Recombinant Saccharomyces cerevisiae strains for enzymatic hydrolysis of bioplastic polymers

Abstract

The present invention relates to a method for producing a cutinase-like enzyme (CLE1) in a S. cerevisiae cell, comprising heterologously expressing in the cell a codon-optimized nucleic acid encoding the cutinase-like enzyme and operably linked to an engineered promoter. The invention further relates to recombinant S. cerevisiae cells capable of heterologously expressing the cutinase-like enzyme, and the cutinase-like enzyme obtained from the cells or prepared by the method. Also provided is a method for preparing a cell-free supernatant comprising the cutinase-like enzyme from the recombinant S. cerevisiae cells, and the use of the cell-free supernatant or the recombinant S. cerevisiae cells in the hydrolysis of bioplastic polymers.

Description

[Technical Field]

[0001] The present invention provides a method for producing a cutinase-like enzyme (CLE) in S. cerevisiae cells, comprising heterologous expression of a codon-optimized nucleic acid encoding the cutinase-like enzyme under the control of an engineered yeast constitutive promoter in the cells. The present invention further relates to recombinant S. cerevisiae cells capable of heterologous expression of the cutinase-like enzyme. The cutinase-like enzyme produced by the method or cells is useful for the hydrolysis of bioplastic polymers. Also provided are methods for preparing a cell-free supernatant containing the cutinase-like enzyme from recombinant S. cerevisiae cells, and the use of the cell-free supernatant in the hydrolysis of bioplastic polymers. [Background technology]

[0002] Bioplastics are broadly defined as plastics produced from biomass, plastics that can be decomposed in natural or industrial environments, or plastics that possess both of these properties. While the bioplastics industry accounts for only a small portion of the global plastics industry, its share of the global plastics market is steadily growing and is expected to become a $13.1 billion industry by 2027. Current waste management strategies for biodegradable bioplastics, such as starch blends and polylactic acid (PLA), two of the main drivers of the bioplastics industry, involve forming part of conventional organic waste treatment facilities. PLA is one of the most popular biodegradable bioplastics due to its high mechanical strength, high modulus, biodegradability, biocompatibility, bioresorbability, transparency, energy efficiency, low toxicity, and processability. Its monomer (lactic acid) is produced from plant biomass via microbial fermentation, and the lactic acid is then polymerized to form PLA. Anaerobic digestion and composting are currently the preferred end-of-life treatments for these materials and will continue to play this role for the foreseeable future. Although PLA is considered biodegradable, recent studies have shown that some bioplastics persist during processing, resulting in reduced product value and processing limitations. Furthermore, life cycle analyses have confirmed that new, efficient recycling systems should be prioritized for the long-term sustainable use of these materials.

[0003] Microbial hydrolytic enzymes capable of bioplastic degradation have previously been identified, and their use for large-scale bioplastic processing may have several advantages over current systems. Hydrolytic enzymes can speed up conventional processes for bioplastic processing, reduce the risk of damaging composting and anaerobic digestion systems, and remove these materials from the final product. Furthermore, these enzymes function at mild temperatures and pH levels, have specificity for certain polymers, and can deliver pure monomers, paving the way for cradle-to-cradle recycling systems for bioplastics. However, there are currently no commercially available enzyme preparations specifically designed for large-scale bioplastic processing. Several factors still need to be addressed for enzyme-based strategies to become a viable option for large-scale bioplastic processing, either as a standalone treatment or in combination with existing strategies. First, a cost-effective and scalable enzyme production process is needed to produce cocktails that enable efficient bioplastic degradation. Second, the enzyme-based treatment process needs to be simplified to function efficiently at mild temperatures and pH levels, without requiring additional pretreatment of the material or the use of solvents, emulsifiers, and other chemical catalysts.

[0004] Research papers describing the use of exoenzymes for bioplastic processing have focused on traditional prokaryotic hosts, such as Escherichia coli, in recombinant enzyme production systems or have utilized non-traditional prokaryotic and eukaryotic hosts to produce native exoenzymes. The use of traditional eukaryotic hosts to produce recombinant enzymes offers several advantages over prokaryotic and non-traditional eukaryotic systems. These include established genetic engineering protocols, improved enzyme production titers, complex protein production and secretion mechanisms, ease of protein purification, GRAS status, growth on inexpensive substrates, and the use of existing industrial processes and infrastructure for large-scale cultivation.

[0005] Fungi are known to produce a myriad of hydrolytic enzymes. Indeed, several fungal hydrolases capable of plastic degradation have been characterized, but their applications remain relatively underdeveloped, especially compared to bacterial enzymes. Cutinase-like enzymes (CLEs) are of particular interest because they have been shown to hydrolyze a wide range of bioplastics. These enzymes are typically associated with plant pathogens, including several fungal species. Therefore, traditional industrially applicable fungal hosts are particularly adept at producing extracellular enzymes derived from other eukaryotes, including other fungi. Still, significant hurdles must be overcome when overexpressing native eukaryotic genes in other eukaryotes traditionally used in industry, typically resulting in low expression levels of the native genes. Major bottlenecks in the heterologous expression of native fungal genes include, among others, suboptimal GC content, low codon bias index, the presence of tandem rare codons, the lack of strong promoters, and efficient secretion to drive enzyme production. Therefore, the inventors of the present invention used molecular techniques and genetic engineering approaches in industrially applicable fungal hosts for the improved production of bioplastic-degrading enzymes, including CLEs, for application during the processing of bioplastic polymers.

[0006] Specifically, the present inventors have constructed a genetically modified, industrially applicable eukaryotic host that exhibits enhanced expression of the CLE1 gene from Cryptococcus sp. S-2 and increased hydrolysis of bioplastics. Summary of the Invention

[0007] The present invention relates to a method for producing a cutinase-like enzyme (CLE1) in S. cerevisiae cells, the method comprising heterologously expressing in the cells a codon-optimized nucleic acid encoding the cutinase-like enzyme and operably linked to an engineered promoter. The present invention also relates to recombinant S. cerevisiae cells capable of heterologously expressing the cutinase-like enzyme, and the cutinase-like enzyme obtained from the cells or prepared by the described methods. The present invention further relates to a method for preparing a cell-free supernatant containing the cutinase-like enzyme from recombinant S. cerevisiae cells, and the use of the cutinase-like enzyme in the hydrolysis of bioplastic polymers.

[0008] According to a first aspect of the present invention, there is provided a method for producing a cutinase-like enzyme (CLE1) in an S. cerevisiae cell, the method comprising heterologously expressing a nucleic acid encoding CLE1 (CLE1 gene) in the cell, wherein the nucleic acid encoding CLE1 is codon-optimized for expression in S. cerevisiae, and further wherein the nucleic acid encoding CLE1 is operably linked to an engineered promoter.

[0009] In a first embodiment of the method, the cutinase-like enzyme may have the amino acid sequence of SEQ ID NO:2.

[0010] According to a second embodiment of the method, the nucleic acid encoding CLE1 has a nucleotide sequence having at least about 80%, at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 1. In one embodiment of the method, the nucleic acid encoding CLE1 has a nucleotide sequence substantially identical to SEQ ID NO: 1.

[0011] In a third embodiment of the method of the present invention, the engineered promoter may be a TDHi engineered promoter having at least about 80%, at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:9, or a nucleotide sequence substantially identical to SEQ ID NO:9, or a TEF1i engineered promoter having at least about 80%, at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:7, or a nucleotide sequence substantially identical to SEQ ID NO:7. It will be appreciated by those skilled in the art that other engineered promoters may be suitable for producing the CLE1 enzyme from the CLE1 gene, including, but not limited to, promoters selected from the group consisting of ENO1i, ADH2i, TDH3i, HXT7i, ENO1cxi, TDH3cxi, HXT7cxi, and TEF1cxi engineered promoters.

[0012] According to a fourth embodiment of the method of the present invention, the CLE1 enzyme may comprise a secretion signal. Suitably, the secretion signal may have the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

[0013] In further embodiments of the method of the invention, the S. cerevisiae cells may be of the S. cerevisiae strain Y294, S. cerevisiae strain Ethanol Red V1, S. cerevisiae strain M2n, or S. cerevisiae strain YI30.

[0014] In one embodiment of the method of the present invention, the method may further comprise culturing the S. cerevisiae cells to obtain a population of S. cerevisiae cells.

[0015] In yet another embodiment of the method of producing the CLE1 enzyme of the present invention, the method may further comprise preparing a cell-free supernatant from the population of S. cerevisiae cells, wherein the cell-free supernatant comprises the CLE1 enzyme.

[0016] According to a second aspect of the present invention, there is provided a recombinant S. cerevisiae cell comprising a nucleic acid encoding a cutinase-like enzyme (CLE1), wherein the nucleic acid encoding CLE1 is codon-optimized for expression in S. cerevisiae, and the nucleic acid encoding CLE1 is operably linked to an engineered promoter, and wherein the recombinant S. cerevisiae cell is capable of heterologously expressing CLE1.

[0017] In a first embodiment of the recombinant S. cerevisiae cell, the cutinase-like enzyme may have the amino acid sequence of SEQ ID NO:2.

[0018] According to a second embodiment of the recombinant S. cerevisiae cell of the invention, the nucleic acid encoding CLE1 has a nucleotide sequence having at least about 80%, at least about 90%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to SEQ ID NO: 1. In one embodiment of the recombinant S. cerevisiae cell, the nucleic acid encoding CLE1 has a nucleotide sequence substantially identical to SEQ ID NO: 1.

