Escherichia coli capable of anchoring yarrowia lipolytica cell surface and application method thereof

Abstract

The invention provides escherichia coli capable of anchoring yarrowia lipolytica and an application method of the escherichia coli, and belongs to the field of bioengineering, the escherichia coli genetically engineered bacterium is provided, genes of anchoring proteins agalpha1, egt2, cwp2, dan4 or sed1 and an ice nucleation protein INP gene are subjected to fusion expression and inserted between a SalI restriction enzyme cutting site and a BamHI restriction enzyme cutting site of a nucleotide sequence SEQ ID NO.12, and the escherichia coli genetically engineered bacterium is obtained. The recombinant expression vector is obtained and transformed into escherichia coli BL21 (DE3) to obtain the escherichia coli genetically engineered bacterium, and the escherichia coli genetically engineered bacterium provided by the invention can be used for screening a yarrowia lipolytica high-yield erythritol strain and can also be used for detecting the concentration of erythritol.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to an Escherichia coli capable of anchoring Yarrowia lipolytica and an application method thereof. Background Art

[0002] Erythritol is a white, odorless, non-hygroscopic, heat-stable crystalline powder. It tastes cool and has the advantages of low calories, non-caries, antioxidant, and endothelial protection. Therefore, it is widely used in a variety of foods, such as baked goods, fermented milk, candy, and chocolate. Both fungi and bacteria can synthesize erythritol. At present, the main strain producing erythritol is Yarrowia lipolytica. In view of the important application value and huge market demand of erythritol, breeding high-yield, low-cost, and genetically stable production strains has become a key goal of the industry. However, there is currently no efficient high-throughput breeding method for erythritol production strains. Therefore, breeding erythritol production strains with excellent fermentation performance is still a meaningful task.

[0003] Biosensors are a group of response systems widely found in microorganisms that are used to identify and respond to specific metabolites in cells and convert them into specific response signals, such as fluorescence. Transcription regulators are the most important type of biosensors. They can respond to the concentration of specific metabolites in cells and control the expression of response signal proteins. When transcription factors are induced, the response signal produced by the response signal protein is positively correlated with the metabolite concentration. The commonly used response signal protein is fluorescent protein. The main advantage of using fluorescence as a response signal is that high-throughput screening at the cellular level can be achieved through fluorescence-activated cell sorting, which greatly improves the efficiency in analyzing a large number of mutant enzymes or mutant bacteria. At present, biosensors have been widely used for qualitative and quantitative analysis of intracellular metabolites, and have also been used for the detection of chemical substances in industrial production.

[0004] In order to further improve the response efficiency of the sensor, yeast cells in yeast cell surface engineering have the ability to convert promoters into signals. Based on the advantages of yeast itself, the yeast surface display system is considered to be a safe, efficient and application-worthy system. An important task of yeast cell display technology is to study an efficient anchor protein, which requires cells to produce a large amount of anchor proteins while being efficiently displayed on the cell surface, thereby providing more space for the binding of target proteins to substrates. There are two types of anchor protein-cell connection in yeast cells, one is non-covalent connection between proteins and cell walls, and the other is covalent connection between proteins and cell walls. Covalently linked cell wall anchor proteins can be divided into GPI type and Pir type according to their connection form; non-covalently linked cell wall proteins are mainly Flo1p's N-terminal flocculation system. GPI-type anchor proteins play an important role in the expression of heterologous proteins displayed on the cell surface, and are essential for the study of yeast display technology. GPI-type anchor protein fusion methods are divided into N-terminal fusion and C-terminal fusion, and the representative ones are α-lectin and a-lectin. The glycolipid part of the GPI anchor protein in yeast cells is connected to the C-terminus of the hydrophobic protein to ensure stable binding of the membrane to the protein. After the fusion protein is completed, the protein precursor is anchored in the endoplasmic reticulum, and the remaining protein remains in the endoplasmic reticulum cavity. Finally, the protein with certain functions is fixed on the cell surface through the action of the anchor protein. Saccharomyces cerevisiae, as a high-quality yeast for yeast cell surface display platform, has been used in the display of various enzymes. The mining of new anchor proteins has become a research hotspot. Vaart has developed many cell wall proteins, such as Cwp1p, Cwp2p, Tip1p, Sed1p, Ycr89w, Tirl, etc. These proteins have been confirmed to be used as connecting proteins for N-terminal display platforms. Yang et al. studied new surface display systems, including Dan4p, Sed1p, Pry3p, Tos6p, Cwp2p, Srp2p and α-lectin, and studied the display efficiency of these display systems. The results showed that Aga1p and Dan4p are more suitable for fixing larger proteins. At present, there are more studies on the construction of biosensors in phage and bacterial display systems, but relatively few in yeast. For example, glutamate dehydrogenase is displayed in Escherichia coli to construct a biosensor, and the concentration of the enzyme is detected by assembling it with carbon nanomaterials into positive and negative electrodes.

