A strain for efficiently synthesizing d-tagatose and a preparation method and application thereof

Abstract

This invention discloses a strain for the efficient synthesis of D-tagatose, its preparation method, and its applications. Through strategies such as the discovery of novel galactitol dehydrogenase and promoter engineering, this invention initially increased tagatose yield by 28.48%. Simultaneously, a highly efficient and sensitive tagatose biosensor was developed, which was used as a screening tool for xylose reductase. Ss XR molecularly modified the strain to obtain the F128M-Q219K double mutant, which achieved a tagatose yield of 5.39 g / L in the producing strain. Based on this, amino acid complementation optimization was performed on the high-yielding strain, and the final engineered strain achieved a tagatose yield of 44.61 g / L in a 5 L bioreactor. This study provides a new strategy and core technology support for the efficient biosynthesis of tagatose.

Description

Technical Field

[0001] This invention relates to a strain for the efficient synthesis of D-tagatose, its preparation method, and its application, belonging to the fields of bioengineering and food additive synthesis technology. Background Technology

[0002] Tagatose is the C4 epimer of fructose, a novel functional monosaccharide found in trace amounts naturally in fruits, cocoa, and dairy products. Its sweetness can reach 92% of an equivalent amount of sucrose, and its taste is very similar to sucrose, without any off-flavor or bitterness. It is not only a high-quality low-energy food sweetener but also possesses multiple physiological benefits, including inhibiting hyperglycemia, regulating intestinal flora, and preventing tooth decay. It is widely used in food, pharmaceuticals, and cosmetics, with significant market potential. Regarding safety and compliance, tagatose has obtained global authoritative certifications. It received GRAS certification from the US FDA in 2003, was approved as a novel food ingredient by the EU in 2006, and has been approved for civilian use in South Korea, Australia, New Zealand, and other regions. In 2014, it was also approved as a national new food ingredient in China.

[0003] Traditional chemical synthesis of tagatose has drawbacks such as harsh reaction conditions, numerous byproducts, difficulty in separation and purification, and poor environmental friendliness. In contrast, biocatalysis, with its mild conditions, high selectivity, and green sustainability, has become the mainstream direction for large-scale production and is also the core path to solve the problems of yield and cost. Currently, there are four classic routes for the biosynthesis of tagatose, but all of them have insurmountable technical shortcomings: The L-arabinose isomerase route, as the most classic isomerization route, uses galactose as a substrate. Due to thermodynamic equilibrium limitations, the conversion rate is only around 60%, and conventional enzyme modification has reached a bottleneck. Although there are optimization methods such as immobilization and arginine catalysis, the room for improvement is limited, hindering industrialization; The tagatose-4-epimerase route uses inexpensive fructose as a substrate, which has an advantage in raw material cost, but the current conversion rate is only 30%. The key enzyme catalytic efficiency and substrate specificity are insufficient, and it is still in the laboratory optimization stage; The phosphorylation multi-enzyme route uses inexpensive polysaccharides such as maltodextrin and starch as raw materials, and the conversion rate can reach 48.1%, but the reaction steps are lengthy, the multi-enzyme synergistic regulation is complex, and the many by-products lead to a sharp increase in separation and purification costs, making industrialization extremely difficult. The redox pathway uses galactose as a substrate to synthesize tagatose through a two-step redox reaction. This pathway completely avoids the limitations of thermodynamic equilibrium and has a theoretical conversion rate of up to 90%. It is far superior to the traditional route in terms of conversion efficiency and cost control, and has significant industrialization potential. However, this route is still in the exploratory stage and has not yet formed a mature whole-cell catalytic system. The core bottlenecks are concentrated on problems such as low catalytic activity of key redox enzymes and mismatch of intracellular metabolic flux. Single enzyme modification or metabolic regulation is difficult to achieve overall optimization.

[0004] Based on the aforementioned industry needs and research gaps, this invention takes the oxidoreductase pathway as the core route, addresses existing key bottlenecks, constructs a high-throughput screening platform, and uses protein engineering to directionally modify key oxidoreductases to overcome the bottlenecks in enzyme activity and stability. It also couples protein engineering and metabolic engineering to construct a high-yield tagatose cell factory. Summary of the Invention

[0005] To overcome the technical bottlenecks in existing D-tagatose biosynthesis processes, such as low substrate conversion efficiency, lack of efficient functional enzymes, poor adaptability of gene expression regulation, inefficient screening methods, and imperfect fermentation process regulation, this paper proposes an efficient and stable D-tagatose biosynthesis technology.

