Escherichia coli capable of anchoring to the surface of Yarrowia lipolytica cells and application method thereof

Abstract

The invention provides an Escherichia coli capable of anchoring Yarrowia lipolytica and an application method thereof, belonging to the field of bioengineering. The invention provides an Escherichia coli genetically engineered bacterium, wherein genes of anchoring proteins agα1, egt2, cwp2, dan4 or sed1 are fused with an ice nucleation protein INP gene and are inserted between the SalⅠ and BamHI restriction sites of the nucleotide sequence SEQ ID NO.12 to obtain a recombinant expression vector, which is transformed into Escherichia coli BL21 (DE3) to obtain the Escherichia coli genetically engineered bacterium. The Escherichia coli genetically engineered bacterium provided by the invention can be used for screening high-erythritol-producing strains of Yarrowia lipolytica and can also be used for detecting the concentration of erythritol.

Description

Technical Field

[0001] The present 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, and heat-stable crystalline powder. It has a refreshing taste and is low in calories, non-caries-causing, antioxidant, and endothelial-protective. Therefore, it is widely used in a variety of foods, such as baked goods, fermented milk, candies, and chocolate. Both fungi and bacteria can synthesize erythritol, and currently the main strain producing erythritol is Yarrowia lipolytica. Given the important application value and huge market demand for erythritol, breeding high-yield, low-cost, and genetically stable production strains has become a key goal for the industry. However, there is currently no efficient high-throughput breeding method for erythritol-producing strains. Therefore, breeding erythritol-producing strains with excellent fermentation performance remains a significant task.

[0003] Biosensors are a group of response systems that are widely present in microorganisms. They are used to identify and respond to specific metabolites in cells and convert them into specific response signals, such as fluorescence. Transcriptional regulatory factors 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 the qualitative and quantitative analysis of intracellular metabolites, and have also been used for the detection of chemical substances in industrial production.

[0004] To further improve sensor response efficiency, yeast cells possess the ability to convert promoters into signals in yeast cell surface engineering. Due to yeast's inherent advantages, yeast surface display systems are considered safe, efficient, and promising. A key challenge in yeast cell display technology is developing efficient anchoring proteins. This requires cells to produce large quantities of anchoring proteins while efficiently displaying them on the cell surface, thereby providing more space for target proteins to bind to substrates. There are two types of anchoring proteins in yeast: non-covalent attachment to the cell wall and covalent attachment. Covalently attached anchoring proteins can be divided into GPI-type and Pir-type anchoring proteins, depending on the type of attachment. Non-covalently attached cell wall proteins primarily utilize the N-terminal flocculation system of Flo1p. GPI-type anchoring proteins play a crucial role in the expression of heterologous proteins on the cell surface and are essential for the study of yeast display technology. GPI-type anchoring proteins can be fused to the cell wall in two ways: N-terminal and C-terminal fusions, with α-lectin and α-agglutinin being representative examples. In yeast, the glycolipid portion of a GPI-anchored protein is linked to the C-terminus of a hydrophobic protein, ensuring stable membrane binding. After the fusion protein is complete, the protein precursor is anchored in the endoplasmic reticulum, while the remaining protein remains within the lumen. Ultimately, the anchoring protein immobilizes proteins with specific functions on the cell surface. Saccharomyces cerevisiae, a yeast strain known for its excellent surface display platforms, has been used to display a variety of enzymes. The discovery of new anchoring proteins has become a research hotspot. Vaart has developed numerous cell wall proteins, such as Cwp1p, Cwp2p, Tip1p, Sed1p, Ycr89w, and Tirl, which have been shown to function as linker proteins for N-terminal display platforms. Yang et al. investigated new surface display systems, including Dan4p, Sed1p, Pry3p, Tos6p, Cwp2p, Srp2p, and α-lectin, and studied their display efficiency. The results revealed that Aga1p and Dan4p are more suitable for immobilizing larger proteins. Currently, there is more research on constructing biosensors using phage and bacterial display systems, while research in yeast is relatively limited. For example, a biosensor was constructed by displaying glutamate dehydrogenase in Escherichia coli, and by assembling it with carbon nanomaterials to form positive and negative electrodes, it can detect the concentration of the enzyme.

[0005] Chinese patent document CN 111996132 A (application number 202010708696.9) provides a method for displaying the target protein on the surface of Yarrowia lipolytica cells, which relates to the field of biochemical 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 target protein to be secreted extracellularly, 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 realize the surface display of the target protein on the Yarrowia lipolytica cell.

[0006] Chinese patent document CN 117737066 A (application number 202311769795.8) discloses a transcriptional regulatory sequence and its application. The transcriptional regulatory sequence is obtained by mutating the transcriptional regulatory sequence disclosed in the prior art. Compared with the prior art, the recombinant Escherichia coli prepared using the mutated transcriptional regulatory sequence can effectively reduce the background fluorescence intensity during the screening of erythritol-producing strains, and the fluorescence response signal is 6.5 times that of the prior art, significantly improving 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 have identified a genetically engineered bacterium capable of anchoring to the cell surface of Yarrowia lipolytica and displaying the anchoring protein cwp2. The recombinant E. coli BL21 / pET-22b(+)-eryD-INP-cwp2, containing the anchoring protein, was co-cultured with Yarrowia lipolytica and used as a biosensor to detect erythritol production in the yeast. Within 24 hours, the erythritol concentration response signal was 10.5 times higher than that of the original strain. Using the recombinant E. coli as a biosensor, they screened for high-erythritol-producing mutants of Yarrowia lipolytica, ultimately yielding 178 g / L of erythritol.