[0005] Chinese patent document CN 111996132 A (application number 202010708696.9) provides a method for displaying the surface of Yarrowia lipolytica cells, which relates to the field of biochemical engineering technology. Yarrowia lipolytica is used as a chassis cell, pYCSD is used as a vector, XPR2 pre secretion signal peptide is used to guide the secretion of the target protein to the extracellular space, YlCWP1 (110) endogenous to Yarrowia lipolytica is used as a cell surface anchoring signal, and a flexible connecting peptide (G4S) 2 is used to connect the target gene and YlCWP1 (110), thereby reducing the impact on the expression and folding of the target protein. The target gene is cloned into the cloning site of pYCSD, and Yarrowia lipolytica is transformed to achieve the surface display of the target protein on the Yarrowia lipolytica cells.

[0006] Chinese patent document CN 117737066 A (application number 202311769795.8) invention provides a transcription regulatory sequence and its application. The transcription regulatory sequence is obtained by mutating the transcription regulatory sequence disclosed in the prior art. Compared with the prior art, the recombinant Escherichia coli prepared by using the mutated transcription regulatory sequence can effectively reduce the background fluorescence intensity in the process of screening erythritol-producing strains, and the fluorescence response signal is 6.5 times that of the prior art, which significantly improves the screening efficiency of erythritol-producing strains.

[0007] Research and develop an anchor protein genetically engineered bacterium that can quickly detect erythritol concentration and efficiently screen erythritol-producing strains. Summary of the invention

[0008] In view of the deficiencies of the prior art, the present invention provides an Escherichia coli capable of anchoring to the surface of Yarrowia lipolytica cells and an application method thereof.

[0009] The inventors screened out a genetically engineered bacterium that can anchor to the cell surface of Yarrowia lipolytica and can display the anchor protein cwp2 on the surface. The anchor protein recombinant E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 was embedded and co-cultured with Yarrowia lipolytica, and the recombinant E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 was used as a biosensor to detect the production of erythritol by Yarrowia lipolytica. The concentration response signal of erythritol detected within 24 hours was 10.5 times that of the original strain. The recombinant E. coli was used as a biosensor to screen high-yield erythritol mutants of Yarrowia lipolytica, and finally a mutant strain Y.lipolytica 5-14-E6 was obtained, and the erythritol production reached 178g / L.

[0010] The technical solution of the present invention is as follows:

[0011] The invention discloses an application of anchoring proteins agα1, egt2, cwp2, dan4 or sed1 in screening erythritol producing strains, wherein the nucleotide sequences of the anchoring proteins are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively.

[0012] Preferably according to the present invention, in the application, the anchoring protein agα1, egt2, cwp2, dan4 or sed1 is fused with the ice nucleation protein INP respectively, and the amino acid sequence of the ice nucleation protein INP is SEQ ID NO.7.

[0013] A recombinant plasmid comprising the nucleotide sequence of anchoring protein agα1, egt2, cwp2, dan4 or sed1;

[0014] The nucleotide sequences of the anchoring proteins are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 respectively;

[0015] The nucleotide sequence of the anchoring protein cwp2 is not limited to SEQ ID NO.3, and also includes other nucleotide sequences encoding the amino acid sequence of the anchoring protein cwp2 SEQ ID NO.6.

[0016] According to the preferred embodiment of the present invention, the recombinant plasmid further comprises the nucleotide sequence of ice nucleation protein INP;

[0017] The nucleotide sequence is a nucleotide sequence encoding the ice nucleation protein INP amino acid sequence SEQ ID NO.7;

[0018] Further preferably, the nucleotide sequence of the ice nucleation protein INP is SEQ ID NO.13.