[0006] This invention is achieved through the following technical solution: The first objective of this invention is to provide a strain that efficiently synthesizes D-tagatose, wherein the strain expresses xylose reductase in a Saccharomyces cerevisiae host. Ss XR and galactitol dehydrogenase Rr GDH.

[0007] In one embodiment of the present invention, the xylose reductase Ss XR has the following amino acid sequence: (1) The amino acid sequence as shown in SEQ ID NO.2; or, (2) The amino acid sequence obtained by mutating phenylalanine at position 128 of the amino acid sequence shown in SEQ ID NO.2 to methionine; or, (3) The amino acid sequence obtained by mutating phenylalanine at position 128 of the amino acid sequence shown in SEQ ID NO.2 to methionine and glutamine at position 219 to lysine.

[0008] In one embodiment of the present invention, the galactitol dehydrogenase Rr The amino acid sequence of GDH is shown in SEQ ID NO.4.

[0009] In one embodiment of the present invention, the galactitol dehydrogenase Rr GDH adopts P HXT7 Promoter startup expression.

[0010] In one embodiment of the present invention, the brewer's yeast host is a knockout strain of brewer's yeast BY4741. GAL1 Genes, and replenish histidine genes. 。

[0011] In one embodiment of the present invention, the strain has restored the methionine gene, uracil gene, and leucine gene at the reduction site.

[0012] A second objective of this invention is to provide a method for increasing the yield of D-tagatin in brewer's yeast, comprising the following steps: Xylose reductase expression in Saccharomyces cerevisiae host Ss XR and galactitol dehydrogenase Rr GDH, wherein the xylose reductase Ss XR has the following amino acid sequence: (1) The amino acid sequence as shown in SEQ ID NO.2; or, (2) The amino acid sequence obtained by mutating phenylalanine at position 128 of the amino acid sequence shown in SEQ ID NO.2 to methionine; or, (3) The amino acid sequence obtained by mutating phenylalanine at position 128 of the amino acid sequence shown in SEQ ID NO.2 to methionine and glutamine at position 219 to lysine.

[0013] In one embodiment of the present invention, the galactitol dehydrogenase Rr The amino acid sequence of GDH is shown in SEQ ID NO.4.

[0014] In one embodiment of the present invention, the galactitol dehydrogenase Rr GDH adopts P HXT7 Promoter startup expression.

[0015] In one embodiment of the present invention, the brewer's yeast host is a knockout strain of brewer's yeast BY4741. GAL1 Genes, and replenish histidine genes. 。

[0016] In one embodiment of the present invention, the strain also replenished the methionine gene, uracil gene, and leucine gene.

[0017] A third objective of this invention is to provide the application of the strain in the fermentation production of tagatose.

[0018] In one embodiment of the present invention, the application is to inoculate the strain into a fermentation medium for fermentation to prepare the tagatose.

[0019] In one embodiment of the present invention, the fermentation medium contains 10-30 g / L glucose, 10-30 g / L galactose, 10-30 g / L tryptone, and 5-15 g / L yeast extract.

[0020] In one embodiment of the present invention, the fermentation culture medium further includes 8-12 mL / L of trace element solution and 10-15 mL / L of vitamin solution.

[0021] A fourth objective of this invention is to provide a high-throughput screening method based on tagatose fluorescence response, the method comprising the following steps: S1, will P GAL Promoter and green fluorescent protein reporter gene GFP The strain was tandemly integrated into the Ph expression plasmid, transformed into the recipient bacterium BY4741, and a screening strain was constructed. S2. Introduce genes related to the tagatose metabolism pathway into the screened strains to construct strains capable of producing tagatose; S3. The tagatose-producing strain constructed in step S2 was cultured in YPD screening medium containing galactose, and high-throughput screening was performed by detecting the change in its fluorescence value relative to biomass.

[0022] In one embodiment of the present invention, the amount of galactose in the YPD screening medium is 2-10 g / L.

[0023] In one embodiment of the present invention, the YPD screening medium comprises 50-70 g / L glucose, 2-10 g / L galactose, 10-30 g / L tryptone, and 5-15 g / L yeast extract.

[0024] The beneficial effects of this invention are: This invention, through strategies such as discovering novel galactitol dehydrogenases and promoter engineering, initially increased tagatose yield by 28.48%; simultaneously, it developed a highly efficient and sensitive tagatose biosensor, which was used as a screening tool for xylose reductase. Ss XR molecularly modified the strain to obtain the F128M-Q219K double mutant, which achieved a tagatose yield of 5.39 g / L in the producing strain. Based on this, amino acid complementation optimization was performed on the high-yielding strain, and the final engineered strain achieved a tagatose yield of 44.61 g / L in a 5 L bioreactor. This study provides a new strategy and core technology support for the efficient biosynthesis of tagatose. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 To screen for novel oxidoreductases in Saccharomyces cerevisiae.

