CN102492636B - Saccharomyces cerevisiae strain and method for selecting saccharomyces cerevisiae strains expressing activated xylose translocator - Google Patents
Saccharomyces cerevisiae strain and method for selecting saccharomyces cerevisiae strains expressing activated xylose translocator Download PDFInfo
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Abstract
The invention discloses a saccharomyces cerevisiae strain expressing beta-xylosidase in cells, which is BSW2AB. The classification is named saccharomyces cerevisiae and filed in 'China General Microbiological Culture Collection Center' with the filed number CGMCC No.5465 on November 17, 2011. The saccharomyces cerevisiae strain is capable of expressing activated beta-xylosidase in cells. The invention provides a method for selecting saccharomyces cerevisiae strainsexpressing activated xylose translocator, and the reaction substrate for selecting the translocator is xylose analogue, namely p-nitrophenyl-beta-D-xyloside. The invention further provides a recombination saccharomyces cerevisiae strain BSW2ABT which has high xylose transfer capability and is obtained by the above method. The classification is named saccharomyces cerevisiae and filed in 'China General Microbiological Culture Collection Center' with the filed number CGMCC No.5464 on November 17, 2011.
Description
Technical field
The present invention relates to a strain and can express the Wine brewing yeast strain of xylobiase and the Wine brewing yeast strain that efficient wood sugar translocator is expressed in a strain in the born of the same parents, and be used for the method that the Wine brewing yeast strain of activated wood sugar translocator has been expressed in screening.
Background technology
At present, resource, energy situation problem have been subjected to worldwide concern, and seeking promising eco-friendly resource, the energy is the prerequisite that satisfies the Future Society Sustainable development.The annual production of lignocellulose resource is huge, can be used as potential fuel and Chemicals raw materials for production, and various relevant Study on Production Technology are very extensive, and has obtained a lot of technical breakthroughs in recent years.The main component of lignocellulose is: Mierocrystalline cellulose (cellulose), hemicellulose (hemicellulose) and xylogen (lignin) three parts.Mierocrystalline cellulose is through not branch crystalloid polymer that β-1,4 glycosidic link connects into by glucosyl residue; Hemicellulose is the noncrystalline heterogeneous polymer that comprises multiple sugars such as wood sugar, pectinose, semi-lactosi and seminose.After the lignocellulosic material hydrolysis, wood sugar content in hydrolyzed solution can reach 30% of total reducing sugar, and it effectively utilizes is to improve the xylogen raw material availability, reduces one of key of products production cost.Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is the production bacterial strain that is widely used in food and chemical engineering industry, have strong stress resistance, fermentation condition is easy to control, genetic background is clear, food grade waits many good characteristics safely, is that favorable industrial is produced bacterial strain.Can not ferment in lignocellulose content of glucose although yeast saccharomyces cerevisiae can effectively ferment in the ligno-cellulose hydrolysate, natural bacterial strain is only second to the wood sugar of glucose, must carry out further genetic modification to yeast saccharomyces cerevisiae.The introducing of xylose metabolism approach can make yeast saccharomyces cerevisiae utilize wood sugar, and still, the lower and capture process of wood sugar ingestion efficiency is suppressed by glucose, makes capture process become the restriction yeast saccharomyces cerevisiae to one of major obstacle of xylose utilization.Natural yeast saccharomyces cerevisiae is by non-specific hexose translocator (mainly containing Hxt1, Hxt2, Hxt4, Hxt5 and HXt7) and galactose permease (Gal2) picked-up wood sugar, these translocators all belong to Major Facilitator Superfamily (MFS) translocator, affinity to wood sugar is lower, and its Km value is 10～100 times with respect to glucose.The wood sugar translocator of part external source is in Expression in Saccharomyces Cerevisiae, can promote bacterial strain to the utilization of wood sugar, for example, derive from the Gxf1 of a type candiyeast (Candida intermedia), the Sut1 of pichia stipitis (Pichia stipitis) and the At5g5920 in the Arabidopis thaliana (Arabidopsis thaliana) etc., but they still much smaller than the avidity to glucose, can not satisfy the demand that makes up efficient xylose utilization recombinant Saccharomyces cerevisiae to the avidity of wood sugar.Screening is high-affinity wood sugar specificity translocator in yeast saccharomyces cerevisiae, and expresses corresponding protein in yeast saccharomyces cerevisiae, still is one of important enforcement means that improve the yeast saccharomyces cerevisiae xylose utilization.
Mainly contain the dual mode screening before this and study the wood sugar translocator that function is arranged in yeast saccharomyces cerevisiae.A kind of method is the translocator storehouse to be introduced have in the Wine brewing yeast strain of xylose metabolism approach, by bacterial strain energy for growth on wood sugar whether improvement is arranged, judge the function power of translocator, still, the false positive results that this method obtains is too much, and can't be quantitative.Another kind method is that the bacterial strain of introducing the translocator storehouse is hatched in isotope-labeled wood sugar, measures bacterial strain to the transportcapacity of wood sugar, this being potentially dangerous property of method, complicated operation is all very high to the requirement of operator and equipment.Therefore, it is very necessary setting up a kind of high-throughput wood sugar translocator screening method that can detection by quantitative.
