Enhancing the activity of a plastic-degrading enzyme

Abstract

In a method of enhancing the activity of a plastic-degrading enzyme (32) such as a PETase, a biofilm-producing micro-organism (30) is modified to exhibit elevated levels of cyclic di-GMP. The micro- organism (30), such as E. coli modified to express and secrete the plastic-degrading enzyme (32), can be modified to express a heterologous diguanylate cyclase such as DdcC or WspR. An expression construct for use in the method encodes an enzyme that modulates levels of cyclic di-GMP, such as diguanylate cyclase, and also a plastic- degrading enzyme (32), such as a PETase. The diguanylate cyclase can be an E. coli diguanylate cyclase or a P. aeruginosa diguanylate cyclase. A biofilm-forming micro-organism (30) is transformed by the expression construct.

Description

[0001] ENHANCING THE ACTIVITY OF A PLASTIC-DEGRADING ENZYME

[0002] This application relates to a method of enhancing the activity of a plastic-degrading enzyme, to a method of biodegrading plastic, and to an expression construct and a micro-organism for use in such methods.

[0003] Globally, plastic pollution is an increasing problem. 12,000 million metric tonnes of plastic waste are predicted to end up in the environment and landfill by 2050, even with increasing recycling trends. Recycling is only part of the solution, not all plastic types can be recycled and recycled plastic can often have fewer uses and limited reuses.

[0004] Most collected plastic waste is incinerated, which increases toxic fumes and carbon emissions, or is taken to landfill, which pollutes the surrounding environment and endangers wildlife.

[0005] An alternative plastic processing could involve micro-organisms that degrade the plastic and therefore remove it completely, whilst also converting it to useful bi-products, such as polyethylene terephthalate (PET), which when degraded can release polyhydroxyalkanoate (PHA), a bioplastic (Kenny et al. (2008)).

[0006] Plastic-biodegrading enzymes produced by such micro-organisms can either be purified and used directly or the micro-organisms themselves that express the enzymes (usually bacteria), whether native or heterologous, can be grown in batch culture to degrade plastic in bioreactors. To date several species of bacteria and their enzymes have been found to degrade plastics, mainly PET, but none have yet transitioned into real world applications (Herrero Acero et aL, (2011);

[0007] Ribitsch et aL (2012)).

[0008] Plastic-degrading enzymes (such as PETases) represent a potential solution to the plastic waste crisis. However, an issue that has arisen with many of the enzymes that have been identified so far is that their efficiency is too low, limiting their potential to be developed as plastic bioremediation tools.

[0009] Many research groups have focussed on modifying the enzymes to improve efficacy and stability or creating enzymatic chimeras that can be expressed in an autologous host. For example, several research groups have looked at improving enzyme efficiency, through targeted mutations or machine learning to create a new synthetic enzyme (Lu et at. (2022)).

[0010] The present invention seeks to provide an improved method of biodegrading plastic.

[0011] According to an aspect of the present invention, there is provided a method of enhancing the activity of a plastic-degrading enzyme expressed by a biofilm-forming micro-organism, including modifying the micro-organism to exhibit elevated levels of cyclic di-GMP.

[0012] According to another aspect of the present invention, there is provided a method of enhancing the activity of a plastic-degrading enzyme expressed by a biofilm-forming micro-organism, wherein the microorganism is modified to express the plastic-degrading enzyme or at least an active fragment thereof or wherein the micro-organism is modified to enhance expression of a native plastic-degrading enzyme or at least an active fragment thereof, including modifying the microorganism to exhibit elevated levels of cyclic di-GMP.

[0013] The micro-organism may have been genetically modified to express or have enhanced expression of the plastic-degrading enzyme by way of introduction of a vector carrying exogenous genetic material. In another embodiment, the micro-organism may have been modified by undergoing a process of directed evolution to select for organisms able to express or having enhanced expression of the plastic-degrading enzyme.

[0014] According to another aspect of the present invention, there is provided a method of biodegrading plastic including : providing a biofilm-forming micro-organism that expresses a plastic-degrading enzyme, wherein the micro-organism is modified to exhibit elevated levels of cyclic di-GMP; contacting the micro-organism with plastic to be degraded; and allowing the plastic to be at least partially biodegraded by the plastic-degrading enzyme.

[0015] The step of allowing may include incubating the micro-organism with the plastic to be degraded for a time sufficient at least partially to biodegrade the plastic.

[0016] According to another aspect of the present invention, there is provided a method of biodegrading waste plastic including: providing a biofilmforming micro-organism that expresses a plastic-degrading enzyme, wherein the micro-organism is modified to exhibit elevated levels of cyclic di-GMP; contacting the micro-organism with waste plastic to be degraded; and allowing the waste plastic to be at least partially biodegraded by the plastic-degrading enzyme.