[0027] Figure 2To screen for strong expression promoters in Saccharomyces cerevisiae.

[0028] Figure 3 To validate the high-throughput screening mechanism of tagatose in Saccharomyces cerevisiae, A is a schematic diagram of the fluorescence response mechanism of the screening strain; B shows the fluorescence response characteristics of the screening strain in different culture media (a, glucose; b, galactose; c, galactitol; d, tagatose); C shows the residual glucose, residual galactose, and tagatose yields and FLU / OD of the screening strain during fermentation. 600 Dynamic changes; D represents the FLU / OD ratio of the model strain at different galactose concentrations. 600 The differences.

[0029] Figure 4 Xylose reductase Ss The tagatose yield of the XR mutant in the screening strain and the production strain, where A is the tagatose yield of the screening strain containing the mutant plasmid after 48 hours of fermentation; B is the tagatose yield of the production strain containing the mutant plasmid after 48 hours of fermentation.

[0030] Figure 5 OD of amino acid-replenishing strain fermented for 0-120 h 600 The results showed that A was the tagatose yield after 48 h of shake-flask fermentation of the amino acid-added strain; B was the tagatose yield after 120 h of shake-flask fermentation of the YXCY-031 strain; and C was the tagatose yield after 120 h of fermentation of the YXCY-031 strain in a 5 L fermenter. Detailed Implementation

[0031] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0032] Biochemical materials: Saccharomyces cerevisiae BY4741 has been described in the article "BAKER BRACHMANN C, DAVIES A, COST GJ, et al. Designer deletion strains derived from..." Saccharomyces cerevisiae S288C: Auseful set of strains and plasmids for PCR-mediated gene disruption and other applications[J]. Yeast, 1998, 14(2): 115-132. DOI: 10.1002 / (sici)1097-0061(19980130)14:2<115::Aid-yea204>3.0.Co;2-2"; The plasmid pCEV-G1-Km has been used in the article "VICKERS CE, BYDDER SF, ZHOU Y, NIELSEN L K. Dual gene expression cassette vectors with antibiotic selection markers for engineering in..." Saccharomyces cerevisiae [J]. Microbial Cell Factories, 2013, 12(1): 96. DOI: 10.1186 / 1475-2859-12-96; Restriction endonuclease, T4 DNA ligase, high-fidelity DNA polymerase, and DNA marker were purchased from Takara; Geninomycin G418 and 5-fluoroorotic acid (FOA) were purchased from Yisheng Biotechnology Co., Ltd.; 4S green nucleic acid dye was purchased from Shanghai Sangon Biotech Co., Ltd.; Yeast nitrogen source YNB, plasmid extraction kit, gel extraction kit, washing kit, and yeast genome extraction kit were purchased from Tiangen Biotech Co., Ltd.; Tryptone, glucose, sodium chloride, galactose, glycerol, and amino acids were purchased from Sinopharm Chemical Reagent Co., Ltd.; Yeast extract was purchased from Angel Yeast Co., Ltd.; Bleomycin was purchased from Shanghai Xin'aosheng Biotechnology Co., Ltd.

[0033] Culture medium: YPD medium: glucose (20 g / L), tryptone (20 g / L), yeast extract (10 g / L), sterilized at 115℃ for 30 min. For solid medium, add agar powder (20 g / L); adding antibiotics will produce the corresponding antibiotic-selective medium.

[0034] Based on YPD medium, the types and amounts of sugars were adjusted to obtain YPD-D (20 g / L glucose), YPD-G (10 g / L glucose, 10 g / L galactose), YPD-GT (10 g / L glucose, 10 g / L galactitol), and YPD-T (10 g / L glucose, 10 g / L tagatose).

[0035] YPD selection medium: glucose (60 g / L), galactose (5 g / L), tryptone (20 g / L), yeast extract (10 g / L), sterilized at 115℃ for 30 min. Adding antibiotics will produce the corresponding antibiotic selective medium.

[0036] YPD production medium: glucose (20 g / L), galactose (20 g / L), tryptone (20 g / L), yeast extract (10 g / L), sterilized at 115℃ for 30 min. Adding antibiotics will produce the corresponding antibiotic selective medium.

