WO2016015021A1 - Dégradation oxydative de l'amidon par une nouvelle famille de pmos - Google Patents

Dégradation oxydative de l'amidon par une nouvelle famille de pmos Download PDF

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WO2016015021A1
WO2016015021A1 PCT/US2015/042126 US2015042126W WO2016015021A1 WO 2016015021 A1 WO2016015021 A1 WO 2016015021A1 US 2015042126 W US2015042126 W US 2015042126W WO 2016015021 A1 WO2016015021 A1 WO 2016015021A1
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seq
amino acid
acid sequence
starch
polypeptide
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PCT/US2015/042126
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Michael A. Marletta
William T. Beeson
Van V. VU
Elise A. SPAN
Christopher M. Phillips
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The Regents Of The University Of California
Bp Corporation North America Inc.
The Scripps Research Institute
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Publication of WO2016015021A1 publication Critical patent/WO2016015021A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present disclosure relates generally to methods for degrading starch containing material, and more specifically to methods of degrading starch containing material by providing a polypeptide and incubating the polypeptide with the starch containing material in the presence of oxygen and a reductant.
  • Starch is a carbohydrate polymer that has been shown to be a good source for food products, biofuels and other applications. It is the major energy reserve in plants and the most important energy source in the human diet (Christiansen et ah, FEBS J, 2009). There are many products that rely on the use of starch, which include food for humans, cattle and other animals. Starch can also be used in the paper industry and to produce dextrin - a substance commonly used in making syrup. Furthermore starch can be fermented to produce biofuel alcohol. [0005] Developing a sustainable energy industry is of key importance to achieve energy security, large-scale substitution of petroleum-based fuels and reduce carbon footprint (Farrell et al, Science, 2006). Biofuels are under intensive investigation due to increasing concerns about energy security, sustainability, and global climate change (Lynd et al, Science, 1991).
  • Bioconversion has been regarded as an attractive alternative to chemical production of fossil fuels (Lynd et al, Nat Biotech, 2008; Hahn-Hagerdal et al, Biotechnol Biofuels, 2006). Enzymes and organisms that can degrade simple sugars have been known and used for many years.
  • Starch has a complex structure composed of two distinct glucose polymers:
  • amylose comprising essentially unbranched a-(l ⁇ 4)-linked glucose residues
  • amylopectin comprising a-(l ⁇ 6) linkages between adjoining straight glucan chains on an a-(l ⁇ 4) backbone
  • methods for degrading starch containing material including, but not limited to the steps of providing starch containing material, providing a polypeptide comprising SEQ ID NO: 1, wherein the polypeptide is bound to a copper cofactor; and incubating the starch containing material and polypeptide in the presence of oxygen and a reductant, thereby degrading the starch containing material, are provided.
  • the polypeptide used in the method includes an amino acid sequence of SEQ ID NO: 6.
  • the polypeptide includes the amino acid sequence of any of SEQ ID NOs: 8- 37 or SEQ ID No; 87.
  • the polypeptide described above further include a carbohydrate binding module 20 (CBM20).
  • the polypeptide used in the method includes the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of any of SEQ ID NOs: 38-86.
  • the polypeptide used in the method includes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 6, an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, an amino acid sequence encoded by a polynucleotide that hybridizes under medium stringency conditions with the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 or the complementary strand of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6, an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6 or the complementary strand of the polynucleotide encoding the amino acid sequence of SEQ ID NO: 6, an amino acid sequence having a substitution, deletion, and/or insertion of up to 40 amino acids of the amino acid sequence of SEQ ID NO: 6, an amino acid sequence having
  • the reductant used in the method may be an iron-containing compound.
  • This iron-containing compound may comprise a heme domain and in some embodiments is cellobiose dehydrogenase (CDH).
  • CDH cellobiose dehydrogenase
  • the iron- containing compound is ferrous sulfate.
  • the reductant is an arene or ascorbic acid.
  • the method may include additional steps, such as the further step of providing copper, providing amylase, or providing oxygen.
  • the incubating takes places at a temperature ranging from 25 °C to 70°C.
  • the methods described herein relate to producing a fermentation product, including the steps of degrading starch-containing material according to the methods described above to form glucose; and culturing the glucose with one or more fermentative microorganisms or a chemical solution under conditions sufficient to produce a fermentation product.
  • compositions including a polypeptide including the amino acid sequence of SEQ ID NO: 6 and copper; and a reductant are provided.
  • the reducatnt is an iron-containing compound.
  • the iron-containing compound may include a heme domain and may be cellobiose dehydrogenase (CDH).
  • CDH cellobiose dehydrogenase
  • the iron-containing compound is ferrous sulfate.
  • the reductant is an arene.
  • the reductant is ascorbic acid.
  • the patent or application file contains at least one drawing executed in color.
  • FIG. 1A is representative of overall and active site structures of fungal PMOs
  • PMOs PDB ID 2YET
  • histidine brace coordinate the copper center.
  • the N-terminal histidine ligand binds in a bidentate mode, and its imidazole ring is methylated at the ⁇ position in fungal PMOs.
  • FIG. IB illustrates the structure of cellulose as previously described in Nishiyama et ah, JAmChem Soc. 2002 and Nishiyama et ah, J Am Chem Soc, 2003. As illustrated,
  • Cellulose and chitin contain long linear chains of ⁇ (1 ⁇ 4) linked glucose units and N- acetylglucosamine units, respectively.
  • FIG. 1C illustrates the model structure of amylopectin (Perez and Bertoft, Starke,
  • FIG. 2A and FIG. 2B illustrate common domain architecture of 43 predicted starch-active PMOs from different fungal species (SEQ ID NOs: 1-2). Thirty one have the CBM20 domain.
  • FIG. 3 illustrates data collected from the activity assays of NCU08746.
  • the data show the high performance anion exchange chromatographic (HPAEC) traces of NCU08746 assays under various conditions.
  • Assays contained 5 ⁇ NCU08746 with 2 mM ascorbic acid and atmospheric oxygen.
  • Traces A-B Maltodextrins (1 -7 units) and soluble portion of amylose (average molecular weight ⁇ 2.8 kDa), respectively, oxidized with Lugol's solution.
  • Traces C-E Assays with 50 mg/niL amylopectin, 5 mg/ml PASC, and 50 mg/niL chitin, respectively. The assays were carried out in 50 mM sodium acetate buffer at pH 5.0 and 42 ° C.
  • FIG. 4A illustrates the effect of NCU08746 (5 ⁇ ) on the rate of oxidation of
  • MiCDH-2 (1 ⁇ ) incubated with 6 ⁇ cellobiose at room temperature. The data indicates that this oxidation occurs slowly in the presence of atmospheric oxygen but was significantly enhanced in the presence of NCU08746.
  • FIG. 4B illustrates NCU08746 (5 ⁇ ) activity assays on amylopectin (50 mg/niL) with 2 mM ascorbic acid (A), with 0.5 ⁇ MiCDH-2 and 5 mM cellobiose (B), and with 0.5 ⁇ MiCDH-2 only (C).
  • A ascorbic acid
  • B cellobiose
  • C 0.5 ⁇ MiCDH-2 only
  • FIG. 5 illustrates B- weighted EXAFS data of Cu(II)-NCU08746 and its Fourier transform.
  • the boxes highlight the features that arise from the outer-shell atoms of the imidazole ligands. Best fit parameters are provided in Table 5 (Fit 9).
  • the third shell features in the Fourier transform correspond to the double-humped feature centered at ⁇ 4 A-l in the EXAFS spectrum, which arises from the multiple scattering paths of several imidazole moieties as found in the spectra of many other metalloproteins and model complexes (Vu et ah, J Am Chem Soc, 2011, Pellei et ah, Dalton Trans, 2011, Sanyal et ah, J Am Chem Soc, 1993, Costello et ah, J Biol Inorg Chem, 2006, D'Angelo et ah, Biochemistry, 2005). Fitting progress is shown in Table 5.