[0019] Preferably, according to the present invention, the recombinant plasmid further comprises a transcriptional regulatory sequence, a gene sequence encoding a repressor protein, and a marker gene sequence encoding a fluorescent protein;

[0020] The nucleotide sequence of the transcriptional regulatory sequence is SEQ ID NO.9, the gene sequence encoding the repressor protein is SEQ ID NO.10, and the marker gene sequence encoding the fluorescent protein is SEQ ID NO.11.

[0021] Preferably, according to the present invention, the recombinant plasmid is a fusion of the genes of the anchoring protein agα1, egt2, cwp2, dan4 or sed1 and the ice nucleation protein INP, and is inserted between the SalⅠ and BamHI restriction sites of the nucleotide sequence SEQ ID NO.12.

[0022] A recombinant engineering bacterium comprises the above-mentioned recombinant plasmid.

[0023] Preferably according to the present invention, the host bacteria of the recombinant engineered bacteria is Escherichia coli.

[0024] More preferably, the host bacteria is Escherichia coli BL21 (DE3).

[0025] A method for constructing a genetically engineered Escherichia coli comprises the following steps:

[0026] (1) The genes of anchoring proteins agα1, egt2, cwp2, dan4 or sed1 were fused with the ice nucleation protein INP gene and inserted between the SalⅠ and BamHI restriction sites of the nucleotide sequence SEQ ID NO.12 to obtain recombinant expression vectors pET-22b(+)-eryD-INP-sed1, pET-22b(+)-eryD-INP-agα1, pET-22b(+)-eryD-INP-egt2, pET-22b(+)-eryD-INP-cwp2, and pET-22b(+)-eryD-INP-dan4;

[0027] The nucleotide sequences of the anchoring proteins agα1, egt2, cwp2, dan4, and sed1 are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively, and the nucleotide sequence of the ice nucleation protein INP is SEQ ID NO.13;

[0028] (2) The recombinant expression vector pET-22b(+)-eryD-INP-sed1, pET-22b(+)-eryD-INP-agα1, pET-22b(+)-eryD-INP-egt2, pET-22b(+)-eryD-INP-cwp2 or pET-22b(+)-eryD-INP-dan4 obtained in step (1) is transformed into Escherichia coli BL21(DE3), and after screening and identification, genetically engineered Escherichia coli bacteria E. coli BL21 / pET-22b(+)-eryD-INP-sed1, E. coli BL21 / pET-22b(+)-eryD-INP-agα1, E. coli BL21 / pET-22b(+)-eryD-INP-egt2, E. coli BL21 / pET-22b(+)-eryD-INP-cwp2, E. coli BL21 / pET-22b(+)-eryD-INP-dan4.

[0029] The application of the recombinant plasmid, the recombinant engineering bacteria and the genetically engineered Escherichia coli bacteria constructed by the method in screening erythritol production strains.

[0030] A method for screening erythritol-producing strains comprises the following steps:

[0031] The above-mentioned recombinant engineered bacteria or genetically engineered Escherichia coli bacteria are inoculated into a culture medium for cultivation, and the strain to be screened is inoculated into a culture medium for cultivation. The fermentation liquids of the two are mixed, and the erythritol production capacity of the strain to be screened can be confirmed according to the fluorescence intensity of the fermentation liquid.

[0032] Preferably, according to the present invention, the above-mentioned recombinant engineered bacteria or genetically engineered Escherichia coli are inoculated into LB medium for culture, and Yarrowia lipolytica is inoculated into YPD medium for culture, and the fermentation broths of the two are mixed, and the erythritol production capacity of the strain to be screened can be confirmed based on the fluorescence intensity of the fermentation broth.

[0033] Preferably according to the present invention, the erythritol producing strain is Yarrowia lipolytica.

[0034] The application of the recombinant plasmid, the recombinant engineering bacteria, and the genetically engineered Escherichia coli bacteria constructed by the method in detecting the concentration of erythritol.

[0035] Preferably, according to the present invention, the above-mentioned recombinant plasmid, recombinant engineered bacteria, and genetically engineered Escherichia coli bacteria constructed by the method are used as biosensors for detecting erythritol concentration.

[0036] The steps not described in detail in the present invention are all performed according to conventional operations in the art.

