Construction method of double-immobilized recombinant yeast engineering bacteria, engineering bacteria, catalyst and application in synthesis of rebaudioside

Abstract

The invention discloses a construction method of double-immobilized recombinant yeast engineering bacteria, engineering bacteria, a catalyst and application of the engineering bacteria in rebaudioside synthesis, and relates to the technical field of double-immobilized recombinant yeast strain preparation and rebaudioside synthesis. According to the invention, yeast is taken as a chassis cell, an orderly self-assembled multi-enzyme-level conjoined surface display system is constructed, and a carrier-free immobilization and UDPG in-situ regeneration technology is coupled, so that a novel enzyme and thallus dual-immobilized yeast whole-cell catalyst is obtained. According to the catalyst, cheap stevioside (St) and Rebaudioside A (Rebaudioside A) are used as substrates, expensive UDPG does not need to be additionally added, and the Rebaudioside (Rebaudioside D / M) can be efficiently and economically synthesized through a one-pot method. The complex operations such as cell disruption, enzyme separation and purification and immobilization are not needed in the synthesis process, and the bottleneck problems of substance transmembrane transport resistance, mass transfer resistance, hydrolysis of intracellular enzyme to products and the like in the prior art are avoided. The catalyst has reusability, continuous and high-strength rebaudioside biosynthesis can be realized through strain rejuvenation, and a powerful technical support is provided for industrial application of the catalyst.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of double-immobilized recombinant yeast strains and synthesis of rebaudioside, and more specifically to double-immobilized recombinant yeast engineering bacteria and their application in synthesis of rebaudioside. Background Art

[0002] In the past few decades, in order to reduce the intake of high-calorie sugars, low-calorie or zero-calorie artificial sweeteners have been developed and commercialized as functional substitutes for sugars, among which acesulfame potassium, sucralose and aspartame are the most widely used. Although artificial sweeteners meet the human need for sweetness, they are often reported to have potential health risks (destroying the balance of intestinal flora in the human body and Class 2B carcinogens, etc.), which have been questioned by consumers and their market share has declined year by year. Therefore, natural sweeteners with high safety, low calories and high sweetness have become the most popular new sugar substitutes among consumers.

[0003] Steviol glycosides (SGs), derived from natural extracts from stevia leaves, are known as the "three major sugar sources in the world" along with cane sugar and beet sugar. SGs are recognized as GRAS (Generally Recognized As Safe) and have been recognized as food additives by organizations such as FAO and WHO. They have also been included in my country's "National Food Safety Standard for the Use of Food Additives" (GB2760). In stevia leaves, stevioside (St) and rebaudioside A (RebA) are the most abundant, accounting for 5-10% (w / w) and 2-4% (w / w) of the dry weight of the leaves, respectively, but their taste is poor (bitter and aftertaste), which affects their wide application. Reb D / M blends have the advantages of high sweetness (about 300 times that of sucrose), fast sweetening speed, long sweetness retention time, good thermal stability, low calories and good taste (no odor and aftertaste), and are the best sucrose substitutes. However, the content of Reb D / M only accounts for 0.4-0.5% (w / w) of the weight of dry stevia leaves. Using the traditional plant extraction method, the extraction amount in 1 ton of dry stevia leaves is about 2 kg. The process is cumbersome, highly polluting and expensive, which seriously restricts the large-scale production of stevia leaf extracted rebaudioside (Reb D / M) for commercial purposes.

[0004] The use of bioenzymatic conversion is the most technically advantageous sustainable alternative strategy for achieving the biosynthesis of rebaudioside. The existing Reb D / M bioenzymatic conversion method still has the following common problems:

[0005] (1) High production cost: Using expensive Reb D or Reb E as substrate (content far below 1%) and expensive uridine diphosphate glucose (UDPG) as the glucose donor substrate.

[0006] (2) Low substrate conversion rate and product accumulation. When using whole-cell catalysts that express enzymes intracellularly, product synthesis is hindered by the resistance of substrate or product transmembrane transport; and intracellular hydrolases (such as endogenous glycoside hydrolase SCW2) hydrolyze the product, reducing product accumulation.

