Recombinant escherichia coli for producing d-mannose and preparation method therefor

Abstract

Provided are a recombinant Escherichia coli for producing D-mannose, and a preparation method therefor, belonging to the field of food chemical engineering. A recombinant strain is provided, which is a recombinant strain obtained by performing the following modifications A) and B) in Escherichia coli; A) inhibiting or reducing expression of the pfkA gene, pfkB gene, zwf gene and / or lpxM gene in the Escherichia coli; B) increasing the expression of a D-mannose-6-phosphate isomerase encoding gene, a phosphomannomutase encoding gene, a phosphomannose guanylyltransferase encoding gene, and a GDP-mannose hydrolase encoding gene in the Escherichia coli. The recombinant strain uses D-glucose, glycerol, etc. as carbon sources to support microbial growth, and can also grow by utilizing mixed carbon sources containing the described carbon source components. After microbial growth reaches a certain biomass, D-glucose is used as a raw material to synthesize D-mannose.

Description

Recombinant Escherichia coli for D-mannose production and its preparation method Technical Field

[0001] This invention belongs to the field of food chemical industry, and specifically relates to recombinant Escherichia coli for the production of D-mannose and its preparation method. Background Technology

[0002] D-Mannose, a simple reducing monosaccharide abundant in nature, is not only a key component of various polysaccharides and glycoproteins but is also increasingly becoming an emerging dietary supplement. Its applications span food, health products, skincare products, pharmaceuticals, and livestock farming. In the food and beverage industry, demand for D-Mannose as a natural sweetener and functional ingredient continues to rise. Consumer preferences for natural and healthy foods drive market demand for D-Mannose. Compared to sucrose and D-glucose, its sweetness reaches 60% and 86% respectively, while its low-calorie and non-toxic properties have led to its widespread use in the food, pharmaceutical, cosmetic, and food additive industries. D-Mannose can be used in beverages, pastries, candies, dairy products, and other foods to enhance sweetness, optimize taste, and provide functional properties. Current domestic research on the functions of D-Mannose mainly focuses on regulating the immune system, promoting wound healing, anti-inflammation, inhibiting tumor growth, and preventing bacterial infections, with a particularly large number of publications on its role in urinary tract infections.

[0003] There are three main traditional methods for producing D-mannose: extraction, chemical synthesis, and biological methods. Extraction utilizes diverse raw materials such as palm kernels, coffee grounds, acai berry seeds, and jujube powder to extract D-mannose, involving steps such as hydrolysis, filtration, separation, and purification. While this method stands out due to the availability of raw materials and high yields, it faces environmental pollution problems due to the use of high temperatures and strong acids and alkalis, and is also limited by geographical and seasonal factors. Chemical synthesis produces D-mannose through a molybdate-catalyzed D-glucose isomerization reaction, requiring temperatures of 100-150°C and a pH of approximately 3.0. Its challenges lie in the stringent reaction conditions, low conversion rates, and high difficulty in separation and purification, despite the stable availability of raw materials. Meanwhile, biological methods convert D-fructose or D-glucose into D-mannose through enzymatic reactions, demonstrating advantages such as stable raw material sources, mild reaction conditions, and lower costs. Although the mild conditions and lower costs of biological methods give them an advantage, their low conversion rates (20%–30%) and separation difficulties limit their further application. Invention Overview

[0004] The technical problem this invention aims to solve is to provide a recombinant strain that utilizes D-glucose, glycerol, and other raw materials to supply microbial growth and to prepare D-mannose from D-glucose. The recombinant strain provided by this invention can achieve microbial growth using a mixed carbon source containing glycerol and other components, and can also utilize a combination of carbon sources containing specific components of the aforementioned carbon sources. After the microorganisms reach a certain biomass, D-mannose is synthesized from D-glucose, thereby establishing a corresponding technical route for the synthesis of D-mannose. Technical solutions

[0005] The first aspect of the present invention provides a recombinant bacterium, which is a recombinant bacterium obtained by modifying Escherichia coli according to the following A) and B);

[0006] A) Inhibit or reduce the expression of the pfkA gene, pfkB gene, zwf gene and / or lpxM gene in the Escherichia coli;

[0007] B) Increase the expression of the genes encoding D-mannose 6-phosphate isomerase, mannose phosphate mutase, mannose phosphate guanylate transferase, and GDP-mannose hydrolase in the *E. coli*.

