CN117646011A - Yarrowia lipolytica genetically engineered bacterium for heterologous expression of lipase and application thereof - Google Patents

Abstract

The invention discloses a yarrowia lipolytica genetically engineered bacterium for heterologous expression of lipase and application thereof. Specifically, a lipase gene lipRs is disclosed, and the nucleotide sequence of the lipRs is shown as SEQ ID NO. 2. The invention obtains the yarrowia lipolytica genetic engineering strain by transferring the recombinant plasmid into the wild yarrowia lipolytica strain, which expresses lipase by a plasmid vector, and has stronger stability compared with mutagenesis; the recombinant plasmid can be transferred into any strain with stronger lipase production capability, and has stronger expansibility and applicability. The yarrowia lipolytica genetic engineering bacterium provided by the invention has important significance on the application of yarrowia lipolytica in grease wastewater treatment.

Description

Yarrowia lipolytica genetically engineered bacterium for heterologous expression of lipase and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering biology, and particularly relates to a yarrowia lipolytica genetic engineering strain capable of heterologously expressing lipase and application thereof.

Background

Lipase (Lipase, EC3.1.1.3, glyceroyl hydrolase) belongs to the family of carboxyester hydrolases, catalyzing long chains (. Gtoreq.C) ₁₀ ) Hydrolysis of triglycerides. The glyceroyl hydrolase is characterized in that the hydrolysis reaction catalyzed by the glyceroyl hydrolase is a heterogeneous system, and the water-soluble enzyme catalyzes the substrate to react on the interface of the water-insoluble substrate and water, but does not catalyze the water-soluble substrate. In high concentrations of oily wastewater, lipases degrade oils into fatty acids and glycerol, which have wide application in oily wastewater pretreatment, but they are generally soluble and unstable.

In the studies reported so far, it was found that several tens of microorganisms from different sources contain lipase genes, such as Candida sp, rhizopus sp, penicillium sp, pseudomonas sp, etc. Yarrowia lipolytica (Yarrowia lipolytica) is an effective microorganism for treating oily wastewater, is capable of surviving in oily wastewater for a long period of time, and is recognized as a safe class microorganism. Yarrowia lipolytica can be added directly to activated sludge for oily wastewater treatment and become a microbial population in the activated sludge. However, the endogenous lipase secreted by yarrowia lipolytica is too low in activity to effectively degrade the oil. How to increase the yield of yarrowia lipolytica lipase has become a hotspot in current research.

Existing research methods are broadly divided into two types. The first is genetic engineering. The heterologous lipase genes are synthesized into plasmids by metabolic engineering and synthetic biology technology, and then transferred into yarrowia lipolytica for over-expression or the genome of the yarrowia lipolytica is directly modified to improve the lipase expression quantity. For example, as disclosed in Chinese patent document CN108517329A, by establishing the expression of yarrowia lipolytica lipase lip2 in bacillus subtilis WB800N, the efficient production of lipase for feed is realized, and the yield of recombinant lipase is verified to be 109.3U/mL; destin et al have used a mutation to increase the enzyme productivity of yarrowia lipolytica by approximately 30-fold. The second is culture environment optimization. Gerardo et al found that yarrowia lipolytica increased lipase production by using olive oil or corn oil as the carbon source and inducer, adding Tween 80 to the medium, and reducing aeration.

Studies on the lipRs lipase gene are relatively mature. Rhizopus is the main producer of microbial lipase. Nowadays, more than 30 rhizopus lipases are commercially produced, and most rhizopus lipases have the advantages of good stability, high conversion efficiency and the like. In 2007 Zhang Yinbo et al, a high-yield lipase strain, namely Rhizopus delemar YF6, was studied and identified, and a novel lipase gene lipRs (GenBank: DQ 139862) was cloned, and the characteristics of the lipase gene lipRs are suitable for the treatment of oily wastewater. However, the concentration of oily wastewater is generally high, and the strain with good degradation effect on the wastewater with high concentration (such as 100 g/L) of grease is still lacking in the market at present.

