CN110452847A - A kind of YH-76S bacterial strain of high yield isobutanol and preparation method thereof - Google Patents

A kind of YH-76S bacterial strain of high yield isobutanol and preparation method thereof Download PDF

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CN110452847A
CN110452847A CN201910765514.9A CN201910765514A CN110452847A CN 110452847 A CN110452847 A CN 110452847A CN 201910765514 A CN201910765514 A CN 201910765514A CN 110452847 A CN110452847 A CN 110452847A
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isobutanol
strain
medium
preparation
bacterial strain
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张沨
王新飞
王兴春
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Shandong Huiguan Kangbo Biotechnology Co Ltd
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Shandong Huiguan Kangbo Biotechnology Co Ltd
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    • C12P7/00—Preparation of oxygen-containing organic compounds
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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    • C12R2001/19—Escherichia coli

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Abstract

The invention discloses a kind of preparation methods of the YH-76S bacterial strain of high yield isobutanol, comprising the following steps: goes out the activation of bacterium germination, ARTP mutagenic treatment is in the medium transformed isobutanol superior strain in the medium to isobutanol Screening of strain with high productivity.YH-76S bacterial strain prepared by the present invention is to be mutated by ARTP (normal temperature and pressure plasma) mutagenesis system to Escherichia coli MG1655, then it is screened using BmoR bio-sensor system, it can be by fermenting and producing high concentration isobutanol, and the isobutyl alcohol content in fermentation liquid can achieve as 50-70g/L.