[0008] Among the recombinant bacteria mentioned above,

[0009] The modification A is any of the following:

[0010] A-1) Inhibit or reduce the expression of the pfkA, pfkB, zwf, and lpxM genes in the *E. coli*.

[0011] A-2) Inhibit or reduce the expression of the pfkA, pfkB and zwf genes in the *E. coli*.

[0012] A-3) Inhibit or reduce the expression of the pfkA and pfkB genes in the *E. coli*;

[0013] A-4) Inhibit or reduce the expression of the pfkA gene in the Escherichia coli.

[0014] In the recombinant bacteria mentioned above, the Escherichia coli is Escherichia coli MG1655.

[0015] The recombinant bacteria mentioned above have the accession number CGMCC No. 30520.

[0016] In a second aspect, the present invention provides a method for preparing the recombinant bacteria described in the first aspect, comprising the following steps: performing the modification as described in the first aspect.

[0017] In the above method, the modification A-1) is to knock out or replace the pfkA gene, pfkB gene, zwf gene and lpxM gene;

[0018] Alternatively, the modification A-2) may involve knocking out or replacing the pfkA, pfkB, and zwf genes;

[0019] Alternatively, the modification A-3) may involve knocking out or replacing the pfkA and pfkB genes;

[0020] Alternatively, the modification A-4) may involve knocking out or replacing the pfkA gene;

[0021] Alternatively, modification B) may involve introducing the D-mannose 6-phosphate isomerase encoding gene, the mannose phosphate mutase encoding gene, the mannose phosphate guanylate transferase encoding gene, and the GDP-mannose hydrolase encoding gene into the Escherichia coli.

[0022] In embodiments of the present invention, the method described above is any one of the following:

[0023] Modification A-1)+B specifically involves replacing the pfkA, pfkB, and zwf genes in the *E. coli* MG1655 genome with the fragment shown in sequence 1 (1414-6382) – the FRT site – Ptrc promoter – MCG sequence – TrrnB terminator, and replacing the lpxM gene with the FRT site shown in sequence 1 (1414-1447). The resulting recombinant bacterium is MS03.

[0024] Modification A-2)+B specifically involves replacing the pfkA, pfkB, and zwf genes on the *E. coli* MG1655 genome with the fragment shown in sequence 1 (1414-6382) – FRT site – Ptrc promoter – MCG sequence – TrrnB terminator. The resulting recombinant bacterium is MS02.

[0025] Modification A-3)+B specifically involves replacing both the pfkA and pfkB genes in the *E. coli* MG1655 genome with the fragment shown in sequence 1 (1414-6382) – FRT site – Ptrc promoter – MCG sequence – TrrnB terminator. The resulting recombinant bacterium is MS01.

[0026] Modification A-4)+B specifically involves replacing the pfkA gene in the E. coli MG1655 genome with a fragment shown in sequence 1 (1414-6382) that represents the FRT site-Ptrc promoter-MCG sequence-TrrnB terminator. The resulting recombinant bacterium is MS00.

[0027] Thirdly, the present invention provides recombinant bacteria prepared by the method described in the second aspect.

[0028] The recombinant bacteria mentioned above can be the bacteria with accession number CGMCC No.30520.

[0029] Fourthly, the present invention provides the application of the recombinant bacteria described in the first or third aspects in the preparation of whole-cell catalysts.

[0030] Fifthly, the present invention provides a whole-cell catalyst, which is prepared by a method comprising the following steps: culturing the recombinant bacteria described in the first or third aspect in a growth medium, followed by induction culture, collecting the bacterial cells, and obtaining the whole-cell catalyst;

[0031] The growth medium is a medium containing any one, two, three, or four of the following: glycerol, acetic acid, fatty acids, triglycerides, and D-glucose.

[0032] In the above text, the growth medium is specifically a glycerol growth medium.

[0033] In the above text, the induction culture refers to the addition of an inducing agent to the growth medium followed by continued culturing.

[0034] The aforementioned inducer is specifically IPTG.

[0035] The whole-cell catalyst mentioned above is prepared according to the following steps: the recombinant bacteria described in the first or third aspect are inoculated at a 1% inoculum in 200 ml of glycerol growth medium and cultured at 37°C until OD reaches 100%. 600nm After adding 0.8 to a final concentration of 0.5 mM IPTG, the cells were cultured at 37°C for 12 h, centrifuged at 8000g for 10 min to collect the whole-cell catalyst.

