Double-enzyme expression plasmid, double-enzyme expression recombinant strain and method for co-producing mannitol and gluconic acid through whole-cell catalysis

Abstract

The invention provides a double-enzyme expression plasmid, a double-enzyme expression recombinant strain and a method for co-producing mannitol and gluconic acid through whole-cell catalysis, the plasmid co-expresses heat-resistant glucose dehydrogenase and heat-resistant mannitol dehydrogenase, the plasmid is transferred into escherichia coli engineering bacteria to obtain the double-enzyme expression recombinant strain, glucose is used as a raw material, and glucose and gluconic acid are co-produced through whole-cell catalysis. Under the action of glucose isomerase, the mannitol and gluconic acid are catalytically produced by using a whole-cell biological catalytic system consisting of co-expressed mannitol dehydrogenase and glucose dehydrogenase by using a double-enzyme expression recombinant strain. The method adopts a brand new catalytic path, has the advantages of low production cost, low pollution, high yield and the like, selects high-temperature source enzyme, is high in reaction rate and high in substrate conversion rate, is a new preparation path for co-production of mannitol and gluconic acid, and is suitable for large-scale production of mannitol and gluconic acid.

Description

Technical Field

[0001] This invention relates to the field of mannitol and gluconic acid co-production technology, and in particular to a dual-enzyme expression plasmid, a dual-enzyme expression recombinant strain, and a whole-cell catalytic method for co-producing mannitol and gluconic acid. Background Technology

[0002] Mannitol (D-Mannitol) is a naturally occurring hexose alcohol widely used in the food, pharmaceutical, chemical, and feed industries. Currently, the main production methods for mannitol include natural extraction, chemical synthesis, and bioconversion. Extracting mannitol from seaweed is a commercial method for small-scale mannitol production in China, but this process suffers from high water and energy consumption. The main industrial method for mannitol production involves the chemical hydrogenation of a mixture of approximately 50% fructose and 50% glucose, using Raney nickel as a catalyst and hydrogen as a reducing agent. Glucose is converted to sorbitol, while fructose is converted to a mixture of mannitol and sorbitol. This method suffers from extreme reaction conditions, complex purification steps, and low product yield. In recent years, bioconversion, with its advantages of high selectivity and low energy consumption, has attracted widespread attention for mannitol production. Many microorganisms in nature can synthesize mannitol from carbohydrates, such as yeast, fungi, and lactic acid bacteria. In the bio-fermentation method for mannitol production, glucose is converted into inexpensive acetic acid and lactic acid, while simultaneously providing energy for the conversion of fructose to mannitol. However, the production of mannitol by microbial fermentation has disadvantages such as long growth cycle, many by-products and complex culture medium composition. This necessitates the development of new strategies to produce mannitol in a simpler and more efficient way.

[0003] Currently, numerous studies have explored mannitol production using whole-cell catalysis or multi-enzyme synergistic catalysis systems. However, cofactors remain a major limiting factor in mannitol yield. In 1981, Wichmann et al. proposed a two-enzyme system in which mannitol dehydrogenase and formate dehydrogenase are coupled, converting fructose and formic acid into mannitol and CO2, respectively, achieving continuous regeneration of cofactors. In 2004, Kaup et al. developed a whole-cell catalytic system for converting fructose to mannitol in *E. coli* using genetic engineering methods. This strain expressed mannitol-2-dehydrogenase from *L. pseudodomesenteroides* and formate dehydrogenase from *Mycobacterium vaccae*. This recombinant strain was able to produce 362 mM mannitol within 8 hours, with a yield of 84 mol%. In 2005, Kaup et al. added extracellular glucose isomerase or co-expressed glucose isomerase to the aforementioned recombinant *E. coli* strain, enabling the cells to synthesize D-mannitol using D-glucose as a substrate. However, this method uses formic acid or formate as one of the substrates and produces carbon dioxide, which is not only detrimental to the environment but also wastes raw materials.

