WO2023115997A1 - 一种用于生产异麦芽酮糖的重组谷氨酸棒杆菌及其应用 - Google Patents

一种用于生产异麦芽酮糖的重组谷氨酸棒杆菌及其应用 Download PDF

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WO2023115997A1
WO2023115997A1 PCT/CN2022/112647 CN2022112647W WO2023115997A1 WO 2023115997 A1 WO2023115997 A1 WO 2023115997A1 CN 2022112647 W CN2022112647 W CN 2022112647W WO 2023115997 A1 WO2023115997 A1 WO 2023115997A1
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isomaltulose
corynebacterium glutamicum
recombinant
gene
molasses
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French (fr)
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苏慧慧
张平军
黄俊生
郭艺山
胡双岚
黄冬婷
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广东省科学院生物与医学工程研究所
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    • 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
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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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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    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Definitions

  • the present invention belongs to the field of metabolic engineering and fermentation engineering, and specifically relates to a recombinant Corynebacterium glutamicum capable of high-yield and high-purity isomaltulose and its application.
  • Isomaltulose is also known as palatinose, isomerized sucrose, isomalt, and barose. Its molecular formula is C 12 H 22 O 11 ⁇ H 2 O, and its molecular weight is 360. It is the ⁇ -1,6 Dextrose (6-o- ⁇ -D glucopyranosyl-D-fructose) linked by glycosidic bonds is a new type of natural functional sugar.
  • Isomaltulose not only has excellent properties such as pure sweet taste, low calorific value, low hygroscopicity, high safety and no dental caries, but also its metabolism in the body does not depend on insulin, so it can be eaten by people with high blood sugar, diabetes and obesity. Therefore, isomaltulose is considered to be an "ideal substitute" for sucrose, and has great application potential and market prospects in the fields of food, pharmaceuticals and chemicals.
  • Corynebacterium glutamicum (Corynebacterium glutamicum) is a Gram-positive bacterium, which is rod-shaped under the microscope, and the colony on the plate is round and light yellow. It has been widely used in the production of amino acids. Compared with Escherichia coli, Corynebacterium glutamicum is a food-grade safe strain certified by the US FDA, which has the advantages of high safety, low allergenicity, high stress resistance, and a relatively low probability of being contaminated by phages, so it is used in The field of genetic engineering plays an important role. Because there isomaltulose synthase in Corynebacterium glutamicum, there is no pathway to synthesize isomaltulose via inexpensive substrates. Therefore, only by highly expressing exogenous isomaltulose synthase can Corynebacterium glutamicum have the ability to synthesize isomaltulose.
  • the present invention expresses isomaltulose synthase in Corynebacterium glutamicum, introduces the signal peptide sequence at the multi-cloning site of the expression vector and does not affect the growth of Corynebacterium glutamicum on the basis of using molasses as the starting energy At the same time, the production of isomaltulose was improved.
  • the first object of the present invention is to provide a recombinant Corynebacterium glutamicum, which integrates the isomaltulose synthase gene, and the isomaltulose synthase gene is shown in NCBI Gene ID: AY223549.1.
  • the recombinant Corynebacterium glutamicum is integrated with a signal peptide gene, and the nucleotide sequence of the signal peptide gene is shown in SEQ ID NO.1.
  • the starting strain of the recombinant Corynebacterium glutamicum is Corynebacterium glutamicum ATCC13032.
  • the second object of the present invention is to provide a method for constructing the above-mentioned Corynebacterium glutamicum engineering bacteria, the nucleotide sequence is as shown in SEQ ID NO.1 signal peptide gene and as shown in NCBI Gene ID: AY223549.1
  • the indicated isomaltulose synthase genes were respectively connected to expression vectors to obtain recombinant vectors, and the recombinant vectors were transformed into Corynebacterium glutamicum, and recombinant Corynebacterium glutamicum was obtained through screening.
  • the expression vector is pEC-XK99E, and the expression of the isomaltulose synthase gene is controlled by the promoter P trc .
  • the isomaltulose synthase gene is inserted between the BamHI and XbaI enzyme sites of pEC-XK99E, and the signal peptide gene is inserted between the EcoRI and KpnI enzyme sites of pEC-XK99E.
  • the isomaltulose synthase is derived from Pantoea dispersus.
  • the steps of constructing the above-mentioned Corynebacterium glutamicum engineering bacteria are as follows: construct pEC-XK99E-UQ68J SIase (express isomaltulose synthase gene); construct pEC-XK99E-cgR0949 (express cgR0949 signal peptide); construct pEC- XK99E-cgR0949-UQ68J SIase (simultaneous expression of cgR0949 signal peptide and isomaltulose synthase gene); the expression vectors pEC-XK99E-UQ68J SIase and pEC-XK99E-cgR0949-UQ68J SIase were respectively transferred into the host Corynebacterium glutamicum ATCC13032 , respectively to obtain the Corynebacterium glutamicum strains that finally produce isomaltulose.
  • the third object of the present invention is to provide the application of the recombinant Corynebacterium glutamicum in the preparation of any of the following:
  • a single colony of the recombinant Corynebacterium glutamicum is inoculated into a CGXII medium, cultured at 25-35°C and 200-250rpm for 18-24h to obtain a seed liquid, and then the seed liquid is pressed into the medium Add 5-10% of the volume to CGXII medium, and culture at 25-35°C, 200-250rpm for no less than 18-24h.