[0017] The micro-organism may be capable of expressing the plastic-degrading enzyme in its native state, or it may have been modified to do so.

[0018] The micro-organism may be modified to express an enzyme that modulates levels of cyclic di-GMP. For example, it can have increased diguanylate cyclase activity.

[0019] In an embodiment, the micro-organism is modified to express a heterologous diguanylate cyclase or at least an active fragment thereof. An active fragment of a DGC will, in general, include at least a functional GGDEF or GGEEF domain, and will be capable of increasing intracellular levels of CdiGMP.

[0020] The diguanylate cyclase may be an E. coli diguanylate cyclase, such as DgcC.

[0021] The diguanylate cyclase may be a P. aeruginosa diguanylate cyclase, such as WspR.

[0022] The micro-organism may be a bacterium, such as E. coli. The bacterium may be, for example, E. coli BL21 (DE3), BL21 (DE3) plysS, Rosetta (DE3), Tuner DE3, E. coli HMS174 (DE3), E. coli NiCo21(DE3). In other embodiments, the bacterium may be Lactococcus lactis, or B. subtilis. The plastic-degrading enzyme may a PETase. In some embodiments, the plastic-degrading enzyme may additionally / alternatively be an MHETase, an alkane hydroxylase, a hydrodroquinone peroxidase, an esterase, laccase or a mixture of any two or more of these.

[0023] According to another aspect of the present invention, there is provided an expression construct for use in any of the methods as disclosed above or herein, the expression construct for expression of at least an active fragment of the diguanylate cyclase by a biofilm-forming microorganism able to express a plastic-degrading enzyme, wherein the expression construct encodes a diguanylate cyclase or at least an active fragment of a diguanylate cyclase.

[0024] According to another aspect of the present invention there is provided an expression construct for use in any of the methods specified above, the expression construct for expression of at least an active fragment of an enzyme that modulates levels of cyclic di-GMP by a biofilm-forming micro-organism able to express a plastic-degrading enzyme, wherein the expression construct encodes the enzyme that modulates levels of cyclic di-GMP or at least an active fragment of the enzyme that modulates levels of cyclic di-GMP, wherein the expression construct also encodes at least an active fragment of a plastic-degrading enzyme.

[0025] An expression construct for use in any of the method disclosed above, the expression construct for expression of at least an active fragment of diguanylate cyclase by a biofilm-forming micro-organism able to express a plastic-degrading enzyme, wherein the expression construct encodes the diguanylate cyclase or at least an active fragment of the diguanylate cyclase, wherein the diguanylate cyclase is an E. coli diguanylate cyclase or a P. aeruginosa diguanylate cyclase.

[0026] The DGC encoded by the expression construct may have been codon optimised for optimised expression by the host micro-organism.

[0027] The expression construct may be a plasmid. The plasmid may enable inducible or constitutive expression of the DGC encoded theron.

[0028] The diguanylate cyclase may be an E. coli diguanylate cyclase, such as DgcC

[0029] The diguanylate cyclase may be a P. aeruginosa diguanylate cyclase, such as WspR.

[0030] The expression construct may also encode a plastic-degrading enzyme, or at least an active fragment thereof.

[0031] According to another aspect of the present invention, there is provided a biofilm-forming micro-organism for use in a method as disclosed above or herein, the micro-organism being able to express a plasticdegrading enzyme, wherein the micro-organism is modified to exhibit elevated levels of cyclic di-GMP.

[0032] According to another aspect of the present invention there is provided a biofilm-forming micro-organism transformed by any of the expression constructs disclosed above. The micro-organism may be modified to have increased diguanylate cyclase activity. For example, it may have been modified to express a heterologous diguanylate cyclase or at least an active fragment thereof.

[0033] The micro-organism may have been modified to express a heterologous plastic-degrading enzyme or at least an active fragment thereof.

[0034] The micro-organism may have been transformed by an expression construct as disclosed above or herein.

[0035] The micro-organism may have been transformed by an expression construct as disclosed above or herein, and also by an expression construct encoding at least an active fragment of a plastic-degrading enzyme.

[0036] The micro-organism may be a bacterium, such as E. coli. The bacterium may be, for example, E. coli BL21 (DE3), BL21 (DE3) plysS, Rosetta (DE3), Tuner DE3, E. coli HMS174 (DE3), E. coli NiCo21(DE3). In other embodiments, the bacterium may be Lactococcus lactis, or B. subtilis.

[0037] The plastic-degrading enzyme may be a PETase. In some embodiments, the plastic-degrading enzyme may additionally / alternatively be an MHETase, an alkane hydroxylase, a hydrodroquinone peroxidase, an esterase, a laccase or a mixture of any two or more of these. The micro-organism may secrete the plastic-degrading enzyme or may express the plastic-degrading enzyme on its surface whereby the plastic-degrading enzyme is attached to the cell surface.