[0004] Glucoic acid is a naturally occurring organic acid with wide applications in various fields. Currently, the main methods for producing gluconic acid both domestically and internationally include: chemical oxidation, electrolytic oxidation, bio-fermentation, and enzymatic conversion. Chemical oxidation and electrolytic oxidation both use glucose as raw material, producing gluconic acid through sodium hypochlorite or electrolytic oxidation. Both methods suffer from drawbacks such as high production costs and cumbersome industrial processes. Bio-fermentation utilizes the oxidation process of bacteria and fungi to oxidize glucose into gluconic acid. Currently, the most widely used method in industry is the fermentation of *Aspergillus niger* to produce gluconic acid. Pande et al. achieved a conversion rate of 87% and a selectivity of 38% after 20 hours at 33℃ and pH = 6. However, microbial fermentation suffers from long production cycles, numerous byproducts, and complex wastewater treatment. Enzymatic production of gluconic acid mainly utilizes the coupled reaction of glucose oxidase and catalase, offering advantages such as mild reaction conditions and a single product. However, the H₂O₂ produced during glucose oxidation and decomposition can damage the enzymes.

[0005] Currently, there are two main pathways for the co-production of mannitol and gluconic acid. One is to use glucose and fructose as raw materials and achieve co-production of the two through the catalysis of mannitol dehydrogenase and glucose dehydrogenase. In 2022, the team led by Rao Zhiming of Jiangnan University (Pan Shan, Hu Mengkai, Pan Xuewei, et al. Efficient catalytic synthesis of D-mannitol based on dual-enzyme cascade coordinated expression strategy. Chinese Journal of Biotechnology, 2022, 38(07): 2549-2565.) constructed a whole-cell catalytic system that co-expresses glucose dehydrogenase from Bacillus amyloliquefaciens and mannitol dehydrogenase from Leuconostoc mesenteroides. Using glucose and fructose in equal proportions as substrates, mannitol and gluconic acid were co-produced. The highest yield of D-mannitol was 81.9 g / L in a 5L fermenter at 30℃ for 24 h. Another method, invented in 2015 by Zhao Yong's team at the Dalian Institute of Chemical Physics (CN106811488A), uses sucrose as a raw material to produce glucose and fructose via sucrase. Glucose and fructose are then co-produced into mannitol and gluconic acid by glucose dehydrogenase and mannitol dehydrogenase, respectively. In a 1L reaction system, using 100g / L sucrose as the substrate, 0.3U / mL sucrase, 1U / mL glucose dehydrogenase and mannitol dehydrogenase, 100mM NAD+ and NADH, and 0.6M calcium chloride were added. The reaction was carried out at 30℃ for 12 hours, yielding 39g / L mannitol and 31g calcium gluconate. However, both of these methods suffer from high raw material costs and the use of room-temperature enzymes.

[0006] Therefore, there is an urgent need to develop a new method for the large-scale co-production of mannitol and gluconic acid that is low-cost and high-yield. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a dual-enzyme expression plasmid.

[0008] Another technical problem to be solved by the present invention is to provide a recombinant strain for dual-enzyme expression obtained from the above-mentioned dual-enzyme expression plasmid.

[0009] Another technical problem to be solved by the present invention is to provide a method for the whole-cell catalytic co-production of mannitol and gluconic acid using the above-mentioned dual-enzyme expression recombinant strain.

[0010] The technical solution adopted in this invention is:

[0011] A dual-enzyme expression plasmid that co-expresses the mannitol dehydrogenase gene and the glucose dehydrogenase gene on the same plasmid.

[0012] In some technical solutions of the present invention, the dual-enzyme expression plasmid comprises a first promoter, a mannitol dehydrogenase gene, a first terminator, a second promoter, a glucose dehydrogenase gene, and a second terminator; or comprises a first promoter, a glucose dehydrogenase gene, a first terminator, a second promoter, a mannitol dehydrogenase gene, and a second terminator; or comprises a first promoter, a mannitol dehydrogenase gene, a second promoter, a glucose dehydrogenase gene, and a terminator; or comprises a first promoter, a glucose dehydrogenase gene, a second promoter, a mannitol dehydrogenase gene, and a terminator; preferably, the promoter is a T7 promoter, and the terminator is a T7 terminator. The above dual-enzyme expression plasmid is transformed into engineered Escherichia coli to obtain recombinant strains, and recombinant strain expression is induced.

[0013] A recombinant strain expressing two enzymes was obtained by transforming the above-mentioned two-enzyme expression plasmid into engineered Escherichia coli.

[0014] Preferably, the above-mentioned dual-enzyme expression recombinant strain is Escherichia coli expression strain BL21(DE3).