[0019] In a third embodiment of the recombinant S. cerevisiae cell of the present invention, the engineered promoter may be a TDHi engineered promoter having at least about 80%, at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:9, or a nucleotide sequence substantially identical to SEQ ID NO:9, or may be a TEF1i engineered promoter having at least about 80%, at least about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:7, or a nucleotide sequence substantially identical to SEQ ID NO:7. It will be appreciated by those skilled in the art that other engineered promoters may be suitable for producing the CLE1 enzyme from the CLE1 gene, including, but not limited to, promoters selected from the group consisting of ENO1i, ADH2i, TDH3i, HXT7i, ENO1cxi, TDH3cxi, HXT7cxi, and TEF1cxi engineered promoters.

[0020] According to a fourth embodiment of the recombinant S. cerevisiae cell of the present invention, the CLE1 enzyme may comprise a secretion signal. Suitably, the secretion signal may have the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 5.

[0021] In further embodiments of the recombinant S. cerevisiae cell of the invention, the S. cerevisiae cell may be of the S. cerevisiae strain Y294, S. cerevisiae strain Ethanol Red V1, S. cerevisiae strain M2n, or S. cerevisiae strain YI30.

[0022] According to a third aspect of the present invention, there is provided a cutinase-like enzyme produced by the method of the invention described herein or heterologously produced by the recombinant S. cerevisiae cell of the invention described herein. In one embodiment, the cutinase-like enzyme may have the amino acid sequence of SEQ ID NO:2.

[0023] A fourth aspect of the present invention provides a method for preparing a cell-free supernatant comprising a cutinase-like enzyme, the method comprising culturing a recombinant S. cerevisiae cell of the present invention as described herein to obtain a population of S. cerevisiae cells, and preparing a cell-free supernatant from the population of S. cerevisiae cells.

[0024] In one embodiment of the method for preparing a cell-free supernatant, the method may further comprise concentrating the cell-free supernatant. Suitable methods for concentrating the cell-free supernatant include, but are not limited to, concentrating the cell-free supernatant by lyophilization, filtration, and / or chromatography.

[0025] According to a fifth aspect of the present invention there is provided a cell-free supernatant prepared by the methods described herein.

[0026] Another aspect of the invention contemplates the use of a cutinase-like enzyme produced by the methods of the invention described herein, or heterologously produced by a recombinant S. cerevisiae cell of the invention described herein, or the use of a cell-free supernatant prepared by the methods described herein, to hydrolyze a polymer.

[0027] The present invention also provides a method for hydrolyzing bioplastic polymers, comprising incubating the polymer with a cutinase-like enzyme produced by the methods of the present invention described herein, or a cutinase-like enzyme heterologously produced by a recombinant S. cerevisiae cell of the present invention described herein, or a cell-free supernatant prepared by the methods of the present invention described herein. Suitable bioplastics hydrolyzed by the cell-free supernatant prepared by the methods described herein include, but are not limited to, poly(L-lactide) (PLLA), poly(D-lactide) (PDLA), and poly(DL-lactide) (PDLLA), polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polyhydroxyalkanoates (PHAs), and polylactic acid (PLA), including thermoplastic starch (TPS) blends, and any combination or mixture thereof.

[0028] According to further embodiments of the method for hydrolyzing a bioplastic polymer, incubating the polymer with the cutinase-like enzyme or cell-free supernatant can be performed at about 42°C to about 45°C (such as at about 42°C to about 45°C and a pH of about 6.8 to 7).

[0029] In a further aspect of the invention, there is provided a method for hydrolyzing a bioplastic polymer, comprising incubating the bioplastic polymer with a recombinant S. cerevisiae cell of the invention or an S. cerevisiae cell expressing a cutinase-like enzyme of the invention. In one embodiment, the recombinant S. cerevisiae cell may be a mutant strain that is unable to consume the hydrolysis products of the method for hydrolyzing the bioplastic polymer.

[0030] Also contemplated in accordance with the present invention is a bioreactor comprising the recombinant S. cerevisiae cells of the invention described herein, wherein the bioreactor is configured for the heterologous production of cutinase-like enzymes by the recombinant S. cerevisiae cells and subsequent hydrolysis of bioplastic polymers by the cutinase-like enzymes.

[0031] Non-limiting embodiments of the present invention will now be described, by way of example only, with reference to the following figures: [Brief explanation of the drawings]

