Application of overexpression of genes encoding alkane hydroxylases and endogenous transporter proteins in yeast for polyethylene degradation

Abstract

The present invention relates to the field of bioengineering, and in particular to the use of genes encoding alkane hydroxylase and yeast endogenous transporter in the degradation of polyethylene. The present invention provides the use of overexpressing any of the following genes in the degradation of polyethylene: a gene encoding an endogenous transporter in yeast; or a gene encoding an alkane hydroxylase; or a gene encoding an alkene monooxygenase; or a gene encoding an alkane hydroxylase and a gene encoding an alkane hydroxylase coenzyme. The chassis cell selected by the present invention is a Yarrowia lipolytica cell that can utilize a variety of oily substances as a carbon source, can very effectively degrade and oxidize hydrophobic substrates, and has developed powerful tools for genetics and molecular biology, so the yeast has recently been used as a model organism to study metabolic pathways involved in metabolism. Therefore, the present invention transforms Yarrowia lipolytica by a synthetic biology method to obtain a Yarrowia lipolytica with enhanced transporter and expression of membrane protein alkane hydroxylase.

Description

[0001] This application claims the priority of a Chinese patent application filed with the China Patent Office on June 6, 2023, with application number 202310663991.0 and invention name “Application of overexpression of genes encoding alkane hydroxylase and genes endogenous transport proteins in yeast in the degradation of polyethylene”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The invention relates to the field of bioengineering, and in particular to the application of overexpressing genes encoding alkane hydroxylase and genes of yeast endogenous transport proteins in degrading polyethylene. Background Art

[0003] Plastic is one of the most widely used and important materials in the modern world. However, the huge production and abuse of plastics has brought a huge burden to the environment. It is reported that about 58% of plastic waste is placed in landfills or directly discharged into the environment, 24% is burned, and only 18% of plastic waste is recycled. Some researchers estimate that by 2050, there will be about 120 million tons of plastic waste in the natural environment. Compared with conventional chemical recycling and physical recycling, recycling waste plastics through biological methods has the advantages of low energy consumption, low pollution and low cost. In recent years, there have been many precedents for transforming various model organisms or non-model organisms to degrade plastics through systems biology methods. Polyethylene is one of the most difficult types of plastics to degrade. At present, many wild bacteria have been isolated from environments containing polyethylene waste, including Rhodococcus, Pseudomonas, Bacillus, Acinetobacter, etc. Some wild bacteria can initially degrade polyethylene into a mixture of long-chain aliphatic compounds (alcohols, aldehydes, acids, olefins, alkanes, etc.). For example, after incubation with Pseudomonas aeruginosa PAO1, the LDPE sample turned into long-chain fatty acids, esters, hydrocarbons, and oxygen-containing compounds (mainly containing compounds such as aldehydes, ketones, esters and ethers, unsaturated fatty acids and some unknown compounds). Some researchers have isolated a mixed bacterial system containing Bacillus brevis and Bacillus neuroalkali from a landfill. After the mixed bacterial system was incubated on the PE sample for 140 days, the mass losses of LDPE and HDPE membranes were 58.2% and 46.6%, respectively, and the mass losses of LDPE and HDPE balls were 45.7% and 37.2%, respectively. In addition, the results of gas chromatography-mass spectrometry analysis showed that the PE sample was degraded into cis-2-chlorovinyl acetate, tridecanoic acid and octadecanoic acid. The first step of microbial degradation of olefins, one of the intermediate products of polyethylene degradation, is generally that olefin monooxygenase or alkane hydroxylase first oxidizes olefins to epoxides, which are basic and common intermediate structural units in the pharmaceutical, fragrance and polymer industries. Epoxides can also be oxidized to vicinal diols in microbial cells, which enter the TCA cycle in microbial cells and are then metabolized by microorganisms. Some researchers have selected fungal peroxidases from several different sources to achieve the epoxidation of long-chain terminal olefins of different lengths. In addition to epoxides, the oxidation products of terminal olefins also include terminal aldehydes. Some researchers have performed site-directed mutagenesis on the alkane hydroxylase gene alkB and obtained a mutant V91W in which the proportion of terminal aldehydes in the product is higher than that of epoxides. In addition, some researchers have identified wild bacteria that can metabolize olefins, but the specific metabolic mechanism is not clear enough.