[0015] A whole-cell catalytic co-production method for mannitol and gluconic acid is disclosed, which uses glucose as raw material, applies the above-mentioned dual-enzyme expression recombinant strain under the action of glucose isomerase, and utilizes a whole-cell biocatalytic system composed of co-expressed mannitol dehydrogenase and glucose dehydrogenase to catalyze the production of mannitol and gluconic acid.

[0016] Preferably, in the above-described whole-cell catalytic co-production method of mannitol and gluconic acid, the dual-enzyme expression recombinant strain, along with glucose isomerase and glucose, undergoes a whole-cell catalytic reaction.

[0017] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid, glucose is used as a single substrate. Under the action of glucose isomerase, some glucose is converted into fructose. Then, the dual-enzyme expression recombinant strain uses glucose and fructose as substrates to carry out the reaction. That is, the dual-enzyme expression recombinant strain carries out the whole-cell catalytic reaction together with glucose and fructose.

[0018] The above-described whole-cell catalytic co-production method of mannitol and gluconic acid can utilize mannitol dehydrogenase, glucose dehydrogenase, and glucose isomerase from various sources.

[0019] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid, the mannitol dehydrogenase is derived from *Thermotoga maritima*, *Thermotoganeapolitana*, or *Thermoanaerobacterium thermosaccharolyticum*.

[0020] Preferably, in the above-mentioned whole-cell catalytic co-production method of mannitol and gluconic acid, the mannitol dehydrogenase is derived from *Thermophyton floccosum*.

[0021] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid, the glucose dehydrogenase is derived from Sulfolobus solfataricus, Thermoplasma acidophilum, or Sulfolobus tokodaii.

[0022] Preferably, in the above-described whole-cell catalytic co-production method of mannitol and gluconic acid, the glucose dehydrogenase is derived from *Leymus sulphureus*.

[0023] Preferably, in the above-described whole-cell catalytic co-production method of mannitol and gluconic acid, the glucose isomerase is derived from *Thermus thermophilus*, *Thermoanaerobacter ethanolicus*, or *Streptomyces flavogriseus*.

[0024] Preferably, in the above-described whole-cell catalytic co-production method of mannitol and gluconic acid, the glucose isomerase is derived from thermophilic bacteria.

[0025] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid method, the glucose isomerase is added by means of nickel column purification or by means of permeation treatment.

[0026] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid method, the glucose isomerase and the dual-enzyme expression recombinant strain are added sequentially to establish a whole-cell catalytic reaction system.

[0027] Preferably, in the above-described whole-cell catalytic co-production method for mannitol and gluconic acid, the concentration of glucose is 2-600 g / L.

[0028] Preferably, in the above-described whole-cell catalytic co-production method for mannitol and gluconic acid, the concentration of glucose is 10-400 g / L.

[0029] Preferably, in the above-described whole-cell catalytic co-production method for mannitol and gluconic acid, the concentration of glucose is 100-300 g / L.

[0030] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid, the temperature of the reaction system is 40-90℃.

[0031] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid, the temperature of the reaction system is 50-80℃.

[0032] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid, the temperature of the reaction system is 60-70℃.

[0033] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid, the cell volume of the dual-enzyme expression recombinant strain is 0.001-0.3 g DCW / mL.

[0034] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid method, the cell volume of the dual-enzyme expression recombinant strain is 0.005-0.2 g DCW / mL.

[0035] Preferably, in the above-mentioned whole-cell catalytic co-production of mannitol and gluconic acid, the cell volume of the dual-enzyme expression recombinant strain is 0.01-0.05 g DCW / mL.

[0036] The beneficial effects of this invention are:

[0037] The aforementioned dual-enzyme expression plasmid co-expresses thermostable glucose dehydrogenase and thermostable mannitol dehydrogenase. The plasmid is then transformed into engineered *E. coli* bacteria to obtain a recombinant strain expressing both enzymes. A whole-cell catalytic method for the co-production of mannitol and gluconic acid is employed. Using glucose as a raw material, and under the action of glucose isomerase, the recombinant strain expressing both enzymes utilizes a whole-cell biocatalytic system composed of co-expressed mannitol dehydrogenase and glucose dehydrogenase to catalyze the production of mannitol and gluconic acid. This method employs a novel catalytic pathway, offering advantages such as low production cost, low pollution, high yield, and high efficiency. The use of enzymes derived from high temperatures results in a fast reaction rate and high substrate conversion rate, making it a novel preparation route for the co-production of mannitol and gluconic acid, suitable for large-scale production. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the pathway for the co-production of mannitol and gluconic acid by the whole cell; where: XI is glucose isomerase, GDH is glucose dehydrogenase, and MDH is mannitol dehydrogenase.

