Field of Invention

The invention relates generally to the field of protein engineering. In particular, the disclosure teaches a modified cyclic polypeptide that has enhanced thermostability.

Background

Commercial bottle grade polyethylene terephthalate (cPET) is extremely recalcitrant to enzymatic degradation due its highly crystalline state. Efficient hydrolysis will be likely achievable at elevated temperature close to its glass transition temperature (60 to 65 ºC). At this temperature changes in cPET structure facilitate enzyme access and improve degradation efficiency.

Polyethylene terephthalate hydrolases (or PETases) refer to a class of hydrolases that are able to catalyze the cleavage of PET into mono (2-hydroxyethyl) terephthalic acid. However, naturally occurring PETase enzymes are unstable at high temperatures that are required for efficient hydrolysis of PET. This makes them unsuitable for use in degrading PET.

Accordingly, it is generally desirable to overcome or ameliorate one or more of the above mentioned difficulties.

Summary

Disclosed herein is modified cyclic polypeptide comprising: a) a first amino acid sequence comprising a PETase enzyme or active fragment thereof; and b) a second and third amino acid sequence; wherein the first amino acid sequence is positioned between the second and third amino acid sequences, and wherein the second and third amino acid sequences are joined by a covalent bond such that the polypeptide forms a cyclic polypeptide. Brief Description of Drawings

Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which:

Figure 1 Amino acid sequence of thermostable cPETase: Spycatcher and Spytag components (N and C termini respectively) are underlined. PETase sequence is in bold. A polyhistidine linker connects Spycatcher to PETase whilst a protease cutting site/linker connects PETase to SpyTag ( italicized ). A lysine (K) residue (in bold) on the SpyCatcher sequence is able to form an isopeptide bond with an aspartate (D) residue (in bold) on the Spytag sequence.

Figure 2 Thermostability of purified PETase and thermostable cPETase:

Thermostability was tested by heating the purified proteins at RT, 40, 50, 60, 70 and 80 °C for 10 minutes. The samples were cooled down and the precipitates were separated by centrifugation. Supernatants were analysed by SDS-PAGE. Results show that cPETase protein is partially thermostable even up to 80 °C while PETase is not stable above 40 °C.

Figure 3. Melting point detection of purified PETase and cPETase by thermofluor assay. The results indicate two Tms for cPETase at 36 and 78.8 °C while PETase has only one Tm at 38 °C.

Figure 4. Thermostability study of PETase and cPETase using p-nitrophenyl acetate (PNPA). PCR tubes containing 100 μl of 1 mg/ml PETase or equivalent molar concentration of cPETase were heated in a thermocycler at 40, 50, 60, 70, 80 and 90 °C for 10 minutes, cooled down and precipitates were removed after spinning in microcentrifuge. 10 μl supernatant were taken in an eppendorf tube and the reaction was started by adding 990 pi master mix [containing 980 pi 50 mM phosphate buffer (pH 8) pre-mixed with 10 μl substrate (5 mM PNPA stock in absolute ethanol)] and incubated at 37 °C for 10 minutes. The samples were transferred to ice and OD405 were measured immediately (within 5 minutes) against water as blank. Blanks were made without adding any enzyme. The result shows the cPETAse retains >50% activity at 75 °C and >35% activity at 90 °C while the wild type PETase was almost inactivated upon treatment at 50 °C. Same data is presented in two graph formats.

Figure 5. Amino acid sequence of ePETase-G4S: Spycatcher and Spytag components (N and C termini respectively) are underlined. PETase sequence is in bold. A polyhistidine followed by a GGGGS linker connects Spycatcher to PETase while a protease cutting site connects PETase to SpyTag (italicized). A lysine (K) residue (in bold) on the SpyCatcher sequence is able to form an isopeptide bond with an aspartate (D) residue (in bold) on the Spytag sequence.

Figure 6. Thermostability of purified PETase, cPETase and cPETase-G4S: Thermostability was tested by heating the purified proteins at 70 °C for 10 minutes. The samples were cooled down and the precipitates were separated by centrifugation. Supernatants were analysed by SDS-PAGE. Results show that cPETase and cPETase - G4S proteins are thermostable at 70 °C while PETase is completely denatured at this temperature.