[0037] SD medium: glucose (20 g / L), yeast nitrogen base YNB (1.7 g / L), ammonium sulfate (5 g / L), amino acid mixture (0.71 g / L), sterilized by filtration. For solid media, agar powder (20 g / L) is added; adding antibiotics or inducers creates the corresponding antibiotic / inducer selective medium, such as SD-FOA medium (5-fluoroorotic acid 1 g / L).

[0038] LB medium: NaCl (10 g / L), tryptone (10 g / L), yeast extract (5 g / L), sterilized at 121℃ for 20 min. For solid media, add agar powder (20 g / L). Adding antibiotics creates the corresponding antibiotic-selective medium.

[0039] Trace element solution: 15 g / L EDTA, 10.2 g / L ZnSO4·7H2O, 0.5 g / L MnCl2·4H2O, 0.5 g / L CuSO2, 0.86 g / L CoCl2·6H2O, 0.56 g / L Na2MoO4·2H2O, 3.84 g / L CaCl2·2H2O, 5.12 g / L FeSO4·7H2O, autoclaved at 115℃ for 30 min.

[0040] Vitamin solution: 0.05 g / L biotin, 1 g / L calcium pantothenate, 1 g / L niacin, 25 g / L inositol, 1 g / L thiamine hydrochloride, 1 g / L pyridoxine, 0.2 g / L para-aminobenzoic acid. Sterilize by filtration through a 0.22 μm syringe filter and store at 4°C.

[0041] 5 L bioreactor culture medium: Initial fermentation tank volume 2 L, YPD fermentation basal culture medium with substrate galactose 20 g / L added, sterilized and cooled, then add 10 mL / L trace element solution and 12 mL / L vitamin solution.

[0042] Strains, plasmids, and primers: Table 1. Strains involved in this patent

[0043] Note: The plasmids carried in Table 1 are described in detail in Table 2. Table 2 Plasmids and their genotypes

[0044] Table 3 Primers

[0045] Basic operations in molecular biology: (1) Unless otherwise specified, the procedures for E. coli plasmid extraction, genome extraction, PCR product purification, and gel recovery shall be performed in accordance with the instructions of the corresponding kit.

[0046] (2) Use PCR technology to amplify DNA fragments separately, and use different reaction systems and procedures according to different experimental requirements.

[0047] (3) Enzyme digestion and ligation: Unless otherwise specified, the double DNA digestion and T4 ligase reaction system and reaction conditions shall be performed according to the Takara company website (https: / / www.takarabiomed.com.cn / ).

[0048] (4) Homologous recombination: The Gibson assembly reaction system and reaction conditions were performed according to the kit instructions.

[0049] (5) The heat shock method was used for the transformation of competent Escherichia coli: the transformation method was performed in accordance with the instructions.

[0050] (6) The transformation of competent cells of Saccharomyces cerevisiae was carried out using the lithium acetate / single-stranded DNA / polyethylene glycol method: the transformation method was carried out in accordance with the instructions.

[0051] (7) Strain culture and fermentation methods: Single colonies were picked from agar plates and inoculated into 5 mL of YPD / LB liquid medium. Yeast was cultured overnight in a constant temperature shaker at 30°C and a rotation speed of 220 rpm. Escherichia coli was cultured overnight in a constant temperature shaker at 37°C and a rotation speed of 200 rpm. For the expansion culture of Saccharomyces cerevisiae, 2% inoculum was transferred to fresh liquid YPD medium (with 20 g / L galactose added during fermentation) for expansion culture. Appropriate antibiotics were added according to the strain requirements.

[0052] (8) 5 L bioreactor fed-batch fermentation: Preparation of primary seed culture: Pick the corresponding single colony from the plate and inoculate it into 5 mL of YPD liquid medium (containing 200 μg / mL G418), and incubate overnight at 30℃ and 220 rpm.

[0053] Fermenter preparation: Activate dissolved oxygen electrode 24 h in advance; sterilize fermenter at 115℃ for 30 min, add 2 LYPD fermentation basal medium, and after sterilization and cooling, add 10 mL / L trace element solution and 12 mL / L vitamin solution, and add defoamer as needed.

[0054] Secondary seed culture: Take the primary seed culture and inoculate it at a 2% inoculum into two 100 mL bottles of YPD (containing 200 μg / mLG418) medium. Incubate at 30℃ and 220 rpm for 10-12 h until OD reaches the target value. 600 Achieve a score of 8-10 to obtain secondary seed solution.

[0055] Formal inoculation and fermentation: Introduce 200 mL of secondary seed culture into the fermenter and set the parameters as follows: temperature 30℃, initial aeration 1 vvm, stirring 100 rpm, and initial DO 100%; adjust the pH to 5.5 with 6 M ammonia water, and manually add defoamer if there is too much foam.

