CN101130782B - Construction method for producing 1,3-trimethylene glycol regrouping saccharomyces cerevisiae with glucose as substrate - Google Patents
Construction method for producing 1,3-trimethylene glycol regrouping saccharomyces cerevisiae with glucose as substrate Download PDFInfo
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Abstract
The invention discloses a constructing method for producing 1, 3-methyl ethylene glycol recombinant Saccharomyces cerevisiae with glucose as substrate in the gene engineering technique field. The invention constructs recombinant plasmid pGAPZB-yqhD, pGAPZB-dhaB and the vector pYX212-zeocin firstly, which constructs corpus expression vector pYX212-zeocin-pGAP-yqhD-pGAP-dhaB of Saccharomyces cerevisiae and expresses in Saccharomyces cerevisiae W303-1A. The recombinant Saccharomyces cerevisiae applies glucose as the substrate, and the content of 1, 3-methyl ethylene is about 1. 5g/l after fermenting 70 hours. The invention achieves the expression of yqhD and dhaB in series successfully in Saccharomyces cerevisiae, which creates the basis for constructing gene engineering bacterial with high-producing 1, 3-methyl ethylene with glucose as the substrate.
Description
Technical field
With glucose is that substrate produces 1, and the construction process of ammediol recombinant Saccharomyces cerevisiae belongs to gene engineering technology field.
Background technology
1, ammediol (1,3-Propanediol, 1,3-PD) be a kind of important chemical material, be used for industries such as food, makeup and pharmacy, 1 as the medicine and the intermediate of organic synthesis, the most important purposes of ammediol is exactly as synthesizing new polyester such as poly terephthalic acid-1, the monomer of ammediol ester (PTT).1, the ammediol production method mainly contains chemical synthesis and microbe transformation method.Have only chemical synthesis to realize industrialization at present, but chemical method synthesizes 1, the ammediol facility investment is big, technical difficulty height, separation and purification of products difficulty; Microbe transformation method produces 1, and ammediol still is in laboratory and pilot scale research stage, but microbe transformation method is environmentally friendly, the reaction conditions gentleness, and raw material is easy to get, thereby has more meaning.
That finds at present can produce 1, and the natural bacterial strain of ammediol (as klebsiella, Fu Shi lemon bacterium, clostridium butylicum) can only be substrate with glycerine all, and through following two-step reaction, promptly glycerine generates intermediate product 3-hydroxy propanal under the effect of glycerol dehydratase; The 3-hydroxy propanal is 1, and the catalysis of ammediol oxydo-reductase generates 1 down, ammediol.
At home, use nature bacterial strain fermentative production 1, ammediol has obtained paying close attention to widely, and klebsiella is to transform 1, and ammediol is bacterial classification preferably, but because they are as possible pathogenic of entero-bacte, so be subjected to certain restriction in practical application.There are problems such as fermentation condition harshness, the production cycle is long, glycerol conversion yield is low in Fu Shi lemon bacterium and clostridium butylicum, can't carry out large-scale production application.The two-step fermentation of Tsing-Hua University is glycerine with yeast with conversion of glucose earlier, is 1 with bacterium with transformation of glycerol again, and also there is the equipment requirements height in ammediol, operates comparatively shortcomings such as complexity.It is that substrate produces 1 that this breadboard doctor Zhang Xiaomei has made up with glycerine, the recombination bacillus coli of ammediol, but still do not solve the too high problem of raw materials cost.
U.S. Dupont company utilizes genetic engineering bacterium at first conversion of glucose to be glycerine, and then is converted into 1, ammediol, and successfully having made up a strain directly is substrate high yield 1 with glucose, the genetic engineering bacterium of ammediol.
Because glycerine is compared with other cheap carbon sources such as glucose, price is higher, has increased fermentation costs.And occurring in nature not have to be 1 with conversion of glucose directly, therefore the microorganism of ammediol utilizes molecular biotechnology to make up and carbohydrate such as glucose can be converted into 1, the bacterial classification of ammediol becomes the important means that reduces fermentation costs.
The work of being done from Dupont company as can be seen, the method exploitation of using pathways metabolism is that the biological catalyst of raw material will have competitive power more in future with carbohydrate (as glucose).
The present invention will utilize Protocols in Molecular Biology to being that the yeast saccharomyces cerevisiae that substrate produces glycerine is transformed with glucose, use 1 of future in the hope of intravital glycerine further being converted into more have, ammediol, thereby realize with the glucose of cheapness but not glycerine is substrate direct production 1, the target of ammediol.
Summary of the invention
The objective of the invention is to make up with glucose is that substrate produces 1, the ammediol recombinant Saccharomyces cerevisiae.Recombinant Saccharomyces cerevisiae of the present invention can be earlier that substrate produces glycerine with glucose, and the glycerine with cylinder accumulation further is converted into 1 again, and ammediol is a substrate high yield 1 for further making up with glucose, and the genetic engineering bacterium of ammediol lays the first stone.