Figure 7. Thermostability study of PETase, cPETase and cPETase-G4S using p- nitrophenyl acetate (PNPA): The purified proteins were heated at 70 °C for 10 minutes, cooled down and precipitates were removed. 10 μl supernatant were taken in a Eppendorf tube and tire reaction was started by adding 990 μl master mix (containing 980 μl 50 niM phosphate buffer (pH 8) pre-mixed with 10 μl substrate (5 mM PNPA stock in absolute ethanol)) and incubated at 37 °C for 10 minutes. The samples were transferred to ice and OD405 were measured immediately (within 5 minutes) against water as blank. Blanks were made without adding any enzyme. The result show _' s cPETAse and cPETase-G4S retains >55% activity while the wild type PETase was inactivated upon the heat treatment. However, the activity of cPETase was improved by addition of the GGGGS linker.

Detailed Description

The specification teaches a modified cyclic polypeptide comprising a first amino acid sequence comprising a PETase (or polyethylene terephthalate (PET) hydrolase) enzyme or active fragment thereof. The modified cyclic polypeptide may comprise a second amino acid sequence and a third amino acid sequence, wherein the first amino acid sequence is positioned between the second and third amino acid sequences. The second and third amino acid sequences may be joined by a covalent bond (such as a peptide or an isopeptide bond) such that the polypeptide forms a cyclic polypeptide.

Disclosed herein is a modified cyclic polypeptide comprising: a first amino acid sequence comprising a PETase enzyme or active fragment thereof; and a second and third amino acid sequence; wherein the first amino acid sequence is positioned between the second and third amino acid sequences, and wherein the second and third amino acid sequences are joined by a covalent bond such that the polypeptide forms a cyclic polypeptide.

In one embodiment, the covalent bond is a peptide bond. The peptide bond may be an isopeptide bond.

Without being bound by theory, the highly crystalline nature of cPET likely mandates enzymatic degradation to be carried out at elevated temperatures. This will require identification and development of thermostable PET hydrolase enzymes. In one embodiment, the inventors have engineered the prototypical hydrolase (PETase) enzyme to increase its melting temperature (Tm) by ~ 40 degrees Celsius. This variant has significant potential for industrial applications. The disclosure teaches a thermostable engineered version of the polyethylene terephthalate (PET) hydrolysing enzyme PETase. The native enzyme was discovered in I. sakaiensis, a recently discovered bacterium that attaches to PET surface and metabolises PET through PETase secretion. The endogenous enzyme shows poor activity on commercial grade PET (cPET), and many efforts are under way to improve its activity for commercial purposes. A major bottleneck is the highly crystalline nature of cPET which renders enzymatic activity sub-optimal for industrial usage. Efficiency of degradation could be improved by carrying out reactions closer to the glass transition temperature of cPET (60-65 ºC) as this facilitates improved enzyme access to the substrate. Accordingly, there is a need for thermostable PET hydrolase enzymes. The inventors have therefore engineered the PETase enzyme to increase its Tm from ~38 ºC to ~79ºC. This significant increase in thermostability will facilitate degradation of substrates at higher temperature and therefore has industrial importance. In addition, the inventors have surprisingly found that the inclusion of a flexible linker at a particular position can significantly improve the enzymatic activity of the thermally stabilized enzyme (see, for example, Figure 7).

The term “PETase” or “polyethylene terephthalate hydrolases” refer to a class of hydrolases that have the property of catalyzing the cleavage of PET (polyethylene terephthalate) into mono (2-hydroxyethyl) terephthalic acid.

In one embodiment, there is provided an engineered thermostable PETase enzyme with a melting temperature (Tm) that is ~ 40 degrees Celsius higher than wild type enzyme. The engineered PETase enzyme may be active at higher temperatures (70-90 ºC) enabling more efficient degradation of substrates. The thermostable PETase enzyme may also have longer shelf life at ambient temperatures.

In one embodiment, the modified cyclic polypeptide has improved thermal stability.