[0038] It is particularly envisaged that modification of the micro-organism be achieved by engineering using genetic techniques.

[0039] Embodiments of the present invention are described below, by way of example only, with reference to the accompanying drawings, in which:

[0040] Figure 1 is a schematic illustration of how biofilm formation could impact enzyme activity;

[0041] Figure 2 is a schematic of the cyclic di GMP signalling system;

[0042] Figure 3 is a schematic illustration of an embodiment of a method of biodegrading waste plastic;

[0043] Figure 4 shows the results of a biofilm formation assay; and Figures 5 and 6 show the results of polycaprolactone weight loss experiments.

[0044] The present inventors set out to identify novel plastic-degrading enzymes.

[0045] Several known enzymes have previously been identified that can degrade plastics such as PET. Sequence similarity between these enzymes is quite high. The inventors collated known plastic-degrading enzymes and their protein sequences were used to search for novel potentially plastic-degrading enzymes using NCBI Protein Blast. BLAST hits were selected if their identity was between ~50-80% to ensure they were distinct proteins but closely related enough that function could be maintained.

[0046] Novel enzyme Dh3 was identified and had the following amino acid sequence (SEQ ID No. 1):

[0047] MSS I IKRKLA S LEAFS AAML LSTSVWSMNP GDVPDTCEGD CGYARGPDPT ESFLEADSGP YTIATSNVSS LVRGFGGGTI YYPVNAEGTM AAIWIPGYM SYQSS ISFWG PRLASHGFW MTIDTNRISD QPPSRRDQIE AALEYLVDQS NSSRSPINGM VDPNRLGAVG WSMGGGGTLR LAADDGIQAA IGLAPWNTSS LGFRS IETPT LI FACERDS I APVRSHASPF YNAIPSSTDK AYFEISGGNH YCANGSNRYD ALLGKYGVAW MKLHLDQDQR YAPFLCGPNH ERDRQISEHR

[0048] STCPF

[0049] Dh3 was predicted to have activity based on sequence conservation and tertiary structure homology. The predicted structure of Dh3 had 87%, confidence

[0050] Dh3 is a dienelactone hydrolase family protein (WP_116302305.1) from Alkalilimnicola ehrlichii and has a query cover of 98% and identity of 71.58% to a well known PET degrading enzyme, PET2. A. ehrlichii is a Gram-negative arsenite-oxidising haloalkaliphilic gammaproteobacteria that was originally isolated from a lake. Alignment of the predicted structures with the closest known plastic-degrading enzyme showed that the majority had a root mean square deviation (R.MSD) of 0.904, which is considered a good alignment.

[0051] However, when tested, the efficiency of plastic-degradation by Dh3 was found to be low. This led the inventors to seek methods of enhancing plastic-degradation by Dh3 and other plastic-degrading enzymes. The present inventors investigated a novel approach to enhancing the activity of plastic-degrading enzymes, involving modulating the behaviour of the host bacteria.

[0052] Biofilm formation is a method of growth where bacteria are attached to an abiotic or biotic surface and encased in a protective polysaccharide matrix, as opposed to being "free" (planktonic) in a liquid medium. Figure 1 schematically illustrates these two modes of existence.

[0053] The inventors hypothesised that increased biofilm formation could enhance the rate of plastic degradation. Figure 1 schematically illustrates how biofilm formation could impact enzyme activity.

[0054] The principal benefit of driving the host bacteria towards forming biofilms is that it brings the bacteria into closer proximity with the surface of waste plastic and increases attachment of the bacteria to the plastic, which in turn will increase the local concentration of secreted plastic-degrading enzymes around their target substrate. The other major benefit is that the polysaccharide matrix will prevent the enzymes from being washed off the plastic.

[0055] To test this hypothesis, the inventors manipulated the cyclic di-GMP (CdiGMP) signalling system of bacteria. This signalling system is universal in eubacterial species and it has been shown that high levels of CdiGMP lead to high levels of biofilm formation. The levels of CdiGMP within a bacterial cell are controlled by two sets of enzymes.

[0056] Diguanylate cyclases (DGC) which have a GGDEF or GGEEF domain, synthesise CdiGMP, whereas phosphodiesterases (PDE) which have an EAL / HD-GYP domain break it down (McCarthy et al. (2017)). This process is illustrated schematically in Figure 2.

[0057] Experiments carried out by the inventors demonstrated that bacteria expressing DGCs exhibit increased biofilm formation. Furthermore, the inventors have shown that bacteria that secrete plastic-degrading enzymes biodegrade plastic more efficiently if they also express DGCs compared to bacteria that do not express DGCs.

