Breeding method for recombinant escherichia coli by utilizing cellobiose
By expressing the cellobiose phosphorylase gene in E. coli and performing gradient acclimation, the problem of low utilization efficiency of cellobiose in traditional E. coli is solved, and the effect of efficient utilization of cellobiose is achieved, and biomass energy and biology is improved. Production efficiency of base materials.
Patent Information
- Application Number
- CN202411571759.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-06
- Publication Date
- 2025-01-14
AI Technical Summary
Traditional E. coli has low efficiency in utilizing cellobiose, resulting in low efficiency and complex production processes of biomass energy and bio-based materials, making it difficult to efficiently utilize cellulose-derived sugars.
By cloning the cellobiose phosphorylase gene celA into the plasmid pMD19-PldhA-TldhA and expressing it in E. coli, the cellobiose utilization efficiency was improved. The recombinant plasmid was transferred into the strain by electroshock or chemical transformation methods, and multiple gradients were performed Domestic culture, optimize enzyme activity and temperature conditions, and improve the strain's metabolic ability to cellobiose.
The utilization efficiency of E. coli on cellobiose was significantly improved, and the growth rate was increased by 66.67-83.33%. The utilization rate of cellobiose in the fermentation stage reached more than 98%, and the domestication success rate exceeded 93.33%, achieving more efficient and sustainable biomass Production of energy and bio-based materials.
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Abstract
Description
Technical Field
[0001] This patent relates to the fields of molecular biology, genetic engineering and industrial fermentation technology, and specifically to a recombinant Escherichia coli strain obtained through genetic modification and domestication breeding, which can effectively use cellobiose as a carbon source for growth. Background Art
[0002] Cellobiose is one of the main products of cellulose degradation. It is a disaccharide formed by two glucose molecules connected by a β-1,4-glycosidic bond. In the production of biomass energy and bio-based materials, the hydrolysis of cellulose is a key step, and the utilization efficiency of cellobiose directly affects the economy and sustainability of the entire bioconversion process. When using these raw materials, traditional Escherichia coli has a low affinity for cellobiose and the presence of other easily available carbon sources (such as glucose), resulting in low efficiency and complex processes. Therefore, the development of recombinant strains that can efficiently utilize cellobiose is the key to achieving a transition from a "hydrocarbon economy" to a "sugar economy."
[0003] Glucose produced by cellulose hydrolysis can be utilized by microorganisms, but it is more challenging to utilize other sugars produced by cellulose hydrolysis, such as cellobiose, xylose, and arabinose (Intracellular cellobiosemetabolism and its applications in lignocellulose-based biorefineries, Bioresour Technol, 2017, 239: 496-506.). The utilization efficiency of these sugars directly affects the economy and sustainability of the entire bioconversion process. As an industrial production host, Escherichia coli has the characteristics of rapid growth and efficient metabolism, but its natural strain cannot effectively utilize cellulose-derived sugars (New insights into transport capability of sugars and its impact on growth from novel mutants of Escherichia coli, Applied Microbiology and Biotechnology, 2020, 104(4): 1463-1479.).
[0004] In order to overcome these limitations, the present invention chooses to regulate the expression of cellobiose phosphorylase. The cellobiose phosphorylase gene celA is cloned into the plasmid pMD19 through the promoter PldhA and the terminator TldhA to obtain the recombinant plasmid pMD19-PldhA-celA-TldhA, and the recombinant plasmid pMD19-PldhA-celA-TldhA is transformed into the above-mentioned Escherichia coli strain host to overexpress the key enzyme encoding gene for cellobiose utilization, and the domestication is completed, so that the growth rate of the strain is increased by 66.67-83.33%. When using cellulosic material as raw material (or cellobiose as the only carbon source), the recombinant strain can use cellobiose to grow and the OD 600 On this basis, the iterative domestication of strains improves the utilization efficiency of cellobiose, so that cellobiose can be used by E. coli to the maximum extent. 600 The highest value can reach 6.27, the utilization rate of cellobiose in the fermentation stage is above 98%, and the domestication success rate is above 93.33%, thus achieving a more efficient and sustainable conversion process in the production of biomass energy and bio-based materials. Summary of the invention
[0005] The present invention aims to provide a method for domesticating and breeding recombinant Escherichia coli that can efficiently utilize cellobiose, transforming Escherichia coli to be able to directly metabolize and utilize cellobiose as a carbon source, and improving the utilization efficiency of cellobiose through domestication and breeding.