[0007] (3) Low enzyme utilization. The use of free pure enzymes for co-catalysis requires many steps to prepare pure enzymes, which is difficult and easy to inactivate. Free enzymes are difficult to recycle and reuse, and the separation and purification of downstream products are difficult, resulting in high production costs. The use of traditional enzyme immobilization methods requires additional immobilization steps, which consumes immobilization materials and reduces enzyme activity. In addition, the activity of immobilized enzymes decreases irreversibly with the increase in usage time.

[0008] Therefore, how to provide a green biomanufacturing technology for rebaudioside that is efficient, sustainable, low-cost and high-strength and break through the bottleneck of rebaudioside industrialization is an urgent problem that technicians in this field need to solve. Summary of the invention

[0009] In view of this, the present invention is proposed.

[0010] One of the purposes of the present invention is to provide a dual-immobilized recombinant yeast engineering bacterium.

[0011] The second object of the present invention is to provide a method for producing a dual-immobilized whole-cell catalyst using dual-immobilized recombinant yeast engineering bacteria.

[0012] The third object of the present invention is to provide a method for producing rebaudioside using dual immobilized whole-cell catalysts.

[0013] In order to achieve the above object, the present invention adopts the following technical solution:

[0014] In one aspect, an embodiment of the present invention provides a method for constructing a dual-immobilized recombinant yeast engineering bacterium, the process comprising:

[0015] 1) Construction of a multi-enzyme complex immobilization module: Fragment 1 and fragment 2 are connected to a PESC-URA vector to obtain a recombinant PESC-URA vector; fragment 3 is connected to a PRS424 vector to obtain a recombinant PRS424 vector; wherein, fragment 1 is a surface display scaffold protein expression frame composed of a signal peptide, a yeast cell wall anchor protein and an adhesion domain Cohesion; fragment 2 is a UDPG in situ regeneration system expression frame composed of a signal peptide, a yeast cell wall anchor protein and a sucrose synthase; fragment 3 is a multi-enzyme complex expression frame composed of a promoter, a signal peptide and an anchor domain Dockerin carrying an enzyme subunit;

[0016] 2) Construction of a vector-free self-immobilization module for bacteria: Integrate fragment 4 into the yeast genome, wherein fragment 4 is a vector-free self-immobilization expression frame consisting of a promoter and a flocculation gene;

[0017] 3) Transformation: The recombinant PESC-URA vector and the recombinant PRS424 vector were transferred into yeast cells to complete the construction of the dual-immobilized recombinant yeast engineering bacteria.

[0018] In a preferred embodiment, in fragment 1, the signal peptide is an α-factor signal peptide, the yeast cell wall anchor protein is an α-lectin Agα1, and the adhesion domain Cohesion scaffold proteins are three Cohesions, namely, Cohesion1 derived from Clostridiu m acetobutylicum, Cohesion2 derived from Ruminiclostridium cellulolyticum, and Cohesion3 derived from Clostridium cellulovorans, connected by 3×G4S Linker; the nucleotide sequences of the α-factor signal peptide, Cohesion1, Cohesion2, Cohesion3 and Agα1 are shown in SEQ ID NOs. 1, 9, 10, 11 and 12, respectively;

[0019] In fragment 2, the signal peptide is Aga2 signal peptide, the yeast cell wall anchor protein is α-lectin Aga2p, the sucrose synthase is SUS01, and Aga2p and SUS01 are connected using 3×G4S Linker; the nucleotide sequences of the Aga2 signal peptide and Aga2p are shown in SEQ ID NO.2; the amino acid sequence of sucrose synthase SUS01 is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.13.