[0032] [Figure 1]Plasmid maps of the S. cerevisiae expression vectors described herein. A = Plasmid pBBH4 is used herein as a control and does not contain a gene encoding the CLE1 enzyme; B = pBBH4-CLExs plasmid was constructed to contain a codon-optimized gene (SEQ ID NO: 1) encoding a cutinase-like enzyme from Cryptococcus sp. S-2 with an XYNSEC (SEQ ID NO: 5) secretion signal; C = pBBH4-CLEns plasmid was constructed to contain a codon-optimized gene encoding a cutinase-like enzyme from Cryptococcus sp. S-2 with a native secretion signal (SEQ ID NO: 3) and an engineered TEF1i promoter (SEQ ID NO: 7) driving expression. D = Plasmid pBBH4-CLEwt was constructed to contain the native cutinase-like enzyme from Cryptococcus sp. S-2 (SEQ ID NO: 8) with its native secretion signal (SEQ ID NO: 3) and an engineered TEF1i promoter (SEQ ID NO: 7) driving its expression; E = pBBH4-CLEns-TDHi was constructed to contain the codon-optimized gene encoding the cutinase-like enzyme from Cryptococcus sp. S-2 (SEQ ID NO: 1) with its native secretion signal (SEQ ID NO: 3) and an engineered TDHi promoter (SEQ ID NO: 9) driving its expression. [Figure 2] SDS-PAGE gel showing that the CLE1 protein species (black arrow) is present in the supernatants of the Y294[CLExs] and Y294[CLEns] strains, while the protein species is absent in the supernatant of the Y294[BBH] strain. Molecular markers are indicated on the far left. [Figure 3] Photograph showing hydrolysis halo formation by Y294[CLExs], Y294[CLEns], and Y294[BBH] strains on tributyrin-containing agar plates. A larger hydrolysis halo is observed for the Y294[CLExs] strain compared to Y294[CLEns], and no halo is observed around the Y294[BBH] control. [Figure 4]A turbidity-based assay was developed to assess whether secreted CLE1 protein species were active in hydrolyzed, emulsified PLA substrates (solid lines = PLLA; dashed lines = PDLA, representing polymers derived from L- and D-lactic acid isomers, respectively). Proteinase-K (0.5 mg / mL in 0.1 M KH2PO4 buffer) (hereafter referred to as commercially available ProK (●)) was used as a positive control during the PLLA assay. Y294[BBH] (◆), Y294[CLEns] (■), and Y294[CLExs] (▲) yeast strains were cultured in 2x SC-URA medium, and activity was measured after 24, 48, and 72 hours of growth. Error bars represent the standard deviation from the mean of three replicates. The ProK positive control delivered consistent activity across multiple assay rounds on PLLA substrates, whereas the Y294[BBH] control showed no significant activity on either substrate. Both recombinant yeast strains showed increasing activity over time on both PLA substrates. [Figure 5] Graph showing the percentage of hydrolysis of PDLA (dotted bars) and PLLA (striped bars) emulsions after 72 hours of hydrolysis using cell-free supernatants from various yeast strains and commercial ProK. Error bars represent standard deviations from the mean values ​​of three replicates. Complete hydrolysis of PDLA emulsions was achieved by the recombinant Y294[CLEns] and Y294[CLExs] strains after 72 hours of hydrolysis, whereas the Y294[BBH] control and commercial ProK did not show a significant decrease in turbidity. Near-complete hydrolysis of PLLA emulsions was also observed after 72 hours of hydrolysis for both the recombinant Y294[CLEns] and Y294[CLExs] strains and the commercial ProK positive control. [Figure 6] HPLC analysis results showing lactate production from various substrate loadings of PLLA (solid line) and PDLA (dashed line) powder. A = 4 g / L; B = 10 g / L, and C = 25 g / L PLA powder substrate loadings. Error bars represent standard deviation from the mean of three replicates. It is clear that lactate production titers increase with increasing substrate concentration. The Y294[CLEns] strain outperformed the Y294[CLExs] control strain with both substrates. [Figure 7]Lactic acid production ratio and productivity during cell-free hydrolysis of PLA powder. Cn represents the lactic acid concentration at each time point, and Cf is the final concentration of lactic acid produced in each setup. The ratio of lactic acid produced at each time point to the final concentration (%) as well as the lactic acid productivity (g / L / h) is given. Shading indicates the performance of the strain at each time point (darker shading = better performance). NA = not available. [Figure 8] HPLC analysis results showing lactic acid production from PLLA (solid line) and PDLA (dashed and dotted lines) films over a 10-day hydrolysis period at 37°C. Error bars represent standard deviation from the mean of three replicates. Significant amounts of lactic acid are produced by hydrolyzing PLA films with supernatants from recombinant Y294[CLEns] and Y294[CLExs] strains. [Figure 9] Lactic acid production ratio and productivity during cell-free hydrolysis of PLA film. Cn represents the lactic acid concentration at each time point, and Cf is the final concentration of lactic acid produced in each setup. The ratio of lactic acid produced at each time point to the final concentration (%) as well as lactic acid productivity (g / L / h) is given. Shading indicates strain performance at each time point (darker shading = better performance). [Figure 10] SEM images of PDLA film surfaces during hydrolysis tests. A = PDLA film before treatment with cell-free supernatant; B = PDLA film surface incubated with cell-free supernatant from Y294[BBH] cells for 168 hours; C = PDLA film surface incubated with cell-free supernatant from Y294[CLEns] cells for 168 hours. A clear hydrolysis pattern was observed in the film incubated with the supernatant from the recombinant Y294[CLEns] strain, but no signs of hydrolysis were observed in the Y294[BBH4] strain. [Figure 11] SEM images of PDLA film surfaces during hydrolysis tests. A = PDLA film surface incubated with cell-free supernatant from Y294[CLEns] cells for 24 hours; B = PDLA film surface incubated with cell-free supernatant from Y294[CLEns] cells for 48 hours; white arrows indicate hydrolyzed amorphous regions, while black arrows indicate non-hydrolyzed crystalline regions. [Figure 12] SEM images of PLLA film surfaces during hydrolysis tests. A = PLLA film surface incubated with cell-free supernatant from Y294[CLEns] cells for 24 hours; white arrows indicate amorphous regions with clear pit formation, and black arrows indicate non-hydrolyzed crystalline regions. B = PLLA film surface incubated with commercial ProK (0.5 mg / mL in 0.1 M KH2PO4 buffer) for 24 hours, showing more general attack on the film surface. [Figure 13] SEM images of PLLA film surfaces during hydrolysis tests. A = PLLA film surface incubated with cell-free supernatant from Y294[CLEns] cells for 24 hours; B = PLLA film surface incubated with commercial ProK (0.5 mg / mL in 0.1 M KH2PO4 buffer) for 24 hours. [Figure 14] Figure 1 shows PLLA film crystallinity during hydrolysis by cell-free supernatants from strains Y294[CLEns] (■) and Y294[BBH] (◆) based on differential scanning calorimetry analysis. The significant decrease in crystallinity followed by a sharp increase confirms hydrolysis of amorphous regions before more crystalline regions by the recombinant strains. [Figure 15] Graph showing data from a small-scale hydrolysis study of PLA films after 240 hours of incubation. A = percent weight loss compared to the Y294[BBH] control; B = lactic acid concentration determined by HPLC analysis. Error bars represent standard deviation from the mean of three replicates. Substantial weight loss and lactic acid production are observed at the end of hydrolysis using cell-free supernatant from the recombinant yeast strain. [Figure 16] Photographs showing the effect of using cell-free supernatants from strains Y294[BBH] and Y294[CLEns] for 240-hour scale-up hydrolysis of various PLA films. PDLAHMW and PLLAHMW refer to the higher molecular weight PLA materials. Excessive fragmentation is observed in films incubated with the supernatant from strain Y294[CLEns]. [Figure 17]Graph showing data from scale-up hydrolysis tests on various PLA films after 240 hours of incubation. A = percent weight loss compared to the Y294[BBH] control; B = lactic acid concentration determined by HPLC analysis. Error bars represent standard deviation from the mean of three replicates. After scaling up the reaction volume, similar weight loss and lactic acid concentrations are observed as in the small-scale hydrolysis tests. [Figure 18] SDS-PAGE gel showing the presence of CLE1 protein species (black brackets) in the supernatants of the Y294[CLEns]-TDHi and Y294[CLEns] strains, while the protein species is absent from the supernatant of the Y294[BBH] strain. Molecular markers are indicated on the far left. It is clear that the Y294[CLEns]-TDHi strain delivered substantially more CLE1 in the extracellular fraction than the previously constructed Y294[CLEns] strain. [Figure 19] Photograph showing hydrolysis halo formation by Y294[CLEns]-TDHi, Y294[CLEns], and Y294[BBH] strains on 0.035 g / L polycaprolactone containing agar plates. A larger hydrolysis halo is observed for the Y294[CLEns]-TDHi strain compared to Y294[CLEns], and no halo is observed around the Y294[BBH] control. [Figure 20] A turbidity-based assay was used to evaluate the extracellular PLA hydrolysis activity of CLE1-producing S. cerevisiae strains under the control of different engineered promoters, including the codon-optimized CLE1 gene and the wild-type gene. The Y294[BBH] (▲), Y294[CLEwt] (x), Y294[CLEns]-TDHi (●), and Y294[CLEns] (■) yeast strains were cultured in 2x SC-URA medium, and extracellular PLA hydrolysis activity was measured after 24, 48, and 72 hours of growth. Error bars represent the standard deviation from the mean of three replicates. The Y294[CLEwt] strain yielded 1.4-fold lower activity than the Y294[CLEns] strain, while the Y294[CLEns]-TDHi strain exhibited a 1.5-fold increase compared to the Y294[CLEns] strain. [Figure 21]The optimal temperature for PLA hydrolysis using recombinant CLE1 was determined using a turbidity-based enzyme activity assay performed at various incubation temperatures. After culturing the Y294[BBH] (▲) and Y294[CLEns]-TDHi (●) strains in 2×SC-URA medium for 72 h, the supernatants were collected by centrifugation and used in the assay at different temperatures. The extracellular PLA hydrolysis activity from the Y294[CLEns]-TDHi strain at 42°C (69 U / mL) was 1.6-fold higher than the previously reported optimum of 37°C (44 U / mL). [Figure 22] The stability of recombinant CLE1 in the supernatant of strain Y294[CLEns]-TDHi was determined by incubating the supernatant at 37, 42, 45, and 50°C for a total of 120 hours. Residual activity at each temperature was determined using a turbidity-based enzyme assay after 72 and 120 hours of incubation. Recombinant CLE1 in the supernatant was highly stable at 37 and 42°C after 120 hours of incubation, with 100% and 94% residual activity detected after incubation at the two respective temperatures. Significant decreases in residual activity were detected after 120 hours of incubation at 45°C (75% residual activity) and 50°C (28% residual activity). [Figure 23] HPLC analysis results showing the lactic acid released from 10 g / L PLLA films over a 10-day hydrolysis period at 37 °C in small-scale hydrolysis studies using supernatants from the Y294[CLEns] (■), Y294[CLEns]-TDHi (●), and Y294[BBH] (▲) strains. Error bars represent the standard deviation from the mean value of three replicates. Significant amounts of lactic acid are released by hydrolyzing PLA films with supernatants from the recombinant Y294[CLEns] and Y294[CLEns]-TDHi strains. After 24 h of hydrolysis, the supernatant from the Y294[CLEns]-TDHi strain released nearly 3 g / L more lactic acid than the supernatant from the Y294[CLEns] strain. [Figure 24]HPLC analysis results showing lactic acid released from a 10 g / L PLLA film over a 7-day hydrolysis period in a bioreactor experiment using 700 mL of supernatant from strain Y294[CLEns]-TDHi. After 72 hours of incubation, the supernatant from strain Y294[CLEns]-TDHi was collected by centrifugation. Three hydrolysis settings were investigated for improved PLA hydrolysis: the first hydrolysis setting was performed at 37°C and non-neutralized conditions (no pH control) (▲); the second hydrolysis setting was performed at 37°C and neutralized conditions (pH maintained at 6.8-7 by the addition of 3 M KOH) (●); and the third hydrolysis setting was performed at 42°C and neutralized conditions (pH maintained at 6.8-7 by the addition of 3 M KOH) (■). Error bars represent the standard deviation from the mean value of three replicates. The effect of neutralized conditions is evident in the release of higher lactic acid concentrations compared to non-neutralized conditions at 37°C. The higher PLA hydrolysis activity of CLE1 at 42 °C versus 37 °C resulted in the release of 11.7 g / L of lactic acid after 48 h, which is 4.5 g / L higher than that found at 37 °C. The maximum released lactic acid concentration reached 14.9 g / L after 7 days of hydrolysis and neutralization conditions at 42 °C, resulting in a 78% weight loss of the PLA film.

[0033] Sequence Listing The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and standard three-letter abbreviations for amino acids. Those skilled in the art will understand that while only one strand of each nucleic acid sequence is shown, the complementary strand is included in any reference to the displayed strand. The accompanying sequence listing is incorporated herein by reference in its entirety. In the accompanying sequence listing:

[0034] SEQ ID NO:1 - Nucleotide sequence of the codon-optimized CLE1 gene.

[0035] SEQ ID NO:2 - Amino acid sequence of CLE1.

[0036] SEQ ID NO:3 - Amino acid sequence of the native secretion signal of CLE1.

[0037] SEQ ID NO:4—Nucleotide sequence encoding the codon-optimized native secretion signal of CLE1.

[0038] SEQ ID NO:5—Amino acid sequence of the secretion signal from Trichoderma reesei xyn2.

[0039] SEQ ID NO: 6: Nucleotide sequence encoding the secretion signal from Trichoderma reesei xyn2.

[0040] SEQ ID NO:7—Nucleotide sequence of codon-optimized TEF1i promoter.

[0041] SEQ ID NO:8 - Nucleotide sequence of the native (non-codon optimized) CLE1 gene.

[0042] SEQ ID NO: 9—Nucleotide sequence of TDHi promoter. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

[0044] The invention as described should not be limited to the particular embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0045] As used throughout this specification and the claims that follow, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0046] The terms and terminology used herein are for purposes of description and should not be regarded as limiting. As used herein, the terms "comprising," "containing," "having," and "including," and variations thereof, are intended to encompass the items listed thereafter and equivalents thereof, as well as additional items.