[0006] To achieve the above purpose, the present invention discloses the following technical contents: A method for breeding a recombinant Escherichia coli strain that utilizes cellobiose, characterized in that the recombinant strain is an Escherichia coli carrying a recombinant plasmid pMD19-PldhA-celA-TldhA, wherein the cellobiose phosphorylase gene celA from natural cellulose-decomposing bacteria, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence of which is shown in SEQ ID NO.2, is cloned into the promoter downstream and terminator upstream of the pMD19-PldhA-TldhA plasmid, and is expressed in Escherichia coli, including but not limited to Escherichia coli W3110, K12, MG1655, and other Escherichia coli strains used for fermentation production.
[0007] Proceed as follows: a. Using Escherichia coli as the starting strain, the recombinant plasmid pMD19-PldhA-celA-TldhA was transferred into the starting strain by electroporation or chemical transformation; b. Preparation of bacterial suspension: A single colony of Escherichia coli containing plasmid pMD19-PldhA-celA-TldhA is inoculated into 1-2 bottles of sterilized LB medium, and activation culture is continuously performed at 30-37°C and 150-200 r / min for 8-12 h to obtain a preliminary activated bacterial suspension, the preliminary activated bacterial suspension is centrifuged in a centrifuge, the supernatant is discarded to obtain a bacterial liquid, the bacterial liquid is washed with an inorganic salt medium or ultrapure water, the centrifugation and washing steps are repeated three times, the bacterial liquid is placed in 2-3 bottles of inorganic salt medium, additional trace elements and 3-5 g / L cellobiose carbon source are supplemented, and the OD of the bacterial liquid is measured. 600 The value was adjusted to 0.050, and activation culture was continued at 30-37°C and 150-200 r / min; c. Gradient acclimation: Transfer the primary bacterial suspension: Take 1 portion of the primary bacterial suspension and transfer it to 2-3 bottles of inorganic salt culture medium without a carbon source, add 5-8 g / L cellobiose solution to the inorganic salt culture medium, and continue stirring at 33-37°C and 180-200 r / min for acclimation and culture for 1 week to obtain a secondary bacterial suspension; d. Repeat the transfer operation of steps b and c five times, and make the culture temperature after each transfer correspond to 28, 30, 37, and 42°C, respectively. After the last transfer is completed, select the group with the best bacterial density and continue to acclimate and culture for 24 hours to obtain six bacterial suspensions containing plasmid pMD19-PldhA-celA-TldhA. Take out one portion of the six bacterial suspensions, mix it with 30% glycerol in a volume ratio of 1:1, and store it under ultra-low temperature conditions to obtain a successfully acclimated recombinant bacterial freeze-dried preservation. The Escherichia coli is W3110, K12, MG1655, or other Escherichia coli used for fermentation production.
[0008] The components and mass concentrations of the inorganic salt culture medium are: 0.5% to 1.0% cellobiose, 0.5% to 2.5% disodium hydrogen phosphate dodecahydrate, 0.02% to 0.1% sodium chloride, 0.1% to 1.0% potassium dihydrogen phosphate, and 0.01% to 0.5% ammonium chloride; the solvent is deionized water, and the pH of the inorganic salt culture medium is 7.0; the components and mass concentrations of the LB culture medium are: 5g / L yeast extract, 10g / L tryptic peptone, 10g / L sodium chloride, and 1.5g / L agar powder, the solvent is ultrapure water, the pH of the LB culture medium is 7.0, the sterilization temperature is 120-125°C, and the sterilization time is 20-30min.