[0004] In existing polyethylene degradation research, most of the strains with better degradation effects are wild fungi or wild fungal communities isolated from the natural environment. However, the genomic information and metabolic networks of most wild fungi are unclear, making it difficult to perform genetic manipulation. Summary of the invention

[0005] In view of this, the present invention provides the use of genes encoding alkane hydroxylase and yeast endogenous transporter in degrading polyethylene. The chassis cells selected by the present invention are Yarrowia lipolytica cells that can use a variety of oily substances as carbon sources, can very effectively degrade and oxidize hydrophobic substrates, and have developed powerful tools for genetics and molecular biology, so the yeast has recently been used as a model organism to study metabolic pathways involved in metabolism. Therefore, the present invention transforms Yarrowia lipolytica by synthetic biology methods to obtain Yarrowia lipolytica with enhanced transporter and expression of membrane protein alkane hydroxylase.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of overexpressing any one of the following genes in degrading polyethylene;

[0008] (I), a gene encoding an endogenous yeast transporter; or

[0009] (II), a gene encoding an alkane hydroxylase; or

[0010] (III), a gene encoding olefin monooxygenase; or

[0011] (IV), a gene encoding an alkane hydroxylase and a gene encoding an alkane hydroxylase coenzyme.

[0012] In some embodiments of the present invention, in the above application, the gene encoding the yeast endogenous transporter includes: ABC1;

[0013] The genes encoding alkane hydroxylase include: AlkB1 and / or AlkB2 from Pseudomonas aeruginosa PAO1;

[0014] The genes encoding the alkane hydroxylase coenzyme include: rubA1 and / or rubB from Pseudomonas aeruginosa PAO1;

[0015] The gene encoding olefin monooxygenase includes: antF from Streptomyces sp.

[0016] In some embodiments of the present invention, in the above application, the gene encoding the yeast endogenous transporter has:

[0017] (1), the nucleotide sequence shown in SEQ ID NO: 1; or

[0018] (2) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (1), and having the same or similar function as the nucleotide sequence shown in (1); or

[0019] (3) a nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (1) or (2); and / or

[0020] The gene encoding alkane hydroxylase has:

[0021] (4), the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3; or

[0022] (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or

[0023] (6) a nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (4) or (5); and / or

[0024] The gene encoding the alkane hydroxylase coenzyme has:

[0025] (7), the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5; or

[0026] (8) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as the nucleotide sequence shown in (7); or

[0027] (9) a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (7) or (8); and / or

[0028] The gene encoding olefin monooxygenase has:

[0029] (10), the nucleotide sequence shown in SEQ ID NO: 6; or

[0030] (11) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (10), and having the same or similar function as the nucleotide sequence shown in (10); or

[0031] (12) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (10) or (11).

[0032] In some embodiments of the present invention, in the above application, the sequence of ABC1, SEQ ID NO: 1, is:

[0033]

[0034]

[0035] In some embodiments of the present invention, in the above application, the sequence of AlkB1 SEQ ID NO: 2 is:

[0036]

[0037]

[0038] In some embodiments of the present invention, in the above application, the sequence of AlkB2 SEQ ID NO:3 is:

[0039]

[0040]

[0041] In some embodiments of the present invention, in the above application, the sequence of rubA1, SEQ ID NO: 4, is:

[0042]

[0043] In some embodiments of the present invention, in the above application, the sequence of rubB SEQ ID NO:5 is:

[0044]

[0045] In some embodiments of the present invention, in the above application, the sequence of antF SEQ ID NO:6 is:

[0046]

[0047]

[0048] The present invention also provides an expression module, comprising any one of the following genes;

[0049] (I), a gene encoding an endogenous yeast transporter; and / or

[0050] (II), a gene encoding an alkane hydroxylase; or

[0051] (III), a gene encoding olefin monooxygenase; or

[0052] (IV), a gene encoding an alkane hydroxylase and a gene encoding an alkane hydroxylase coenzyme.