The shortcomings of the culture environment optimization means are very remarkable. Time consuming, laborious, requiring extensive prior conditioning fumbling. In addition, the transfer from laboratory pilot test to pilot test requires a condition search again, which additionally increases reagent cost and is costly. The strain obtained by ultraviolet mutagenesis has the remarkable characteristic of instability, and along with the increase of passage number, the genome can generate self-repair to cause mutation failure. Recombinant plasmids are relatively stable in strain state, but currently there are few commercially available lipases that are mature in the market, and require scientific researchers to search for the optimal lipase sequence again and to perform stability identification. The commercial lipase with strong stability and high conversion efficiency cannot be rapidly formed.

Therefore, the search for a fast and simple method for efficiently preparing lipase is of great significance to the application of yarrowia lipolytica in grease wastewater treatment.

Disclosure of Invention

The invention provides a yarrowia lipolytica genetically engineered bacterium for heterologously expressing lipase and application thereof, in order to overcome the defect that high-concentration grease wastewater cannot be treated efficiently

In order to solve the technical problems of the prior art, the first aspect of the invention provides a lipase gene lipRs, wherein the nucleotide sequence of the lipRs is shown as SEQ ID NO. 2.

In a second aspect, the invention provides a recombinant expression vector comprising a lipase gene lipRs according to the first aspect of the invention.

In certain embodiments, one or more of a promoter lac, a resistance gene such as the ampicillin gene Amp, and an auxotroph gene, preferably a leucine auxotroph gene such as leu 2; the skeleton plasmid of the recombinant expression vector is pCAS1yl.

The third aspect of the invention provides a genetically engineered bacterium comprising a recombinant expression vector according to the second aspect of the invention. Preferably, the chassis bacteria of the genetically engineered bacteria are yarrowia lipolytica strains. More preferably, the chassis fungus is yarrowia lipolytica strain ATCC 90811.

According to a fourth aspect of the present invention, there is provided a method for preparing a genetically engineered bacterium according to the third aspect of the present invention, comprising transducing a recombinant expression vector according to the second aspect of the present invention into a yeast such as yarrowia lipolytica. Preferably, the yarrowia lipolytica is yarrowia lipolytica strain ATCC 90811.

In certain embodiments, the yarrowia lipolytica is cultured on a leucine-deficient solid medium.

The leucine-deficient solid medium described above may be an SD-LEU medium as conventionally defined in the art. The main components can be yeast nitrogen source foundation (YNB), glucose, adenine sulfate, L-arginine hydrochloride, L-aspartic acid, L-glutamic acid, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and uracil.

In a fifth aspect, the present invention provides a method for producing a lipase by fermenting a genetically engineered bacterium according to the third aspect of the present invention.

In a sixth aspect, the present invention provides a method for treating wastewater containing high-concentration grease, comprising mixing the genetically engineered bacterium according to the third aspect of the present invention with the wastewater to degrade the high-concentration grease in the wastewater.

The waste water can be living waste water or industrial waste water, and can comprise kitchen grease waste water and harmful organic pollutant waste water.

In certain embodiments, the genetically engineered bacterium according to the third aspect of the invention is cultured in an oil-containing sugarless YPD medium.

Preferably, the sugarless YPD medium has an oil content of 100g/L.

The formula of the sugarless YPD medium is as follows: 2% peptone, 1% yeast extract, said% being mass to volume ratio.

More preferably, the temperature of the cultivation is 25-32 ℃, e.g. 28 ℃, and/or,

the duration of the incubation is 40-80 hours, for example 60 hours, and/or,

the cultivation is carried out in shaking at a rotational speed of 180-300rpm, for example 200rpm.

The seventh aspect of the invention provides an application of the recombinant expression vector according to the second aspect of the invention or the genetically engineered bacterium according to the third aspect of the invention in grease degradation.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

compared with the method of unsteady expression of mutagenesis, the expression of lipase by plasmid vector has strong stability; the recombinant plasmid can be transferred into any strain with stronger lipase production capacity, and has strong expansibility; the lipRs lipase gene has the potential of strong stability and commercial application and strong applicability. Compared with the method for optimizing the culture environment, the method has the advantages of short treatment time and no need of adding additional reagent cost.

Drawings

FIG. 1 shows a recombinant vector LipRs_pCAS1yl containing a lipRs gene fragment, full length 7714bp.