[0009] The initial OD of the inoculated bacterial suspension 600The pH value is controlled at 0.05-0.12, the acclimation temperature is 30-37°C, the stirring speed is 150-200 rpm, and the ultra-low temperature is -80°C. When the acclimated cellobiose phosphorylase activity is higher than 7 U / mL, the acclimation success rate is high. The centrifugal speed of the resuspension treatment is 4000 rpm, and the centrifugal treatment time is 5 minutes.
[0010] The promoter and terminator for intracellular expression of cellobiose phosphorylase (CelA) described in the present invention include but are not limited to the promoter (PldhA, nucleotide sequence such as SEQ ID NO.3) and terminator (TldhA, nucleotide sequence such as SEQ ID NO.4) of D-lactate dehydrogenase.
[0011] The present invention further discloses the application of the method for domestication and breeding of domesticated recombinant Escherichia coli strains for improving the efficiency of cellobiose utilization. Experimental results show that the domesticated recombinant Escherichia coli strains have significantly improved the efficiency of cellobiose utilization. Compared with the undomesticated strains, the domesticated strains showed a higher substrate conversion rate in the fermentation experiment. This result shows that the domestication process effectively enhances the strain's ability to metabolize cellobiose and optimizes its fermentation characteristics.
[0012] The more detailed transformation of the present invention includes the following aspects: Genetic Engineering: The exogenous cellobiose phosphorylase gene (CelA) was introduced by selecting and cloning the CelA gene from natural cellulose-decomposing bacteria. The codon preference principle of Escherichia coli was used to optimize the original CelA sequence to codons preferred by Escherichia coli using codon optimization software. The restriction endonuclease exclusion sites were BamHI and EcoRI. After full gene synthesis, the CelA gene was cloned into the pMD19-PldhA-TldhA plasmid and transferred into the starting Escherichia coli strain by electroporation to construct a recombinant strain.
[0013] Acclimation process optimization: Cultivate in an inorganic salt medium with cellobiose as the sole carbon source, and screen and domesticate a CelA overexpression strain that can efficiently utilize cellobiose. Cultivate at 30-37℃ for 24-110 h, OD 600 Reached 4.65-6.22, compared with the OD of the untamed strain 600 1.62-2.89, and the growth rate increased by 66.67-93.33%.
[0014] Furthermore, a control group was set up, and the starting strain without the celA plasmid was used as a blank control for synchronous domestication. This strain could not grow in an inorganic salt medium with cellobiose as the sole carbon source.
[0015] Optimization, the effect of different promoters on celA expression: There are significant differences in the expression level of celA under different promoters. By comparing different promoters including but not limited to T7 promoter, PldhA (D-lactate dehydrogenase promoter), LacI promoter (lactose promoter), AraC promoter (arabinose promoter), using the extracellular PhoA promoter, it was found that the extracellular expression enzyme activity of CelA was poor, only 0.57 U / mL. When the PhoA promoter was removed, the enzyme activity of CelA increased by 1.10 U / mL. After changing to the D-lactate dehydrogenase promoter, it was found that the enzyme activity of celA was the highest when expressed intracellularly, reaching 16.26 U / mL, Furthermore, by optimizing the CelA enzyme activity level, it was found that when the cellobiose phosphorylase activity was lower than 2 U / mL, the acclimation period was prolonged and the utilization efficiency was reduced. When the cellobiose phosphorylase activity was higher than 7 U / mL, the acclimation period was shortened and the acclimation success rate was increased to more than 90%.