[0053] In some embodiments of the present invention, the above-mentioned expression module further comprises a rigid connecting peptide and / or a flexible connecting peptide.

[0054] In some embodiments of the present invention, in the above expression module, the rigid connecting peptide comprises: EAAAK (as shown in SEQ ID NO: 34); the flexible connecting peptide comprises: GGGGS (as shown in SEQ ID NO: 35).

[0055] The present invention also provides a strain, into which the above expression module is transformed and / or introduced.

[0056] In some embodiments of the present invention, in the above-mentioned strains, the chassis strain includes: Yarrowia lipolytica ATCC201249 (Y).

[0057] In some embodiments of the present invention, in the above-mentioned strains, the source of the Yarrowia lipolytica ATCC201249 (Y) is: Jia D, Xu S, Sun J, et al. Yarrowia lipolytica construction for heterologous synthesis of-santalene and fermentation optimization [J]. Applied Microbiology and Biotechnology, 2019, 103 (8): 3511-3520.

[0058] The present invention also provides a culture medium, comprising: 10-50 g / L n-hexadecene, 0.1-1 g / L sophorolipid and 22 g / L glucose.

[0059] In some embodiments of the present invention, the above-mentioned culture medium comprises: 10-50 g / L n-hexadecene, 500 mg / L sophorolipids and 22 g / L glucose.

[0060] The present invention also provides application of the above expression module, the above strain and / or the above culture medium in degrading polyethylene.

[0061] The present invention also provides a method for degrading polyethylene, which comprises culturing the above-mentioned strain and mixing it with the polyethylene.

[0062] In some embodiments of the present invention, in the above method, the polyethylene uses n-hexadecene, a degradation intermediate product of the polyethylene.

[0063] In some embodiments of the present invention, in the above method, the culturing uses the above culture medium.

[0064] The present invention provides the use of overexpressing any one of the following genes in degrading polyethylene;

[0065] (I), a gene encoding an endogenous yeast transporter; or

[0066] (II), a gene encoding an alkane hydroxylase; or

[0067] (III), a gene encoding olefin monooxygenase; or

[0068] (IV), a gene encoding an alkane hydroxylase and a gene encoding an alkane hydroxylase coenzyme.

[0069] The chassis cells selected in the present invention are Yarrowia lipolytica cells that can utilize a variety of oily substances as carbon sources, can very effectively degrade and oxidize hydrophobic substrates, and have developed powerful tools for genetics and molecular biology, so the yeast has recently been used as a model organism to study metabolic pathways involved in metabolism. Therefore, the present invention transforms Yarrowia lipolytica through a synthetic biology method to obtain Yarrowia lipolytica with enhanced transport proteins and expression of membrane protein alkane hydroxylase. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0071] Figure 1 This indicates the enhancement of the transport pathway in Yarrowia lipolytica;

[0072] Figure 2 The effect of different concentrations of n-hexadecene on the growth of Yarrowia lipolytica is shown;

[0073] Figure 3 Shows the metabolic diagram of polyolefin degradation intermediates;

[0074] Figure 4 The figure shows the construction of a multi-copy expression strain of alkane hydroxylase;

[0075] Figure 5 The figure shows the construction of a multi-copy expression strain of alkane hydroxylase;

[0076] Figure 6 The growth of n-hexadecene-degrading bacteria is shown; wherein: (a) shows bacteria expressing alkane hydroxylase and alkene monooxygenase; (b) shows bacteria expressing alkane hydroxylase and its coenzyme;

[0077] Figure 7 It shows the degradation rate of n-hexadecene, the intermediate product of polyethylene degradation;

[0078] Figure 8 It shows the degradation rate of n-hexadecene, the intermediate product of polyethylene degradation;

[0079] Fig. 9 It shows the degradation rate of n-hexadecene, the intermediate product of polyethylene degradation;

[0080] Fig.10 The relative expression levels of alkane hydroxylase genes in polyethylene degradation intermediate degrading bacteria at different times; wherein: (a) shows the control strain YAE02; (b) shows the control strain YAE03. DETAILED DESCRIPTION

[0081] The invention discloses application of over-expressing genes encoding alkane hydroxylase and genes of yeast endogenous transport proteins in degrading polyethylene.