FIG. 2 is an agarose gel electrophoresis image of recombinant vector LipRs_pCAS1yl.

FIG. 3 shows the results of coating leucine auxotroph medium (SD-LEU) on yeast cells transformed with LipRs_pCAS1yl recombinant vector using a yeast transformation package.

FIG. 4 shows the results of gel electrophoresis PCR amplification using the yeast plasmid extraction kit.

FIG. 5 shows the results of coating on leucine auxotrophic medium (SD-LEU) after 3 passages of yarrowia lipolytica into recombinant vectors.

FIG. 6 shows the residual oil content of the centrifuge tube after lipid degradation of experimental bacteria and genetically engineered bacteria (3 single clones are selected and two replicates per group).

FIG. 7 is a comparison of the production of hydrolytic circles by yarrowia lipolytica ATCC90811 and ATCC90811-lipRS genetically engineered bacteria on rhodamine plates; A. b is a wild strain; C. d is genetically engineered bacteria.

Fig. 8 shows the residual oil amount of the centrifuge tube after the oil degradation of the experimental bacteria and the genetically engineered bacteria GPD 1.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 plasmid System construction

The recombinant vector LipRs_pCAS1yl is obtained by using yarrowia lipolytica plasmid pCAS1yl as a vector and connecting the in vitro synthesized lipRs gene fragment to the vector. lipRs gene synthesis was performed by the company gold sri biotechnology. The synthesis length of the lipRs gene is 1179bp (shown as SEQ ID NO: 2). The genetic constitution of the plasmid is shown in FIG. 1, and the recombinant vector structure comprises the following gene fragments: the promoter lac, the lipase gene lipRs, the resistance gene ampicillin gene Amp and the leucine auxotroph gene leu2.

Original sequence (SEQ ID NO: 1)

ATGGTTTCATTCATTTCCATTACTCAAGGTGTAAGTCTTTGTCTTCTTGTCTCTTCCATGATGATGGGCTCTTCTGCTGTTCCCGTTTCTGGTAAAACTGGGGCTTCAAGTGATGCTGTATCCACATCTGGCAACTTTACTCTCCCTCCTATCATCTCTAGTCGTTGTGCGCCTCCTTCTGGAAAAGGATCCAGCAGCGATCTTCAATCCGAGCCTTACTTTGTCAAGAAAAACACCGAATGGTACACTGCCCATGGTGGAGATCTGGCTGCCATTGGTAAACGTGACGATAATCAAGTCGGTGGCATGACATTGGATTTACCCGAAAATGCTCCTCCTATCGCAAGCTCTTATTCCCTCGACAGTGTCTCTGATAGCTCAGTCGTTGCTGCTACTGCCGCTCAAGTTCAAGAGCTTACCAAGTATGCTGGTGTCGCCGCCACTGCCTATTGTCGTAGTGTTGTTCCTGGAAACAAATGGGACTGCAAGCAATGTTTGAAGTGGGTTCCTGATGGTAAAATTATCACAACATTCACTTCTATTCTCTCAGACACAAATGGTTATGTCTTAAGAAGTGATAAGCAGAAGACTATCTATCTTGTTTTTCGTGGTACCAACTCCTTCAGAAGTGCTATCACAGACATCGTCTTCAATTTCTCAAATTATAAGCCTGTTTCTGGTGCCAAGGTACACACTGGCTTCCTCTCTTCTTACGAACAAGTCGTCAATGATTACTTCCCTGTTATCCAAGCCCAGTTAACAGCCAATCCTAGCTACCAAGTTATTGTTACTGGTCACTCACTTGGTGGAGCCCAAGCTTTGCTTGCTGGTATGGACTTATACCAACGCGAAAAGAGACTGTCTCCCAAGAACTTGAGTATTTTCACCATCGGTGGTCCTCGTGTCGAAAACCCTACCTTTGCTTACTATGTTGAATCTACTGGTATTCCTTTTCATCGTACTGTCCACAAGAGAGATATCGTCCCTCACGTCCCCCCTCAAGCAATGGGATTCCTTCATCCTGGTGTTGAGTCTTGGATCAAGTCTGGTGATTCTAATGTTCAAATCTGTAACTCTCAAATCGAGACCAAGGCTTGTAGTAACTCAATCGTTCCTTTCACTTCTATTGCTGACCACTTGTCTTACTTTGGTATCAACGAAGGAAGCTGTTTGTAATGA