[0010] The technical solutions of the present invention are as follows:

[0011] The invention relates to 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 anchoring protein cwp2 amino acid sequence SEQ ID NO. 6.

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

[0017] The nucleotide sequence is the 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] According to the present invention, preferably, 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 anchoring protein agα1, egt2, cwp2, dan4 or sed1 with the ice nucleation protein INP gene, and is inserted between the SalⅠ and BamHI restriction sites of the nucleotide sequence SEQ ID NO.12.

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

[0023] According to the present invention, preferably, 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 bacterium 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 vectors 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) were transformed into Escherichia coli BL21(DE3), and after screening and identification, the 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, recombinant engineering bacteria, and genetically engineered Escherichia coli bacteria constructed by the method in screening erythritol-producing 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 based on 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 bacteria 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.

[0033] According to the present invention, preferably, the erythritol-producing strain is Yarrowia lipolytica.

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

[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 for 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 and displaying the anchoring protein cwp2 on the surface. The anchoring protein recombinant Escherichia coli E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 is embedded and co-cultured with Yarrowia lipolytica. The recombinant E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 is used as a biosensor to detect erythritol production by Yarrowia lipolytica. The concentration response signal of erythritol detected within 24 hours is 10.5 times that of the original strain. The recombinant E. coli is used as a biosensor to screen high-erythritol-producing mutants of Yarrowia lipolytica, and finally a mutant strain Y. lipolytica 5-14-E6 is obtained, and the erythritol production reaches 178 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0041] Figure 2 Schematic diagram of the construction of the anchoring 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 fluorescence expression vector pET-22b(+)-eryD-INP-dan4.

[0045] Figure 6 This is the agarose gel electrophoresis diagram of the PCR products of the anchoring 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 Agarose gel electrophoresis diagram of the PCR products of the anchoring protein fluorescence expression vectors 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 This 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] Figure 9 This is a diagram showing the anchoring effect of E. coli BL21 / pET-22b(+)-eryD-INP-sed1 on Y. lipolytica.

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

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

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

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

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

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

[0057] The technical solution of the present invention is further described below with reference to the examples, but the scope of protection of the present invention is not limited thereto. The operating methods not described in detail in the examples are all conventional operating methods well 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, with the nucleotide sequence being 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, with a nucleotide sequence of SEQ ID NO. 3, which contains a 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 20 g / L agar to LB liquid medium.

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

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

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

[0075] Example 1: Construction of anchor protein vector

[0076] Synthesis of anchoring protein fluorescent 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] 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; a gene sequence encoding a repressor protein, shown in SEQ ID NO. 10; a marker gene sequence encoding a fluorescent protein, shown in SEQ ID NO. 11; and a recombinant plasmid vector containing the gene, the nucleotide sequence of which is shown in SEQ ID NO. 12. This plasmid vector is named "pET-22b(+)-eryD" in this application.

[0078] The nucleotide sequences of the anchoring proteins agα1, egt2, cwp2, dan4 or sed1 and the nucleotide sequence of the 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 nucleation 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, and are double-digested with FastDigest SalⅠ and FastDigest BamHI. After dephosphorylation, they are purified by 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 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 was constructed as shown in the 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] ① Take 10 μL of the 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 incubate on ice for 30 minutes;

[0084] ②Heat shock at 42°C for 90 seconds, then quickly cool in an ice bath for 3 minutes;

[0085] ③ The competent cells were inoculated into 500 μL LB medium and cultured at 37°C with shaking at 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 supplemented with 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 aspirated dry, invert the plate and incubate at 37℃ for 12-16 hours.

[0088] (2) Identification of positive clones:

[0089] Colony PCR identification

[0090] Pick a single colony cultured above and inoculate the E. coli into 1 mL of liquid LB medium containing 100 μg / mL ampicillin. Incubate the culture at 37°C and 200 rpm with shaking for 6-8 h. Pipette 2 μL of the bacterial solution and perform colony PCR identification using a 50 μL PCR reaction system. The presence of a single target band indicates that 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 designed primers in this experiment are shown in Table 1. The agarose gel electrophoresis images of the products of PCR amplification of the sed1, agα1, egt2, cwp2, and dan4 genes are shown in Figure 1. 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 anchor proteins

[0097] (1) Strain activation: 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 a 1% inoculum size 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: 1 mL of the activated bacterial solution of the above-mentioned anchor protein fluorescence expression strain was inoculated into 50 mL of liquid LB medium containing 100 μg / mL ampicillin, and cultured at 37°C and 200 rpm until the bacterial solution OD 600The mixture was inoculated with Yarrowia lipolytica 5-14 at a concentration of 10% (v / v) into a 500 mL conical flask containing 50 mL of fermentation medium, and the mixture was shaken in a shaking incubator at 30°C and 200 rpm for 12 hours to obtain a fermentation broth.