[0056] Sampling and feeding: OD measured after 14 h 600 Glucose content; the initial glucose feeding rate was 2 mL / h, adjusted according to the test results, and yeast extract (200 g / L) was added every 10 h for a total of 4 times; galactose was added in a timely and appropriate amount according to the liquid phase results.

[0057] Subsequent monitoring: Samples were taken every 2-4 hours during the feeding period. After the strain stabilized, the testing interval was extended until fermentation was completed.

[0058] The technical solution of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the reagents, materials and equipment used can be purchased commercially, prepared by conventional methods, or commonly used in the industry.

[0059] Example 1: Construction of the tagatose redox pathway (1) Screening of novel galactitol dehydrogenases: Based on literature review and screening of the UniProt database, a total of 10 oxidoreductase genes from different sources were obtained: namely Ss XR (UniProt ID: P31867), Sc XR (UniProt ID: P38715) An XR (UniProt ID: Q9P8R5), SgXR (UniProt ID: A0A173DUJ9), Ao XR (UniProtID: Q2UKD0) Rl GDH (UniProt ID: Q1MLL4), Rr GDH (UniProt ID: A0A546XM76), Ro GDH (UniProt ID: A0A7L5BLH5), As GDH (UniProt ID: A0A9X3ZA10), Af GDH (UniProt ID: A9CES4). These 10 genes were integrated into the PCEV-G1-Km plasmid and knocked out. GAL1 The gene was heterologously expressed in the engineered Saccharomyces cerevisiae strain YXCY-001. Fermentation with 20 g / L galactose as a substrate was performed for 48 h to verify the result. Figure 1 As shown. By comparing the tagatose production of different strains, it was found that strain YXCY-003 performed best, and its two key enzyme genes expressed heterologously originated from different microorganisms: xylose reductase gene. SsXR From Scheffersomyces stipitis (P31867), galactitol dehydrogenase gene RrGDH From Rhizobium radiobacter (A0A546XM76).