[0020] In a preferred embodiment, in fragment 3, the promoters are PGK, TEF1 and ADH1, the signal peptides are all α-factor signal peptides, and the anchor domain Dockerin interacting proteins are Dockerin1 carrying glycosyltransferase UGT01 from C.acetobu tylicum, Dockerin2 carrying glycosyltransferase UGT02 from R.cellulolyticum, and Dockerin3 glycosyltransferase UGT01 from C.cellulovorans; UGT01 and Dockerin1, UGT02 and Dockerin2, and UGT01 and Docker in3 are connected using 3×G4S Linker; the nucleotide sequences of the PGK_α-factor secretion signal_UGT01_Dockerin1, TEF1_α-factor secretion signal_UGT02_Dockerin2 and ADH1_α-factor secretion signal_UGT01_Dockerin3 are shown in SEQ ID NO.3, 14, and 15, respectively;

[0021] The amino acid sequence of the glycosyltransferase UGT01 is shown in SEQ ID NO.5;

[0022] The amino acid sequence of the glycosyltransferase UGT02 is shown in SEQ ID NO.6.

[0023] In a preferred embodiment, in fragment 4, the promoter is PGK, the flocculation gene is FLO1s, the nucleotide sequence of which is shown as SEQ ID NO.4, and the amino acid sequence of the flocculation protein FLO1s is shown as SEQ ID NO.8.

[0024] In a preferred embodiment, the yeast is any one of yeast hosts such as Pichia pastoris, Saccharomyces cerevisiae and Kluyveromyces marxianus;

[0025] In a more preferred embodiment, the yeast is Saccharomyces cerevisiae AWY100.

[0026] The second aspect of the present invention provides a dual-immobilized recombinant yeast engineering bacterium constructed by the above-mentioned method.

[0027] The third aspect of the embodiments of the present invention provides the use of the dual-immobilized recombinant yeast engineering bacteria in the preparation of dual-immobilized recombinant yeast whole-cell catalysts and the synthesis of rebaudioside.

[0028] The fourth aspect of the embodiment of the present invention provides a method for preparing a dual-immobilized recombinant yeast whole-cell catalyst using the dual-immobilized recombinant yeast engineering bacteria, the process comprising:

[0029] The activated double-immobilized recombinant yeast engineering bacteria were inoculated into the culture medium 1, and cultured at 20-40° C. and 150-300 rpm for 48-72 hours; then fresh culture medium 2 was added, and cultured at 20-40° C. and 150-300 rpm for 48-72 hours.

[0030] A fifth aspect of the embodiments of the present invention provides a method for synthesizing rebaudioside RebD / M, the process comprising:

[0031] Adding a final concentration of 1-50mmol / L St or 1-25mmol / L Reb A, 10-500mmol / L sucrose, 1-50mmol / L UDP and 1-25mmol / L CaCl2 to the dual immobilized whole-cell catalyst, 20-40°C, using 1-5mol / L HCl and 10-50% ammonia water to maintain pH stability in the range of 5.0-8.5, 50-200rpm catalysis for 1-5h, repeating the feeding operation 1-5 times; finally, the whole-cell catalyst is quickly separated from the reaction solution by bacterial self-sedimentation to terminate the reaction, thereby realizing a one-pot synthesis of rebaudioside RebD / M;

[0032] After the whole-cell catalyst settles by its own weight, fresh reaction solution can be added to directly enter the next round of rebaudioside biosynthesis. When the catalytic activity of the whole-cell catalyst decreases to about 50-60% of the initial value, the whole-cell catalyst can be rejuvenated by culturing for 1 / 3-1 / 2 of the culture time described in claim 8.

[0033] Preferably, medium 1 is SD-SCAA-Leu-Trp-Ura medium, containing 13.4 g / L YNB, 0.6 g / L amino acid deficiency mixture DO Supplement-Leu / -Trp / -Ura, and 20 g / L glucose;

[0034] Medium 2 is a high-concentration medium containing 67.0 g / L YNB, 3 g / L amino acid deletion mixture, 50 g / L glucose, 100 g / L galactose, and supplemented at 20% of the volume of medium 1.

[0035] It can be seen from the above technical solutions that, compared with the prior art, the technical effects achieved by the present invention are:

[0036] 1) The raw materials used in the present invention are St and Reb A, which are abundant and cheap in stevia, and the glycosyl donor used is cyclically supplied through the UDPG in-situ regeneration system, without the need to add expensive UDPG. Compared with the expensive substrates used in the prior art, the present invention greatly reduces the production cost of rebaudioside (RebD / M), has strong market competitiveness, and is conducive to occupying more market share.

