AU2022422808A1 - Recombinant corynebacterium glutamicum for producing high-purity isomaltulose at high yield, and application thereof - Google Patents
Recombinant corynebacterium glutamicum for producing high-purity isomaltulose at high yield, and application thereof Download PDFInfo
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- AU2022422808A1 AU2022422808A1 AU2022422808A AU2022422808A AU2022422808A1 AU 2022422808 A1 AU2022422808 A1 AU 2022422808A1 AU 2022422808 A AU2022422808 A AU 2022422808A AU 2022422808 A AU2022422808 A AU 2022422808A AU 2022422808 A1 AU2022422808 A1 AU 2022422808A1
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- isomaltulose
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- corynebacterium glutamicum
- culture medium
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/12—Disaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y504/00—Intramolecular transferases (5.4)
- C12Y504/99—Intramolecular transferases (5.4) transferring other groups (5.4.99)
- C12Y504/99011—Isomaltulose synthase (5.4.99.11)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to the field of metabolic engineering and fermentation engineering. Disclosed are a recombinant corynebacterium glutamicum for producing isomaltulose, and an application thereof. According to the present invention, isomaltulose synthetase is expressed in corynebacterium glutamicum, the coding gene sequence of the isomaltulose synthetase is shown in NCBI Gene ID: AY223549.1, and a signal peptide sequence as shown in SEQ ID NO. 1 is introduced at multiple cloning sites of an expression vector. The recombinant bacterium of the present invention can improve the yield of isomaltulose without affecting the growth condition of corynebacterium glutamicum on the basis of using molasses as a starting energy source.
Description
[0001] The present invention belongs to the fields of metabolic engineering and fermentation engineering, and in particular, relates to a recombinant Corynebacterium glutamicum for producing high-purity isomaltulose at a high yield, and an application thereof.
[0002] Obesity, arteriosclerosis, hypertension, diabetes, dental caries, and other diseases caused by excessive use of sucrose have become a growing concern in society. Isomaltulose (IsomaltuloseTM, palatinose@, Lylose), also known as palatinose, isomeric sucrose, isomalt, or Batang, with a molecular formula of C 12 H 2 2 0 1 1H20 and a molecular mass of 360, is a new type of natural functional sugar, chemically known as dextrose (6-o-a-D pyranoglucosyl-D-fructose) composed of glucose and fructose with a a-1,6 glycosidic bond ligated therebetween. Isomaltulose has excellent properties such as pure sweetness, low caloric value, low hygroscopicity, high safety and no cariogenicity, and it is metabolized in vivo without depending on insulin. Hence, it can be consumed by people with high blood sugar, diabetes and obesity. Consequently, isomaltulose is regarded as an "ideal substitute" for sucrose, and shows great application potential and market prospects in fields such as food, pharmaceutics and chemicals.
[0003] At present, the use of cheap waste biomasses as a carbon source for fermentation to produce high value-added products has become a research hotspot. Compared with other wastes such as straws or other lignocellulosic raw materials, molasses as the main by-products in the sugar industry contain a large amount of fermentable sugar (the vast majority of which is sucrose) without any pretreatment. Meanwhile, the molasses contains inorganic salts, vitamins and other components, which can provide influential substances required for microbial growth and metabolism. Hence, it is a cheap biomass resource with great potential.
[0004] Corynebacteriumglutamicum is a gram-positive bacterium, which exists as a rod under the microscope and has round yellowish colonies on plates. At present, it has been widely used in the production of amino acids. Compared with Escherichiacoli, Corynebacteriumglutamicum is an FDA-approved food-grade safety strain having the advantages of high safety, low allergenicity, high stress resistance, and low probability of contamination by bacteriophages. Therefore, it plays an important role in the field of genetic engineering. Due to the absence of isomaltulose synthetase in Corynebacteriumglutamicum, there is no pathway for synthesis of isomaltulose from cheap substrates. Therefore, it is possible to endow Corynebacterium glutamicum with the ability to synthesize isomaltulose only by allowing it to efficiently express an exogenous isomaltulose synthetase.
[0005] In the present invention, an isomaltulose synthetase is expressed in Corynebacterium glutamicum, and a signal peptide sequence is introduced to multiple cloning sites of an expression vector, whereby the isomaltulose output is increased without affecting the growth of Corynebacteriumglutamicum on the basis of taking molasses as a starting energy source.
[0006] The first object of the present invention is to provide a recombinant Corynebacterium glutamicum, in which an isomaltulose synthetase gene as shown in in NCBI Gene ID: AY223549.1 is integrated.
[0007] Preferably, a signal peptide gene is integrated in the recombinant Corynebacterium glutamicum, and the signal peptide gene has a nucleotide sequence as shown in in SEQ ID NO.1.
[0008] Preferably, a starting strain for the recombinant Corynebacterium glutamicum is Corynebacteriumglutamicum ATCC13032.
[0009] The second object of the present invention is to provide a construction method of the engineered Corynebacteriumglutamicum defined above, including ligating a signal peptide gene as shown in in SEQ ID NO. 1 and an isomaltulose synthetase gene as shown in in NCBI Gene ID: AY223549.1 separately to an expression vector to obtain a recombinant vector, transferring the recombinant vector into Corynebacterium glutamicum, and carrying out screening to obtain the recombinant Corynebacteriumglutamicum.