[0037] Beneficial Effects

[0038] 1. The recombinant Escherichia coli provided by the present invention can exhibit a good anchoring state on the surface of Yarrowia lipolytica cells. The recombinant Escherichia coli can be used as a biosensor to detect the production of erythritol by Yarrowia lipolytica, can be used to quickly detect the concentration of erythritol, and can also be used to screen high-erythritol-producing mutants of Yarrowia lipolytica.

[0039] 2. The present invention provides a genetically engineered bacterium capable of anchoring to the cell surface of Yarrowia lipolytica, capable of displaying an anchoring protein cwp2 on the surface, and embedding and co-culturing the anchoring protein recombinant Escherichia coli E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 with Yarrowia lipolytica, using the recombinant Escherichia coli E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 as a biosensor to detect the production of erythritol by Yarrowia lipolytica, and the concentration response signal of erythritol detected within 24 hours is 10.5 times that of the original strain, and using the recombinant Escherichia coli as a biosensor to screen high-erythritol-producing mutants of Yarrowia lipolytica, finally obtaining a mutant strain Y. lipolytica 5-14-E6 with an erythritol production of 178 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Schematic diagram for the construction of the anchor protein fluorescent expression vector pET-22b(+)-eryD-INP-sed1.

[0041] Figure 2 Schematic diagram of the construction of the anchor protein fluorescent expression vector pET-22b(+)-eryD-INP-agα1.

[0042] Figure 3 Schematic diagram of the construction of the anchor protein fluorescent expression vector pET-22b(+)-eryD-INP-egt2.

[0043] Figure 4 Schematic diagram of the construction of the anchor protein fluorescence expression vector pET-22b(+)-eryD-INP-cwp2.

[0044] Figure 5 Schematic diagram of the construction of the anchor protein fluorescent expression vector pET-22b(+)-eryD-INP-dan4.

[0045] Figure 6 It is the agarose gel electrophoresis diagram of the PCR products of the anchor protein fluorescence expression vector pET-22b(+)-eryD-INP-sed1 and pET-22b(+)-eryD-INP-agα1 genes; among them, lanes 1 and 2 are sed1 fragments, 1887 bp; lanes 3 and 4 are agα1, 1887 bp.

[0046] Figure 7 It is the agarose gel electrophoresis diagram of the PCR products of the anchor protein fluorescence expression vector pET-22b(+)-eryD-INP-egt2 and pET-22b(+)-eryD-INP-dan4 genes;

[0047] In the figure: Lane 5 is egt2, 3990 bp; Lanes 6 and 7 are dan4, 4353 bp.

[0048] Figure 8 It is the agarose gel electrophoresis diagram of the PCR product of the anchor protein fluorescence expression vector pET-22b(+)-eryD-INP-cwp2 gene;

[0049] In the figure: Lanes 8 and 9 are cwp2, 1143 bp.

[0050] Fig. 9 This is a diagram showing the anchoring effect of E.coli BL21 / pET-22b(+)-eryD-INP-sed1 on Y.lipolytica.

[0051] Fig.10 This is a diagram showing the anchoring effect of E.coli BL21 / pET-22b(+)-eryD-INP-agα1 on Y.lipolytica.

[0052] Fig.11 This is a diagram showing the anchoring effect of E.coli BL21 / pET-22b(+)-eryD-INP-egt2 on Y.lipolytica.

[0053] Fig.12 This is a diagram showing the anchoring effect of E.coli BL21 / pET-22b(+)-eryD-INP-cwp2 on Y.lipolytica.

[0054] Fig.13 This is a diagram showing the anchoring effect of E.coli BL21 / pET-22b(+)-eryD-INP-dan4 on Y.lipolytica.

[0055] Fig.14 This is a comparison of the fluorescence intensity of the Escherichia coli biosensor fermented for 24 hours without and with erythritol.

[0056] Fig.15 is the erythritol yield of Y.lipolytica 5-14-E6. DETAILED DESCRIPTION

[0057] The technical scheme of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The operating methods not described in detail in the embodiments are all conventional operating methods known to those skilled in the art.

[0058] The reagents and drugs used in the present invention are all common commercially available products.

[0059] Sources of biological materials:

[0060] Anchoring protein agα1 gene: derived from Brewer's yeast, the nucleotide sequence is SEQ ID NO.1.

[0061] Anchoring protein egt2 gene: derived from Brewer's yeast, the nucleotide sequence is SEQ ID NO.2.

