CN118703409A - A WYH-1 strain with high isobutanol production - Google Patents
A WYH-1 strain with high isobutanol production Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to an isobutanol high-yield strain WYH-1 and a yield achieved in a production process thereof. The isobutanol high-yield strain is obtained by taking JCL16 as an initial strain to carry out ARTP mutation and screening through BmoR sensing system. The strain has higher isobutanol tolerance, and the isobutanol concentration reaches 70-80g/L after 100 hours of detection by a 5L fermentation tank experiment. The high-yield strain can be used for industrial production of isobutanol, and improves economic benefit.
Description
Technical field:
The invention belongs to the technical field of bioengineering, and particularly relates to a high-yield strain WYH-1 of isobutanol and application thereof in a production process.
The background technology is as follows:
Isobutanol (2-Methyl-1-Propanol) is a colorless, flammable, organic compound with a specific odor. The isomers are n-butanol, sec-butanol and tert-butanol. In nature, isobutanol can be obtained by natural fermentation of carbohydrates, and can also be used as a byproduct in the organic matter degradation process. The n-butyraldehyde and the iso-butyraldehyde are obtained from the oxo synthesis of propylene in industry, and the product is hydrogenated and separated to obtain the iso-butanol.
Isobutanol plays an important role in the chemical industry, and can be used as an important prodrug, an intermediate of various chemicals, and an important raw material for biofuels due to the reproducibility and chemical stability. In the current dual carbon policy context, the market for isobutanol will continue to expand in the future as the demand for clean and alternative energy sources increases, as well as the demand for environmentally friendly, low volatility solvents in the industry.
The traditional chemical synthesis of isobutanol adopts the oxo synthesis method of isobutanol propylene. The high pressure oxo process has been replaced by the low pressure oxo process with rhodium as catalyst because of poor selectivity and high byproducts such as propane and high boiling point substances. The current industrial production is highly dependent on petroleum industry, and has the problems of serious environmental pollution, large price fluctuation, limited scale and the like. Meanwhile, when the isobutanol is produced by fermentation in a cell factory, if the butanol concentration in the fermentation broth reaches 13-14g/L, the growth of the cells is inhibited, resulting in extremely low product concentration at the end of fermentation. Therefore, screening out an excellent strain with high butanol tolerance is one of the effective methods for solving this phenomenon.
The invention utilizes BmoR biological sensor to screen and obtain the high-yield strain of the isobutanol from the ARTP mutagenesis library, and provides an effective way for modeling and producing the isobutanol by using biological regulation.
The invention comprises the following steps:
the invention aims to provide a method for producing high-concentration isobutanol bacterial strain, which utilizes BmoR biological sensors to screen from an ARTP mutagenesis library, and finally passes fermentation tank tests, and the concentration of isobutanol reaches 70-80g/L after fermentation for 100 hours.
Description of the drawings:
FIG. 1 production of isobutanol by fermentation of isobutanol producing strain WYH-1
FIG. 2 construction of fermentation tank for isobutanol production
The specific embodiment is as follows:
The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
The isobutanol high-yield strain provided by the invention is a mutant strain starting from escherichia coli JCL16, and the genes KivD and AdhA from 2-ketoisovalerate to isobutanol are expressed in a heterologous mode, and meanwhile, genes alsS, ilvC and ilvD from pyruvate to 2-ketoisovalerate synthesis paths are expressed in a coexpression mode.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The invention will be further illustrated by the following examples.
EXAMPLE 1 production of isobutanol with WYH-1 Strain Using fermenter
Preparation of a culture medium: 2L of fermentation medium, feed medium and metal ion mixed solution are prepared. The fermentation medium consists of: 40g/L glucose, 3g/L (NH 4) 2SO4, 14.6g/L K2HPO4,4g/L KH2PO4,2.2g/L sodium citrate, 8g/L yeast extract, 1.25g/L MgSO4.7H2O, 0.1g/L Amp,0.05g/L Kana,0.025g/L Cm and 1mL/L trace metals solution (containing 14.1g/L EDTA,2.5g/L CoCl2·6H2O,15g/L MnCl2·4H2O,1.5g/L CuCl2·2H2O,3g/L H3BO3,,2.1g/L Na2MoO4·2H2O,33.8g/L Zn(CH3COO)2·2H2O and 80g/L FeCl3.6H2O). The feed medium included 500g/L glucose, 1.25g/L MgSO4.7H2O, sterilized at 115℃for 20 minutes, and then added with 0.1g/L Amp,0.05g/L Kana,0.025g/L Cm and 0.1mM IPTG at the super clean bench.