[0010] Preferably, the expression vector is pEC-XK99E, with a promoter Ptre to control expression of the isomaltulose synthetase gene.
[0011] Preferably, the isomaltulose synthetase gene is inserted between enzyme sites BamHI and XbaI of the pEC-XK99E, and the signal peptide gene is inserted between enzyme sites EcoRI and KpnI of the pEC-XK99E.
[0012] Preferably, the isomaltulose synthetase gene is derived from Pantoea dispersa.
[0013] Preferably, constructing the engineered Corynebacterium glutamicum defined above includes the following steps: constructing pEC-XK99E- UQ68J Slase (expressing the isomaltulose synthetase gene); constructing pEC-XK99E-cgR0949 (expressing a cgR0949 signal peptide); constructing pEC-XK99E-cgR949-UQ68J Sase (expressing the cgR0949 signal peptide and the isomaltulose synthetase gene at the same time); and transferring expression vectors pEC-XK99E- UQ68J Slase and pEC-XK99E-cgR0949-UQ68J Slase separately into host Corynebacterium glutamicum ATCC13032 to obtain Corynebacterium glutamicum strains for finally producing isomaltulose, respectively.
[0014] The third object of the present invention is to provide an application of the recombinant Corynebacteriumglutamicum as defined for preparation of any of the following:
[0015] (1) an isomaltulose synthase or a preparation thereof;
[0016] (2) an isomaltulose and a derivative thereof; and
[0017] (3) isomaltulose-containing functional food.
[0018] In an embodiment of the present invention, a single colony of the recombinant Corynebacteriumglutamicum is inoculated into a CGXII culture medium, and cultured for 18-24 h at 25-35°C and 200-250 rpm to obtain a seed solution, and then the seed solution is added to the CGXII culture medium at a volume of 5-10% of the culture medium, and cultured for not less than 18-24 h at 25-35°C and 200-250 rpm.
[0019] In another embodiment of the present invention, the recombinant Corynebacterium glutamicum is inoculated into a CGXII culture medium, cultured for 18-24 h at 25-35°C and 200-250 rpm, then added to a fermentation culture medium in a fermenter containing molasses and corn step liquor, at a volume of 5-10% of the culture medium, and fermented at 25-35°C and a pH value of 5-7, with dissolved oxygen controlled to be greater than 30%.
[0020] Preferably, the fermenter contains ingredients including 100-500 g/L of molasses and 1-25 g/L of corn steep liquor.
[0021] Preferably, the fermentation culture medium contains 350 g/L of cane molasses and 12 g/L of corn steep liquor, or 400 g/L of beet molasses and 15 g/L of corn steep liquor.
[0022] More preferably, the molasses are waste molasses in sugar industry, including but not limited to cane molasses, beet molasses, sweet sorghum stalk juice.
[0023] Compared with the prior art, the present invention has the following advantageous effects.
[0024] (1) The recombinant Corynebacteriumglutamicum provided by the present invention can allow the extracellular accumulation of isomaltulose. In a 7L fermenter, with the cane molasses as a carbon source, the isomaltulose output is up to 169.29 g/L, the sucrose conversion rate is 96.7%, the space-time yield is 2.26 g/(L-h), and the purity can reach 98%; and with the beet molasses as a carbon source, the isomaltulose output is up to 166.76 g/L, the sucrose conversion rate is 97%, the space-time yield is 2.32 g/(L -h), and the purity can reach 98%. It achieves the highest value in both yield and purity with respect to engineering bacteria reported so far.
[0025] (2) The construction method of the recombinant Corynebacteriumglutamicum as provided by the present invention is simple and convenient, and is applicable to industrial production applications.
[0026] (3) The present invention recycles molasses from industrial wastes to achieve low-cost and efficient production of isomaltulose.
[0027] FIG. 1 shows a profile for construction of a recombinant pEC-XK99E-UQ68J Slase plasmid;
[0028] FIG. 2 shows a profile for construction of a recombinant pEC-XK99E-cgR0949 plasmid;
[0029] FIG. 3 shows a profile for construction of a recombinant pEC-XK99E-cgR949-UQ68J Slase plasmid;
[0030] FIG. 4 shows cell growth of different strains fermented in shaker flasks;
[0031] FIG. 5 shows a procedure chart of outputs of isomaltulose in supernatants of different strains fermented in shaker flasks;
[0032] FIG. 6 shows cell growth of different strains fermented in shaker flasks after the preliminary optimization of fermentation conditions;
[0033] FIG. 7 shows a procedure chart of outputs of isomaltulose in supernatants of different strains fermented in shaker flasks after the preliminary optimization of fermentation conditions;
[0034] FIG. 8 shows optimization results of a cane molasses concentration and a corn steep liquor concentration;
[0035] FIG. 9 shows optimization results of a beet molasses concentration and a corn steep liquor concentration;
[0036] FIG. 10 shows a process of producing isomaltulose by fermenting the recombinant Corynebacteriumglutamicum of the present invention at the optimal cane molasses concentration in a 7L fermenter; and
[0037] FIG 11 shows a process of producing isomaltulose by fermenting the recombinant Corynebacteriumglutamicum of the present invention at the optimal beet molasses concentration in a 7L fermenter.