[0062] Anchoring protein cwp2 gene: derived from Brewer's yeast, the nucleotide sequence is SEQ ID NO.3, and the sequence contains the lactose operon sequence.

[0063] Anchoring protein dan4 gene: derived from Brewer's yeast, the nucleotide sequence is SEQ ID NO.4.

[0064] Anchoring protein sed1 gene: derived from Brewer's yeast, the nucleotide sequence is SEQ ID NO.5.

[0065] The amino acid sequence of the anchoring protein cwp2 gene is SEQ ID NO.6.

[0066] The amino acid sequence of the ice nucleation protein INP gene is SEQ ID NO.7.

[0067] E. coli BL21 (DE3) cells were purchased from Vazyme.

[0068] Yarrowia lipolytica 5-14 is Yarrowia lipolytica (CICC1457).

[0069] The culture medium involved in the embodiment:

[0070] LB liquid medium: sodium chloride 10 g / L, peptone 10 g / L, yeast powder 5 g / L.

[0071] LB solid medium: Add 20g / L agar to LB liquid medium.

[0072] YPD liquid culture medium: glucose 20 g / L, peptone 20 g / L, yeast powder 10 g / L.

[0073] YPD solid medium: Add 20g / L agar to YPD liquid medium.

[0074] Fermentation medium: glucose 300g / L, peptone 10g / L, yeast powder 5g / L.

[0075] Example 1: Construction of anchor protein vector

[0076] Synthesis of anchor protein fluorescence expression vectors pET-22b(+)-eryD-INP-sed1, pET-22b(+)-eryD-INP-agα1, pET-22b(+)-eryD-INP-egt2, pET-22b(+)-eryD-INP-cwp2, and pET-22b(+)-eryD-INP-dan4:

[0077] The Chinese patent document CN 117737066 A (application number 202311769795.8) discloses a transcriptional regulatory sequence, the nucleotide sequence of which is shown in SEQ ID NO.9; the gene sequence encoding the repressor protein is shown in SEQ ID NO.10; the marker gene sequence encoding the fluorescent protein is shown in SEQ ID NO.11; and the nucleotide sequence of the recombinant plasmid vector containing the gene is shown in SEQ ID NO.12. In this application, the plasmid vector is named "pET-22b(+)-eryD".

[0078] The nucleotide sequences of anchoring proteins agα1, egt2, cwp2, dan4 or sed1 and the nucleotide sequence of ice nucleation protein INP were obtained by gene synthesis.

[0079] The nucleotide sequences of five anchoring proteins agα1, egt2, cwp2, dan4, and sed1 from Saccharomyces cerevisiae are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. The nucleotide sequence of anchoring protein gene sed1, agα1, egt2, cwp2 or dan4 and ice nuclease protein INP is shown in SEQ ID NO.13. They are inserted between the SalⅠ and BamHI restriction sites of the nucleotide sequence SEQ ID NO.12 for fusion expression, double-digested with FastDigest SalⅠ and FastDigest BamHI, dephosphorylated, and then cleaved with T4 DNALigase was connected to the fusion gene fragments of sed1, agα1, egt2, cwp2 or dan4 and INP respectively; the structural diagram of the anchor protein fluorescence expression vector pET-22b(+)-eryD-INP-sed1, pET-22b(+)-eryD-INP-agα1, pET-22b(+)-eryD-INP-egt2, pET-22b(+)-eryD-INP-cwp2, pET-22b(+)-eryD-INP-dan4 was constructed as shown in the following figure Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 shown.

[0080] The amino acid sequence of the fusion protein of cwp2 and INP is shown in SEQ ID NO.8.

[0081] Example 2: Construction of an anchor protein fluorescence expression strain

[0082] (1) Transformation of recombinant plasmid

[0083] ①Take 10 μL of recombinant plasmids pET-22b(+)-eryD-INP-sed1, pET-22b(+)-eryD-INP-agα1, pET-22b(+)-eryD-INP-egt2, pET-22b(+)-eryD-INP-cwp2, and pET-22b(+)-eryD-INP-dan4, respectively, and add them to 100 μL of E.coli BL21(DE3) competent cells, mix gently, and place on ice for 30 min;

[0084] ②Heat shock at 42℃ for 90s, then quickly cool in an ice bath for 3min;

[0085] ③Inoculate the competent cells into 500 μL LB medium and culture at 37°C, 200 rpm for 60 min;

[0086] ④ Take 200 μL of the above bacterial solution and spread the E. coli bacterial solution on LB solid medium containing 50 μg / mL kanamycin, 100 μg / mL ampicillin and 40 μg / mL streptomycin;

[0087] ⑤ Place the solid culture medium upright in a 37℃ incubator for 30 minutes. After the bacterial liquid is absorbed dry, invert the plate and incubate at 37℃ for 12-16 hours.