  • the best fit includes 4 Cu-O/N paths at 1.97 A, 1 Cu-O/N path at 2.22 A, 1 Cu-O/N path at 2.42 A, 2 Cu-C paths at 3.23 A, and paths from 2.3 imidazole moieties (Table 5).
  • FIG. 6 illustrates a proposed mechanism of NCU08746, analogous to that of cellulose-active PMOs and chitin-active PMOs.
  • NCU08746 is shown here to oxidize the CI position in both amylose and amylopectin. Illustrated are the mechanisms of NCU08746 involving the cleavages of a(l ⁇ 4) (top) and a(l ⁇ 6) (bottom) linkages via hydroxylation at the CI position.
  • FIG. 7 illustrates SDS-PAGE analysis of fractions eluted from an amylose resin column (left) and subsequent size exclusion column (S75, GE Healthcare) (right). As illustrated, SDS-PAGE analysis of the fractions from the amylose resin column shows that NCU08746 has relatively strong affinity for amylose.
  • FIG. 8 illustrates fragment mass spectrum of the N-terminal peptide. This data is consistent with a methylated N-terminal histidine. The following were used: Monoisotopic mass of neutral peptide Mr(calc): 1203.6400; Fixed modifications: Carbamidomethyl (C) (apply to specified residues or termini only; Variable modifications: HI : Methyl-Histidine; Ions Score: 46 Expect: 0.00026; Matches : 28/80 fragment ions using 74 most intense peaks.
  • FIG. 9 illustrates activity assays of Cu-NCU08746 (5 ⁇ , Enz) with amylopectin
  • Traces A-B Maltodextrins (1-7 units) and soluble portion of amylose (average molecular weight ⁇ 2.8 kDa), respectively, oxidized with Lugol's solution.
  • Trace C AmPe+Enz+Asc+0 2 .
  • Trace D AmPe-Enz+Cu+Asc+0 2 .
  • Trace E AmPe+Enz-Asc+0 2 .
  • Trace F AmPe+Enz+Asc-0 2 .
  • G AmPe+Enz-Asc-0 2 .
  • Traces H-I PASC and chitin, respectively, +Enz+Asc+0 2 .
  • the assays were carried out in 50 mM sodium acetate buffer at pH 5.0 and 42°C. When both ascorbic acid and oxygen were present, the chromatogram exhibits a set of new peaks (trace C) that are not observed in the absence of either reductant or oxygen.
  • FIG. 10 illustrates NCU08746 activity assays with amylopectin from maize.
  • FIG. 11 illustrates NCU08746 activity assays with corn starch.
  • CoSt 50 mg/niL corn starch.
  • E 5 ⁇ Cu-NCU08746.
  • Asc 2 mM ascorbic acid.
  • Cu 5 ⁇ CuS0 4 .
  • the addition of one equivalent of CuS04 to the assays of holo NCU08746 had no effect on activity.
  • FIG. 12 illustrates A: The soluble portion of a 50 mg/niL suspension of amylose with average molecular weight of 2.8 kDa (AM2.8).
  • B AM2.8 oxidized with Lugol's solution.
  • C A mixture of maltodextrins with 1-7 glucose units.
  • D C oxidized with Lugol's solution.
  • E NCU08746 assay with AM2.8 (50 mg/niL suspension).
  • F NCU08746 assay with amylopectin (injected 20 times more than the assay with AM2.8). The product peaks of NCU08746 do not overlay with the peaks of maltodextrins.
  • FIG. 13 illustrates amylopectin activity assays of partially-apo NCU08746 in the presence of various metal ions. Among the metal ions tested, only Cu(II) increased the oxidative activity of this partially apo NCU08746.
  • FIG. 14 illustrates X-ray absorption near edge spectrum (XANES) of Cu(II)-
  • FIG. 15 illustrates FEFF model for EXAFS fitting.
  • Theoretical scattering paths were calculated with FEFF8.4 (Rehr et al , J Am Chem Soc, 1991) using this model.
  • the r and ⁇ 2 values of all significant single and multiple scattering paths of the imidazole moiety were linked together in the fit.
  • FIG. 16 illustrates the activity of truncated NCU08746 lacking the CBM20 domain (Truncl): Line A: 4 ⁇ full length NCU08746 Line B: 4 ⁇ Truncl and 10 ⁇ CuS04. Line C: 4 ⁇ Truncl only; Line D: lOuM CuS0 4 only.
  • FIG. 18 illustrates the X-band EPR spectra of wild type NCU08746 and two truncation mutants (Trunc 1 and Trunc 2).
  • FIG. 19 illustrates the activity of full-length NCU08746 enzyme on amylose
  • amylose trace is a 1/10 dilution.
  • the present disclosure relates to methods for degrading starch containing material and more specifically, to methods for degrading such material using a new family of
  • PMOs polysaccharide monooxygenases
  • PMOs are secreted by a variety of fungal and bacterial species and have been found to degrade chitin and cellulose by oxidizing either the CI or C4 atom of the ⁇ (1 ⁇ 4) glycosidic bond.
  • fungal PMOs that oxidize cellulose
  • bacterial PMOs that are active either on chitin or cellulose
  • fungal PMOs that oxidize chitin.
  • Sequence homology between these three families is very low. Nevertheless, the available structures of PMOs from all three families reveal a conserved fold, including an antiparallel ⁇ -sandwich core and a highly conserved monocopper active site on a flat protein surface.
  • the N-terminal histidine ligand binds in a bidentate mode, and its imidazole ring is methylated at the ⁇ position in fungal PMOs.
  • PMOs act on substrates with structurally similar structures.
  • Cellulose and chitin contain long linear chains of ⁇ (1 ⁇ 4) linked glucose units and N- acetylglucosamine units, respectively.
  • the polymer chains form extensive hydrogen bonding networks, which result in insoluble and very stable crystalline structures.
  • PMOs are thought to bind to the substrate with their flat active site surface, which orients the copper center for selective oxidation at the CI or C4 position.
  • Some bacterial chitin-binding proteins are cellulose- active PMOs, further suggesting that the set of PMO substrates is restricted to ⁇ (1 ⁇ 4) linked carbohydrate polymers.
  • Applicants have discovered that the oxidative mechanism of glycosidic bond cleavage used by PMOs on chitin and cellulose is more widespread than initially expected, also acting on starch despite its structural differences from chitin and cellulose.
  • Starch is made up of amylose and amylopectin. Both amylose and amylopectin contain linear chains of a(l ⁇ 4) linked glucose, while the latter also contains a(l ⁇ 6) glycosidic linkages at branch points in the otherwise a(l ⁇ 4) linked polymer. Unlike cellulose and chitin, amylose and amylopectin do not form microcrystals; instead, they exist in disordered, single helical, and double helical forms. Starch can exist partially in nanocrystalline form, but lacks the flat molecular surfaces as those found in chitin and cellulose.
  • Described herein are methods for degrading starch-containing material including the steps of providing a starch-containing material and providing a polypeptide having starch- degrading activity and a copper cofactor. The method further comprises incubating the polypeptide with the starch containing material in the presence of oxygen and a reductant.
  • polypeptide is an amino acid sequence comprising a plurality of consecutive polymerized amino acid residues (i. e. , at least about 15 consecutive polymerized amino acid residues, optionally at least about 30 consecutive polymerized amino acid residues, at least about 50 consecutive polymerized amino acid residues).