The terms "protein" and "polypeptide" are used interchangeably and refer to any polymer of amino acids (dipeptide or greater) linked through peptide bonds or modified peptide bonds. Polypeptides of less than about 10-20 amino acid residues are commonly referred to as "peptides." The polypeptides of the invention may comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a polypeptide by the cell in which the polypeptide is produced, and will vary with the type of cell. Polypeptides are defined herein, in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

The term "modified" as used herein refers to any changes made to a given polypeptide, such as changes to the length of the polypeptide, the amino acid sequence, chemical structure, co-translational modification, or post-translational modification of a polypeptide. The modification of a given polypeptide may include addition of amino acid sequences to either ends of the polypeptide to allow for cyclisation of the polypeptide when expressed in a bacterial cell.

The term “fragment” when referring to a PETase enzyme, means a polypeptide which has an amino acid sequence which is the same as part of but not all of the amino acid sequence of a full-length PETase polypeptide. An “active fragment” refers to a PETase fragment that retains the enzymatic activity of full-length PETase.

The SpyCatcher/SpyTag bioconjugation system was selected which employs a fibronectin-binding protein (FbaB) from Streptococcus pyogenes which is a split protein that employs two subunit domains referred to as the SpyCatcher and SpyTag sequences. Unlike many split protein systems, the SpyCatcher/SpyTag system provides for the formation of an isopeptide bond between proximal aspartic acid and lysine amino acid residues. This interaction and bond formation happens spontaneously as it does not require the addition of chaperone proteins, catalytic enzymes, or cofactors. The reaction occurs at room temperature and over a wide range of physiologically relevant conditions. The reaction can also occur in a cell.

In one embodiment, the second amino acid sequence is positioned at the N terminus of the first amino acid sequence and the third amino acid sequence is positioned at the C terminus of the first amino acid sequence. In one embodiment, the second and first amino acid sequences are contiguous with one another. In another embodiment, the first and third amino acid sequences are contiguous with one another.

The second and third amino acid sequences may comprise a pair of tag and catcher sequences. The tag and catcher sequences may be positioned at the C and N termini of the enzyme, or vice versa, and generally form a stable isopeptide bond when they react. The tag and catcher sequences may help in refolding of the enzyme following exposure to high temperatures, and improving thermal stability.

The second and third amino acid sequences may comprise a pair of SpyCatcher and SpyTag sequences. In one embodiment, the second amino acid sequence comprises a SpyCatcher or SpyTag sequence. In one embodiment, the second amino acid sequence comprises a SpyCatcher sequence. In one embodiment, the third amino acid sequence comprises a SpyCatcher or SpyTag sequence. In one embodiment, the third amino acid sequence comprises a SpyTag sequence.

In one embodiment, the second amino acid sequence comprises an amino acid sequence having at least 70% (or 80%, 90%, 95%, 99% or 100%) sequence identity to SEQ ID NO: 2 or 9. In one embodiment, the third amino acid sequence comprises an amino acid sequence having at least 70% (or 80%, 90%, 95%, 99% or 100%) sequence identity to SEQ ID NO: 3.

In one embodiment, the second amino acid sequence comprises an amino acid sequence having at least 70% (or 80%, 90%, 95%, 99% or 100%) sequence identity to SEQ ID NO: 2 or 9 and the third amino acid sequence comprises an amino acid sequence having at least 70% (or 80%, 90%, 95%, 99% or 100%) sequence identity to SEQ ID NO: 3.

In one embodiment, there is provided a modified cyclic polypeptide comprising a) a first amino acid sequence comprising a PETase enzyme or active fragment thereof; b) a second amino acid sequence having at least 70% (or 80%, 90%, 95%, 99% or 100%) sequence identity to SEQ ID NO: 2 or 9; and c) a third amino acid sequence having at least 70% (or 80%, 90%, 95%, 99% or 100%) sequence identity to SEQ ID NO: 3; wherein the first amino acid sequence is positioned between the second and third amino acid sequences, and wherein the second and third amino acid sequences are joined by an isopeptide bond such that the polypeptide forms a cyclic polypeptide.

