGRGLLIEGQTIWLYGTGVEHHSLYQYQFSGAS SVVAGFIQTETPYYQPNPDAANGPYPSNPDLKDPDYSSCLSGNCDSLGLRVLDSSDIVIY
GAGLYSFFNNYSTDCSTFPVPENCQSEIFSIEGDTSNLVVYALSTVGTTNMIVKDGTSLA VVSDNLATYAATIAYFTL (SEQ ID NO: 112) b) MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNN GLLFINTTIASIAAKEEGVSLEKREAEAAAQLLEIPAVDELVSSALQPLEAWTDYQGPTGI ASSALSKSTHAIVANVAVEAADASYWLADISHQGKAAFNPNPSSYKVFRNVKDYGAKG DGVTDDTAAINSAISDGGRYGPSSRQSSTTTPAIVYFPAGTYLISTPIIDYYFTQLIGNPNS MPVIKATAGFSGLGLIDGDQYQSDGNQGWTSTNVFFRQIRNLKLDLTNIPASSAATGIH WPTGQATSIQNVDIVMSSASGTQHQGIFIENGSGGFLADITITGGLYGANVGNQQFTMR NLVITDAVTAISQIWDWGWTYQGLTVTNCSTALSVDNGGAGNQLVGSVIVLDSTIQDCS TFVTSAWQASTFSNGSLILENISLENVPVAVKGPSGTVLVGGTMTISAWGQGHKYTPNG PTNFQGTFTAPTRPSSLLASGSSRYYTKSKPQYETLSQSSFVSTRSAGATGDGSTDDTSAI QSALNSAASSGKIVFFDQGTYKVTDTIYVPPGSRITGEAYPVIMASGSAFSSISKPVPVVQ VGKSGESGSVEWSDMIVSTQGSTPGAVLIEWNLAANSGSGMWDVHTRIGGFSGSQQQV AQCPTSAAVSAACEVAYMSMHITDSASGVYLDNVWLWTADHDLDSADNTRISVYSGR GLLIEGQTIWLYGTGVEHHSLYQYQFSGASSVVAGFIQTETPYYQPNPDAANGPYPSNP DLKDPDYSSCLSGNCDSLGLRVLDSSDIVIYGAGLYSFFNNYSTDCSTFPVPENCQSEIFSI 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 CGGAAAACGGTACAGATCATAATGTCGCGAATTGGGAAttaggtaagttgagaatggaccaactgtga tttttgtgtggtgacttatattgtgctgTTAGTCATGACCGAAGCAGCAATACTTATGGCAGTCTTTCTT 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)MVFLASVLYLVTATIIPIVTAGQSVSATAPTTWAHPGAMISENQLSFIRKKVQNKEQPW 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 × 108 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) MgSO4, 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) K2HPO4, 0.14% (w/v) (NH4)2SO4, 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: H2O] 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× 103 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,
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Claims
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.
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