[0088] (2) Identification of positive clones:

[0089] Colony PCR identification

[0090] Pick the single colony cultured above, inoculate the E. coli into 1 mL of liquid LB medium containing 100 μg / mL ampicillin, and culture at 37°C and 200 rpm for 6-8 h. Pipette 2 μL of the bacterial solution and perform colony PCR identification according to the 50 μL PCR reaction system. If the target band appears and the band is single, the colony is a positive clone.

[0091] The sed1, agα1, egt2, cwp2, and dan4 genes on the pET-22b(+)-eryD-INP-sed1, pET-22b(+)-eryD-INP-agα1, pET-22b(+)-eryD-INP-egt2, pET-22b(+)-eryD-INP-cwp2, and pET-22b(+)-eryD-INP-dan4 plasmids were verified by colony PCR. The sequences of all the primers designed in this experiment are shown in Table 1. The agarose gel electrophoresis of the products of PCR amplification of the sed1, agα1, egt2, cwp2, and dan4 genes is shown in Figure 6 , 7, 8 as shown:

[0092] Table 1: Primer sequences

[0093]

[0094] Finally, the anchor protein fluorescence expression strain was constructed:

[0095] That is, E.coli BL21 / pET-22b(+)-eryD-INP-sed1, E.coli BL21 / pET-22b(+)-eryD-INP-agα1, E.coli BL21 / pET-22b(+)-eryD-INP-egt2, E.coli BL21 / pET-22b(+)-eryD-INP-cwp2, E. coli BL21 / pET-22b(+)-eryD-INP-dan4.

[0096] Example 3: Cell surface display of fluorescent expression strains of anchored proteins

[0097] (1) Activation of strains: The anchor protein fluorescence expression strain constructed in Example 2 was inoculated into 50 mL of liquid LB medium containing 100 μg / mL ampicillin at an inoculum size of 1%, and cultured at 37° C. and 200 rpm for 12 h. Yarrowia lipolytica 5-14 was cultured in a shaker at 30° C. and 200 rpm for 24 h.

[0098] (2) Bacterial transfer: Take 1 mL of the activated bacterial solution of the above anchor protein fluorescent expression strain and inoculate it into 50 mL of liquid LB medium containing 100 μg / mL ampicillin, and culture it at 37°C and 200 rpm until the bacterial solution OD reaches 0. 600The concentration of IPTG in the culture medium was 0.5 mM, and the culture was carried out at 37°C and 200 rpm for 12 h to obtain a fermentation broth. The Yarrowia lipolytica 5-14 was inoculated at a rate of 10% (v / v) into a 500 mL conical flask containing 50 mL of fermentation medium, and the flask was continued to be placed in a shaking incubator at 30°C and 200 r / min for 12 h to obtain a fermentation broth.

[0099] (3) Collecting bacterial broth: Take 1 mL of the fermentation broth of the recombinant E. coli strain and 1 mL of the fermentation broth of Y. lipolytica and co-culture them at 30°C for 12 h. Observe and compare the effects of the five recombinant E. coli cell surface displays under a microscope;

[0100] The glycolipids on the surface of Yarrowia lipolytica can form a covalent bond with the anchoring proteins expressed by recombinant Escherichia coli. Through observation under a microscope, it was found that the anchoring proteins sed1, agα1, egt2, cwp2 and dan4 successfully anchored the recombinant Escherichia coli on the surface of Yarrowia lipolytica cells. Figure 9-13 As shown, the anchoring efficiency of the anchoring protein CWP2 is the highest.

[0101] Fig. 9 This is the anchoring effect of E. coli BL21 / pET-22b(+)-eryD-INP-sed1 on Y. lipolytica.

[0102] Fig.10 This is the anchoring effect of E.coli BL21 / pET-22b(+)-eryD-INP-agα1 on Y.lipolytica.