Codon optimized sequence (SEQ ID NO: 2)

ATGGTGTCGTTCATTTCTATTACCCAAGGCGTGTCTCTCTGTCTCCTCGTCAGCTCCATGATGATGGGCTCTTCCGCTGTTCCTGTATCTGGAAAGACTGGTGCTTCCTCGGATGCAGTCTCAACGTCAGGCAACTTTACGCTACCACCCATTATCAGTAGTCGATGCGCGCCGCCTTCGGGCAAAGGTTCCAGTTCGGACCTGCAGTCCGAGCCTTACTTTGTCAAGAAGAACACCGAGTGGTACACCGCACATGGCGGAGATCTGGCCGCCATTGGAAAACGGGACGACAACCAGGTTGGAGGAATGACCCTGGATCTCCCCGAAAATGCTCCTCCTATTGCGTCATCATATTCGCTTGACTCCGTCTCTGATTCTTCGGTGGTGGCCGCGACAGCTGCTCAGGTCCAGGAGCTCACTAAGTACGCCGGCGTTGCTGCCACTGCCTACTGCCGTTCGGTGGTTCCCGGAAACAAGTGGGACTGCAAGCAATGTTTGAAATGGGTGCCCGATGGCAAGATCATCACTACCTTTACAAGCATTCTTAGCGACACCAACGGCTACGTCTTGCGCTCCGACAAGCAGAAGACCATCTACCTGGTTTTCCGAGGTACAAACTCTTTCAGATCTGCTATCACCGATATTGTCTTCAACTTCTCCAACTACAAGCCCGTTTCGGGTGCCAAAGTGCACACGGGTTTCCTGTCATCCTACGAGCAGGTGGTCAACGACTACTTCCCCGTCATCCAGGCACAACTGACAGCCAACCCTTCTTATCAAGTTATCGTGACTGGTCACAGCCTGGGAGGCGCCCAGGCACTTCTTGCTGGCATGGACCTCTACCAGCGAGAGAAGCGGCTGTCTCCCAAGAATCTGAGCATCTTCACTATAGGGGGCCCCCGAGTGGAGAACCCCACCTTCGCTTACTACGTCGAATCGACTGGAATCCCCTTCCACAGAACGGTCCACAAGCGAGACATTGTGCCTCATGTGCCACCACAGGCCATGGGTTTTCTGCATCCTGGTGTCGAGTCGTGGATCAAGTCTGGAGACTCTAATGTGCAGATCTGCAACTCCCAGATTGAGACCAAGGCCTGTTCCAATTCTATTGTTCCGTTTACCTCCATTGCCGACCACTTGTCTTATTTTGGCATCAACGAAGGATCCTGTTTATGATGA

Example 2 plasmid transformed E.coli DH 5. Alpha. Bacteria protection and screening

100ng of the recombinant vector synthesized in example 1 was added to 100. Mu.L of E.coli DH 5. Alpha. Competent cells, gently flicked and mixed, placed on ice for 30min, heat-shocked for 45s at 42℃for 2min on ice, added with 700. Mu.L of LB liquid medium, shaken for 1h at 37℃and centrifuged, suspended with 100. Mu.L of supernatant, and then spread on Amp plates (Amp final concentration 50. Mu.g/mL); the transformants grown on the plates were then subjected to plasmid gel running verification as shown in FIG. 2. The results showed that the lipRs gene fragment was successfully ligated to the plasmid vector, and the transformant strain was simultaneously subjected to bacterial protection.

Example 3 Yeast transformation, plasmid extraction and validation

The recombinant vector LipRs_pCAS1yl in the above transformant was extracted, and the plasmid in the transformant was transformed into a chassis strain (yarrowia lipolytica ATCC90811, available from Ningbo Minnean BioCo., ltd.) using a yeast transformation package (Frozen-EZ Yeast Transformation kit). Yeast seed solution was spread on leucine-deficient solid medium (SD-LEU), cultured at 28℃for 48 hours, and colony growth was observed, as shown in FIG. 3.