[0038] The present invention is further illustrated by the embodiments below, which are not intended to limit the present invention.
[0039] Measurement method for isomaltulose:
[0040] High-performance liquid chromatography (HPLC) assay: Waters 2695; RED detector; Sugar-ParkTMI (Waters, USA); mobile phase: ultrapure water; flow rate: 0.5 mL/min; column temperature: 65°C; and injection volume: 20 uL.
[0041] Seed-activation culture medium (LBB) (g/L): yeast powder: 5; peptone: 10; sodium chloride: 5; brain heart infusion: 10; and liquid volume in flask: 30 mL liquid in a 250 mL Erlenmeyer flask.
[0042] Seed-activation solid culture medium (LBB) (g/L): yeast powder: 5; peptone: 10; sodium chloride 5; brain heart infusion 10; and agar powder: 20.
[0043] Competent culture medium (LBHI) (g/L): yeast powder: 5; peptone: 10; sodium chloride 5; glycine: 25; isoniazid: 5; Tween 80: 5 mL; and liquid volume in flask: 50 mL of liquid in a 250 mL Erlenmeyer flask.
[0044] Post-electroporation recovery culture medium LBHIS (g/L): yeast powder: 5; peptone: 10; sodium chloride: 5; brain heart infusion: 20; and sorbitol: 91.
[0045] Transformant-detection solid culture medium (g/L): yeast powder: 5; peptone: 10; sodium chloride: 5; brain heart infusion: 20; sorbitol: 91; and agar powder: 20.
[0046] Seed culture medium (g/L): molasses: 20, corn steep liquor: 15, and KH 2 PO 4 : 1.5; and a pH value of 6.8-7.2.
[0047] Fermentation culture medium (g/L): molasses: 50, corn steep liquor: 25, KH 2 PO 4 : 1.5, MgSO4: 0.8, CaCO3: 30, and FeSO4: 0.2; and a pH value of 6.8-7.2.
[0048] Optimized fermentation culture medium (g/L): molasses: 150, corn steep liquor: 20, KH 2 PO 4 : 1.5, MgSO4: 0.8, CaCO3: 30, and FeSO4: 0.2; and a pH value of 6.8-7.2.
[0049] Sterilization conditions: 120°C; 20 min; and 25 mg/L kanamycin added when all media are used for transformant detection or for recombinant Corynebacteriumglutamicum culture.
[0050] Embodiment 1: Construction of Recombinant Plasmid pEC-XK99E- UQ68J Slase
[0051] UQ68J Slase was expressed with the plasmid pEC-XK99E as an expression vector. Taking the plasmid pET-28a-UQ68J-Sase as a template (for its sequence, see Su H. H., Xu R. Y., Yang Z. D., et al. Green Synthesis of Isomaltulose from Cane Molasses by an Immobilized Recombinant Escherichia coli Strain and Its Prebiotic Activity[J]. LWT- Food Science and Technology, 2021, 143(43):111054.) and with primers pF (5'-GGATCCGCAACGAATATACAAAAGT-3') and pR (5'-TCTAGATCAGTTC AGCTTATAGATCCCG -3'), the sequence UQ68J Slase was amplified using PrimeSTAR Max DNA polymerases. PCR conditions were as follows: pre-denaturation for min at 95°C; denaturation for 10 s at 98°C; annealing for 15 s at 55°C; extension for 120 s at 72°C, for 35 cycles of reaction; and finally, extension for 7 min at 72°C. A DNA purification kit was used to recover PCR products, and the obtained PCR products have a nucleotide sequence as follows: gcaacgaatatacaaaagtccgctgattttcccatttggtggaaacaggcagtattttaccagatttatccccgctcatttaaagatagcaatggt gatggtatcggcgatattcccggtatcattgagaaactggactatttaaaaatgctgggagttgatgctatctggataaacccgcactatgagt ctcctaacaccgacaatggttacgatattagtgattatcgtaaaatcatgaaggagtacggcagcatggctgactttgaccgtctggttgccga aatgaataaacgtggtatgcgcctgatgattgatattgttatcaatcataccagcgatcgtcaccgctggtttgtgcagagccgttcaggtaaa gataatccttaccgcgactattatttctggcgtgatggtaaacagggacaggctcccaataactatccctctttctttggcggttcagcctggca actggataaacagactgaccagtattatctgcactattttgcaccacagcagccggatctgaactgggataacccaaaagttcgggctgaact ctacgatattctgcgtttctggctggataaaggcgtatccggactacgttttgataccgtggctactttctccaaaattcctggcttcccggacct gtcaaaagcgcagctgaagaattttgccgaagcttatactgaggggccgaatattcataaatatatccatgaaatgaaccgccaggtactgtc taaatataatgttgccaccgctggtgaaatcttcggtgtgccagtgagtgctatgccggattattttgaccggcggcgtgaagaactcaatatt gctttcacctttgatttgatcaggctcgatcgttatcccgatcagcgctggcgtcgtaaaccatggacattaagccagtttcgtcaagttatctct cagactgaccgtgccgccggtgaatttggctggaacgcctttttccttgataaccatgataacccgcgccaggtctcacactttggtgacgac agcccacaatggcgcgaacgctcggcaaaagcactggcaacgctgctgctgacgcagcgtgccacgccgtttatctttcagggggcgga gttgggaatgactaattacccctttaaaaatatagaggaatttgatgatattgaggttaaaggcttctggaacgactatgtagccagcggaaaa gtaaacgctgctgaatttttacaggaggttcgcatgaccagccgcgataacagccgaacaccaatgcagtggaacgactctgttaatgccg gattcacccagggcaaaccctggtttcacctcaatcccaactataagcaaatcaatgccgccagggaggtgaataaacccgactcggtattc agttactaccgtcaactgatcaacctgcgtcaccagatcccggcactgaccagtggtgaataccgtgatctcgatccgcagaataaccaggt ctatgcctatacccgtatactggataatgaaaaatatctggtggtagttaattttaaacctgagcagctgcattacgctctgccagataatctgac tattgccagcagtctgctggaaaatgtccaccaaccatcactgcaagaaaatgcctccacgctgactcttgctccgtggcaagccgggatct ataagctgaactga.