[0037] 2) The present invention uses a highly active and highly substrate-selective glycosyltransferase mutant UGT01, which significantly changes the substrate selectivity of the enzyme, and is more inclined to convert St and Reb A into Reb D / M, with almost no byproduct Reb I generated. The relative enzyme activity of the glycosyltransferase mutant used is 5.4 times that of the wild type, achieving efficient and green synthesis of rebaudioside (Reb D / M).

[0038] 3) The present invention constructs an ordered self-assembled multi-enzyme cascade catalytic system based on the substrate channel strategy of the cellulosome scaffold composed of adhesion proteins. Compared with the single enzyme mixed co-catalysis or multi-enzyme disordered fixed catalysis in the prior art, the reaction intermediates of the present invention can be quickly transferred from one active center to another, achieving efficient and orderly multi-enzyme catalytic reactions, reducing the competitive by-products and main product losses caused by the disordered diffusion of reaction intermediates, reducing substrate transportation time, and improving catalytic efficiency.

[0039] 4) The present invention couples the enzyme immobilization technology of surface-displayed multi-enzyme complexes and the flocculation carrier-free bacterial self-immobilization technology to construct a new yeast whole-cell catalyst with dual immobilization of multi-enzyme complexes and bacterial cells. Compared with the existing technology, it has the advantages of avoiding the transmembrane transport resistance of substrates / or products, the diffusion or mass transfer resistance of traditional enzymes / or bacterial cell immobilization, the hydrolysis of products by intracellular hydrolases, and complicated and cumbersome operation steps; at the same time, static sedimentation can achieve rapid separation of whole-cell catalysts and reaction products, can be reused many times, and its catalytic activity is regenerative after rejuvenation. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0041] Figure 1 The attached figure shows a carrier for constructing a dual-immobilized recombinant yeast whole-cell catalyst.

[0042] Figure 2The attached figure shows the SDS-PAGE of glycosyltransferases UGT01, UGT02 and sucrose synthase SUS01.

[0043] Figure 3 The attached figure shows the substrate interaction relationship of UGT76G1 and the comparison of the catalytic activities of UGT76G1 and UGT01.

[0044] Figure 4 The attached figure is a comparison of the synthesis of rebaudioside by free enzyme co-catalysis and multi-enzyme cascade catalysis.

[0045] Figure 5 The attached figure is a comparison of the relative activities of free, embedded and flocculated self-immobilized whole-cell catalysts.

[0046] Figure 6 The attached figure shows the effects of single feeding and batch feeding on the synthesis of rebaudioside.

[0047] Figure 7 The attached figure shows the reuse and rejuvenation of the dual-immobilized recombinant yeast whole-cell catalyst. DETAILED DESCRIPTION

[0048] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1 Construction of dual immobilized recombinant yeast engineering bacteria

[0050] 1) Construction of a multi-enzyme complex immobilization module: Fragment 1 and fragment 2 were obtained by full gene synthesis and connected to the PESC-URA vector. The constructed recombinant vector 1 is as follows Figure 1 As shown in A.

[0051] Fragment 3 was obtained by total gene synthesis and connected to the pRS424 vector. The recombinant vector 2 was constructed as shown in Figure 1 As shown in B.

[0052] 2) Construction of a vector-free self-immobilization module for bacteria: Fragment 4 with upper and lower homologous arms at both ends is obtained by total gene synthesis, and a vector-free self-immobilization integration fragment consisting of a promoter and a flocculation gene is constructed, such as Figure 1 As shown in C;

[0053] 3) Transformation: After integrating fragment 4 into the genome of Saccharomyces cerevisiae AWY100, recombinant vector 1 and recombinant vector 2 were introduced by electroporation to construct the dual-immobilized recombinant yeast engineering strain AWY100-PESC-C 1-3 -GAL1 / 10-SUS01-PRS424-UGT01 / D1-UGT02 / D2-UGT01 / D3-FLO1s, abbreviated as A 絮凝 .

