CN103773791A - Molecular regulation method for improving heat resistance of escherichia coli - Google Patents
Molecular regulation method for improving heat resistance of escherichia coli Download PDFInfo
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- CN103773791A CN103773791A CN201410020497.3A CN201410020497A CN103773791A CN 103773791 A CN103773791 A CN 103773791A CN 201410020497 A CN201410020497 A CN 201410020497A CN 103773791 A CN103773791 A CN 103773791A
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Abstract
For a problem of high energy consumption in a microorganism temperature-controlled fermentation production process of modern biomedicine and light-industry food industry systems, the invention provides a molecular regulation method for improving heat resistance of escherichia coli, and belongs to the field of biochemical engineering. Heat shock protein, ubiquitin and thermal induced transcription factor genes of thermophilic bacteria and thermoduric bacteria are used as core functional elements and promoters differing in strength are taken as regulating elements; heat-resisting components are assembled through three ways, i.e., single-functional, cluster and multifunctional series connection, and are integrated to a substrate host, namely the escherichia coli. Heat resistance of the components is represented through gradient temperature rising and constant high-temperature culture fermentation, and the heat-resisting components are divided into strong, medium and weak classes depending on various heat-resisting temperature ranges of engineering bacteria, thus regulating escherichia coli in different heat-resisting degrees on molecular level. The invention provides a novel method for an efficient and low-energy consumption process of producing bio-based products through industrial fermentation.
Description
Technical field
The present invention relates to the construction process of the heat-resisting components and parts of intestinal bacteria and integrated with chassis host thereof, realize on molecular level and regulate and control intestinal bacteria thermotolerance, belong to biological chemical field.
Background technology
Microorganism thermotolerance is the important factor that determines fermenting process energy expenditure and product combined coefficient.Utilizing in the process of microorganism fermentation to produce biological goods, cell metabolism discharges a large amount of heat, and fermentation system heats up gradually, cannot drop to voluntarily the required optimum temperuture of reaction, so need to consume a large amount of water coolant temperature controls, cause the problem of power charge increase, fermenting process high energy consumption.But contradiction with it, in born of the same parents, most of enzyme optimal reactive temperatures, all higher than optimum growth temperature, from thermodynamics of reactions angle analysis, improve temperature priming reaction process effectively, accelerate metabolism mass transfer, improve cell combined coefficient.So improve fermentation strain thermotolerance, the scope of widening optimum growth temperature will well solve this contradiction, can significantly reduce costs simultaneously, enhance productivity.
Colibacillary genetic manipulation is simple, is easy to cultivate, and production cost is low, is one of most widely used industrial producing strain of industrial biotechnology.Therefore, improving colibacillary thermotolerance is the effective way that obtains low-cost bio goods.In research, the mode of mainly being screened or being tamed by high temperature improves intestinal bacteria thermotolerance at present, but this method can only improve optimum growth temperature, cannot widen optimum growth temperature scope, can not meet the demand that industrial fermentation heats up gradually.For the research in molecule aspect, mainly improve thermotolerance by regulation and control some transcription factors of intestinal bacteria self or protein expression at present, this method is limited to the heat-resisting mechanism of intestinal bacteria itself, and effect is also not obvious.Though and on external source heat-resisting machine-processed molecular level, transform colibacillary thermotolerance and have been reported by introducing, study not system, be applied to industrial gradient increased temperature fermentation and have not been reported.
The most of ancient bacterium territory that belongs to of thermophilic microorganism, in the very hot environment generally living on earth, represent the limit of life temperature, the heat-resisting mechanism of part is reasonably set forth, such as special cells film moiety, protein steric structure, thermal induction transcription factor, heat shock protein (HSP) etc. more closely.Wherein producing a large amount of heat shock proteins is a kind of important molecular regulation means, and it is effective Cell protection under heat stress not only, coerces and also can bring into play its biochemical functions for organic solvent, sour environment etc.At present, the molecule components and parts that the crucial Data mining in hot protection mechanism become to have to heat-resisting function are transformed colibacillary thermotolerance, and are applied to industrial fermenting process and yet there are no bibliographical information.
Based on synthetic biology fast development in recent years, the heat-resisting components and parts of having excavated varying strength pedestrian's work design and optimization of going forward side by side, carries out the regulation and control of molecular level to intestinal bacteria, improved its thermotolerance.Be applied to microorganism fermentation production process, will increase substantially production intensity, reduce costs.
Summary of the invention
The object of the invention is by improving intestinal bacteria thermotolerance, solve it causes production process high energy consumption in industrial fermentation process problem due to control normal temperature fermentation, reduce production costs.