  • the recombinant Corynebacterium glutamicum is inoculated into a medium containing CGXII, cultured at 25-35°C and 200-250rpm for 18-24h, and then 5-10% of the medium volume % added to the fermentation medium in the fermenter containing molasses and corn steep liquor, fermented at 25-35 ° C, pH 5-7, controlled dissolved oxygen greater than 30%.
  • the components of the fermenter contain 100-500 g/L of molasses and 1-25 g/L of corn steep liquor.
  • the fermentation medium contains 350 g/L of sugarcane molasses and 12 g/L of corn steep liquor; or 400 g/L of beet molasses and 15 g/L of corn steep liquor.
  • the molasses is waste molasses from the sugar industry, including but not limited to cane molasses, beet molasses, and sweet sorghum stalk juice.
  • the recombinant Corynebacterium glutamicum provided by the present invention can realize the extracellular accumulation of isomaltulose, and in a 7L fermenter, its isomaltulose output using sugarcane molasses as a carbon source reaches 169.29g/L, and sucrose
  • the conversion rate is 96.7%, its space-time yield rate is 2.26g/(L h), and the purity can reach 98%;
  • the isomaltulose output using sugar beet sucrose molasses as the carbon source reaches 166.76g/L, and the sucrose conversion rate is 97%, its space-time yield is 2.32g/(L h), and the purity can reach 98%, which is the highest yield and purity among the currently reported engineering bacteria;
  • the present invention recycles industrial waste molasses to realize low-cost and high-efficiency production of isomaltulose.
  • Figure 1 is a map of the recombinant pEC-XK99E-UQ68J SIase plasmid.
  • Fig. 2 is the map of constructing recombinant pEC-XK99E-cgR0949 plasmid.
  • Figure 3 is a map of the recombinant pEC-XK99E-cgR0949-UQ68J SIase plasmid.
  • Fig. 4 is the cell growth situation of different bacterial strains shake flask fermentation.
  • Fig. 5 is a graph showing the production process of isomaltulose in the supernatant of the shake flask fermentation of different strains.
  • Figure 6 shows the cell growth of different strains of shake flask fermentation after initial optimization of fermentation conditions.
  • Fig. 7 is a graph showing the production process of isomaltulose in the supernatant of shake flask fermentation of different strains after the initial optimization of fermentation conditions.
  • Fig. 8 is the optimization result of sugarcane molasses concentration and corn steep liquor concentration.
  • Figure 9 shows the optimization results of beet molasses concentration and corn steep liquor concentration.
  • Fig. 10 is the process of fermenting isomaltulose by the recombinant bacteria of the present invention in a 7L fermenter under the optimal concentration of sugarcane molasses.
  • Fig. 11 is the process of fermenting isomaltulose by the recombinant bacteria of the present invention in a 7L fermenter under the optimal concentration of sugar beet molasses.
  • High performance liquid chromatography (HPLC) detection method Waters 2695, RID detector, Sugar-ParkTMI (Waters, USA), mobile phase: ultrapure water, flow rate 0.5mL/min, column temperature 65 °C, injection volume is 20uL.
  • Seed activation medium (LBB) (g/L): Yeast powder 5, peptone 10, sodium chloride 5, brain heart infusion 10, the liquid volume is 30mL liquid/250mL conical flask.
  • Seed activation solid medium yeast powder 5, peptone 10, sodium chloride 5, brain heart extract 10, agar powder 20.
  • Competent medium (LBHI) (g/L): Yeast powder 5, peptone 10, sodium chloride 5, glycine 25, isoniazid 5, Tween 80 5mL, liquid volume is 50mL liquid/250mL conical flask.
  • Recovery medium LBHIS (g/L) after electric shock: yeast powder 5, peptone 10, sodium chloride 5, brain heart infusion 20, sorbitol 91.
  • Transformant detection solid medium yeast powder 5, peptone 10, sodium chloride 5, brain heart infusion 20, sorbitol 91, agar powder 20.
  • Seed medium g/L: molasses 20, corn steep liquor 15, KH 2 PO 4 1.5, pH 6.8-7.2.
  • Fermentation medium molasses 50, corn steep liquor 25, KH 2 PO 4 1.5, MgSO 4 0.8, CaCO 3 30, FeSO 4 0.2, pH6.8-7.2.
  • Optimized fermentation medium molasses 150, corn steep liquor 20, KH 2 PO 4 1.5, MgSO 4 0.8, CaCO 3 30, FeSO 4 0.2, pH6.8-7.2.
  • Sterilization conditions 120°C, 20min, all mediums are used for the detection of transformants or for the cultivation of recombinant bacteria, add 25mg/L kanamycin.
  • UQ68J SIase was expressed using plasmid pEC-XK99E as an expression vector.
  • plasmid PET-28A-UQ68J-SIASE Take the plasmid PET-28A-UQ68J-SIASE as the template (its granular sequences see SU H H, xu Ry, yang Z D, et al.Green synthesis of Isomaltulose from Cane Molasses by An Immobilized Recombinant Esch ERICHIA COLI Strain and Its Prebiotical [J] .LWT-Food Science and Technology,2021,143(43):111054.), using primers pF(5'-GGATCCGCAACGAATATACAAAAGT-3') and pR(5'-TCTAGATCAGTTCAGCTTATAGATCCCG-3'), using PrimeSTAR Max DNA polymerase
  • the PCR conditions were pre-denaturation at 95°C for 5 minutes; denaturation at 98°C for 10 seconds; anne
  • Digest the PCR product of the UQ68J SIase gene sequence obtained in the previous step with restriction endonucleases BamHI and XbaI, and the digestion system for recovery is: 10 ⁇ L of PCR product, 0.6 ⁇ L of BamHI, 0.6 ⁇ L of XbaI, 5 ⁇ L of 10X buffer, and 23.8 ⁇ L of double distilled water. Perform 1% agarose gel electrophoresis to detect the digested product and recover the target fragment. At the same time, the plasmid pEC-XK99E was subjected to the same double digestion treatment, and then the digested product was recovered by gel.