[0036] Sixthly, the present invention provides the use of the recombinant bacteria described in the first or third aspect or the whole-cell catalyst described in the fifth aspect in any of the following:

[0037] C1. Production of D-mannose;

[0038] C2, catalyzes the conversion of D-glucose to D-mannose;

[0039] C3. Preparation of products for producing D-mannose;

[0040] C4. Preparation of products that catalyze the conversion of D-glucose to D-mannose.

[0041] The above products are compositions, microbial agents, or other biological products.

[0042] In a seventh aspect, the present invention provides a composition which may be D1 or D2).

[0043] D1. Contains the recombinant bacteria described in the first or third aspect;

[0044] D2, containing the whole-cell catalyst described in the fifth aspect.

[0045] The active ingredient of the above composition may be the recombinant bacteria and / or the metabolites of the recombinant bacteria and / or the culture of the recombinant bacteria.

[0046] The culture can be a substance obtained by culturing the recombinant bacteria in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the recombinant bacteria and a substance secreted into a liquid culture medium, or a solid fermentation product containing the recombinant bacteria and a substance secreted into a solid culture medium).

[0047] In the above text, the metabolite may be a product obtained by removing the recombinant bacteria from the culture, such as culturing the recombinant bacteria in a liquid fermentation medium, collecting the fermentation broth (containing the recombinant bacteria and substances secreted into the liquid culture medium), removing the recombinant bacteria from the fermentation broth, collecting the remaining components of the fermentation broth, and obtaining the metabolite of the recombinant bacteria.

[0048] The active ingredients of the above composition may also contain other biological or non-biological components, and those skilled in the art can determine the other active ingredients of the above composition based on the effects of the composition.

[0049] The above composition may be the culture described above. The above composition may also be a microbial agent.

[0050] The aforementioned microbial agents refer to live microbial preparations made by using a carrier as an adsorbent to adsorb the fermentation broth or solid fermentation products of the target microorganisms after propagation.

[0051] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0052] Depending on the needs, the microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0053] Eighthly, the present invention provides a method for producing D-mannose, comprising the following steps: using D-glucose as a substrate, catalyzing a reaction with the recombinant bacteria described in the first or third aspect, the whole-cell catalyst described in the fifth aspect, or the composition described in the seventh aspect, to obtain D-mannose.

[0054] In the above-described method, before the recombinant bacteria described in the first or third aspect are used for catalytic reaction, the method further includes the following step: culturing the recombinant bacteria described in the first or third aspect in a growth medium and then inducing them, wherein the growth medium is a medium containing any one, two, three or four of glycerol, acetic acid, fatty acids, triglycerides and D-glucose.

[0055] In some embodiments of this application, the growth medium may be as follows:

[0056] 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4, 3% (g / 100mL) glycerol, 0.5% (g / 100mL) molasses, 0.5% (g / 100mL) gluten, trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3, balance water.

[0057] The inducing agent used in the above induction was IPTG. Beneficial effects

[0058] The production process of D-mannose using this invention significantly shortens the production cycle, laying a solid foundation for its widespread application and development.

[0059] Preservation Instructions

[0060] Bacterial species name: Escherichia coli

[0061] Latin name: Escherichia coli

[0062] Strain number: MS03

[0063] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0064] Collection institution abbreviation: CGMCC

[0065] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0066] Deposit date: May 7, 2024

[0067] Registered with the China Museum Collection Center (CGMCC) No. 30520

[0068] Classification and nomenclature: Escherichia coli Attached Figure Description

[0069] Figure 1 shows the HPLC detection results. Embodiments of the present invention

[0070] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0072] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0073] Escherichia coli MG1655 (CGSC#: 6300), plasmid pKD46 (CGSC#: 7739), pKD4 (CGSC#: 7682), and pCP20 (CGSC#: 7629) were purchased from the Escherichia coli Genetic Collection Center (CGSC) at Yale University, USA.