Preparing a fermentation tank: firstly, cleaning each part of the fermentation tank, adding distilled water to about 2/3 volume, then setting a program to sterilize the fermentation tank in situ, and sucking out the distilled water after 20 minutes at 121 ℃. The feeding culture medium, ammonia water (pH value is regulated) bottle, feeding bottle, pH electrode and dissolved oxygen electrode are connected, and after sealing with newspaper, the sterilizing is carried out. After sterilization, ammonia water and feed supplement liquid are respectively added into the corresponding bottles in an ultra clean bench, and the newspaper seal is detached and connected to the fermentation tank device. First, the pH electrode is corrected by a pH standard solution, then the corrected pH electrode and the dissolved oxygen electrode are inserted into a fermentation tank, and 2L of culture medium is added. Starting the fermentation system and ventilating the fermentation tank, wherein the ventilation rate is 1vvm (air volume/min, ventilation rate/total volume/min of the fermentation tank), performing in-situ sterilization again, sterilizing at 115 ℃ for 20 min, correcting dissolved oxygen to be 100% at the rotating speed of 1000rpm and the temperature of 100 ℃, and preparing for inoculation when the temperature is reduced to about 40 ℃.
Preparing seed liquid: first, the strain WYH-1 was streaked on a plate, and then single colonies were picked up in 5mL of LB (plus antibiotics Amp, kana and Cm), and cultured overnight at 37℃and 220rpm to obtain a first seed solution. 1mL of the primary seed solution was inoculated to 100mL of LB (Amp, kana and Cm), cultured at 37℃and 220rpm for 2 to 3 hours to reach an OD600 of about 0.8, and 50mL of the seed solution was inoculated to 2L of medium.
Inoculating and fermenting: firstly, igniting alcohol cotton at an inoculation port to ensure that flames are distributed at the inoculation port, then opening the inoculation port, sequentially inoculating 50mL of seed liquid, 2mL of amp, kana, cm and metal ion mother liquor into a fermentation tank, and fermenting for 2 hours at the speed of 800rpm at the temperature of 37 ℃. Then inoculated with IPTG (final concentration 0.1 mM) and the temperature was adjusted to 30℃and rotated to 600rpm. The timer was started with the time of addition of IPTG being 0 hours. The whole process maintains the pH at 6.8 by automatically supplementing ammonia water, and maintains the glucose concentration at more than 5g/L by manually adjusting a feeding peristaltic pump. After the fermentation time reaches 12 hours, the air flow rate is increased to 3vvm, so that the isobutanol in the fermentation broth is blown out to be condensed and recovered.
Sampling and processing: sampling the fermentation tank bacterial liquid every two hours from 0 hour, blocking the air outlet during sampling, and collecting about 2mL of fermentation tank liquid from the sampling tube. The collection bottles a, B, C were sampled every 4 hours, about 2mL was sampled and the volume of collection bottle a was measured and recorded while distilled water from collection bottles B and C was changed. The OD600 of the fermentation broth was measured, the conversion coefficient of OD600 to biomass (g/L) was 0.23, and the remaining broth was centrifuged at 12000rpm for 10 minutes. After centrifugation, the glucose content of the supernatant was determined by DNS, 197.5. Mu.L of supernatant was collected, 2.5. Mu.L of 10% n-amyl alcohol was added, and samples from flasks A, B, and C were collected, 197.5. Mu.L of 10% n-amyl alcohol was directly collected, and gas chromatography was performed. The isobutanol content measured in flasks a, B, C was converted to a yield corresponding to 2L fermentation volume, total yield at each time point = isobutanol yield of fermentation broth + sum of yields in flasks at all times before this time point.
Yield determination: isobutanol was quantified using a Gas Chromatograph (GC) using a DB-FFAP capillary column (30 m 0.32mm 0.25 μm; agilent technology). The GC oven temperature was initially maintained at 80 ℃ for 3 minutes and after increasing to 230 ℃ with a gradient of 115 ℃ per minute, maintained for 1 minute. The carrier gas was nitrogen and the injector and detector temperatures were maintained at 250 ℃ and 280 ℃, respectively. The sample injection amount of the sample injector is 0.2 mu L, and the split ratio is 30:1. the isobutanol content of the fermentation broth is measured to be 70-80g/L, and the result is shown in figure 1.
Claims (6)
1. A WYH-1 strain for high yield of isobutanol is an Escherichia coli JCL16 mutant, and comprises genes alsS, ilvC and ilvD from pyruvic acid to 2-ketoisovalerate and genes Kivd and AdhA from 2-ketoisovalerate to isobutanol.
2. The WYH-1 strain capable of producing isobutanol at a high yield according to claim 1, wherein the strain is characterized by:
(1) Bacterial strain preparation: the method is characterized in that the escherichia coli JCL16 is mutated through a normal temperature and pressure plasma mutagenesis system, and then the escherichia coli JCL16 is obtained through screening by using a BmoR biosensor system.
(2) Capacity of production: the isobutanol content in the fermentation broth is 70-80g/L as analyzed by gas chromatography.
