Yeast engineering strain for high-efficiency production of plastic depolymerase and application thereof

Abstract

This invention relates to the fields of microbial fermentation and genetic engineering technology, specifically disclosing a highly efficient engineered yeast strain for producing plastic depolymerization enzymes and its applications. The engineered yeast strain is *Pichia pastoris* (…). Pichia pastoris Using a specific yeast strain as the expression host, a molecular chaperone was co-expressed, and the vacuolar sorting receptor gene was knocked out or knocked down. Furthermore, this application combines regulatory element optimization and target gene copy number regulation to construct a yeast cell factory for efficiently expressing plastic depolymerase. The regulatory elements include any one or more combinations of strong promoters, signal peptides, and C-terminal tags. The engineered yeast strain provided by this invention can achieve efficient accumulation and secretion of plastic depolymerase, significantly increasing enzyme yield and reducing the production cost per unit enzyme activity. It provides a stable and efficient yeast expression system for the industrial-scale, low-cost production of plastic depolymerase, and has significant application prospects in the field of biodegradation and resource utilization of waste plastics.

Description

Technical Field

[0001] This invention relates to the field of microorganisms and genetic engineering, specifically to a yeast strain that efficiently produces plastic depolymerase and its applications. Background Technology

[0002] Plastic pollution is one of the major environmental challenges facing the world, especially polyester plastics, represented by polyethylene terephthalate (PET), which are chemically stable and degrade extremely slowly. Bioenzymatic hydrolysis, as an environmentally friendly and mild plastic recycling strategy, has received widespread attention in recent years. The core of this technology lies in using plastic depolymerization enzymes (such as PETase and keratinase) to catalytically depolymerize plastic polymers into reusable monomers or oligomers, thereby achieving the recycling and "upgrading" of plastic waste. However, one of the key aspects of realizing the industrial application of this technology is how to produce highly active plastic depolymerization enzymes on a large scale at low cost and high efficiency.

[0003] Pichia pastoris ( Pichia pastoris As a highly efficient eukaryotic protein expression system, it has been widely used in the industrial production of recombinant proteins. It possesses significant advantages such as rapid growth, high-density fermentation capability, potent and tightly regulated promoters (e.g., AOX1), the ability to perform correct post-translational modifications, and low background protein secretion, making it considered an ideal "cell factory" for producing exocrine industrial enzymes. Using expression strategies, target proteins can be directly transported to extracellular culture media, which not only helps maintain enzyme activity but also greatly simplifies downstream separation and purification processes, thereby significantly reducing production costs. Although... P. pastoris While the system has obvious advantages, the expression of exogenous proteins also faces many bottlenecks, such as protein misfolding, low endoplasmic reticulum transport efficiency, abnormal post-secretory processing, or easy degradation by host proteases. As a result, the actual yield and activity often fail to meet the economic requirements of industrial production, which seriously restricts the economic feasibility of enzyme production.

[0004] In existing technologies, researchers typically modify recombinant proteins from a single perspective to increase their yield. This includes screening or optimizing signal peptides to improve secretion efficiency, using strong promoters to enhance transcription levels, or overexpressing a single molecular chaperone to assist protein folding. However, these single strategies often fail to fundamentally address the simultaneous folding pressures and degradation issues faced by exogenous proteins within host cells. For example, a significant increase in transcription levels may exceed the capacity of the host's folding and transport systems, triggering endoplasmic reticulum stress; and relying solely on molecular chaperone overexpression still cannot prevent some incorrectly folded proteins from being directed to vacuoles and degraded.

[0005] Therefore, a new engineering strategy is urgently needed to synergistically optimize both protein folding quality control and degradation pathway regulation in the Pichia pastoris expression system, in order to reduce misfolding and non-specific degradation of plastic depolymerase in host cells, thereby significantly increasing its effective accumulation and secretion level. This is of vital significance for breaking through the production bottleneck of plastic depolymerase and promoting the practical application of plastic biorecycling technology. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a yeast strain that produces plastic depolymerization enzymes and its applications, in order to address the shortcomings of the prior art.

[0007] To address the aforementioned technical problems, this invention discloses a highly efficient engineered yeast strain for producing plastic depolymerization enzymes and its applications. The specific technical solution is as follows:

[0008] In a first aspect, the present invention provides a yeast strain that efficiently produces plastic depolymerization enzymes, using Pichia pastoris (… Pichia pastoris The host bacterium co-expressed the gene encoding plastic depolymerase and a molecular chaperone, and knocked out or knocked down the vacuole sorting receptor gene VPS10. The molecular chaperone is used to assist protein folding, processing or secretion, and the vacuole sorting receptor gene VPS10 is used to reduce the misfolding and vacuole degradation of plastic depolymerase in cells, thereby achieving efficient expression and secretion of plastic depolymerase.