[0016] Set the temperature gradient. After transferring the bacterial suspension to the inorganic salt medium, the culture temperature was set to 28, 30, 37, and 42°C, and three parallels were made for each group. Under the conditions of 28 and 30°C, the bacterial cells grew slowly. After 48 h, the OD 600 At about 1.2, although the bacterial growth rate at 42°C was faster, the plasmid stability was poor. At 37°C, the bacterial growth rate was most suitable, and the OD 600 At about 3.03, the plasmid was stable after the second transfer, which was the optimal acclimation temperature. After establishing the optimal promoter, the best optimized temperature, and the most suitable acclimation enzyme activity, the iterative temperature control acclimation experiment began. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the plasmid map of pMD19-PldhA-celA-TldhA; Figure 2 This is the plate verification picture after the recombinant bacteria were domesticated (cellobiose is the only carbon source); Figure 3 The growth of cellobiose transformants of the CelA series domesticated strains (cellobiose is the only carbon source); Figure 4 This is the growth of a series of Escherichia coli strains after cellobiose acclimation (cellobiose is the only carbon source); Figure 5 is the conversion rate of cellobiose (cellobiose is the only carbon source) of domesticated E. coli strains; Figure 6 is the success rate of cellobiose domestication of E. coli strains (cellobiose is the only carbon source); Figure 7This is the growth status of blank control of cellobiose acclimation of Escherichia coli strains (cellobiose is the only carbon source). DETAILED DESCRIPTION
[0018] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available.
[0019] The cellobiose phosphorylase (CelA) used in the present invention is selected and cloned from natural cellulose decomposing bacteria. The original sequence of CelA (nucleotide sequence information SEQ ID NO.1, amino acid sequence SEQ ID NO.2) is optimized to the preferred codons of Escherichia coli by codon optimization software according to the codon preference principle of Escherichia coli. The restriction endonuclease exclusion site is Bam HI and Eco RI, whole gene synthesis.
[0020] The plasmids used in the present invention include, but are not limited to, pet28a, pSK, pMD18, and pMD19; preferably, pMD19-PldhA-TldhA plasmid.
[0021] Using the codon preference principle of E. coli, the original sequence of celA (nucleotide sequence SEQ ID NO. 1, amino acid sequence SEQ ID NO. 2) was optimized to the preferred codons of E. coli by codon optimization software, and the restriction endonuclease exclusion site was Bam HI and Eco RI; plasmid pMD19- PldhA-TldhA Bam HI+ Eco The recombinant transformants were obtained by double digestion with RⅠ and ligation with the optimized codon celA, followed by heat shock transformation with T4 DNA enzyme at 16 ℃ for 4 h.
[0022] The present invention and embodiments relate to promoter and terminator nucleotide sequences.
[0023] PldhA promoter sequence: SEQ ID NO.3; TldhA promoter sequence: SEQ ID NO.4 Example
[0024] The main experimental methods used in the present invention are as follows: Gene amplification DNA amplification was performed in 200 μL PCR thin-wall tubes. PCR amplification conditions were: 1× (95°C for 5 min); 30× (94°C for 30 s, 60°C for 30 s, 72°C for 300 s); 1× (72°C for 10 min). Unless otherwise specified, all PCR reactions were performed using LA Taq DNA polymerase.
[0025] The gene and plasmid were connected by T4 DNA enzyme at 16 ℃ for 4 h and then heat-shocked for transformation.
[0026] Extraction of plasmid DNA Plasmid DNA was extracted using a plasmid extraction kit from Sigma after cell walls were lysed with a certain concentration of lysozyme.
[0027] Electrotransformation: Electrotransform the celA plasmid into the expression strain (1) Inoculation: Streak three zones in the glycerol tube onto LB plates (Amp resistance, 100 mg / mL) and incubate at 37 °C for 12 h. (2) Primary shake flask: Select a single colony and inoculate it into 50 mL LB liquid medium (inoculate 2 flasks, and 1 flask is used to monitor bacterial growth OD 600 The other bottle was used to inoculate the secondary shake flask) and cultured at 37 °C for 12 h (3) Secondary shake flask: Take one of the flasks and test its OD value. When the OD value is between 3.0 and 5.0, take 1 mL of bacterial solution from another flask and transfer it to new LB liquid medium (Amp resistance). Inoculate two flasks and use one flask to monitor bacterial growth OD. 600 , and the other bottle was used for preparation and cultured at 37 ℃ for 1.5 h (4) When OD 600 When the value reaches about 0.70, place the flask in an ice bath for 15 min, and then heat at 4 °C, 8000 r / min, for 10 min. (5) Clean the electrophoresis cup, wash it three times with sterile water and three times with anhydrous ethanol, place the electrophoresis cup on ice to pre-cool, and evaporate for 30 minutes. (6) Wash with 25 mL pre-cooled sterile water, 4°C, 6000 r / min, 5 min, repeat once (7) Wash with 25 mL of pre-cooled 10% glycerol, 4°C, 6000 r / min, 5 min, repeat once (8) Pipette 95 μL of bacterial cells and 5 μL of plasmid mixture and transfer to an electroporation cup (1800 V, 5 ms). (9) Immediately after electroporation, add 900 μL LB liquid for recovery and transfer to a 1.5 mL EP tube. (10) Place the EP tube in a 37°C incubator and incubate for 2 h. (11) Take out the EP tube and put it into the centrifuge, centrifuge at 4000 r / min for 5 min, and keep 100 μL for coating. (12) Place the coated resistant plate in an incubator at 37 °C for 12 h, select transformants and streak them densely, extract the plasmid to verify the plasmid size, inoculate the correct plasmid onto the LB plate, and subculture until the single colony size is consistent.