[0099] (3) Collecting bacterial broth: 1 mL of fermentation broth of the recombinant E. coli strain and 1 mL of fermentation broth of Y. lipolytica were co-cultured at 30°C for 12 h. The effects of the five recombinant E. coli cell surface displays were observed and compared under a microscope.

[0100] The glycolipids on the surface of Yarrowia lipolytica can form covalent bonds with the anchor proteins expressed by recombinant Escherichia coli. Microscopic observation revealed that the anchor 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] Figure 9 This is the anchoring effect of E. coli BL21 / pET-22b(+)-eryD-INP-sed1 on Y. lipolytica.

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

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

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

[0105] Figure 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] Escherichia coli was used 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. The culture was then inoculated at a 10% (v / v) inoculum 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 h. The fluorescence intensity in the fermentation broth was detected every 12 h by a microplate reader during the fermentation process. Figure 14 It shows that the E. coli biosensor E. coli BL21 / pET-22b(+)-eryD-INP-cwp2 showed low background fluorescence intensity at 24 hours. 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 rpm for 24 hours. The inoculum was then transferred to a Yarrowia lipolytica fermentation medium at a 10% (v / v) inoculum concentration, and the culture was continued in a shaking incubator at 30°C and 200 rpm for 12 hours to obtain a Yarrowia lipolytica fermentation broth. A Yarrowia lipolytica mutant culture was obtained.

[0110] The recombinant Escherichia coli fermentation broth prepared without adding erythritol in Example 4 was mixed evenly with the diluted fermentation broth of 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 3 The mixture was fermented at 30°C for 75 h in a fermenter, and the erythritol production was detected by HPLC. Figure 15 The mutant strain Y.lipolytica 5-14-E6 was shown to be 3 The fermentation results of erythritol production in a fermenter showed that after 75 hours of fermentation, the erythritol production of the engineered 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 erythritol concentration and can also be used to screen strains of Yarrowia lipolytica that produce high erythritol.

Claims

1. Use of a recombinant engineered bacterium in any of the following: ① Screening an erythritol-producing strain, wherein the erythritol-producing strain is Yarrowia lipolytica; ② Detect erythritol concentration; The recombinant engineering bacteria comprises a host bacteria and a recombinant plasmid; The recombinant plasmid is an anchor protein cwp2 Fused with the gene of ice nucleation protein INP, inserted into the nucleotide sequence SEQ ID NO.12 Sal Ⅰ and Bam HI restriction site; The nucleotide sequence of the ice nucleation protein INP is the nucleotide sequence encoding the ice nucleation protein INP amino acid sequence SEQ ID NO.7; Ankyrin cwp2 The nucleotide sequence is SEQ ID NO.3; The host bacteria is Escherichia coli BL21 (DE3); Ankyrin cwp2 The gene was fused with the ice nucleation protein INP gene and inserted into the nucleotide sequence SEQ ID NO.12 Sal Ⅰ and Bam HI enzyme cutting site to obtain the recombinant expression vector pET-22b (+) - eryD -INP- cwp2 ; Recombinant expression vector pET-22b (+) - eryD -INP- cwp2 , transformed into Escherichia coli BL21 (DE3) to obtain recombinant engineered bacteria.

2. The use according to claim 1, characterized in that The nucleotide sequence of ice nucleation protein INP is SEQ ID NO.

13.

3. The use according to claim 1, characterized in that The method for constructing the recombinant engineered bacteria is characterized by comprising the following steps: (1) Anchoring protein cwp2 The gene was fused with the ice nucleation protein INP gene and inserted into the nucleotide sequence SEQID NO.12 Sal Ⅰ and Bam HI enzyme cutting site to obtain the recombinant expression vector pET-22b (+) - eryD -INP- cwp2 ; The anchoring protein cwp2 The nucleotide sequence of is SEQ ID NO.3, and the nucleotide sequence of ice nucleation protein INP is SEQ ID NO.13; (2) The recombinant expression vector pET-22b (+) - obtained in step (1) eryD -INP- cwp2 , transformed into Escherichia coli BL21 (DE3), and after screening and identification, the genetically engineered Escherichia coli was obtained. E. coli BL21 / pET-22b (+) - eryD - INP -cwp2 .

4. A method for screening erythritol-producing strains, characterized in that: The steps include: The recombinant engineered bacteria according to any one of claims 1 to 3 are inoculated into a culture medium for cultivation, the strain to be screened is inoculated into a culture medium for cultivation, 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; The erythritol producing strain is Yarrowia lipolytica.

5. The method according to claim 4, wherein The recombinant engineered bacteria were inoculated into LB medium for culture, and Yarrowia lipolytica was inoculated into YPD medium for culture. The fermentation broths of the two were mixed, and the erythritol production capacity of the strain to be screened was confirmed based on the fluorescence intensity of the fermentation broth.

6. The use according to claim 1, wherein The recombinant engineered bacteria is used as a biosensor to detect the concentration of erythritol.

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