  • the insert and plasmid were ligated using T4 ligase. Mix the vector and the insert at a molar ratio of 1:1 to 1:9, add an equal amount of ligation mixing solution, and ligate at 22°C for 1 hour or overnight at 4°C. Then transform E.coli DH5 ⁇ competent cells (see TransGen Biotechn Escherichia coli Competent Kit Instructions for detailed preparation methods). The correct transformants of colony PCR were selected for sequencing verification to obtain recombinant plasmid pEC-XK99E-UQ68J SIase (Fig. 1).
  • PCR conditions were pre-denaturation at 95°C for 5 min; denaturation at 98°C for 10 s; annealing at 55°C for 15 s; extension at 72°C for 15 s, and 35 cycles of reaction; finally, extension at 72°C for 7 min.
  • the PCR product was recovered with a DNA purification kit to obtain the cgR0949 signal peptide sequence, the nucleotide sequence of which was: Atgggtaagcaccgtcgcaacaattcaaacgcaactcgcaaggctgtagcagcatctgcagttgcgcttggaccaaccgcagctatcgcctccccagcacaggcagctgaggtt (SEQ ID NO.1).
  • the digestion system for recovery is: 10 ⁇ L of PCR product, 0.6 ⁇ L of EcoRI, 0.6 ⁇ L of KpnI, 5 ⁇ L of 10X buffer, and 23.8 ⁇ L of double distilled water. Perform 2% agarose gel electrophoresis to detect the digested product and recover the target fragment. At the same time, the plasmid pEC-XK99E was subjected to the same double digestion treatment, and then the digested product was recovered by gel.
  • the insert and plasmid were ligated using T4 ligase. Mix the vector and the insert at a molar ratio of 1:1 to 1:15, add an equal amount of ligation mixing solution, and ligate at 22°C for 1 hour or overnight at 4°C. Then transform E.coli DH5 ⁇ competent cells (see TransGen Biotechn Escherichia coli Competent Kit Instructions for detailed preparation methods). The transformants with correct colony PCR were selected for sequencing verification to obtain the recombinant plasmid pEC-XK99E-cgR0949 ( FIG. 2 ).
  • Example 2 Using the method described in Example 1, using the plasmid pET-28a-UQ68J-SIase as a template, using primers pF and pR to perform PCR amplification to obtain the UQ68J SIase gene sequence.
  • the UQ68J SIase gene sequence PCR product was digested with restriction endonucleases BamHI and XbaI. Perform 1% agarose gel electrophoresis to detect the digested product and recover the target fragment.
  • the plasmid pEC-XK99E-cgR0949 was subjected to the same double digestion treatment, and then the digested product was recovered by gel.
  • the insert and plasmid were ligated using T4 ligase. Mix the vector and the insert at a molar ratio of 1:1 to 1:9, add an equal amount of ligation mixing solution, and ligate at 22°C for 1 hour or overnight at 4°C. Then transform E.coli DH5 ⁇ competent cells (see TransGen Biotechn Escherichia coli Competent Kit Instructions for detailed preparation methods). The correct transformant of colony PCR was selected for sequencing verification to obtain recombinant plasmid pEC-XK99E-cgR0949-UQ68J SIase (Fig. 3).
  • Embodiment 3 Preparation of recombinant Corynebacterium glutamicum
  • the recombinant plasmid pEC-XK99E-UQ68J SIase obtained in Example 1 and the recombinant plasmid pEC-XK99E-cgR0949-UQ68J SIase obtained in Example 2 were transformed into Corynebacterium glutamicum ATCC13032 bacterial strain by electric shock transformation method to obtain different recombinant Corynebacterium glutamicum.
  • Corynebacterium glutamicum ATCC13032 was inoculated in LBB medium, placed on a reciprocating shaker (180 rpm), cultured at 30°C for 20 hours, and the OD 562 reached 3.0.
  • Competent Corynebacterium glutamicum stored at -80°C, thawed in an ice bath.
  • the bacteria were cultured on a reciprocating shaker at 150 rpm at 30° C. for 1.5-2.0 h.
  • Verification of competence efficiency add 10 ⁇ L of sterile water as a negative control, no colonies, and 1 ⁇ L of plasmid pEC-XK99E as a positive control, a large number of colonies grow.
  • Example 4 Effect of signal peptide on the production of isomaltulose by recombinant Corynebacterium glutamicum
  • Corynebacterium glutamicum strains containing plasmids pEC-XK99E-UQ68J SIase and pEC-XK99E-cgR0949-UQ68J SIase with correct sequencing results were inoculated on streaked LBB plates in glycerol tubes (adding kanamycin sulfate 50mg/mL ), cultured at 200 rpm at 30°C for 20 hours, then picked a single colony and re-streaked the LBB plate until a large number of colonies grew.
  • the seed culture liquid was inoculated into the fermentation medium at an inoculation amount of 10%, and the recombinant bacteria containing the plasmid pEC-XK99E-UQ68J SIase was used as a control, and 6 repetitions were set up, and cultured at 200 rpm at 30°C for 80 hours.
  • the 80h isomaltulose output of the strain containing the signal peptide sequence plasmid pEC-XK99E-cgR0949-UQ68J SIase was 64g/L, compared to the control strain isomaltulose output of 56g/L , an increase of 14.28%.
