CN103820345B - A kind of molecular regulation method improving saccharomyces cerevisiae thermostability - Google Patents
A kind of molecular regulation method improving saccharomyces cerevisiae thermostability Download PDFInfo
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- CN103820345B CN103820345B CN201410022595.0A CN201410022595A CN103820345B CN 103820345 B CN103820345 B CN 103820345B CN 201410022595 A CN201410022595 A CN 201410022595A CN 103820345 B CN103820345 B CN 103820345B
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Abstract
For modern biotechnology medicine with light industry food industries system in, the problem of microorganism temperature controlled fermentation production process high energy consumption.The present invention provides a kind of molecular regulation method improving saccharomyces cerevisiae thermostability, belongs to biological chemical field.With Thermophilic Bacteria, in heatshock protein in thermoduric bacteria, ubiquitin, ATP synthase gene and saccharomyces cerevisiae, heat shock protein gene is as function element, in saccharomyces cerevisiae, varying strength promoter is as controlling element, by single function, multi-functional, 3 kinds of different assembling modes heat-resisting components and parts of structure of synergetic, the heat-resisting components and parts built import in yeast cells with carrier and genome conformity 2 kinds strategy again, it is achieved heat-resisting components and parts are integrated with chassis host's saccharomyces cerevisiae.The thermostability of components and parts is characterized, it is achieved the regulation and control of saccharomyces cerevisiae different temperature capability on a molecular scale, efficient, the low energy consumption process that produce biobased products for industrial fermentation provide new method by gradient increased temperature and constant high temperature cultivation and fermentation.
Description
Technical field
The present invention relates to the use of the heat-resisting components and parts of structure and improve thermostability and the commercial Application thereof of industrial microorganism saccharomyces cerevisiae, belong to biological chemical field.
Background technology
Microorganism thermostability is the key factor determining sweat energy expenditure and Product formation efficiency.Fermentable is utilized to produce in the process of biomass product, cell metabolism discharges substantial amounts of heat, and fermentation system heats up gradually, it is impossible to drop to the optimum temperature that reaction is required voluntarily, so a large amount of cooling water control temperature, the problem causing power cost increase, sweat high energy consumption need to be consumed.But contradiction with it, in born of the same parents, the optimal reactive temperature of most of enzymes is all higher than optimum growth temperature, from thermodynamics of reactions angle analysis, improve temperature can priming reaction process effectively, accelerate metabolism mass transfer, improve cell combined coefficient.So improving fermentation strain thermostability, the scope widening optimum growth temperature will well solve this contradiction, it is possible to cost is greatly lowered, improves production efficiency simultaneously.
The thermostability of saccharomyces cerevisiae is always up yeast research and the hot issue of fermentation industry production.It is a kind of generally acknowledged safe microorganisms that saccharomyces cerevisiae is not only due to it, more because its fast growth, be prone to genetic manipulation, fermentability strong, without endotoxin, the advantage that the toleration of ethanol and other mortifiers is also stronger so that it is become one of most widely used industrial producing strain in industrial biotechnology.Therefore, the thermostability improving saccharomyces cerevisiae is the effective way obtaining low-cost bio goods.
At present, research worker both domestic and external mainly includes the methods such as hybridization, random mutagenesis, evolution engineering and genome rearrangement mainly through adaptability domestication strategy or genetic manipulation strategies yeast carries out the transformation of heat resistance.But on the whole, utilize the successful example of heat-resisting saccharomyces cerevisiae that engineered method builds little, DeGrain.And these methods can only improve optimum growth temperature, it is impossible to widen optimum growth temperature scope, it is impossible to meet the demand that industrial fermentation heats up gradually.Though and by introduce external source heat-resisting mechanism molecular level on transform saccharomyces cerevisiae thermostability it has been reported that but study not system, be applied to industry gradient fermentation at elevated temperatures have not been reported.
Extreme microorganism represents the limit of life, the unknown that is richly stored with process and function, is abundant specific function protein resource treasure-house, has immeasurable biotechnology DEVELOPMENT PROSPECT.For Thermophilic Bacterium, can live in the environment of nearly 100 DEG C, having the adaptation mechanism of its uniqueness, main relevant with spore, the constituent of cell membrane, Zimadzhunt L 340, protein steric structure, heatshock protein and hereditary material, wherein producing a large amount of heatshock protein is a kind of important molecule control measures.It not only can be effectively protected cell under heat stress, coerces for organic solvent, sour environment etc. and also can play its biochemical functions.At present, the crucial Data mining in thermal protection mechanism is become to have the molecule components and parts of heat-resisting function to transform the thermostability of saccharomyces cerevisiae, and be applied to industrial sweat and yet there are no bibliographical information.
