Escherichia coli engineering bacteria for high-efficiency production of d-tagatose, and construction method and application thereof

Abstract

The present application relates to a kind of high-efficiency production D-tagatose engineering bacteria of escherichia coli and its construction method and application.The escherichia coli is with escherichia coli as starting strain, overexpresses fructose-6-phosphate-4 epimerase gene gatz, phosphoglycolate phosphatase gene pgp and lipoic acid protein ligase A gene lplA, simultaneously knock out fructose-6-phosphate kinase I gene pfkA and mannose-6-phosphate isomerase gene manA.This strain blocks glycolysis pathway and mannose metabolism, so that fructose-6-phosphate is accumulated in large quantities;While greatly strengthening the energy metabolism efficiency of downstream TCA cycle, provide sufficient energy support for the biosynthesis and correct folding of heterologous double-enzyme system, improve the conversion rate of D-tagatose.In using the engineering bacteria with glucose as substrate fermentation production D-tagatose, greatly improve the yield of D-tagatose, 24h The yield reaches 3.42g / L, conversion rate reaches 34.2%, provides important reference and guidance for the industrial synthesis of D-tagatose.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an engineered Escherichia coli strain that efficiently produces D-tagatose, its construction method, and its application. Background Technology

[0002] D-Tagatose is a rare, naturally occurring ketose (chemical formula C6H4H2O). 12 D-tagatose (O6) has been a research hotspot in the food, pharmaceutical, and cosmetic fields since its discovery in the 1990s due to its unique low calorie content (1.5 kcal / g, only 38% of sucrose), slow glycemic index (low GI value), and antioxidant and probiotic-promoting functions. In the food industry, D-tagatose has obtained GRAS certification from the US FDA and sweetener approval from the EU, and is widely used in low-sugar beverages, functional health products, and baked goods. In the pharmaceutical field, it can regulate gut microbiota, inhibit gluconeogenesis, assist in diabetes treatment, and has the potential to serve as a drug carrier. In cosmetics, its antioxidant properties can alleviate skin oxidative damage. However, the feasibility of natural extraction of D-tagatose is low, and its large-scale application still faces multiple technical bottlenecks. D-tagatose exists only in trace amounts in dairy products and fruits (such as apples and pineapples) in nature, and direct extraction is costly and inefficient. Current industrial production mainly relies on biological methods, using lactose as a raw material. Lactose is hydrolyzed by β-galactosidase to produce D-glucose and D-galactose. Subsequently, D-galactose is isomerizes with L-arabinose isomerase (L-AI) to produce D-tagatose. This method suffers from high raw material costs, low utilization rates, high production costs, low yields, and requires multiple conversions or complex product separations. The short lifespan of enzymes increases enzyme consumption costs, and the complex separation process further drives up the overall cost.

[0003] Despite significant progress in the optimization of enzyme catalysis processes in recent years—for example, obtaining L-AI mutants through directed evolution (such as the D390V / V468L double mutant derived from Lactobacillus fermentum, which extends the half-life to 72 hours at 40°C and increases enzyme activity by 36.68%), and employing multi-enzyme cascade self-assembly systems (such as the SpyTag / SpyCatcher system, which increases the conversion rate of fructose to tagatose to 75%), the core bottleneck in tagatose biosynthesis has not yet been fundamentally overcome.

[0004] Specifically: Regarding substrate specificity, natural L-AI enzymes generally exhibit low catalytic specificity for D-galactose, with only about 38% of L-AIs showing high selectivity for D-galactose. Furthermore, most enzymes require high temperatures of 60–75°C to maintain activity, highlighting the persistent problem of insufficient thermostability. In terms of byproduct formation, glucose byproducts from the lactose pathway limit conversion rates (below 50%), while multi-enzyme cascade systems face the problem of intermediate product inhibition (e.g., D-glucose inhibits β-galactosidase activity by up to 40%).

[0005] Therefore, there is an urgent need to develop an efficient D-tagatose production process to overcome existing technological bottlenecks and meet the rapidly growing demand for functional rare sugars in the food, pharmaceutical, and cosmetic industries. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an engineered Escherichia coli strain for the efficient production of D-tagatose, its construction method, and its application.

[0007] The technical solution of the present invention is as follows:

[0008] A highly efficient engineered Escherichia coli strain for producing D-tagatose is described, wherein the engineered Escherichia coli strain is based on Escherichia coli strain, overexpressing the fructose-6-phosphate-4 epimerase gene gatz, the phosphate glycolate phosphatase gene pgp, and the lipoic acid protein ligase A gene lplA, while simultaneously knocking out the fructose-6-phosphate kinase I gene pfkA and the mannose-6-phosphate isomerase gene manA.

[0009] According to a preferred embodiment of the present invention, the fructose-6-phosphate-4 epimerase gene gatz is derived from Agrobacterium rhizogenes, and its nucleotide sequence is shown in SEQ ID NO.1.

[0010] According to a preferred embodiment of the present invention, the phosphoglycolate phosphatase gene pgp is derived from Archaeococcus, and its nucleotide sequence is shown in SEQ ID NO.2.