[0052] The PCR products of UQ68J Slase gene sequence resulting from the previous step were digested with restriction endonuclease BamHI and XbaI. A recovery and digestion system included: 10 pL of the PCR products, 0.6 pL of BamHI, 0.6 pL of XbaI, 5 pL of loX buffer, and 23.8 tL of double distilled water added. The digestion products were detected by 1% agarose gel electrophoresis, and the fragments of interest were recovered. At the same time, the plasmid pEC-XK99E was similarly digested with double enzymes, and the digestion products were then recovered from the gel.
[0053] An insertion fragment and the plasmid were ligated with a T4 ligase. The vector and the insertion fragment were mixed at a molar ratio of 1:1 to 1:9; an equal amount of a ligation mix solution was added to the resultant for ligation at 22°C for 1 h or at 4°C overnight. Then, E. coli DH5a competent cells were transformed (for the preparation method for the competent cells, see the instructions of TransGen Biotechn E. coli competent kit). The correct transformants for colony PCR were selected for sequencing validation, and the recombinant plasmid pEC-XK99E- UQ68J Slase was obtained (FIG. 1).
[0054] Embodiment 2: Construction of Recombinant Plasmid pEC-XK99E-cgR949-UQ68J Slase
[0055] Taking the purchased plasmid pAU3 as a template (for the sequence of the plasmid pAU3, see Zhang L., Jia H., Daqing X. U. Construction of a novel twin-arginine translocation (Tat)-dependent type expression vector for secretory production of heterologous proteins in Corynebacterium glutamicum. Plasmid, 2015, 82:50-55.) and with primers CRgF (5'-GAATTCatgggtaagcaccgtcgcaacaat-3') and CRgR (5'-GGTACCaacctcagctgcctgtgctgggga-3'), the cgR0949 sequence was amplified using PrimeSTAR Max DNA polymerases. PCR conditions were as follows: pre-denaturation for 5 min at 95°C; denaturation for 10 s at 98°C; annealing for 15 s at 55°C; extension for 15 s at 72°C, for cycles of reaction; and finally, extension 7 min at 72°C. PCR products were recovered with a DNA purification kit to obtain a cgR0949 signal peptide sequence, with a nucleotide sequence as follows: Atgggtaagcaccgtcgcaacaattcaaacgcaactcgcaaggctgtagcagcatctgcagttgcgcttggac caaccgcagctatcgcctccccagcacaggcagctgaggtt (SEQ ID NO. 1).
[0056] The PCR products of cgR0949 signal peptide sequence resulting from the previous step were digested with restriction endonuclease EcoRI and KpnI. A recovery and digestion system included: 10 pL of the PCR products, 0.6 pL of EcoRI, 0.6 pL of KpnI, 5 pL of OX buffer, and 23.8 tL of double distilled water added. The digestion products were detected by 2% agarose gel electrophoresis, and the fragments of interest were recovered. At the same time, the plasmid pEC-XK99E was similarly digested with double enzymes, and the digestion products were then recovered from the gel.
[0057] An insertion fragment and the plasmid were ligated with a T4 ligase. The vector and the insertion fragment were mixed at a molar ratio of 1:1 to 1:15; an equal amount of a ligation mix solution was added to the resultant for ligation at 22°C for 1 h or at 4°C overnight. Then, E. coli DH5a competent cells were transformed (for the preparation method for the competent cells, see the instructions of TransGen Biotechn E. coli competent kit). The correct transformants for colony PCR were selected for sequencing validation, and the recombinant plasmid pEC-XK99E-cgR0949 was obtained (FIG. 2).
[0058] With the method described in Embodiment 1, the UQ68J Slase gene sequence was obtained by PCR using the primers pF and pR by taking the plasmid pET-28a-UQ68J-Slase as a template. The PCR products of UQ68J Sase gene sequence was digested with restriction endonucleases BamHI and XbaI. The digestion products were detected by 1% agarose gel electrophoresis, and the fragments of interest were recovered. At the same time, the plasmid pEC-XK99E-cgR949 was similarly digested with double enzymes, and the digestion products were then recovered from the gel.