To further verify the transfer of the plasmid, the present invention performed PCR amplification of specific fragments on the proposed plasmid. The results of gel electrophoresis are shown in FIG. 4, and the gel is cut, purified and sequenced. The plasmid transfer was confirmed to be successful. After transferring into yeast, extracting, the nucleotide sequence of plasmid is shown as SEQ ID NO. 3 and SEQ ID NO. 4.

Plasmid lipRs M13F (SEQ ID NO: 3)

AGGCCGGTACGCTTTTCGTAGATAATGGAATACAAATGGATATCCAGAGTATACACATGGATAGTATACACTGACACGACAATTCTGTATCTCTTTATGTTAACTACTGTGAGGCGTTAAATAGAGCTTGATATATAAAATGTTACATTTCACAGTCTGAACTTTTGCAGATTACCTAATTTGGTAAGATATTAATTATGAACTGAAAGTTGATGGCATCCCTAAATTTGATGAAAGGGGGATCACGCGTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGACTCGAGGTCCAATGACTCGAGGTCCAAAATCATCATAAACAGGATCCTTCGTTGATGCCAAAATAAGACAAGTGGTCGGCAATGGAGGTAAACGGAACAATAGAATTGGAACAGGCCTTGGTCTCAATCTGGGAGTTGCAGATCTGCACATTAGAGTCTCCAGACTTGATCCACGACTCGACACCAGGATGCAGAAAACCCATGGCCTGTGGTGGCACATGAGGCACAATGTCTCGCTTGTGGACCGTTCTGTGGAAGGGGATTCCAGTCGATTCGACGTAGTAAGCGAAGGTGGGGTTCTCCACTCGGGGGCCCCCTATAGTGAAGATGCTCAGATTCTTGGGAGACAGCCGCTTCTCTCGCTGGTAGAGGTCCATGCCAGCAAGAAGTGCCTGGGCGCCTCCCAGGCTGTGACCAGTCACGATAACTTGATAAGAAAGGTTGGCTGTCAGTTGTGCCTGGATGACGGGGAAGTAGTCGTTGACCACCTGCTCGTAGATGACAGGAAACCGTGTGCACTTTGGCACCCGAAACGGGCTTGTAGTTGGAAGAGTTGAAGACAATATCGTGATAGCAGATCTGAAAGGAGTTTGTACCTCGGAAACAAGGTAGATGTCTCTGCTGTCGAGCGCAGACGTAGCGTGTTGTCGCTAGATGCTGTAAGTATGATGATCTTGCCATCGGCACCAATTCAACATTGCTGAGTCACCTTGTTGCGGAGCATCGAACGTAGTA

Plasmid lipRs M13R (SEQ ID NO: 4)

CTCATGATTGTTTAACAGAGACCGGGTTGGCGGCGTATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCAAATTGACCCAGTAGCGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCGTTAAGGGCGTAGGGTACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACCCATGCCGGACGCAAAATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCCCCGAATTACCTTTCCTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCTTCTGAGTATAAGAATCATTCAAAATGGTGAGTTTCAGAGGCAGCAGCAATTGCCACGGGCTTTGAGCACACGGCCGGGTGTGGTCCCATTCCCATCGACACAAGACGCCACGTCATCCGACCAGCACTTTTTGCAGTACTAACCGCAGATGGTGTCGTTCATTTCTATTACCCAAGGCGTGTCTCTCTGTCTCCTCGTCAGCTCCATGATGATGGGCTCTTCCGCTGTTCCTGTATCTGGAAAGACTGGTGCTTCCTCGGATGCAGTCTCAACGTCAGGCAACTTTACGCTACCACCCATTATCAGTAGTCGATGCGCGCCGCCTTCGGGCAAAGGTTCCAGTTCGGACCTGCAGTCCGAGCCTTACTTTGTCAAGAAGAACACCGAGTGGTACACCGCACATGGCGGAGATCTGGCCGCCATTGGAAAACGGGACGACAACCAGGTTGGAGGAATGACCCTGGATCTCCCCGAAAATGCTCCTCCTATTGCGTCATCATATTCGCTTGACTCCGTCTCTGATTCTTCGGTGGTGGCCGCGACAGCTGCTCAAGTCCAGGAGCTCACTACGTACGCCGGCGTTGCTGCACTGCCTACTGCCGTTCGGTGGTCCCGAACAGTGGGACTGCCAGCATGTTTGAACTGGTGCACGATGGCAGATCATCACTACTTTACAGCATCTAGCGACCCACCGCTACGTCTGCGCTCGACAGCAAAGACATCTACCTGGTTTCGAGGTCACTCT