[0054] Specifically, in fragment 1: the signal peptide is an α-factor signal peptide, the yeast cell wall anchor protein is an α-lectin Agα1, and the adhesion domain Cohesion scaffold proteins are Cohesion1 derived from Clostridium acetobutylic um, Cohesion2 derived from Ruminiclostridium cellulolyticum, and Cohesion3 derived from Clostridium cellulovorans, which are connected by 3×G4S Linker; the nucleotide sequences of the α-factor signal peptide, Cohesion1, Cohesion2, Cohesion3 and Agα1 are shown in SEQ ID NOs. 1, 9, 10, 11 and 12, respectively;

[0055] In fragment 2: the signal peptide is Aga2 signal peptide, the yeast cell wall anchor protein is α-lectin Aga2p, the sucrose synthase is SUS01, and Aga2p and SUS01 are connected using 3×G4S Linker; the nucleotide sequences of the Aga2 signal peptide and Aga2p are shown in SEQ ID NO.2; the amino acid sequence of sucrose synthase SUS01 is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.13.

[0056] In fragment 3: the promoters are PGK, TEF1 and ADH1, the signal peptides are all α-factor signal peptides, the anchor domain Dockerin interacting proteins are Dockerin1 carrying glycosyltransferase UGT01 from C.acetobutylicum, Dockerin2 carrying glycosyltransferase UGT02 from R.cellulolyticum and Dockerin3 glycosyltransferase UGT01 from C.cellulovorans; the nucleotide sequences of PG K_α-factor secretion signal_UGT01_Dockerin1, TEF1_α-factor secretion signal_UGT02_Dockerin2 and ADH1_α-factor secretion signal_UGT01_Dockerin3 are shown in SEQ ID NO.3, 14 and 15 respectively; UGT01 and Dockerin1, UGT 02 and Dockerin2, UGT01 and Dockerin3 are connected by 3×G4S Linker; the amino acid sequence of the glycosyltransferase UGT01 is shown in SEQ ID NO.5; the amino acid sequence of the glycosyltransferase UGT02 is shown in SEQID NO.6.

[0057] In fragment 4: the promoter is PGK, the flocculation gene is FLO1s, the nucleotide sequence of which is shown in SEQ ID NO.4, and the amino acid sequence of the flocculation protein FLO1s is shown in SEQ ID NO.8.

[0058] Example 2 Protein expression of biological enzymes UGT01, UGT02 and SUS01

[0059] Characterize protein expression by SDS-PAGE electrophoresis and compare the positions of protein bands with those of protein marker reference bands.

[0060] According to the technical solution provided by the present invention, the heterologous soluble expression of sucrose synthase SUS01, glycosyltransferase UGT01 and UGT02 in yeast host was successfully achieved, and almost no insoluble inclusion bodies were generated. The molecular weight of SUS01 was about 85kDa, and the molecular weights of UGT01 and UGT02 were about 50kDa and 55kDa, respectively, which were basically consistent with the theoretical molecular weight. The results are as follows Figure 2 shown.

[0061] Example 3 Comparison of glycosyltransferase UGT01 mutant and UGT76G1 wild type

[0062] In the enzymatic biosynthesis of rebaudioside, glycosyltransferase UGT76G1 is the most critical rate-limiting enzyme. In order to improve the yield of rebaudioside, the wild-type UGT76G1 was subjected to directed evolution and multiple rounds of rational design transformation. According to the reported crystal structure-substrate interaction relationship of UGT76G1, the uridine ring of the substrate forms a π-π stacking with W338; forms a series of hydrogen bond positioning networks with D27, S283, V339, N360, S361, and E364 to assist the positioning of the substrate UDP; and forms a salt bridge with H356. In view of the above functional relationships, a series of mutant libraries were designed using the rational mutation method. W338 was replaced with F and H that produce similar effects to fine-tune the π-π interaction; the six amino acids D27, S283, V339, N360, S361, and E364 were iteratively saturated mutated using the NNK codon method to adjust the substrate positioning method to improve the catalytic activity; and H356 was verified by sequence alignment to be highly conserved and was not mutated. The rational design strategy and the mutants of error-prone PCR random mutations were combined and superimposed to obtain the glycosyltransferase mutant UGT01.