(p-nitrophenyl-β-D-xylopyranoside pNPX) is a kind of xylose structure analogue to the p-nitrophenol xyloside.Xylobiase can produce equimolar p-NP (pNP) and wood sugar molecule with the pNPX hydrolysis.PNP shows yellow under alkaline condition, and at 405nm place performance maximum absorption band.Because the speed of reaction of xylobiase catalysis and the speed that pNP diffuses out cell all are higher than the ability of cell transportation wood sugar or pNPX far away, so the generating rate of pNP has reflected the uptake rate of the pNPX that enters cell.By at the expression in escherichia coli xylobiase, the method for this screening wood sugar translocator is set up in intestinal bacteria.But because the different and cyto-architectural difference of expression system, there is certain difficulty in related work in yeast saccharomyces cerevisiae: yeast saccharomyces cerevisiae is fungi, and its cell walls and cell membrane component may cause the discrepancy born of the same parents difficulty of molecule than bacterium complexity; Yeast saccharomyces cerevisiae is different with colibacillary wood sugar transporting mechanism; Brewing yeast cell is than the big order of magnitude of intestinal bacteria, and settling ratio is bigger, the value of reading is disturbed big, causes to be difficult to carrier and to monitor in real time; There is not effective xylose metabolism downstream gene in the wild-type yeast saccharomyces cerevisiae, it is to the transhipment of wood sugar and utilize speed to be starkly lower than intestinal bacteria, cause the difficulty of pNPX trace detection, comparatively harsh to the requirement of operator and equipment, the successful foundation of this method in yeast does not appear in the newspapers so far.
Summary of the invention
At above-mentioned prior art, the invention provides a strain and can express the Wine brewing yeast strain of xylobiase, and provide a kind of and expressed the method for the Wine brewing yeast strain of activated wood sugar translocator for screening, the expression that also provides a strain to obtain through this method screening has the Wine brewing yeast strain of highly active wood sugar translocator.
The present invention is achieved by the following technical solutions:
The Wine brewing yeast strain of xylobiase is expressed in one strain, this bacterial strain is BSW2AB, classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae, be preserved on November 17th, 2011 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number are CGMCC No.5465.Its mycology is characterized as: CEN.PK102-3A derivative, MATa, leu2-3, ura3-52, pYX242-PGKt-TEFp TPIp-xynB-PGKt, this yeast strain is unicellular eukaryotic microorganisms, ellipse does not move, the complete synthesis auxotroph substratum (SC-Leu of solid or liquid, this substratum is existing in the prior art) culture condition under for gemmation, exist with unicellular form under the liquid culture condition, during solid culture, be bacterium colony and exist, bacterium colony smooth surface, moistening, thickness are creamy white.
This bacterial strain can be expressed activated xylobiase in the high-caliber born of the same parents, compares with common Wine brewing yeast strain, and it is the two defective type haploid strains of leu and ura, can realize leu
-And ura
-The coexpression of the plasmid episomal of mark, this bacterial strain pYX242 (leu
-) plasmid episomal efficiently expressed the xylosidase gene, can with the ura that has cloned transporter gene
-The plasmid episomal coexpression of mark reaches the purpose of screening transporter gene.This bacterial strain is that the contriver obtains by recombination to construct and screening.
A kind of screening can be expressed the method for the Wine brewing yeast strain of activated wood sugar translocator, and step is as follows:
(1) strain culturing: bacterial strain to be detected and conventional bacterial strain are carried out the routine cultivation respectively, get bacterium liquid;
(2) the centrifugal substratum that removes of the bacterium liquid that above-mentioned cultivation is obtained uses phosphate buffered saline buffer resuspended then, gets the resuspended liquid of cell;
(3) in the resuspended liquid of above-mentioned cell, add xylose structure analogue pNPX (p-nitrophenyl-β-D-xyloside), hatch back centrifuging and taking supernatant, add isopyknic Na
2CO
3Solution is measured the 405nm absorbance value OD of place
405nm
(4) set up pNP at 405nm place absorbance value-pNP content standard curve and bacterium liquid OD
600nmValue-dry cell weight typical curve, the OD that integrating step (3) records
405nm, calculate translocator to the transport velocity (living with the enzyme that intact cell records) of pNPX;
(5) judge: contrast the transport velocity of bacterial strain to be detected and the transport velocity of conventional bacterial strain, if the transport velocity of bacterial strain to be detected is higher than the transport velocity of conventional bacterial strain, prove that then the wood sugar translocator that this bacterial strain to be detected is expressed is the albumen that can improve Wine brewing yeast strain wood sugar turn-over capacity, difference is more big, illustrates that the ability of the wood sugar translocator transhipment wood sugar that this bacterial strain to be detected is expressed is more high; If the transport velocity of bacterial strain to be detected is not higher than the transport velocity of conventional bacterial strain, prove that then this bacterial strain to be detected do not express the albumen that can improve yeast saccharomyces cerevisiae wood sugar turn-over capacity.
Described bacterial strain to be detected can make up by the following method:
(1) expression vector establishment: set up the recombinant vectors that includes transporter gene to be screened;
(2) recombinant bacterial strain makes up: the recombinant vectors of setting up in the step (1) is transformed in the Wine brewing yeast strain of expressing xylobiase in the born of the same parents, and obtains transforming successful transformant by screening, be bacterial strain to be detected.
Described transporter gene to be screened can be from various sources, such as: from the transporter gene AraT of plant lactobacillus (Lactobacillus plantarum).
The used plasmid of described construction of expression vector is pJFE3 (YEplac195, TEFp-PGKt, Ura
-, Amp
+) empty plasmid (this plasmid is existing in the prior art).
Described method for transformation is the Lithium Acetate conversion method.
The mode of the successful transformant of described screening is: with the correct transformant of complete synthesis auxotroph substratum SC-Leu-Ura (this substratum is existing in the prior art) screening that adds 2% glucose (massfraction).
Described conventional bacterial strain is for expressing the Wine brewing yeast strain of xylobiase in the born of the same parents of not containing transporter gene to be screened.
The described pNP of foundation in the method for 405nm place absorbance value-pNP content standard curve is: prepare the pNP of gradient concentration according to the PBS liquid of pH6.5, and measure the 405nm absorbance value OD of place
405nm, set up typical curve; Gained typical curve equation is: y
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735, x span is preferably at 1.5-7.5.