[0059] An insertion fragment and the plasmid were ligated with a T4 ligase. The vector and the insertion fragment were mixed at a molar ratio of 1:1 to 1:9; an equal amount of a ligation mix solution was added to the resultant for ligation at 22°C for 1 h or at 4°C overnight. Then, E. coli DH5a competent cells were transformed (for the preparation method for the competent cells, see the instructions of TransGen Biotechn E. coli competent kit). The correct transformants for colony PCR were selected for sequencing validation, and the recombinant plasmid pEC-XK99E-cgR0949-UQ68J Slase was obtained (FIG. 3).
[0060] Embodiment 3: Preparation of Recombinant Corynebacterium Glutamate
[0061] The recombinant plasmid pEC-XK99E- UQ68J Slase obtained in Embodiment 1 and the recombinant plasmid pEC-XK99E-cgR949-UQ68J Sase obtained in Embodiment 2 were transferred into the strains of Corynebacterium glutamicum ATCC13032 by electroporation transformation to obtain different recombinant Corynebacteriumglutamicum.
[0062] Preparation of competent cells from Corynebacterium glutamicum by electroporation transformation:
[0063] (1) The Corynebacterium glutamicum ATCC13032 was inoculated in the LBB culture medium, placed on a reciprocating shaker (at 180 rpm), and cultured for 20 h at 30°C, with OD5 62
up to 3.0.
[0064] (2) 10% of the Corynebacteriumglutamicum was transferred into the competent culture medium (LBHI) (with OD 562 up to 0.3), placed on the reciprocating shaker (at 200 rpm), and cultured for 4 h at 30°C, with OD5 62 up to 0.8-1.0.
[0065] (3) The bacterial solution was collected, treated in an ice bath for 20 min, and centrifuged for 10 min at 6000 rpm at 4°C, and the supernatant was discarded.
[0066] (4) The bacteria were fully suspended with 50 mL of pre-chilled 10% glycerol, centrifuged for 10 min at 6000 rpm at 4°C, and after the supernatant was discarded, washed 3 times.
[0067] (5) The cells were resuspended with 500 gL of pre-chilled 10% glycerol, and aliquoted into 1.5 mL sterile centrifuge tubes, approximately 100 pL per tube.
[0068] (6) The cells were stored at -80°C for later use. In order to ensure the transformation efficiency of the competent cells, preparation on site was preferred, and the storage time should be less than 1 week.
[0069] Electroporation transformation of Corynebacteriumglutamicum:
[0070] (1) The competent cells of Corynebacteriumglutamicum stored at -80°C were melted in an ice bath.
[0071] (2) 0.5-2 L of each of the above two plasmids was added and mixed well, and the resulting mixture was treated in an ice bath for 20 min.
[0072] (3) The mixture was added to a pre-chilled 0.2 cm electroporation cup and electroporated 1 time in an EcN2 mode.
[0073] (4) 0.5 mL of LBHIS pre-warmed at 46 °C was quickly added and mixed well, then transferred to a new 1.5 mL sterile centrifuge tube, treated for 6 min in a water bath at 46°C, and then immediately treated for 10 min in ice.
[0074] (5) The bacteria were placed on the reciprocating shaker and cultured for 1.5-2.0 h at 150 rpm and 30°C.
[0075] (6) The bacteria were centrifuged for1 min at 8000 rpm, spread onto a kanamycin-resistant transformant detection plate, and cultured for 24 h in a constant-temperature incubator at 30°C.
[0076] (7) Verification of competent cell efficiency: with 10 L of sterile water added as a negative control, no colonies grew; and with 1 pL of plasmid pEC-XK99E as a positive control, a large number of colonies grew.
[0077] Embodiment 4: Effect of Signal Peptide on Isomaltulose Output of Recombinant Corynebacterium Glutamate
[0078] The recombinant Corynebacterium glutamicum strains, with correct sequencing results, respectively containing the plasmids pEC-XK99E- UQ68J Sase and pEC-XK99E-cgR949 UQ68J Slase were added to a glycerol tube, inoculated and streaked on an LBB plate (with 50 mg/mL kanamycin sulfate added), and then cultured for 20 h at 200 rpm at 30°C; then, single-colonies were selected; and then, streaking was conducted on the LBB plate again, until a large number of colonies grew.
[0079] A ring of single colony was inoculated into the seed culture medium and cultured for 20 h at 200 rpm at 30 °C. The seed culture solution was inoculated into the fermentation culture medium at 10% inoculation volume, and cultured for 80 h at 200 rpm at 30°C, with 6 replicates set up and the recombinant strain containing plasmid pEC-XK99E- UQ68J Slase as a control. The bacterial solution was taken every 10 h to measure OD 562 and isomaltulose output. For the measurement methods, a reference was made to Su H. H., Xu R. Y., Yang Z. D., et al. Green synthesis of Isomaltulose from Cane Molasses by an Immobilized Recombinant Escherichia coli Strain and Its Prebiotic Activity[J]. LWT- Food Science and Technology, 2021, 143(43):111054.
[0080] From FIG. 4, it can be seen that different strains fermented in shaker flasks show similar cell growth tendency within 80 h.