[0074] The *Escherichia coli* JW1176 was kindly provided by the Tao Yong group at the Institute of Microbiology, Chinese Academy of Sciences, and originated from the KEIO collection, as disclosed in the literature “Xiaoyu Piao, Lei Wang, Baixue Lin, Hao Chen, Weifeng Liu, Yong Tao. Metabolic engineering of *Escherichia coli* for production of L-aspartate and its derivative β-alanine with high stoichiometric yield. Metabolic Engineering. 2019 54:244-254.” The public can obtain the above-mentioned biological material from the applicant. The obtained biological material is only for repeating the experiments of this invention and may not be used for other purposes.

[0075] The primer sequences in the following examples are as follows:

[0076]

[0077]

[0078]

[0079] In this invention, the NCBI Reference Sequence number of 6-phosphofructokinase 1 (pfkA) is NP_418351.1 (09-MAR-2022), and the NCBI Reference Sequence number of its encoding gene is Gene ID: 948412 (02-May-2024).

[0080] In this invention, the NCBI Reference Sequence number of 6-phosphofructokinase 2 (pfkB) is NP_416237.3 (09-MAR-2022), and the NCBI Reference Sequence number of its encoding gene is Gene ID: 946230 (02-May-2024).

[0081] In this invention, the NCBI Reference Sequence number of glucose-6-phosphate dehydrogenase (zwf) is NP_416366.1 (09-MAR-2022), and the NCBI Reference Sequence number of its encoding gene is Gene ID: 946370 (02-May-2024).

[0082] In this invention, the NCBI Reference Sequence number of the lipid A biosynthesis myristyltransferase (lpxM) is NP_416369.1 (09-MAR-2022), and the NCBI Reference Sequence number of its encoding gene is Gene ID: 945143 (02-May-2024).

[0083] Unless otherwise specified, all substitutions in the embodiments are complete substitutions.

[0084] Example 1: Construction of recombinant Escherichia coli MS03

[0085] This embodiment prepared a basic bacterium, MS03, which can use glycerol as a carbon source for bacterial growth and can convert D-glucose into D-mannose. The preparation method of this strain is as follows, and the primers used in the preparation process are shown in Table 1.

[0086] 1. Construction of strain MS00

[0087] 1) Preparation of ManA-CpsG-CpsB-Gmm gene fragment

[0088] The genes for D-mannose 6-phosphate isomerase (ManA), phospmannose mutase (CpsG), phospmannose guanylate transferase (CpsB), and GDP-mannose hydrolase (Gmm) were artificially synthesized, and RBS were designed between adjacent genes to form a fragment MCG containing the sequences ManA, RBS, CpsG, RBS, CpsB, RBS, and Gmm in sequence. The nucleotide sequence of the entire MCG sequence is from position 1522 to 6042 of sequence 1. The whole sequence was synthesized by Suzhou Genewise Biotechnology Co., Ltd.

[0089] In sequence 1, positions 1522-2697 are ManA, positions 2705-2709 are RBS, positions 2717-4087 are CpsG, positions 4095-4099 are RBS, positions 4107-5543 are CpsB, positions 5551-5555 are RBS, and positions 5563-6042 are Gmm.

[0090] Using the MCG fragment (positions 1522-6042) in Sequence 1 as a template, PCR amplification was performed using primers CMG-1 / CMG-2 to obtain the amplified product MCG. Then, using primers Trc-1 / Trc-2 and the commercial plasmid pTrc-99A (Shanghai Zeye Biotechnology Co., Ltd., catalog number ZY-64292) as a template, PCR amplification was performed to obtain the vector fragment pTrc. The MCG fragment was constructed on pTrc using the Gibson assembly method, and the result was verified by sequencing to obtain the pTrc-MCG plasmid.

[0091] 2) Preparation of the pfkA site-targeting fragment

[0092] Using primers KD-BB-R and pfkA-F, PCR amplification was performed with plasmid pKD4 as a template. The target band of about 1500 bp was recovered, yielding fragment A1. Fragment A1 contains a kanamycin resistance gene fragment with FRT sites at both ends (positions 1-1447 in sequence 1, where FRT-1 is positions 21-54, the kanamycin resistance gene is positions 429-1223, and FRT-2 is positions 1414-1447).

[0093] Using primers KD-BB-F and pfkA-R, PCR amplification was performed with pTrc-MCG plasmid as a template. The target band of about 5000 bp was recovered, yielding fragment A2. The MCG gene cluster in fragment A2, regulated by the trc promoter and rrnB terminator (positions 1448-6382 in sequence 1, of which the trc promoter is position 1448-1521 and the rrnB terminator is position 6296-6382), is located in fragment A2.