[0011] According to a preferred embodiment of the present invention, the phospholipoic acid protein ligase A gene lplA is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.3.

[0012] According to a preferred embodiment of the present invention, the nucleotide sequence of the fructose-6-phosphokinase I gene pfkA is shown in SEQ ID NO. 4.

[0013] According to a preferred embodiment of the present invention, the nucleotide sequence of the mannose-6-phosphate isomerase gene manA is shown in SEQ ID NO. 5.

[0014] The above-mentioned method for constructing the engineered Escherichia coli strain for efficient D-tagatose production includes the following steps:

[0015] (1) Using the Escherichia coli genome as a template, PCR amplification was performed to obtain the upstream and downstream homologous arms of the gene pfkA; then, the upstream and downstream homologous arms of the gene pfkA were ligated by overlap extension PCR to obtain the pfkA-Donor DNA fragment.

[0016] (2) Using the Escherichia coli genome as a template, PCR amplification was performed to obtain the upstream and downstream homologous arms of the gene manA; then, the upstream and downstream homologous arms of the gene manA were connected by overlap extension PCR to obtain the manA-Donor DNA fragment.

[0017] (3) Design the CRISPR target N20 sequence and ligate it into the BsaI-HF digested plasmid vector pEcgRNA to obtain the recombinant plasmid pEcgRNA-N20.

[0018] (4) The pEcCas plasmid was transformed into E. coli BL21(DE3) to obtain E. coli expressing Cas9 protein; the pfkA-Donor DNA fragment and the recombinant plasmid pEcgRNA-N20 were electroporated into E. coli expressing Cas9 protein to obtain engineered bacteria ΔpfkA with pfkA gene knocked out; the manA-Donor DNA fragment and the recombinant plasmid pEcgRNA-N20 were electroporated into engineered bacteria ΔpfkA / pEcCas with pfkA gene knocked out to obtain engineered bacteria ΔpfkA-ΔmanA with pfkA gene knocked out and manA gene knocked out.

[0019] (5) Insert the gene gatz and the gene pgp into the plasmid vector pRSF-Duet1 to obtain the recombinant plasmid pRSF-Duet1-gatz-pgp;

[0020] (6) Insert the gene lplA into the plasmid vector pCD-Duet1 to obtain the recombinant plasmid pCD-Duet1-lplA;

[0021] (7) The recombinant plasmid pRSF-Duet1-gatz-pgp and recombinant plasmid pCD-Duet1-lplA were sequentially transformed into the engineered bacteria ΔpfkA-ΔmanA with the pfkA gene and manA gene knocked out. Positive recombinants were selected to obtain the engineered Escherichia coli that produces D-tagatose efficiently.

[0022] According to a preferred embodiment of the present invention, in step (1), pfkA-frw / pfkA-rev and pfkA-olfrw / pfkA-olrev are used as primers to amplify the upstream and downstream homologous arms of the gene pfkA.

[0023] The sequence is as follows:

[0024] pfkA-frw: 5′-ctgtttgcattgggaatcggcatctatattttatatagcgcgttacgc-3′,

[0025] pfkA-rev:5′-gcttcaataccgttgattaccgacgtcgcttgtttacct-3′;

[0026] pfkA-olfrw: 5′-aagttcagaggtagtctgatttcggaaaaaggcagattcctttaccc-3′,

[0027] pfkA-olrev: 5′-ctgcctttttccgaaatcagactacctctgaactttggaatgcaa-3′.

[0028] According to a preferred embodiment of the present invention, in step (2), manA-frw / manA-rev and manA-olfrw / manA-olrev are used as primers to amplify the upstream and downstream homologous arms of the gene pfkA.

[0029] The sequence is as follows:

[0030] manA-frw: 5′-CGACTCTGCTCGGAGAGGCCGTTGCCAAA-3′,

[0031] manA-rev: 5′-CTGTCAGCATTTAACTGGAACTCGCCGCCGCTAAAG-3′;

[0032] manA-olfrw: 5′-ACCGCACGCTTACGTAGCGGTAGGCGTCATTGTT-3′,

[0033] manA-olrev: 5′-GACGCCTACCGCTACGTAAGCGTGCGGTGTTTCAG-3′.

[0034] According to a preferred embodiment of the present invention, in step (3), the CRISPR target N20 sequence is: 5′-TTCGACGCAGAACAAAATGG-3′.

[0035] According to a preferred embodiment of the present invention, in steps (3), (5), and (6), the genes gatz, pgp, and lplA are all artificially synthesized by Qingke Biotechnology Co., Ltd. according to the sequence information.

[0036] The application of the above-mentioned engineered Escherichia coli strain in the production of D-tagatose.

[0037] A method for producing D-tagatose includes the following steps:

[0038] The engineered *E. coli* strain that efficiently produces D-tagatose was activated and then inoculated into TB liquid medium at a volume percentage of 1-3%, and cultured at 35-40℃ and 200-220 rpm until OD500 was reached. 600 The concentration of the culture medium was 0.6-0.8 to obtain the fermentation broth. Glucose, isopropyl-β-D-thiogalactoside and MgSO2 were added to the fermentation broth, and the culture was induced at 25-30℃ and 200-220 rpm for 6-24 h. Then, the culture was centrifuged at 10000-12000 rpm for 10-15 min, the supernatant was collected, filtered and purified to obtain D-tagatose.