[0009] The plastic depolymerization enzyme includes an enzyme with polyethylene terephthalate (PET) degradation activity; the molecular chaperone is selected from any one of YDJ1, UBC1, HRD1, CPR5, ERO1, PDI, PDI1, HAC1, SSA4, SSO1, or KEX2; wherein the Gene ID of YDJ1 is 855661, the Gene ID of UBC1 is 8200711, the Gene ID of HRD1 is 854149, the Gene ID of CPR5 is 851898, the Gene ID of ERO1 is 8197528, the Gene ID of PDI is 8197593, the Gene ID of PDI1 is 8201243, the Gene ID of HAC1 is 8196642, the Gene ID of SSA4 is 856840, the Gene ID of SSO1 is 855844, and the Gene ID of KEX2 is 855483. In some embodiments of the present invention, the molecular chaperone is HRD1.

[0010] The vacuole sorting receptor gene VPS10 includes VPS10-1 or VPS10-2, and the nucleotide sequences of VPS10-1 and VPS10-2 are shown in SEQ ID NO.32 and SEQ ID NO.33, respectively. In some embodiments of the present invention, the vacuole sorting receptor gene VPS10 is VPS10-2.

[0011] The gene encoding the plastic depolymerase is expressed under the regulation of a regulatory element, which includes any one or more combinations of the following (1) to (3):

[0012] (1) Strong promoter;

[0013] (2) Signal peptide;

[0014] (3) C-end tags.

[0015] The strong promoters include any one of AOX1 (methanol-induced alcohol oxidase promoter), MOX1 (methanol oxidase gene promoter from Hansenula polymorpha), or FLD1 (methanol or methylamine-induced formaldehyde dehydrogenase promoter), and the nucleotide sequences of AOX1, MOX1, and FLD1 are shown in SEQ ID NO.13~14 and SEQ ID NO.16, respectively.

[0016] The signal peptide includes any one of the sequences shown in SEQ ID NO. 17~20, SEQ ID NO. 23~24, SEQ ID NO. 26 or SEQ ID NO. 28;

[0017] The C-end label mentioned includes the 6×His label.

[0018] In some embodiments of the present invention, the coding gene for the plastic depolymerase and the regulatory element are integrated or transformed into the engineered yeast in the form of an expression cassette, wherein the expression cassette sequentially comprises: a promoter, a signal peptide coding sequence (optional), a coding gene for the plastic depolymerase, a C-terminal tag coding sequence (optional), and a terminator. In other embodiments of the present invention, at least one of the expression cassettes is integrated or transformed into the engineered yeast.

[0019] The regulatory element includes a strong promoter, a signal peptide, and a C-terminal tag. The strong promoter includes MOX1, the signal peptide includes the sequence shown in SEQ ID NO.24, and the C-terminal tag includes a 6×His tag.

[0020] The engineered yeast strain contains a gene encoding a plastic depolymerase with 3 copies. In some embodiments of the present invention, the gene encoding the plastic depolymerase is optimized for a specific copy number through resistance screening, preferably by screening with 3 g / L of genimycin (G418) to obtain transformants with 3 copies.

[0021] The amino acid sequence of the plastic depolymerase includes any one of SEQ ID NO. 1 to 6, which are respectively encoded by the nucleotide sequences shown in SEQ ID NO. 7 to 12.

[0022] The host bacteria mentioned include Pichia pastoris (Pichia pastoris) Pichia pastoris GS115, X-33, SMD1168H, or KM71 are all selected from these strains. In some embodiments of the present invention, the host strain includes Pichia pastoris (…). Pichia pastoris GS115 or KM71.

[0023] This invention provides an engineered yeast strain, using Pichia pastoris (Pichia pastoris) Pichia pastoris GS115 is the host strain, which co-expresses the gene encoding plastic depolymerase and a molecular chaperone, while knocking out the vacuolar sorting gene VPS10-2. The expression of the plastic depolymerase gene is regulated by the strong promoter MOX1, the signal peptide shown in SEQ ID NO.24, and a 6×His tag. The engineered yeast strain contains 3 copies of the plastic depolymerase gene. The molecular chaperone is HRD1 (Gene ID: 854149). The sequence of the vacuolar sorting gene VPS10-2 is shown in SEQ ID NO.33, and the nucleotide sequence of the strong promoter MOX1 is shown in SEQ ID NO.14.