[0094] Using primers pfkA-F and pfkA-R, overlap PCR amplification was performed using a mixture of A1 and A2 modules. A target band of approximately 4700 bp was recovered, yielding the target fragment KA. Fragment KA sequentially contains the upstream homologous arm of the pfkA gene, kanamycin resistance genes flanked by FRT sites, the Ptrc promoter, the MCG sequence, the TrrnB terminator, and the downstream homologous arm of the pfkA gene.

[0095] 3) The MCG fragment integrates into the pfkA site of MG1655.

[0096] Escherichia coli MG1655 was prepared using the CaCl2-MgCl2 method. The pKD46 plasmid was transformed into MG1655 and cultured overnight at 30 °C on LB agar plates containing 50 μg / mL ampicillin. Single colonies were picked and transferred at a 1% inoculum to 100 mL of liquid LB medium containing 50 μg / mL ampicillin. The culture was carried out at 30 °C with shaking at 220 rpm. The bacterial OD was measured periodically. 600 When the OD600 value is around 0.2, L-arabinose is added to the culture medium to a final concentration of 10 mM to induce full expression of λ-red recombinase protein on pKD46. Culture continues until the OD600 value reaches 0.2. 600 When the concentration reaches 0.4-0.6, stop culturing; prepare MG1655 electrocompetent cells. Then transform the KA targeting fragment prepared in step 2 into MG1655 electrocompetent cells, plate them on LB agar plates containing 50 μg / mL kanamycin, and incubate overnight at 37 °C until single colonies grow.

[0097] 4) Screening and identification of positive transformants

[0098] Single colonies from the overnight culture in step 3 were randomly selected and cultured at 37 °C. Positive recombinant bacteria were screened by PCR identification. Initial screening was performed using 2×Taq Mix from Ossab: using the bacterial culture as a template, amplification was performed using primers pfkA-U (Table 1) for the external sequence of the knockout site and primers PK for the internal sequence of the kanamycin resistance fragment used for the knockout selection marker. A 1200 bp target band was amplified as a positive transformant, which was used for further resistance elimination.

[0099] Competent cells were prepared from positive clones. The pCP20 plasmid was transferred into the competent cells and plated on LB agar plates containing 50 μg / mL ampicillin, incubated overnight at 30°C. Single clones were picked and cultured in LB liquid medium containing 50 μg / mL ampicillin at 37°C for 5 h, then streaked onto antibiotic-free LB agar plates and incubated overnight at 37°C. Single clones were picked and transferred to antibiotic-free LB agar and cultured at 37°C for 5 h. If the culture did not grow in LB agar containing 50 μg / mL kanamycin, further PCR identification was performed: PCR amplification of the bacterial culture was performed using primers (pfkA-U / pfkA-A). Single clones amplifying a target band of approximately 5000 bp were considered positive.

[0100] The positive clone with the resistant fragment eliminated was named recombinant bacteria MS00. This recombinant bacteria was obtained by replacing the pfkA gene on the genome of Escherichia coli MG1655 with the fragment shown in sequence 1 (1414-6382) which is the FRT site-Ptrc promoter-MCG sequence-TrrnB terminator. Other genes remained unchanged.

[0101] 2. Construction of strain MS01

[0102] The recombinant strain MS01 was obtained by replacing the pfkA and pfkB genes in the genome of Escherichia coli MG1655 with the fragment shown in sequence 1 (1414-6382) – FRT site – Ptrc promoter – MCG sequence – TrrnB terminator, while keeping other genes unchanged.

[0103] The preparation method of recombinant strain MS01 is basically the same as that of recombinant strain MS00 in section 1 above, except that MG1655 electrotransfer competent cells are replaced with MS00 electrotransfer competent cells.

[0104] The target fragment KA was replaced with KB. The method for constructing the target fragment KB was as follows: using primers KD-BB-R and pfkB-F, PCR amplification was performed using plasmid pKD4 as a template, and the target band of about 1500bp was recovered to obtain fragment B1.

[0105] Using primers KD-BB-F and pfkB-R, PCR amplification was performed with pTrc-MCG plasmid as a template. The target band of about 5000 bp was recovered, and fragment B2 was obtained.