[0028] Functional verification: Inoculate the recombinant bacteria on LB plates by streaking or spotting, culture at 37°C for 12-18h, then pick a single colony and inoculate it on an M9 plate with cellobiose as a carbon source, divide the plate into two and inoculate the control group (the starting strain without celA plasmid) in the other half, observe the growth of the two strains at 37°C for 24-72 hours, the strain containing celA can grow on the cellobiose plate, and no colonies are generated in the control group. Secretory expression and enzyme activity determination of cellobiose phosphorylase in the recombinant bacteria, and select the most suitable enzyme activity for domestication.
[0029] The recombinant bacteria were inoculated on LB plates by streaking or spotting, cultured at 37°C for 12-18 h, and then a single colony was picked and inoculated into LB liquid culture medium at 150-200 r / min and cultured at 37°C for 12 h. 600 The inoculum size was 0.05 and transferred to M9 medium with glycerol as carbon source. The culture was carried out at 150-200 r / min and 37°C for 12 h. The bacterial solution was centrifuged to obtain the precipitate and the cells were broken to determine the enzyme activity.
[0030] Add ddH2O to a 50mL centrifuge tube for washing. Place the bacteria in an EP tube in an ice box. Turn on the ultrasonic disruptor and set the time to 15-30 min and the power to 150-300W. Clean the probe (ultrasonicate in ddH2O for about 10 s). After drying, insert the probe deep into the bacteria and start ultrasonication. End the ultrasonication after observing the color change of the bacteria.
[0031] Cellobiose phosphorylase (CelA) is an enzyme that can catalyze the reaction of cellobiose with inorganic phosphate to generate glucose-1-phosphate and glucose. The following is a method for determining the enzymatic activity of cellobiose phosphorylase of the present invention, covering reagents, equipment, steps and result analysis.
[0032] The chemical equation for the reaction catalyzed by cellobiose phosphorylase is: Cellobiose + Pi ⇌ Glucose-1-phosphate + Glucose. In this reaction, the measurement of the generated glucose-1-phosphate or consumed cellobiose can be used to calculate the enzyme activity.
[0033] Enzyme activity assay reagents: cellobiose; inorganic phosphate (Pi); enzyme solution: extracted from recombinant bacteria or purified CelA; enzyme inhibitors: if necessary, known inhibitors can be used to confirm enzyme activity; indicator: DNS is dinitrosalicylic acid, DNS undergoes redox reaction with reducing sugar to generate 3-amino-5-nitrosalicylic acid, which is brown-red under boiling conditions.] Enzyme activity definition: The activity of cellobiose phosphorylase is defined as the rate of converting cellobiose into glucose and glucose-1-phosphate (G1P) per hour at 35°C and pH 6.5. This reaction usually uses inorganic phosphate (Pi) as the donor of the phosphate group.
[0034] Prepare the reaction system: 100 µL of cellobiose substrate; 100 µL of inorganic phosphate; enzyme solution: crude cellobiose phosphorylase solution obtained after cell disruption; mix well and stir gently.