  • Example 5 Effect of optimization of fermentation conditions on the production of isomaltulose by recombinant Corynebacterium glutamicum
  • composition ratio of fermentation medium and the inoculum ratio have a great influence on the accumulation of strain growth and metabolites, so on the basis of Example 4, the composition of the medium is changed into (g/L): molasses 150, corn steep liquor 20, pH 6.8-7.2, the inoculum size of the fermentation medium was adjusted so that the OD 562 was 1.8 after inoculation.
  • the recombinant Corynebacterium glutamicum containing pEC-XK99E-cgR0949-UQ68J SIase was tested, and the recombinant bacteria containing plasmid pEC-XK99E-UQ68J SIase was used as a control.
  • Other culture conditions and post-fermentation detection methods are consistent with Example 4.
  • the isomaltulose yields of the recombinant strains increased at 80 hours.
  • the isomaltulose yield of the strain containing the signal peptide sequence pEC-XK99E-cgR0949-UQ68J SIase was 73g/L at 80h, which was 17.72% higher than that of the control strain, which was 62g/L.
  • Example 6 Effects of different types of molasses on the growth of recombinant Corynebacterium glutamicum and the yield of isomaltulose
  • compositions distribution ratio and the inoculum ratio of the fermentation medium have a great impact on the growth of the strain and the accumulation of metabolites, so on the basis of Example 4, the composition of the medium is changed to (g/L): sugarcane molasses or beet molasses, corn pulp.
  • sugarcane molasses+corn steep liquor group the initial content of sugarcane molasses was 350g/L, and the initial content of corn steep liquor was 10g/L; for the beet molasses+corn steep liquor group, the initial content of beet molasses was 400g/L, and the initial content of corn steep liquor was 10g/L.
  • the initial contents are replaced by gradient contents.
  • the inoculum size of the fermentation medium was adjusted to make its OD 562 1.8 after inoculation.
  • Recombinant C. glutamicum containing pEC-XK99E-cgR0949-UQ68J SIase was tested. After 72 hours of fermentation, samples were taken to measure OD 600 and isomaltulose production.
  • the optimal concentration of sugarcane molasses is 350g/L
  • the concentration of corn steep liquor is 12g/L
  • the yield of recombinant bacteria isomaltulose of the present invention reaches 168.88g/L
  • the conversion rate of sucrose is 96.5%
  • its space-time yield is 2.82g/(L ⁇ h).