The described bacterium liquid OD that sets up
600nmThe method of value-dry cell weight typical curve is: get the bacterium liquid of cultivation, and centrifugal washing, dry weight is measured in suction filtration, oven dry back; Dilute six levels, calculate dry weight and measure corresponding OD according to extension rate
600That directly measure in experiment is the OD of bacterium liquid
600, can specifically calculate the transport velocity of every milligram of dry weight cell according to this formula; Gained typical curve equation is: y
(mgdw/ml)=0.2365x
(oD600nm)+ 0.1149, x span is preferably at 1-22.
Described " transport velocity of bacterial strain to be detected is higher than the transport velocity of conventional bacterial strain " refers to that bacterial strain transport velocity to be detected exceeds 18.5% of conventional bacterial strain transport velocity, and this moment is for being significantly higher than.
Above-mentioned screening can be expressed the method for the Wine brewing yeast strain of activated wood sugar translocator, and also can be used for screening can be at the translocator of yeast saccharomyces cerevisiae high reactivity transportation wood sugar.
Pass through aforesaid method, the present invention also obtains the recombinant Saccharomyces cerevisiae bacterial strain of a plant height wood sugar turn-over capacity, this bacterial strain is BSW2ABT, classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae, be preserved on November 17th, 2011 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number are CGMCC No.5464.This bacterial strain has been expressed the transporter gene AraT of clone from plant lactobacillus (Lactobacillus plantarum), and the more common Wine brewing yeast strain of its wood sugar turn-over capacity is more than doubled.Can predict, this bacterial strain contains in other product scopes of wood sugar raw material production in utilization and has wide purposes, such as being starting strain with this bacterial strain, introduces the xylose metabolism approach, can make up to have the recombinant bacterial strain that high level utilizes the wood sugar ability.
In the method for the present invention, the reaction substrate that is used for the screening translocator is the xylose structure analogue---and p-nitrophenyl-β-D-xyloside (p-nitrophenyl-β-D-xylopyranoside, pNPX), as shown in Figure 1; Transporter gene to be screened is introduced Wine brewing yeast strain by the yeast saccharomyces cerevisiae expression vector; The product nitrophenols that translocator is obtained by the xylobiase hydrolysis by pNPX the transport velocity of pNPX (p-nitrophenol, pNP) generating rate defines; The pNP generating rate is calculated by the reaction solution absorbancy rate of change that pNP causes; Translocator defines the pNPX conveying efficiency by translocator indirectly to the transportcapacity of wood sugar.Method of the present invention can effectively be avoided the appearance of false positive situation, and can the quantitative description bacterial strain ability of transhipment wood sugar, operate not dangerous property, simple operating steps, not harsh to the requirement of operator and equipment, have versatility.
Description of drawings
Bacterial strain BSW2AB, classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 17th, 2011, deposit number is CGMCC No.5465, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101.
Bacterial strain BSW2ABT, classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 17th, 2011, deposit number is CGMCC No.5464, the preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101.
Fig. 1 is the high-throughput screening method principle schematic of yeast saccharomyces cerevisiae wood sugar translocator.Present method is associated the xylosidase active and that express of wood sugar translocator in born of the same parents, (p-nitrophenyl-β-D-xylopyranoside pNPX) replaces wood sugar to carry out quantitative analysis to utilize xylose structure analogue p-nitrophenol xyloside.The pNPX of hatching with the yeast saccharomyces cerevisiae intact cell enters in the born of the same parents under the effect of wood sugar translocator, be hydrolyzed to p-NP (pNP) and wood sugar rapidly by xylosidase, pNP can quickly diffuse to outside the born of the same parents, shows yellow under alkaline condition, and at 405nm place performance maximum absorption band.
Fig. 2 is yeast saccharomyces cerevisiae episome shuttle plasmid pYX242-PGKt-TEFp-xynB synoptic diagram.Wherein xylosidase gene (xynB) clone expresses under the control of yeast saccharomyces cerevisiae TPIp promotor from bacillus pumilus (Bacilluspumilus).
Fig. 3 is yeast saccharomyces cerevisiae episome shuttle plasmid pJFE3-araT synoptic diagram.Wherein, translocator araT clones from plant lactobacillus (Lactobacillus plantarum), expresses under the control of yeast saccharomyces cerevisiae TEFp promotor.
Fig. 4 be BSW2ABP (CEN.PK102-3A derivative, MATa, leu2-3, ura3-52, pYX242-PGKt-TEFpTPIp-xynB-PGKt, pJFE3) the xylosidase enzyme of measuring under the inside and outside and intact cell state of the born of the same parents of genetic engineering modified bacterial strain is lived.Wherein represents the BSW2ABP bacterial strain; 1,2,3 represents 10 hours (initial OD of cultivation respectively
600nmBe 0.5) time xylosidase enzyme that records in the cytoclasis liquid live (, xylosidase activity in the cell); After cellular segregation, the xylosidase enzyme in the nutrient solution (that is, emiocytosis is to extracellular xylosidase activity) alive; After nutrient solution separates, without the xylosidase enzyme that the cell of fragmentation can be measured to live (that is the xylosidase activity that can measure when, utilizing intact cell to measure).The xylosidase enzyme is lived: 1U is defined as micromole's number (μ mol) of the pNP that decomposes the pNPX generation in the 1min.Xylosidase activity is higher in the cell, proves xylosidase efficiently expressing in yeast saccharomyces cerevisiae; Emiocytosis to extracellular xylosidase activity very low, almost detect less than, prove that this xylosidase is intracellular enzyme; Can detect the xylosidase enzyme when utilizing intact cell to measure lives, proved the operability of this kind detection method, simultaneously, its result is much smaller than (differing more than 100 times) intracellular xylosidase activity, prove that the pNPX that enters cell can be by the rapid hydrolysis of xylosidase, enzymolysis process is non-rate-limiting step.