[0039] According to a preferred embodiment of the present invention, the activation refers to: inoculating the frozen, high-efficiency D-tagatose-producing engineered Escherichia coli into LB selective medium containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin, and incubating at 35-40°C with constant temperature shaking at 200-220 rpm for 10-12 h; wherein the formulation of the LB selective medium is: 1% tryptone, 0.5% yeast extract, 1% NaCl, and the pH is adjusted to 7.5 with NaOH.

[0040] According to a preferred embodiment of the present invention, the formulation of the TB liquid culture medium is as follows: 1.2% tryptone, 2.4% yeast extract, 0.94% K2HPO4, 0.22% KH2PO4, and 0.4% glycerol; the concentration of glucose is 8-12 g / L, the concentration of isopropyl-β-D-thiogalactoside is 0.4-0.8 mM, and the concentration of MgSO2 is 3-7 mM.

[0041] Technical features and beneficial effects of the present invention:

[0042] 1. This invention uses *Escherichia coli* as the starting strain, overexpressing the fructose-6-phosphate-4 epimerase gene *gatz*, the phosphate glycolate phosphatase gene *pgp*, and the lipoic acid protein ligase A gene *lplA*, while simultaneously knocking out the fructose-6-phosphate kinase I gene *pfkA* and the mannose-6-phosphate isomerase gene *manA*, to construct a novel, highly efficient *E. coli* engineered strain, *E. coli*-ΔPM-GPL. This engineered *E. coli* strain blocks the glycolysis pathway and mannose metabolism, resulting in a large accumulation of fructose-6-phosphate; it also significantly enhances the energy metabolism efficiency of the downstream TCA cycle, providing sufficient energy support for the biosynthesis and correct folding of the heterologous dual-enzyme system, thus improving the conversion rate of D-tagatose.

[0043] 2. The *E. coli* engineered strain *E. coli*-ΔPM-GPL*, constructed in this invention, successfully established a metabolic pathway of "glucose → glucose-6-phosphate → fructose-6-phosphate → tagatose-6-phosphate → D-tagatose" and exhibited good growth. Therefore, when using this engineered strain to ferment D-tagatose using glucose (10 g / L) as a substrate, the yield of D-tagatose was significantly increased, reaching 3.42 g / L in 24 hours with a conversion rate of 34.2%, providing important reference and guidance for the industrial synthesis of D-tagatose. Attached Figure Description

[0044] Figure 1 This is a route diagram for the synthesis of D-tagatose.

[0045] Figure 2 This is a plasmid map of the recombinant plasmid pRSF-duet1-gatz-pgp.

[0046] Figure 3 This is the plasmid map of the recombinant plasmid pCD-duet1-lplA.

[0047] Figure 4 Shake-flask fermentation curves of engineered strain E. coli-ΔPM-GPL in different culture media;

[0048] In the diagram, A represents LB liquid culture medium, and B represents TB liquid culture medium.

[0049] Figure 5 The effects of induction temperature and IPTG concentration on the catalytic synthesis of D-tagatose from E. coli-ΔPM-GPL;

[0050] In the figure, A represents the effect of induction temperature, and B represents the effect of IPTG concentration.

[0051] Figure 6 The effect of metal ion concentration on the synthesis of D-tagatose catalyzed by E. coli-ΔPM-GPL.

[0052] Figure 7 The curves show cell growth, substrate consumption, and product accumulation during shake-flask fermentation of the engineered strain E. coli-ΔPM-GPL under optimal conditions. Detailed Implementation

[0053] The technical solution of the present invention will be further described below with reference to the embodiments and accompanying drawings, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the reagents and medicines involved in the embodiments are all commercially available products; unless otherwise specified, the experimental operations involved in the embodiments are all conventional operations in the art.

[0054] This invention achieves the biosynthesis and industrial production of D-tagatose by constructing a metabolic pathway in *E. coli* that follows the sequence "glucose → glucose-6-phosphate → fructose-6-phosphate → tagatose-6-phosphate → tagatose," specifically as follows: Figure 1 As shown.

[0055] The following are the key enzyme systems in this metabolic pathway:

[0056] The glucose phosphotransferase system (PTS system) transports glucose into the cell and phosphorylates it to glucose-6-phosphate.

[0057] Glucose-6-phosphate isomerase (GPI) catalyzes the conversion of glucose-6-phosphate (G6P) to fructose-6-phosphate (F6P).

[0058] Fructose-6-phosphate kinase I (pfkA) catalyzes the conversion of fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (FDP), and participates in the glycolysis metabolic pathway. Knocking out pfkA can lead to the blockage of this glycolysis process.

[0059] Mannose-6-phosphate isomerase (manA) catalyzes the conversion of fructose-6-phosphate to mannose-6-phosphate and participates in the mannose metabolic pathway. Knocking out the manA gene blocks this pathway.