[0039] Figure 2 This is a graph showing the reaction process of whole-cell co-production of mannitol and gluconic acid using glucose as a substrate at high substrate concentrations. Detailed Implementation

[0040] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0041] Example 1

[0042] Construction of a whole-cell catalytic system with co-expression of two enzymes

[0043] Four dual-enzyme expression plasmids containing glucose dehydrogenase and mannitol dehydrogenase genes were constructed. The constructed prokaryotic expression plasmids were then transformed into engineered *E. coli* bacteria to obtain recombinant strains, and expression was induced in the recombinant strains.

[0044] In this embodiment, glucose dehydrogenase was derived from *Sulfolobus solfataricus*, with the gene number AJ012093.1 on NCBI; mannitol dehydrogenase was derived from *Thermotoga maritima*, with the gene number TM0298 on KEGG. Both gene DNAs are available from the ATCC official website (www.atcc.org).

[0045] Construction of plasmid pET28a-TmMDH: Using a pair of primers, pET28a-MDH-IF: 5'-GTTTAACTTTAAGAAGGAGATATACCATGAAAGTACTTTTGATAGAAAAACCCGG-3' and pET28a-MDH-IR: 5'-GATCTCAGTGGTGGTGGTGGTGGTGAGAAAAAATTCCCTTCATCAATGCC-3', plasmids were extracted from Thermotoga by PCR. The mdh gene (KEGG: TM0298) was obtained from the maritima genome. Using a pair of primers, pET-28a-MDH-VF: 5'-CATTGATGAAGGGAATTTTTTCTCACCACCACCACCACCACTGAGATCCGGCTG-3' and pET-28a-MDH-VR: 5'-CAACACTCGCAACACCGGGTTTTTCTATCAAAAGTACTTTCATGGTATATCTCCTTCTTAAAGTTAAAC-3', the pET28a vector backbone was obtained from plasmid pET28a (Novagen, Madison, WI) via PCR. Simple cloning was employed (You C, Zhang X-Z, Zhang YHP. 2012. Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis. Applied and Environmental Microbiology). 78(5):1593-1595.) The recombinant plasmid pET28a-TmMDH was obtained.

[0046] Construction of plasmid pET28a.1-GDH-MDH: A gene fragment containing RBS (5'-AAGGAG-3'), mannitol dehydrogenase gene, and T7 terminator was obtained from plasmid pET28a-TmMDH by PCR using a pair of primers pET28a.1-GDH-MDH-IF: 5'-CCCAAGCTTAAGAAGGAGATATACATATGAAAGTACTTTTGATAG-3' and pET28a.1-GDH-MDH-IR: 5'-CGCGGATCCCGGATATAGTTCCTCCTTTCAGC-3'. Another gene fragment containing the T7 promoter, RBS, glucose dehydrogenase gene, and pET28a vector backbone was obtained from plasmid pET28a-SsGDH (Xie Leipeng. Construction and optimization of L-lactic acid synthesis pathway using glucose as substrate in vitro multi-enzyme catalysis system [D]. Henan Agricultural University, 2018.) via PCR.

[0047] Construction of plasmid pET28a.R1-MDH-GDH: A gene fragment containing RBS and glucose dehydrogenase was obtained from plasmid pET28a-SsGDH by PCR using a pair of primers: pET28a.R1-MDH-GDH-IF: 5'-CCCAAGCTTAAGAAGGAGATATACCATGGGCAG-3' and pET28a.R1-MDH-GDH-IR: 5'-CGCGGATCCCGGATATAGTTCCTCCTTTCAGC-3'. The gene and T7 terminator were obtained by PCR from plasmid pET28a-TmMDH using a pair of primers pET28a.R1-MDH-GDH-VF: 5'-CGCGGATCCATTGGCGAATGGGACGCG-3' and pET28a.R1-MDH-GDH-VR: 5'-CCCAAGCTTGTTAGCAGCCGGATCTCAGTGG-3'. The fragment contained the T7 promoter, RBS, mannitol dehydrogenase gene, and pET28a vector backbone.