[0082] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0083] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0084] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0085] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0086] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0087] The chassis cells selected in the present invention are Yarrowia lipolytica cells that can utilize a variety of oily substances as carbon sources, can very effectively degrade and oxidize hydrophobic substrates, and have developed powerful tools for genetics and molecular biology, so the yeast has recently been used as a model organism to study metabolic pathways involved in metabolism. Therefore, the present invention transforms Yarrowia lipolytica through a synthetic biology method to obtain Yarrowia lipolytica with enhanced transport proteins and expression of membrane protein alkane hydroxylase.

[0088] In Examples 1 to 7 of the present invention, the source of Yarrowia lipolytica ATCC201249 (Y) is: Jia D, Xu S, Sun J, et al. Yarrowia lipolytica construction for heterologous synthesis of-santalene and fermentation optimization [J]. Applied Microbiology and Biotechnology, 2019, 103 (8): 3511-3520.

[0089] In Examples 1 to 7 of the present invention, the SC culture medium includes: 22 g / L glucose, 6.7 g / L YNB and 2 g / L drop-out mixed powder (adenine 0.5, asparagine 2.0, alanine 2.0, threonine 2.0, arginine 2.0, cysteine ​​2.0, glutamine 2.0, methionine 2.0, glutamic acid 2.0, p-aminobenzoic acid 0.2, inositol 2.0, serine 2.0, lysine 2.0, tyrosine 2.0, isoleucine 2.0, aspartic acid 2.0, glycine 2.0, proline 2.0, all in g), 0.002 g / L histidine, 0.01 g / L leucine, 0.002 g / L uracil and 0.002 g / L tryptophan.

[0090] Table 1 Primer sequences

[0091]

[0092]

[0093]

[0094] All the raw materials and reagents used can be purchased from the market.

[0095] The present invention will be further described below in conjunction with embodiments:

[0096] Example 1 Construction of engineered Yarrowia lipolytica

[0097] 1. In this example, Yarrowia lipolytica ATCC201249 (Y) was used as the chassis cell. First, the endogenous ABC1 transporter of Yarrowia lipolytica was overexpressed. The construction process was as follows: Figure 1 As shown. ABC1 was codon optimized and synthesized with Yarrowia lipolytica, and PCR amplification was performed with the primers shown in Table 1. After recovery, the ABC1 gene fragment containing the homology arm of PINA1269 was obtained. The vector PINA1269 was double-digested with BamHI and KpnI, and the linear vector PINA1269 was obtained after recovery. The linear vector and the target gene fragment were assembled using SE ligase and transferred into the competent state of Escherichia coli. The Escherichia coli transformants were verified by colony PCR with the primers shown in Table 1. After the verification result was correct, the sequencing was sent. If the sequencing result was correct, the engineered Escherichia coli was obtained. The engineered Escherichia coli plasmid was extracted and digested with NotI, and the linearized recombinant vector was recovered. The linearized recombinant vector was transformed into Y, and the Yarrowia lipolytica colony PCR was verified with the same primers as the Escherichia coli colony PCR. After the verification result was correct, it was sent for sequencing. If the sequencing result was correct, YAE01 was obtained.

[0098] Example 2

[0099] In order to determine whether Yarrowia lipolytica can utilize n-hexadecene as the sole carbon source, and also to explore the optimal concentration of n-hexadecene in the culture medium, this example uses SC medium without added glucose (SC-G medium) as the n-hexadecene degradation medium, and adds different concentrations of n-hexadecene as the sole carbon source thereto to measure the growth of Yarrowia lipolytica.

[0100] Wild-type Yarrowia lipolytica Y and the engineered strain YAE01 overexpressing the transporter gene ABC1 constructed in the laboratory were cultured in SC-G medium with 10 g / L, 20 g / L, 30 g / L, 40 g / L and 50 g / L n-hexadecene as the sole carbon source, and the OD600 value was measured at appropriate time intervals. Figure 2 , as shown in Table 2 and Table 3.