  • protein refers to an amino acid sequence, oligopeptide, peptide, polypeptide, or portions thereof whether naturally occurring or synthetic.
  • Polypeptide refers to an amino acid sequence, oligopeptide, peptide, protein, or portions thereof, and the terms “polypeptide” and “protein” are used interchangeably.
  • the polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, and non-naturally occurring amino acid residues.
  • the polypeptide can be recombinant or purified.
  • a recombinant polypeptide or recombinant protein refers to polypeptides produced by recombinant DNA techniques. These techniques are known to one skilled in the art and include recombinant polypeptides obtained from cells transformed by an exogenous DNA construct encoding the desired polypeptide.
  • polypeptides of the disclosure may include conserved motifs specific to starch-active PMOs, such as SEQ ID NOs: 3-5, either individually or in combination, and a copper cofactor.
  • the polypeptide includes a conserved sequence of a starch-active PMO catalytic domain, SEQ ID NO: 1 or SEQ ID NO: 2.
  • the polypeptide comprises a fragment of the conserved sequence of a starch-active PMO catalytic domain.
  • the polypeptide includes the amino acid sequence of the
  • NCU08746 protein without the signal sequence (SEQ ID NO: 7).
  • the polypeptide includes the amino acid sequence of a truncated version of the NCU08746 protein lacking its CBM20 domain (SEQ ID NO: 6 or SEQ ID NO: 87).
  • the polypeptide includes the amino acid sequence of a truncated version of the NCU08746 hypothetical protein lacking its CBM20 domain (SEQ ID NO: 6) along with a carbohydrate binding domain (CBM) other than the one found in the native sequence.
  • CBM carbohydrate binding domain
  • Carbohydrate binding domains present in starch- active enzymes that function as attachment modules between the enzymes and the starch granules or other high molecular weight substrates and have also been suggested to distort the conformation and packing of the polymers, thereby facilitating their degradation.
  • the polypeptides described herein include CBM21, CBM48, and/or CBM53 domain(s).
  • polypeptide includes the amino acid sequence of a
  • polypeptides can display structural homology and sequence homology. Either or both can be used to predict common mechanisms of action between the polypeptides. Proteins are three dimensional structures for which structure and function are very tightly related.
  • Structure is much more evolutionarily conserved than sequence. Amino acids which are part of the same catalytic domain tend to be conserved, even when not sequential. Likewise, secondary structural elements in a polypeptide are highly conserved as are their arrangement in tertiary structural motifs. Structural alignment programs such as DALI can use the 3D structure of a polypeptide to find proteins with similar folds (Holm et ah, Protein Science, 1992). Sequence homology can be used to determine conserved sequential amino acids. Methods for the alignment of sequences and for the analysis of similarity and identity of polypeptide sequences are well-known in the art.
  • the polypeptide includes the amino acid sequence of a NCU08746 homolog provided in SEQ ID NOs: 8 - 86.
  • SEQ ID NOs 8 to 37 include a CBM20 domain.
  • SEQ ID NOs: 38-86 lack a CBM20 domain.
  • polypeptides described herein include the amino acid sequence of a homolog lacking a CBM20 domain coupled with a carbohydrate binding motif.
  • polypeptide sequence includes the amino acid sequence of the homologue of NCU08746 found in Myceliophthora thermophile, GL347014680 (SEQ ID NO: 34).
  • Starch has a complex structure composed of two distinct glucose polymers:
  • amylose comprising essentially unbranched a-(l ⁇ 4)-linked glucose residues
  • amylopectin comprising a-(H6) linkages between adjoining straight glucan chains on an a-(H4) backbone
  • a starch-active PMO catalytic domain has starch glycosidic activity.
  • the starch-active PMO catalytic domain cleaves the CI position in both amylase and amylopectin.
  • the polypeptide cleaves the C4 and/or C5 positions in amylase and amylopectin.
  • the polypeptide having a starch- active PMO catalytic domain or a fragment of a starch-active PMO catalytic domain can cleave only the a(l ⁇ 4) bond.
  • the polypeptide having a starch-active PMO catalytic domain can attack both a(l ⁇ 4) and a(l ⁇ 6) linkages.
  • a starch-active PMO catalytic domain might cleave glycosidic bonds in starch in other similar ways not described herein.
  • sequence identity refers to the percentage of residues that are identical in the same positions in the sequences being analyzed.
  • sequence similarity refers to the percentage of residues that have similar biophysical / biochemical characteristics in the same positions ⁇ i.e. charge, size, hydrophobicity) in the sequences being analyzed.
  • the polypeptide comprises and amino acid sequence having at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% sequence identity to the sequences described herein including SEQ ID NOs: 1-2 and 6-87.
  • the determination of percent sequence identity and/or similarity between any two polypeptide sequences can be accomplished using a mathematical algorithm.
  • mathematical algorithms are the algorithm of Myers and Miller, CABIOS, 1988; the local homology algorithm of Smith et al., Adv. Appl. Math, 1981; the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. ,1970; the search-for-similarity-method of Pearson and Lipman, Proc. Natl. Acad. Scz.,1988; the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA,1990 , modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. i/SA, 1993.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity and/or similarity.
  • Such implementations include, for example: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the AlignX program, versionl0.3.0 (Invitrogen, Carlsbad, CA) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters.
  • the CLUSTAL program is well described by Higgins et al. Gene, 1988; Higgins et al.
  • the present disclosure also relates to variations in the polypeptide occurring naturally or by exogenous actions. Variations may be a substitution, deletion or insertion of one or more amino acids resulting in a different sequence when compared with the polypeptide sequence claimed. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains. Amino acid substitutions can be the result of replacing one amino acid with another amino acid. Insertions result when a new amino acid is introduced to the sequence and deletion result when an amino acid is deleted from the native sequence.
  • amino acids can be divided into groups based on their chemical properties. For example, one in the art would recognize that hydrophobic amino acids ⁇ i.e. He, Leu, Met, Phe, Trp, Tyr, Val, Ala, Cys, or Pro) are functionally more similar to each other that they are to hydrophilic amino acids (i.e. Gly, Asn, Gin, Ser, Thr, Asp, Glu, Lys, Arg, or His).
  • hydrophilic amino acids i.e. Gly, Asn, Gin, Ser, Thr, Asp, Glu, Lys, Arg, or His.
  • a substitution is considered conservative when it minimally disrupts the biochemical properties of the polypeptide. Conservative substitution tables providing
  • the polypeptide comprises an amino acid sequence with substitutions, deletions and/or insertions of up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, up to 5 or up to 2 amino acids when compared to the sequences described herein including SEQ ID NOs: 1-2 and 6-87.
  • the polypeptides described herein include a copper cofactor.
  • the polypeptide is incubated with copper prior to being provided in methods of the disclosure.
  • methods of the disclosure include a step of providing copper.
  • the polypeptide of the present disclosure may be at least 100%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10% or at least 5% bound by copper.
  • the present disclosure further relates to polynucleotides that encode the polypeptides described herein.
  • Polynucleotides as used herein can refer to, among other things, genomic DNA, isolated DNA, cDNA, and any and all forms of RNA partaking in the coding and making of the polypeptide.
  • the terms "polynucleotide,” nucleic acid sequence,” “nucleic acid,” and variations thereof shall be generic to polydeoxyribonucleotides (containing 2-deoxy-D- ribose), to polyribonucleotides (containing D-ribose), to any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, and to other polymers containing non-nucleotidic backbones, provided that the polymers contain nucleobases in a configuration that allows for base pairing and base stacking, as found in DNA and RNA.
  • nucleic acid sequence modifications for example, substitution of one or more of the naturally occurring nucleotides with analog and inter-nucleotide modifications.
  • symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature.