[0063] The enzyme was added to a final concentration of 1-50mmol / L St or 1-25mmol / L Reb A, 10-500mmol / L sucrose, 1-50mmol / L UDP and 1-25mmol / L CaCl2 at 20-40°C. 1-5mol / L HCl and 10-50% ammonia were used to maintain the pH stable in the range of 5.0-8.5. The reaction was catalyzed at 50-200rpm for 1-5h. The concentration of rebaudioside was detected by HPLC.

[0064] HPLC: Liquid chromatography detection method: C18 chromatographic column (length: 250 mm; inner diameter: 4.6 mm; particle size: 5 μm). The mobile phase is a 68:32 (v / v) mixture of 1.38 g / L sodium phosphate buffer and acetonitrile (pH 2.6), the flow rate is 1.0 mL / min, the column temperature is 40 ° C, the detection wavelength is 210 nm, and the injection volume is 10 μL.

[0065] The mutant UGT01 synthesized rebaudioside at a yield of 37.1% within 30 minutes, while the yield of the wild-type UG T76G1 was only 6.9%. The catalytic activity of UGT01 was 5.4 times higher than that of the wild-type. At the same time, UGT01 changed the substrate selectivity, reduced the synthesis of the byproduct Reb I, and made the catalytic reaction process proceed towards the synthesis of the main product Reb D / M.

[0066] Example 4 Comparison of free enzyme co-catalysis and multi-enzyme cascade catalysis

[0067] According to the technical solution provided by the present invention, the following recombinant yeast engineering bacteria were cultured. Other culture conditions were kept consistent. The recombinant bacteria without the addition of inducers were unable to express the surface display scaffold protein to self-assemble to form a multi-enzyme cascade complex, and this group was the free enzyme co-catalysis experimental group; while the recombinant bacteria with the addition of inducers were the multi-enzyme cascade catalysis experimental group. UDPG was directly added during the reaction without enabling the UDPG in situ regeneration system to reduce interference factors. Other reaction conditions were as described above. The results are shown in Figure 4 As shown, the yield of rebaudioside catalyzed by the multi-enzyme cascade was increased by 46.6% compared with the yield co-catalyzed by the free enzyme.

[0068] Example 5 Comparison of free, embedded and flocculated self-immobilized whole cell catalysts

[0069] According to the technical solution provided by the present invention, the following recombinant yeast engineering bacteria were cultured. The recombinant PESC-URA vector and the recombinant PRS424 vector were introduced into the AWY100 host of Saccharomyces cerevisiae without the integration of the flocculation gene to construct a free recombinant bacteria with a surface display of the multi-enzyme cascade complex, named A 游离 Then, 1.5% sodium alginate was used to make it into an embedded immobilized ball, named A 包埋 , and the high-performance dual-immobilized recombinant bacteria A constructed in Example 1 of the present invention 絮凝 .

[0070] Activity detection conditions: add a final concentration of 1-50mmol / LSt or 1-25mmol / L Reb A, 10-500mmol / L sucrose, 1-50mmol / L UDP and 1-25mmol / L CaCl2 to the dual immobilized whole-cell catalyst, 20-40°C, use 1-5mol / L HCl and 10-50% ammonia water to maintain pH stability in the range of 5.0-8.5, 50-200rpm catalysis for 1-5h, and repeat the feeding operation 1-5 times; finally, the whole-cell catalyst is quickly separated from the reaction solution by self-sedimentation of the bacteria to terminate the reaction, thereby realizing a one-pot synthesis of rebaudioside Reb D / M.

[0071] The results are as follows Figure 5 As shown, A 絮凝 and A 游离 The relative activities were basically similar, indicating that the flocculation self-immobilization method did not have a negative impact on the catalytic functional domain, and there was almost no mass transfer resistance; on the contrary, flocculation aggregation increased the bacterial density (group effect) and gave the strain better environmental stress tolerance; while A 包埋 Affected by mass transfer resistance, the reaction substrates / intermediates cannot quickly pass through the encapsulation material to enter the enzyme active center, resulting in a significant slowdown in the catalytic rate.