[0060] When the glycolysis and mannose metabolic pathways are blocked, fructose-6-phosphate can accumulate in the cell and be used to synthesize D-tagatose.

[0061] Fructose-6-phosphate-4 epimerase (Gatz) catalyzes the isomerization of fructose-6-phosphate to tagatose-6-phosphate, and overexpression can improve its conversion efficiency.

[0062] Phosphoglycolate phosphatase (Pgp) catalyzes the dephosphorylation of tagatose-6-phosphate to produce D-tagatose, and overexpression can improve its conversion efficiency.

[0063] In the examples, the formulation of TB liquid culture medium was: 1.2% tryptone, 2.4% yeast extract, 0.94% K2HPO4, 0.22% KH2PO4, and 0.4% glycerol.

[0064] Example 1: Construction of Escherichia coli genetically engineered bacteria with pfkA and manA genes knocked out

[0065] The pfkA and manA genes in the engineered E. coli BL21 (DE3) were knocked out using CRISPR / Cas9 technology, resulting in the engineered E. coli E. coli-BL21 (DE3)-ΔpfkA-ΔmanA with the pfkA and manA genes knocked out.

[0066] 1. Select a 500 bp fragment from the pfkA gene as the knockout region, and use SnapGene software to design upstream and downstream homologous arms of the pfkA gene, each approximately 500 bp in length, as well as corresponding validation primers.

[0067] Using E. coli BL21(DE3) genomic DNA as a template, and pfkA-frw / pfkA-rev and pfkA-olfrw / pfkA-olrev as primers, the upstream and downstream homologous arms of the pfkA gene were obtained by PCR amplification.

[0068] The primer sequences are as follows:

[0069] pfkA-frw: 5′-ctgtttgcattgggaatcggcatctatattttatatagcgcgttacgc-3′,

[0070] pfkA-rev:5′-gcttcaataccgttgattaccgacgtcgcttgtttacct-3′;

[0071] pfkA-olfrw: 5′-aagttcagaggtagtctgatttcggaaaaaggcagattcctttaccc-3′,

[0072] pfkA-olrev: 5′-ctgcctttttccgaaatcagactacctctgaactttggaatgcaa-3′.

[0073] The PCR amplification system was as follows: total volume 50 μL, 2 μL upstream primer, 2 μL downstream primer, 2 μL template, 25 μL 2×phanta enzyme, and 19 μL ddH2O.

[0074] The PCR amplification conditions were: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s; 67℃ annealing for 5s; 72℃ extension for 5s; 32 cycles; and 72℃ complete extension for 1min.

[0075] Then, using the upstream and downstream homologous arms of the gene pfkA as templates, and pfkA-olfrw / pfkA-rev as primers, the two were ligated by overlap extension PCR to obtain the pfkA-Donor DNA fragment.

[0076] The overlap extension PCR amplification system was as follows: total volume 50 μL, 2 μL upstream primer, 2 μL downstream primer, 2 μL template 1, 2 μL template 2, 25 μL 2×phanta enzyme, and 17 μL ddH2O.

[0077] The conditions for overlap extension PCR amplification were: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s; 67℃ annealing for 30s; 72℃ extension for 10s; 32 cycles; and 72℃ complete extension for 1min.

[0078] 2. Select a 500 bp fragment in gene manA as the knockout region, and use SnapGene software to design upstream and downstream homologous arms of gene manA, each about 500 bp in length, and corresponding verification primers.

[0079] Using E. coli BL21(DE3) genomic DNA as a template, and manA-frw / manA-rev and manA-olfrw / manA-olrev as primers, the upstream and downstream homologous arms of the manA gene were obtained by PCR amplification.

[0080] The primer sequences are as follows:

[0081] manA-frw: 5′-CGACTCTGCTCGGAGAGGCCGTTGCCAAA-3′,

[0082] manA-rev: 5′-CTGTCAGCATTTAACTGGAACTCGCCGCCGCTAAAG-3′;

[0083] manA-olfrw: 5′-ACCGCACGCTTACGTAGCGGTAGGCGTCATTGTT-3′,

[0084] manA-olrev: 5′-GACGCCTACCGCTACGTAAGCGTGCGGTGTTTCAG-3′.

[0085] The PCR amplification system was as follows: total volume 50 μL, 2 μL upstream primer, 2 μL downstream primer, 2 μL template, 25 μL 2×phanta enzyme, and 19 μL ddH2O.

[0086] The PCR amplification conditions were: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s; 67℃ annealing for 5s; 72℃ extension for 5s; 32 cycles; and 72℃ complete extension for 1min.

[0087] Then, using the upstream and downstream homologous arms of the gene manA as templates, and manA-olfrw / manA-rev as primers, the two were ligated by overlap extension PCR to obtain the manA-Donor DNA fragment.

[0088] The overlap extension PCR amplification system was as follows: total volume 50 μL, 2 μL upstream primer, 2 μL downstream primer, 2 μL template 1, 2 μL template 2, 25 μL 2×phanta enzyme, and 17 μL ddH2O.