[0024] This invention also provides a method for constructing the engineered yeast strain described in the first aspect, comprising amplifying the coding gene for plastic depolymerase and its molecular chaperone, cloning them into a plasmid vector, and introducing them into a host cell to obtain the engineered yeast strain. In some embodiments of this invention, when cloning the coding gene for plastic depolymerase into the plasmid vector, regulatory elements (any one or a combination of promoters, signal peptides, or C-terminal tags) are also cloned into the plasmid vector; the coding gene for plastic depolymerase and the regulatory elements are cloned into the plasmid vector pPIC9K, and the molecular chaperone is cloned into the plasmid vector pGAPZA; in other embodiments of this invention, the vacuolar sorting receptor gene is also knocked out in the host cell.

[0025] Secondly, this invention provides the application of the aforementioned engineered yeast strain in the production of plastic depolymerization enzymes, thereby providing low-cost and efficient enzyme preparation production technology support for the biological recycling of plastics.

[0026] Thirdly, the present invention provides a method for producing plastic depolymerase using the engineered yeast strain, comprising the following steps: inoculating the engineered yeast strain into a fermentation medium and fermenting it at 25-32°C for 96-144 h.

[0027] The fermentation culture involves first inoculating the culture medium into a first fermentation medium and culturing it at 25-32°C for 16-32 hours, followed by inoculation into a second fermentation medium and culturing it at 25-32°C for another 80-112 hours. The first fermentation medium uses glycerol as the carbon source, and the second fermentation medium uses methanol as the carbon source. In some embodiments of the invention, the first fermentation medium includes BMGY medium (using glycerol as the sole carbon source), and the second fermentation medium includes BMMY medium (using methanol as the sole carbon source). In other embodiments of the invention, the fermentation culture involves culturing in the first fermentation medium (using glycerol as the sole carbon source) until the carbon source is depleted, then adding methanol (to obtain the second fermentation medium) for induction culture.

[0028] Beneficial effects:

[0029] (1) This invention, through the synergistic effect of molecular chaperone co-expression and vacuole sorting receptor gene regulation, simultaneously acts on two key limiting links in the expression process of plastic depolymerase: protein folding and intracellular degradation, significantly improving the correct folding ratio of plastic depolymerase and its effective extracellular accumulation level. Compared with the technical solutions that only use molecular chaperone co-expression or only regulate the vacuole pathway, this invention can more effectively reduce the ineffective consumption of plastic depolymerase during the expression process, increase the enzyme yield per unit fermentation volume, and show a more stable and reproducible expression effect.

[0030] (2) Based on the above core engineering strategy, this invention combines signal peptide optimization, promoter regulation, protein C-terminal engineering and copy number regulation and other optimization methods to further improve the expression efficiency and industrial adaptability of plastic depolymerase, and is suitable for large-scale production;

[0031] (3) The yeast engineered strain constructed in this invention has a clear genetic background, mild culture conditions, and easy-to-control fermentation process, which significantly reduces the production cost of plastic depolymerization enzyme. The obtained enzyme preparation can be directly applied to the biodegradation of polyester plastics and has good industrial application prospects. Attached Figure Description

[0032] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0033] Figure 1 Expression of different plastic depolymerases in Pichia pastoris strains.

[0034] Figure 2 Comparison of the secretion activities of plastic depolymerase expressed in different Pichia pastoris host strains.

[0035] Figure 3 To enhance the activity of plastic depolymerase by knocking out the vacuole sorting receptor gene.

[0036] Figure 4 This study demonstrates the synergistic effect of molecular chaperone co-expression and vacuole sorting receptor gene knockout, where A represents the effect of different molecular chaperones on PETase 1 expression, and B represents the effect of different plastic depolymerases on co-expressed molecular chaperones. HRD1 Synergistic effect with vacuolar sorting receptor gene knockout.

[0037] Figure 5 To investigate the effect of signal peptide screening on enhancing the secretion activity of plastic depolymerase.

[0038] Figure 6 The diagram shows the enzyme activity of plastic depolymerase expression for the C-terminal tag and promoter, respectively, where A is the C-terminal tag and B is the promoter screening. Detailed Implementation

[0039] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0040] In the following examples, the transformant screening plate (g / L) contains: 20g peptone, 10g yeast extract, 20g glucose, 20g agar powder, 0.1g bleomycin or 3.0g genimycin (G418).

[0041] The Minimal Dextrose Medium (MD) deficient culture medium contains: yeast basal nitrogen source 6.7, glucose 20, and agar powder 20.

[0042] The BMGY fermentation medium (g / L) contained: peptone 20g, yeast extract 10g, dipotassium hydrogen phosphate 3g, potassium dihydrogen phosphate 11.8g, yeast basal nitrogen source 13.4g, and biotin 4×10⁻⁶. -4 10g glycerol.