Fast-developing based on synthetic biology in recent years, excavate the heat-resisting components and parts of varying strength and carried out engineer and optimization, saccharomyces cerevisiae has been carried out the regulation and control of molecular level, having improve its thermostability.It is applied to fermentable production process, production intensity will be increased substantially, reduce cost.
Summary of the invention
It is an object of the invention to as solving in industrial microorganism incubation owing to controlling the problem that normal temperature fermentation causes production process high energy consumption.A kind of molecular regulation method improving saccharomyces cerevisiae thermostability is provided.
For achieving the above object, technical scheme provides a kind of method building heat-resisting components and parts and heat-resisting saccharomyces cerevisiae, regulates and controls the thermostability of saccharomyces cerevisiae from molecular level.By cloning or synthetic obtains a series of gene that cell has heat protection function as function element from Thermophilic Bacteria and thermoduric bacteria; such as heatshock protein; ubiquitin and ATP synthase gene etc.; in saccharomyces cerevisiae, varying strength promoter is as controlling element, builds heat-resisting components and parts by following assembling mode 3 kinds different: (1) is assembled into single heat-resisting components and parts of functional type with individual feature element and controlling element: promoter-function element-terminator;(2) that make mutually for function or there is the function element of different heat protection function be assembled into the heat-resisting components and parts of Mobyneb: promoter 1-function element 1-terminator 1-promoter 2-function element 2-terminator 2-promoter n-function element n-terminator n (n 2);(3) using the heat-resisting gene excavated from the host's saccharomyces cerevisiae of chassis as endogenous function element, carry out being assembled into the heat-resisting components and parts of synergetic with above-mentioned external source function element: in promoter 1-external source function element 1-terminator 1-promoter 2-endogenous function element 2-terminator 2-promoter n-(, outer) function element n-terminator n (n 2).The all heat-resisting components and parts built are imported in saccharomyces cerevisiae with 2 kinds of strategies, the first strategy is to import with the form of carrier, the second strategy is that the method utilizing gene to assemble is integrated directly in genes of brewing yeast group, realize the integrated of heat-resisting components and parts and saccharomyces cerevisiae, to widen the optimum growth temperature scope of saccharomyces cerevisiae, improve its thermostability, as heat-resisting chassis host truly for industrial microorganism sweat.Fermented by gradient increased temperature and constant high temperature fermentation characterizes engineering bacteria thermostability, and carry out the functional verification of heat-resisting components and parts and β-Amyrin synthetic approach, realize the molecular regulation of saccharomyces cerevisiae difference temperature capability so that it is the yield of β-Amyrin is significantly improved under high-temperature cultivation.In addition the heat-resisting components and parts built have been carried out the toleration analysis of organic solvent, it has been found that this molecular level regulation and control method makes saccharomyces cerevisiae have both heat-resisting and organic solvent-resistant dual-use function.
The molecular regulation method improving saccharomyces cerevisiae thermostability of the present invention, has the advantage that
1, the heat-resisting components and parts that the present invention builds achieve the molecular regulation of saccharomyces cerevisiae thermostability in various degree, meet different fermentations production requirement.
2, the heat-resisting saccharomyces cerevisiae engineered yeast that the present invention builds, simplifies fermentation technology, reduces production cost, reach the purpose of energy-saving and emission-reduction.
Accompanying drawing explanation
Fig. 1 is that three engineering bacteria T.te-TTE2469, T.te-GroS2, T.te-IbpA and the WT/Vector control strains built are respectively at 6%(v/v) and the 8%(v/v) OD under concentration of alcohol660nmThe change (a) (b) of value and cell survival rate (c) (d) figure.
Fig. 2 is the heat-resisting β-Amyrin engineering bacteria Sg-sb-T.te-TTE2469 built and Sg-sb control strain Yield mapping of β-Amyrin under 40 DEG C of cultivations.
Detailed description of the invention
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.