[0060] Codon optimization SsXR optThe nucleotide sequence is as shown in SEQ ID NO.1: ATGCCATCTATTAAATTGAATTCTGGTTATGATATGCCTGCTGTTGGTTTTGGTTGTTGGAAAGTTGATGTTGATACTTGCTCAGAACAAATTTATAGAGCTATTAAAACTGGTTATAGATTGTTTGATGGTGCTGAAGATTATGCTAATGAAAAATTGGTTGGAGCTGGTGTCAAAAAAGCTATTGACGAGGGTATCGTTAAAAGGGAAGACTTGTTTTTGACTTCTAAATTGTGGAATAACTACCATCATCCTGATAATGTTGAAAAAGCTTTGAATAGAACATTGTCTGATTTGCAAGTCGATTATGTCGATTTGTTTTTGATCCATTTTCCTGTTACTTTCAAATTTGTTCCATTGGAAGAAAAATATCCACCTGGTTTTTATTGTGGTAAAGGTGATAATTTTGATTATGAAGATGTTCCAATTTTGGAAACTTGGAAAGCTTTGGAGAAATTGGTCAAAGCTGGTAAAATTAGATCTATTGGTGTTTCTAATTTTCCTGGTGCTTTGTTATTGGATTTGTTGAGAGGTGCTACTATTAAACCATCTGTTTTGCAAGTCGAACATCATCCATATTTGCAACAACCAAGATTGATTGAATTTGCTCAATCTAGGGGTATTGCTGTTACTGCTTATTCTTCTTTTGGTCCACAATCTTTTGTTGAATTGAATCAAGGTAGAGCTTTGAATACTTCTCCATTGTTTGAAAATGAAACTATTAAAGCTATTGCTGCTAAACATGGTAAATCTCCTGCTCAAGTTTTGTTGAGATGGTCTTCTCAAAGAGGTATCGCTATTATCCCAAAATCTAATACTGTTCCAAGATTGTTGGAAAACAAGGATGTCAATTCTTTCGATTTGGATGAACAAGATTTTGCTGATATTGCTAAATTGGATATTAATTTGAGATTTAATGATCCATGGGATTGGGATAAAATTCCAATTTTTGTTTAA SsXR The amino acid sequence is as shown in SEQ ID NO.2: MPSIKLNSGYDMPAVGFGCWKVDVDTCSEQIYRAIKTGYRLFDGAEDYANEKLVGAGVKKAIDEGIVKREDLFLTSKLWNNYHHPDNVEKALNRTLSDLQVDYVDLFLIHFPVTFKFVPLEEKYPPGFYCGKGDNFDYEDVPILETWKALEKLVKAGKIRSIGVSNFPGALLLDLLRGATIKPSVLQVEHHPYLQQPRLIEFAQSRGIAVTAYSSFGPQSFVELNQGRALNTSPLFENETIKAIAAKHGKSPAQVLLRWSSQRGIAIIPKSNTVPRLLENKDVNSFDLDEQDFADIAKLDINLRFNDPWDWDKIPIFV* After codon optimization RrGDH optThe nucleotide sequence is shown in SEQ ID NO.3: ATGAGATTGAATAATAAAGTTGCTTTGATTACTGGTGCTGCTAGAGGTATTGGTTTGGGTTTTGCTCAAGCTTTTGCTGATGAAGGTGCTAAAGTTATTATTGCTGATATTGATATTGCTAGAGCTACTGCTTCTGCTGCAGCTATTGGTCCATCTGCTAAAGCTGTTAGATTGGATGTTACTGATTTGGCTCAAATTGATGCTGTTGTTAAAGCTGTTGATGAAGAATTTGGTGGTATTGATATTTTGGTTAATAATGCAGCTATTTTTGATATGGCTCCTATCAATGGTATTACTGAAGAATCTTATGAAAGGGTTTTCGACATCAATTTGAAAGGTCCATTGTTTATGATGAAAGCTGTTTCTAATGTTATGATTGAAAGAGCTAGAGGTGGTAAAATTATTAATATGGCTTCTCAAGCTGGTAGAAGAGGTGAAGCTTTGGTTACTTTGTATTGTGCTTCTAAAGCTGCTATTATTTCTGCTACTCAATCTGCTGCTTTGGCTTTGGTTAAACATGGTATTAATGTTAATGCTATTGCTCCTGGTGTTGTTGATGGTGAACATTGGGAAGTTGTTGATGCTCATTTTGCTAAATGGGAAGGTTTGAAACCTGGTGAAAAAAAAGCTGCTGTTGCTAAATCTGTTCCAATTGGTAGATTTGCTACTCCTGATGATATTAAAGGTTTGGCTGTTTTTTTGGCTTCTGCTGATTCTGATTATATTTTGGCTCAAACTTATAATGTTGATGGTGGTAATTGGATGTCTTAA RrGDH optThe amino acid sequence such as SEQ ID Shown in NO.4: MRLNNKVALITGAARGIGLGFAQAFADEGAKVIIADIDIARATASAAAIGPSAKAVRLDVTDLAQIDAVVKAVDEEFGGIDILVNNAAIFDMAPINGITEESYERVFDINLKGPLFMMKAVSNVM IERARGGKIINMASQAGRRGEALVTLYCASKAAIISATQSAALALVKHGINVNAIAPGVVDGEHWEVVDAHFAKWEGLKPGEKKAAVAKSVPIGRFATPDDIKGLAVFLASADSDYILAQTYNVDGGNWMS* Example 2: Screening for strong expression promoters in yeast (1) Promoter engineering: Five strong promoters of brewing yeast were screened. P HXT7 , P ACT1 , P TEF2 , P TDH1 , P TDH3 Promoter gene fragments were obtained by PCR amplification of the Saccharomyces cerevisiae genome, and the original promoter was modified by homologous recombinase. P PGK1 The recombinant plasmid was introduced into YXCY-001 and fermented in 20 ml of production medium at 30°C and 220 rpm for 48 h. The results are as follows. Figure 2 As shown, tagatose production varied significantly among strains with different promoter replacements, with the strain using the most effective replacements yielding the highest yields. P HXT7 The recombinant strain YXCY-011, constructed with the promoter, performed best. This strain achieved a tagatose yield of 3.88 g / L after 48 h, representing a 28.48% increase compared to the initial strain YXCY-003.

[0061] Example 3: Construction of a high-throughput screening mechanism based on tagatose fluorescence response (1) Construction of screening strains: P GAL Promoter and green fluorescent protein reporter gene GFP The strain was tandemly integrated into the Ph expression plasmid and transformed into the recipient bacterium BY4741, successfully constructing the screening strain YXCY-016. (2) Verification of the specificity of the screening mechanism: To eliminate the interference of other sugars in the fermentation system on the screening specificity, YXCY-016 was inoculated into four different culture media: YPD-D (20 g / L glucose), YPD-G (10 g / L glucose, 10 g / L galactose), YPD-GT (10 g / L glucose, 10 g / L galactitol), and YPD-T (10 g / L glucose, 10 g / L tagatose). After culturing at 30℃ and 220 rpm for 24 h, the fluorescence response was observed using an inverted fluorescence microscope. The results are as follows: Figure 3 As shown in Figure B, a significant fluorescence signal was observed only in the YPD-G medium containing galactose, while no obvious fluorescence response was observed in the other media, confirming that the screening method has good specificity and that other coexisting sugars in the system do not interfere with the screening results.