[0047] In its broadest sense, the present invention relates to the construction of recombinant eukaryotic hosts that exhibit enhanced heterologous expression of cutinase-like enzymes (CLEs) using molecular engineering techniques, such as codon-optimized and engineered yeast promoters, resulting in improved recombinant enzyme production. The use of culture supernatants from recombinant yeast strains in processes involving the processing of bioplastics, such as polylactic acid (PLA), in various forms and concentrations is also described. Quantitative data from PLA hydrolysis is provided, as well as insights into the degradation mechanism of the CLE1 enzyme on PLA films.

[0048] The present inventors have constructed a genetically engineered, industrially applicable eukaryotic host that exhibits enhanced expression of the CLE1 gene from Cryptococcus sp. S-2. Following in silico evaluation of the expressibility of the CLE1 gene in the S. cerevisiae host, the inventors identified key points where heterologous expression could be improved, and were able to significantly enhance CLE1 expression and recombinant production in S. cerevisiae, with concomitant improvements in PLA hydrolysis using the recombinant S. cerevisiae. While the use of crude supernatant without purification or concentration resulted in improved PLA hydrolysis compared to previously reported data, substantial supporting results are also provided herein. Thus, this study represents a potentially important step toward commercial CLE1 production by recombinant eukaryotic strains for the degradation of bioplastic materials, such as commercially available PLA and starch blends, in a simple yet efficient processing process.

[0049] In one embodiment of the present invention, a method for constructing a recombinant S. cerevisiae strain for overexpressing a bioplastic-degrading enzyme at very high titers is provided. The present invention involves the use of molecular techniques, such as codon optimization, in conjunction with the use of an expression-enhancing engineered yeast promoter, e.g., the TEF1i (Myburgh et al., 2020) (SEQ ID NO: 7) or TDHi promoter (SEQ ID NO: 9), resulting in improved expression and production of the CLE1 enzyme in yeast host cells. Here, the inventors demonstrate that codon optimization significantly improves extracellular protein production, resulting in higher hydrolytic activity against PDLA emulsions in a turbidity-based enzyme assay. Specifically, the inventors demonstrate that strains containing the engineered TDHi promoter produced significantly more CLE1 in the extracellular fraction than strains containing the TEF1i promoter.

[0050] In another embodiment of the present invention, the use of the extracellular fraction of the developed recombinant strain for the degradation of bioplastics is provided, which allows significant fragmentation, weight loss, and monomer production from commercially available PLA material. In one embodiment, a method for the processing of PLA involves the use of a recombinant yeast strain producing CLE1 in its extracellular fraction, which can be used directly at mild temperatures and pH levels without any material pretreatment, addition of solvents, activity-enhancing emulsifiers, and / or other catalysts.

[0051] The data presented herein provide the first quantitative analysis of PLA hydrolysis using a recombinant yeast strain with CLE1 activity. In one embodiment, the CLE1 enzyme is encoded by a codon-optimized gene under the control of a genetically engineered yeast promoter, significantly improving heterologous CLE1 expression in S. cerevisiae, affecting the recombinant strain to produce the bioplastic-degrading CLE1 enzyme at very high titers in the extracellular fraction. The effect of codon optimization on CLE1 expression potential in S. cerevisiae is demonstrated by in silico analysis of the native and codon-optimized genes (Table 1) and the presented experimental data. In another embodiment, the recombinant strain of the present invention is demonstrated to exhibit improved hydrolysis performance on different types of PLA materials (PLLA and PDLA) through hydrolysis tests at various substrate loadings of PLA emulsions, powders, and films, quantifying turbidity, lactic acid production, film weight loss, and fragmentation. Notably, hydrolysis of various PLA substrates is achieved without any additional pretreatment, solvents, emulsifiers, or other pH levels, or purification or concentration of the culture supernatant. Thus, the process by which PLA materials are degraded using the recombinant strains of the present invention is clearly simplified compared to processes using known CLE1-expressing strains. Furthermore, tests using PLA emulsions show complete hydrolysis of higher loadings of PLA in similar hydrolysis times compared to known CLE1-producing strains. In addition, scanning electron micrographs and differential scanning calorimetry on hydrolyzed PLA films provide insight into the degradation mechanism of the CLE1 enzyme on PLA films, which is distinct from proteinase K, referred to herein as ProK, a commercially available enzyme typically used in commercial PLA hydrolysis.

[0052] In a further embodiment, optimized process parameters for the hydrolysis of PLA substrates using cell-free supernatants obtained from recombinant S. cerevisiae strains are provided. Specifically, recombinant codon-optimized CLE1 in the crude supernatant showed a 1.6-fold increase in activity on emulsified PDLA at 42°C compared to activity at 37°C. The enzyme in the crude supernatant was confirmed to be stable at higher temperatures, retaining 94% activity after 120 h at 42°C, 75% at 45°C, and 28% at 50°C. Thus, the optimal temperature for hydrolysis of PLA substrates with codon-optimized CLE1 at 42°C is higher than the 37°C previously reported for the wild-type enzyme. In addition, we demonstrated that maintaining a pH between 6.8 and 7.0 did not improve lactate release within the first 48 h, but the benefit of pH control was evident from 72 h onward, with lactate reaching 12.7 g / L after 168 h, correlating with a 1.4-fold increase compared to unneutralized conditions at 37°C. Running the process under neutral conditions and at the newly identified optimum temperature for PLA hydrolysis (42°C) significantly increased lactic acid release within the first 48 hours (11.7 g / L released), which was 1.6-fold higher than that at 37°C and correlates to a productivity of 0.25 g / L / h. The final lactic acid concentration after 168 hours of hydrolysis at 42°C with pH control was 14.92 g / L, as opposed to 9.36 g / L at 37°C without pH control.

[0053] The term "bioplastic polymer" refers to any material, either biodegradable or non-biodegradable, derived from bio-based or fossil fuel-based resources. These include, but are not limited to, polylactic acid (PLA), including poly(L-lactide) (PLLA), poly(D-lactide) (PDLA), and poly(DL-lactide) (PDLLA), polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polyhydroxyalkanoates (PHAs), thermoplastic starch (TPS) blends, and polyethylene terephthalate (bio-PET).

[0054] A "protein," "peptide," or "polypeptide" is any chain of two or more amino acids, including natural or unnatural amino acids or amino acid analogs, regardless of post-translational modification (e.g., glycosylation or phosphorylation).

[0055] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein and encompass both ribonucleotides (RNA) and deoxyribonucleotides (DNA), including cDNA, genomic DNA, and synthetic DNA. A nucleic acid may be double-stranded or single-stranded. If the nucleic acid is single-stranded, it may be the sense strand or the antisense strand. A nucleic acid molecule may be any strand of two or more covalently linked nucleotides, including naturally occurring or non-naturally occurring nucleotides, or nucleotide analogs or derivatives. "RNA" refers to a sequence of two or more covalently linked, naturally occurring, or modified ribonucleotides. The term "DNA" refers to a sequence of two or more covalently linked, naturally occurring, or modified deoxyribonucleotides.

[0056] The term "isolated" is used herein to mean removed from its natural environment.

[0057] The term "purified" refers to the isolation of a molecule or compound in a form that is substantially free of contaminants or contaminants that are normally associated with the molecule or compound in its natural environment, and thus purified refers to increased purity as a result of separation from other components of the original composition. The term "purified nucleic acid" refers to a nucleic acid sequence that has been separated from other compounds, including but not limited to polypeptides, lipids, and carbohydrates, with which it is normally associated in its natural state.

[0058] The term "cell-free supernatant," as used herein, refers to the culture broth from which the yeast cells have been removed but which contains all extracellular products, including recombinant proteins, and which is primarily collected at the end of a particular culture period. Cell-free supernatant typically refers to the broth in its unconcentrated form, in which the products it contains have not been purified by dialysis, chromatography, filtration, or any other method. Cell-free supernatant can be collected by centrifugation (typically, for yeast, at 4000 rpm for 5 minutes) and / or filtration of the culture.

[0059] The term "complementary" refers to two nucleic acid molecules, e.g., DNA or RNA, that can form Watson-Crick base pairs to create a double-stranded region between the two nucleic acid molecules. Those skilled in the art will understand that it is not necessary for every nucleotide in a nucleic acid molecule to form a matching Watson-Crick base pair with a nucleotide in the opposing complementary strand to form a duplex. Thus, one nucleic acid molecule is "complementary" to a second nucleic acid molecule if it hybridizes with the second nucleic acid molecule under high stringency conditions. Nucleic acid molecules according to the present invention include both complementary molecules.

[0060] As used herein, a "substantially identical" sequence is an amino acid or nucleotide sequence that differs from a reference sequence only by one or more conservative substitutions, or one or more non-conservative substitutions, deletions, or insertions located at positions in the sequence that do not disrupt or substantially reduce the activity of one or more of the expressed polypeptides or polypeptides encoded by the nucleic acid molecule. Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways within the knowledge of those skilled in the art. These include, for example, the use of computer software such as ALIGN, Megalign (DNASTAR), CLUSTALW, or BLAST software. Those skilled in the art can readily determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared. In one embodiment of the present invention, polypeptide or polynucleotide sequences are provided that have at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, e.g., about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity, with the sequences described herein.