Embodiment 1: single heat-resisting components and parts of functional type assemble
Completed the thermophilic microorganism of order-checking by database lookup such as NCBI, HSPIA and relevant there is heat protection function gene, having chosen the heatshock protein in Thermophilic Bacteria and thermoduric bacteria, ubiquitin and ATP synthase gene as function element.For T. tengcongensis bacterium HSP10 family gene GroS2, perform twice at OE-PCR with promoter and terminator and assemble heat-resisting components and parts: promoter-GroS2-terminator.After all heat-resisting components and parts built are connected with shuttle vector, convert escherichia coli, after positive-selecting, choose single bacterium colony and cultivate extraction plasmid, it is transformed in saccharomyces cerevisiae, or utilize the method that gene assembles to be integrated directly in genes of brewing yeast group by heat-resisting components and parts, it is achieved integrated with chassis host's saccharomyces cerevisiae.
Embodiment 2: the heat-resisting components and parts of Mobyneb assemble
In the function element that the data bases such as NCBI, HSPIA obtain; for function make mutually such as heatshock protein GroES/GroEL system or there is the function element such as (ubiquitin, heatshock protein and ATP synzyme) of different heat protection function be assembled into the heat-resisting components and parts of Mobyneb.First for GroES/GroEL system, the polymer of 14 monomer GroEL molecules and 7 GroES molecule formation interposition cavitys, help protein correctly to fold.First pass through twice OE-PCR and respectively obtain single heat-resisting components and parts promoter 1-GroES-terminator 1 of function element and promoter 2-GroEL-terminator 2.Then the method assembled with gene is transformed into two heat-resisting components and parts of single function and shuttle vector in saccharomyces cerevisiae by certain concentration ratio, or two heat-resisting components and parts of single function are integrated directly in genes of brewing yeast group, after positive-selecting, obtain new heat-resisting components and parts: promoter 1-GroES-terminator 1-promoter 2-GroEL-terminator 2.Cell is had different protective effects by different heat-resisting function element; with ubiquitin; heatshock protein and ATP synzyme are example; the major function of ubiquitin is to be degraded by the carrying out of Denatured protein in cell under high temperature stress; after generating a series of small peptide and free amino acid; continue to utilize for cell as nutrient substance, make to reach in cyton the balance of a kind of albumen, maintain its continued growth.And the most basic function of heatshock protein is and the protein bound of the degeneration under hot environment, repairs the albumen of false folding and maintain space conformation and function, it is prevented that it is subject to the infringement of environment.ATP synzyme participates in oxidative phosphorylation, synthesizes ATP, provide ability for cell under the promotion of cross-film proton motive force.Implementation is ibid, first pass through twice OE-PCR and respectively obtain single heat-resisting components and parts of function: promoter 1-ubiquitin-terminator 1, promoter 2-heatshock protein-terminator 2 and promoter 3-ATP synzyme-terminator 3, then the method assembled with gene is transformed into three heat-resisting components and parts of single function and shuttle vector in saccharomyces cerevisiae by certain concentration ratio, or three heat-resisting components and parts of single function are integrated directly in genes of brewing yeast group, after positive-selecting, obtain new heat-resisting components and parts: promoter 1-ubiquitin-terminator 1-promoter 2-heatshock protein-terminator 2-promoter 3-ATP synzyme-terminator 3, what achieve with chassis host's saccharomyces cerevisiae is integrated.That other functions are made mutually or difference in functionality element is adopted and is built heat-resisting components and parts in the same way.
Embodiment 3: the heat-resisting components and parts of synergetic assemble
From the host's saccharomyces cerevisiae of chassis, cell is had the gene such as HSP104 of better heat protection function as endogenous function element by clone or synthetic, and with above-mentioned good external source function element such as ubiquitin, HSP10 etc. is assembled into the heat-resisting components and parts of synergetic again.First pass through twice OE-PCR and respectively obtain single heat-resisting components and parts of function: promoter 1-HSP104-terminator 1, promoter 2-ubiquitin-terminator 2, promoter 3-HSP10-terminator 3, then the method assembled with gene is transformed into three heat-resisting components and parts of single function and shuttle vector in saccharomyces cerevisiae by certain concentration ratio, or three heat-resisting components and parts of single function are integrated directly in genes of brewing yeast group, after positive-selecting, obtain new heat-resisting components and parts: promoter 1-HSP104-terminator 1-promoter 2-ubiquitin-terminator 2-promoter 3-HSP10-terminator 3, what achieve with chassis host's saccharomyces cerevisiae is integrated.In other difference in functionalitys or function are made mutually, external source function element is adopted and is built heat-resisting components and parts in the same way.