Fig. 5 be bacterial strain BSW2ABP under the intact cell state, the OD that measures
405nmCorresponding relation between the value of reading and the incubation time.Prove that the data of getting two time points characterize the reasonableness of whole dynamic process trend indirectly.Wherein, BSW2ABP strain culturing 15h.
Fig. 6 be bacterial strain BSW2ABP under the intact cell state, the pNP generating rate of different incubation times (being the transport velocity of translocator).Different incubation times (in the 60min) are the pNP generating rate basically identical of gained down, proves to characterize speed just with the pNP generating rate of certain time point.Wherein, BSW2ABP strain culturing 15h.
Fig. 7 be bacterial strain BSW2ABP under the intact cell state, in the born of the same parents and diffuse to the outer pNP Determination on content result of born of the same parents.Wherein, 1,2 represent the experimental result that the BSW2ABP bacterial strain is hatched 10min and 30min respectively; represents the pNP amount in the born of the same parents, and the ■ representative diffuses to the outer pNP amount of born of the same parents; BSW2ABP strain culturing 15h.PNP amount in the born of the same parents is measured much smaller than the pNP that (differing nearly 20 times) diffuses to outside the born of the same parents, proves that the pNP that generates in the born of the same parents can go out born of the same parents rapidly, and this step is non-rate-limiting step.
Fig. 8 is bacterial strain BSW2ABT (CEN.PK102-3A derivative, MATa, leu2-3, ura3-52, pYX242-PGKt-TEFp TPIp-xynB-PGKt, pNPX transportcapacity measurement result pJFE3-araT).Wherein, represents the BSW2ABP bacterial strain, and ■ represents the BSW2ABT bacterial strain; The strain culturing time is 10h.Its wood sugar transportcapacity of the bacterial strain of overexpression araT significantly strengthens, and has improved 1.06 times than the contrast bacterial strain.
Fig. 9 is the competitive relation that detects between bacterial strain BSW2ABP and BSW2ABT transhipment pNPX and the different concns wood sugar.Wherein, represents the BSW2ABP bacterial strain, and ■ represents the BSW2ABT bacterial strain; The strain culturing time is 10h.Raising along with the outer xylose concentration of born of the same parents, the pNPX of two strain bacterium utilizes speed on a declining curve, and the downtrending of BSW2ABT bacterial strain is more obvious, no matter proof still is for the AraT for yeast saccharomyces cerevisiae endogenous wood sugar translocator, all there is competitive relation between pNPX and the wood sugar, be substrate analogue, therefore, the efficient of translocator transhipment pNPX can be reacted the efficient of its transhipment wood sugar.
Figure 10 is in two bacterial strain BSW2ABP and BSW2ABT competitive relation determination experiment, the linear relationship between the first speed inverse of transhipment pNPX and the different concns wood sugar.In the Michaelis-Menton equation of competitive relation, there is linear relationship between the first speed inverse of specific substrates and the competitive concentration of substrate.Linear relationship match between the BSW2ABP that measures in the experiment and the first speed inverse of BSW2ABT bacterial strain transhipment pNPX and the different concns wood sugar is all better, R
2=0.925 and 0.942, proved that further pNPX is the competitive substrate of wood sugar.
Wherein, ▲ represent the BSW2ABT bacterial strain; ◆ represent the BSW2ABP bacterial strain; The strain culturing time is 10h.
Embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1: structure and the xylosidase enzyme activity determination of expressing the bacterial strain BSW2ABP of xylosidase
Method is as follows:
(1) bacterial classification is selected: select for use heterogenous expression in the born of the same parents of structure that laboratory strains BSW2AB (the CEN.PK102-3A derivative of xylosidase is arranged, MATa, leu2-3, ura3-52, pYX242-PGKt-TEFp TPIp-xynB-PGKt) does acceptor strain bacterium, this strain classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae, be preserved on November 17th, 2011 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number are CGMCC No.5465.Its mycology is characterized as: CEN.PK102-3A derivative, MATa, leu2-3, ura3-52, pYX242-PGKt-TEFpTPIp-xynB-PGKt, this yeast strain is unicellular eukaryotic microorganisms, ellipse does not move, the complete synthesis auxotroph substratum (SC-Leu of solid or liquid, this substratum is existing in the prior art) culture condition under for gemmation, exist with unicellular form under the liquid culture condition, during solid culture, be bacterium colony and exist, bacterium colony smooth surface, moistening, thickness are creamy white.The construction process of this bacterial strain is summarized as follows: (gene order and aminoacid sequence are existing in the prior art to the xylosidase gene, and after the blast, consistence is immediate with it to be 96% to gene order on NCBI; Aminoacid sequence is on NCBI after the blast, consistence is immediate with it to be 97%, sequence is seen sequence table) be Bp-xynB, the clone is from Bacilluspumilus AS1.940, making up the used upstream primer XynBWup of recombinant vectors is: 5 '-CGGCAATTGATGAAGATTATCAATCCAGTGC-3 ', downstream primer XynBWdown is: 5 '-CATGCCATGGTTATTCGTCTGTTTCCTCATAG-3 '.The recombinant vectors that makes up is: pYX242-PGKt-TEFp TPIp-xynB-PGKt heterogenous expression carrier, as shown in Figure 2;
(2) carrier is selected: the pJFE3 (YEplac195, TEFp-PGKt, the Ura that adopt the laboratory to make up in earlier stage
-, Amp
+) empty plasmid;
(3) strain construction: plasmid pJFE3 transforms the BSW2AB bacterial strain with the Lithium Acetate conversion method, screens correct transformant with the complete synthesis auxotroph substratum SC-Leu-Ura that adds 2% glucose;
(4) strain culturing: 40ml is in the little triangular flask of 100ml with the complete synthesis auxotroph substratum SC-Leu-Ura that adds 2% glucose, switching for the transformant list bacterium colony that checking is correct in will step (3), cultivates after 1 day and transfers once, initial OD