  • Polynucleotides encoding the polypeptides of the present disclosure may be prepared by various suitable methods known in the art, including, for example, direct chemical synthesis or cloning.
  • direct chemical synthesis formation of a polymer of nucleic acids typically involves sequential addition of 3 '-blocked and 5 '-blocked nucleotide monomers to the terminal 5'-hydroxyl group of a growing nucleotide chain, wherein each addition is effected by nucleophilic attack of the terminal 5'-hydroxyl group of the growing chain on the 3 '-position of the added monomer, which is typically a phosphorus derivative, such as a phosphotriester, phosphoramidite, or the like.
  • the desired sequences may be isolated from natural sources by splitting DNA using appropriate restriction enzymes, separating the fragments using gel electrophoresis, and thereafter, recovering the desired polynucleotide sequence from the gel via techniques known to those of ordinary skill in the art, such as utilization of polymerase chain reactions (PCR; i.e., U.S. Pat. No. 4,683,195).
  • PCR polymerase chain reactions
  • Polynucleotides homologous to the polynucleotide encoding the polypeptides described herein can be identified by hybridization to each other under stringent or under highly stringent conditions. Single stranded polynucleotides hybridize when they associate based on a variety of well characterized physical-chemical forces, such as hydrogen bonding, solvent exclusion, base stacking and the like.
  • the stringency of a hybridization reflects the degree of sequence identity of the nucleic acids involved, such that the higher the stringency, the more similar are the two polynucleotide strands. Stringency is influenced by a variety of factors, including temperature, salt concentration and composition, organic and non-organic additives, solvents, etc.
  • polynucleotide sequences that are capable of hybridizing to the disclosed polynucleotide sequences and fragments thereof under various conditions of stringency (see, for example, Wahl and Berger, Methods Enzymol., 1987; and Kimmel, Methods Enzymo., 1987).
  • Full length cDNA, homologs, orthologs, and paralogs of polynucleotides of the present disclosure may be identified and isolated using well-known polynucleotide hybridization methods.
  • the polypeptide includes an amino acid sequence encoded by a polynucleotide that hybridizes under medium stringency conditions with the polynucleotide encoding the amino acid sequence of the polypeptides described herein or the complementary strand of the polynucleotide encoding the amino acid sequence of the polypeptides described herein.
  • the polypeptide includes an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with the polynucleotide encoding the amino acid sequence of the polypeptides described herein or the complementary strand of the polynucleotide encoding the amino acid sequence of the polypeptides described herein.
  • Hybridization experiments are generally conducted in a buffer of pH between 6.8 to 7.4, although the rate of hybridization is nearly independent of pH at ionic strengths likely to be used in the hybridization buffer (Anderson and Young, 1985, (supra)).
  • one or more of the following may be used to reduce non-specific hybridization: sonicated salmon sperm DNA or another non-complementary DNA, bovine serum albumin, sodium pyrophosphate, sodium dodecylsulfate (SDS), polyvinylpyrrolidone, ficoll and Denhardt's solution.
  • Dextran sulfate and polyethylene glycol 6000 act to exclude DNA from solution, thus raising the effective probe DNA concentration and the hybridization signal within a given unit of time.
  • conditions of even greater stringency may be desirable or required to reduce non-specific and/or background hybridization. These conditions may be created with the use of higher temperature, lower ionic strength and higher concentration of a denaturing agent such as formamide.
  • Stringency conditions can be adjusted to screen for moderately similar fragments such as homologous sequences from distantly related organisms, or to highly similar fragments such as genes that duplicate functional enzymes from closely related organisms.
  • the stringency can be adjusted either during the hybridization step or in the post-hybridization washes.
  • Salt concentration, formamide concentration, hybridization temperature and probe lengths are variables that can be used to alter stringency.
  • high stringency is typically performed at T m -5°C to T m -20°C, moderate stringency at T m -20°C to T m -35°C and low stringency at T m -35°C to T m -50° C for duplex >150 base pairs.
  • Hybridization may be performed at low to moderate stringency (25-50°C below T m ), followed by post-hybridization washes at increasing stringencies. Maximum rates of hybridization in solution are determined empirically to occur at T m -25°C for DNA-DNA duplex and T m -15°C for RNA-DNA duplex. Optionally, the degree of dissociation may be assessed after each wash step to determine the need for subsequent, higher stringency wash steps.
  • High stringency conditions may be used to select for nucleic acid sequences with high degrees of identity to the disclosed sequences.
  • An example of stringent hybridization conditions obtained in a filter-based method such as a Southern or northern blot for hybridization of complementary nucleic acids that have more than 100 complementary residues is about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • Hybridization and wash conditions that may be used to bind and remove polynucleotides with less than the desired homology to the nucleic acid sequences or their complements that encode the present transcription factors include, for example: 6X SSC and 1% SDS at 65°C; 50% formamide, 4X SSC at 42°C; 0.5X SSC to 2.0 X SSC, 0.1% SDS at 50°C to 65°C; or 0.1X SSC to 2X SSC, 0.1% SDS at 50°C - 65°C; with a first wash step of, for example, 10 minutes at about 42°C with about 20% (v/v) formamide in 0.1X SSC, and with, for example, a subsequent wash step with 0.2 X SSC and 0.1% SDS at 65°C for 10, 20 or 30 minutes.
  • wash steps may be performed at a lower temperature, i.e., 50° C.
  • An example of a low stringency wash step employs a solution and conditions of at least 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS over 30 min. Greater stringency may be obtained at 42°C in 15 mM NaCl, with 1.5 mM trisodium citrate, and 0.1% SDS over 30 min. Wash procedures will generally employ at least two final wash steps. Additional variations on these conditions will be readily apparent to those skilled in the art (see, for example, US Patent Application No. 20010010913).
  • wash steps of even greater stringency including conditions of 65°C -68°C in a solution of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS, or about 0.2X SSC, 0.1% SDS at 65° C and washing twice, each wash step of 10, 20 or 30 min in duration, or about 0.1 X SSC, 0.1% SDS at 65° C and washing twice for 10, 20 or 30 min.
  • Hybridization stringency may be increased further by using the same conditions as in the hybridization steps, with the wash temperature raised about 3°C to about 5°C, and stringency may be increased even further by using the same conditions except the wash temperature is raised about 6°C to about 9°C.
  • Polynucleotide probes may be prepared with any suitable label, including a fluorescent label, a colorimetric label, a radioactive label, or the like.
  • Labeled hybridization probes for detecting related polynucleotide sequences may be produced, for example, by oligo- labeling, nick translation, end- labeling, or PCR amplification using a labeled nucleotide.
  • Phylogenetic trees may be created for a gene family by using a program such as
  • Evolutionary distances may be computed using the Poisson correction method (Zuckerkandl and Pauling, pp. 97-166 in Evolving Genes and Proteins, edited by V. Bryson and H.J. Vogel.
  • evolutionary information may be used to predict gene function.
  • consensus sequences can not only be used to define the sequences within each clade, but define the functions of these genes; genes within a clade may contain paralogous sequences, or orthologous sequences that share the same function (see also, for example, Mount, Bioinformatics: Sequence and Genome Analysis Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., page 543, 2001).
  • Gapped BLAST in BLAST 2.0 can be utilized as described in Altschul et al., Nucleic Acids Res., 1997.
  • PSI- BLAST in BLAST 2.0
  • PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al., 1997 supra.
  • the default parameters of the respective programs i.e., BLASTN for nucleotide sequences, BLASTX for proteins
  • BLASTN for nucleotide sequences
  • BLASTX for proteins
  • sequence identity refers to the percentage of residues that are identical in the same positions in the sequences being analyzed.