[0043] The BMMY induction medium (g / L) consisted of: peptone 20 g, yeast extract 10 g, dipotassium hydrogen phosphate 3 g, potassium dihydrogen phosphate 11.8 g, yeast basal nitrogen source 13.4 g, and biotin 4 × 10⁻⁶ g / L. -4 And add 2% v / v methanol.

[0044] The BSM fermentation medium (g / L) consists of: 85% v / v phosphate solution (26.7 mL / L), calcium sulfate dihydrate 0.93, potassium sulfate 18.2, magnesium sulfate dihydrate 14.9, potassium hydroxide 4.13, glycerol 40, and 4 mL / L filtered sterilized PTM1, adjusted to pH 5.5 with ammonia. The PTM1 (g / L) consists of: sulfuric acid 5 mL / L, sodium iodide 0.08, copper sulfate 6, manganese sulfate 3, boric acid 0.02, cobalt chloride 0.5, zinc chloride 20, ferrous sulfate 65, sodium molybdate 0.2, and D-biotin 0.25.

[0045] Example 1: Validation of the expression of exogenous plastic depolymerase in Pichia pastoris

[0046] First, the codons of the PET depolymerase gene were optimized and an expression vector was constructed. This example includes the following PET depolymerases:

[0047] PET depolymerase 1 (PETase 1, amino acid sequence as shown in SEQ ID NO. 1, nucleotide sequence as shown in SEQ ID NO. 7); PET depolymerase 2 (FAST-PETase, amino acid sequence as shown in SEQ ID NO. 2, nucleotide sequence as shown in SEQ ID NO. 8); PET depolymerase 3 ( Mu PETase (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.9); PET depolymerase 4 (PES-H1, amino acid sequence as shown in SEQ ID NO.4, nucleotide sequence as shown in SEQ ID NO.10); PET depolymerase 5 (PES-H1 mutant 1, amino acid sequence as shown in SEQ ID NO.5, nucleotide sequence as shown in SEQ ID NO.11); PET depolymerase 6 (PES-H1 mutant 2, amino acid sequence as shown in SEQ ID NO.6, nucleotide sequence as shown in SEQ ID NO.12).

[0048] The above gene sequences were inserted into the multiple cloning site of the Pichia pastoris expression vector pPIC9K, using the following restriction enzyme sites: EcoR I and Not I. Recombinant plasmids carrying the target gene (named pPIC9K-PETase 1 and pPIC9K-Fast, respectively) - PETase, pPIC9K- Mu PETase, pPIC9K-PES-H1, pPIC9K-PES-H1 Mut1 and pPIC9K-PES-H1 Mut2 )use SalI. Restriction endonucleases were linearized and transformed into the expression host strain *Pichia pastoris* GS115. Auxotrophic transformants were obtained by screening on MD auxotrophic medium. The transformants were further screened on transformant selection plates containing 3.0 g / L G418. qPCR identification yielded engineered strains with 3 copies of the target gene stably integrated, named GS115 PETase 1-00, GS115 FAST-PETase, GS115 PES-H1, and GS115 PES-H1, respectively. Mut1 GS115 PES-H1 Mut2 .

[0049] Single-clone engineered bacteria were picked and inoculated into BMGY medium, and cultured with shaking at 30 °C until OD reached. 660 Approximately 15 cells were collected by centrifugation and resuspended in BMMY induction medium. Expression was induced at 30 °C for 96 hours, with methanol added every 24 hours until a final concentration of 2% v / v was reached. After induction, the fermentation supernatant was collected by centrifugation as the crude enzyme solution.

[0050] Enzyme activity was determined using the pNPO method. The reaction was carried out at 37℃ and pH 8 for 3 minutes, and the amount of p-nitrophenol produced was measured. The results showed that ( Figure 1 The plastic depolymerase described herein can be effectively expressed in Pichia pastoris and exhibits detectable enzymatic activity. Protein concentration was detected using a protein assay kit.

[0051] Example 2: Effects of different Pichia pastoris hosts on basal expression

[0052] Based on Example 1, taking PET depolymerase 1 as an example, the effect of different Pichia pastoris hosts on the expression of plastic depolymerase was evaluated. Any one of Pichia pastoris GS115, X33, SMD1168H or KM71 was selected as the expression host, and the corresponding engineered strains were constructed respectively.

[0053] The recombinant plasmid pPIC9K-PETase 1 constructed in Example 1 was transformed into different hosts described in this example, and cultured, induced, and had its enzyme activity measured according to the method described in Example 1. The results showed ( Figure 2 Under the same culture and induction time, the enzyme activities of different host strains showed significant differences: the KM71 engineered strain had the highest enzyme activity at 18.29 mg / L; the GS115 engineered strain had 10.64 mg / L; the SMD1168H engineered strain had 5.99 mg / L; and the X33 engineered strain had 3.96 mg / L. The results indicate that the host strain type has a significant impact on the expression of plastic depolymerase, with KM71 being the superior expression host and GS115 the second-best.