[0062] (3) Validation of the screening mechanism: The screened strain YXCY-020 was cultured for 48 h in a screening medium, a 48-well plate high-throughput culture system, at 30℃ and 220 rpm. The changes in its sugar content and fluorescence value were detected. The results are as follows: Figure 3 As shown in Figure C, in the early stage of fermentation, the glucose concentration is relatively high. P GAL The promoter is in a state of transcriptional repression, which drives... GFP No gene expression was observed, and the system showed no fluorescent pattern. As fermentation progressed, glucose was rapidly consumed, and galactose-specific induction occurred. P GAL Promoter activation, downstream GFP Gene expression begins, and the fluorescence signal gradually increases; subsequently, as galactose is continuously consumed and tagatose is synthesized, the fluorescence signal shows a decreasing trend.

[0063] (4) Optimization of the screening mechanism: [This will allow] devices carrying different sources to be screened. XR and GDH The Km expression plasmid of the gene was transformed into YXCY-016 to construct three model strains representing high, medium, and low gradients of tagatose synthesis capacity. A 48-well plate high-throughput culture system was used, with a series of galactose concentration gradients set at 30℃ and 220 rpm. FLU / OD ratios were measured after 48 h of culture. 600 The result is as follows Figure 3 As shown in Figure D, the FLU / OD ratios of the three model bacteria were observed when the galactose concentration was too low or too high. 600 The differences were not significant; however, within the 2-5 g / L galactose concentration range, the three strains exhibited significant fluorescence differences consistent with theoretical expectations, i.e., the higher the tagatose production, the higher the FLU / OD ratio. 600The lower the value, the better. Therefore, 5 g / L galactose was selected as the optimal substrate concentration for the YPD screening medium. In summary, a highly efficient and sensitive tagatose biosensor was successfully developed.

[0064] Example 4: Xylose reductase Ss XR molecular modification (1) Selection of saturation mutation sites: Select Ss Twenty-one non-conserved sites near the XR substrate and cofactor channels were selected as mutation targets, including nine sites near the substrate binding site (W20, D47, F111, F128, F221, L224, N306, D307, W311) and twelve sites near the cofactor binding site (C19, Q187, S215, F216, Q219, S220, E223, F236, A253, N272, R276, E279). An iterative saturation mutagenesis strategy was employed to target these sites, utilizing the degeneracy of the NNK codon to construct a small and efficient mutant library.

[0065] (2) High-throughput fluorescence screening: The mutated PCR product was introduced into the screening strain YXCY-016. After 48 h of plate culture, 200 strains from each site were selected and transferred to 48-well plates. The plates were cultured in 1 mL of screening medium at 30℃ and 220 rpm for 48 h. After centrifugation at 4000 rpm for 15 min, the supernatant was discarded, and the plates were washed twice with 900 μL of sterile water. The fluorescence value of the strains was detected using a microplate reader with an excitation wavelength of 480 nm and an emission wavelength of 510 nm. The absorbance was measured at 600 nm. The FLU / OD ratio was screened. 600 Positive strains with values ​​lower than the starting strain underwent a second round of screening, followed by shake-flask fermentation of the target strain, and product detection was performed using liquid chromatography. After fluorescence screening and sequencing verification, mutants at the F128, Q219, and E223 sites exhibited a significantly decreased fluorescence phenotype. Shake-flask fermentation was then performed on the above single mutants to verify the tagatose yield (…). Figure 4 (A) The tagatose yield of the F128M mutant was 93.08% higher than that of the control strain YXCY-020, 43.57% higher than that of the Q219K mutant, and 40.94% higher than that of the E223A mutant.

[0066] (3) Validation of positive strains: The three effective mutation sites mentioned above were combined and superimposed. To accurately determine the tagatose synthesis capacity of the mutants, the superimposed mutant plasmid was transformed into the high-efficiency production strain YXCY-001. The shake-flask fermentation results showed that the F128M-Q219K double mutant had the highest tagatose yield, reaching 5.39 g / L, which was 38.92% higher than that of the control strain YXCY-011. Figure 4 (B)

[0067] Example 5: Amino acid replenishment and optimization of fermentation conditions (1) Amino acid supplementation: Based on strain YXCY-001, methionine, uracil, and leucine genes were in situ supplemented to successfully construct a auxotrophic complementary strain YXCY-030; subsequently, the F128M-Q219K double mutant plasmid was transformed into YXCY-030 to obtain the recombinant strain YXCY-031. High-performance liquid chromatography (HPLC) analysis of the fermentation broth after 48 h yielded the following results: Figure 5 As shown in Figure A, the results indicate that the addition of the three amino acid genes significantly increased the tagatose synthesis yield. Among them, the tagatose yield of strain YXCY-031 reached 6.77 g / L, which is the highest yield obtained in this study to date. This yield is 25.53% higher than that of the control strain YXCY-027 without amino acid addition and 124.04% higher than that of the starting strain YXCY-003.