[0048] Construction of plasmid pET28a.2-GDH-MDH: A gene fragment containing the T7 promoter, RBS, and mannitol dehydrogenase group was obtained from plasmid pET28a-TmMDH by PCR using a pair of primers: pET28a.2-GDH-MDH-IF: 5'-CCCAAGCTTCTCGATCCCGCGAAATTAATACG-3' and pET28a.2-GDH-MDH-IR: 5'-CGCGGATCCCGGATATAGTTCCTCCTTTCAGC-3'. Due to the T7 terminator, another gene fragment was obtained from the plasmid pET28a-SsGDH by PCR using a pair of primers pET28a.2-GDH-MDH-VF: 5'-CGCGGATCCATTGGCGAATGGGACGC-3' and pET28a.2-GDH-MDH-VR: 5'-CCCAAGCTTCGGATATAGTTCCTCCTTTCAGC-3'. This fragment contained the T7 promoter, RBS, glucose dehydrogenase gene, T7 terminator, and pET28a vector backbone.

[0049] Construction of plasmid pET28a.R2-MDH-GDH: A gene fragment containing the T7 promoter, RBS, and glucose dehydrogenase was obtained from plasmid pET28a-SsGDH by PCR using a pair of primers pET28a.R2-MDH-GDH-IF:5'-CCCAAGCTTCTCGATCCCGCGAAATTAATACG-3' and pET28a.R2-MDH-GDH-IR:5'-CGCGGATCCCGGATATAGTTCCTCCTTTCAGC-3'. The gene and T7 terminator were obtained by PCR using a pair of primers pET28a.R2-MDH-GDH-VF: 5'-CGCGGATCCATTGGCGAATGGGACGC-3' and pET28a.R2-MDH-GDH-VR: 5'-CCCAAGCTTCGGATATAGTTCCTCCTTTCAGC-3'. Another gene fragment containing the T7 promoter, RBS, mannitol dehydrogenase gene, T7 terminator, and pET28a vector backbone was obtained.

[0050] All primers were synthesized by Suzhou Genewise Biotech Co., Ltd. The PCR conditions for the gene were: denaturation at 98℃ for 2 min, followed by 30 cycles of the following parameters: 98℃ denaturation for 30 s, annealing at 60℃ for 15 s, extension at 72℃ for 1 min, and final extension at 72℃ for 10 min. The PCR products were analyzed by 1% agarose gel electrophoresis. After confirming the correct fragment size using a gel imaging system, the target fragment was recovered using a DNA purification and recovery kit (Tiangen Biotech Co., Ltd., China) for the construction of the recombinant expression vector.

[0051] The PCR products were double-digested with HindIII and BamHI (NEB). The digested products were recovered using a PCR product recovery kit. The PCR products (two gene fragments) were ligated with T4 DNA ligase (NEB) to obtain recombinant plasmids. The recombinant plasmids were transformed into Escherichia coli BL21(DE3) (Invitrogen, Carlsbad, CA) competent cells by heat shock and fermented to obtain whole cells expressing the corresponding enzymes. These cells were named Case1 double-enzyme co-expression strain, Case2 double-enzyme co-expression strain, Case3 double-enzyme co-expression strain, and Case4 double-enzyme co-expression strain, respectively.

[0052] Example 2

[0053] Case 1: Whole-cell catalytic reaction of dual-enzyme co-expression strain

[0054] A 40 mL reaction system containing 200 g / L glucose, 200 g / L fructose, and 0.2 g DCW / mL Case1 dual-enzyme co-expression strain was used for the catalytic reaction at 80 °C and pH 7.0 for 12 hours.

[0055] Depending on the retention time, the Sugar-Pak (Waters) RID detector can be used to analyze and distinguish gluconic acid, glucose, fructose or mannitol in the reaction solution. The analysis conditions are: column temperature 80℃, flow rate 0.5 mL / min, mobile phase is ultrapure water, and single sample run time is 20 min.

[0056] After the reaction was completed, the yield of mannitol was 125 g / L, the yield of gluconic acid was 148 g / L, and the conversion rate was 65%.

[0057] Example 3

[0058] Case 2: Whole-cell catalytic reaction of dual-enzyme co-expression strains

[0059] A 3 mL reaction system containing 5 g / L glucose, 5 g / L fructose, and 0.005 g DCW / mL Case2 dual-enzyme co-expression strain was used for the catalytic reaction at 60 °C and pH 7.0 for 2 hours.