[0089] The conditions for overlap extension PCR amplification were: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s; 67℃ annealing for 30s; 72℃ extension for 10s; 32 cycles; and 72℃ complete extension for 1min.

[0090] 3. The CRISPR target N20 sequence was designed using CHOPCHOP, and then a double linker was formed by annealing. This linker was then ligated to a linearized pEcgRNA vector with the ccdB virulence gene removed by BsaI-HF restriction enzyme digestion, and the recombinant plasmid pEcgRNA-N20 was successfully constructed.

[0091] The CRISPR target N20 sequence is: 5′-TTCGACGCAGAACAAAATGG-3′.

[0092] 4. The pEcCas plasmid was transformed into E. coli BL21(DE3) to obtain E. coli expressing the Cas9 protein; the pfkA-Donor DNA fragment and the recombinant plasmid pEcgRNA-N20 were co-electroplated into competent E. coli cells expressing the Cas9 protein to obtain engineered bacteria ΔpfkA with the pfkA gene knocked out; the manA-Donor DNA fragment and the recombinant plasmid pEcgRNA-N20 were then co-electroplated into engineered bacteria ΔpfkA / pEcCas competent cells with the pfkA gene knocked out. After revival, the cells were plated on LB solid medium containing spectinomycin and kanamycin. Positive clones were verified by colony PCR and sequencing to obtain engineered E. coli ...

[0093] Example 2: Construction of an engineered Escherichia coli strain for efficient D-tagatose production

[0094] 1. Referring to the sequence characteristics of the fructose-6-phosphate-4 epimerase gene gatz and the phosphate glycolate phosphatase gene pgp, according to... Figure 2 The recombinant plasmid pRSF-Duet1-gatz-pgp was constructed using the plasmid structure shown.

[0095] The fructose-6-phosphate-4 epimerase gene gatz (SEQ ID NO.1) and phosphoglycolate phosphatase gene pgp (SEQ ID NO.2) were artificially synthesized by Qingke Biotechnology Co., Ltd. Then, using these as templates and gatz-YF / R and pgp-YF / R as primers, respectively, PCR amplification was performed to obtain gatz and pgp gene sequence fragments.

[0096] The primer sequences are as follows:

[0097] gatz-YF: 5′-CACAGCCAGGATCCGATGAGCACTCTGC-3′,

[0098] gatZ-YR: 5′-GCGGCCGCAAGCTTTTAACCCAGCAGAGTAG-3′;

[0099] pgp-YF: 5′-AGAAGGAGATATACATATGTTCAAAGCGCTGGTGGTTG-3′,

[0100] pgp-YR: 5′-TTCTTTACCAGACTCGAGGATCAAGCCCAGAAATTC-3′.

[0101] The PCR amplification system was as follows: total volume 20 μL, 1 μL upstream primer, 1 μL downstream primer, 1 μL template, 10 μL 2×phanta enzyme, and 7 μL ddH2O.

[0102] PCR amplification conditions were as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 5-10 s, 32 cycles, followed by 72℃ final extension for 1 min.

[0103] The plasmid vector pRSF-Duet1 was double-digested at the BamhI and HindIII restriction sites and the NdeI and XhoI restriction sites. The digested plasmid vector pRSF-Duet1, gatz and pgp gene sequence fragments were mixed evenly at a molar ratio of 1:1:1, and ligated with T4 DNA ligase to obtain the recombinant plasmid pRSF-Duet1-gatz-pgp.

[0104] 2. Based on the sequence characteristics of the alpha-lipoic acid protein ligase A gene lplA, ... Figure 3 The recombinant plasmid pCD-Duet1-lplA was constructed using the plasmid structure shown.

[0105] The lipoic acid protein ligase A gene lplA (SEQ ID NO.3) and phosphoglycolate phosphatase gene pgp (SEQ ID NO.2) were artificially synthesized by Qingke Biotechnology Co., Ltd. Then, using them as templates and lplA-YF / R as primers, PCR amplification was performed to obtain the lplA gene sequence fragment.

[0106] The primer sequences are as follows:

[0107] lplA-YF:5′-ATGTCTACTCTGCGTCTGCTGATCTCCGACAGC-3′;

[0108] lplA-YR: 5′-TTAACGCACAGCACCTGCGATCCAGGTGCTCA-3′.

[0109] The PCR amplification system was as follows: total volume 20 μL, 1 μL upstream primer, 1 μL downstream primer, 1 μL template, 10 μL 2×phanta enzyme, and 7 μL ddH2O.

[0110] The PCR amplification conditions were as follows: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 5 s, for 32 cycles, followed by 72℃ final extension for 1 min.

[0111] The EcoRI and HindIII restriction sites of the plasmid vector pCD-Duet1 were cut. The digested plasmid vector pCD-Duet1 and the lplA gene sequence fragment were mixed evenly at a 1:1 molar ratio, and T4 DNA ligase was added for ligation to obtain the recombinant plasmid pCD-Duet1-lplA.