[0058] It is therefore envisaged that micro-organisms able to express plasticdegrading enzymes can be modified to exhibit elevated levels of CdiGMP, and thus increased biofilm formation. This can be done, for example, by modifying or engineering the micro-organism to have increased DGC activity, for example by engineering it to express a heterologous DGC, or at least an active fragment thereof.

[0059] Such bacteria can then be used in a method of biodegrading plastic as schematically illustrated in Figure 3. For example, the modified microorganisms can be grown in batch culture to degrade plastic in bioreactors. It is also envisaged that the modified bacteria could be deployed in the field. For example, they could be deployed in landfill sites to degrade waste plastic discarded therein.

[0060] In an embodiment, a method of biodegrading waste plastic includes providing a biofilm-forming micro-organism 30 that expresses a plasticdegrading enzyme 32, wherein the micro-organism is modified to exhibit elevated levels of cyclic di-GMP; contacting the micro-organism with waste plastic to be degraded 34; and allowing the waste plastic 34 to be at least partially biodegraded by the plastic-degrading enzyme 32. In a specific embodiment, the micro-organism 30 has been modified by an expression construct such as a plasmid for expression of at least an active fragment of an enzyme that modulates levels of cyclic di-GMP. The expression construct encodes the enzyme that modulates levels of cyclic di-GMP or at least an active fragment of the enzyme that modulates levels of cyclic di-GMP. In some embodiments, the microorganism is one that is easy to grow in culture, such as E. coli, and has been genetically modified to express the plastic-degrading enzyme 32. The plastic-degrading enzyme 32 is any suitable plastic-degrading enzyme such as a PETase, an MHETase, an alkane hydroxylase, a hydrodroquinone peroxidase, an esterase, laccase or a mixture of any two or more of these

[0061] The micro-organism 30 in specific embodiments has been modified to exhibit elevated levels of cyclic di-GMP by being modified to have increased diguanylate cyclase activity, for example by being modified to express at least an active fragment of a heterologous diguanylate cyclase such as DgcC or WspR.

[0062] Example 1 - Transformation of E. co / / ' with plasmids expressing DGCs and plastic degrading enzymes

[0063] To validate that CdiGMP levels could be manipulated in a standard E. coli protein expression system (BL21), the inventors tested two different DGCs, DgcC (a native E. coli diguanylate cyclase) and WspR. (a high potency P. aeruginosa diguanylate cyclase). Both enzymes have previously been shown to increase biofilm formation when overexpressed (McCarthy et al. (2017); Leech (2017)). Plasmids containing either the coding sequence of dgcC or wspR were designed and synthesised commercially using the vector pCOLADuet-1 as the expression vector with the DGC sequence cloned into the pCOLADuet-1 multiple cloning site. This vector has a ColA replicon, a kanamycin resistance gene, a T7 promoter for IPTG inducible expression of cloned in genes and is commercially available from Merck / Sigma.

[0064] To enable this expression system to be able to degrade plastic, two PETases with known plastic-degrading activity, Dh3 and Tfcut2, were synthesised commercially and cloned into the multiple cloning site of the pET20b expression vector (GenScript). This vector has a pBR.322 replicon, an ampicillin resistance gene (a different selection marker to pCOLADuet-1), a T7 promoter for IPTG inducible expression of cloned in genes, SEC signal peptide (PelB) at start of ORF. Two different enzymes were selected to demonstrate the versatility of the approach for different plastic-degrading enzymes.

[0065] Bacterial strains with plasmids expressing DGCs and plastic-degrading enzymes were prepared using the following steps:

[0066] 1) Chemically competent BL21 (DE3) cells (Invitrogen) were thawed on ice.

[0067] 2) 1-5 pl plasmid DNA at a concentration >20 ng / pL of the following plasmid combinations pdgcC + pdh3, pdgc + ptfcut2, pwspR + pdh3, pwspR + ptfcut2, empty vector + pdgC, empty vector + pwspR was mixed with 50 pl chemically competent BL21 (DE3) cells (Invitrogen) and incubated on ice for 30 minutes. The empty plasmid was also transformed as a control. 3) Heat shock was performed at 42°C for 45 seconds, then incubated on ice for 2 minutes.

[0068] 4) Cells were then mixed with 250 pl LB and grown at 37 °C with agitation for 1 hour and plated on LB agar plates supplemented with 50 pg / mL kanamycin and 50 pg / mL ampicillin and were grown overnight at 37 °C.

[0069] 5) A single colony growing on this selective media was then used to inoculate 5mL of LB broth with 50 pg / mL kanamycin and 50 pg / mL ampicillin, which was allowed to grow at 37 °C overnight. This solution was then used to prepare bacterial stocks which involved 800pL culture being mixed with 200pL of 80% glycerol in a cryovial. This cryovial was then stored at -80 °C until further use.

[0070] Example 2 - Expression of DGCs and effect on biofilm formation

[0071] To determine if expression of these enzymes could impact biofilm formation a biofilm assay was performed.