[0061] Alternatively, or in addition, two nucleic acid sequences can be "substantially identical" if they hybridize under high stringency conditions. The "stringency" of a hybridization reaction can be easily determined by one of ordinary skill in the art and is generally an empirical calculation dependent on probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. A typical example of such "stringent" hybridization conditions is hybridization at 65°C for 18 hours with gentle shaking, a first wash in wash buffer A (0.5% SDS; 2X SSC) at 65°C for 12 minutes, and a second wash in wash buffer B (0.1% SDS; 0.5% SSC) at 65°C for 10 minutes.

[0062] Those skilled in the art will appreciate that polypeptides, peptides, or peptide analogs comprising the CLE1 enzyme of the present invention can be prepared from their corresponding nucleic acid molecules using recombinant DNA technology. Polypeptides, peptides, and peptide analogs can also be synthesized using standard chemical techniques, for example, by automated synthesis using solution or solid-phase synthesis methods. Automated peptide synthesizers are commercially available and use techniques known in the art.

[0063] As used herein, the term "gene" refers to a nucleic acid that encodes a functional product, e.g., an RNA, a polypeptide, or a protein. A gene can include regulatory sequences upstream or downstream of the sequence that encodes the functional product.

[0064] As used herein, the term "coding sequence" refers to a nucleic acid sequence that encodes a specific amino acid sequence. "Regulatory sequence," on the other hand, refers to a nucleotide sequence located either upstream, downstream, or within a coding sequence. Generally, regulatory sequences affect the transcription, RNA processing or stability, or translation of an associated coding sequence. Regulatory sequences include, but are not limited to, effector binding sites, enhancers, introns, polyadenylation recognition sequences, promoters, RNA processing sites, stem-loop structures, and translation leader sequences.

[0065] In some embodiments, the genes used in the methods of the present invention may be operably linked to other sequences. "Operably linked" means that the nucleic acid molecule encoding the cutinase-like enzyme of the present invention and the regulatory sequences are linked in a way that allows expression of the protein when the appropriate molecules are bound to the regulatory sequences. Such operably linked sequences may be contained in a vector or expression construct that can be transformed or transfected into a host cell for expression. It should be understood that any vector or multiple vectors can be used for the purpose of expressing the cutinase-like enzyme of the present invention.

[0066] The term "promoter" refers to a DNA sequence capable of controlling the expression of a nucleic acid coding sequence or functional RNA. A promoter can be based entirely on a native gene promoter or can be composed of different elements from different promoters found in nature. Different promoters can direct the expression of a gene in different cell types, at different developmental stages, or in response to different environmental or physiological conditions. A "constitutive promoter" is a promoter that most often directs the expression of a gene of interest in most host cell types. An "engineered promoter" refers to a promoter that does not naturally occur in the form used for recombinant expression of a heterologous gene. An engineered promoter can be composed of various regulatory elements, such as a 3'-UAS, a 5'-UTR intron, and a native promoter, to improve promoter-driven expression of a heterologous gene.

[0067] The term "recombinant" means something has been engineered. When used with respect to a nucleic acid construct, the term refers to a molecule comprising nucleic acid sequences linked together or produced by molecular biological techniques. When referring to a protein or polypeptide, the term "recombinant" refers to a protein or polypeptide molecule that is not isolated from a natural source (e.g., a biological sample), e.g., expressed from a recombinant nucleic acid construct made by means of molecular biological techniques. A recombinant nucleic acid construct can contain a nucleotide sequence that is ligated or engineered to be ligated to a nucleic acid sequence to which it is not naturally ligated or to which it is naturally ligated at a different location. Thus, a recombinant nucleic acid construct indicates that a nucleic acid molecule has been manipulated using genetic engineering, i.e., by human intervention. A recombinant nucleic acid construct can be introduced into a host cell by transformation. Such a recombinant nucleic acid construct can contain sequences from the same host cell species or from different host cell species.

[0068] The term "vector" refers to a means by which a polynucleotide or gene sequence can be introduced into a cell. There are various types of vectors known in the art, including plasmids, viruses, bacteriophages, and cosmids. Generally, a polynucleotide or gene sequence is introduced into a vector via a cassette. The term "cassette" refers to a polynucleotide or gene sequence expressed from a vector, such as a polynucleotide or gene sequence encoding a cutinase-like enzyme of the present invention. A cassette generally comprises a gene sequence inserted into a vector, which in some embodiments provides regulatory sequences for expression of the polynucleotide or gene sequence. In other embodiments, the vector provides regulatory sequences for expression of the cutinase-like enzyme. In further embodiments, the vector provides some regulatory sequences and the nucleotide or gene sequence provides other regulatory sequences. "Regulatory sequences" include, but are not limited to, promoters, transcription termination sequences, enhancers, splice acceptors, donor sequences, introns, ribosome binding sequences, poly(A) addition sequences, and / or origins of replication.

[0069] The terms "heterologous expression" or "heterologously expressing" refer to the expression of a gene or portion of a gene in a host organism that does not naturally possess this gene or gene fragment. Insertion of a gene into a heterologous host is accomplished by recombinant techniques. In one non-limiting example, Cryptococcus sp. S-2 can be heterologously expressed in a non-native host cell, such as a S. cerevisiae cell, including, but not limited to, S. cerevisiae Y294, S. cerevisiae Ethanol Red V1, S. cerevisiae M2n, or S. cerevisiae YI30.

[0070] In some embodiments, recombinant yeast strains expressing cutinase-like enzymes, cell-free supernatants of such strains, or the cutinase-like enzymes of the present invention are well suited for hydrolysis of different bioplastics, bioplastic blends, and biocompounds made from bioplastics, which may include, but are not limited to, poly(L-lactide) (PLLA), poly(D-lactide) (PDLA), and poly(DL-lactide) (PDLLA), as well as any combination or blend of polylactic acid (PLA), including polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), polyhydroxyalkanoates (PHAs), and thermoplastic starch (TPS) blends.

[0071] The following examples are offered by way of illustration and not by way of limitation.

[0072] Example 1 Strain structure The native cutinase-like enzyme (CLE1) gene (Genbank accession number AB671329.1 - SEQ ID NO: 8), previously known as lipase from Cryptococcus sp. S-2, was evaluated through an in silico approach to determine its expressibility in S. cerevisiae. As shown in Table 1, the native gene has a high GC content (65%), very low codon bias (CBI, 0.07) and codon adaptation index (CAI, 0.51), and a high number of repetitive tandem rare codons (13%). While these features may be important for proper expression in the native host, they may also adversely affect expression in S. cerevisiae. Transcription can be affected by the high GC content. Translation efficiency is influenced by codon usage, while tandem rare codons can cause ribosome pausing and bottlenecks during protein secretion. Codon optimization of the CLE1 gene (SEQ ID NO: 1) for expression in S. cerevisiae using the Optimumgene™ algorithm (GeneScript) reduced the GC content (44%), significantly increased the CBI (0.55) and CAI (0.93), and removed tandem rare codons (Table 1).

[0073] The codon-optimized CLE1 gene, with either its native secretion signal (SEQ ID NO: 3), encoded by the nucleotide sequence of SEQ ID NO: 4, or the secretion signal from the Trichoderma reesei xyn2 gene (XYNSEC (SEQ ID NO: 5)), encoded by the nucleotide sequence of SEQ ID NO: 6, was inserted downstream of the TEF1i engineered yeast promoter (SEQ ID NO: 7) on the pBBH4 yeast episomal plasmid (FIG. 1A) to deliver the pBBH4-CLEx and pBBH4-CLEns vectors (FIGS. 1B and 1C, Table 2). The engineered TEF1i promoter was constructed and is fully described in Myburgh et al. (see FEMS Yeast Res. 2020.20(6), foaa047), which is incorporated herein by reference in its entirety. This promoter has been shown to strongly enhance heterologous expression of amylase genes in S. cerevisiae. The episomal vector was transformed into S. cerevisiae strain Y294 to obtain recombinant Y294[CLExs] and Y294[CLEns] strains.

[0074] In a subsequent series of experiments, an additional vector was constructed to compare expression of the native CLE1 gene with a codon-optimized version. Another vector was also constructed to compare expression of the codon-optimized CLE1 gene under the control of the engineered TEF1i promoter with expression under another engineered promoter, TDHi (Figure 1). This vector was also used in experiments to compare expression of the native CLE1 gene with the codon-optimized version.

[0075] The native CLE1 gene (SEQ ID NO: 8), i.e., the native CLE1 gene (SEQ ID NO: 8) without S. cerevisiae codon usage optimization and with its native secretion signal (SEQ ID NO: 4), was inserted downstream of the engineered TEF1i promoter (SEQ ID NO: 7) on the episomal pBBH4 plasmid to obtain the plasmid pBBH4-CLEwt (Figure 1D). The codon-optimized CLE1 gene with its native secretion signal (SEQ ID NO: 3), encoded in its entirety by the nucleotide sequence of SEQ ID NO: 1, was inserted downstream of the TDHi engineered yeast promoter (SEQ ID NO: 9) on the pBBH4 yeast episomal plasmid to produce the pBBH4-CLEns-TDHi vector (Figure 1E). The engineered TDHi promoter was constructed and is fully described in Myburgh et al. (see FEMS Yeast Res. 2020.20(6), foaa047), which is incorporated herein by reference in its entirety. Because previous experiments showed that the use of the alternative secretion signal XYNSEC (SEQ ID NO: 5) did not increase CLE1 protein production, the native secretion signal (SEQ ID NO: 3) was used in this subsequent vector. The episomal vector was transformed into S. cerevisiae strain Y294 to obtain recombinant Y294[CLEwt] and Y294[CLEns]-TDHi strains (Table 2).