[0035] Reaction conditions: In a constant temperature water bath, keep the reaction system at 35°C and pH 6.5 for 30-60 minutes. The specific time can be adjusted according to the actual situation. Reaction termination: After the reaction is completed, the reaction is terminated by heating or adding inhibitors. Product analysis: The generated glucose-1-phosphate is analyzed by DNS method or biosensor method.
[0036] If the DNS method is used, add the reaction mixture to the DNS reagent, heat for 5-7 minutes, cool, and then measure the absorbance at 550 nm using a spectrophotometer.
[0037] Calculate enzyme activity: Calculate the concentration of glucose-1-phosphate generated in the reaction based on the relationship between the sample absorbance and the standard curve. Calculate enzyme activity (unit: U / mL) using the following formula:
[0038] According to the calculated enzyme activity results, the enzyme activities under different experimental conditions were compared, and the strains under six groups of enzyme activities, 0-1 U / mL, 2-3 U / mL, 4-5 U / mL, 5-6 U / mL, 6-7 U / mL, and 7-8 U / mL, were selected for domestication experiments to analyze the factors affecting enzyme activity on domestication. The experiment found that when the cellobiose phosphorylase activity was lower than 2 U / mL, the domestication period was extended, the utilization efficiency was reduced, and the domestication success rate was low. When the cellobiose phosphorylase activity was higher than 7 U / mL, the domestication period was shortened and the domestication success rate was increased to more than 90%.
[0039] Note: All experimental equipment and reagents should be kept as free of contamination as possible. Enzyme activity is significantly affected by temperature and pH and needs to be strictly controlled. Reaction time and substrate concentration should be adjusted according to the experimental design and enzyme activity.
[0040] Optimal acclimation temperature selection: set the temperature gradient, the culture temperature was set to 28, 30, 37, 42 ° C, and three parallels were made for each group. Under 28 and 30 ° C conditions, the bacterial cells grew slowly, and the OD after 48 h was 600 At about 1.2, although the growth rate of the bacteria at 42°C is faster, it is not conducive to the stability of the plasmid, and there are problems such as plasmid loss after secondary transfer. The above findings show that the growth rate of the bacteria is most suitable at 37°C. After 48 h, OD 600 At around 3.03, the plasmid was stably present after the second transfer, which was the optimal acclimation temperature.
[0041] Acclimation experiment: Inoculate the recombinant bacteria on LB plates by streaking or spotting, culture at 37°C for 12-18 h, then pick a single colony and inoculate it into LB liquid culture medium, continue to culture for 12-18 h, wait until the OD reaches above 3.0 (enzyme activity 7 U / mL), resuspend the bacteria 2-3 times with inorganic salt culture medium or sterile water, 4000 r / min, and transfer to M9 medium with cellobiose as carbon source (cellobiose 0.5%-1.0%, disodium hydrogen phosphate dodecahydrate 0.5%-2.5%, sodium chloride 0.02%-0.1%, potassium dihydrogen phosphate 0.1%-1.0%, ammonium chloride 0.01%-0.5%; pH 7.0) at 150-200 rpm, 37°C to start acclimation culture, take samples every 12 h to measure the cell density and residual cellobiose (such as Figures 4 and 5 ).
[0042] Several common Escherichia coli W3110, K12, MG1655, production strain 1, production strain 2, production strain 3, etc. were selected for domestication experiments. The domestication success rate was as follows: Figure 6 As shown, a control group without celA was set up to conduct the acclimation experiment simultaneously. The results are shown in Figure 7 shown.
[0043] The CelA strain is obtained by electroporating the cellobiose phosphorylase plasmid pMD19-PldhA-CelA-TldhA into the starting strain. It is Amp-resistant and has a cellobiose shake flask growth cycle of 108 h.
[0044] CelA1 is the first generation of domesticated strain for cellobiose adaptation experiment using CelA as the starting strain. It is Amp-resistant and has a cellobiose shake flask growth cycle of 96 h.
[0045] CelA2 is the second generation domesticated strain for cellobiose adaptation experiment based on CelA1. It is Amp-resistant and has a cellobiose shake flask growth cycle of 60 h.