[0060] After the reaction was completed, the yield of mannitol was 2.20 g / L, the yield of gluconic acid was 2.18 g / L, and the conversion rate was 43.8%.

[0061] Example 4

[0062] Case 4: Whole-cell catalytic reaction of dual-enzyme co-expression strains

[0063] A 40 mL reaction system containing 100 g / L glucose, 100 g / L fructose, and 0.1 g DCW / mL Case4 dual-enzyme co-expression strain was used for the catalytic reaction at 70 °C and pH 7.0 for 8 hours.

[0064] After the reaction was completed, the yield of mannitol was 100 g / L, the yield of gluconic acid was 108 g / L, and the conversion rate was 99.6%.

[0065] Example 5

[0066] Case 3: A dual-enzyme co-expression strain and a nickel-coated glucose isomerase underwent a whole-cell catalytic reaction using glucose as a substrate.

[0067] In this embodiment, the glucose isomerase was derived from *Thermus thermophilus*, with the gene number D90256.1 on NCBI. Genomic DNA was obtained from the ATCC website (www.atcc.org). The gene was obtained by PCR and cloned into the pET28a vector (Novagen, Madison, WI) to obtain the corresponding expression vector pET28a-TtXI. This plasmid was then transformed into *E. coli* expression strain BL21(DE3) (Invitrogen, Carlsbad, CA) for protein expression and purification.

[0068] A 3 mL reaction system containing 10 g / L glucose, 3 U / mL nickel column purified glucose isomerase, 5 mM divalent manganese ions, and 0.006 g DCW / mL Case3 dual-enzyme co-expression strain was used to catalyze the reaction at 80 °C and pH 7.0 for 2 hours.

[0069] After the reaction was completed, the yield of mannitol was 5.08 g / L, the yield of gluconic acid was 4.83 g / L, and the conversion rate was 99.6%.

[0070] Example 6

[0071] Case 4: A dual-enzyme co-expression strain and a nickel-coated glucose isomerase underwent a whole-cell catalytic reaction using glucose as a substrate.

[0072] A 3 mL reaction system containing 20 g / L glucose, 6 U / mL nickel column purified glucose isomerase, 5 mM divalent manganese ions, and 0.012 g DCW / mL Case4 dual-enzyme co-expression strain was carried out at 80 °C and pH 7.0 for 4 hours.

[0073] After the reaction was completed, the yield of mannitol was 9.98 g / L, the yield of gluconic acid was 9.82 g / L, and the conversion rate was 99.0%.

[0074] Example 7

[0075] Case 4: A dual-enzyme co-expression strain and permeabilized glucose isomerase performed a whole-cell catalytic reaction using glucose as a substrate.

[0076] A 3 mL reaction system containing 10 g / L glucose, 0.01 g DCW / mL permeabilized glucose isomerase, 5 mM divalent manganese ions, and 0.006 g DCW / mL Case4 dual-enzyme co-expression strain was subjected to a catalytic reaction at 80 °C and pH 7.0 for 3 hours.

[0077] After the reaction was completed, the yield of mannitol was 5.08 g / L, the yield of gluconic acid was 4.83 g / L, and the conversion rate was 98.7%.

[0078] Example 8

[0079] A whole-cell catalytic reaction system was established by sequentially adding strains co-expressing glucose isomerase and Case4.

[0080] In a 40 mL reaction system containing 300 g / L glucose, 20 U / mL nickel column purified glucose isomerase, and 5 mM divalent manganese ions, the reaction was catalyzed at 70 °C and pH 7.0 for 1 hour. Then, 0.03 g DCW / mL of the Case4 dual-enzyme co-expression strain was added, and the reaction was carried out at 70 °C for 16 hours.

[0081] The test results are as follows: Figure 2 As shown, after the reaction was completed, the yield of mannitol was 130 g / L, the yield of gluconic acid was 174 g / L, and the conversion rate was 96%.

[0082] The co-production pathways used in Examples 2-8 above are as follows: Figure 1As shown, the final products mannitol and gluconic acid can be further separated by existing techniques, such as the method described in Kulbe KD, Schwab U, Gudernatsch W. Enzyme-catalyzed production of mannitol and gluconic acid. Product recovery by various procedures [J]. Annals of the New York Academy of Sciences, 1987, 506: 552-568.