[0076] All sequences referred to herein are provided in Table 3. [Table 1] [Table 2] [Table 3-1] [Table 3-2]

[0077] Example 2 Extracellular CLE1 production As seen in Figure 2, SDS-PAGE analysis confirmed the extracellular production of CLE1 protein by both the Y294[CLExs] and Y294[CLEns] strains. The protein species, easily identified (indicated by the black arrowhead), is larger (25 kDa) than the expected 22 kDa for the CLE1 protein. This is most likely due to glycosylation of the protein species by the yeast cells during secretion of the enzyme into the extracellular matrix. Indeed, signs of differential glycosylation were evident in samples from both strains, observed as a slight smear or several protein signals for the single CLE1 protein. Glycosylation of enzymes is known to affect their stability, which may be an important factor during processing of bioplastic materials. Furthermore, from the intensity of the protein bands, it is clear that the Y294[CLEns] strain, which produces CLE1 with a native secretion signal, delivered more recombinant enzyme to the extracellular fraction than the Y294[CLExs] strain.

[0078] Figure 3 shows hydrolysis halo formation surrounding the Y294[CLEns] and Y294[CLExs] recombinant strains on SC-URA agar plates containing tributyrin (1% v / v). The Y294[BBH] control strain showed no halo formation. It is also evident that a larger hydrolysis halo was formed around Y294[CLEns] compared to Y294[CLExs].

[0079] Figure 4 shows the results from a turbidity-based enzyme assay involving incubating PLLA (represented by the solid line) and PDLA (represented by the dashed line) substrate solutions with supernatants collected from 24-, 48-, and 72-hour yeast cultures.

[0080] The ProK positive control showed consistent activity across multiple assay rounds on the PLLA substrate, indicating that measurements were accurate and reproducible at different time points. The Y294[BBH] control showed no significant activity on either the PLLA or PDLA substrates, confirming that the decrease in turbidity was due to the effect of the recombinant (or commercially available) enzyme, rather than autohydrolysis of the PLA polymer. It is clear that the secreted protein species observed during SDS-PAGE analysis (Figure 2) were active on both PLLA and PDLA substrates.

[0081] The Y294[CLEns] strain delivered substantial activity levels onto the PLLA substrate, reaching 10 U / mL after 48 h of incubation. The Y294[CLExs] strain exhibited significantly lower activity levels compared to the Y294[CLEns] strain, reaching 3.12 U / mL of activity after 72 h of incubation.

[0082] Higher activity levels were detected for both recombinant strains on PLLA substrates compared to PDLA. The Y294[CLEns] and Y294[CLExs] strains reached 20 U / mL and 7.22 U / mL, respectively, after 72 hours. These results indicate that active CLE1 enzyme is produced by both recombinant S. cerevisiae strains. However, it is clear that the Y294[CLEns] strain produces a greater amount of extracellular protein, resulting in better activity levels during the enzyme assay.

[0083] Example 3 Small-scale cell-free hydrolysis in PLA powder A small-scale cell-free hydrolysis test was performed using an emulsified PLA substrate with a final concentration of 0.075% w / v incubated with 2.5 mL of cell-free supernatant at 37 °C. Figure 5 shows the results of the small-scale hydrolysis test using PLA emulsion. Complete (98%) hydrolysis of emulsified PDLA was achieved after 72 h of hydrolysis using supernatant from Y294[CLEns], while nearly complete (91%) hydrolysis of PLLA was achieved with the same strain. The control and commercial ProK did not show a decrease in turbidity of the PDLA substrate, whereas commercial ProK resulted in complete hydrolysis of the PLLA substrate. This indicates a similar time for complete hydrolysis of the PLA emulsion compared to a known purified and concentrated CLE1 preparation from a native Cryptococcus sp. strain S-2. Importantly, a higher concentration of PLA substrate (0.075% w / v) was completely hydrolyzed using the present recombinant strain compared to the known strain (0.04% w / v substrate), suggesting improved hydrolysis when using supernatant from the recombinant S. cerevisiae strain from the present invention directly (without purification or concentration). Further strengthening the evidence that the present strains exhibit improved PLA hydrolysis is the fact that no surfactant was used during the hydrolysis of emulsified PLA by the present strains, whereas previous hydrolysis studies included Plysurf A210G in the assay. Surfactants such as Plysurf have been shown to increase PLA hydrolysis by various hydrolytic enzymes.

[0084] HPLC analysis was performed on samples collected at several time points during the cell-free hydrolysis experiments to demonstrate the hydrolysis of PLLA and PDLA powders, as reflected by the residual lactate concentration. Figure 6 shows the production of lactate during the cell-free hydrolysis of PLLA (solid line) and PDLA (dashed line) powders using various substrate concentrations (A = 4; B = 10; and C = 25 g / L PLA powder loading). In all experiments performed, there was substantial production of free lactate by samples incubated with the supernatants of the Y294[CLEns] and Y294[CLExs] strains. Again, it is clear that improved CLE1 secretion by the Y294[CLEns] strains improves the hydrolysis of PLA powder compared to the Y294[CLExs] samples. Increasing the powder concentration (substrate loading) resulted in an increase in the final concentration of lactate produced from the PLLA and PDLA polymers. The highest concentrations of lactic acid produced using the supernatant from strain Y294[CLEns] were 5.73 and 5.50 g / L from 25 g / L PLLA and PDLA powders, respectively ( Figure 6C ).

[0085] Figure 7 shows the ratio of lactic acid produced at each time point to the final concentration (%) and lactic acid productivity (g / L / h). Shading indicates strain performance at each time point (darker shading = better performance). Considering the concentration of lactic acid produced at each time point (Cn) compared to the final concentration of lactic acid produced (Cf) (expressed as a percentage in Figure 7), the fraction of final lactic acid concentration was significantly higher from 10 g / L PLA powder compared to 4 and 25 g / L. From 10 g / L PLLA powder, 87% of the final lactic acid concentration was produced within the first 72 hours of hydrolysis. Similarly, 86% of the final lactic acid concentration was produced within the same time period using 10 g / L PDLA powder. This indicates that most of the lactic acid was produced within 72 hours. Lactate production rates for hydrolysis tests using Y294[CLEns] supernatant on PLLA and PDLA powders showed maximum rates of 0.155 and 0.120 g / L / h, respectively. The data show that free lactic acid is produced using crude enzyme extracts from recombinant strains and that the substrate loading affects the efficiency of PLA hydrolysis.

[0086] Example 4 Small-scale cell-free hydrolysis on PLA films PLA thin films provide a better representation of real-world PLA substrates. Therefore, small-scale hydrolysis studies were performed using 10 mL of cell-free supernatant (crude enzyme) incubated with PLA films. Figure 8 shows the lactic acid release from PLLA (solid line) and PDLA (dashed and dotted lines) films over a 10-day hydrolysis period at 37 °C.

[0087] Substantial amounts of free lactic acid were produced from 10 g / L PLLA and PDLA films. Surprisingly, hydrolysis of PLA films resulted in increased levels of lactic acid release compared to powdered PLA (Figure 8). Powdered substrates have a larger surface area and are expected to undergo better enzymatic hydrolysis than films. At the end of the hydrolysis period (240 h), samples treated with supernatant from the Y294[CLEns] strain yielded 6.84 and 9.44 g / L of lactic acid from 10 g / L PLLA and PDLA films, respectively. This counterintuitive result may be due to the creation of more hydrophobic surfaces on the powder particles by the treatment of the PLA material. The enhanced effect observed throughout previous experiments for the Y294[CLEns] strain compared to the Y294[CLExs] strain was even more pronounced in the experiments using PLA films, likely due to better secretion and therefore higher enzyme concentrations in the extracellular compartment. Although higher final lactic acid concentrations were achieved from the hydrolysis of PLA films, a lower lactic acid production ratio (Cn / Cf) (expressed as a percentage in Figure 9) was observed within the first 72 hours of hydrolysis compared to using powders. Nevertheless, 49% and 76% of the final lactic acid concentrations were produced within the first 72 hours of hydrolysis for PLLA and PDLA films, respectively. Furthermore, a higher lactic acid production rate was reported from PDLA films, with 0.175 g / L / h of lactic acid produced from 10 g / L PDLA films in the first 24 hours of hydrolysis.

[0088] To identify specific hydrolysis patterns, scanning electron microscopy (SEM) was performed on PLA films incubated with cell-free supernatant from the Y294[CLEns] strain. SEM analysis of PLA films treated with supernatant from the Y294[CLEns] strain showed clear signs of degradation (Figure 10C), whereas no degradation pattern was evident in films incubated with supernatant from the Y294[BBH] control strain (Figure 10B). Degradation patterns were evident on PDLA films after 24 h of hydrolysis and were more pronounced after 48 h (Figures 11A and 11B, respectively). Hydrolysis initiated in specific regions of the film and steadily extended outward to include larger areas as well as the interior within the film matrix (white arrows in Figure 11B). While degradation of PLLA films was generally lower than that of PDLA films, specific pit formation was more pronounced when PLLA films were treated with supernatant from the Y294[CLEns] strain (Figures 12A and 13A).