  • Combination 1 the concentration of sugarcane molasses is 350g/L, the concentration of corn steep liquor is 12g/L, combination 2: the concentration of beet molasses is 400g/L, and the concentration of corn steep liquor is 15g/L as the culture medium components of 7L fermenter, and the fermentation conditions are: 30°C, pH 6.0, dissolved oxygen level 1.0vvm, rotation speed 200rpm, take the bacterial liquid every 6h, measure OD 600 and isomaltulose production.

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Abstract

本发明公开了一种用于生产异麦芽酮糖的重组谷氨酸棒杆菌及其应用，属于代谢工程和发酵工程领域。本发明在谷氨酸棒杆菌中表达异麦芽酮糖合成酶，所述异麦芽酮糖合成酶的编码基因序列如NCBI Gene ID:AY223549.1所示，并在表达载体多克隆位点处引入SEQ ID NO.1所示的信号肽序列。本发明的重组菌能够在以糖蜜为出发能源的基础上，不影响谷氨酸棒杆菌生长情况的同时，提高异麦芽酮糖的产量。

Description

[根据细则37.2由ISA制定的发明名称]　一种用于生产异麦芽酮糖的重组谷氨酸棒杆菌及其应用 技术领域：
本发明本发明属于代谢工程和发酵工程领域，具体涉及一种高产高纯度异麦芽酮糖的重组谷氨酸棒杆菌及其应用。
背景技术：
蔗糖过量使用所带来的肥胖、动脉硬化、高血压、糖尿病、龋齿等疾病，已成为社会越来越关注的问题。异麦芽酮糖(Isomaltulose,Isomaltulose TM,
Figure PCTCN2022112647-appb-000001
Lylose)亦称帕拉金糖、异构蔗糖、益寿糖、巴糖，其分子式为C 12H 22O 11·H 2O，其分子质量为360，是葡萄糖和果糖以α-1，6糖苷键相连的右旋糖(6-o-α-D吡喃葡糖基-D-果糖)是一种天然的新型功能性糖。异麦芽酮糖不仅具有甜味纯正、热值低、吸湿性低、安全性高和不致龋齿等优异性质，而且其在体内的代谢不依赖于胰岛素，可供高血糖、糖尿病及肥胖人群食用。因此，异麦芽酮糖被认为是蔗糖的“理想替代品”，在食品、制药及化工等领域拥有巨大的应用潜力和市场前景。
目前利用廉价废弃生物质作为碳源发酵生产高附加质产品成为研究热点。而制糖工业中主要的副产物糖蜜，与其他废弃物如秸秆等木质纤维素原料相比，不需要进行预处理即含有大量的可发酵糖，其中绝大多数为蔗糖，同时含有无机盐、维生素等成分，可提供微生物生长代谢所需的影响物质，是十分具有潜力的廉价生物质资源。
谷氨酸棒杆菌(Corynebacterium glutamicum)是一种革兰氏阳性菌，在显微镜下呈现出棒杆状，在平板上菌落为圆形呈淡黄色，目前已经广泛用于氨基酸的生成。相比于大肠杆菌，谷氨酸棒杆菌属于美国FDA认证食品级安全菌株，其具有高安全性，低致敏性，高抗逆性， 而且受噬菌体污染的概率比较低等优点，因此它在基因工程领域发挥着重要作用。在谷氨酸棒杆菌中因为不存在异麦芽酮糖合成酶，不存在通过廉价底物合成异麦芽酮糖的途径。因此只有高效表达外源异麦芽酮糖合成酶才能使得谷氨酸棒杆菌具有合成异麦芽酮糖的能力。
发明内容：
本发明通过在谷氨酸棒杆菌中表达异麦芽酮糖合成酶，并在表达载体多克隆位点处引入信号肽序列后且在以糖蜜为出发能源的基础上不影响谷氨酸棒杆菌生长情况的同时也提高了异麦芽酮糖的产量。
本发明的第一个目的是提供一种重组谷氨酸棒杆菌，整合有异麦芽酮糖合成酶基因，所述的异麦芽酮糖合成酶基因如NCBI Gene ID:AY223549.1所示。
优选地，所述的重组谷氨酸棒杆菌，整合有信号肽基因，所述信号肽基因的核苷酸序列如SEQ ID NO.1所示。
优选地，所述的重组谷氨酸棒杆菌的出发菌株为谷氨酸棒杆菌ATCC13032。
本发明的第二个目的是提供一种构建上述谷氨酸棒杆菌工程菌的方法，将核苷酸序列如SEQ ID NO.1所示的信号肽基因和如NCBI Gene ID:AY223549.1所示的异麦芽酮糖合成酶基因分别连接至表达载体上获得重组载体，将所述重组载体转入谷氨酸棒杆菌中，经过筛选得到重组谷氨酸棒杆菌。
优选地，所述的表达载体为pEC-XK99E，以启动子P trc控制异麦芽酮糖合成酶基因的表达。
优选地，所述异麦芽酮糖合成酶基因插入至pEC-XK99E的BamHI与XbaI酶位点间，所述信号肽基因插入至pEC-XK99E的EcoRI与KpnI酶位点间。
优选地，所述的异麦芽酮糖合成酶来源于分散泛菌。
优选地，构建上述谷氨酸棒杆菌工程菌的步骤如下：构建pEC-XK99E-UQ68J SIase(表达异麦芽酮糖合成酶基因)；构建pEC-XK99E-cgR0949(表达cgR0949信号肽)；构建pEC-XK99E-cgR0949-UQ68J SIase(同时表达cgR0949信号肽和异麦芽酮糖合成酶基因)；将表达载体pEC-XK99E-UQ68J SIase和pEC-XK99E-cgR0949-UQ68J SIase分别转入宿主谷氨酸棒杆菌ATCC13032，分别得到最终生成异麦芽酮糖的谷氨酸棒杆菌菌株。
本发明的第三个目的是提供所述的重组谷氨酸棒杆菌在制备以下任一所述中的应用：
(1)异麦芽酮糖合成酶或其制剂；
(2)产异麦芽酮糖及其衍生物；
(3)含异麦芽酮糖的功能性食品。
本发明的一种实施方式中，将所述的重组谷氨酸棒杆菌单菌落接种至CGXII培养基，25-35℃、200-250rpm培养18-24h得到种子液，然后将种子液按培养基体积的5-10％添加至CGXII培养基中，在25-35℃、200-250rpm下培养不少于18-24h。