EXAMPLE 4 identification of Lipase by rhodamine B plate

100mL of YPD medium (containing 1.5% agar) was placed in a flask, and sterilized at 121℃for 20min. Rapeseed oil emulsion was prepared with 25% triton-X100 solution in PBS buffer (ph=7.4) for use. A25% Triton-X100 solution was diluted to 5% with PBS buffer (pH=7.4), 20mL of an emulsion containing 40% (V/V) rapeseed oil was prepared, and the mixture was sonicated in an ice bath on a cell disruptor for 10min (50%, 2 s). And storing at 4 ℃ after the emulsification is completed. After sterilizing the above-mentioned Erlenmeyer flasks, 250. Mu.L of 1M MgCl was added to each Erlenmeyer flask while it was hot ₂ ，125μL 1M CaCl ₂ And 250. Mu.L of 20% dextrose solution, incubated at 60 ℃. 100mL of the oil emulsion and rhodamine B solution (5 mg/mL, final concentration 0.001%) were added to different Erlenmeyer flasks, and 1.5% agar was added, and incubated at 60℃for 10min to prepare rhodamine B plates, which were stored at 4 ℃. Diluting the bacterial liquid obtained by culturing the bacterial body transferred into the recombinant vector by 100 times, and OD ₆₀₀ The value is 0.1-0.3, and 100 μl is coated on rhodamine B plate culture medium. The transparent ring size was observed by incubation at 28℃for 96 h.

The invention further verifies the capability of generating lipase by identifying the size of the hydrolysis circle generated by rhodamine plate culture medium. Compared with the wild strain, the genetically engineered bacterium generates more remarkable lipase hydrolysis circle in 48 hours, which means that the genetically engineered bacterium has stronger lipase generating capacity than the wild strain (as shown in figure 7).

Example 5 measurement of degradation Rate of oil

And (3) carrying out a grease degradation test by using high-concentration oily wastewater (oil content of 100 g/L). The oil degradation rate is quantitatively measured by adopting a centrifugal volumetric method. 10mL of rapeseed oil, 2mL of bacterial liquid and 88mL of sugar-free YPD (peptone 20g/L, yeast extract 10 g/L) liquid medium are added into each conical flask, and the mixture is placed on a shaking table for shake culture at 28 ℃ at 200rpm for 60 hours. After the cultivation is finished, the conical flask is taken out of the shaking table, the solution in the conical flask is evenly shaken and transferred into two 50mL centrifuge tubes, and the solution is centrifuged at a low speed of 4000rpm for 10min. At this time, the 50mL centrifuge tube is divided into four layers, the uppermost layer is an oil layer in a clear oil state, the second layer is an emulsion state of an oil degradation product layer, the third layer is a light brown liquid state of a fungus liquid layer, and the bottommost layer is a fungus layer in a flocculent state. The uppermost layer of oil in the centrifuge tube was transferred to a 15mL measuring centrifuge tube using a pipette gun, and the second layer of solution was transferred to a plurality of 2mL centrifuge tubes. All 2mL centrifuge tubes were high speed centrifuged at 12000rpm for 5min. At this time, the 2mL centrifuge tube is divided into three layers, the uppermost layer is an oil layer which is in a clear oil state, the second layer is an oil degradation product layer which is in a compact solid state, and the third layer is a fungus liquid layer which is in a light brown liquid state. The uppermost layer of oil in the 2mL centrifuge tube was transferred to the corresponding measuring centrifuge tube. All remaining oil that was not degraded (to the nearest 0.1 mL) was collected in the measurement centrifuge tube. At this time, we define the lipid degradation rate of yeast to be κ, and the calculation method is shown in formula (1.1):

wherein V is ₀ Initial refueling volume (mL); v (V) ₁ Remaining volume (mL) of oil for control group; v (V) ₂ Residual volume (mL) for experimental group oil.