[0089] It is also evident that hydrolysis initiated in the amorphous regions (white arrows in Figures 11A and 12A) before moving to more crystalline regions (black arrows in Figures 11A and 12A), which showed no degradation after 24 h (Figure 12). This contrasts with the more typical hydrolysis pattern observed for PLLA films incubated with commercial ProK (Figures 12B and 13B). The commercial enzyme appears to hydrolyze the film surface at the same rate, rather than preferentially hydrolyzing the amorphous regions (Figures 12B and 13B). Hydrolysis of the amorphous regions prior to the crystalline regions may result in an initial increase in film crystallinity (i.e., the film becomes more brittle), and therefore, film fragmentation may be more pronounced during this initial hydrolysis. This observation was confirmed by DSC analysis of PLLA films collected at 24 h intervals throughout hydrolysis using supernatant from Y294[CLEns] (Figure 14). The initial increase in crystallinity in the first 24 h indicates a significant decrease in crystallinity at 72 h due to the initial hydrolysis of the amorphous regions, followed by the hydrolysis of the crystalline regions, confirming the mechanism of PLA film hydrolysis by CLE1 observed during SEM.

[0090] Small-scale film hydrolysis was performed to determine the film weight loss after 240 hours of hydrolysis using a 10 mL reaction volume (10 g / L thin film). Briefly, PLA films were weighed before adding the enzyme, filtered through Whatmann filter paper after hydrolysis, and the total sample was reweighed after drying. HPLC analysis was performed to determine the final lactic acid concentration produced from the hydrolysis. Films treated with each of the recombinant strain supernatants showed significant weight loss at the end of the hydrolysis period (Figure 15A). Compared to the BBH control, final weight losses of approximately 35% and 45% were achieved for the PLLA and PDLA film samples, respectively. This is higher than the 31% weight loss reported for the commercially available ProK enzyme.

[0091] Again, substantial lactic acid production was observed from 10 g / L film hydrolysis for both the recombinant strain and the PLLA and PDLA films. Final lactic acid concentrations of 4.30 g / L and 4.79 g / L were produced after incubation of PLLA with Y294[CLExs] and Y294[CLEns] supernatant samples, respectively (Figure 15B). Meanwhile, final lactic acid concentrations of 6.64 g / L and 7.75 g / L were produced after incubation of PDLA with Y294[CLExs] and Y294[CLEns] supernatant samples, respectively (Figure 15B). To our knowledge, this is the first report demonstrating such extensive film hydrolysis using a recombinant S. cerevisiae strain expressing the CLE1 enzyme.

[0092] Example 5 Scale-up of cell-free hydrolysis on PLA films The hydrolysis reaction was scaled up to a working volume of 50 mL. For these experiments, a substrate loading of 10 g / L was maintained. In addition to the low molecular weight PDLA and PLLA films, two high molecular weight PLA polymers (PDLA HMW and PLA HMW ) were included in these experiments.

[0093] Scaling up the hydrolysis reaction resulted in trends similar to those observed in the small-scale experiments: films incubated with supernatant from the Y294[BBH] control strain showed little or no signs of degradation, and the higher molecular weight PLLA HMW and PDLA HMW The polymers showed less signs of degradation than the lower molecular weight PLLA and PDLA samples (Figure 16). Low molecular weight PLLA and PDLA films treated with supernatant from strain Y294[CLEns] showed extreme fragmentation after 240 hours of hydrolysis, whereas the higher molecular weight PLLA HMW and PDLA HMW Less fragmentation was observed with the polymer, but still clearly more than with the Y294[BBH] control.

[0094] The weight loss data for the two PDLA polymers are shown in Figure 16. Despite increased fragmentation of the lower molecular weight polymer, PDLA and PDLA HMW sample (37% and 34%, respectively), and PLLA and PLLA HMW The weight loss rates were surprisingly similar for the polymers (27% and 24%, respectively) (Figure 17A). Lactic acid concentrations were similar to those observed during small-scale hydrolysis of the films (Figure 17B). As expected, the highest concentration of lactic acid was produced from the PDLA film (9.66 g / L), with the PDLA HMW (8.16g / L), PLLA(6.09g / L) and PLLA HMW (5.48 g / L). Higher molecular weight PDLA than low molecular weight PLLA material. HMW It is interesting to note that a higher concentration of lactic acid was produced from the polymer.

[0095] The study reported herein provides comprehensive data on the construction of a recombinant S. cerevisiae strain that produces high levels of extracellular CLE1, enabling enhanced PLA degradation by conducting an extracellular fraction without the need for purification, concentration, or surfactant supplementation. This is the first report demonstrating such positive results in the hydrolysis of PLA material to generate an active agent used during PLA degradation using a recombinant S. cerevisiae strain. Furthermore, it also provides the most thorough evaluation of the use of recombinantly produced CLE1 in hydrolysis tests, where reduced turbidity, lactic acid production, and film weight loss all suggest an improved system for PLA degradation. Insights into the mechanism of PLA film hydrolysis are also provided.

[0096] Example 6 Extracellular CLE1 production As seen in Figure 18, SDS-PAGE analysis confirmed the extracellular production of CLE1 protein by both the Y294[CLEns] and Y294[CLEns]-TDHi strains. The protein species were easily identified (indicated by black arrowheads), and it is clear from the intensity of the protein bands that the Y294[CLEns]-TDHi strain, with expression driven by the engineered TDHi promoter, delivered more recombinant enzyme in the extracellular fraction than the Y294[CLEns] strain.

[0097] Figure 19 shows hydrolysis halo formation surrounding the Y294[CLEns] and Y294[CLEns]-TDHi recombinant strains on SC-URA agar plates containing 0.035 g / L polycaprolactone (MW 80,000). The Y294[BBH] control strain showed no halo formation, whereas both Y294[CLEns] and Y294[CLEns]-TDHi produced hydrolysis halos. It is also evident that a larger hydrolysis halo formed around Y294[CLEns]-TDHi compared to Y294[CLEns].

[0098] Figure 20 shows results from a turbidity-based enzyme assay. The assay involves incubating an emulsified PDLA substrate solution with supernatants harvested from 24-, 48-, and 72-hour yeast cultures. The Y294[BBH] control showed no significant activity, confirming that any decrease in turbidity under the assay conditions was due to the effect of the recombinant enzyme and not autohydrolysis of the PLA polymer. All three strains expressing CLE1 under the control of the engineered promoter showed activity, and both strains containing the codon-optimized sequence showed increased activity at all time points. The Y294[CLEwt] strain reached a maximum activity of 21 U / mL, 10 U / mL less than that detected in the Y294[CLEns] strain. Y294[CLEns]-TDHi was the best strain, reaching 45 U / mL after 72 hours, a 1.5-fold increase compared to Y294[CLEns], demonstrating the benefit of using the TDHi engineered promoter for expression of the codon-optimized CLE1 gene.

[0099] The optimum temperature for PLA hydrolysis was investigated by conducting turbidity-based assays at various incubation temperatures using the supernatant of strain Y294[CLEns]-TDHi (Figure 21). This appears to be the best-performing strain based on previous results. It is clear that recombinant CLE1 produced by strain Y294[CLEns]-TDHi has better PLA hydrolysis activity (1.6-fold increase) at 42 °C than the previously reported optimum of 37 °C. Performing enzymatic hydrolysis at temperatures close to the glass transition (Tg) temperature of PLA can significantly improve enzymatic recycling of solid PLA films.

[0100] The stability of the recombinant CLE1 species in the supernatant of strain Y294[CLEns]-TDHi was investigated by incubating the supernatant at 37, 42, 45, and 50°C for 120 h. The enzyme activity remained very stable at 37°C (100% residual activity) and 42°C (94% residual activity), whereas a decrease in the residual activity was detected after 120 h of incubation at 45°C and 50°C, which decreased to 75% and 28%, respectively (Figure 22). Therefore, since the recombinant enzyme is very stable at 42°C, performing PLA film hydrolysis at this temperature is a viable option.

[0101] Example 7 Small-scale cell-free hydrolysis on PLA films PLA thin films provide a better representation of real-world PLA substrates. Therefore, small-scale hydrolysis studies using 10 mL of supernatant and 10 g / L PLA film were performed at 37°C. Figure 23 shows the lactic acid released from the PLLA film over a 10-day hydrolysis period.

[0102] A significant amount of free lactic acid was released from the 10 g / L PLLA film. After 72 hours of hydrolysis, samples treated with the supernatants from the Y294[CLEns] and Y294[CLEns]-TDHi strains yielded 5.22 g / L and 7.87 g / L of lactic acid, respectively. At the end of hydrolysis (240 hours), treatment with the supernatant from the Y294[CLEns]-TDHi strain delivered 9.10 g / L of lactic acid, 3.31 g / L more than that obtained with the supernatant from the Y294[CLEns] strain.

[0103] Example 8 Scale-up and process improvement of cell-free hydrolysis of PLA films PLA hydrolysis reactions were scaled up to a 700 mL working volume in a control bioreactor setup using supernatant from the best-performing strain, Y294[CLEns]-TDHi. A PLA film loading of 10 g / L was used for these experiments, but the effects of different temperatures (37°C and 42°C) and pH control were investigated. Agitation of the hydrolysis mixture was maintained at 200 rpm using a central impeller, and addition of 3 M KOH was used in setups requiring pH control. The pH was set to 6.8 and maintained between 6.8 and 7 through a hysteresis setting of 0.2. Lactic acid release was monitored at 24-hour intervals for a total of 168 hours using HPLC analysis (Figure 24).