[0046] CelA3 is the third generation domesticated strain for cellobiose adaptation experiment based on CelA2. It is Amp-resistant and has a cellobiose shake flask growth cycle of 48 h.
[0047] CelA4 is the fourth generation domesticated strain for cellobiose adaptation experiment based on CelA3. It is Amp-resistant and has a cellobiose shake flask growth cycle of 36 h.
[0048] CelA5 is the fifth generation domesticated strain for cellobiose adaptation experiment based on CelA4. It is Amp-resistant and has a cellobiose shake flask growth cycle of 24 h.
[0049] For the plate verification experiment of domestication results, the domestication first generation strain CelA1 and the domestication fifth generation strain CelA5 were selected.
[0050] Set the starting strain CelA1 and the untamed strain CelA as the control group, divide a plate into four parts, draw lines in three areas and make giant colonies in parallel. The results are as follows Figure 2 100 uL of the deglycerolized tube bacteria was spread on four cellobiose plates and cultured for 48 hours. The growth was shown in Figure 3 shown.
[0051] The fermentation method in the fermenter is as follows: the pretreated biomass (Arundo donax) is used as the carbon source of the fermentation medium, the seed liquid of the production strain is inoculated into the fermentation medium at an inoculation rate of 1-10%, the temperature is controlled at 37°C, the stirring speed is adjusted to 200-1000r / min (gradually increased according to the dissolved oxygen), ammonia water is added to maintain the pH at 7.0, and the fermentation cycle is 100-110h.
[0052] The pretreatment method of Phragmites australis is as follows: the absolute dry weight of Phragmites australis is mixed with 1.5% sodium hydroxide solution at a solid-liquid ratio of 1:10, sterilized at 115°C for 1 h, the pH is adjusted to 6.0-7.5, deionized water with a volume of 1-1.5 times that of the sodium hydroxide solution is added, mixed, and sterilized again to obtain a fermentation medium for a shake flask; the pretreatment method of Phragmites australis for a fermenter is as follows: the absolute dry weight of Phragmites australis is mixed with 1.5% sodium hydroxide solution at a solid-liquid ratio of 1:10, sterilized at 115°C for 1 h, the pH is adjusted to 5.0, and sterilized again to obtain a fermentation medium for a fermenter.
[0053] The fermentation results showed that the utilization efficiency of cellobiose by the domesticated strain was significantly improved, showing a higher conversion rate, realizing the utilization of cellulose and the conversion of biomass by Escherichia coli, and is expected to promote the optimization and development of related industrial fermentation processes.
[0054] The present invention provides a recombinant Escherichia coli that efficiently utilizes cellobiose, a domestication method thereof, and an application idea, provides a method for domesticating Escherichia coli that utilizes cellobiose with a simple process, mild conditions, and low experimental requirements, and provides an application of Escherichia coli fermenting non-food biomass materials with a simple process and mild conditions. There are many methods and approaches to specifically implement the technical solution, and the above is only a preferred embodiment of the present invention.
Claims
1. A method for breeding a recombinant Escherichia coli strain that utilizes cellobiose, characterized in that The recombinant strain is an Escherichia coli carrying the recombinant plasmid pMD19-PldhA-celA-TldhA. The strain is a cellobiose phosphorylase gene celA from natural cellulose-decomposing bacteria, the nucleotide sequence of which is shown in SEQ ID NO.1 and the amino acid sequence of which is shown in SEQ ID NO.2; cloned into the downstream of the promoter and the upstream of the terminator of the pMD19-PldhA-TldhA plasmid, and expressed in Escherichia coli, including but not limited to Escherichia coli W3110, K12, MG1655, and other Escherichia coli strains used for fermentation production.
2. A promoter and terminator for intracellular expression of cellobiose phosphorylase (CelA) including but not limited to a promoter (PldhA, nucleotide sequence such as SEQ ID NO.3) and a terminator (TldhA, nucleotide sequence such as SEQ ID NO.4) of D-lactate dehydrogenase.