[0072] Glycerol stocks from Example 1 of BL21 (DE3) pc / gcC + pdh3, BL21 (DE3) pdgc + ptfcut2, BL21 (DE3) pwspR + pdh3fBL21 (DE3) pwspR + ptfcut2, BL21 (DE3) empty vector + pdgC, BL21 (DE3) empty vector + pwspR were used to inoculate 5ml of LB broth in a 25ml glass vial and grown in LB broth at 37 °C overnight.

[0073] Overnight cultures were then used to inoculate 3ml fresh LB at OD6oo 0.1 in glass test tubes then grown with agitation at 37 °C. The cultures were induced with ImM IPTG at ~ODeoo 0.6 and grown for a total of 24 hours. The biofilm assay was performed with 5ml pipettes to reduce biofilm dislodging. The culture was removed, then the test tube was washed 3 times with distilled water, 5ml 0.1% crystal violet was added for 10 minutes then removed and the test tube was washed 5 times with distilled water. The test tubes were fully air dried at room temperature then photographed for visual inspection.

[0074] Figure 4 is a series of images demonstrating the increased levels of biofilm formation (visible as the darker (purple) ring in each test tube) induced by each diguanylate cyclase (DgcC and WspR).

[0075] Each image is of a different plastic-degrading and diguanylate cyclase expressing enzyme combination, with appropriate empty vector controls. The first image is BL21 (DE3) empty vector control. The culture used to prepare the left-most tube in each image expressed no DGC but does have the empty pCOLADuet-1 plasmid as a control. The middle tube in each image expressed DgcC, and the right-most tube in each image expressed WspR.. In each image, a darker (purple) ring can be seen in the tubes that had the bacteria expressing a DGC, indicating higher levels of biofilm formation in these strains.

[0076] The results of this assay showed that expression of either of DgcC or WspR increased biofilm formation in E. coli BL21. This demonstrates that both can influence biofilm formation in a standard bacterial protein expression host E. coli BL21 that has another plasmid expressing a plastic-degrading enzymes Example 3 - Degradation of PCL by E. coli expressing DGCs

[0077] To determine if the increased levels of biofilm formation, observed in Example 2, could have an impact on the plastic-degrading activity of the known plastic degrading enzymes Tfcut2 and Dh3, a weight loss assay was performed. Weight loss of the model plastic substrate polycaprolactone (PCL) was measured after 5 days incubation (with media refreshed after 3 days due to Ampicillin instability) with E. coli BL21 (DE3) expressing the enzyme + / - DgcC or WspR. in combination with either pdh3, ptfcut2 or the pET20b empty vector.

[0078] For the PCL weight loss assay, overnight cultures of E. coli BL21 (DE3) grown in LB broth at 37 °C were used to inoculate 10 ml fresh LB at OD6OO 0.1 containing sterilised PCL beads. The cultures were grown at 37 °C with agitation until ODeoo 0.6, then were induced with ImM IPTG and grown for 5 days. The culture media was refreshed every 3 days due to Ampicillin instability (culture collected, centrifuged at 5000 x g for 15 minutes, pellet resuspended in fresh media and returned to flask). The PCL beads were collected using a fine mesh sieve (63 pm), rinsed with distilled water, washed on a room temperature rocker in 2% sodium dodecyl sulphate overnight to remove biofilm, rinsed with distilled water and dried before weighing. Statistical analysis and graph construction were performed in Prism (version 9.4.1). Control weight loss flasks with uninoculated media and plastic were used to correct the weight loss measurements to account for instrument error so that the control had 0% weight loss.

[0079] Figure 5 is a graph showing PCL weight loss after 5 days when incubated with BL21 (DE3) empty vector (EV), Dh3 or Tfcut2 with and without co-expression of DgcC or WspR. Mean and StEM of seven independent experiments, * is p <0.05, ** is p <0.01, one-way ANOVA was used for statistical comparison.

[0080] Figure 6 shows examples of PCL beads from one experiment for Dh3 (top) and Tfcut2 (bottom) with and without DgcC and WspR.

[0081] In each case, the presence of the DGC and therefore a strain with increased biofilm exhibited better PCL degradation (Figure 5). Representative images of the degraded PCL beads after 5 days are included to show the difference in PCL degradation between the enzymes with normal levels of biofilm and the enzymes with increased biofilm (Figure 6).

[0082] It is notable that the improvement was seen even for a plasticdegrading enzyme (Dh3) having relatively low efficiency when not enhanced by DGC expression.