[0106] Using primers pfkB-F and pfkB-R, overlap PCR amplification was performed with a mixture of B1 and B2 modules, yielding a target band of approximately 4700 bp. The target fragment KB was obtained. The KB fragment sequentially contains the upstream homologous arm of the pfkB gene, the kanamycin resistance gene flanked by FRT sites, the Ptrc promoter, the MCG sequence, the TrrnB terminator, and the downstream homologous arm of the pfkB gene.

[0107] The screening primers for positive transformants were pfkB-U (Table 1) and primer PK, yielding a 1200bp positive transformant. The primers for identifying the resistant positive monoclonal were (pfkB-U / pfkB-A), yielding a 5000bp positive monoclonal, namely MS01.

[0108] 3. Construction of strain MS02

[0109] The recombinant strain MS02 was obtained by replacing the pfkA, pfkB, and zwf genes in the genome of Escherichia coli MG1655 with the fragment shown in sequence 1 (1414-6382) – FRT site – Ptrc promoter – MCG sequence – TrrnB terminator, while keeping other genes unchanged.

[0110] The preparation method of recombinant strain MS02 is basically the same as that of recombinant strain MS00 in section 1 above, except that MG1655 electrotransfer competent cells are replaced with MS01 electrotransfer competent cells.

[0111] The target fragment KA was replaced with KZ. The method for constructing the target fragment KZ was as follows: using primers KD-BB-R and zwf-F, PCR amplification was performed using plasmid pKD4 as a template. The target band of about 1500bp was recovered, and fragment Z1 was obtained.

[0112] Using primers KD-BB-F and zwf-R, PCR amplification was performed with pTrc-MCG plasmid as a template. The target band of about 5000 bp was recovered, and fragment Z2 was obtained.

[0113] Using primers zwf-F and zwf-R, overlap PCR amplification was performed with a mixture of Z1 and Z2 modules, yielding a target band of approximately 4700 bp. The target fragment KZ was obtained. The KZ fragment sequentially contains the upstream homologous arm of the zwf gene, the kanamycin resistance gene flanked by FRT sites, the Ptrc promoter, the MCG sequence, the TrrnB terminator, and the downstream homologous arm of the zwf gene.

[0114] The selection primers for positive transformants were zwf-U (Table 1) and primer PK, yielding a 1200bp positive transformant. The primers for identifying the resistant positive monoclonal were (zwf-U / zwf-A), yielding a 5000bp positive monoclonal, which was identified as MS02.

[0115] 4. Construction of strain MS03

[0116] The recombinant strain MS03 was obtained by replacing the pfkA, pfkB, and zwf genes in the genome of Escherichia coli MG1655 with the fragment shown in sequence 1 (1414-6382) – the FRT site – Ptrc promoter – MCG sequence – TrrnB terminator, and replacing the lpxM gene with the FRT site shown in sequence 1 (1414-1447). All other genes remained unchanged.

[0117] The preparation method of recombinant strain MS03 is basically the same as that of recombinant strain MS00 in section 1 above, except that MG1655 electrotransfer competent cells are replaced with MS02 electrotransfer competent cells.

[0118] The target fragment KA was replaced with KL. The target fragment KL was amplified by PCR using primers lpxM-F and lpxM-R with plasmid pKD4 as a template. The target band of about 1500 bp was recovered, and fragment KL was obtained. The upstream homologous arm of the lpxM gene - the kanamycin resistance gene with FRT sites on both sides - the downstream homologous arm of the lpxM gene.

[0119] The selection primers for positive transformants were lpxM-U (Table 1) and primer PK, yielding a 1200bp positive transformant. The primers for identifying the resistant positive monoclonal were (lpxM-U / lpxM-A), yielding a 200bp positive monoclonal, which was identified as MS03.

[0120] The recombinant strain MS03 was deposited on May 7, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the accession number is CGMCC No. 30520, and the strain is classified as Escherichia coli.

[0121] Example 2: Application of recombinant Escherichia coli MS03 in the preparation of D-mannose using D-glucose as a substrate

[0122] 1. Components of glycerol growth medium

[0123] The components and final concentration of the growth medium are as follows:

[0124] 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4, 3% (g / 100mL) glycerol, 0.5% (g / 100mL) molasses, 0.5% (g / 100mL) gluten, trace elements: 50μM FeCl3, 20μM CaCl2, 10μM MnCl2, 10μM ZnSO4, 2μM each of CoCl2, NiCl2, Na2MO4, Na2SeO3 and H3BO3, balance water.