3. The method for breeding a recombinant Escherichia coli strain that utilizes cellobiose according to claim 1, characterized in that Proceed as follows: a. Using Escherichia coli as the starting strain, the recombinant plasmid pMD19-PldhA-celA-TldhA was transferred into the starting strain by electroporation or chemical transformation; b. Preparation of bacterial suspension: A single colony of Escherichia coli containing plasmid pMD19-PldhA-celA-TldhA is inoculated into 1-2 bottles of sterilized LB medium, and activation culture is continuously performed at 30-37°C and 150-200 r / min for 8-12 h to obtain a preliminary activated bacterial suspension, the preliminary activated bacterial suspension is centrifuged in a centrifuge, the supernatant is discarded to obtain a bacterial liquid, the bacterial liquid is washed with an inorganic salt medium or ultrapure water, the centrifugation and washing steps are repeated three times, the bacterial liquid is placed in 2-3 bottles of inorganic salt medium, additional trace elements and 3-5 g / L cellobiose carbon source are supplemented, and the OD of the bacterial liquid is measured. 600 The value was adjusted to 0.050, and activation culture was continued at 30-37°C and 150-200 r / min; c. Gradient acclimation: Transfer the primary bacterial suspension: Take 1 portion of the primary bacterial suspension and transfer it to 2-3 bottles of inorganic salt culture medium without a carbon source, add 5-8 g / L cellobiose solution to the inorganic salt culture medium, and continue stirring at 33-37°C and 180-200 r / min for acclimation and culture for 1 week to obtain a secondary bacterial suspension; d. Repeat the transfer operation of steps b and c five times, and make the culture temperature after each transfer correspond to 28, 30, 37, and 42°C, respectively. After the last transfer is completed, select the group with the best bacterial density and continue to culture for 24 hours to obtain six bacterial suspensions containing plasmid pMD19-PldhA-celA-TldhA. Take out one portion of the six bacterial suspensions, mix them with 30% glycerol in a volume ratio of 1:1, and store them under ultra-low temperature conditions to obtain a freeze-dried preservation of successfully domesticated recombinant bacteria.
4. The method for domesticating and breeding a recombinant strain of Escherichia coli according to claim 1, characterized in that: The Escherichia coli is W3110, K12, MG1655, or other Escherichia coli used for fermentation production.
5. The method for domesticating and breeding a recombinant strain of domesticated Escherichia coli according to claim 3, characterized in that: The components and mass concentrations of the inorganic salt culture medium are: 0.5% to 1.0% cellobiose, 0.5% to 2.5% disodium hydrogen phosphate dodecahydrate, 0.02% to 0.1% sodium chloride, 0.1% to 1.0% potassium dihydrogen phosphate, and 0.01% to 0.5% ammonium chloride; the solvent is deionized water, and the pH of the inorganic salt culture medium is 7.0; the components and mass concentrations of the LB culture medium are: 5g / L yeast extract, 10g / L tryptic peptone, 10g / L sodium chloride, and 1.5g / L agar powder, the solvent is ultrapure water, the pH of the LB culture medium is 7.0, the sterilization temperature is 120-125°C, and the sterilization time is 20-30min.
6. The method for domesticating and breeding a recombinant strain of domesticated Escherichia coli according to claim 3, characterized in that: The initial OD of the inoculated bacterial suspension 600 The acclimation temperature is 30-37°C, the stirring speed is 150-200 rpm, and the ultra-low temperature is -80°C.
7. The method for domesticating and breeding a recombinant strain of domesticated Escherichia coli according to claim 3, characterized in that: When the activity of the domesticated cellobiose phosphorylase is higher than 7 U / mL, the domestication success rate is higher.
8. The method for domesticating and breeding a recombinant strain of domesticated Escherichia coli according to claim 3, characterized in that: The centrifugal speed of the resuspension treatment is 4000 rpm, and the centrifugal treatment time is 5 minutes.
9. Application of the method for domesticating and breeding the recombinant strain of domesticated Escherichia coli according to claim 1 in improving the efficiency of cellobiose utilization.