[0083] The results shown in Figures 5 and 6 demonstrate that increased biofilm formation increases the average weight loss of PCL and therefore that increasing levels of biofilm formation leads to increased levels of plastic degradation. In view of these results, it can be expected that increasing biofilm improves access to and degradation of plastic so would have the same effect on PET. Plastic biodegradation systems, whether batch culture degradation systems or systems involving deployment in the field (for example, application of bacteria to landfill sites) could therefore benefit from this method by co-expression of DGCs. Example 4 - Preparation of plastic-degrading bacterial strains

[0084] Methods for preparation of plastic-degrading bacterial strains with plasmids expressing DGCs can include the steps of:

[0085] 1) Competent cells of a suitable bacterial expression host, such as such as BL21 (DE3), are thawed on ice. To ensure secretion of the plastic-degrading enzyme a secretion signal should be added to the coding sequence if one is not already present and the sequence of the plastic-degrading enzyme should be codon optimised for expression in the selected host.

[0086] 2) Plasmid (for example pCOLADuet-1, as above) encoding a desired DGC (for example pc / gcC or pwspR which have both shown to be active in E. coli expression systems) is added to the competent cells, for example, at a concentration of 1-5 pl of plasmid DNA at a concentration > 20 ng / pL .

[0087] 3) Cells and plasmid are incubated on ice for 20 minutes before being heated to 43 °C for 30 secs.

[0088] 4) 1ml of a nutrient rich broth is then be added and cells allowed to recover a 37°C for 1 hour before being plated on selective media.

[0089] The above method is an example only. Depending on the specific requirements, other bacterial strains could be used. For example, BL21 (DE3) plysS (which facilitates lower background expression of target genes), Rosetta (DE3) (designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli), Tuner DE3 (enables adjustable levels of protein expression throughout all cells in a culture), E. coli HMS174 (DE3), E. coli NiCo21(DE3), Lactococcus lactis pl70 expression system (for expression of proteins derived from a Gram positive organism), Lactococcus lactis NICE expression system (for expression of proteins derived from a Gram positive organism), B. subtilis Secretory Protein Expression System (for expression of proteins derived from a Gram positive organism - the strain secretes protein directly into culture media and is particularly amenable to large-scale fermentation).

[0090] The skilled person would appreciate that other suitable plasmids are available for expression of the DGC as long as they can facilitate the inducible or constitutive expression of the desired DGC and is capable of replicating in the selected host organism. Possible plasmids include pBluescript II KS(-),pBluescript II KS(+), pBluescript II SK(-), pBluescript II SK(+), pET-3a, pET-3b, pET-3c, pET-3, pET-9a, pET-lla, pET-llb, pET-llc, pET-lld, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b(+), pET-21a(+), pET-21b(+), pET-21d(+), pET- 22b(+), pET-23a(+), pET-24a(+), pET-24b(+), pET-24c(+), pET- 24d(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET- 28b(+), pET-28c(+), pET-29a(+), pET-29b(+), pET-29c(+), PET- 30a(+), PET-30b(+), PET-30c(+), PET-31b(+), pET-32a(+), pET- 32b(+), pET-41a(+), pET-41b(+), pET-41c(+), pET-42a(+), pET- 42b(+), pET-42c(+), pET-43. la(+), pET-43. lb(+), pET-45b(+), pET- 50b(+), pET-51b(+), pET-52b(+), pET-24a(+)-TEV, pET-28a(+)-TEV, pGEX-2TK, pGEX-4T-l, pGEX-4T-2, pGEX-4T-3, pGEX-5X-l, pGEX-5X- 2, pGEX-5X-3, pGEX-6P-l,pGEX-6P-2, pGEX-6P-3, pMAL-c4x, pMAL- c5E, pMAL-c5x, pMAL-p5E, pMAI-p5g, pMAI-p5x, pQE-1, pQE-60, pGS- 21a, pETDuet-1, pCDFDuet-1, pRSFDuet-1, pGEX-4T-l-H(RBS), pGEX- 4T-l-M(RBS),pGEX-5X-l-H(RBS), pGEX-5X-l-M(RBS), pGEX-6P-l-

[0091] H(RBS), pGEX-6P-l-M(RBS), pMAL-c4x-l-H(RBS), pMAL-c4x-l-M(RBS) all of which are commercially available.

[0092] The antibiotic selection marker should be different to the selection marker used for the plasmid expressing the plastic-degrading enzyme.

[0093] The skilled person will appreciate that each of the expression systems described above will have optimal transformation conditions which should be followed for best transformation efficiencies.

[0094] Example 5 - Use of bacteria expressing a DGC in a method of biodegrading plastic

[0095] Bacteria expressing DGC can be used in a batch reactor to degrade waste plastics, for example using a method such as the following:

[0096] 1) Inoculate 50ml LB in a 250ml flask with glycerol stocks of host bacteria containing IPTG inducible DGC and plastic-degrading enzyme, and grow in LB broth at 37 °C with appropriate antibiotics added (for example for pdgcC and ptfcut2, 50 pg / mL ampicillin and kanamycin would be added).