[0054] Example 3: Co-expression of molecular chaperones

[0055] This embodiment further investigates the effect of molecular chaperone co-expression on the expression of plastic depolymerase. A co-expression vector was constructed using pGAPZA as the expression backbone. EcoR I and Sal Using enzyme I as the cleavage site, the molecular chaperone is integrated into the pGAPZA vector, resulting in a recombinant vector expressing the molecular chaperone. Avr II. After linearization, the enzyme was electroporated into the host Pichia pastoris GS115 PETase 1-00. Recombinant strains expressing PET depolymerase 1 and co-expressing different molecular chaperones were obtained by screening transformants on bleomycin-containing plates. These molecular chaperones included YDJ1 (Gene ID: 855661), UBC1 (Gene ID: 8200711), HRD1 (Gene ID: 854149), and CPR5 (Gene ID: 851898) related to protein folding and quality; ERO1 (Gene ID: 8197528), PDI (Gene ID: 8197593), PDI1 (Gene ID: 8201243), HAC1 (Gene ID: 8196642), AFT1 (Gene ID: 8196669), and SSA4 (Gene ID: 856840) related to control systems; and BMH2 (Gene ID: 851676) and SSO1 (Gene ID: 851676) related to protein transport. The study included either the protein processing and secretion-related KEX2 (Gene ID: 855844) or the GS115 PETase1-00 strain, which does not co-express a molecular chaperone, as a control.

[0056] Enzyme expression was induced under the conditions described in Example 1, and enzyme activity was measured. The results (Table 1) showed that, compared with the control strain GS115PETase 1-00 (protein concentration of 10.64 mg / L), the co-expression control system generally had a more significant promoting effect. Among them, the molecular chaperone that assists in protein folding and quality control had a more significant enhancing effect, with enzyme activity increasing by 2.25-3.72 times: the highest protein concentration in the fermentation supernatant obtained from the engineered strain co-expressing the HRD1 molecular chaperone reached 39.60 mg / L.

[0057] Table 1. Enzymatic activity enhancement of co-expressed molecular chaperones on plastic depolymerization enzymes

[0058]

[0059] Example 4: Vacuole sorting receptor knockout

[0060] This embodiment provides a method for improving the secretion efficiency of exogenous proteins based on vacuole sorting receptor gene editing. Specifically, the vacuole sorting receptor genes VPS10-1 (nucleotide sequence shown in SEQ ID NO. 32) and / or VPS10-2 (nucleotide sequence shown in SEQ ID NO. 33) in Pichia pastoris are knocked out using a CRISPR / Cas9 system to construct single or double knockout mutants, and their effects on the secretory expression of target proteins are verified.

[0061] (1) Construction of CRISPR / Cas9 editing system. The sgRNA sequences sgRNAVPS10-1 (SEQ ID NO.42) and sgRNAVPS10-2 (SEQ ID NO.43) designed for the VPS10-1 and VPS10-2 genes were cloned into the pCas9 vector to obtain recombinant editing plasmids pCas9-sgRNA-VPS10-1 and pCas9-sgRNA-VPS10-2. Homologous arms of the VPS10-1 and VPS10-2 genes, 1000 bp upstream and downstream, were amplified from the Pichia pastoris genome using primers VPS10-1-upF / R (SEQ ID NO.34~35), VPS10-1-downF / R (SEQ ID NO.36~37), VPS10-2-upF / R (SEQ ID NO.38~39), and VPS10-2-downF / R (SEQ ID NO.40~41), respectively. The Donor fragments Donor-VPS10-1 and Donor-VPS10-2 were constructed by overlap PCR and used as homology repair templates.

[0062] (2) Transformation, screening and identification of VPS10 gene-deficient strains. The above recombinant plasmid and the corresponding homologous repair template were co-electrotransduced into the host strain GS115 PETase 1-00 constructed in Example 1, and transformants were obtained by screening with a hygromycin YPD resistance plate containing 200 µg / mL. The transformants were verified by colony PCR using primers V1test F / R (SEQ ID NO.44~45) and V2test F / R (SEQ ID NO.46~47). The single gene knockout mutants GS115 PETase1-00 VPS10-1△ and GS115 PETase 1-00 VPS10-2△, as well as the double gene knockout mutant GS115 PETase1-00 VPS10△, were successfully obtained and named GS115 PETase 1-10, GS115 PETase 1-20 and GS115 PETase 1-30, respectively.