[0112] 3. The recombinant plasmid pRSF-Duet1-gatz-pgp was transformed into the E. coli-ΔPM competent cells constructed in Example 1 to obtain E. coli-BL21(DE3)-ΔpfkA-ΔmanA / pRSFDuet-gatz-pgp; then the recombinant plasmid pCD-Duet1-lplA was transformed into E. coli-BL21(DE3)-ΔpfkA-ΔmanA / pRSFDuet-gatz-pgp competent cells. After revival, the cells were plated on LB solid medium containing spectinomycin and kanamycin. Positive clones were verified by colony PCR and sequencing to obtain the highly efficient D-tagatose-producing engineered E. coli strain, abbreviated as E. coli-ΔPM-GPL.

[0113] Example 3: Method for producing D-tagatin

[0114] 1. Optimization of culture medium

[0115] The *E. coli*-ΔPM-GPL constructed in Example 3 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin, and cultured at 37°C and 220 rpm with constant temperature shaking for 12 hours to complete activation. The activated engineered bacteria were then inoculated into LB liquid medium at a volume percentage of 2%, and glucose was added to a final concentration of 10 g / L. The medium was then cultured at 37°C and 220 rpm for 24 hours. The fermentation curve is shown below. Figure 4 As shown in Figure A.

[0116] Replace LB liquid medium with TB liquid medium, and the fermentation curve is as follows. Figure 4 As shown in B.

[0117] Depend on Figure 4 A indicates that, in LB liquid medium, the OD of the engineered strain E. coli-ΔPM-GPL was [value missing] after 24 hours. 600 The titer was 3.25, and the D-tagatose titer reached 1.15 g / L. However, due to the low nutrient level and lack of effective pH buffering capacity of the LB liquid medium, the growth of the engineered bacteria slowed down significantly after 16 h, and 6.00 g / L of glucose remained unutilized in the system after 24 h.

[0118] Depend on Figure 4 As shown in Figure B, in TB liquid medium, the engineered strain E. coli-ΔPM-GPL exhibited better growth and catalytic performance, and the LplA-mediated enhancement of energy metabolism was fully demonstrated. At the end of 24 h fermentation, the OD of the engineered strain E. coli-ΔPM-GPL... 600With the concentration increased to 18.50, glucose in the system was further utilized, with a residual concentration of only 0.50 g / L, and D-tagatose production increased to 2.75 g / L. In TB liquid medium with better nutritional and buffering conditions, the engineered strain E. coli-ΔPM-GPL could effectively coordinate cell growth, substrate consumption, and target product synthesis, thus exhibiting superior overall fermentation performance. This indicates that TB liquid medium can provide a more favorable culture environment for LplA-mediated energy metabolism enhancement, thereby alleviating the growth limitation problem of double-knockout engineered strains to some extent.

[0119] 2. Optimization of induction temperature and IPTG concentration

[0120] The E. coli-ΔPM-GPL constructed in Example 3 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin, and cultured at 37°C and 220 rpm with constant temperature shaking for 12 hours to complete activation. The activated engineered bacteria were then inoculated into LB liquid medium at a volume percentage of 2% and cultured at 37°C and 220 rpm until OD... 600 The concentration of the culture medium was 0.6-0.8 to obtain the fermentation broth. Glucose at a final concentration of 10 g / L and isopropyl-β-D-thiogalactoside at concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mM were added to the fermentation broth. The mixture was then induced and cultured for 24 h at 200 rpm and temperatures of 25, 28, 30, 33, and 37 °C. The mixture was then centrifuged at 12000 rpm for 10 min, and the supernatant was collected. After purification by filtration through a 0.22 μm aqueous microporous membrane, D-tagatose was obtained.

[0121] Two mL samples were aseptically collected from different culture conditions and analyzed by HPLC. The tagatose yield was calculated, and the results are as follows: Figure 5 As shown.

[0122] The HPLC conditions were as follows: calcium column (300×7.7 mm), mobile phase (ultrapure water), column temperature 80℃, flow rate 0.6 mL / min, time 20 min, and injection volume 10 μL.

[0123] Depend on Figure 5 It was found that the accumulation of D-tagatose was highest during induction within the temperature range of 25℃ to 30℃; when the temperature exceeded 30℃, the yield of D-tagatose decreased significantly. Then, the yield of D-tagatose peaked at an IPTG concentration of 0.6 mM; when the concentration exceeded 0.6 mM, the yield of D-tagatose declined. Therefore, the optimal conditions for induction fermentation were culture at 30℃ with the addition of 0.6 mM IPTG.

[0124] 4. Optimization of metal ion concentration

[0125] The E. coli-ΔPM-GPL constructed in Example 3 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin, and cultured at 37°C and 220 rpm with constant temperature shaking for 12 hours to complete activation. The activated engineered bacteria were then inoculated into LB liquid medium at a volume percentage of 2% and cultured at 37°C and 220 rpm until OD... 600 The concentration was set at 0.6-0.8 to obtain the fermentation broth. Glucose (final concentration 10 g / L), 0.6 mM isopropyl-β-D-thiogalactoside, and MgSO4 at concentrations of 1, 3, 5, 7, and 10 mM were added to the fermentation broth. The mixture was induced and cultured at 200 rpm and 30 °C for 24 h. Then, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. After purification by filtration through a 0.22 μm aqueous microporous membrane, D-tagatose was obtained.