600Connecing is 0.5, and culture condition is 30 ℃, 200rmin
-1
(5) intact cell suspension preparation: get the bacterium liquid of cultivating in the step (4) 10 hours, centrifugal (3000rmin
-1, it is resuspended 5min) to remove behind the former substratum 50mmol/L PBS liquid with pH6.5;
(6) measuring the xylosidase enzyme under the intact cell state lives: get intact cell suspension in an amount of step (5) with 96 hole flat boards or EP pipe, the adding final concentration is the pNPX of 6mmol/L, and both totally are 120 μ l, with being placed on 30 ℃, 200rmin
-1Hatched in the shaking table 30 minutes, centrifuging and taking supernatant 100 μ l add in the 96 hole flat boards, add the Na of isopyknic 0.4mol/L again
2CO
3The aqueous solution behind the mixing, uses microplate reader in OD
405nmThe following absorbance value of hatching the p-nitrophenol (pNP) that generates after 30 minutes of measuring.Hatch initial value when measuring, do not add pNPX earlier, only add bacteria suspension and experimental group is hatched jointly, add the pNPX of final concentration 6mmol/L behind an amount of volume supernatant of centrifuging and taking, both totally are 100 μ l, add the aqueous sodium carbonate of the 0.4M of 100 μ l again, behind the mixing, use microplate reader in OD
405nmUnder measure absorbance value; PNP typical curve y according to the preparation of the PBS liquid of pH6.5
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735 and the bacterium liquid OD of laboratory strains
600nmThe corresponding relation y of value and dry cell weight
(mgdw/ml)=0.2365x
(OD600nm)+ 0.1149, the xylosidase enzyme that calculates under the intact cell state is lived, and 1U is defined as micromole's number (μ mol) of the pNP that decomposes the pNPX generation in the 1min, sees Fig. 4;
(7) the inside and outside xylosidase enzyme of born of the same parents mensuration alive: get the bacteria suspension 4ml that cultivated 10 hours, centrifuged supernatant is the outer crude enzyme liquid of born of the same parents; With the 50mmol/L PBS suspension sedimentation cell of 1ml pH6.5, add the 0.6g granulated glass sphere, use tissue homogenizer smudge cells: 5500rmin
-1, 20s, 8 times, sample ice bath 4min between per twice fragmentation; 13000rmin
-1, centrifugal 10min, supernatant are crude enzyme liquid in the born of the same parents; Mensuration system in the 96 hole flat boards: get the pH6.5 phosphate buffered saline buffer that exoenzyme liquid configuration in an amount of born of the same parents contains 6mmol/LpNPX, totally 100 μ l measure after leaving standstill the yellow soda ash mixing of hatching the 0.4mol/L that adds 100 μ l behind the 10min for 30 ℃; Control group and experimental group are taked same treatment, just do not add earlier pNPX, reserve volume, add pNPX after the termination again and measure; Be used in the clean absorbance value at 405nm place, according to the pNP typical curve y of the PBS liquid of pH6.5 preparation
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735 and the bacterium liquid OD of laboratory strains
600nmThe corresponding relation y of value and dry cell weight
(mg dw/ml)=0.2365x
(OD600nm)+ 0.1149, the enzyme that calculates the inside and outside xylosidase of born of the same parents is lived, and 1U is defined as micromole's number (μ mol) of the pNP that decomposes the pNPX generation in the 1min, sees Fig. 4.
The described pNP of foundation in the method for 405nm place absorbance value-pNP content standard curve is: prepare the pNP of gradient concentration according to the PBS liquid of pH6.5, and measure the 405nm absorbance value OD of place
405nm, set up typical curve; Gained typical curve equation is: y
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735, x span is at 1.5-7.5.
The described bacterium liquid OD that sets up
600nmThe method of value-dry cell weight typical curve is: get the bacterium liquid of cultivation, and centrifugal washing, dry weight is measured in suction filtration, oven dry back; Dilute six levels, calculate dry weight and measure corresponding OD according to extension rate
600That directly measure in experiment is the OD of bacterium liquid
600, can specifically calculate the transport velocity of every milligram of dry weight cell according to this formula; Gained typical curve equation is: y
(mg dw/ml)=0.2365x
(OD600nm)+ 0.1149, x span is at 1-22.
Embodiment 2: the relation between incubation time and the transport velocity measured value is measured
Method is as follows:
(1) selects the bacterium liquid of cultivating in embodiment 1 step (4) 15 hours, centrifugal (3000rmin for use
-1, it is resuspended 5min) to remove behind the former substratum 50mmol/L PBS liquid with pH6.5;
(2) relation between incubation time and the transport velocity measured value is measured: get intact cell suspension in an amount of step (1) with 96 hole flat boards or EP pipe, the adding final concentration is the pNPX of 6mmol/L, with being placed on 30 ℃, 200rmin
-1In the shaking table, hatch 10min, 20min, 30min, 40min, 50min and 60min respectively, centrifuging and taking supernatant 100 μ l add in the 96 hole flat boards, add the Na of isopyknic 0.4mol/L again
2CO
3The aqueous solution behind the mixing, uses microplate reader in OD
405nmThe following absorbance value of measuring the p-nitrophenol (pNP) of hatching the back generation.Hatch initial value when measuring, add behind the pNPX pNPX that adds final concentration 6mmol/L behind an amount of volume supernatant of centrifuging and taking immediately, both totally are 100 μ l, add the aqueous sodium carbonate of the 0.4mol/L of 100 μ l again, behind the mixing, with microplate reader in OD
405nmUnder measure absorbance value.Mensuration obtains OD
405nmCorresponding relation between the value of reading and the incubation time is seen Fig. 5; PNP typical curve y according to the preparation of the PBS liquid of pH6.5
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735 and the bacterium liquid OD of laboratory strains
600nmThe corresponding relation y of value and dry cell weight
(mgdw/ml)=0.2365x
(OD600nm)+ 0.1149, calculate, in 60 minutes, incubation time does not influence the transport velocity value, sees Fig. 6.