  • sequence similarity refers to the percentage of residues that have similar biophysical / biochemical characteristics in the same positions (i.e., charge, size, hydrophobicity) in the sequences being analyzed.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity and/or similarity.
  • Such implementations include, for example: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the AlignX program, versionl0.3.0 (Invitrogen, Carlsbad, CA) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters.
  • the CLUSTAL program is well described by Higgins et al. Gene, 1988; Higgins et al.
  • the present disclosure relates to methods for degrading starch containing material, including the steps of providing a starch-containing material and providing a polypeptide having starch-degrading activity and a copper cofactor. The method further comprises incubating the polypeptide with the starch containing material in the presence of oxygen and a reductant.
  • Starch-containing materials include the endosperm of many plants, including the cereal grains.
  • starch containing materials may include, but are not limited to, whole grains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, rice, peas, beans, or sweet potatoes.
  • Starch-containing materials should also be understood to include starches with a high amylopectin content sometimes called waxy starches, as well as chemically and physically modified starches, such as for example starches whose acid values have been reduced, starches in which the type and concentration of cations associated with the phosphate groups have been modified, ethoxylated starches, starch acetates, cationic starches, oxidated starches and cross- linked starches.
  • the polypeptides described herein are incubated with starch-containing material in the presence of a reductant and oxygen under conditions suitable to degrade the starch-containing material.
  • the methods include a step of providing reductants and/or oxygen.
  • a reductant is an element or compound that donates (or loses) an electron to another chemical species in a reaction.
  • reductants can be successfully used.
  • any iron containing compound, arene, or any other functionally similar reductant can be used.
  • reductants can be used, both organic and inorganic. Examples include metals ⁇ i.e., iron, zinc, and magnesium), metal alloys and organic materials ⁇ i.e., hydrides).
  • the reductant used is an iron containing compound. More specifically, this iron containing compound can comprise a heme domain.
  • the iron containing compound can be cellobiose dehydrogenase.
  • the iron containing compound can be ferrous sulfate.
  • any iron containing compound, in which iron is able to transfer an electron can serve as an iron containing reductant.
  • metal reductants can also be used.
  • other metal reductants include lithium, sodium, magnesium, iron, zinc, and aluminum.
  • Other reductants include formaldehyde, formic acid, Hantzsch ester, hydrazine, isopropanol, lithium aluminum hydride, lithium
  • the reducant is either ascorbic acid or gallic acid.
  • the reductant can also be any arene.
  • Incubation may occur for any period of time sufficient to degrade the starch- containing material.
  • the incubating step may be 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, or 1 hour.
  • Incubation may occur for a period of time sufficient to achieve the desired amount of degradation of starch-containing material. For example, incubation may occur for a period of time sufficient to achieve 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% degradation of the starch-containing material.
  • Incubation may occur with any amount of starch-containing material.
  • the amount of starch-containing material may be 1 mg/niL, 5mg/mL, lOmg/mL, 15 mg/niL, 20 mg/niL, 25 mg/mL,30 mg/niL, 35 mg/niL, 40 mg/niL, 45 mg/niL, 50 mg/niL, 55 mg/niL, 60 mg/niL, 65 mg/niL, 70 mg/niL, 75 mg/niL, 80 mg/niL, 85 mg/niL, 90 mg/niL, 95 mg/niL, 100 mg/niL.
  • Incubation may occur with any amount of polypeptide.
  • the amount of polypeptide used may be 0.001 ⁇ , 0.005 ⁇ , 0.01 ⁇ , 0.05 ⁇ , 0.1 ⁇ , 0.5 ⁇ , 1 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , 10 ⁇ , 15 ⁇ , 20 ⁇ , 25 ⁇ , 30 ⁇ , 35 ⁇ , 40 ⁇ , 45 ⁇ , 50 ⁇ , 55 ⁇ , 60 ⁇ , 65 ⁇ , 70 ⁇ , 75 ⁇ , 80 ⁇ , 85 ⁇ , 90 ⁇ , 95 ⁇ , or 100 ⁇ .
  • Incubation may occur with any amount of reductant.
  • the amount of ascorbic acid used may be 0.001 mM, 0.005 mM, 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 2 ⁇ ⁇ , 3 niM, 4 niM, 5 niM, 6 niM, 7 niM, 8 niM, 9 niM, 10 niM, 15 niM, 20 niM, 25 niM, 30 niM, 35 niM, 40 niM, 45 niM, 50 niM, 55 niM, 60 niM, 65 mM, 70 niM, 75 niM, 80 mM, 85 niM, 90 mM, 95 mM, or 100 mM.
  • the amount of CDH used may be 0.001 ⁇ , 0.005 ⁇ , 0.01 ⁇ , 0.05 ⁇ , 0.1 ⁇ , 0.5 ⁇ , 1 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , 10 ⁇ , 15 ⁇ , 20 ⁇ , 25 ⁇ , 30 ⁇ , 35 ⁇ , 40 ⁇ , 45 ⁇ , 50 ⁇ , 55 ⁇ , 60 ⁇ , 65 ⁇ , 70 ⁇ , 75 ⁇ , 80 ⁇ , 85 ⁇ , 90 ⁇ , 95 ⁇ , or 100 ⁇ .
  • Incubation may occur at a temperature ranging from 25 °C to 70°C, for example,
  • the methods include a further step of providing amylases.
  • Amylases are enzymes used to break starches into dextrins and to break dextrins into glucose. Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha- amylases obtained from Bacillus, i.e., a special strain of Bacillus icheniformis, described in more detail in GB 1 ,296,839.
  • the fermentable sugar obtained from the starch-containing material can be fermented by one or more fermenting microorganisms capable of fermenting the sugars directly or indirectly into a desired fermentation product.
  • Fermentation products may include, alcohols (i.e., ethanol, methanol, butanol); organic acids (i.e., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (i.e., acetone); amino acids (i.e., glutamic acid); gases (i.e., H 2 and C0 2 ); antibiotics (i.e., penicillin and tetracycline); enzymes; vitamins (i.e., riboflavin, Bi 2 , beta-carotene); and hormones.
  • alcohols i.e., ethanol, methanol, butanol
  • organic acids i.e., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid
  • ketones i.e., acetone
  • amino acids i.e., glutamic acid
  • gases i.e., H 2 and C
  • the fermentation product can be ethanol (i.e., fuel ethanol, drinking ethanol, potable neutral spirits, industrial ethanol), products used in the consumable alcohol industry (i.e., beer and wine), leather industry and tobacco industry.
  • the fermentation product can be used in many different ways.
  • the fermentation product, such as ethanol, obtained according to the method may be used as fuel, whether or not blended with gasoline or it may also be used as potable ethanol.
  • “Fermentation” or “fermentation process” refers to any fermentation process or any process comprising a fermentation step. Fermentation processes include those used in the consumable alcohol industry, leather industry, tobacco industry and biofuel industry.
  • the fermentation conditions depend on the desired fermentation product and proper fermenting organism and can be easily determined by one skilled in the art.
  • sugars released from the starch- containing material as a result of enzymatic glycolysis, are fermented to a fermentation product by a fermenting organism, such as yeast.
  • the fermentation product is then further isolated and/or purified.
  • the fermentation can also be carried out simultaneously with the enzymatic saccharification in the same vessel, again under controlled pH, temperature, and mixing conditions.
  • saccharification and fermentation are performed simultaneously in the same vessel, the method is generally termed simultaneous saccharification and fermentation.
  • Any suitable starch-containing material may be used in the fermentation method (2010/059413 PCT/US2009/062955).
  • “Fermenting microorganism” refers to any microorganism suitable for use in a desired fermentation method. Suitable fermenting microorganisms are able to ferment, which is, convert, sugars, such as glucose, maltose, or maltodextrins directly or indirectly into the desired fermentation product.