[0104] Similar lactic acid release was observed across all three settings during the first 24 hours of hydrolysis. The benefit of conducting hydrolysis at higher temperatures is evident, with 42°C releasing 4.5 g / L more lactic acid after 48 hours compared to 37°C. From 72 hours onward, the benefit of pH control is also evident, as lactic acid release increased more in the pH-controlled setting than in the setting without added base. Ultimately, the best conditions for PLA film hydrolysis using the recombinant CLE1 enzyme were found to be 42°C with pH control between 6.8 and 7. Under these conditions, 14.9 g / L of lactic acid was released after 168 hours of hydrolysis, resulting in a 78% weight loss in the PLA film. The present disclosure also includes the following aspects. <1> 1. A method for producing cutinase-like enzyme (CLE1) in S. cerevisiae cells, comprising: The method includes heterologously expressing a nucleic acid encoding the CLE1 in the cell, wherein the nucleic acid encoding the CLE1 is codon-optimized for expression in S. cerevisiae, and further wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter. <2> The cutinase-like enzyme has the amino acid sequence of SEQ ID NO: 2. <1> The method described below. <3> the nucleic acid encoding CLE1 has a nucleotide sequence substantially identical to SEQ ID NO: 1; <1> or <2> The method described below. <4> the engineered promoter is a TDHi engineered promoter having a nucleotide sequence substantially identical to SEQ ID NO:9, or a TEF1i engineered promoter having a nucleotide sequence substantially identical to SEQ ID NO:7; <1> ~ <3> 10. The method according to claim 9, wherein <5> the CLE1 comprises a secretion signal; <1> ~ <4> 10. The method according to claim 9, wherein <6> the S. cerevisiae cells are of the S. cerevisiae Y294 strain, S. cerevisiae Ethanol Red V1 strain, S. cerevisiae M2n strain, or S. cerevisiae YI30 strain; <1> ~ <5> 10. The method according to claim 9, wherein <7> further comprising culturing the S. cerevisiae cells to obtain a population of S. cerevisiae cells. <1> ~ <6> 10. The method according to claim 9, wherein <8> and further comprising preparing a cell-free supernatant from the population of S. cerevisiae cells, wherein the cell-free supernatant comprises the CLE1 enzyme. <7> The method described below. <9> 1. A recombinant S. cerevisiae cell comprising: a nucleic acid encoding a cutinase-like enzyme (CLE1), wherein the nucleic acid encoding the CLE1 is codon-optimized for expression in S. cerevisiae, and wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter; A recombinant S. cerevisiae cell, wherein the recombinant S. cerevisiae cell is capable of heterologously expressing the CLE1. <10> The cutinase-like enzyme has the amino acid sequence of SEQ ID NO: 2. <9> Recombinant S. cerevisiae cells as described in. <11> the nucleic acid encoding CLE1 has a nucleotide sequence substantially identical to SEQ ID NO: 1; <9> or <10> Recombinant S. cerevisiae cells as described in. <12> the engineered promoter is a TDHi engineered promoter having a nucleotide sequence substantially identical to SEQ ID NO:9, or a TEF1i engineered promoter having a nucleotide sequence substantially identical to SEQ ID NO:7; <9> ~ <11> 10. The recombinant S. cerevisiae cell according to any one of claims 1 to 9. <13> the CLE1 comprises a secretion signal; <9> ~ <12> 10. The recombinant S. cerevisiae cell according to any one of claims 1 to 9. <14> the recombinant S. cerevisiae cells are of the S. cerevisiae Y294 strain, the S. cerevisiae Ethanol Red V1 strain, the S. cerevisiae M2n strain, or the S. cerevisiae YI30 strain; <9> ~ <13> 10. The recombinant S. cerevisiae cell according to any one of claims 1 to 9. <15> <1> ~ <8> or a method according to any one of <9> ~ <14> A cutinase-like enzyme produced by the recombinant S. cerevisiae cell according to any one of claims 1 to 4. <16> 1. A method for preparing a cell-free supernatant containing a cutinase-like enzyme, comprising: <9> ~ <14> Culturing the recombinant S. cerevisiae cell according to any one of the preceding claims to obtain a population of S. cerevisiae cells; and preparing a cell-free supernatant from said population of S. cerevisiae cells. <17> further comprising concentrating the cell-free supernatant. <16> The method described below. <18> the concentrating is by lyophilization, filtration, precipitation, and / or chromatography; <17> The method described below. <19> <8> or <16> ~ <18> 1. A cell-free supernatant prepared by any one of the methods described above. <20> for hydrolyzing bioplastic polymers, <15> or a cutinase-like enzyme according to <19> Use of the cell-free supernatant described in 1. <21> 1. A method for hydrolyzing a bioplastic polymer, comprising: <15> or a cutinase-like enzyme according to <19> 2. A method comprising incubating the cells with the cell-free supernatant described in claim 1. <22> The incubation of the polymer with the cutinase-like enzyme or the cell-free supernatant is performed at about 42°C to about 45°C and a pH of 6.8 to 7. <21> The method described below. <23> 1. A method for hydrolyzing a bioplastic polymer, comprising: <9> ~ <14> 10. A method comprising incubating a recombinant S. cerevisiae cell of claim 1 with the recombinant S. cerevisiae cell of claim 9.

Claims

1. 1. A method for producing cutinase-like enzyme (CLE1) in S. cerevisiae cells, comprising: heterologously expressing in the cell a nucleic acid encoding the CLE1, wherein the nucleic acid encoding the CLE1 is codon-optimized for expression in S. cerevisiae, and further wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5'-UTR intron.

2. 2. The method of claim 1, wherein the cutinase-like enzyme has the amino acid sequence of SEQ ID NO:

2.

3. 3. The method of claim 1 or 2, wherein the nucleic acid encoding CLE1 has the nucleotide sequence of SEQ ID NO:

1.

4. 4. The method of any one of claims 1 to 3, wherein the engineered promoter is a TDHi engineered promoter having the nucleotide sequence of SEQ ID NO: 9 or a TEF1i engineered promoter having the nucleotide sequence of SEQ ID NO:

7.

5. The method of any one of claims 1 to 4, wherein the CLE1 comprises a secretion signal.

6. 6. The method of any one of claims 1 to 5, wherein the S. cerevisiae cells are of the S. cerevisiae strain Y294, S. cerevisiae strain Ethanol Red V1, S. cerevisiae strain M2n, or S. cerevisiae strain YI30.

7. 7. The method of any one of claims 1 to 6, further comprising culturing the S. cerevisiae cells to obtain a population of S. cerevisiae cells.

8. 8. The method of claim 7, further comprising preparing a cell-free supernatant from the population of S. cerevisiae cells, wherein the cell-free supernatant comprises the CLE1 enzyme.

9. A recombinant S. cerevisiae cell comprising: a nucleic acid encoding a cutinase-like enzyme (CLE1), wherein the nucleic acid encoding the CLE1 is codon-optimized for expression in S. cerevisiae, and further wherein the nucleic acid encoding the CLE1 is operably linked to an engineered promoter comprising a 5'-UTR intron; A recombinant S. cerevisiae cell, wherein the recombinant S. cerevisiae cell is capable of heterologously expressing the CLE1.

10. 10. The recombinant S. cerevisiae cell of claim 9, wherein the cutinase-like enzyme has the amino acid sequence of SEQ ID NO:

2.

11. 11. The recombinant S. cerevisiae cell of claim 9 or 10, wherein the nucleic acid encoding CLE1 has the nucleotide sequence of SEQ ID NO:

1.

12. 12. The recombinant S. cerevisiae cell of any one of claims 9 to 11, wherein the engineered promoter is a TDHi engineered promoter having the nucleotide sequence of SEQ ID NO:9 or a TEF1i engineered promoter having the nucleotide sequence of SEQ ID NO:

7.

13. 13. The recombinant S. cerevisiae cell of any one of claims 9 to 12, wherein the CLE1 comprises a secretion signal.

14. The recombinant S. cerevisiae cell of any one of claims 9 to 13, wherein the recombinant S. cerevisiae cell is of the S. cerevisiae strain Y294, S. cerevisiae strain Ethanol Red V1, S. cerevisiae strain M2n, or S. cerevisiae strain YI30.

15. A cutinase-like enzyme produced by the method of any one of claims 1 to 8 or by the recombinant S. cerevisiae cell of any one of claims 9 to 14.

16. 1. A method for preparing a cell-free supernatant containing a cutinase-like enzyme, comprising: Culturing the recombinant S. cerevisiae cell of any one of claims 9 to 14 to obtain a population of S. cerevisiae cells; and preparing a cell-free supernatant from said population of S. cerevisiae cells.

17. 17. The method of claim 16, further comprising concentrating the cell-free supernatant.

18. 18. The method of claim 17, wherein said concentrating is by lyophilization, filtration, precipitation and / or chromatography.

19. A cell-free supernatant prepared by the method of any one of claims 8 or 16 to 18.

20. 20. Use of the cutinase-like enzyme of claim 15 or the cell-free supernatant of claim 19 for hydrolyzing bioplastic polymers.

21. 20. A method for hydrolyzing a bioplastic polymer, comprising incubating the polymer with the cutinase-like enzyme of claim 15 or the cell-free supernatant of claim 19.

22. 22. The method of claim 21, wherein the incubation of the polymer with the cutinase-like enzyme or the cell-free supernatant is at 42°C to 45°C and a pH of 6.8 to 7.

23. 15. A method for hydrolyzing a bioplastic polymer, comprising incubating said polymer with a recombinant S. cerevisiae cell according to any one of claims 9 to 14.

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