[0101] The growth of Y improved with the increase of n-hexadecene concentration. When the concentration of n-hexadecene increased to 20g / L, the OD600 value of Y could reach about 5.5 after 120h, which was about 1.7 times higher than that at 10g / L. When the concentration of n-hexadecene increased to 30g / L, the OD600 value of Y could reach about 7.0 after 120h, which was about 2.2 times higher than that at 10g / L. When the concentration of n-hexadecene increased to 40g / L and 50g / L, the OD600 value of Y after 120h was not much different from that at 30g / L (such as Figure 2 shown).

[0102] Unlike Y, the growth of YAE01 first increased with the increase of n-hexadecene concentration, and the growth was the best at 30 g / L, and then decreased with the increase of n-hexadecene concentration. When the n-hexadecene concentration was 10 g / L, the OD600 value of YAE01 reached about 8.2 after 120 hours, which was about 2.5 times that of Y. When the n-hexadecene concentration was 10 g / L, the OD600 value of YAE01 reached about 11.5 after 120 hours, which was about 3.6 times that of 10 g / L and about 1.6 times that of the wild type at the same concentration. Therefore, 30 g / L was initially selected as the degradation concentration of n-hexadecene in this study.

[0103] Table 2

[0104]

[0105] Table 2 corresponds to Figure 2 (a) data;

[0106] Table 3

[0107]

[0108]

[0109] Table 3 corresponds to Figure 2 (b) Data.

[0110] Example 3

[0111] Metabolic pathways of fatty compounds in polyethylene degradation intermediates by Yarrowia lipolytica Figure 3 As shown in the figure, the polyethylene degradation intermediates first enter the cell through the Yarrowia lipolytica transporter, and the alkanes are gradually oxidized to fatty alcohols, fatty aldehydes and fatty acids under the action of alkane hydroxylases, alcohol dehydrogenases and aldehyde dehydrogenases, and enter the β-oxidation pathway of fatty acids. Olefins can be oxidized to fatty aldehydes or epoxides under the action of alkane hydroxylases, or to epoxides under the action of olefin monooxygenases. Epoxides can be oxidized to vicinal diols under the action of epoxide hydrolases, and then gradually oxidized to long-chain acyl-CoA and enter the β-oxidation pathway.

[0112] In this embodiment, exogenous alkane hydroxylase genes AlkB1 and AlkB2 are overexpressed on the basis of optimizing the ABC1 transporter of the starting bacteria Y, and on this basis, the rubredoxin gene and the rubredoxin genes rubA1 and rubB that can help the alkane hydroxylase to transfer electrons are overexpressed. The alkane hydroxylase encoded by AlkB1 and AlkB2 is a membrane protein, so it can directly contact with the polyethylene degradation intermediate and undergo oxidation reaction. The alkane hydroxylase encoded by AlkB1 and AlkB2 can oxidize alkanes to fatty alcohols and oxidize alkenes to aldehydes or epoxides.

[0113] In order to improve the ability of Yarrowia lipolytica to degrade n-hexadecene, in this embodiment, the alkane hydroxylase genes AlkB1, AlkB2 and their coenzyme genes rubA1 and rubB and the olefin monooxygenase gene antF are cloned into a single-copy expression vector PUC57-K8FB containing a strong promoter, and transferred into Escherichia coli competent cells. The Escherichia coli transformants are verified by colony PCR using the primers shown in Table 1. After the verification results are correct, they are sent for sequencing. If the sequencing results are correct, the engineered Escherichia coli is obtained. The engineered Escherichia coli plasmid is extracted and digested with NotI, and the linearized recombinant vector is recovered. The linearized recombinant vector is transformed into the obtained YAE02, YAE03, YAE04, YAE05, YAE06, YAE07 and YAE08 construction process. The specific construction process is as follows Figure 4 and Figure 5 The corresponding relationship between strains and genes is shown in Figure 6 shown.

[0114] Example 4

[0115] Sophorolipids (Hangyuan Biotechnology Food Grade Sophorolipids) as biosurfactants can enhance the contact between enzymes and hydrophobic substrates. In order to explore the effect of sophorolipids on the growth of Yarrowia lipolytica, this example uses SC-G medium with 30 g / L of n-hexadecene to culture n-hexadecene-degrading strains in shake flasks, and compares the effect of adding sophorolipids (500 mg / L) on the growth of Yarrowia lipolytica. The results are as follows: Figure 6 , as shown in Table 4 and Table 5.