[0103] Fig.11 This is the anchoring effect of E. coli BL21 / pET-22b(+)-eryD-INP-egt2 on Y. lipolytica.

[0104] Fig.12 This is the anchoring effect of E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 on Y. lipolytica.

[0105] Fig.13 This is the anchoring effect of E.coli BL21 / pET-22b(+)-eryD-INP-dan4 on Y.lipolytica.

[0106] Example 4: Using recombinant Escherichia coli as a biosensor to detect erythritol concentration

[0107] Using Escherichia coli as a biosensor, E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 was cultured in a shaker at 37°C and 200 r / min for 24 h, and then inoculated as a seed solution at a 10% (v / v) inoculation amount into LB liquid culture medium containing 0.5 mg / mL IPTG at concentrations of 0 mmol / L and 500 mmol / L erythritol, respectively. The culture was carried out in a shaker at 37°C and 200 r / min for 24 hours. The fluorescence intensity in the fermentation broth was detected every 12 h by an enzyme reader during the fermentation process. Fig.14 It was shown that the E. coli biosensor E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 exhibited low background fluorescence intensity at 24 h. When erythritol was added, its fluorescence intensity was stronger, and the concentration response signal of erythritol was 10.5 times that of the original strain.

[0108] Example 5: Using recombinant Escherichia coli as a biosensor to screen high-erythritol-producing mutants of Yarrowia lipolytica

[0109] The Yarrowia lipolytica was irradiated under ultraviolet light for 120 seconds, inoculated into YPD liquid medium, and cultured in a shaking incubator at 30°C and 200 r / min for 24 hours, and then transferred to the Yarrowia lipolytica fermentation medium at a 10% (v / v) inoculation amount, and continued to be fermented in a shaker at 30°C and 200 r / min for 12 hours to obtain the fermentation liquid of Yarrowia lipolytica. The mutant bacterial liquid of Yarrowia lipolytica was obtained;

[0110] The fermentation broth of recombinant Escherichia coli prepared without adding erythritol in Example 4 was evenly mixed with the fermentation broth of diluted Yarrowia lipolytica in a ratio of 1:10, and the formed microdroplets were cultured for 48 hours using a microfluidic cell culture and sorting system. The fluorescence intensity of the microdroplets was detected, and the strains with increased fluorescence intensity were sorted. The mutant bacteria with strong fluorescence were inoculated onto a YPD solid plate and cultured in a constant temperature incubator at 30°C for 2 days. Two mutant bacteria of different shapes were picked up with a sterile inoculation loop, inoculated into YPD liquid culture medium, and cultured at 30°C and 200r / min for 24 hours. They were inoculated into the fermentation medium as seed liquid and cultured at 100m / min. 3 The mixture was fermented in a fermenter at 30°C for 75 h, and the erythritol production was detected by HPLC. Fig.15 The mutant strain Y.lipolytica 5-14-E6 showed that the 3 The fermentation results of producing erythritol in a fermenter showed that after 75 hours of fermentation, the erythritol production of the engineered bacteria mutant strain Y.lipolytica 5-14-E6 was 178g / L.

[0111] The genetically engineered Escherichia coli provided by the present invention can be used to quickly detect the concentration of erythritol, and can also be used to screen strains of Yarrowia lipolytica that produce high erythritol.

Claims

1. Use of anchoring proteins agα1, egt2, cwp2, dan4 or sed1 in screening erythritol-producing strains, wherein the nucleotide sequences of the anchoring proteins are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively.

2. The use according to claim 1, characterized in that In the application, the anchoring protein agα1, egt2, cwp2, dan4 or sed1 is fused with the ice nucleation protein INP respectively, and the amino acid sequence of the ice nucleation protein INP is SEQ ID NO.

7.

3. A recombinant plasmid, characterized in that: including the nucleotide sequence of the anchoring protein agα1, egt2, cwp2, dan4 or sed1; The nucleotide sequences of the anchoring proteins are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5 respectively; The nucleotide sequence of the anchoring protein cwp2 is not limited to SEQ ID NO.3, and also includes other nucleotide sequences encoding the amino acid sequence of the anchoring protein cwp2 SEQ ID NO.

6.