[0125] 2. Components of the transformation culture medium

[0126] 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM MgCl2, 12% (g / 100mL) D-glucose, balance water.

[0127] 3. Bacterial cell culture and enzyme induction

[0128] Wild-type strain MG1655, recombinant strain MS03, and control strains MG1655, MS00, MS01, and MS02, cultured overnight, were inoculated at a 1% inoculation rate into shake flasks containing 200 ml of glycerol growth medium and incubated at 37°C until OD500. 600nm After adding 0.8 to a final concentration of 0.5 mM IPTG, the cells were cultured at 37°C for 12 h, centrifuged at 8000g for 10 min to collect the whole-cell catalyst.

[0129] 4. Whole-cell catalysis of D-mannose

[0130] The 150 mg of collected bacterial cells obtained in step 3 were resuspended in shake flasks containing 10 mL of transformation medium and reacted at 37 °C and 220 rpm. The pH was monitored periodically using pH test paper and adjusted to 7.0 with 0.1 M NaOH and HCl solutions. After 24 h of reaction, the reaction product was centrifuged at 8000 g for 10 minutes, and the supernatant was filtered through a 0.22 μm filter membrane. The D-mannose content in the supernatant was determined by HPLC. The HPLC was performed using a Hi-Plex Ca column (300 mm × 7.7 mm, 8 μm); the mobile phase was ultrapure water; the flow rate was 0.5 mL / min; the column temperature was 78 °C; the detector was a differential refractive index detector; and the temperature was 40 °C.

[0131] D-mannose standard was purchased from Shanghai Yuanye Biotechnology (catalog number: S48276). An HLPC standard curve was constructed using the D-mannose standard, and the equation for D-mannose concentration was obtained (Figure 1A shows the standard curve, and Figure 1B shows the HPLC results of the standard).

[0132] The conversion rate is the ratio of D-mannose production to D-glucose consumption in the whole-cell catalytic stage (step 4) multiplied by 100%.

[0133] The results are shown in Table 2. The wild-type strain MG1655 produced zero D-mannose, while the yield and conversion rate of the engineered strains MS00, MS01, and MS02 gradually increased. The engineered strain MS03 produced 97.70 ± 3.56 g / L of D-mannose with a conversion rate of 81.42 ± 2.97%. This indicates that this method can achieve highly efficient whole-cell catalytic synthesis of D-mannose from D-glucose.

[0134]

[0135] Furthermore, the supernatant of the engineered strain MS03 showed high purity of D-mannose, and no other sugars were detected under the same HPLC conditions (Figure 1C), which helps to simplify the downstream separation and purification process.

[0136] 5. Compare the effects of different growth media on the culture of engineered strain MS03 and whole-cell catalysis of D-mannose.

[0137] Following steps 1-4 of Example 2 above, whole-cell catalysis of D-mannose was performed using the engineered strain MS03. The difference was that in step 3, in addition to using glycerol growth medium, growth medium 0 and growth medium 1 were used instead of glycerol growth medium. The yield and conversion rate of whole-cell catalysis of D-mannose were compared after using the above three growth media.

[0138] The composition of growth medium 0 is: based on glycerol growth medium, but without glycerol, with other components remaining unchanged. The composition of growth medium 1 is: based on glycerol growth medium, but without glycerol, and containing 2% (g / 100mL) acetic acid, 1% (g / 100mL) triglycerides, 0.01% (g / 100mL) lipase and 0.5% D-glucose, with other components remaining unchanged.

[0139] The results showed that the strain could not grow and the D-mannose yield was zero when using growth medium 0. When using growth medium 1, the D-mannose yield reached 84.53±5.20 g / L, and the conversion rate reached 70.44±4.33%.

[0140] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. Industrial applicability

[0141] This invention addresses the aforementioned challenges in the large-scale production of D-mannose by developing a novel D-mannose production route based on microbial fermentation. This method utilizes cheaper raw materials such as D-glucose, employing microbial fermentation to synthesize high-purity D-mannose in a single step. The D-mannose production process using this invention significantly shortens the production cycle, laying a solid foundation for its widespread application and development.