[0116] Whether it is wild bacteria or engineered bacteria, the growth after adding sophorolipids is better than without adding sophorolipids. Among them, the change of YAE03 is the most obvious. When sophorolipids are not added to the medium containing 30g / L n-hexadecene, the OD600 value after 120h is 20.482. After adding sophorolipids, the OD600 value increases by about 1.5 times. When other strains add different substrates, the OD600 values ​​after 120h are slightly increased by about 1 to 2 after adding sophorolipids. The above results show that the addition of sophorolipids is beneficial to the growth of Yarrowia lipolytica in the medium with olefins and alkanes. Therefore, the n-hexadecene degradation experiments in the subsequent examples of the invention are all carried out in the medium with sophorolipids added.

[0117] Table 4

[0118]

[0119] Table 4 corresponds to Figure 6 (a) data;

[0120] Table 5

[0121]

[0122]

[0123] Table 5 corresponds to Figure 6 (a) Data.

[0124] Table 6

[0125]

[0126] Table 6 corresponds to Figure 6 (b) data;

[0127] Table 7

[0128]

[0129] Table 7 corresponds to Figure 6 (b) data;

[0130] Example 5 Degradation of polyethylene degradation intermediate n-hexadecene by engineered Yarrowia lipolytica and optimization of degradation conditions

[0131] In order to verify the effect of Yarrowia lipolytica on the degradation of n-hexadecene, in this example, the polyolefin degradation intermediate product degradation strain was first cultured in a SC-G + sophorolipid (500 mg / L) medium supplemented with 30 g / L n-hexadecene for 120 h, and then extracted according to the process and then subjected to gas phase detection. The results are as follows Figure 7 and as shown in Table 8.

[0132] Table 8

[0133]

[0134] Table 8 corresponds to Figure 7 data.

[0135] The degradation rate of polyethylene degradation intermediate n-hexadecene (30 g / L) by wild-type Y. lipolytica Y was 14.846%, while that of YAE02, YAE03 and YAE04 were 24.713%, 13.189% and 24.389%, respectively. Among them, YAE02 and YAE04 were improved compared with YAE01, which were 1.80 times and 1.78 times of YAE01, and 2.82 times and 2.78 times of Y.

[0136] After co-expression of rubredoxin gene and rubredoxin reductase gene (rubA1 and rubB), the degradation rate of polyethylene degradation intermediate n-hexadecene was highest in strain YAE05, reaching 44.055%, which was 1.78 times and 3.34 times higher than that of YAE02 and YAE01, respectively. In addition, YAE07 was 1.65 times higher than that of YAE03.

[0137] The strains that co-expressed erythrodoxin and erythrodoxin reductase and improved the degradation rate of polyethylene degradation intermediates all used GGGGS as the connecting peptide. GGGGS is a flexible connecting peptide that is easy to fold and has little or no effect on the spatial structure of alkane hydroxylase, erythrodoxin and erythrodoxin reductase. The possible reason why YAE06 and YAE08 using the rigid connecting peptide EAAAK did not improve the degradation effect of polyethylene degradation intermediates is that the rigid connecting peptide is not easy to fold, which affects the spatial structure of alkane hydroxylase, erythrodoxin and erythrodoxin reductase.

[0138] Example 6

[0139] Adding glucose to the culture medium is beneficial to the growth of Yarrowia lipolytica, so in this example, 22 g / L of glucose was added to the hexadecene degradation medium to verify whether the hexadecene degradation rate was improved. The polyolefin degradation intermediate product degradation strain was cultured in a shake flask in a SC+sophorolipid (500 mg / L) culture medium with 30 g / L hexadecene for 120 h, and gas phase detection was performed after extraction according to the process. The results are as follows Figure 8 and as shown in Table 9.

[0140] Table 9

[0141]

[0142] Table 9 corresponds to Figure 8 data.