4. The recombinant plasmid according to claim 3, characterized in that The recombinant plasmid also includes the nucleotide sequence of ice nucleation protein INP; The nucleotide sequence is a nucleotide sequence encoding the ice nucleation protein INP amino acid sequence SEQ ID NO.7; Preferably, the nucleotide sequence of the ice nucleation protein INP is SEQ ID NO.

13.

5. The recombinant plasmid according to claim 4, characterized in that The recombinant plasmid also includes a transcriptional regulatory sequence, a gene sequence encoding a repressor protein, and a marker gene sequence encoding a fluorescent protein; The nucleotide sequence of the transcriptional regulatory sequence is SEQ ID NO.9, the gene sequence encoding the repressor protein is SEQ ID NO.10, and the marker gene sequence encoding the fluorescent protein is SEQ ID NO.11; Preferably, the recombinant plasmid is a fusion of the anchoring protein agα1, egt2, cwp2, dan4 or sed1 and the ice nucleation protein INP gene, and is inserted between the SalⅠ and BamHI restriction sites of the nucleotide sequence SEQ ID NO.

12.

6. A recombinant engineered bacterium comprising the recombinant plasmid according to any one of claims 3 to 5; Preferably, the host bacteria of the recombinant engineered bacteria is Escherichia coli; Preferably, the host bacteria is Escherichia coli BL21 (DE3).

7. A method for constructing a genetically engineered Escherichia coli, characterized in that: The steps include: (1) The genes of anchoring proteins agα1, egt2, cwp2, dan4 or sed1 were fused with the ice nucleation protein INP gene and inserted between the SalⅠ and BamHI restriction sites of the nucleotide sequence SEQ ID NO.12 to obtain recombinant expression vectors pET-22b(+)-eryD-INP-sed1, pET-22b(+)-eryD-INP-agα1, pET-22b(+)-eryD-INP-egt2, pET-22b(+)-eryD-INP-cwp2, and pET-22b(+)-eryD-INP-dan4; The nucleotide sequences of the anchoring proteins agα1, egt2, cwp2, dan4, and sed1 are SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively, and the nucleotide sequence of the ice nucleation protein INP is SEQ ID NO.13; (2) The recombinant expression vector pET-22b(+)-eryD-INP-sed1, pET-22b(+)-eryD-INP-agα1, pET-22b(+)-eryD-INP-egt2, pET-22b(+)-eryD-INP-cwp2 or pET-22b(+)-eryD-INP-dan4 obtained in step (1) is transformed into Escherichia coli BL21(DE3), and after screening and identification, genetically engineered Escherichia coli strains E. coli BL21 / pET-22b(+)-eryD-INP-sed1, E. coli BL21 / pET-22b(+)-eryD-INP-agα1, E. coli BL21 / pET-22b(+)-eryD-INP-egt2, E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 or E.coliBL21 / pET-22b(+)-eryD-INP-dan4.

8. Use of the recombinant plasmid according to any one of claims 3 to 5, the recombinant engineered bacteria according to claim 6, or the genetically engineered Escherichia coli bacteria constructed by the method according to claim 7 in screening erythritol-producing strains.

9. A method for screening erythritol-producing strains, comprising the following steps: The recombinant engineered bacteria according to claim 6 or the genetically engineered Escherichia coli bacteria constructed by the method according to claim 7 are inoculated into a culture medium for cultivation, and the strain to be screened is inoculated into a culture medium for cultivation, and the fermentation liquids of the two are mixed, and the erythritol production capacity of the strain to be screened can be confirmed according to the fluorescence intensity of the fermentation liquid; Preferably, the erythritol producing strain is Yarrowia lipolytica; Preferably, the above-mentioned recombinant engineered bacteria or genetically engineered Escherichia coli are inoculated into LB medium for culture, and Yarrowia lipolytica is inoculated into YPD medium for culture. The fermentation broths of the two are mixed, and the erythritol production capacity of the strain to be screened can be confirmed based on the fluorescence intensity of the fermentation broth.

10. Use of the recombinant plasmid according to any one of claims 3 to 5, the recombinant engineered bacteria according to claim 6, or the genetically engineered Escherichia coli bacteria constructed by the method according to claim 7 in detecting erythritol concentration; Preferably, the recombinant plasmid according to any one of claims 3 to 5, the recombinant engineered bacteria according to claim 6 or the genetically engineered Escherichia coli constructed by the method according to claim 7 is used as a biosensor for detecting erythritol concentration.

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