3. The WYH-1 strain for high isobutanol production according to claim 2, wherein the strain is obtained by the steps of:
(1) Activation of the starting strain JCL 16: streaking the original strain JCL16 in a fresh LB solid culture medium, culturing a single colony at 37 ℃, inoculating the single colony into an LB liquid culture medium, and culturing to obtain the activated JCL16 thallus.
(2) Art mutagenesis: washing the activated thalli with sterile physiological saline, re-suspending thalli, properly diluting thalli to proper concentration, and constructing a mutation library of the original strain JCL16 under a treatment source to obtain mutant monoclonal.
(3) Screening, modifying and identifying isobutanol high-yield strains: and (3) carrying out fluorescence screening on the monoclonal by utilizing BmoR biosensor system, and screening a mutant with fluorescence intensity being more than 2 times higher than that of the original strain.
(4) Optimization of pathways and fermentation conditions: plasmids expressing enzymes alsS, ilvC and ilvD of pyruvic acid to 2-ketoisovalerate are transformed in the mutant, and an isobutanol high-yield strain WYH-1 is constructed. After optimizing the medium, isobutanol was produced by fermenting WYH-1 strain through a fermenter and the yield of isobutanol was detected by GC.
4. A strain WYH-1 for high isobutanol production according to claim 3, characterized in that the fermentation medium used in step (4) consists of: 40g/L glucose, 3g/L (NH 4) 2SO4, 14.6g/L K2HPO4,4g/L KH2PO4,2.2g/L sodium citrate, 8g/L yeast extract, 1.25g/L MgSO4.7H2O, 0.1g/L Amp,0.05g/L Kana,0.025g/L Cm and 1mL of a trace metal solution comprising 14.1g/L EDTA,2.5g/L CoCl2·6H2O,15g/L MnCl2·4H2O,1.5g/L CuCl2·2H2O,3g/L H3BO3,,2.1g/LNa2MoO4·2H2O,33.8g/L Zn(CH3COO)2·2H2O and 80g/L FeCl3.6H2O.
5. A strain WYH-1 for high isobutanol production according to claim 3, wherein the seed liquid required for fermentation in the 5L fermentor in step (4) is treated as follows: firstly, activating strain WYH-1, inoculating to 5mL of LB culture medium, culturing overnight, inoculating 1mL of primary seed solution to 100mL of LB, culturing at 37 ℃ and 220rpm for 2 to 3 hours to ensure that the OD600 reaches about 0.8, and inoculating 50mL of seed solution to 2L of culture medium.
6. A strain WYH-1 for high isobutanol production according to claim 3, wherein the fermentation process in step (4) is as follows: after seed liquid inoculation, the temperature was set at 37℃and the rotational speed was 800rpm for fermentation for 2 hours, and then IPTG was inoculated at a final concentration of 0.1mM (and the temperature was adjusted to 30℃and the rotational speed was adjusted to 600rpm, and the pH was maintained at 6.8 throughout the course and the glucose concentration was maintained at 5g/L or more).
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Citations (6)
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| US20110262982A1 (en) * | 2010-04-27 | 2011-10-27 | The Regents Of The University Of California | Acetolactate synthase having 2-ketoisovalerate decarboxylase activity and uses thereof |
| WO2013192237A1 (en) * | 2012-06-18 | 2013-12-27 | The Regents Of The University Of California | Escherichia coli engineered for isobutyraldehyde production |
| CN110452847A (en) * | 2019-08-19 | 2019-11-15 | 山东汇冠康博生物科技有限公司 | A kind of YH-76S bacterial strain of high yield isobutanol and preparation method thereof |
| CN110885777A (en) * | 2019-08-19 | 2020-03-17 | 山东汇冠康博生物科技有限公司 | Method for screening isobutanol high-yield strain by using Bmor biosensor |
| JP2023019310A (en) * | 2021-07-29 | 2023-02-09 | 国立研究開発法人産業技術総合研究所 | Acetolactate synthase and method for producing isobutanol using the same |
| CN116023447A (en) * | 2021-10-25 | 2023-04-28 | 北京理工大学 | A wide-area biosensor based on BmoR mutants |
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- 2024-05-27 CN CN202410661343.6A patent/CN118703409A/en active Pending
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|---|---|---|---|---|
| US20110262982A1 (en) * | 2010-04-27 | 2011-10-27 | The Regents Of The University Of California | Acetolactate synthase having 2-ketoisovalerate decarboxylase activity and uses thereof |
| WO2013192237A1 (en) * | 2012-06-18 | 2013-12-27 | The Regents Of The University Of California | Escherichia coli engineered for isobutyraldehyde production |
| CN110452847A (en) * | 2019-08-19 | 2019-11-15 | 山东汇冠康博生物科技有限公司 | A kind of YH-76S bacterial strain of high yield isobutanol and preparation method thereof |
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| JP2023019310A (en) * | 2021-07-29 | 2023-02-09 | 国立研究開発法人産業技術総合研究所 | Acetolactate synthase and method for producing isobutanol using the same |
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