[0143] After adding glucose, the degradation rate of Y increased to 16.816%, which is 1.13 times that of not adding glucose. Except for YAE04, the degradation rates of all engineered yeasts increased. The degradation rate of YAE01 increased by about 1%. The strain with the most obvious improvement in degradation rate was YAE06, which increased by 1.56 times, followed by YAE03, which increased by 1.07 times. The strain with the highest degradation rate of n-hexadecene after adding glucose was YAE05, reaching 46.809%, which was 1.28 times higher than that without adding glucose, and 3.61 times higher than that of the wild type without adding glucose. The strain with the highest degradation rate was still YAE05, which was 46.809%, which was 2.78 times and 3.21 times that of Y and YAE01, respectively. The degradation rate of YAE01 did not increase relative to Y in either the medium with or without adding glucose. The possible reason is that the concentration of n-hexadecene, an intermediate product of polyethylene degradation, added to the medium is too high, and the transport protein of the optimized strain increases the metabolic burden of the strain, resulting in no improvement in the degradation rate. In addition, at a concentration of 30 g / L, the degradation rate of n-hexadecene did not exceed 50% at most. Therefore, the present invention attempts to reduce the concentration of n-hexadecene to 10 g / L, and selects YAE02 and YAE05 with higher degradation rates as well as Y and YAE01 as control strains to test the degradation effect.

[0144] like Fig. 9 As shown in Table 10, when the concentration of n-hexadecene was 10 g / L, the degradation rate of wild-type Y was 39.998%, and the degradation rate of YAE01 with optimized ABC1 transporter was 43.347%, which was higher than that of Y. The degradation rates of YAE02 and YAE05 were 48.045% and 63.876%, respectively, which were 1.20 times and 1.60 times that of Y, respectively.

[0145] Table 10

[0146]

[0147] Table 10 corresponds to Fig. 9 data.

[0148] Example 7

[0149] After co-expression of erythroreductase and erythroreductase, the degradation rate of n-hexadecene in the engineered Yarrowia lipolytica YAE06 and YAE08 did not increase but decreased. In order to determine whether the co-expression of erythroreductase and erythroreductase has an effect on the transcription level of alkane hydroxylase, the transcription level of alkane hydroxylase genes AlkB1 and AlkB2 in Yarrowia lipolytica was analyzed, and YAE02 and YAE03 were used as control groups, and YAE05, YAE06, YAE07 and YAE08 were used as experimental groups. The culture time was 120h, and the culture medium was SC liquid medium supplemented with sophorolipids. The total RNA of Yarrowia lipolytica was extracted every 24h, and qPCR reaction was performed after reverse transcription. Actin was used as the internal reference gene to analyze the transcription level of alkane hydroxylase genes in the control group and the experimental group. The results are shown in Fig.10 , as shown in Table 11 and Table 12.

[0150] The control strain for YAE05 and YAE06 was YAE02. Fig.10 As shown in Figure 1a and Table 11, the transcription level of AlkB1 in YAE05 was significantly lower than that in YAE02 in the first 72 hours. The transcription level of AlkB1 in YAE05 at 96 hours increased to 339.08 times that of YAE02, and the transcription level of AlkB1 at 120 hours was not much different from that of YAE02. The transcription level of AlkB1 in YAE06 was always significantly lower than that of YAE02 within 120 hours. Although the transcription level of YAE06 at 96 hours was significantly higher than that of YAE02, the degradation rate was not significantly improved. The possible reason is that the three genes in YAE06 were connected by rigid connecting peptides, which are not easy to fold, which may further affect the spatial structure or positional relationship of alkane hydroxylase, erythroredoxin and erythroredoxin, thereby limiting the activity of alkane hydroxylase.

[0151] Table 11

[0152]

[0153] Table 11 corresponds to Fig.10 (a) Data.

[0154] The control strain for YAE07 and YAE08 was YAE03, Fig.10 b and Table 12, the transcription level of alkB2 in YAE07 was always higher than that in YAE03, which was 84.59 times, 3.53 times, 52.84 times, 47.38 times and 52.76 times that of the control strain YAE03 at 24h, 48h, 72h, 96h and 120h, respectively. Figure 2-1 The results of 2-2 showed that the degradation effect of YAE07 was improved compared with that of YAE03. The transcription level of alkB2 of YAE08 at 48h was 9.09 times that of YAE03, and it was lower than that of YAE02 at other times. The possible reason that the degradation rate was not improved compared with YAE03 was similar to that of YAE06. The three genes were connected by rigid linker peptides, which were not easy to fold, and may further affect the spatial structure or position relationship of alkane hydroxylase, erythroredoxin and erythroredoxin, resulting in the restriction of alkane hydroxylase activity, thereby limiting the degradation rate of YAE08 to n-hexadecene.