  • Fermentation systems and culture conditions which can be used are described in WO2009/076676, WO2010/003007, WO 2009/132220, WO 2010/031062, WO2010/031068, WO 2010/031076, WO2010/031077, WO2010/031079, WO2010/148150,
  • WO2010/005525 WO 2010/078457
  • WO2010/124146 WO2010/148144
  • WO2010/148256 and U.S. Patent Application Nos. 12/496,573, 12/560,390, 12/560,317, 12/560,370, 12/560,305, and 12/560,366.
  • fermenting microorganisms are grown using any known mode of fermentation, such as batch, fed-batch, continuous, or continuous with recycle methods.
  • the polypeptide is used to degrade starch to form glucose, which is then cultured with fermentative organisms to produce a fermentation product as described. In another embodiment of the present invention, the polypeptide is used to degrade starch to form glucose, which is then cultured with a chemical solution to produce a fermentation product as described.
  • Amylases are enzymes used to break starches into dextrins and to break dextrins into glucose. Suitable amylases (alpha and/or beta) include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha- amylases obtained from Bacillus, i.e., a special strain of Bacillus icheniformis, described in more detail in GB 1 ,296,839.
  • all the processes described above can be performed in the presence of any amylase. In another embodiment all the processes described above can be performed in the presence of any amylase and a reductant. In another embodiment all the processes described above can be performed in the presence of any amylase and an iron containing compound. In another embodiment all the processes described above can be performed in the presence of a polypeptide and an amylase and a reductant comprising a heme domain. In yet another embodiment all the processes described herein can be performed in the presence of any amylase and cellobiose dehydrogenase. In another embodiment all the processes described above can be performed in the presence of any amylase and ferrous sulfate. In another embodiment all the processes described above can be performed in the presence of an amylase and an arene. For example, all the processes described above can be performed in the presence of any amylase and ascorbic acid.
  • the present disclosure also relates to a composition including a polypeptide having conserved motifs specific to starch-active PMOs, including SEQ ID NOs: 3-5, either individually or in combination, a copper cofactor, and a reductant.
  • the polypeptide in the composition includes the amino acid sequence of SEQ ID NO: 1 or 2.
  • the polypeptide in the composition includes any of the following amino acid sequences: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 87 or SEQ ID NOs: 8-86.
  • the composition includes the polypeptide, copper and an iron containing reductant.
  • the iron containing reductant can be a compound with a heme reducing domain, cellobiose dehydrogenase or ferrous sulfate.
  • the iron containing reductant can be a compound with a heme reducing domain, cellobiose dehydrogenase or ferrous sulfate.
  • composition may comprise, any concentration of the polypeptide, whether in its entirety or truncated form including, 0.001 ⁇ , 0.005 ⁇ , 0.01 ⁇ , 0.05 ⁇ , 0.1 ⁇ , 0.5 ⁇ , 1 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , 10 ⁇ , 15 ⁇ , 20 ⁇ , 25 ⁇ , 30 ⁇ , 35 ⁇ , 40 ⁇ , 45 ⁇ , 50 ⁇ , 55 ⁇ , 60 ⁇ , 65 ⁇ , 70 ⁇ , 75 ⁇ , 80 ⁇ , 85 ⁇ , 90 ⁇ , 95 ⁇ , and 100 ⁇ ; any concentration of CDH, including 0.001 ⁇ , 0.005 ⁇ , 0.01 ⁇ , 0.05 ⁇ , 0.1 ⁇ , 0.5 ⁇ , 1 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , 10 ⁇ , 15 ⁇ , 20
  • the composition includes the polypeptide, copper and an arene.
  • the composition can comprise a polypeptide, copper and ascorbic acid.
  • the composition may comprise any concentration of the polypeptide, whether in its entirety or truncated form including, 0.001 ⁇ , 0.005 ⁇ , 0.01 ⁇ , 0.05 ⁇ , 0.1 ⁇ , 0.5 ⁇ , 1 ⁇ , 2 ⁇ , 3 ⁇ , 4 ⁇ , 5 ⁇ , 6 ⁇ , 7 ⁇ , 8 ⁇ , 9 ⁇ , 10 ⁇ , 15 ⁇ , 20 ⁇ , 25 ⁇ , 30 ⁇ , 35 ⁇ , 40 ⁇ , 45 ⁇ , 50 ⁇ , 55 ⁇ , 60 ⁇ , 65 ⁇ , 70 ⁇ , 75 ⁇ , 80 ⁇ , 85 ⁇ , 90 ⁇ , 95 ⁇ , and 100 ⁇ ; any concentration of ascorbic acid including 0.001 mM, 0.005 mM, 0.01 mM, 0.05 mM, 0.1 mM,
  • compositions described above further include an amylase.
  • PMOs Polysaccharide monooxygenases
  • PMOs have been shown to oxidize either the CI or C4 atom of the ⁇ (1 ⁇ 4) glycosidic bond on the surface of chitin (Vaaje-Kolstad et al, 2010 Science, 2010, Hemsworth et al., Nat Chem Biol, 2014) or cellulose (Quinlan et al, Proc Natl Acad Sci USA, 2011, Phillips et al, ACS Chem Biol, 2011, Beeson et al, J Am Chem Soc, 2012), resulting in the cleavage of this bond and the creation of new chain ends that can be subsequently processed by hydrolytic chitinases and cellulases.
  • the N- terminal histidine ligand binds in a bidentate mode, and its imidazole ring is methylated at the ⁇ position in fungal PMOs (FIG. 1A).
  • PMOs act on substrates with structurally similar structures.
  • Cellulose and chitin contain long linear chains of ⁇ (1 ⁇ 4) linked glucose units and N- acetylglucosamine units, respectively (FIG. IB).
  • the polymer chains form extensive hydrogen bonding networks, which result in insoluble and very stable crystalline structures (Nishiyama et al., JAmChem Soc. 2002, Nishiyama et al., J Am Chem Soc, 2003, Nishiyama et al.,
  • PMOs are thought to bind to the substrate with their flat active site surface, which orients the copper center for selective oxidation at the CI or C4 position (Vaaje-Kolstad et al., 2010 Science, 2010, Li et al., Structure, 2012, Vu et al., J Am Chem Soc, 2014).
  • Some bacterial chitin-binding proteins are cellulose- active PMOs, further suggesting that the set of PMO substrates is restricted to ⁇ (1 ⁇ 4) linked carbohydrate polymers.
  • amylose and amylopectin contain linear chains of a(l ⁇ 4) linked glucose, while the latter also contains a(l ⁇ 6) glycosidic linkages at branch points in the otherwise a(l ⁇ 4) linked polymer.
  • amylose and amylopectin do not form microcrystals; instead, they exist in disordered, single helical, and double helical forms (Perez and Bertoft, Starke, 2010, Popov et al., Macromolecules, 2009, Imberty et al., J Mol Biol. 1988) (see FIG. 1C for example).
  • Starch can exist partially in nanocrystalline form, but lacks the flat molecular surfaces as those found in chitin and cellulose.
  • the discovery of starch-active PMOs shows that this oxidative mechanism of glycosidic bond cleavage is more widespread than initially expected.
  • cellulose active PMOs in light blue; formerly known as GH61
  • GH61 cellulose active PMOs
  • Three sequences belonging to a family annotated as chitinases are also shown (in aqua); a member of this family was recently characterized as a chitin-active PMO (Hemsworth et al., Nat Chem Biol, 2014).