Description

A kind of YH-76S bacterial strain of high yield isobutanol and preparation method thereof
Technical field
The present invention relates to the preparation technical field of coli strain, in particular to a kind of YH-76S bacterium of high yield isobutanol Strain and preparation method thereof.
Background technique
Isobutanol (2-Methyl-1-Propanol) is that one kind is colourless inflammable, there is the organic compound of special odor.Its is different Structure body is n-butanol, sec-butyl alcohol and the tert-butyl alcohol.It is listed in alcohols, and therefore, it is widely used as the solvent of chemical reaction, simultaneously It is also a useful raw material of organic synthesis.In nature, isobutanol can be obtained by the spontaneous fermentation of carbohydrate.It It is also likely to be a byproduct during organic matter degradation.N-butanal, isobutylaldehyde industrially are obtained from the carbonylation synthesis of propylene, Repeated hydrogenation, separation product just obtain isobutanol.
Traditional chemical synthesizes isobutanol and uses isobutanol propenecarbonyl synthetic method.High pressure carbonylation synthesis technology is due to selectivity The byproducts such as poor, propane and high-boiling components are more, by using rhodium as replaced the low pressure carbonylation synthesis technology of catalyst.There is research Show that the growth of the thallus when butanol concentration reaches 13-14g/L in fermentation liquid will receive inhibiting effect, so as to cause fermentation ends When production concentration it is extremely low.Therefore, the strain excellent for filtering out high butanol tolerance is one of the effective ways for solving this phenomenon.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of preparation methods of coli strain, to filter out high butanol The strain excellent of tolerance.
In order to solve the above-mentioned technical problem, the technical solution of the present invention is as follows:
A kind of preparation method of the YH-76S bacterial strain of high yield isobutanol, comprising the following steps:
Step 1, the activation for going out bacterium germination: it using the Escherichia coli MG1655 of laboratory preservation as bacterium germination is gone out, sets out described Bacterium is crossed in solid medium, and turns out single colonie in the environment of 30 DEG C -37 DEG C, by the single colonie in Liquid Culture It is cultivated in base, obtains the thallus of activation；
Step 2, ARTP mutagenic treatment: the sterile slide glass for being coated with thallus liquid is placed in progress mutagenic treatment under processing source and obtains Obtain mutant monoclonal；
Step 3, in the medium to isobutanol Screening of strain with high productivity: by BmoR bio-sensor system in step 2 The mutant monoclonal of acquisition carries out fluorescent screening, screens the bacterial strain that fermented verifying fluorescence intensity is higher than bacterium germination 2 times or more As aimed strain；
Step 4 is in the medium transformed isobutanol superior strain: will convert plasmid pYH15, expression pyruvic acid to 2- ketone Enzyme alsS, ilvC and the ilvD of isovaleric acid, building obtain isobutanol superior strain YH-76S.
Optional: the culture medium includes seed culture medium and fermentation medium.
Optional: the seed culture medium and fermentation medium include: NaH2PO41-6g/L, KH2PO42-6g/L, NaCl 0.2-2g/L, NH4Cl 0.5-5g/L, MgSO40.5-1mM, CaCl20.1-2mM, vitamin B1 5-20mg/L and Portugal Grape sugar 20-60g/L.
According to another aspect of the present invention, a kind of YH-76S bacterial strain of high yield isobutanol is additionally provided, deposit number is SYH015。
By adopting the above technical scheme, by ARTP (normal temperature and pressure plasma) mutagenesis system to Escherichia coli MG1655 into Row mutation, is then screened to obtain isobutanol superior strain YH-76S using BmoR bio-sensor system, can pass through fermentation High concentration isobutanol is produced, the isobutyl alcohol content in fermentation liquid can achieve as 50-70g/L.
Detailed description of the invention
The case where Fig. 1 is isobutanol production bacterial strain YH-76S Fermentive production of isobutanol, and biomass and sugar utilize relationship is schemed.
Specific embodiment
Specific embodiments of the present invention will be further explained with reference to the accompanying drawing.It should be noted that for The explanation of these embodiments is used to help understand the present invention, but and does not constitute a limitation of the invention.In addition, disclosed below The each embodiment of the present invention involved in technical characteristic can be combined with each other as long as they do not conflict with each other.
Embodiment 1
A kind of preparation method of the YH-76S bacterial strain of high yield isobutanol, comprising the following steps:
Step 1, the activation for going out bacterium germination: it using the Escherichia coli MG1655 of laboratory preservation as bacterium germination is gone out, sets out described Bacterium is crossed in LB solid medium, and turns out single colonie in the environment of 30 DEG C -37 DEG C, by the single colonie in LB liquid It is cultivated in culture medium, obtains the thallus of activation.
Step 2, ARTP mutagenic treatment: the sterile slide glass for being coated with thallus liquid is placed in progress mutagenic treatment under processing source and obtains Obtain mutant monoclonal.
ARTP (Atmospheric and Room Temperature Plasma) mutagenesis, that is, normal temperature and pressure plasma lures Become: thallus being resuspended after the thallus after washing activation with sterile saline, and suitably dilution thallus is placed it in suitable concentration The building for carrying out mutated library under processing source to starting strain, obtains mutant monoclonal.
Step 3, in the medium to isobutanol Screening of strain with high productivity: by BmoR bio-sensor system in step 2 The mutant monoclonal of acquisition carries out fluorescent screening, screens the bacterial strain that fermented verifying fluorescence intensity is higher than bacterium germination 2 times or more As aimed strain.
BmoR is a kind of transcription factor, by it in conjunction with isobutanol molecule after, can be withRNAP constitutes complex, to make For the biosensor of isobutanol, it is fluorescin that mesocomplex, which originates gfp,.
Step 4 is in the medium transformed isobutanol superior strain: will convert plasmid pYH15, expression pyruvic acid to 2- ketone Enzyme alsS, ilvC and the ilvD of isovaleric acid, building obtain isobutanol superior strain YH-76S.
Above-mentioned culture medium includes seed culture medium and fermentation medium, in an embodiment of the present invention, seed culture medium and Fermentation medium includes: NaH2PO41-6g/L, KH2PO42-6g/L, NaCl 0.2-2g/L, NH4Cl 0.5-5g/L, MgSO4 0.5-1mM, CaCl20.1-2mM, vitamin B1 5-20mg/L and glucose 20-60g/L.
The identification of step 5, isobutanol superior strain:
The output increased of isobutanol to 5-20g/L is passed through into ferment tank isobutanol Producing Strain by medium optimization Strain YH-76S generates isobutanol, and detects isobutanol yield using GC.
The volume of above-mentioned fermentor is 2-4L, and inoculum concentration is 0.1%-4% (V/V), and fermentation temperature is 30-37 DEG C, stirring Revolving speed is 200-300rpm, and for fermentation process using hydrochloric acid and ammonium hydroxide control pH in 6.5-7.5, fermentation time is 72-144 hours.
In an embodiment of the present invention, ferment tank culture volume is 3.5L, first sterilizes 20 in 121 DEG C of environment Minute, then inoculation fermentation, wherein inoculum concentration is 0.2% (V/V), and fermentation temperature is 37 DEG C, speed of agitator 250rpm, fermentation For process using hydrochloric acid and ammonium hydroxide control pH 7.0 or so, fermentation time is 120 hours；Finally using in GC analysis fermentation liquid Isobutyl alcohol content is 60g/L, as shown in Figure 1, Fig. 1 a ordinate is biomass concentration, abscissa is the time, and Fig. 1 b ordinate is Concentration of glucose, permanent coordinate are the time, and dot broken line represents biomass data, and rectangular broken line is glucose.
In conjunction with attached drawing, the embodiments of the present invention are described in detail above, but the present invention is not limited to described implementations Mode.For a person skilled in the art, in the case where not departing from the principle of the invention and spirit, to these embodiments A variety of change, modification, replacement and modification are carried out, are still fallen in protection scope of the present invention.