[0063] (3) The engineered strains were fermented and cultured according to the method described in Example 1, and their enzyme activity was measured. Three parallel experiments were set up for each strain. The results showed that ( Figure 3 The enzyme activities of the single knockout mutants GS115 PETase 1-00 VPS10-1△ and GS115PETase 1-00 VPS10-2△ reached 13.48 mg / L and 17.81 mg / L, respectively, which were approximately 1.26-fold and 1.67-fold higher than the starting strain (GS115PETase 1-00, 10.64 mg / L). However, the enzyme activity of the double knockout mutant GS115 PETase 1-00 VPS10△ decreased to 5.95 mg / L. This indicates that knocking out VPS10-1 or VPS10-2 genes alone can increase the secretion yield of the target protein in Pichia pastoris, while knocking out both genes simultaneously leads to a significant decrease in secretion capacity.

[0064] Example 5: Synergistic Implementation of Molecular Chaperones and Vacuole Sorting Regulation

[0065] Based on Examples 3 and 4, a synergistic engineered expression system was further constructed by combining vacuole sorting receptor knockout with molecular chaperones that differentiate protein folding and quality class. The results showed that ( Figure 4 In the A group, there is a synergistic effect between the co-expression of molecular chaperones and vacuole sorting regulation. This was observed in the GS115 PETase 1-00 strain. HRD1 The preferred strain with the VPS10-2 gene knocked out was named GS115 PETase 1-21 (expressed in GS115 PETase 1-00 strain according to the method described in Example 3). HRD1 Then, the VPS10-2 gene was knocked out again according to the method described in Example 4. This was done on strain GS115 where only the gene expressed... HRD1 Furthermore, the chassis cells that did not express the target PET depolymerase after the VPS10-2 gene was knocked out were named GS115 PET-00 (expressed in GS115 strain according to the method described in Example 3). HRD1 Then, the VPS10-2 gene was knocked out again using the method described in Example 4.

[0066] This embodiment further verifies the applicability and universal promoting effect of the synergistic engineering strategy combining molecular chaperone co-expression and vacuole sorting receptor knockout in different plastic depolymerization enzymes.

[0067] The six plasmids pPIC9K-PETase 1 and pPIC9K-Fast from Example 1 were used. - PETase, pPIC9K- MuPETase, pPIC9K-PES-H1, pPIC9K-PES-H1 Mut1 and pPIC9K-PES-H1 Mut2 Through respectively Sal I. Restriction endonucleases were linearized and transformed into Pichia pastoris GS115 PET-00. Auxotrophic transformants were obtained by MD plate selection. The transformants were further inoculated onto transformant selection plates containing 3.0 g / L G418 for selection. Copy number verification was performed by qPCR, yielding an engineered strain stably integrating 3 copies of the target gene.

[0068] Single clones of the engineered bacteria corresponding to the different plastic depolymerases were selected and fermented according to the method described in Example 1. Enzyme activity and expression levels were analyzed. The results showed that (…) Figure 4 In the context of the same co-engineered host, plastic depolymerases from different sources, with different sequences and structural features can all achieve stable secretory expression.

[0069] Compared with the corresponding basal expression strains without the introduction of molecular chaperone co-expression and vacuole sorting regulation strategies, the secretion levels and enzyme activities of each plastic depolymerase were significantly improved under the background of the synergistic engineered bacteria, and no obvious expression instability or cell growth inhibition was observed.

[0070] The above results demonstrate that the synergistic engineering strategy combining molecular chaperone co-expression and vacuole sorting receptor knockout is not only applicable to the expression optimization of a single plastic depolymerase, but also has a good universal promoting effect on a variety of different types of plastic depolymerases, thus verifying the universal applicability of this strategy in constructing engineered bacteria for efficient plastic depolymerase production.

[0071] Example 6 Systematic Screening and Optimization of Plastic Depolymerase Expression Regulatory Elements under a Cooperative Engineering Strategy

[0072] Based on the synergistic engineered bacteria GS115 PETase 1-21 described in Example 5, the secretory expression of plastic depolymerase was further systematically optimized from multiple expression regulation levels, including signal peptide, protein C-terminal engineering, and promoter regulation.

[0073] (1) Screening of different signal peptides

[0074] Based on the aforementioned synergistic engineered bacteria, different secretion signal peptides were systematically screened. Using the Pichia pastoris secretory expression vector pPIC9K as the backbone, a differential design was performed on the signal peptide in the recombinant vector pPIC9K-PETase 1 constructed in Example 1: The α-factor secretion signal peptide (α-factor, whose amino acid sequence is shown in SEQ ID NO. 31) inherent in this recombinant vector was used as a control. The signal peptide was then replaced with one of the following: D1SS, D2SS, Mss, Fss, Wss, Gss, Hss, Iss, Kss, Inss, Lss, Apss, hss, or Alss, respectively, to obtain new signal peptides. The amino acid sequences of each signal peptide are shown in SEQ ID NO. 17~30.