  • NCU08746 in yellow
  • CBM20 C-terminal carbohydrate binding module 20
  • Table 1 Predicted N. crassa PMOs, based on secreted proteins with a N-terminal histidine residue
  • FIG. 2 shows the domain architecture and the consensus sequence of NCU08746 and homologues. Forty three sequences of this family were found with an iterative protein database search using a Hidden Markov Model algorithm (Christiansen et al., FEBS J, 2009). They are all found in the genomes of fungi in the Ascomycota phylum. Unlike chitin- and cellulose-active PMOs, the NCU08746 family has very high sequence identity. The N-terminal domain suggested by this sequence alignment contains 204-233 amino acids; this finding is inconsistent with its original annotation. Given biochemical characterization reported herein, this catalytic domain is designated as a starch active PMO domain.
  • NCU08746 was obtained by expression in N. crassa as previously reported (Vu et al.,J Am Chem Soc, 2014) using the following primer sets:
  • NCU08746 (SEQ ID NO: 7) was purified using an amylose resin column, followed by a size exclusion column. SDS-PAGE analysis of the fractions from the amylose resin column shows that NCU08746 has relatively strong affinity for amylose (FIG. 8), supporting a functional role for the predicted C-terminal CBM20 domain.
  • MS/MS analysis of the trypsin-digested purified NCU08746 yielded nearly 50% sequence coverage, including both the N- and C-termini, confirming the predicted sequence of this protein.
  • Trypsin-digested purified NCU08746 was analyzed with nano LC MS/MS at the Scripps Center for Metabolomics and Mass Spectrometry of The Scripps Research Institute.
  • the mass spectrometry data is consistent with a methylated N-terminal histidine (FIG. 8 and Table 2), a feature previously observed for PMOs natively purified from or recombinantly expressed in fungi (Quinlan et al. , Proc Natl Acad Sci USA , 2011 , Phillips et al.
  • the CuS04-treated sample was then concentrated to approximately 5 mL and purified further with a size exclusion column (Superdex 75, GE Healthcare).
  • a size exclusion column Superdex 75, GE Healthcare.
  • FIG. 3 shows the high performance anion exchange chromatographic (HPAEC) traces of NCU08746 assays under various conditions.
  • the reaction with amylopectin is dependent on reductant (ascorbic acid) and oxygen.
  • reductant ascorbic acid
  • oxygen oxygen
  • the chromatogram exhibits a set of new peaks (trace C) that are not observed in the absence of either reductant or oxygen (FIG. 9).
  • the same set of peaks is also found in the assays with corn starch or amylose (FIGS. 10-12), but not in the assays with phosphoric acid swollen cellulose (PASC) or chitin (traces D and E in FIG. 3, respectively).
  • PASC phosphoric acid swollen cellulose
  • NCU08746 specifically cleaves starch substrates via an oxygen-dependent mechanism.
  • the products of NCU08746 assays were identified by comparison with synthetic standards via HPAEC and mass spectrometry analysis.
  • the product peaks of NCU08746 overlay with the peaks of malto-aldonic acids (traces A and B in FIG. 3), but not with those of maltodextrins (FIG. 12).
  • the mass spectrum of the assay with amylose exhibits a series of peaks 16 amu higher than the corresponding maltodextrin peaks (Table 3), which is consistent with the presence of an additional oxygen atom.
  • the HPAEC and mass spectrometry data indicate that NCU08746 oxidizes the CI position of starch substrates.
  • FIG. 19 shows that the full-length NCU08746 enzyme exhibits significantly higher activity on amylose (source: potato; Sigma A0512) than starch (amylose + amylopectin) or amylopectin from corn.
  • Amylopectin from maize (Sigma, 11020), potato amylose with average molecular weight of 2.8 kDa (AM2.8, TCI Chemicals, A0846) or potato amylose from Sigma (A0512) , and corn starch (Sigma, S4126), and chitin (Sigma, C9752) were washed with water by centrifugation several times prior to addition to assays.
  • Phosphoric acid swollen cellulose was prepared from Avicel PHI 01 (Sigma, 11365) as previously described (Zhang et ah,
  • Activity assays of the polypeptide were carried out in 100 ⁇ L total volume using 96 well plates, which were shaken at 1000 rpm - 1200 rpm at 42 °C for 4 hours using a microplate shaker. All assays contained 50 mg/mL substrate suspension, except for assays with PASC that contained 5 mg/mL substrate. Typical assays contained 5 ⁇ holo NCU08746, 2 mM ascorbic acid, and 50 mM sodium acetate pH 5.0. Ascorbic acid was substituted by 0.5 ⁇ MtCDH-2 in some assays. Cellobiose (5 mM) was added to an assay with MtCDH-2.
  • NCU08746 reconstituted with excess copper(II) sulfate and purified with size exclusion chromatography contained approximately one copper atom per protein molecule based on ICPAES analysis (Table 4). The addition of one equivalent of CuS04 to the assays of holo NCU08746 had no effect on activity (FIGS. 10 and 11). Treatment of holo NCU08746 with lOmM EDTA resulted in a partially apo form containing approximately 0.12 copper atom per protein molecule. This form was assayed with the addition of one equivalent of various second row metal salts. Among the metal ions tested, only Cu(II) increased the oxidative activity of this partially apo NCU08746 (FIG. 13). This data supports that copper is the native metal cofactor of
  • NCU08746 accepts electrons from cellobiose dehydrogenase
  • CDH utilizes FAD and heme cofactors to rapidly oxidize cellodextrins, including cellobiose, cellotriose, and cellotetraose, while activity on glucose and maltose is minimal (Henriksson et al, J Biotechnol, 2000). Oxidation of reduced MtCDH-2, obtained by incubation with cellobiose, was measured as the decrease in absorbance at 430 nm of the reduced heme cof actor. The rate of oxidation was measured by monitoring the decrease in the absorption at 430 nm.
  • NCU08746 activity assays on amylopectin were carried out with MtCDH-2 as the electron donor.
  • MtCDH-2 the electron donor
  • NCU08746 exhibited very weak activity on amylopectin (FIG. 4B, trace C), which is consistent with the low activity of CDH on glucose and maltodextrins (Henriksson et al, J Biotechnol, 2000).
  • NCU08746 activity (FIG. 4B, trace B) was comparable to that in the presence of excess ascorbic acid (FIG. 4B, trace A).
  • Truncl includes the NCU08746 catalytic domain but lacks CBM20.
  • Trunc2 contains the catalytic domain plus the linker region, but lacks the CBM20.
  • Truncl exhibits approximately half the activity of the full-length enzyme on amylose (FIG. 17). The activity of Trunc2 is similar to Truncl, about half as active as full-length enzyme (data not shown).
  • Truncl truncated protein
  • FIG. 18 shows X-band EPR spectra of wild type NCU08746 and the two truncation mutants.
  • the three spectra are essentially identical, suggesting that the coordination environment around Cu is the same for the three samples.
  • the EPR experiments were performed with 50 mM MES (pH 5.0), 150 mM NaCl, 20% glycerol.
  • the NCU08746 is -0.5 mM and is fully reconstituted with copper.
  • NCU08746 contains two domains with a flexible linker region, it is not readily amenable to crystallization.
  • the copper active site was characterized using X-ray absorption spectroscopy, which provides information on the local structure of the copper center.
  • NCU08746 is typical of a five- or six-coordinate copper(II) species containing oxygen/nitrogen ligands, which does not exhibit any ls ⁇ 3d or ls ⁇ 4p pre-edge transition features (FIG. 14) (Sarangi et ah, Coord Chem Rev, 2013).