[0081] As shown in FIG. 5, under the same conditions, the strain containing the plasmid pEC-XK99E-cgR949-UQ68J Sase of the signal peptide sequence has the isomaltulose output of 64 g/L within 80 h, which was 14.28% higher than that of the control strain (56 g/L).
[0082] Embodiment 5: Effect of Fermentation Condition Optimization on Isomaltulose Output of Recombinant Corynebacterium Glutamate
[0083] The composition ratio and inoculation volume of the fermentation culture medium greatly affected the growth of strains and the accumulation of metabolites. Hence, on the basis of Embodiment 4, the components of the culture medium were changed as follows (g/L): molasses: 150, corn steep liquor: 20, and a pH value of 6.8-7.2; and the inoculation volume for the fermentation culture medium was adjusted such that OD5 62 was 1.8 after inoculation. The recombinant Corynebacterium glutamicum containing the plasmid pEC-XK99E-cgR949 UQ68J Slase was tested against the recombinant Corynebacterium glutamicum containing the plasmid pEC-XK99E- UQ68J Slase. Other culture conditions and post-fermentation detection methods were the same as those in Embodiment 4.
[0084] From FIG. 6, it can be seen that different strains fermented in shaker flasks show similar cell growth tendencies within 80 h.
[0085] As shown in FIG. 7, after the fermentation conditions were optimized, the isomaltulose outputs of the recombinant strains within 80 h were all increased. Under the same conditions, the strain containing the plasmid pEC-XK99E-cgR0949-UQ68J Slase of the signal peptide sequence had the isomaltulose output of 73 g/L within 80 h, which was 17.72% higher than that (62 g/L) of the control strain.
[0086] Embodiment 6: Effect of Different Types of Molasses on Growth and Isomaltulose Output of Recombinant Corynebacterium Glutamate
[0087] The composition ratio and inoculation volume of the fermentation culture medium greatly affected the growth of strains and the accumulation of metabolites. Hence, on the basis of Embodiment 4, the components of the culture medium were changed as follows (g/L): cane molasses or beet molasses and corn steep liquor. In the group of cane molasses plus corn steep liquor, the initial content of the cane molasses was set to 350 g/L, and the initial content of the corn steep liquor was set to 10 g/L; and in the group of beet molasses plus corn steep liquor, the initial content of the beet molasses was set to 400 g/L, and the initial content of the corn steep liquor was set to 10 g/L. When the component content was optimized, the initial content was replaced with a gradient content.
[0088] The inoculation volume for the fermentation culture medium was adjusted such that OD5 62
was 1.8 after inoculation. The recombinant Corynebacteriumglutamicum containing the plasmid
I1 pEC-XK99E-cgR949- UQ68J Sase was tested. After 72 h of fermentation, samples were taken to measure OD 6oo and isomaltulose output.
[0089] From FIG. 8, it can be seen that, with the optimal cane molasses concentration of 350 g/L and the corn steep liquor concentration of 12 g/L, the isomaltulose output of the recombinant Corynebacteriumglutamicum according to the present invention is up to 168.88 g/L, with the cane conversion rate of 96.5%, and the space-time yield of 2.82 g/(L-h).
[0090] From FIG. 9, it can be seen that, with the optimal beet molasses concentration of 400 g/L and the corn steep liquor concentration of 15 g/L, the isomaltulose output of the recombinant Corynebacterium glutamicum of the present invention is up to 166.88 g/L, with the cane conversion rate of 97%, and the space-time yield of 2.78 g/(L-h).
[0091] OD 6 0 0 and the isomaltulose output were measured by collecting the bacterial solution every 6 h from the 7L fermenters respectively taking Group 1 (including cane molasses with a concentration of 350 g/L and corn steep liquor with a concentration of 12 g/L) and Group 2 (including beet molasses with a concentration of 400 g/L and corn steep liquor with a concentration of 15 g/L) as culture medium components, under the fermentation conditions of °C, a pH value of 6.0, dissolved oxygen at 1.0 vvm, and 200 rpm.
[0092] From FIG. 10, it can be seen that, in the 7L fermenters, the isomaltulose output of the recombinant Corynebacteriumglutamicum of the present invention is up to 169.29 g/L, with the cane conversion rate of 96.7%, the space-time yield of 2.26g/ (L-h), and the purity up to 98%.
[0093] From FIG. 11, it can be seen that, in the 7L fermenters, the isomaltulose output of the recombinant Corynebacteriumglutamicum of the present invention is up to 166.76 g/L, with the cane conversion rate of 97%, the space-time yield of 2.32g/(L-h), and the purity up to 98%.
[0094] Further, the recombinant Corynebacteriumglutamicum according to the present invention was compared with the existing strains producing isomaltulose, with the results shown in Table 1. Table 1. Comparison of Different Strains Producing Isomaltulose Strain Substrate Enzyme source Output Yield Purity Source (g/L) (g/g) (%) E. coli Sucrose Erwiniasp. 240 0.80 - Z F, C F, J X D, et al. Characterization of a Ejp617 recombinant sucrose isomerase and its application to enzymatic production of isomaltulose [J]. Biotechnology letter, 2021, 43(l):261-269.