The term "sequence identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Methods of aligning amino acid sequences are well known in the art. For example, bioinformatics or computer programs and alignment algorithms such as ClusterW may be used to determine the “% identity” between two amino acid sequences.

The term “at least 70%” as used herein includes at least 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%, 99% or more. The modified cyclic polypeptide may be modified to have insertions, deletions or substitutions, either conservative or non-conservative, provided that such changes allow the modified cyclic polypeptide to retain the enzymatic activity of the PETase enzyme. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence. Such changes may, for example, be made using the methods of protein engineering and site-directed mutagenesis. A "conservative" change is wherein a substituted amino acid has similar structural or chemical properties. A "non- conservative" change is wherein the substituted amino acid is structurally or chemically different.

A “substitution” results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

A “conservative substitution” refers to the substitution of an amino acid in one class by an amino acid in the same class, where a class is defined by common physicochemical amino acid sidechain properties and high substitution frequencies in homologous proteins found in nature (as determined, e.g., by a standard Dayhoff frequency exchange matrix or BLOSUM matrix). Six general classes of amino acid sidechains, categorized as described above, include: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Asp for another class III residue such as Asn, Gin, or Glu, is considered to be a conservative substitution.

In one embodiment, the second and third amino acid sequences are joined by an isopeptide bond between a lysine (K) residue on the second amino acid sequence and an aspartate (D) residue on the third amino acid sequence (see, for example, Figure 1, on SEQ ID NO: 4).

In one embodiment, the PETase enzyme is a bacterial PETase enzyme. The PETase enzyme may be a bacterial PETase from the Ideonella genus. In one embodiment, the PETase enzyme is a bacterial PETase from Ideonella sakaiensis (or I. sakaiensis). An active fragment of the PETase enzyme may also be used in place of the PETase enzyme. In one embodiment, the PETase enzyme or active fragment thereof is an amino acid sequence having at least 70% (or 80%, 90%, 95%, 99% or 100%) sequence identity to SEQ ID NO: 1. The PETase enzyme may be an active fragment of SEQ ID NO: 1. The PETase enzyme or active fragment thereof may also differ from SEQ ID NO: 1 or its active fragment by one or more amino acid residues while retaining its enzymatic property.

In an alternative embodiment, the PETase enzyme or active fragment thereof is positioned between two other peptide motifs (e.g. LPETG and GGG) and is circularised by adding an enzyme (e.g. Sortase) to form a peptide bond.

The modified cyclic polypeptide may comprise a purification tag (such as a polyhistidine tag) positioned between the first and second amino acid sequences. The modified cyclic polypeptide may comprise a protease cutting site (or protease cleavage site) positioned between the first and third amino acid sequences. In one embodiment, the flexible linker comprises the sequence of In one embodiment, the flexible linker comprises the sequence of

The modified cyclic polypeptide may comprise a flexible linker positioned between the first and second amino acid sequences. The flexible linker may improve the enzymatic activity of the modified cyclic polypeptide. The flexible linker may comprise 5 to 30, 5 to 25 or 5 to 20 amino acid residues. The flexible linker may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues. In one embodiment, the flexible linker comprises 16 amino acid residues. In one embodiment, the flexible linker comprises 5 amino acid residues.

In one embodiment, the flexible linker comprises the sequence of GGGGS (SEQ ID NO: 8). The flexible linker may comprise multiple repeats of SEQ ID NO: 8. In one embodiment, the flexible linker comprises the sequence of

The modified cyclic polypeptide may comprise a flexible linker positioned between the first and second amino acid sequences. In one embodiment, the modified cyclic polypeptide comprises or consists of a sequence having at least 70% (or 80%, 90%, 95%, 99% or 100%) sequence identity to SEQ ID NO: 4.

In one embodiment, the modified cyclic polypeptide comprises or consists of a sequence having at least 70% (or 80%, 90%, 95%, 99% or 100%) sequence identity to SEQ ID NO: 5.

In one embodiment, the modified cyclic polypeptide is a recombinant cyclic polypeptide.