[0075] The constructed vectors (containing the plastic depolymerase gene PETase 1) were transformed into the engineered strain GS115 PET-00 to obtain a series of differentially expressed signal peptide strains. Fermentation was carried out under the same culture and induction conditions as in Example 1, and the extracellular plastic depolymerase activity was measured. The results showed that the engineered strain with the signal peptide obtained by replacing Iss (amino acid sequence as shown in SEQ ID NO. 24) had the best secretion efficiency. Figure 5 As shown.

[0076] (2) The impact of C-end engineering on expression

[0077] Based on the co-engineered strain GS115 PETase 1-21, the effects of two construction forms—one with a His tag fused to the C-terminus of the plastic depolymerase and the other without—on expression and secretion performance were further compared. Specifically, using the encoding gene of PETase 1 as a template, the PETase 1 gene was fused with a 6×His tag via overlapping extension PCR using primers 6×His F / R (nucleotide sequences shown in SEQ ID NO. 48~49), and then cloned into the Pichia pastoris expression vector pPIC9K to construct the recombinant plasmid pPIC9K-PETase-6×His. After sequencing verification, this plasmid was introduced into the GS115 PET-00 strain via electroporation. Fermentation was carried out under the same culture and induction conditions as in Example 1, and the extracellular plastic depolymerase activity was measured. The results showed that, as Figure 6 As shown in Figure A, the C-terminal tag has different effects on the expression of plastic depolymerase in the context of GS115 as the host bacterium, indicating that carrying a His tag at the C-terminus can increase the expression level of plastic depolymerase.

[0078] (3) Comparison of the regulatory effects of different strong promoters

[0079] Based on the synergistic engineered bacteria GS115 PETase 1-21, P was used respectively. MOX1 P GAP and P FLD1 The AOX1 promoter (nucleotide sequence shown in SEQ ID NO. 14-16) on the original pPIC9K vector was replaced with a promoter (nucleotide sequence shown in SEQ ID NO. 13) to drive the expression of plastic depolymerase, thus constructing engineered strains regulated by different promoters. The strains were induced and cultured according to the method described in Example 1, and the extracellular enzyme activity was measured. The results are as follows: Figure 6 As shown in B, different promoters have a significant impact on the expression level of plastic depolymerase, among which P MOX1 The promoters exhibited high expression levels in the described co-engineering system.

[0080] (4) Integration and validation of the optimal representation system

[0081] Based on the above step-by-step optimization results, the optimal components were integrated to construct a high-efficiency expression system for plastic depolymerase in Pichia pastoris. The specific implementation method is as follows:

[0082] The optimal recombinant plasmid was constructed as follows: the plastic depolymerase gene PETase 1 was generated by the regulatory element promoter P. MOX1 The expression is guided by the signal peptide Iss and the C-terminal 6×His tag, specifically by replacing the AOX1 promoter of the recombinant plasmid pPIC9K-PETase 1 constructed in Example 1 with P MOX1 The α-factor signal peptide was replaced with the Iss signal peptide, and a 6×His tag was directly linked to the C-terminus of the PETase 1 gene to obtain an optimized recombinant plasmid. This optimized recombinant plasmid was then used... Sal I. Restriction endonucleases were linearized and transformed into the co-engineered strain GS115 PET-00. Following the resistance screening method described in Example 1, an engineered strain with 3 copies (named GS115 PETase 1-40) was obtained. Fermentation culture was then carried out according to the method described in Example 1, and enzyme activity was measured. The results showed that the extracellular protein concentration was 179.07 mg / L, a 16.83-fold increase compared to the original engineered strain (GS115 PETase 1-00). This integrated system achieved significant improvements in both enzyme activity and secretion efficiency.

[0083] Example 7: 5L fermenter-scale production

[0084] Using the above-mentioned preferred strains, high-density fermentation was carried out in a 5 L fermenter. The specific steps are as follows:

[0085] (1) Cell growth stage: Initial batch culture was carried out in BSM medium containing 40 g / L glycerol. The culture conditions were: temperature 30℃, pH 5.5, tank pressure 0.05 MPa, stirring speed adjusted according to dissolved oxygen, and dissolved oxygen (DO) controlled at 30%-35%. The culture time was about 20 hours.

[0086] (2) Feeding fermentation stage: Start feeding with 50% v / v glycerol at a rate of 6-12 mL / L / h, and continue culturing until the cell wet weight of the fermentation broth reaches about 180 g / L (in this example, the feeding fermentation time is 8 h). During this stage, maintain DO at 30%-35%.