[0072] Example 6 Effect of single feeding and batch feeding on the synthesis of rebaudioside

[0073] According to the technical solution provided by the present invention, the high-performance dual-immobilized recombinant yeast engineering bacteria constructed by the present invention are cultured. In this example, one-time feeding and batch feeding were respectively used to study the effects of different substrate addition methods on rebaudioside synthesis.

[0074] One-time feeding: Add 30mmol / L St substrate into the reaction solution at one time.

[0075] Batch feeding: 10 mmol / L St was added into the reaction solution as the initial substrate, and then feeding was performed once every 2.5 h, for a total of 2 feedings.

[0076] The results are as follows Figure 6 As shown in the figure, compared with one-time feeding, batch feeding can significantly increase the yield of rebaudioside. 10mmol / L St is used as the initial substrate, and feeding is performed once every 2.5h. After two feedings, the yield of rebaudioside (Reb D / M) is 33.1g / L.

[0077] Example 7 Reuse and rejuvenation of dual immobilized recombinant yeast whole cell catalyst

[0078] The dual-immobilized recombinant yeast engineering bacteria synthesized rebaudioside in a batch feeding manner as described in Example 6. After each round of catalytic reaction, the reaction liquid was quickly separated by bacterial self-sedimentation. Figure 7 A, and then add fresh reaction solution to enter the next cycle. When the relative enzyme activity of the whole cell catalyst is detected to be less than 60% of the initial value, the cycle is stopped and the strain rejuvenation stage is entered. As described in the technical solution provided by the present invention, only one-third of the initial culture time of the bacteria is required to complete the strain rejuvenation. The results are shown in Figure 7 As shown in B, a multi-enzyme complex dual-immobilized recombinant yeast whole-cell catalyst created by the present invention can maintain good catalytic stability in six cycles, showing the potential as an industrial strain for the efficient and low-cost synthesis of natural sweetener rebaudioside (Reb D / M), which has important practical significance and economic value for promoting my country's international competitiveness in the field of food processing.

[0079] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0080] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for constructing a dual-immobilized recombinant yeast engineering bacterium, characterized in that the process include: 1) Construction of a multi-enzyme complex immobilization module: Fragment 1 and fragment 2 are connected to a PESC-URA vector to obtain a recombinant PESC-URA vector; fragment 3 is connected to a PRS424 vector to obtain a recombinant PRS424 vector; wherein, fragment 1 is a surface display scaffold protein expression frame composed of a signal peptide, a yeast cell wall anchor protein and an adhesion domain Cohesion; fragment 2 is a UDPG in situ regeneration system expression frame composed of a signal peptide, a yeast cell wall anchor protein and a sucrose synthase; fragment 3 is a multi-enzyme complex expression frame composed of a promoter, a signal peptide and an anchor domain Dockerin carrying an enzyme subunit; 2) Construction of a vector-free self-immobilization module for bacteria: Integrate fragment 4 into the yeast genome, wherein fragment 4 is a vector-free self-immobilization expression frame consisting of a promoter and a flocculation gene; 3) Transformation: The recombinant PESC-URA vector and the recombinant PRS424 vector were transferred into yeast cells to complete the construction of the dual-immobilized recombinant yeast engineering bacteria.

2. The method according to claim 1, characterized in that In fragment 1, the signal peptide is an α-factor signal peptide, the yeast cell wall anchor protein is an α-lectin Agα1, and the adhesion domain Cohesion scaffold proteins are Cohesion1 derived from Clostridiumacetobutylicum, Cohesion2 derived from Ruminiclostridium cellulolyticum, and Cohesion3 derived from Clostridium cellulovorans, which are connected by 3×G4S Linker; the nucleotide sequences of the α-factor signal peptide, Cohesion1, Cohesion2, Cohesion3 and Agα1 are shown in SEQ ID NOs. 1, 9, 10, 11 and 12, respectively; In fragment 2, the signal peptide is Aga2 signal peptide, the yeast cell wall anchor protein is α-lectin Aga2p, the sucrose synthase is SUS01, and Aga2p and SUS01 are connected using 3×G4S Linker; the nucleotide sequences of the Aga2 signal peptide and Aga2p are shown in SEQ ID NO.2; the amino acid sequence of sucrose synthase SUS01 is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.