本发明的另一种实施方式中，将所述的重组谷氨酸棒杆菌接种到含有CGXII培养基中，25-35℃、200-250rpm培养18-24h，然后按培养基体积的5-10％添加至含糖蜜和玉米浆的发酵罐中的发酵培养基，在25-35℃、pH为5-7下发酵，控制溶氧大于30％。
优选地，所述发酵罐成分含有糖蜜100-500g/L，玉米浆1-25g/L。
优选地，所述的发酵培养基含有甘蔗糖蜜350g/L，玉米浆12g/L；或甜菜糖蜜400g/L，玉米浆15g/L。
更优选地，所述的糖蜜为制糖工业废弃糖蜜，包括但不限于甘蔗糖蜜、甜菜糖蜜、甜高 粱秸秆汁。
与现有技术相比，本发明的有益效果：
(1)本发明提供的重组谷氨酸棒杆菌可实现异麦芽酮糖在胞外积累，在7L发酵罐中，以甘蔗糖蜜为碳源的其异麦芽酮糖产量达到169.29g/L，蔗糖转化率为96.7％，其时空产率为2.26g/(L·h)，纯度可达98％；以甜菜蔗糖蜜为碳源的其异麦芽酮糖产量达到166.76g/L，蔗糖转化率为97％，其时空产率为2.32g/(L·h)，纯度可达98％，是在目前报道的工程菌中得率和纯度同时最高的；
(2)本发明提供的重组谷氨酸棒杆菌构建方法简便，适用于工业化生产应用；
(3)本发明循环利用工业废弃物糖蜜，实现低成本且高效生产异麦芽酮糖。
附图说明
图1为构建重组pEC-XK99E-UQ68J SIase质粒图谱。
图2为构建重组pEC-XK99E-cgR0949质粒图谱。
图3为构建重组pEC-XK99E-cgR0949-UQ68J SIase质粒图谱。
图4为不同菌株摇瓶发酵的细胞生长情况。
图5为不同菌株摇瓶发酵上清液中异麦芽酮糖的产量过程图。
图6为发酵条件初步优化后不同菌株摇瓶发酵的细胞生长情况。
图7为发酵条件初步优化后不同菌株摇瓶发酵上清液中异麦芽酮糖的产量过程图。
图8为甘蔗糖蜜浓度与玉米浆浓度的优化结果。
图9为甜菜糖蜜浓度与玉米浆浓度的优化结果。
图10为本发明重组菌在甘蔗糖蜜最优浓度条件下的7L发酵罐中发酵产异麦芽酮糖过程。
图11为本发明重组菌在甜菜糖蜜最优浓度条件下的7L发酵罐中发酵产异麦芽酮糖过程。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
异麦芽酮糖的测定方法：
高效液相色谱(HPLC)检测法：Waters 2695，RID检测器，Sugar-ParkTMI(Waters，USA)，流动相：超纯水，流速0.5mL/min，柱温65℃，进样体积为20uL。
种子活化培养基(LBB)(g/L)：酵母粉5，蛋白胨10，氯化钠5，脑心浸液10，装液量为30mL液体/250mL三角锥形瓶。
种子活化固体培养基(LBB)(g/L)：酵母粉5，蛋白胨10，氯化钠5，脑心浸液10，琼脂粉20。
感受态培养基(LBHI)(g/L)：酵母粉5，蛋白胨10，氯化钠5，甘氨酸25，异烟肼5，Tween 80 5mL，装液量为50mL液体/250mL三角锥形瓶。
电击后恢复培养基LBHIS(g/L)：酵母粉5，蛋白胨10，氯化钠5，脑心浸液20，山梨醇91。
转化子检出固体培养基(g/L)：酵母粉5，蛋白胨10，氯化钠5，脑心浸液20，山梨醇91，琼脂粉20。
种子培养基(g/L)：糖蜜20，玉米浆15，KH 2PO 41.5，pH6.8-7.2。
发酵培养基(g/L)：糖蜜50，玉米浆25，KH 2PO 41.5，MgSO 40.8，CaCO 330，FeSO 40.2，pH6.8-7.2。
优化发酵培养基(g/L)：糖蜜150，玉米浆20，KH 2PO 41.5，MgSO 40.8，CaCO 330，FeSO 4 0.2，pH6.8-7.2。
灭菌条件：120℃，20min，所有培养基用于转化子检出或用于重组菌培养时加入25mg/L卡那霉素。
实施例1 构建重组质粒pEC-XK99E-UQ68J SIase
使用质粒pEC-XK99E作为表达载体来表达UQ68J SIase。以质粒pET-28a-UQ68J-SIase为模板(其质粒序列见Su H H,Xu RY,Yang Z D,et al.Green synthesis of Isomaltulose from Cane Molasses by an Immobilized Recombinant Escherichia coli strain and Its PrebioticActivity[J].LWT-Food Science and Technology,2021,143(43):111054.)，使用引物pF(5’-GGATCCGCAACGAATATACAAAAGT-3’)和pR(5’-TCTAGATCAGTTCAGCTTATAGATCCCG-3’)，使用PrimeSTAR Max DNA聚合酶扩增UQ68J SIase序列，PCR条件为95℃预变性5min；98℃变性10s；55℃退火15s；72℃延伸120s，反应35个循环；最后72℃延伸7min。PCR产物用DNA纯化试剂盒回收，获得的PCR产物，其核苷酸序列为：
Figure PCTCN2022112647-appb-000002
Figure PCTCN2022112647-appb-000003
用限制性内切酶BamHI和XbaI酶切上一步所得UQ68J SIase基因序列PCR产物，回收酶切体系为：PCR产物10μL，BamHI 0.6μL，XbaI0.6μL，10X buffer 5μL，加入双蒸水23.8μL。进行1％琼脂糖凝胶电泳检测酶切产物并回收目的片段。同时将质粒pEC-XK99E做同样的双酶切处理，然后胶回收酶切产物。
连接插入片段和质粒，采用T4连接酶进行连接。将载体和插入片段按1：1到1：9的摩尔比混合，加入等量的连接混合溶液，在22℃下连接1h或4℃过夜。然后转化E.coli DH5α感受态细胞(感受态制备方法详见TransGen Biotechn大肠杆菌感受态试剂盒说明书)。挑选菌落PCR正确的转化子进行测序验证，得到重组质粒pEC-XK99E-UQ68J SIase(图1)。