[0126] Two mL samples were aseptically collected from different culture conditions and analyzed by HPLC (HPLC conditions as above). The tagatose yield was calculated, and the results are as follows. Figure 6 As shown.

[0127] Depend on Figure 6 It can be seen that the yield of D-tagatose reaches its maximum when the magnesium ion concentration is 5 mM. Therefore, the optimal metal ion condition is determined to be 5 mM Mg. 2+ .

[0128] 4. A method for producing D-tagatose, comprising the following steps:

[0129] (1) The E. coli-ΔPM-GPL constructed in Example 3 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin and cultured at 37℃ and 220 rpm for 12 hours to complete the activation;

[0130] (2) The activated engineered bacteria were inoculated into TB liquid medium at a volume percentage of 2% and cultured at 37°C and 220 rpm until OD. 600 The concentration was 0.6~0.8, resulting in the fermentation broth;

[0131] (3) Add glucose (10 g / L), isopropyl-β-D-thiogalactoside (IPTG, 0.6 mM), and MgSO4 (5 mM MgSO4) to the fermentation broth. 2+ The D-tagatose was induced and cultured at 30℃ and 200rpm for 24h, then centrifuged at 12000rpm for 10min, the supernatant was collected, and purified by filtration through a 0.22μm aqueous microporous membrane to obtain D-tagatose.

[0132] During the induction expression and product fermentation synthesis stages, 2 mL samples were aseptically taken at 0 h, 4 h, 8 h, 16 h, and 24 h. The OD values ​​of the samples at 0 h, 4 h, and 8 h were measured using a UV spectrophotometer. 600 This study was used to evaluate the shake-flask fermentation level of the engineered strain E. coli-ΔPM-GPL; samples at 16 h and 24 h were analyzed by HPLC to calculate tagatose yield and conversion rate; results of cell growth, substrate consumption, and product accumulation throughout the process are as follows: Figure 7 As shown.

[0133] The HPLC conditions were as follows: calcium column (300×7.7 mm), mobile phase (ultrapure water), column temperature 80℃, flow rate 0.6 mL / min, time 20 min, and injection volume 10 μL.

[0134] Depend on Figure 7 It can be seen that after culturing under optimal conditions for 24 hours, the OD of the engineered strain E. coli-ΔPM-GPL... 600 The concentration reached 18.50 g / L, with a residual glucose concentration of only 0.50 g / L and a D-tagatose concentration increased to 3.42 g / L. Based on this, the conversion rate reached 34.2%.

[0135] In summary, this invention uses *E. coli* as the starting strain, overexpressing the fructose-6-phosphate-4 epimerase gene *gatz*, the phosphate glycolate phosphatase gene *pgp*, and the lipoic acid protein ligase A gene *lplA*, while simultaneously knocking out the fructose-6-phosphate kinase I gene *pfkA* and the mannose-6-phosphate isomerase gene *manA*, to construct a novel, highly efficient *E. coli* engineered strain, *E. coli*-ΔPM-GPL. This engineered *E. coli* strain blocks the glycolysis pathway and mannose metabolism, resulting in a large accumulation of fructose-6-phosphate; it also significantly enhances the energy metabolism efficiency of the downstream TCA cycle, providing sufficient energy support for the biosynthesis and correct folding of the heterologous dual-enzyme system, thus improving the conversion rate of D-tagatose.

Claims

1. An engineered Escherichia coli strain that efficiently produces D-tagatose, characterized in that, The engineered Escherichia coli strain is based on Escherichia coli, which overexpresses the fructose-6-phosphate-4 epimerase gene gatz, the phosphate glycolate phosphatase gene pgp, and the lipoic acid protein ligase A gene lplA, while knocking out the fructose-6-phosphate kinase I gene pfkA and the mannose-6-phosphate isomerase gene manA.

2. The engineered Escherichia coli strain as described in claim 1, characterized in that, The fructose-6-phosphate-4 epimerase gene gatz is derived from Agrobacterium rhizogenes, and its nucleotide sequence is shown in SEQ ID NO.1; the phosphate glycolate phosphatase gene pgp is derived from Archaeococcus, and its nucleotide sequence is shown in SEQ ID NO.2; the phospholipoic acid protein ligase A gene lplA is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.

3.

3. The engineered Escherichia coli strain as described in claim 1, characterized in that, The nucleotide sequence of the fructose-6-phosphokinase I gene pfkA is shown in SEQ ID NO.4; the nucleotide sequence of the mannose-6-phosphoisomerase gene manA is shown in SEQ ID NO.

5.