[0068] (2) Optimization of shake-flask fermentation time: Strain YXCY-031 was the strain with the optimal D-tagatose yield constructed in this study. Shake-flask fermentation was performed for 0-120 h. The culture medium was transferred to fresh liquid YPD at a 2% inoculum and cultured in a constant-temperature shaker at 220 rpm and 30℃. The results are as follows: Figure 5 As shown in Figure B: When fermentation reaches 120 h, the OD of the strain... 600 The value tends to level off, indicating that the strain has entered a stable growth phase; at this time, the D-tagatose yield reaches 10.77 g / L.

[0069] (3) High-density fed-batch fermentation: YXCY-031 was subjected to fed-batch fermentation in a 5 L fermenter, and the results are as follows. Figure 5 As shown in Figure C, the D-tagatose yield of the strain reached 44.61 g / L after 120 h of fermentation.

[0070] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A strain that efficiently synthesizes D-tagatose, characterized in that, The strain expresses xylose reductase in a Saccharomyces cerevisiae host. Ss XR and galactitol dehydrogenase Rr GDH.

2. The strain according to claim 1, characterized in that, xylose reductase Ss XR has the following amino acid sequence: (1) The amino acid sequence as shown in SEQ ID NO.2; or, (2) The amino acid sequence obtained by mutating phenylalanine at position 128 of the amino acid sequence shown in SEQ ID NO.2 to methionine; or, (3) The amino acid sequence obtained by mutating phenylalanine at position 128 of the amino acid sequence shown in SEQ ID NO.2 to methionine and glutamine at position 219 to lysine.

3. The strain according to claim 1, characterized in that, The galactitol dehydrogenase Rr The amino acid sequence of GDH is shown in SEQ ID NO.

4.

4. The strain according to claim 3, characterized in that, The galactitol dehydrogenase Rr GDH adopts P HXT7 Promoter startup expression.

5. The strain according to any one of claims 1-4, characterized in that, The Saccharomyces cerevisiae host is a knockout strain of Saccharomyces cerevisiae BY4741. GAL1 Genes, and replenish histidine genes.

6. The strain according to claim 5, characterized in that, The strain recombined the methionine, uracil, and leucine genes at their reduction sites.

7. A method for increasing the yield of D-tagatin in brewer's yeast, characterized in that, Includes the following steps: Xylose reductase expression in Saccharomyces cerevisiae host Ss XR and galactitol dehydrogenase Rr GDH, wherein the xylose reductase Ss XR has the following amino acid sequence: (1) The amino acid sequence as shown in SEQ ID NO.2; or, (2) The amino acid sequence obtained by mutating phenylalanine at position 128 of the amino acid sequence shown in SEQ ID NO.2 to methionine; or, (3) The amino acid sequence obtained by mutating phenylalanine at position 128 of the amino acid sequence shown in SEQ ID NO.2 to methionine and glutamine at position 219 to lysine; The galactitol dehydrogenase Rr The amino acid sequence of GDH is shown in SEQ ID NO.

4.

8. The use of the strain according to any one of claims 1-6 in the fermentation production of tagatose.

9. The application according to claim 8, characterized in that, The application involves inoculating the strain into a fermentation medium and fermenting it to prepare the tagatose. The fermentation medium contains 10-30 g / L glucose, 10-30 g / L galactose, 10-30 g / L tryptone, and 5-15 g / L yeast extract.

10. A high-throughput screening method based on tagatose fluorescence response, characterized in that, The method includes the following steps: S1, will P GAL Promoter and green fluorescent protein reporter gene GFP The strain was tandemly integrated into the Ph expression plasmid, transformed into the recipient bacterium BY4741, and a screening strain was constructed. S2. Introduce genes related to the tagatose metabolism pathway into the screened strains to construct strains capable of producing tagatose; S3. The tagatose-producing strain constructed in step S2 was cultured in YPD screening medium containing galactose, and high-throughput screening was performed by detecting the change in its fluorescence value relative to biomass.

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