实施例2 构建重组质粒pEC-XK99E-cgR0949-UQ68J SIase
以购买的质粒pAU3质粒为模板(pAU3质粒序列见文献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.)，使用引物CRgF(5’-GAATTCatgggtaagcaccgtcgcaacaat-3’)和CRgR(5’-GGTACCaacctcagctgcctgtgctgggga-3’)，使用PrimeSTAR Max DNA聚合酶扩增cgR0949序列。PCR条件为95℃预变性5min；98℃变性10s；55℃退火15s；72℃延伸15s，反应35个循环；最后72℃延伸7min。PCR产物用DNA纯化试剂盒回收，获得cgR0949信号肽序列，其核苷酸序列为：Atgggtaagcaccgtcgcaacaattcaaacgcaactcgcaaggctgtagcagcatctgcagttgcgcttggaccaaccgcagctatcgcctccccagcacaggcagctgaggtt(SEQ ID NO.1)。
用限制性内切酶EcoRI和KpnI酶切上一步所得cgR0949信号肽序列PCR产物，回收酶切体系为：PCR产物10μL，EcoRI 0.6μL，KpnI 0.6μL，10X buffer 5μL，加入双蒸水23.8μL。进行2％琼脂糖凝胶电泳检测酶切产物并回收目的片段。同时将质粒pEC-XK99E做同样的双酶切处理，然后胶回收酶切产物。
连接插入片段和质粒，采用T4连接酶进行连接。将载体和插入片段按1：1到1：15的摩尔比混合，加入等量的连接混合溶液，在22℃下连接1h或4℃过夜。然后转化E.coli DH5α感受态细胞(感受态制备方法详见TransGen Biotechn大肠杆菌感受态试剂盒说明书)。挑选菌落PCR正确的转化子进行测序验证，得到重组质粒pEC-XK99E-cgR0949(图2)。
使用实施例1所述的方法，以质粒pET-28a-UQ68J-SIase为模板，使用引物pF和pR进行PCR扩增获得UQ68J SIase基因序列。用限制性内切酶BamHI和XbaI酶切UQ68J SIase基因序列PCR产物。进行1％琼脂糖凝胶电泳检测酶切产物并回收目的片段。同时将质粒pEC-XK99E-cgR0949做同样的双酶切处理，然后胶回收酶切产物。
连接插入片段和质粒，采用T4连接酶进行连接。将载体和插入片段按1：1到1：9的摩尔比混合，加入等量的连接混合溶液，在22℃下连接1h或4℃过夜。然后转化E.coli DH5α感受态细胞(感受态制备方法详见TransGen Biotechn大肠杆菌感受态试剂盒说明书)。挑选菌落PCR正确的转化子进行测序验证，得到重组质粒pEC-XK99E-cgR0949-UQ68J SIase(图3)。
实施例3 重组谷氨酸棒杆菌的制备
分别将实施例1获得的重组质粒pEC-XK99E-UQ68J SIase和实施例2获得的重组质粒pEC-XK99E-cgR0949-UQ68J SIase通过电击转化法转化到谷氨酸棒杆菌ATCC13032菌株中，获得不同的重组谷氨酸棒杆菌。
谷氨酸棒杆菌电转化感受态的制备：
(1)谷氨酸棒杆菌ATCC13032接种于LBB培养基，置于往复式摇床(180rpm)上，30℃培养20h，OD 562达到3.0。
(2)10％转接入感受态培养基(LBHI)OD 562达到0.3，置于往复式摇床(200rpm)上，30℃培养4h，OD 562达到0.8-1.0。
(3)收集菌液，并冰浴20min，6000rpm，4℃离心10min，弃上清。
(4)用50mL预冷10％充分悬浮菌体，6000rpm，4℃离心10min，弃上清，重复洗涤3次。
(5)用500μL预冷10％甘油重悬细胞，1.5mL无菌离心管分装，每管约100μL。
(6)-80℃保持备用，为保证感受态的转化效率最好现配现用，且放置时间不超过1周。
谷氨酸棒杆菌的电击转化：
(1)-80℃保存的谷氨酸棒杆菌感受态，冰浴中融化。
(2)分别加入0.5-2μL上述两种质粒与之混匀，冰浴20min。
(3)加入预冷的0.2cm电击杯中，EcN2模式下电击1次。
(4)迅速加入46℃预热的LBHIS 0.5mL混匀并转移到新的1.5mL无菌离心管中，46℃水浴6min，后立即置于冰中10min。
(5)将菌体置于往复式摇床以150rpm，30℃培养1.5-2.0h。
(6)8000rpm，离心1min，涂布到加有卡那霉素抗性的转化子检出平板中，于30℃恒温培养箱中培养24h。
(7)感受态效率验证：加入10μL无菌水作为阴性对照，无菌落，阳性对照1μL质粒pEC-XK99E，长出大量菌落。
实施例4 信号肽对重组谷氨酸棒杆菌生产异麦芽酮糖产量的影响
将测序结果正确的分别含有质粒pEC-XK99E-UQ68J SIase和pEC-XK99E-cgR0949-UQ68J SIase的重组谷氨酸棒杆菌菌株于甘油管分别接种划线LBB平板(添加硫酸卡那霉素50mg/mL)，30℃下200rpm培养20h后挑选单菌落重新划线LBB平板直至长出大量菌落。
接种一环单菌落至种子培养基，30℃下200rpm培养20h。按10％的接种量将种子培养液接种至发酵培养基中，以含有质粒pEC-XK99E-UQ68J SIase的重组菌为对照，并设置6次重复，30℃下200rpm培养80h。每隔10h取一次菌液，测定OD 562及异麦芽酮糖生成量，测定方法及计算公式参考文献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.。
由图4可以看出80h内不同菌株摇瓶发酵的细胞生长趋势相似。
如图5所示，同样条件下，含有信号肽序列的质粒pEC-XK99E-cgR0949-UQ68J SIase的菌株80h异麦芽酮糖产量为64g/L，相比对照菌株异麦芽酮糖的产量56g/L，提高了14.28％。