4. The method for constructing the engineered Escherichia coli strain for efficient D-tagatose production as described in claim 1, characterized in that, Includes the following steps: (1) Using the Escherichia coli genome as a template, PCR amplification was performed to obtain the upstream and downstream homologous arms of the gene pfkA; then, the upstream and downstream homologous arms of the gene pfkA were connected by overlap extension PCR to obtain the pfkA-Donor DNA fragment. (2) Using the Escherichia coli genome as a template, PCR amplification was performed to obtain the upstream and downstream homologous arms of the gene manA; then, the upstream and downstream homologous arms of the gene manA were connected by overlap extension PCR to obtain the manA-Donor DNA fragment. (3) Design the CRISPR target N20 sequence and ligate it into the BsaI-HF digested plasmid vector pEcgRNA to obtain the recombinant plasmid pEcgRNA-N20. (4) The pEcCas plasmid was transformed into E. coli BL21(DE3) to obtain E. coli expressing Cas9 protein; the pfkA-Donor DNA fragment and the recombinant plasmid pEcgRNA-N20 were electroporated into E. coli expressing Cas9 protein to obtain engineered bacteria ΔpfkA with pfkA gene knocked out; the manA-Donor DNA fragment and the recombinant plasmid pEcgRNA-N20 were electroporated into engineered bacteria ΔpfkA / pEcCas with pfkA gene knocked out to obtain engineered bacteria ΔpfkA-ΔmanA with pfkA gene knocked out and manA gene knocked out. (5) Insert the gene gatz and the gene pgp into the plasmid vector pRSF-Duet1 to obtain the recombinant plasmid pRSF-Duet1-gatz-pgp; (6) Insert the gene lplA into the plasmid vector pCD-Duet1 to obtain the recombinant plasmid pCD-Duet1-lplA; (7) The recombinant plasmid pRSF-Duet1-gatz-pgp and recombinant plasmid pCD-Duet1-lplA were sequentially transformed into the engineered bacteria ΔpfkA-ΔmanA with the pfkA gene and manA gene knocked out. Positive recombinants were selected to obtain the engineered Escherichia coli that produces D-tagatose efficiently.

5. The construction method as described in claim 4, characterized in that, In step (1), pfkA-frw / pfkA-rev and pfkA-olfrw / pfkA-olrev are used as primers to amplify the upstream and downstream homologous arms of the gene pfkA. The sequence is as follows: pfkA-frw: 5′-ctgtttgcattgggaatcggcatctatattttatatagcgcgttacgc-3′, pfkA-rev:5′-gcttcaataccgttgattaccgacgtcgcttgtttacct-3′; pfkA-olfrw: 5′-aagttcagaggtagtctgatttcggaaaaaggcagattcctttaccc-3′, pfkA-olrev: 5′-ctgcctttttccgaaatcagactacctctgaactttggaatgcaa-3′.

6. The construction method as described in claim 4, characterized in that, In step (2), the upstream and downstream homologous arms of the gene pfkA are amplified using manA-frw / manA-rev and manA-olfrw / manA-olrev as primers. The sequence is as follows: manA-frw: 5′-CGACTCTGCTCGGAGAGGCCGTTGCCAAA-3′, manA-rev: 5′-CTGTCAGCATTTAACTGGAACTCGCCGCCGCTAAAG-3′; manA-olfrw: 5′-ACCGCACGCTTACGTAGCGGTAGGCGTCATTGTT-3′, manA-olrev: 5′-GACGCCTACCGCTACGTAAGCGTGCGGTGTTTCAG-3′.

7. The construction method as described in claim 4, characterized in that, In step (3), the CRISPR target N20 sequence is: 5′-TTCGACGCAGAACAAAATGG-3′.

8. The use of the engineered Escherichia coli strain described in claim 1 in the production of D-tagatose.

9. A method for producing D-tagatose, characterized in that, The steps include the following: The engineered *E. coli* strain that efficiently produces D-tagatose was activated and then inoculated into TB liquid medium at a volume percentage of 1-3%, and cultured at 35-40℃ and 200-220 rpm until OD500 was reached. 600 The concentration of glucose was 0.6-0.8 to obtain the fermentation broth. Glucose, isopropyl-β-D-thiogalactoside and MgSO2 were added to the fermentation broth to a final concentration of 8-12 g / L. The mixture was induced and cultured at 25-30℃ and 200-220 rpm for 6-24 h. Then, the mixture was centrifuged at 10000-12000 rpm for 10-15 min, and the supernatant was collected. After filtration and purification, D-tagatose was obtained.

10. The production method as described in claim 9, characterized in that, The activation refers to: inoculating the frozen, high-efficiency D-tagatose-producing Escherichia coli engineered bacteria into LB selective medium containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin, and incubating at 35-40℃ and 200-220 rpm for 10-12 h with constant temperature shaking; wherein, the formulation of LB selective medium is: 1% tryptone, 0.5% yeast extract, 1% NaCl, and the pH is adjusted to 7.5 with NaOH; The formulation of the TB liquid culture medium is as follows: 1.2% tryptone, 2.4% yeast extract, 0.94% K2HPO4, 0.22% KH2PO4, and 0.4% glycerol; The concentration of glucose is 8-12 g / L, the concentration of isopropyl-β-D-thiogalactoside is 0.4-0.8 mM, and the concentration of MgSO2 is 3-7 mM.

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