Embodiment 3: under the intact cell mensuration state, and the inside and outside Determination on content of pNP born of the same parents
Method is as follows:
(1) selects the bacterium liquid of cultivating in embodiment 1 step (4) 15 hours, centrifugal (3000rmin for use
-1, it is resuspended 5min) to remove behind the former substratum 50mmol/L PBS liquid with pH6.5;
(2) under the intact cell mensuration state, the outer Determination on content of pNP born of the same parents: get intact cell suspension in an amount of step (1) with 96 hole flat boards or EP pipe, the adding final concentration is the pNPX of 6mmol/L, with being placed on 30 ℃, 200rmin
-1In the shaking table, hatch 10min and 30min respectively; Centrifuging and taking supernatant 100 μ l add in the 96 hole flat boards, add the Na of isopyknic 0.4mol/L again
2CO
3The aqueous solution behind the mixing, uses microplate reader in OD
405nmThe following absorbance value of measuring the p-nitrophenol (pNP) of hatching the back generation.Hatch initial value when measuring, do not add pNPX earlier, only add bacteria suspension and experimental group is hatched jointly, add the pNPX of final concentration 6mmol/L behind an amount of volume supernatant of centrifuging and taking, both totally are 100 μ l, add the aqueous sodium carbonate of the 0.4M of 100 μ l again, behind the mixing, use microplate reader in OD
405nmUnder measure absorbance value; PNP typical curve y according to the preparation of the PBS liquid of pH6.5
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735 and the bacterium liquid OD of laboratory strains
600nmThe corresponding relation y of value and dry cell weight
(mg dw/ml)=0.2365x
(OD600nm)+ 0.1149, calculate born of the same parents' content of pNP outward, see Fig. 7;
(3) under the intact cell mensuration state, the mensuration of pNP born of the same parents' intensive amount: get the bacterium mud after the intact cell of hatching 10min and 30min in (2) is measured, the sodium carbonate solution that after the PBS of 1ml washes fast, adds the 0.2mol/L of 408 μ l, 0.2g granulated glass sphere, 6000rpm, 30s, with tissue homogenizer smudge cells 4 times, centrifugal, get 200 μ l with 96 hole flat boards and measure the value of reading at the 405nm place, the pNP typical curve y for preparing according to the PBS liquid of pH6.5
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735 and the bacterium liquid OD of laboratory strains
600nmThe corresponding relation y of value and dry cell weight
(mg dw/ml)=0.2365x
(OD600nm)+ 0.1149, calculate the pNP content in the born of the same parents, see Fig. 7.
The mensuration of the pNPX transportcapacity of the clone of embodiment 4:AraT and bacterial strain BSW2ABT
Method is as follows:
(1) bacterial classification is selected: select for use heterogenous expression in the born of the same parents of structure that laboratory strains BSW2AB (the CEN.PK102-3A derivative of xylosidase (Bp-xynB) is arranged, MATa, leu2-3, ura3-52, pYX242-PGKt-TEFp TPIp-xynB-PGKt) do acceptor strain bacterium;
(2) expression vector establishment: use downstream primer AraTup:5 '-CGC
GGATCCATGAATAAGCAGAAAAAGGTTT-3 ' and AraTdown:5 '-ACGC
GTCGACTTAATGACTCGCTTGTTGTACG-3 ' is from plant lactobacillus (Lactobacillus plantarum) DNA clone transporter gene AraT, utilize BamH I and SalI two restriction enzyme sites to make up yeast saccharomyces cerevisiae episome shuttle plasmid pJFE3-araT, as shown in Figure 3;
(3) strain construction: plasmid pJFE3-araT transforms the BSW2AB bacterial strain with the Lithium Acetate conversion method, screens correct transformant with the complete synthesis auxotroph substratum SC-Leu-Ura that adds 2% glucose;
(4) strain culturing: 40ml is in the little triangular flask of 100ml with the complete synthesis auxotroph substratum SC-Leu-Ura that adds 2% glucose, switching for the transformant list bacterium colony that checking is correct in will step (3), cultivates after 1 day and transfers once, initial OD
600Connecing is 0.5, and culture condition is 30 ℃, 200rmin
-1
(5) intact cell suspension preparation: get the bacterium liquid of cultivating in the step (4) 10 hours, centrifugal (3000rmin
-1, it is resuspended 5min) to remove behind the former substratum 50mmol/L PBS liquid with pH6.5;
(6) measuring the xylosidase enzyme under the intact cell state lives: get intact cell suspension in an amount of step (5) with 96 hole flat boards or EP pipe, the adding final concentration is the pNPX of 6mmol/L, and both totally are 120 μ l, with being placed on 30 ℃, 200rmin
-1Hatched in the shaking table 30 minutes, centrifuging and taking supernatant 100 μ l add in the 96 hole flat boards, add the Na of isopyknic 0.4mol/L again
2CO
3The aqueous solution behind the mixing, uses microplate reader in OD
405nmThe following absorbance value of hatching the p-nitrophenol (pNP) that generates after 30 minutes of measuring.When hatching initial value mensuration, do not add earlier pNPX, only add bacteria suspension and experimental group is hatched jointly, the pNPX that adds final concentration 6mmol/L behind an amount of volume supernatant of centrifuging and taking, both totally are 100 μ l, the aqueous sodium carbonate termination reaction that adds the 0.4mol/L of 100 μ l again behind the mixing, uses microplate reader in OD
405nmUnder measure absorbance value; PNP typical curve y according to the preparation of the PBS liquid of pH6.5
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735 and the bacterium liquid OD of laboratory strains
600nmThe corresponding relation y of value and dry cell weight
(mgdw/ml)=0.2365x
(OD600nm)+ 0.1149, the xylosidase enzyme that calculates under the intact cell state is lived, and sees Fig. 4;
Embodiment 5: the pNPX of bacterial strain BSW2ABP and BSW2ABT and the competitive relation of wood sugar are measured
Method is as follows:
(1) selects the bacterium liquid of cultivating in embodiment 4 steps (4) 10 hours, centrifugal (3000rmin for use
-1, it is resuspended 5min) to remove behind the former substratum 50mmol/L PBS liquid with pH6.5;
(2) competitive relation checking: get intact cell suspension in an amount of step (1) with 96 hole flat boards or EP pipe, adding final concentration is the pNPX of 6mmol/L, add final concentration in addition and be respectively the wood sugar of 50mmol/L, 100mmol/L, 150mmol/L, 200mmol/L, 250mmol/L, the three totally is 120 μ l, with being placed on 30 ℃, 200rmin
-1Hatched in the shaking table 30 minutes, centrifuging and taking supernatant 100 μ l add in the 96 hole flat boards, add the Na of isopyknic 0.4mol/L again
2CO