[0155] Table 12

[0156]

[0157] Table 12 corresponds to Fig.10 (b) Data.

[0158] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of overexpression of any of the following genes in the degradation of polyethylene; (I), a gene encoding an endogenous yeast transporter; or (II), a gene encoding an alkane hydroxylase and a gene encoding an endogenous yeast transporter; or (III), a gene encoding an alkene monooxygenase and a gene encoding an endogenous yeast transporter; or (IV), a gene encoding an alkane hydroxylase, a gene encoding an alkane hydroxylase coenzyme, and a gene encoding an endogenous yeast transporter; The gene encoding the yeast endogenous transporter is: ABC1 ; The gene encoding alkane hydroxylase is: Pseudomonas aeruginosa PAO1-derived AlkB1 or AlkB2 ; The gene encoding the alkane hydroxylase coenzyme is: Pseudomonas aeruginosa PAO1-derived rubA1 and rubB ; The gene encoding olefin monooxygenase is: Streptomyces sp. Source F ; Said ABC1 The sequence is: the nucleotide sequence shown in SEQ ID NO: 1; Said AlkB1 The sequence is: the nucleotide sequence shown in SEQ ID NO: 2; Said AlkB2 The sequence is: the nucleotide sequence shown in SEQ ID NO: 3; Said rubA1 The sequence is: the nucleotide sequence shown in SEQ ID NO:4; Said rubB The sequence is: the nucleotide sequence shown in SEQ ID NO: 5; Said F The sequence is: the nucleotide sequence shown in SEQ ID NO: 6; The overexpression strain is: Yarrowia lipolytica.

2. A strain characterized in that Transform the expression module in the chassis strain; The expression module includes any one of the following genes: (I), a gene encoding an alkane hydroxylase and a gene encoding an endogenous yeast transporter; or (II), a gene encoding an alkene monooxygenase and a gene encoding an endogenous yeast transporter; or (III), a gene encoding an alkane hydroxylase, a gene encoding an alkane hydroxylase coenzyme, and a gene encoding a rigid linking peptide or a gene encoding a flexible linking peptide; The gene encoding the yeast endogenous transporter is: ABC1 ; The gene encoding alkane hydroxylase is: Pseudomonas aeruginosa PAO1-derived AlkB1 or AlkB2 ; The gene encoding the alkane hydroxylase coenzyme is: Pseudomonas aeruginosa PAO1-derived rubA1 and rubB ; The gene encoding olefin monooxygenase is: Streptomyces sp. Source F ; Said ABC1 The sequence is: the nucleotide sequence shown in SEQ ID NO: 1; Said AlkB1 The sequence is: the nucleotide sequence shown in SEQ ID NO: 2; Said AlkB2 The sequence is: the nucleotide sequence shown in SEQ ID NO: 3; Said rubA1 The sequence is: the nucleotide sequence shown in SEQ ID NO:4; Said rubB The sequence is: the nucleotide sequence shown in SEQ ID NO: 5; Said F The sequence is: the nucleotide sequence shown in SEQ ID NO: 6; The amino acid sequence of the rigid connecting peptide is as shown in SEQ ID NO: 34; The amino acid sequence of the flexible connecting peptide is as shown in SEQ ID NO: 35; The chassis strain is: Yarrowia lipolytica.

3. Use of the strain as claimed in claim 2 in degrading polyethylene.

4. A method for degrading polyethylene, characterized in that: The strain as claimed in claim 2 is cultured and then mixed with the polyethylene.

5. The method according to claim 4, characterized in that The culture adopts a culture medium; the culture medium comprises: 10-30 g / L n-hexadecene, 0.5 g / L sophorolipid and 22 g / L glucose.

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