E. coli Sucrose Klebsiella - 0.768 78.4 A A, N T, S T, et al. Role of several key pneumoniae residues in the catalytic activity of sucrose NK33-98-8 isomerase fromKebsiella pneumoniae NK33-98-8 NK33-98-8 [J]. Enzyme and Microbial Technology, 2007, 40 (5):1221-1227.
E. coli Sucrose K planticola - 0.66 81.5 L W, RG B. Characterization of the highly UQ14S efficient sucrose isomerase from Pantoea dispersaUQ68J and cloning of the sucrose isomerase gene [J]. AppI Environ Microbiol, 2005, 71(3):1581-90.
E. coli Sucrose P dispersa - 0.91 96.8 L W, RG B. Characterization of the highly UQ68J efficient sucrose isomerase from Pantoea dispersaUQ68J and cloning of the sucrose isomerase gene [J]. AppI Environ Microbiol, 2005, 71(3):1581-90.
L. lactis Sucrose Enterobactersp. 36 0.72 <90 J Y P, J H J, D H S, et al. Microbial FMB-1 production of palatinose through extracellular expression of a sucrose isomerase from Enterobacter sp. FMB-1 in Lactococcus lactis MG1363 [J]. Bioresour Technol, 2010, 101(22):8828-33.
S. cereviase Sucrose Enterobactersp. <4 0.074 <10 L G Y, J J H, S D H, et al. Isomaltulose FMB-1 production via yeast surface display of sucrose isomerase from Enterobacter sp. FMB-1 on Saccharomyces cerevisiae [J]. Bioresour Technol. 2011, 102(19):9179-84.
Y lipolytica Sucrose P dispersa 572.1 0.96 97.8 Z P, W Z P, S J, et al. High and efficient UQ68J isomaltulose production using an engineered Yarrowia lipolytica strain [J]. Bioresour Technol. 2018, 265:577-580.
Y lipolytica Sucrose P dispersa 620.7 0.96 - Z P; W Z.P; L, S; et al. Overexpression UQ68J of secreted sucrose isomerase in Yarrowia lipolytica and its application in isomaltulose production after immobilization [J]. it. J. Biol. Macromol. 2019, 121, 97-103.
S. Cane S. plymuthica <33.5 0.84 80.4 D C 0, H H S . Isomaltulose production plymuthica molasses using free and immobilized Serratia plymuthica cells[J]. African Journal of Biotechnology, 2016, 15(20).
B. subtilis Cane E. rhapontici 212.6 0.92 <92.4 W L, W S, Q J, et al. Green synthesis of molasses NX-5 isomaltulose from cane molasses by Bacillus subtilis WB800-pHAO1-pall in a biologic membrane reactor[J]. Food Chemistry, 2017, 229(15):761-768.
Y lipolytica Cane P dispersa 161.2 0.96 97.4 W P, W Q, Liu S, L F, et al. Efficient molasses UQ68J Conversion of Cane Molasses Towards High-Purity Isomaltulose and Cellular Lipid Using an Engineered Yarrowia lipolytica Strain in Fed-Batch Fermentation [J]. Molecules. 2019, 28;24(7):1228.
C. Cane P dispersa 170.1 0.97 98 Present invention glutamicum molasses UQ68J
C. Beet P dispersa 166.98 0.97 98 Present invention glutamicum molasses UQ68J
[0095] From Table 2, it can be seen that the isomaltulose produced from the recombinant Corynebacteriumglutamicum of the present invention shows the highest value in both yield and purity that have been reported so far.
[0096] Sequence Listing <110> Institute of Biological and Medical Engineering, Guangdong Academy of Sciences <120> Recombinant Corynebacterium Glutamicum for Producing High-Purity Isomaltulose at High Yield, and Application Thereof <160> 1 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 114 <212> DNA <213> Pantoeadispersa <400> 1 atgggtaagc accgtcgcaa caattcaaac gcaactcgca aggctgtagc agcatctgca 60 gttgcgcttg gaccaaccgc agctatcgcc tccccagcac aggcagctga ggtt 114
Sequence Listing Sequence Listing 1 1 Sequence Sequence Listing Listing Information Information 1-1 1-1 File Name File Name KYPCT2022024.xml KYPCT2022024.xml 1-2 1-2 DTD Version DTD Version V1_3 V1_3 1-3 1-3 Software Name Software Name WIPOSequence WIPO Sequence 1-4 1-4 Software Version Software Version 2.1.0 2.1.0
1-5 1-5 Production Date Production Date 2023-10-23 2023-10-23 1-6 1-6 Originalfree Original freetext textlanguage language code code 1-7 1-7 NonEnglish Non English freefree texttext
languagecode language code 22 GeneralInformation General Information 2-1 2-1 Currentapplication: Current application: IP IP WO WO Office Office
2-2 2-2 Currentapplication: Current application: PCT/CN2022/112647 PCT/CN2022/112647 Application number Application number 2-3 2-3 Currentapplication: Current application: Filing 2022-08-16 Filing 2022-08-16 date date 2-4 2-4 Currentapplication: Current application: KYPCT2022024 KYPCT2022024 Applicantfile Applicant filereference reference 2-5 2-5 Earliest priority application: Earliest priority application: CN CN IP Office IP Office
2-6 2-6 Earliest priority application: Earliest priority application: 202111579636.2 202111579636.2 Application number Application number 2-7 2-7 Earliestpriority Earliest priority application: application: 2021-12-22 2021-12-22 Filing date Filing date