EXAMPLE 6 plasmid stability test of Strain

The stability of the chassis strain (yarrowia lipolytica ATCC 90811) against the copy number of the recombinant vector pCAS1yl-lipRs was further verified by multiple passages and replications. After multiple generations of replication and passaging, the yeast cells transformed into the recombinant vector were screened again using leucine auxotroph medium (SD-LEU), and the results are shown in FIG. 5. It can be seen that the stability of the strain plasmid after multiple replications and passages is good.

Effect example 1:

wild bacteria (No. ATCC 90811) and genetically engineered bacteria (No. ATCC 90811-lipRs) were added to 100mL of sugar-free YPD medium (peptone 20g/L, yeast extract 10 g/L) containing 100g/L of rapeseed oil in an inoculum size of 2% (V/V), and the mixture was sealed with a sterile sealing film, placed in an air-bath thermostatic shaker, and cultured at 28℃at 200rpm for 60 hours; after the cultivation is finished, the conical flask is taken out of the shaking table, the solution is transferred into two 50mL centrifuge tubes in a shaking mode, the solution is centrifuged at a low speed of 4000rpm for 10min, and the uppermost oil layer in the centrifuge tubes is transferred into a 15mL measuring centrifuge tube by using a pipette. The second layer of solution was then transferred to a plurality of 2mL centrifuge tubes, all 2mL centrifuge tubes were high speed centrifuged at 12000rpm for 5min, and the uppermost layer of oil in the 2mL centrifuge tubes was transferred to the corresponding 15mL measuring centrifuge tube. The graduations are finally measured and recorded as shown in fig. 6. And the oil degradation rate was calculated, and the measurement results are shown in table 1. The results show that the oil degradation rate of the wild bacteria is 20% +/-1%, the oil degradation rate of the genetically engineered bacteria is 30% -34%, and the average oil degradation rate is 31.7% +/-1.7%. The recombinant expression of the heterologous lipase gene lipRs can improve the grease degradation capability of the yeast strain by more than 1.7 times. According to one-way anova, the lipid degradation rates of the genetically engineered bacterial group (accession number: ATCC 90811-lipRs) were significantly statistically different from that of the wild bacterial group (accession number: ATCC 90811), with p < 0.01, as shown in Table 2.

Table 1 test strain and determination of lipid degradation rate of genetically engineered bacteria

TABLE 2 one-way analysis of variance for ATCC90811-lipRS strains and wild-type groups

Analysis of variance
							Source of discrepancy	SS	df	MS	F	P-value	F crit
Inter-group	408.3333	1	408.3333	115.566	8.1597E-07	4.964603
							Within a group	35.33333	10	3.533333
							Totals to	443.6667	11

Effect example 2 verification of Lipase Activity of wild strain and genetically engineered liquid

And (3) performing lipase activity measurement on the bacterial liquid supernatant by a reference titration method, and further identifying the lipolytic capability of the genetically engineered bacteria. Lipase ability is expressed in terms of lipase activity, i.e., the amount of lipase required to decompose olive oil to produce 1. Mu. Mol of free lipase acid per minute at 40℃in 0.025mol/L PBS buffer (pH 7.5)The enzyme amount is one lipase activity, and the unit is U/mL. The results showed that after 48h of cultivation of yarrowia lipolytica ATCC90811 (OD ₆₀₀ When the total amount of the lipase is approximately equal to 1), the lipase activity of the seed solution is 7.75U/mL by using a reference titration method, and the lipase activity of the genetically engineered bacterium is 9.5U/mL, which is improved by 22.7%.