[0142] The recombinant strain provided by this invention can achieve microbial growth using a mixed carbon source containing components such as glycerol, and can also utilize a combination of carbon sources containing specific components of the aforementioned carbon sources. After the microorganisms reach a certain biomass, D-mannose is synthesized from D-glucose, thereby establishing a corresponding technical route for the synthesis of D-mannose.

Claims

1. Recombinant bacteria, which are recombinant bacteria obtained by modifying Escherichia coli according to the following A) and B); A) Inhibit or reduce the expression of the pfkA gene, pfkB gene, zwf gene and / or lpxM gene in the Escherichia coli; B) Increase the expression of the genes encoding D-mannose 6-phosphate isomerase, mannose phosphate mutase, mannose phosphate guanylate transferase, and GDP-mannose hydrolase in the *E. coli*.

2. The recombinant bacteria according to claim 1, characterized in that: The modification A is any of the following: A-1) Inhibit or reduce the expression of the pfkA, pfkB, zwf, and lpxM genes in the *E. coli*. A-2) Inhibit or reduce the expression of the pfkA, pfkB and zwf genes in the *E. coli*. A-3) Inhibit or reduce the expression of the pfkA and pfkB genes in the *E. coli*; A-4) Inhibit or reduce the expression of the pfkA gene in the Escherichia coli.

3. The recombinant bacteria according to claim 1 or 2, characterized in that: The Escherichia coli in question is Escherichia coli MG1655.

4. The recombinant bacteria according to any one of claims 1-3, characterized in that: The recombinant bacteria has the accession number CGMCC No. 30520.

5. A method for preparing the recombinant bacteria according to any one of claims 1-4, comprising the following steps: performing the modification according to any one of claims 1-3.

6. The method according to claim 5, characterized in that: The modification A-1) is to knock out or replace the pfkA gene, pfkB gene, zwf gene, and lpxM gene; Alternatively, the modification A-2) may involve knocking out or replacing the pfkA, pfkB, and zwf genes; Alternatively, the modification A-3) may involve knocking out or replacing the pfkA and pfkB genes; Alternatively, the modification A-4) may involve knocking out or replacing the pfkA gene; Alternatively, modification B) may involve introducing the D-mannose 6-phosphate isomerase encoding gene, the mannose phosphate mutase encoding gene, the mannose phosphate guanylate transferase encoding gene, and the GDP-mannose hydrolase encoding gene into the Escherichia coli.

7. Recombinant bacteria prepared by the method of claim 5 or 6.

8. The use of any of the recombinant bacteria according to claims 1-4 or the recombinant bacteria according to claim 7 in the preparation of whole-cell catalysts.

9. A whole-cell catalyst, prepared by a method comprising the following steps: culturing the recombinant bacteria of any one of claims 1-4 or the recombinant bacteria of claim 7 in a growth medium, followed by induction culture, collecting the bacterial cells, and obtaining the whole-cell catalyst; The growth medium is a medium containing any one, two, three, or four of the following: glycerol, acetic acid, fatty acids, triglycerides, and D-glucose.

10. The use of the recombinant bacteria of any one of claims 1-3, or the recombinant bacteria of claim 7, or the whole-cell catalyst of claim 9, in any of the following: C1. Production of D-mannose; C2, catalyzes the conversion of D-glucose to D-mannose; C3. Preparation of products for producing D-mannose; C4. Preparation of products that catalyze the conversion of D-glucose to D-mannose.

11. A composition comprising, as shown in D1) or D2). D1) Contains any one of the recombinant bacteria according to claims 1-4 or the recombinant bacteria according to claim 7; D2), containing the whole-cell catalyst as described in claim 9.

12. A method for producing D-mannose, comprising the following steps: using D-glucose as a substrate, catalyzing a reaction with any of the recombinant bacteria of claims 1-4 or the recombinant bacteria of claim 7 or the whole-cell catalyst of claim 9 or the composition of claim 11 to obtain D-mannose.

13. The method according to claim 12, characterized in that: The method further includes the following step before catalytic reaction using any of the recombinant bacteria of claims 1-4 or the recombinant bacteria of claim 7: culturing any of the recombinant bacteria of claims 1-4 or the recombinant bacteria of claim 7 in a growth medium and then inducing it, wherein the growth medium is a culture medium containing any one, two, three or four of glycerol, acetic acid, fatty acids, triglycerides and D-glucose.

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