[0097] 2) Inoculate a 100L batch reactor containing 10L of LB medium with the culture of step 1 to an OD6oo of 0.1.

[0098] 3) Add 1 kg of waste PET to the batch reactor. 4) Grow the cultures at 37 °C with agitation until OD6oo of 0.6 is reached, then induce with ImM IPTG and grow for 5 days with regular refreshing with fresh antibiotics.

[0099] 5) After desired incubation period, batch reactor could be stopped and valorisation products such as terephthalic acid (TPA) and Mono-(2- hydroxyethyl)terephthalic acid (MHET) and ethylene glycol (EG) could be separated from undegraded plastic.

[0100] This work has confirmed that using DGCs to manipulate CdiGMP levels, thereby increasing biofilm formation, is a promising approach to enhance plastic degrading activity of different plastic degrading enzymes expressed by bacteria that form biofilm. Of course, the skilled person would appreciate that other ways of modulating CdiGMP levels could also work. For example, inhibiting the action of PDEs, resulting in the accumulation of CdiGMP in the cell.

[0101] The skilled person would appreciate that modifications could be made to the above-described methods.

[0102] For example, plastic-degrading activity by native bacteria (i.e. those that naturally express and secrete a plastic-degrading enzyme) could be enhanced, for example by transforming these native bacteria with a plasmid expressing a DGC as described above. However, it is believed that for most practical purposes, and for ease of culture and scaling-up, transformation of an optimised micro-organism with a construct for expression of an appropriate plastic-degrading enzyme is appropriate. The skilled person would be well aware of how to achieve this. Where a micro-organism is engineered to express the plastic-degrading enzyme, it is possible to have both enzymes (the plastic-degrading enzyme and the DGC) expressed from the same plasmid if the plasmid has two multiple cloning sites.

[0103] Although the method has been exemplified with E. coli DgcC and P. aeruginosa WspR, any other suitable alternative DGC could be used. Essentially any DGC or fragment thereof including GGDEF or GGEEF domain, as these are associated with CdiGMP synthesis and thus increasing intracellular levels of CdiGMP. Another possibility would be to modify the inhibitory site, if one is present, to enhance the activity of the DGC.

[0104] The methods in this application have focussed on the use of PETases. However, they are applicable to any plastic-degrading enzyme. For example, MHETases can, in combination with PETases, degrade PET to high-value end-products such as TPA and EG. Other possibilities include, without limitation, alkane hydroxylases, which have been shown to act on polyethylene, hydrodroquinone peroxidases which have been shown to act on styrenes, laccases, which oxidise a variety of phenolic substrates, and esterases with activity on Low Density PE.

[0105] The inventors have developed a novel methodology to improve the overall performance of plastic-degrading enzymes by modulating the levels of biofilm formation by the host bacteria in which the enzymes are expressed. By modifying the bacteria to exhibit elevated levels of CdiGMP, biofilm formation is increased and the action of the secreted plastic-degrading enzyme is enhanced, thereby providing an improved method of plastic-biodegradation.

[0106] All optional and preferred features and modifications of the described embodiments and dependent claims are usable in all aspects of the invention taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.

[0107] It will be appreciated by those skilled in the art that changes could be made to the embodiments and examples described above without departing from the broad inventive concept. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the claims.

[0108] The disclosures in GB 2219396.5, from which this application claims priority, and in the accompanying abstract are incorporated by reference in their entirety.

[0109] References:

[0110] Herrero Acero E, Ribitsch D, Steinkellner G, Gruber K, Greimel K, Eiteljoerg I, Trotscha E, Wei R, Zimmermann W, Zinn M. Enzymatic surface hydrolysis of PET: effect of structural diversity on kinetic properties of cutinases from Thermobifida. Macromolecules. 2011;44(12):4632-40. Kenny ST, Runic JN, Kaminsky W, Woods T, Babu RP, Keely CM, Blau W, O'Connor KE. Up-Cycling of PET (Polyethylene Terephthalate) to the Biodegradable Plastic PHA (Polyhydroxyalkanoate). Environmental Science & Technology. 2008;42(20):7696-701

[0111] Leech JT. Development of an Escherichia coli Biofilm Platform for use in Biocatalysis: University of Birmingham; 2017.

[0112] Lu H, Diaz DJ, Czarnecki NJ, Zhu C, Kim W, Shroff R, Acosta DJ, Alexander BR, Cole HO, Zhang Y, Lynd NA, Ellington AD, Alper HS. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature. 2022;604(7907):662-7.

[0113] McCarthy RR, Mazon-Moya MJ, Moscoso JA, Hao Y, Lam JS, Bordi C, Mostowy S, Filloux A. Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion. Nat Microbiol. 2017 Mar 6;2: 17027.