Technical scheme of the present invention: with glucose is that substrate produces 1, the construction process of ammediol recombinant Saccharomyces cerevisiae, be the glycerol dehydrase gene dhaB that derives from klebsiella that will obtain and derive from colibacillary 1, ammediol oxidoreductase isozyme gene yqhD, be connected with carrier PGAPZB respectively, i.e. carrier construction PGAPZB-dhaB and PGAPZB-yqhD; Cut with the method for PCR with enzyme and to obtain expression casette PGAP-dhaB-AOX1TT and PGAP-yqhD-AOX1TT; The zeocin resistant gene is inserted yeast saccharomyces cerevisiae expression vector pYX212, carrier construction pYX212-zeocin; PGAP-yqhD-AOX1TT and PGAP-dhaB-AOX1TT are connected into carrier pYX212-zeocin successively, carrier construction pYX212-zeocin-pGAP-yqhD-pGAP-dhaB; Be inserted into yeast saccharomyces cerevisiae W303-1A with the Lithium Acetate conversion method, obtain recombinant Saccharomyces cerevisiae;
(1) structure of plasmid PGAPZB-yqhD:
EcoR I enzyme is cut carrier pHsh-dhaB-yqhD, discharges 1 of 1.2kb size, and ammediol oxidoreductase isozyme encoding gene yqhD reclaims the test kit purifying with PCR fragment glue; Cut carrier PGAPZB with EcoR I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 2.9kb with the PCR fragment; Use T
4Ligase enzyme connects yqhD/EcoR I and PGAPZB/EcoR I, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 25 μ g/mL Zeocin resistances, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 25 μ g/mL Zeocin resistances, extract plasmid, carrying out enzyme with EcoR I and Bgl I cuts, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after PGAPZB-yqhD, positive recombinant called after JM109/PGAPZB-yqhD;
(2) structure of plasmid PGAPZB-dhaB:
Cut carrier pHsh-dhaB-yqhD with Xba I enzyme, discharge the dehydrating glycerin enzyme coding gene dhaB of 2.7kb size, reclaim the test kit purifying with PCR fragment glue; Cut carrier PGAPZB with Xba I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 2.9kb with the PCR fragment; Use T
4Ligase enzyme connects dhaB/Xba I and PGAPZB/Xba I, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 25 μ g/mL Zeocin resistances, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 25 μ g/mL Zeocin resistances, extract plasmid, carrying out enzyme with Xba I and BamH I cuts, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after PGAPZB-dhaB, positive recombinant called after JM109/PGAPZB-dhaB;
(3) structure of plasmid pYX212-zeocin:
With carrier PGAPZB is template, and pcr amplification goes out the resistant gene zeocin of 1.2kb size, reclaims the test kit purifying with PCR fragment glue; Extract plasmid pYX212, cut carrier pYX212 with EcoR I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 8.3kb with the PCR fragment; Use T
4Ligase enzyme connects zeocin/EcoR I and pYX212/EcoR I, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 25 μ g/mL Zeocin resistances, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose are inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 25 μ g/mL Zeocin resistances, extract plasmid, carrying out enzyme with EcoR I cuts, agarose gel electrophoresis is identified positive colony, and positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after pYX212-zeocin, positive recombinant called after JM109/pYX212-zeocin;
(4) structure of plasmid pYX212-zeocin-pGAP-yqhD and pYX212-zeocin-pGAP-yqhD-pGAP-dhaB:
The structure of plasmid pYX212-zeocin-pGAP-yqhD:
Extract pYX212-zeocin, cut carrier pYX212-zeocin with BamH I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 8.3kb with the PCR fragment; Plasmid PGAPZB-yqhD by agarose gel electrophoresis analytical reaction product, target stripe occur at the application of sample swimming lane through BglII and BamH I double digestion, reclaims the target fragment that test kit reclaims out 2.1kb with the PCR fragment; Use T
4Ligase enzyme connects two enzymes and cuts the good fragment of purifying, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose are inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/mL penbritins, extract plasmid, Xba I enzyme is cut, agarose gel electrophoresis is identified positive colony, and positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after pYX212-zeocin-pGAP-yqhD, positive recombinant called after JM109/pYX212-zeocin-pGAP-yqhD;
The structure of pYX212-zeocin-pGAP-yqhD-pGAP-dhaB:
Extracting plasmid PGAPZB-dhaB, is that template is carried out pcr amplification with it, and the primer is as follows:
P1：5’-CGC
AGATCTTTTTTGTAGAAATG-3’
P2：5’-CGC
AGATCTGCACAAACGAAGGTCTCAC-3’
PCR method is as follows: add in the 50 μ L reaction systems: each 1.5 μ L of the primer P1 of 10mol/L and P2, the dNTP 5 μ L of 2mmol/L, 10 * ExTaq Buffer, 5 μ L, the ExTaq archaeal dna polymerase 0.5 μ L of 5U/ μ L, template 1 μ g adds distilled water polishing 50 μ L; The PCR condition is: 95 ℃ of sex change 5 minutes, 52 ℃ 3 minutes, 72 ℃ were extended 5 minutes, 94 ℃ of sex change 1 minute circulate 35 times;