For achieving the above object, technical scheme of the present invention provides a kind of method that builds heat-resisting components and parts and thermotolerant coliform, realizes and regulates and controls colibacillary thermotolerance from molecular level.From thermophile bacteria and thermoduric bacteria, obtain a series of genes that cell is had to heat protection function as functional element by clone or synthetic; as heat shock protein (HSP100, HSP90, HSP70, HSP60, smHSP), ubiquitin and thermal induction transcription factor gene etc.; simultaneously, using intestinal bacteria varying strength promotor and terminator as controlling element, build heat-resisting components and parts by following 3 kinds of different assembling modes.(1) be assembled into heat-resisting components and parts with individual feature element and controlling element: promotor-functional element-terminator.(2) assemble altogether the heat-resisting components and parts of cluster shape for the mutual work energy of heat-resisting function element: promotor-functional element 1-functional element 2 ... functional element n-terminator (n >=2).(3) be assembled into the heat-resisting components and parts of tandem type for the heat-resisting functional element with different heat protection functions: promotor 1-functional element 1-promotor 2-functional element 2 ... promotor n-functional element n-terminator (n >=2).By after all heat-resisting components and parts connection carrier building, transform intestinal bacteria, realize the integrated of heat-resisting components and parts and chassis host e. coli, to widen colibacillary optimum growth temperature scope, improve its thermotolerance.Ferment and constant high temperature fermentation characterizes engineering bacteria thermotolerance by gradient increased temperature, according to heat resisting temperature scope, by all heat-resisting components and parts be divided into by force, in, weak three classes, can realize the molecular regulation of the heat-resisting degree of intestinal bacteria difference.
The stable on heating molecular regulation method of raising intestinal bacteria of the present invention, has the following advantages:
1, the heat-resisting components and parts that the present invention builds have been realized intestinal bacteria stable on heating molecular regulation in various degree, have met different fermentations production requirement.
2, the thermotolerant coliform engineering bacteria that the present invention builds, has simplified zymotechnique, has reduced production cost, reaches the object of energy-saving and emission-reduction.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
Embodiment 1: the heat-resisting components and parts assembling of single functional element
Consult and complete the thermophilic and heat resistant microbe of order-checking and relevant there is heat protection function gene by the database such as NCBI, HSPIA, choose heat shock protein, ubiquitin and thermal induction transcription factor gene as functional element.Take Tengchong thermophile bacteria HSP20 family gene IbpA as example, carry out twice OE-PCR with promotor and terminator and assemble heat-resisting components and parts: promotor-IbpA-terminator.Promotor and terminator two ends arrange respectively Bgl II and the Xho I restriction enzyme site restriction enzyme site as all heat-resisting components and parts, heat-resisting components and parts are connected with after pET-28a (+) carrier double digestion, proceed to intestinal bacteria, realization is integrated with chassis host e. coli.
Embodiment 2: the heat-resisting components and parts assembling of bunch shape
In the functional element that the database such as NCBI, HSPIA obtains, there is the partial function element normal biochemical functions of travelling by cooperating with each other.Take heat shock protein DnaK/DnaJ system as example, DnaJ carries the protein bound of false folding to the ATP site of DnaK monomer, helps protein correctly folding.The mode of cutting connection with enzyme by OE-PCR is connected the heat-resisting components and parts of structure: promotor-DnaK-DnaJ-terminator.Promotor and terminator two ends arrange respectively Bgl II and Xho I restriction enzyme site, and heat-resisting components and parts are connected with after pET-28a (+) carrier double digestion, proceed to intestinal bacteria and obtain heat-resisting engineering bacteria.Other have the element of making mutually function and adopt and build in the same way heat-resisting components and parts.
Embodiment 3: the heat-resisting components and parts assembling of multi-functional series connection type
Different functional element has different provide protections to cell; take thermal induction transcription factor and heat shock protein as example; thermal induction transcription factor can be at transcription and translation under high temperature stress, the special hot protection mechanism in activating cells, the metabolic balance under balance hot environment.The most basic function of heat shock protein be with hot environment under the protein bound of sex change, the albumen that mis repair is folding also maintains space conformation and function, prevents that it is subject to the infringement of environment.The mode of cutting connection with enzyme by OE-PCR is connected the heat-resisting components and parts of structure: promotor 1-thermal induction transcription factor-promotor 2-heat shock protein-terminator.Promotor 1 arranges respectively Bgl II and Xho I restriction enzyme site with terminator two ends, and is connected after carrier double digestion, proceeds to intestinal bacteria and obtains heat-resisting engineering bacteria.The element of other functions is adopted and is built in the same way heat-resisting components and parts.