实施例5 发酵条件优化对重组谷氨酸棒杆菌生产异麦芽酮糖产量的影响
发酵培养基的成分配比及接种量比例对菌株生长和代谢产物的积累影响很大，因此在实施例4的基础上将培养基成分改为(g/L):糖蜜150，玉米浆20，pH6.8-7.2，其发酵培养基接种量调整为接种后使其OD 562为1.8。将pEC-XK99E-cgR0949-UQ68J SIase的重组谷氨酸棒杆菌进行试验，以含有质粒pEC-XK99E-UQ68J SIase的重组菌为对照。其他培养条件及发酵后检出方法同实施例4一致。
由图6可以看出80h内不同菌株摇瓶发酵的细胞生长趋势相似。
如图7所示，发酵条件优化后，重组菌80h异麦芽酮糖产量均有所提高。相同条件下，含有信号肽序列的质粒pEC-XK99E-cgR0949-UQ68J SIase的菌株80h异麦芽酮糖产量为73g/L，相比对照菌株异麦芽酮糖的产量62g/L，提高了17.72％。
实施例6 不同糖蜜种类对重组谷氨酸棒杆菌生长及产异麦芽酮糖产量的影响
发酵培养基的成分配比及接种量比例对菌株生长和代谢产物的积累影响很大，因此在实施例4的基础上将培养基成分改为(g/L):甘蔗糖蜜或甜菜糖蜜，玉米浆。甘蔗糖蜜+玉米浆组设定甘蔗糖蜜初始含量350g/L，玉米浆初始含量10g/L；甜菜糖蜜+玉米浆组设定甜菜糖蜜初始含量400g/L，玉米浆初始含量10g/L。优化组分含量时，其初始含量替换为梯度含量。
其发酵培养基接种量调整为接种后使其OD 562为1.8。将pEC-XK99E-cgR0949-UQ68J SIase的重组谷氨酸棒杆菌进行试验。发酵72h后取样测定OD 600及异麦芽酮糖生成量。
由图8可知，甘蔗糖蜜最优浓度为350g/L，玉米浆浓度为12g/L，本发明重组菌异麦芽酮糖产量达到168.88g/L，蔗糖转化率为96.5％，其时空产率为2.82g/(L·h)。
由图9可知，甜菜糖蜜最优浓度为400g/L，玉米浆浓度为15g/L，本发明重组菌异麦芽酮糖产量达到166.88g/L，蔗糖转化率为97％，其时空产率为2.78g/(L·h)。
分别以组合1：甘蔗糖蜜浓度为350g/L、玉米浆浓度为12g/L，组合2：甜菜糖蜜浓度为400g/L、玉米浆浓度为15g/L作为7L发酵罐培养基成分，发酵条件：30℃，pH6.0，溶氧位1.0vvm，转速为200rpm，每隔6h取一次菌液，测定OD 600及异麦芽酮糖生成量。
图10可知，7L发酵罐中，本发明重组菌异麦芽酮糖产量达到169.29g/L，蔗糖转化率为96.7％，其时空产率为2.26g/(L·h)，纯度可达98％。
图11可知，7L发酵罐中，本发明重组菌异麦芽酮糖产量达到166.76g/L，蔗糖转化率为97％，其时空产率为2.32g/(L·h)，纯度可达98％。
进一步地，将本发明重组菌与现有的产异麦芽酮糖菌株比较，结果如表1所示。
表1 不同产异麦芽酮糖菌株的比较
Figure PCTCN2022112647-appb-000004
Figure PCTCN2022112647-appb-000005
Figure PCTCN2022112647-appb-000006
由表2可知，本发明重组菌生产的异麦芽酮糖是目前报道中得率和纯度同时最高的。
Figure PCTCN2022112647-appb-000007

Claims (10)

  1. 一种高产异麦芽酮糖的重组谷氨酸棒杆菌(Corynebacterium glutamicum)，其特征在于，整合有异麦芽酮糖合成酶基因，所述的异麦芽酮糖合成酶基因如NCBI Gene ID:AY223549.1所示。
  2. 根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于，在异麦芽酮糖合成酶基因前整合有信号肽基因，所述信号肽基因的核苷酸序列如SEQ ID NO.1所示。
  3. 根据权利要求1所述的重组谷氨酸棒杆菌，其特征在于，所述重组谷氨酸棒杆菌的出发菌株为谷氨酸棒杆菌ATCC13032。
  4. 一种高产异麦芽酮糖的重组谷氨酸棒杆菌的构建方法，其特征在于，将核苷酸序列如SEQ ID NO.1所示的信号肽基因和如NCBI Gene ID:AY223549.1所示的异麦芽酮糖合成酶基因分别连接至表达载体上获得重组载体，将所述重组载体转入谷氨酸棒杆菌中，获得高产异麦芽酮糖的重组谷氨酸棒杆菌。
  5. 根据权利要求4所述的构建方法，其特征在于，所述的表达载体为pEC-XK99E，以启动子P trc控制异麦芽酮糖合成酶基因的表达。
  6. 根据权利要求5所述的构建方法，其特征在于，所述异麦芽酮糖合成酶基因插入至pEC-XK99E的BamHI与XbaI酶位点间，所述信号肽基因插入至pEC-XK99E的EcoRI与KpnI酶位点间。
  7. 权利要求1-3任一所述的重组谷氨酸棒杆菌在制备以下任一所述中的应用：
    (1)异麦芽酮糖合成酶或其制剂；
    (2)产异麦芽酮糖及其衍生物；
    (3)含异麦芽酮糖的功能性食品。
  8. 根据权利要求7所述的应用，其特征在于，将所述的重组谷氨酸棒杆菌单菌落接种至CGXII培养基，25-35℃、200-250rpm培养18-24h得到种子液，然后将种子液按培养基体积的5-10％添加至CGXII培养基中，在25-35℃、200-250rpm下培养不少于18-24h；或将种子液按培养基体积的5-10％添加至含糖蜜和玉米浆的发酵罐中的发酵培养基中，在25-35℃、pH为5-7下发酵，控制溶氧大于30％。
  9. 根据权利要求8所述的应用，其特征在于，所述的发酵培养基含有糖蜜100-500g/L，玉米浆1-25g/L。
  10. 根据权利要求9所述的应用，其特征在于，所述的发酵培养基含有甘蔗糖蜜350g/L，玉米浆12g/L；或甜菜糖蜜400g/L，玉米浆15g/L。
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