  • EXAFS Extended X-ray absorption fine structure
  • the third shell features in the Fourier transform correspond to the double-humped feature centered at ⁇ 4 A-l in the EXAFS spectrum, which arises from the multiple scattering paths of several imidazole moieties as found in the spectra of many other metalloproteins and model complexes (Vu et ah, J Am Chem Soc, 2011, Pellei et ah, Dalton Trans, 2011, Sanyal et ah, J Am Chem Soc, 1993, Costello et ah, J Biol Inorg Chem, 2006, D'Angelo et ah, Biochemistry, 2005).
  • the inner shell feature can be fitted with 5 or 6 Cu-O/N paths, which needs to be split into several subshells. Splitting this shell into two subshells significantly improves the fit quality. Including a third subshell of one Cu-O/N path at ⁇ 2.5 A also yields a better fit.
  • the total coordination number deduced by EXAFS analysis is thus consistent with the XANES data.
  • the second shell feature can be simulated with several Cu » "C paths, which may arise from imidazole moieties or backbone atoms of coordinating residues.
  • the third shell features cannot be fitted with several single scattering Cu » "C/N paths around 4.3 A; however, it is well simulated when all significant single and multiple scattering paths associated with an imidazole moiety are included in a rigid body model.
  • the coordination number of the imidazole moiety was floated during the fit and was subsequently optimized to a value between 2 and 3.
  • This result indicates that Cu(II)-NCU08746 contains two or three histidine ligands.
  • the best fit includes 4 Cu-O/N paths at 1.97 A, 1 Cu-O/N path at 2.22 A, 1 Cu-O/N path at 2.42 A, 2 Cu-C paths at 3.23 A, and paths from 2.3 imidazole moieties (Table 5). This result indicates that the copper center of NCU08746 contains two or three histidine ligands.
  • Cu(II)-NCU08746 was prepared in 100 mM MES buffer pH 5.0, concentrated and mixed with 20% glycerol to obtain a final concentration of 1.28 mM enzyme. The sample was transferred to XAS solution sample holder. XAS data was collected at Beamline X3B of National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. To minimize photoreduction of the sample, 8 first scans were collected on 8 different sample spots (0.9 mm x 5.8 mm). Standard procedure was applied to reduce, calibrate, and average data using
  • EXAFSPAK (George and Pickering (Stanford Synchrotron Radiation Laboratory, Stanford Linear Accelerator Center), Stanford University; Stanford, CA, 2000). EXAFS fitting was performed using the OPT function of EXAFSPAK. Theoretical scattering paths were calculated with FEFF8.4 (Rehr et ah, J Am Chem Soc, 1991) using the model shown in FIG. 15. The r and ⁇ 2 values of all significant single and multiple scattering paths of the imidazole moiety were linked together in the fit. ICP-OES analysis of 27 elements was performed at the Research Analytical Laboratory (RAL) at the University of Minnesota-Twin Cities. The samples were prepared according to the instructions from RAL.
  • RAL Research Analytical Laboratory
  • NCU08746 represents a member of a new family of PMOs that cleave starch. Sequence analysis reveals that this family contains the conserved histidine residues that are expected to form the histidine brace motif, as well as the motif N/Q/E-X-F/Y that contains the active site tyrosine residue in PMOs (FIG. 1). MS/MS analysis supports the presence of an N-terminal methylhistidine residue in NCU08746. Activity assays indicate that copper is the native metal cofactor of this enzyme. ICP analysis shows that it contains one Cu atom per protein molecule. Cu K-edge EXAFS analysis indicates that purified NCU08746 contains a copper(II) center with two or three histidine ligands.
  • FIG. 1A in which the elongated hexacoordinate copper(II) site contains two histidine ligands.
  • the coordination number is consistent with both the XANES features and EXAFS analysis.
  • the coordination sphere deduced from EXAFS fitting contains 4 O/N ligands at 1.97 A from the copper center and two additional O/N ligands at longer distances, 2.22 and 2.42 A, which is consistent with the elongated octahedral geometry as predicted by Jahn-Teller effect.
  • the uncertainty in the coordination number deduced by EXAFS analysis does not rule out a third histidine ligand.
  • Starch is known to be degraded mainly by alpha-amylase and glucoamylase, two efficient hydrolytic enzymes currently utilized in the starch-based biofuel industry. Starch-active PMOs may help to further the efficiency of these enzymes.
  • NCU08746 requires both oxygen and a source of electrons. As shown, ascorbic acid is an efficient electron donor for the NCU08746 reaction. It is possible that oligosaccharide dehydrogenases or maltose dehydrogenases can serve as biological electron donors to starch- active PMOs; however, they have not been well characterized (Tessema et al., Anal Chem , 1997, Kobayashi et al., Enzymol, 1980). Cellobiose dehydrogenase (CDH), which is widely accepted as the biological partner of cellulose-active PMOs, can donate electrons to NCU08746 as efficiently as ascorbic acid.
  • CDH Cellobiose dehydrogenase
  • NCU08746 and other family members may possess some protein structural features similar to those in cellulose-active PMOs that allow the transfer of electrons from CDH to the copper center. It is also possible that CDH is the biological partner of starch-active PMOs. In the wild, fungi routinely encounter food sources containing mixed polysaccharides, and co-expression of enzymes that cleave diverse substrates would allow for parallel degradation pathways. Transcriptomic and proteomic analysis of the N. crassa secretome on different growth substrates would shed light on the potential biological relationship between CDH and starch-active PMOs.
  • Amylose contains a(l ⁇ 4) glycosidic linkages and exists in disordered, single helical, and double helical forms.
  • Amylopectin contains a(l ⁇ 6) bonds in addition to a(l ⁇ 4) bonds, which creates branches (FIG. 1C).
  • Double helices can form between amylose and amylopectin in starch, which further complicates the structure (Perez and Bertoft, Starke, 2010, Popov et al., Macromolecules, 2009, Imberty et al., J Mol Biol. 1988). Recently, a cellulose- active PMO from N.
  • NCU08746 is shown in FIG. 6, analogous to that of cellulose- active PMOs and chitin-active PMOs. NCU08746 is shown here to oxidize the CI position in both amylose and amylopectin. For amylopectin, it is unclear if only the a(l ⁇ 4) bond is cleaved or if NCU08746 can attack both a(l ⁇ 4) and a(l ⁇ 6) linkages. In addition, although C4 and C6 oxidized products are not detected for NCU08746, oxidation at these positions is relevant to bond cleavage and may be carried out by other starch-active PMOs.
  • C4 oxidation is known to occur with some cellulose- active PMOs (Phillips et al., ACS Chem Biol, 2011, Beeson et al., J Am Chem Soc, 2012, Vu et al., J Am Chem Soc, 2014, Isaksen et al., J Biol Chem, 2014) and has recently been implicated in fungal chitin-active PMOs (Hemsworth et al., Nat Chem Biol, 2014).
  • [] any one of the characters enclosed in the brackets, e.g., [ED] means one occurrence of E or D
  • Catalytic domain motif version 2 (specifying residues for certain X's) (SEQ ID NO: 2)
  • [] any one of the characters enclosed in the brackets, e.g., [ED] means one occurrence of E or D
  • Truncated NCU08746 without linker and CBM20 (Truncl; SEQ ID NO: 6):
  • Truncated NCU08746 without CBM20 (Trunc2; SEQ ID NO: 87)

Abstract

L'invention concerne des procédés pour dégrader un matériau contenant de l'amidon comprenant les étapes consistant à fournir un matériau contenant de l'amidon et fournir un polypeptide possédant une activité dégradant l'amidon et un cofacteur de cuivre. Le procédé comprend en outre l'incubation du polypeptide avec le matériau contenant de l'amidon en présence d'oxygène et d'un agent réducteur.
PCT/US2015/042126 2014-07-24 2015-07-24 Dégradation oxydative de l'amidon par une nouvelle famille de pmos WO2016015021A1 (fr)

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