2-8en 2-8en Applicant name Applicant name Institute Instituteofof Biological andand Biological Medical Engineering, Medical Guangdong Engineering, Guangdong Academy Academy ofofSciences Sciences 2-8 2-8 Applicant name: Applicant name: NameName Latin Latin
2-9en 2-9en Inventor name Inventor name Huihui Su Huihui Su 2-9 2-9 Inventor name: Inventor name: NameName Latin Latin 2-10en 2-10en Inventiontitle Invention title RECOMBINANTCORYNEBACTERIUM RECOMBINANT CORYNEBACTERIUM GLUTAMICUM GLUTAMICUM FORFOR PRODUCING PRODUCING HIGH-PURITY HIGH-PURITY ISOMALTULOSE ISOMALTULOSE AT AT HIGH HIGH YIELD, YIELD, ANDAND APPLICATION APPLICATION THEREOF THEREOF 2-11 2-11 SequenceTotal Sequence TotalQuantity Quantity 1 1
3-1 3-1 Sequences Sequences 3-1-1 3-1-1 SequenceNumber Sequence Number
[ID][ID] 1 1
3-1-2 3-1-2 MoleculeType Molecule Type DNA DNA 3-1-3 3-1-3 Length Length 114 114 3-1-4 3-1-4 Features Features source1..114 source 1..114 Location/Qualifiers Location/Qualifiers mol_type=genomic DNA mol_type=genomic DNA organism=Pantoea organism=Pantoea dispersa dispersa NonEnglishQualifier Value NonEnglishQualifier Value 3-1-5 3-1-5 Residues Residues atgggtaagcaccgtcgcaa atgggtaage accgtcgcaa caattcaaac caattcaaac gcaactcgca gcaactcgca aggctgtagc aggctgtagc agcatctgca agcatctgca 60 60 gttgcgcttggaccaaccgc gttgcgcttg gaccaaccgc agctatcgcc agctatcgcc tccccagcac tccccagcac aggcagctga aggcagctga ggtt ggtt 114
Claims (10)
- CLAIMS What is claimed is: 1. A recombinant Corynebacteriumglutamicum for producing isomaltulose at a high yield, wherein the recombinant Corynebacterium glutamicum is integrated with an isomaltulose synthetase gene, and the isomaltulose synthase gene is as shown in NCBI Gene ID: AY223549.1.
- 2. The recombinant Corynebacteriumglutamicum according to claim 1, wherein a signal peptide gene is integrated in front of the isomaltulose synthetase gene, and the signal peptide gene has a nucleotide sequence as shown in SEQ ID NO.1.
- 3. The recombinant Corynebacterium glutamicum according to claim 1, wherein a starting strain for the recombinant Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13032.
- 4. A construction method of a recombinant Corynebacterium glutamicum for producing isomaltulose at a high yield, comprising: ligating a signal peptide gene as shown in SEQ ID NO. 1 and an isomaltulose synthetase gene as shown in NCBI Gene ID: AY223549.1 separately to an expression vector to obtain a recombinant vector, and transferring the recombinant vector into Corynebacterium glutamicum to obtain the recombinant Corynebacterium glutamicum for producing the isomaltulose at a high yield.
- 5. The construction method according to claim 4, wherein the expression vector is pEC-XK99E, with a promoter Ptc to control expression of the isomaltulose synthetase gene.
- 6. The construction method according to claim 5, wherein the isomaltulose synthetase gene is inserted between enzyme sites BamHI and XbaI of the pEC-XK99E, and the signal peptide gene is inserted between enzyme sites EcoRI and KpnI of the pEC-XK99E.
- 7. Application of the recombinant Corynebacteriumglutamicum according to any of claims 1 to 3 for preparation of any of the following: (1) an isomaltulose synthase or a preparation thereof; (2) an isomaltulose and a derivative thereof; and(3) isomaltulose-containing functional food.
- 8. The application according to claim 7, wherein a single colony of the recombinant Corynebacteriumglutamicum is inoculated into a CGXII culture medium, and cultured for 18-24 h at 25-35°C and 200-250 rpm to obtain a seed solution, and then the seed solution is added to the CGXII culture medium at a volume of 5 -10% of the culture medium, and cultured for not less than 18-24 h at 25-35°C and 200-250 rpm; or, the seed solution is added to a fermentation culture medium in a fermenter containing molasses and corn step liquor, at a volume of 5-10% of the culture medium, and fermented at 25-35°C and a pH value of 5-7, with dissolved oxygen controlled to be greater than 30%.
- 9. The application according to claim 8, wherein the fermentation culture medium contains 100-500 g/L of molasses and 1-25 g/L of corn steep liquor.
- 10. The application according to claim 9, wherein the fermentation culture medium contains 350 g/L of cane molasses and 12 g/L of corn steep liquor, or 400 g/L of beet molasses and 15 g/L of corn steep liquor.
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| PCT/CN2022/112647 WO2023115997A1 (en) | 2021-12-22 | 2022-08-16 | Recombinant corynebacterium glutamicum for producing isomaltulose, and application thereof |
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