[0114] Ribitsch D, Acero EH, Greimel K, Eiteljoerg I, Trotscha E, Freddi G, Schwab H, Guebitz GM. Characterization of a new cutinase from Thermobifida alba for PET-surface hydrolysis. Biocatalysis and Biotransformation. 2012;30(l):2-9.

Claims

CLAIMS1. A method of biodegrading waste plastic including: providing a biofilm-forming micro-organism that expresses a plastic-degrading enzyme, wherein the micro-organism is modified to exhibit elevated levels of cyclic di-GMP; contacting the micro-organism with waste plastic to be degraded; and allowing the waste plastic to be at least partially biodegraded by the plastic-degrading enzyme.

2. A method as claimed in claim 1, wherein the micro-organism has been modified to express the plastic-degrading enzyme or at least an active fragment thereof or wherein the micro-organism has been modified to enhance expression of a native plastic-degrading enzyme or at least an active fragment thereof.

3. A method of enhancing the activity of a plastic-degrading enzyme expressed by a biofilm-forming micro-organism, wherein the microorganism is modified to express the plastic-degrading enzyme or at least an active fragment thereof or wherein the micro-organism is modified to enhance expression of a native plastic-degrading enzyme or at least an active fragment thereof, including modifying the microorganism to exhibit elevated levels of cyclic di-GMP.

4. A method as claimed in claim 1, 2 or 3, wherein the microorganism is modified to have increased diguanylate cyclase activity.

5. A method as claimed in any preceding claim, wherein the microorganism is modified to express a heterologous diguanylate cyclase or at least an active fragment thereof.

6. A method as claimed in claim 5, wherein the diguanylate cyclase is an E. coli diguanylate cyclase.

7. A method as claimed in claim 5 or 6, wherein the diguanylate cyclase is DgcC.

8. A method as claimed in claim 5, wherein the diguanylate cyclase is a P. aeruginosa diguanylate cyclase.

9. A method as claimed in claim 5 or 8, wherein the diguanylate cyclase is WspR.

10. A method as claimed in any preceding claim, wherein the microorganism is a bacterium.

11. A method as claimed in claim 10, wherein the bacterium is E. coli.

12. A method as claimed in any preceding claim, wherein the plasticdegrading enzyme is a PETase.

13. An expression construct for use in a method as claimed in any preceding claim, the expression construct for expression of at least an active fragment of an enzyme that modulates levels of cyclic di-GMP by a biofilm-forming micro-organism able to express a plastic-degrading enzyme, wherein the expression construct encodes the enzyme thatmodulates levels of cyclic di-GMP or at least an active fragment of the enzyme that modulates levels of cyclic di-GMP, wherein the expression construct also encodes at least an active fragment of a plasticdegrading enzyme.

14. An expression construct as claimed in claim 13, wherein the enzyme that modulates levels of cyclic di-GMP is a diguanylate cyclase.

15. An expression construct for use in a method as claimed in any of claims 1 to 12, the expression construct for expression of at least an active fragment of diguanylate cyclase by a biofilm-forming microorganism able to express a plastic-degrading enzyme, wherein the expression construct encodes the diguanylate cyclase or at least an active fragment of the diguanylate cyclase, wherein the diguanylate cyclase is an E. coli diguanylate cyclase or a P. aeruginosa diguanylate cyclase.

16. An expression construct as claimed in claim 14 or 15, wherein the diguanylate cyclase is an E. coli diguanylate cyclase.

17. An expression construct as claimed in claim 14, 15, or 16, wherein the diguanylate cyclase is DgcC.

18. An expression construct as claimed in claim 14 or 15, wherein the diguanylate cyclase is a P. aeruginosa diguanylate cyclase.

19. An expression construct as claimed in claim 14, 15 or 18, wherein the diguanylate cyclase is WspR.

20. An expression construct as claimed in any of claims 15 to 19, wherein the expression construct also encodes at least an active fragment of a plastic-degrading enzyme.

21. An expression construct as claimed in any of claims 13 to 20, wherein the expression construct is a plasmid.

22. A biofilm-forming micro-organism for use in a method as claimed in any of claims 1 to 12, wherein the micro-organism has been transformed by an expression construct as claimed in any of claims 13 to 21.

23. A biofilm-forming micro-organism for use in a method as claimed in any of claims 1 to 12, wherein the micro-organism has been transformed by an expression construct as claimed in any of claims 15 to 19 or 21 and also by an expression construct encoding at least an active fragment of a plastic-degrading enzyme.

24. A micro-organism as claimed in claim 22 or 23, wherein the micro-organism is a bacterium.

25. A micro-organism as claimed in claim 24, wherein the bacterium is E. coli.

26. A micro-organism as claimed in any of claims 22 to 25, wherein the plastic-degrading enzyme is a PETase.

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