[0087] (3) Starvation stage: Stop all carbon source supplementation and starve culture for 1 hour to deplete the remaining carbon source. During this stage, DO is allowed to rise naturally to the set range of 20%-30%.

[0088] (4) Methanol Induction Stage: Methanol was switched to be the sole carbon source for fed-batch induction. The induction temperature was maintained at 28℃, and the methanol replenishment rate was controlled by dissolved oxygen feedback to stably maintain the methanol concentration in the fermentation broth within the range of 0.5%-1.0% (v / v). Throughout the induction stage, DO was maintained at 20%-30%, and a mixture of vitamin C and casein (at a final concentration of 1% (w / v, g / mL)) was added to the culture system to improve methanol metabolism efficiency. The total induction time was 96 hours.

[0089] After fermentation, the final dry cell weight (DCW) of the GS115 PETase 1-40 fermentation broth reached 545 g / L. The activity of the target depolymerase PETase 1 in the fermentation supernatant was measured to be as high as 475.45 U / mL, and the protein concentration was 5.14 mg / mL. Throughout the fermentation process, the cells showed good growth, stable induced expression, and no significant cell autolysis or excessive release of proteases was observed.

[0090] The results of this embodiment show that, using the fermentation method of the present invention, the multidimensional optimized engineered bacteria exhibits excellent growth stability and high efficiency in producing the target protein in 5 L-scale fermentation, verifying the potential for large-scale application of the strain and the supporting fermentation process.

[0091] Example 8: Application of crude enzyme solution in PET degradation

[0092] Based on the above fermentation results, the degradation activity was verified in a 5 L fermenter. Specifically, the fermentation broth from Example 7 was centrifuged and filtered through a 0.45 μm membrane to obtain a crude enzyme solution. Commercially available amorphous PET film and waste PET pillow cores (crushed and washed) were used as substrates. The degradation reaction system consisted of 400 g of PET substrate, 1.2 g of enzyme, and reacted in 2 L of pure water. The pH was controlled at 8.0 using 4 M sodium hydroxide, and the reaction was carried out at 68°C and 200 rpm for 24 hours. After the reaction, the remaining solids were collected by filtration, dried, weighed, and the mass loss rate was calculated. Simultaneously, the supernatant was taken, and the concentrations of the degradation products terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were determined by HPLC.

[0093] The results showed that the depolymerization rate of amorphous PET film was 80.65%, and that of waste PET pillow cores was 92.25%. This embodiment demonstrates that the crude enzyme solution produced by the engineered bacteria of this invention can efficiently degrade PET plastics of different forms without complex purification.

[0094] This invention provides a highly efficient engineered yeast strain for producing plastic depolymerization enzymes and its application, along with related ideas and methods. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A yeast strain that efficiently produces plastic depolymerization enzyme, characterized in that, Pichia pastoris ( Pichia pastoris The host bacterium co-expressed the gene encoding plastic depolymerase and the molecular chaperone, and knocked out or knocked down the vacuolar sorting receptor gene VPS10. The amino acid sequence of the plastic depolymerase is shown in SEQ ID NO.1; The molecular chaperone is HRD1; the Gene ID of HRD1 is 854149. The vacuole sorting receptor gene VPS10 is VPS10-2; the nucleotide sequence of VPS10-2 is shown in SEQ ID NO.

33. The gene encoding the plastic depolymerase is expressed under the regulation of a regulatory element, which includes a strong promoter, a signal peptide, and a C-terminal tag. The strong promoter is MOX1, the signal peptide is the sequence shown in SEQ ID NO.24, and the C-terminal tag includes a 6×His tag. The nucleotide sequence of MOX1 is shown in SEQ ID NO.

14. The engineered yeast strain contains 3 copies of the gene encoding plastic depolymerase; The host bacteria mentioned is Pichia pastoris (Pichia pastoris) Pichia pastoris GS115.

2. The application of the engineered yeast strain according to claim 1 in the production of plastic depolymerization enzyme.

3. The method for producing plastic depolymerase using engineered yeast according to claim 1, characterized in that, The process includes the following steps: inoculating the engineered yeast strain into a fermentation medium and fermenting it at 25-32°C for 96-144 hours.

4. The method according to claim 3, characterized in that, The fermentation culture is first inoculated into a first fermentation medium and cultured at 25-32℃ for 16-32 h, and then inoculated into a second fermentation medium and cultured at 25-32℃ for another 80-112 h. The first fermentation medium uses glycerol as a carbon source, and the second fermentation medium uses methanol as a carbon source.

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