[0083] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A dual-enzyme expression plasmid, characterized in that: The mannitol dehydrogenase gene and glucose dehydrogenase gene are co-expressed on the same plasmid.

2. The dual-enzyme expression plasmid according to claim 1, characterized in that: The plasmid comprises a first promoter, a mannitol dehydrogenase gene, a first terminator, a second promoter, a glucose dehydrogenase gene, and a second terminator, or comprises a first promoter, a glucose dehydrogenase gene, a first terminator, a second promoter, a mannitol dehydrogenase gene, and a second terminator, or comprises a first promoter, a mannitol dehydrogenase gene, a second promoter, a glucose dehydrogenase gene, and a terminator, or comprises a first promoter, a glucose dehydrogenase gene, a second promoter, a mannitol dehydrogenase gene, and a terminator; preferably, the promoter is a T7 promoter and the terminator is a T7 terminator.

3. The dual-enzyme expression plasmid according to claim 1 or 2, characterized in that: The mannitol dehydrogenase is derived from *Thermotoga maritima*, *Thermotoganea politana*, or thermophilic anaerobic bacteria. (Thermoanaerobacterium thermosaccharolyticum), wherein the glucose dehydrogenase is derived from Sulfolobus solfataricus, Thermoplasma acidophilum, or Sulfolobus tokodaii; preferably, the mannitol dehydrogenase is derived from Thermoanaerobacterium thermosaccharolyticum; and the glucose dehydrogenase is derived from Sulfolobus solfataricus.

4. A recombinant strain expressing two enzymes, characterized in that: It is obtained by transforming the dual-enzyme expression plasmid described in any one of claims 1-3 into engineered Escherichia coli.

5. The recombinant strain expressing dual enzymes according to claim 4, characterized in that: The engineered Escherichia coli strain is Escherichia coli expression strain BL21(DE3).

6. A method for the co-production of mannitol and gluconic acid by whole-cell catalysis, characterized in that: Using glucose as a raw material, and under the action of glucose isomerase, the recombinant strain expressing the dual enzymes as described in claim 4 or 5 is used to catalyze the production of mannitol and gluconic acid using a whole-cell biocatalytic system composed of co-expressed mannitol dehydrogenase and glucose dehydrogenase.

7. The method for whole-cell catalytic co-production of mannitol and gluconic acid according to claim 6, characterized in that: The recombinant strain expressing the dual enzymes underwent a whole-cell catalytic reaction together with glucose isomerase and glucose.

8. The method for whole-cell catalytic co-production of mannitol and gluconic acid according to claim 7, characterized in that: Using glucose as a single substrate, some glucose is converted into fructose under the action of glucose isomerase. Then, the dual-enzyme expression recombinant strain uses glucose and fructose as substrates to carry out the reaction. That is, the dual-enzyme expression recombinant strain carries out whole-cell catalytic reaction together with glucose and fructose.

9. The method for whole-cell catalytic co-production of mannitol and gluconic acid according to any one of claims 6-8, characterized in that: The mannitol dehydrogenase is derived from *Thermus thermophilus*, *Thermus neoapollonius*, or thermophilic glycolytic anaerobic bacteria; preferably, the mannitol dehydrogenase is derived from *Thermus thermophilus*. The glucose dehydrogenase is derived from *Leucobacterium sulfideum*, *Thermoanaerobacter ethanolicus*, or *Streptomyces flavogriseus*; preferably, the glucose dehydrogenase is derived from *Leucobacterium sulfideum*.

10. The method for whole-cell catalytic co-production of mannitol and gluconic acid according to any one of claims 6-8, characterized in that: The glucose isomerase is added after purification by nickel column chromatography or after permeation treatment; the glucose isomerase and the dual-enzyme expression recombinant strain are added sequentially to establish a whole-cell catalytic reaction system; wherein, the concentration of glucose is 2-600 g / L, preferably 10-400 g / L, and more preferably 100-300 g / L. The temperature of the reaction system is 40-90℃, preferably 50-80℃, and even more preferably 60-70℃; The cell volume of the dual-enzyme expression recombinant strain is 0.001-0.3 g DCW / mL, preferably 0.005-0.2 g DCW / mL, and even more preferably 0.01-0.05 g DCW / mL.

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