13.

3. The method according to claim 1, characterized in that In fragment 3, the promoters are PGK, TEF1 and ADH1, the signal peptides are all α-factor signal peptides, and the anchor domain Dockerin interacting proteins are Dockerin1 carrying glycosyltransferase UGT01 from C.acetobutylicum, Dockerin2 carrying glycosyltransferase UGT02 from R.cellulolyticum and Dockerin3 glycosyltransferase UGT01 from C.cellulov orans; the nucleotide sequences of PGK_α-factorsecretion signal_UG T01_Dockerin1, TEF1_α-factor secretion signal_UGT02_Dockerin2 and ADH1_α-factor secretion signal_UGT01_Dockerin3 are shown in SEQ ID NO.3, 14 and 15 respectively; UGT01 and Dockerin1, UGT02 and Dockerin2, UGT01 and Dockerin3 are connected by 3×G4S Linker; The amino acid sequence of the glycosyltransferase UGT01 is shown in SEQ ID NO.5; The amino acid sequence of the glycosyltransferase UGT02 is shown in SEQ ID NO.

6.

4. The method according to claim 1, characterized in that In fragment 4, the promoter is PGK, the flocculation gene is FLO1s, the nucleotide sequence of which is shown in SEQ ID NO.4, and the amino acid sequence of the flocculation protein FLO1s is shown in SEQ ID NO.

8.

5. The method according to claim 1, characterized in that The yeast is any one of yeast hosts such as Pichia pastoris, Saccharomyces cerevisiae and Kluyveromyces marxianus; Preferably, the yeast is Saccharomyces cerevisiae AWY100.

6. The dual-immobilized recombinant yeast engineered bacteria constructed by the method according to any one of claims 1 to 5.

7. Use of the dual-immobilized recombinant yeast engineering bacteria according to claim 6 in the preparation of dual-immobilized recombinant yeast whole-cell catalysts and the synthesis of rebaudioside.

8. A method for preparing a dual-immobilized recombinant yeast whole-cell catalyst using the dual-immobilized recombinant yeast engineered bacteria according to claim 6, characterized in that the process include: The activated double-immobilized recombinant yeast engineering bacteria were inoculated into the culture medium 1, and cultured at 20-40° C. and 150-300 rpm for 48-72 hours; then fresh culture medium 2 was added, and cultured at 20-40° C. and 150-300 rpm for 48-72 hours.

9. A method for synthesizing rebaudioside RebD / M, characterized in that: The process includes: Add a final concentration of 1-50mmol / L St or 1-25mmol / L Reb A, 10-500mmol / L sucrose, 1-50mmol / L UDP and 1-25mmol / L CaCl2 to the dual immobilized whole-cell catalyst of claim 8, 20-40°C, use 1-5mol / L HCl and 10-50% ammonia water to maintain pH stability in the range of 5.0-8.5, 50-200rpm catalysis for 1-5h, and repeat the feeding operation 1-5 times; finally, the whole-cell catalyst is quickly separated from the reaction solution by self-sedimentation of the bacteria to terminate the reaction, thereby realizing a one-pot synthesis of rebaudioside Reb D / M; After the whole-cell catalyst settles by its own weight, fresh reaction solution can be added to directly enter the next round of rebaudioside biosynthesis. When the catalytic activity of the whole-cell catalyst decreases to about 50-60% of the initial value, the whole-cell catalyst can be rejuvenated by culturing for 1 / 3-1 / 2 of the culture time described in claim 8.

10. The method according to claim 9, characterized in that Medium 1 is SD-SCAA-Leu-Trp-Ura medium, containing YNB 13.4 g / L, amino acid deficiency mixture DO Supplement-Leu / -Trp / -Ura 0.6 g / L, and glucose 20 g / L; Medium 2 is a high-concentration medium containing 67.0 g / L YNB, 3 g / L amino acid deletion mixture, 50 g / L glucose, 100 g / L galactose, and supplemented at 20% of the volume of medium 1.

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