3The aqueous solution behind the mixing, uses microplate reader in OD
405nmThe following absorbance value of hatching the p-nitrophenol (pNP) that generates after 30 minutes of measuring.Hatch initial value when measuring, do not add pNPX earlier, only add bacteria suspension and experimental group is hatched jointly, add the pNPX of final concentration 6mmol/L behind an amount of volume supernatant of centrifuging and taking, both totally are 100 μ l, add the aqueous sodium carbonate of the 0.4mol/L of 100 μ l again, behind the mixing, use microplate reader in OD
405nmUnder measure absorbance value; PNP typical curve y according to the preparation of the PBS liquid of pH6.5
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735 and the bacterium liquid OD of laboratory strains
600nmThe corresponding relation y of value and dry cell weight
(mg dw/ml)=0.2365x
(OD600nm)+ 0.1149, calculate this translocator to the transport velocity of pNPX; The wood sugar of checking different concns proves the transport velocity that can reflect wood sugar with the transport velocity of pNPX to the competitive relation of pNPX in two bacterial strains, sees Fig. 8 and Fig. 9.
The high flux screening of embodiment 6:AraT high reactivity wood sugar translocator mutant
Method by above-described embodiment 4, the present invention has obtained the recombinant Saccharomyces cerevisiae bacterial strain of a plant height wood sugar turn-over capacity, this bacterial strain is BSW2ABT, classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae, be preserved on November 17th, 2011 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center ", deposit number are CGMCC No.5464.Its mycology is characterized as: this yeast strain is unicellular eukaryotic microorganisms, oval, do not move, be gemmation under the culture condition of the complete synthesis auxotroph substratum of solid or liquid (SC-Leu, this substratum are existing in the prior art), exist with unicellular form under the liquid culture condition, during solid culture, be bacterium colony and exist, bacterium colony smooth surface, moistening, thickness are creamy white.CEN.PK102-3A?derivative，MATa，leu2-3，ura3-52，pYX242-PGKt-TEFpTPIp-xynB-PGKt，pJFE3-AraT。This bacterial strain has been expressed the clone from the HUCEP-8 Gene A raT of plant lactobacillus (Lactobacillus plantarum), its wood sugar turn-over capacity more common Wine brewing yeast strain be more than doubled (1.06 times are seen Fig. 8).Can predict, this bacterial strain contains in other product scopes of wood sugar raw material production in utilization and has wide purposes, as being starting strain with this bacterial strain, introduces the xylose metabolism approach, can make up to have the recombinant bacterial strain that high level utilizes the wood sugar ability.High-throughput screening method is as follows:
(1) bacterial classification is selected: select for use heterogenous expression in the born of the same parents of structure that laboratory strains BSW2AB (the CEN.PK102-3A derivative of xylosidase (Bp-xynB) is arranged, MATa, leu2-3, ura3-52, pYX242-PGKt-TEFp TPIp-xynB-PGKt) do acceptor strain bacterium;
(2) expression vector establishment: use downstream primer AraTup:5 '-CGC
GGATCCATGAATAAGCAGAAAAAGGTTT-3 ' and AraTdown:5 '-ACGC
GTCGACTTAATGACTCGCTTGTTGTACG-3 ', transporter gene AraT with the last clone of plant lactobacillus (Lactobacillus plantarum) DNA is template, the mode of employing fallibility PCR obtains the sudden change storehouse of AraT, utilizes BamH I and Sal I two restriction enzyme sites to make up sudden change storehouse expression vector at pJFE3 yeast saccharomyces cerevisiae episome shuttle plasmid;
(3) strain construction: the plasmid of structure transforms the BSW2AB bacterial strain with the Lithium Acetate conversion method, with the complete synthesis auxotroph substratum SC-Leu-Ura screening transformant that adds 2% glucose;
(4) strain culturing: with the transformant list bacterium colony dibbling of growth in the step (3) in the 96 hole flat boards that contain 200 μ l substratum, mutant strain is not in contrast for every plate dibbling simultaneously 3 hole BSW2ABT, substratum is the complete synthesis auxotroph substratum SC-Leu-Ura that contains 2% glucose, cultivate and (grew to stationary phase, and guaranteed the OD in each hole in 1-2 days
600nmThe value basically identical) after, the corresponding switching in each hole 10 μ l cultivate 10h in 190 μ l fresh cultures again; Culture condition is 30 ℃, 200rmin
-1
(5) intact cell suspension preparation: get the bacterium liquid of cultivating in the step (4) 10 hours, centrifugal (3000rmin
-1, 5min) remove behind the former substratum resuspended with the 50mmol/L PBS liquid of 102 μ l pH6.5;
The photoabsorption (primary dcreening operation) of the p-nitrophenol generation that (6) mensuration generated after 30 minutes under the intact cell state: measure in the enzyme liquid to the intact cell of step (5), add the pNPX 18 μ l of 40mmol/L, with being placed on 30 ℃, 200rmin
-1Hatched centrifugal (3000rmin in the shaking table 30 minutes
-1, 5min) get supernatant 100 μ l and add in the 96 hole flat boards, add the Na of isopyknic 0.4mol/L again
2CO
3The aqueous solution behind the mixing, uses microplate reader in OD
405nmThe following absorbance value of hatching the p-nitrophenol (pNP) that generates after 30 minutes of measuring.The screening absorbance value is than the do not suddenly change bacterial strain of plant height of BSW2ABT;
(7) mensuration of the wood sugar transport velocity of high mutant strain (multiple sieve): the intact cell suspension of the high mutant strain that obtains in (4) and (5) preparation processes (6) set by step, redeterminate the absorbance value of the p-nitrophenol (pNP) of generation according to the method for step (6), and measure OD
600nmThe value of reading.PNP typical curve y according to the preparation of the PBS liquid of pH6.5
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735 and the bacterium liquid OD of laboratory strains
600nmThe corresponding relation y of value and dry cell weight
(mg dw/ml)=0.2365x
(OD600nm)+ 0.1149, calculate translocator to the transport velocity of pNPX (enzyme that namely utilizes intact cell to record is lived), screening wood sugar transport velocity is than the BSW2ABT bacterial strain that improves of mutant strain not;
(8) the high reactivity bacterial strain that screening obtains in the switching (7), behind the anti-upgrading grain, transformed into escherichia coli spreads cultivation, and order-checking obtains highly active transporter gene sequence.