Claims (4)

1. a kind of preparation method of the YH-76S bacterial strain of high yield isobutanol, it is characterised in that: the following steps are included:
Step 1, the activation for going out bacterium germination: using the Escherichia coli MG1655 of laboratory preservation as bacterium germination is gone out, the bacterium germination out is existed It crosses in solid medium, and turns out single colonie in the environment of 30 DEG C -37 DEG C, in liquid medium by the single colonie Culture, obtains the thallus of activation；
Step 2, ARTP mutagenic treatment: the sterile slide glass for being coated with thallus liquid is placed in progress mutagenic treatment under processing source and is dashed forward Variant monoclonal；
Step 3, in the medium to isobutanol Screening of strain with high productivity: obtained by BmoR bio-sensor system in step 2 Mutant monoclonal carry out fluorescent screening, screen it is fermented verifying fluorescence intensity be higher than bacterium germination 2 times or more bacterial strain conduct Aimed strain；
Step 4 is in the medium transformed isobutanol superior strain: will convert plasmid pYH15, expression pyruvic acid to 2- ketone isoamyl Enzyme alsS, ilvC and the ilvD of acid, building obtain isobutanol superior strain YH-76S.
2. the preparation method of coli strain according to claim 1, it is characterised in that: the culture medium includes seed Culture medium and fermentation medium.
3. the preparation method of coli strain according to claim 2, it is characterised in that: the seed culture medium and hair Ferment culture medium includes: NaH2PO41-6g/L, KH2PO42-6g/L, NaCl 0.2-2g/L, NH4Cl 0.5-5g/L, MgSO4 0.5-1mM, CaCl20.1-2mM, vitamin B1 5-20mg/L and glucose 20-60g/L.
4. a kind of YH-76S bacterial strain of the high yield isobutanol of preparation method preparation according to claim 1 to 3, preservation are compiled Number be SYH015.
CN201910765514.9A 2019-08-19 2019-08-19 A kind of YH-76S bacterial strain of high yield isobutanol and preparation method thereof Pending CN110452847A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009149270A2 (en) * 2008-06-04 2009-12-10 E. I. Du Pont De Nemours And Company A method for producing butanol using two-phase extractive fermentation
CN102533625A (en) * 2011-10-17 2012-07-04 广西科学院 Recombinant strain for producing isobutanol by using tapioca starch as raw materials and construction method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009149270A2 (en) * 2008-06-04 2009-12-10 E. I. Du Pont De Nemours And Company A method for producing butanol using two-phase extractive fermentation
CN102533625A (en) * 2011-10-17 2012-07-04 广西科学院 Recombinant strain for producing isobutanol by using tapioca starch as raw materials and construction method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
林丽华: "大肠杆菌中表达关键基因产异丁醇的研究", 《生物技术》 *

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Application publication date: 20191115