Embodiment 4: the checking of heat-resisting components and parts and classification
Characterize the thermotolerance of all components and parts by engineering bacteria thermophilic fermentation.37 ℃ of culturing engineering bacterium bacterium liquid OD
600reach behind 1.0 left and right, carry out high-temperature cultivation fermentation by two kinds of modes: first method is take 3 ℃ as one thermograde, progressively improve culture temperature to 40 ℃, 43 ℃, 46 ℃, each thermograde is cultivated 3h; Second method is by OD
600the engineering bacteria bacterium liquid that reaches 1.0 left and right is directly put into 40 ℃, 43 ℃, 46 ℃ and is carried out constant high temperature cultivation.With OD
600the variation of value characterizes thermotolerance, determines heat-resisting scope, and heat-resisting components and parts are divided into: strong (37 ℃-46 ℃), in (37 ℃-43 ℃), weak (37 ℃-40 ℃) three classes.Such as, the heat-resisting scope of components and parts " promotor-IbpA-terminator " is 37 ℃-46 ℃, is divided into " by force " heat-resisting components and parts type; The heat-resisting scope of components and parts " promotor-ThiF-terminator " is 37 ℃-43 ℃, be divided into " in " heat-resisting components and parts type.
Claims (3)
1. one kind is improved the stable on heating molecular regulation method of intestinal bacteria; it is characterized in that; the gene that obtains cell to have heat protection function by clone or synthetic from thermophile bacteria and thermoduric bacteria is as functional element; build by different way heat-resisting components and parts; improve intestinal bacteria thermotolerance; by heat-resisting components and parts be divided into by force, in, weak three classes, realize the molecular regulation of the different heat-resisting degree of intestinal bacteria.
2. the structure of the heat-resisting components and parts of realizing molecular regulation as claimed in claim 1, builds heat-resisting components and parts by 3 kinds of modes: (1) is assembled into heat-resisting components and parts with individual feature element and controlling element; (2) assemble altogether the heat-resisting components and parts of cluster shape for the mutual work energy of heat-resisting function element; (3) be assembled into the heat-resisting components and parts of tandem type for the heat-resisting functional element with different heat protection functions.
3. heat-resisting components and parts as claimed in claim 2 and construction process thereof the application aspect raising intestinal bacteria thermotolerance.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105002188A (en) * | 2015-02-10 | 2015-10-28 | 北京理工大学 | Intelligent gradient heat-resistance regulation and control method for microorganisms |
| CN111088281A (en) * | 2020-01-09 | 2020-05-01 | 山东农业大学 | Chinese bee heat-resistant related gene DnaJ1 and application thereof |
| CN112111484A (en) * | 2020-09-17 | 2020-12-22 | 贵州大学 | Method for creating heat-resistant escherichia coli |
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| CN101525588A (en) * | 2008-11-24 | 2009-09-09 | 华南理工大学 | Protein Htp-X-P3 and method of improving enzyme heat stability by using same |
| CN101560249A (en) * | 2009-05-22 | 2009-10-21 | 青岛农业大学 | Amino acid sequence, gene sequence and expression vector of heat shock protein HmHSP 70 of hypsizygus marmoreus |
| CN102286512A (en) * | 2011-06-30 | 2011-12-21 | 复旦大学 | Multi-fragment deoxyribose nucleic acid (DNA) series connection recombination assembly method based on site-specific recombination |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101100668A (en) * | 2007-05-24 | 2008-01-09 | 上海植物园 | Chinese rose RcHsp17.8 coded sequence and application thereof |
| CN101525588A (en) * | 2008-11-24 | 2009-09-09 | 华南理工大学 | Protein Htp-X-P3 and method of improving enzyme heat stability by using same |
| CN101560249A (en) * | 2009-05-22 | 2009-10-21 | 青岛农业大学 | Amino acid sequence, gene sequence and expression vector of heat shock protein HmHSP 70 of hypsizygus marmoreus |
| CN102286512A (en) * | 2011-06-30 | 2011-12-21 | 复旦大学 | Multi-fragment deoxyribose nucleic acid (DNA) series connection recombination assembly method based on site-specific recombination |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105002188A (en) * | 2015-02-10 | 2015-10-28 | 北京理工大学 | Intelligent gradient heat-resistance regulation and control method for microorganisms |
| CN105002188B (en) * | 2015-02-10 | 2018-02-27 | 北京理工大学 | A kind of heat-resisting regulation and control method of microorganism intelligence step |
| CN111088281A (en) * | 2020-01-09 | 2020-05-01 | 山东农业大学 | Chinese bee heat-resistant related gene DnaJ1 and application thereof |
| CN111088281B (en) * | 2020-01-09 | 2022-08-16 | 山东农业大学 | Chinese bee heat-resistant related gene DnaJ1 and application thereof |
| CN112111484A (en) * | 2020-09-17 | 2020-12-22 | 贵州大学 | Method for creating heat-resistant escherichia coli |
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Application publication date: 20140507 |