Claims (8)
1. express the Wine brewing yeast strain of xylobiase in the strain born of the same parents, it is characterized in that: this bacterial strain is BSW2AB, classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 17th, 2011, deposit number is CGMCC No.5465.
2. one kind is screened and can express the method for the Wine brewing yeast strain of activated wood sugar translocator, it is characterized in that step is as follows:
(1) strain culturing: bacterial strain to be detected and conventional bacterial strain are carried out the routine cultivation respectively, get bacterium liquid;
(2) the centrifugal substratum that removes of the bacterium liquid that above-mentioned cultivation is obtained uses phosphate buffered saline buffer resuspended then, gets the resuspended liquid of cell;
(3) in the resuspended liquid of above-mentioned cell, add xylose structure analogue pNPX, hatch back centrifuging and taking supernatant, add isopyknic Na
2CO
3Solution is measured the 405nm absorbance value OD of place
405nm
(4) set up pNP at 405nm place absorbance value-pNP content standard curve and bacterium liquid OD
600nmValue-dry cell weight typical curve, the OD that integrating step (3) records
405nm, calculate translocator to the transport velocity of pNPX;
(5) judge: contrast the transport velocity of bacterial strain to be detected and the transport velocity of conventional bacterial strain, if the transport velocity of bacterial strain to be detected is higher than the transport velocity of conventional bacterial strain, prove that then the wood sugar translocator that this bacterial strain to be detected is expressed is the albumen that can improve Wine brewing yeast strain wood sugar turn-over capacity, difference is more big, illustrates that the ability of the wood sugar translocator transhipment wood sugar that this bacterial strain to be detected is expressed is more high; If the transport velocity of bacterial strain to be detected is not higher than the transport velocity of conventional bacterial strain, prove that then this bacterial strain to be detected do not express the albumen that can improve yeast saccharomyces cerevisiae wood sugar turn-over capacity;
Described conventional bacterial strain refers to that deposit number is the bacterial strain BSW2AB of CGMCC No.5465, classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae, has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 17th, 2011;
Described bacterial strain to be detected makes up by the following method:
(1) expression vector establishment: set up the recombinant vectors that includes transporter gene to be screened;
(2) recombinant bacterial strain makes up: the recombinant vectors of setting up in the step (1) is transformed in the Wine brewing yeast strain of expressing xylobiase in the described born of the same parents of claim 1, and obtains transforming successful transformant by screening, be bacterial strain to be detected.
3. method according to claim 2, it is characterized in that: the used plasmid of described construction of expression vector is the pJFE3 empty plasmid.
4. method according to claim 2 is characterized in that: described conversion adopts the Lithium Acetate conversion method to transform.
5. method according to claim 2, it is characterized in that: the described pNP of foundation in the method for 405nm place absorbance value-pNP content standard curve is: with the pNP of the PBS liquid preparation gradient concentration of pH6.5, and measure the 405nm absorbance value OD of place
405nm, set up typical curve; Gained typical curve equation is: y
(OD405nm)=0.0916x
(pNP/nmol)+ 0.0735, x span is at 1.5-7.5.
6. method according to claim 2 is characterized in that: the described bacterium liquid OD that sets up
600nmThe method of value-dry cell weight typical curve is: get the bacterium liquid of cultivation, and centrifugal washing, dry weight is measured in suction filtration, oven dry back; Dilute six levels, calculate dry weight and measure corresponding OD according to extension rate
600Gained typical curve equation is: y
(mg dw/ml)=0.2365x
(OD600nm)+ 0.1149, x span is at 1-22; That directly measure in experiment is the OD of bacterium liquid
600, specifically calculate the transport velocity of every milligram of dry weight cell according to above-mentioned formula.
7. method according to claim 2, it is characterized in that: the transport velocity that the transport velocity of described bacterial strain to be detected is higher than conventional bacterial strain refers to that bacterial strain transport velocity to be detected exceeds 18.5% of conventional bacterial strain transport velocity.
8. a strain can be expressed the Wine brewing yeast strain of activated wood sugar translocator, it is characterized in that: this bacterial strain is BSW2ABT, classification called after Saccharomyces Cerevisiae in S accharomyces cerevisiae, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 17th, 2011, deposit number is CGMCC No.5464.
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