A “recombinant protein” refers to a protein isolated, purified, or identified by virtue of expression in a heterologous cell, said cell having been transduced or transfected, either transiently or stably, with a recombinant expression vector engineered to drive expression of the protein in the host cell.

The term “expression” means that a protein is produced by a cell, usually as a result of transfection of the cell with a heterologous nucleic acid.

The terms “purified” or “substantially purified” refer to molecules, either polynucleotides or polypeptides, that are removed from their natural environment, isolated or separated, and are at least 90% and more preferably at least 95-99% free from other components with which they are naturally associated. The foregoing notwithstanding, such a descriptor does not preclude the presence in the same sample of splice- or other protein variants (glycosylation variants) in the same, otherwise homogeneous, sample.

In one embodiment, there is provided a modified polypeptide comprising: a first amino acid sequence comprising a PETase enzyme or active fragment thereof; and a second and third amino acid sequence; wherein the first amino acid sequence is positioned between the second and third amino acid sequences, and wherein the second and third amino acid sequences are capable of forming a covalent bond such that the modified polypeptide forms a modified cyclic polypeptide. Disclosed herein is a composition comprising a modified cyclic polypeptide as defined herein.

Table 1 shows the sequences of a PETase enzyme, a SpyCatcher component and a Spytag component. The sequences of the PETase enzyme, SpyCatcher component and Spytag component are indicated as SEQ ID Nos 1-3 respectively.

Disclosed herein is a nucleic acid encoding the modified cyclic polypeptide as defined herein.

Disclosed herein is a nucleic acid encoding a modified polypeptide capable of forming a modified cyclic polypeptide as defined herein.

The term "nucleic acid" includes a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. The terms “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence” and “polynucleotide” are used interchangeably herein unless the context indicates otherwise.

The term “encode" or “encoding” includes reference to nucleotides and/or amino acids that correspond to other nucleotides or amino acids in the transcriptional and/or translational sense.

Disclosed herein is an expression construct comprising a nucleic acid as defined herein.

The term "expression construct" may refer to a nucleic acid molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence (e.g. an insert sequence that codes for a product) in a particular host cell. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter and a ribosome binding site, often along with other sequences.

Disclosed herein is a method of preparing a modified cyclic polypeptide as defined herein, the method comprising a) expressing the modified cyclic polypeptide with the expression construct as defined herein; and b) purifying the modified cyclic polypeptide.

The method may comprise transforming and expressing the expression construct into a bacterial cell (such as E. Coli). The method may further comprise purifying the modified cyclic polypeptide from the bacterial cell.

Disclosed herein is a method of hydrolysing polyethylene terephthalate (PET), the method comprising contacting the PET with a modified cyclic polypeptide as defined herein for a sufficient time and under conditions to hydrolyse the PET.

Disclosed herein is the use of a modified cyclic polypeptide as defined herein for hydrolysing polyethylene terephthalate (PET).

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

Throughout this specification and the statements which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Those skilled in the art will appreciate that the invention described herein in susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described. EXAMPLES

Experimental

Spycatcher-SpyTag technology ¹ was used to cyclise PETase. The genes encoding Spycatcher and SpyTag were respectively fused in-frame to the N- and C- termini of PETase (Figure 1). The resulting construct was transformed into E. coli and cyclic PETase (cPETase) was recombinantly expressed and purified.

Thermostability of purified PETase and cPETase was first tested by heating the proteins over a range of temperatures and measuring soluble protein by gel electrophoresis. The results in Figure 2 show that thermostable cPETase can withstand up to 80°C incubation for ten minutes whilst PETase is completely denatured at 50 ºC incubation for 10 minutes.

The melting temperature (T _m ) of the proteins was further determined using conventional thermofluor analysis. The results in Figure 3 show an additional peak at 78.8 ºC for cPETase that is not present in PETase, again indicating enhanced thermostability.

Validated enzymatic activity of cPETase was next validated after incubation at elevated temperatures using a model substrate. The results in Figure 4 indicate considerable activity of cPETase (40% of non-heated control) even after incubation at 90 ºC whilst PETase is completely inactive at temperatures above 55 ºC.

REFERENCES

1. Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M: Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci USA 2012, 109:E690-697.