The lipase activity of the seed solution was measured by the reference titration method as follows: mixing 4mL of a substrate with 5mL of phosphate buffer solution by taking polyvinyl alcohol olive oil emulsion as a substrate, preheating in a water bath at 40 ℃ for 5min, adding 1mL of supernatant obtained by centrifuging after strain fermentation to form a 10mL reaction system, and heating in a water bath at 40 ℃. The reaction was allowed to proceed for 15min at 200rpm on a shaker, and then quenched by the addition of 10mL of 95% ethanol. The fatty acid amount of the catalytic hydrolysis of lipase is measured by dripping 0.1mol/L sodium hydroxide standard solution, 3 drops of L% phenolphthalein are added as an indicator, the liquid is dripped to be light pink, the 3s is kept unchanged, namely, the titration end point is obtained, the consumption of sodium hydroxide is recorded, and the enzyme activity is calculated by comparing with a control sample. The control sample was not added with fermentation broth, and the other samples were identical, and the measurement was repeated 3 times. The calculation formula of lipase activity is: x= (B-ase:Sub>A) ×5×n. Wherein X represents lipase activity of the sample, and the unit is U/mL; b is the amount of NaOH standard solution required by titration of a sample group, and the unit is mL; a is the amount of NaOH standard solution required by the completion of titration of a blank group, and the unit is mL; n is the dilution multiple of the enzyme solution to be tested in the experiment.

Comparative example

The present invention contemplates other enzymes, such as glycerol-3-phosphate dehydrogenase (encoded by GPD1, also a key enzyme in the glycerol metabolic pathway). The results of the oil degradation are shown in fig. 8 and table 3 below. The results showed that the average oil degradation rate of the genetically engineered bacterium (No. ATCC90811-gpd 1) into which the glycerol-3-phosphate dehydrogenase was transferred was about 31%, which is also significantly different from that of the wild-type bacterium group, and p=0.017, as shown in Table 4 (oil degradation conditions are the same as the present invention, the oil content of the high-concentration oil waste water is 100g/L, the inoculum size is 2%, and the inoculum size is 200rpm at 28 ℃ C., 60 h). The highest oil degradation rate of the genetically engineered bacterium (number: ATCC 90811-lipRs) reaches 34% under the same condition. In comparison, the genetic engineering of lipRs lipase is more advantageous.

TABLE 3 determination of lipid degradation Rate of experimental strains and genetically engineered bacteria

TABLE 4 one-way analysis of variance for ATCC90811-gpd1 Strain and wild type group

Analysis of variance
							Source of discrepancy	SS	df	MS	F	P-value	F crit
Inter-group	182.25	1	182.25	56.07692	0.017369	18.51282
							Within a group	6.5	2	3.25
							Totals to	188.75	3

Claims (10)

1. A lipase gene lipRs is characterized in that the nucleotide sequence of the lipRs is shown in SEQ ID NO. 2.

2. A recombinant expression vector comprising the lipase gene lipRs of claim 1.

3. Recombinant expression vector according to claim 2, characterized in that it further comprises one or more of a promoter lac, a resistance gene such as the ampicillin gene Amp and an auxotroph gene, preferably a leucine auxotroph gene such as leu 2; the skeleton plasmid of the recombinant expression vector is pCAS1yl.

4. A genetically engineered bacterium comprising the recombinant expression vector of claim 2 or 3; preferably, the chassis bacteria of the genetically engineered bacteria are yarrowia lipolytica strains; more preferably, the chassis fungus is yarrowia lipolytica strain ATCC 90811.

5. A method for producing the genetically engineered bacterium of claim 4, comprising transducing the recombinant expression vector of claim 2 or 3 into yarrowia lipolytica; preferably, the yarrowia lipolytica is yarrowia lipolytica strain ATCC 90811.

6. The method of claim 5, wherein the yarrowia lipolytica is cultured on a leucine-deficient solid medium.

7. A method for producing a lipase, which comprises fermenting the genetically engineered bacterium according to claim 4.

8. A method for treating wastewater containing high-concentration grease, comprising mixing the genetically engineered bacterium according to claim 4 with the wastewater to degrade the high-concentration grease in the wastewater.

9. The method according to claim 8, wherein the genetically engineered bacterium according to claim 4 is cultured in an oil-containing sugarless YPD medium;

preferably, the oil content of the sugarless YPD medium is 100g/L;

more preferably, the temperature of the cultivation is 25-32 ℃, e.g. 28 ℃, and/or,

the duration of the incubation is 40-80 hours, for example 60 hours, and/or,

the cultivation is carried out in shaking at a rotational speed of 180-300rpm, for example 200rpm.

10. Use of the recombinant expression vector of claim 2 or 3 or the genetically engineered bacterium of claim 4 in lipid degradation.