Embodiment 4: the biochemical phenotype checking of heat-resisting components and parts
The thermostability of all components and parts is characterized by engineering bacteria hot fermentation.After choosing 30 DEG C of 170rpm cultivations of a single bacterium colony 36 hours, by initial OD660nm0.1 is inoculated in new culture medium, after normal cultivation 12 hours, carry out high-temperature cultivation fermentation by two ways: after (1) fermentation temperature rises to 35 DEG C of continuation cultivations 12, then with 2 DEG C for a thermograde, stepping up cultivation temperature to 45 DEG C, each thermograde cultivates 12h；(2) fermentation temperature directly rises to 42 DEG C, 44 DEG C and 46 DEG C and cultivates 84h continuously.Qualitative characterization's thermostability, OD is come by colonial morphology660nmThe change of value, cell survival rate, content of trehalose carry out quantitatively characterizing thermostability.The alcohol resistance of components and parts is characterized, after choosing 30 DEG C of 170rpm cultivations of a single bacterium colony 36 hours, by initial OD by engineering bacteria normal temperature fermentation660nm0.1 is inoculated into containing 6%(v/v) and 8%(v/v) in the new YPD culture medium under concentration of alcohol, cultivate 84 hours continuously, pass through OD660nmThe change of value and cell survival rate carry out quantitatively characterizing alcohol resistance.Result shows that alcohol resistance is significantly improved by the engineering bacteria built.
Embodiment 5: the functional verification of heat-resisting components and parts
The good heat-resisting components and parts obtained are forwarded in saccharomyces cerevisiae β-Amyrin synthetic system with the form electricity of carrier, builds the β-Amyrin synthetic system with heat-resistant quality.Effect to chassis host and synthetic β-Amyrin system is introduced by the heat-resisting components and parts of Changeement of β-Amyrin yield.
After choosing a heat-resisting β-Amyrin engineering bacteria 30 DEG C 170rpm cultivation 36 hours, by initial OD660nm0.1 is inoculated in new culture medium, then pass through three kinds of modes and carry out high-temperature cultivation fermentation: after (1) normally cultivates 12 hours, after temperature rises to 37 DEG C of continuation cultivations 12, then with 2 DEG C for a thermograde, stepping up cultivation temperature to 45 DEG C, each thermograde cultivates 24h；(2), after normally cultivating 12 hours, temperature directly rises to 40 DEG C and is cultured to 72h continuously；(3) directly cultivate 120 hours respectively at 35 DEG C and 38 DEG C.After cultivation terminates, process culture, extract β-Amyrin, gas chromatograph-mass spectrometer (GC-MS) GCMS-QP2010 is finally utilized to carry out the analysis of heat-resisting saccharomyces cerevisiae engineered yeast fermenting and producing β-Amyrin, yield significantly improves, and result illustrates that the structure of heat-resisting components and parts improves saccharomyces cerevisiae tolerance at high temperature.
Claims (3)
1. the molecular regulation method improving saccharomyces cerevisiae thermostability; it is characterized in that; obtained the gene that cell is had heat protection function by clone or synthetic from Thermophilic Bacteria and thermoduric bacteria; including ubiquitin, heatshock protein and ATP synzyme; as function element; heat-resisting components and parts are built, it is achieved the molecular regulation of saccharomyces cerevisiae difference temperature capability in the way of carrier or genome conformity.
2. a kind of molecular regulation method improving saccharomyces cerevisiae thermostability as claimed in claim 1, wherein assembling mode is following 2 kinds:
(1) that make mutually for function or there is the function element of different heat protection function be assembled into the heat-resisting components and parts of Mobyneb；
(2) using the heat-resisting gene that excavates from the host's saccharomyces cerevisiae of chassis as endogenous function element, the heat-resisting components and parts of synergetic again assembled with above-mentioned external source function element, the heat-resisting components and parts built import in yeast cells with 2 kinds of Different Strategies, the first strategy is to import with the form of carrier, and the second strategy is integrated directly in genes of brewing yeast group.
3. the heat-resisting components and parts built as described in claim 2 application in improving saccharomyces cerevisiae thermostability and alcohol resistance.
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