Analyze the purifying reaction product by agarose gel electrophoresis, target stripe occurs, reclaim the target fragment that test kit reclaims out 3.6kb with the PCR fragment at the application of sample swimming lane; The target fragment that purifying is good is cut through the BglII enzyme, reclaims test kit with the PCR fragment and reclaims; PYX212-zeocin-pGAP-yqhD cuts through BamH I enzyme, reclaims test kit with the PCR fragment and reclaims; Use T
4Ligase enzyme connects two enzymes and cuts the good fragment of purifying, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose are inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/mL penbritins, extract plasmid, cut by Xba I enzyme, agarose gel electrophoresis is identified positive colony, and positive recombinant is kept among the LB that contains 20% glycerine, freezingly is stored in one 80 ℃; Recombinant plasmid called after pYX212-zeocin-pGAP-yqhD-pGAP-dhaB, positive recombinant called after JM109/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;
(5) structure of recombinant Saccharomyces cerevisiae:
Get the JM109/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB of a freezing preservation, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/mL penbritins, extract plasmid, with efficient Lithium Acetate conversion method transformed saccharomyces cerevisiae W303-1A, conversion product is coated SM (YPD+150 μ g/mLZeocin) flat board, in 30 ℃, cultivated 2 days; The bacterium colony that grows is rule again to the SM flat board, the bacterium colony that grows tentatively is defined as positive transformant, extract the plasmid of positive transformant according to the extracting method of yeast plasmid, direct then transformed into escherichia coli JM109, extract plasmid again, cut by PCR and Xba I enzyme, the checking plasmid has changed yeast saccharomyces cerevisiae W303-1A over to, the positive transformant that obtains there be not continuous passage cultivation on the YPD flat board of microbiotic Zeocin, per ten substitute the SM plate screening once, the bacterial classification that deletion can not be grown promptly is the recombinant Saccharomyces cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB of genetic stability through the bacterial classification that still can grow on the SM flat board after 30 generations.
DhaB among the free type expression vector pYX212-zeocin-pGAP-yqhD-pGAP-dhaB and yqhD do not utilize the promotor on the carrier pYX212, but place respectively the source zymic glyceraldehyde 3-phosphate dehydro-genase gene (GAP) the promotor downstream, then on the carrier pYX212-zeocin series connection after expressing.
This recombinant Saccharomyces cerevisiae can detect glycerol dehydratase (DHAB) and 1, the enzyme of two kinds of enzymes of ammediol oxidoreductase isozyme (YQHD) is lived, success is expressed gene dhaB and yqhD series connection back in yeast saccharomyces cerevisiae, and expression of heterologous genes is to not influence of zymic growth, can be that substrate ferments with glucose, behind the fermentation 70h, can detect 1 in the fermented liquid, ammediol.
Recombinant Saccharomyces cerevisiae fermentation product 1, ammediol with structure:
Recombinant Saccharomyces cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB is inoculated in the triangular flask of 250mL of the 30mL seed culture medium that consists of peptone 2%, yeast extract paste 1% and glucose 2%, behind 30 ℃, 150rpm incubated overnight, be inoculated in 10% and consist of peptone 2%, yeast extract paste 1%, glucose 10% and VB
12In the 500mL triangular flask of 0.001% 40mL fermention medium, 30 ℃, 150rpm are cultivated 70h, and centrifugal collection thalline is surveyed dry cell weight; With supernatant liquor detect 1, the content of ammediol, record 1, the content of ammediol is 1.5g/L.
The acquisition of gene dhaB and yqhD, the structure of carrier PGAPZB-dhaB, PGAPZB-yqhD, pYX212-zeocin and pYX212-zeocin-pGAP-yqhD-pGAP-dhaB, gene dhaB and yqhD are that the fermentation of substrate sees embodiment for details in Expression in Saccharomyces Cerevisiae and with glucose.
Glycerine generates the 3-hydroxy propanal under the effect of glycerol dehydratase; The 3-hydroxy propanal is 1, and the catalysis of ammediol oxydo-reductase generates 1 down, ammediol.In the present invention, the dehydrating glycerin enzyme coding gene is that dhaB derives from klebsiella, and 1, ammediol redox enzyme coding gene does not select to derive from the dhaT of klebsiella, but selected to derive from colibacillary non-specific oxydo-reductase-1, the isozyme encoding gene yqhD of ammediol oxydo-reductase, current research is found, it can replace 1 in microbe, ammediol redox enzyme catalysis 3-hydroxy propanal generates 1, ammediol, with 1, the ammediol oxydo-reductase is compared, and non-specific redox enzyme catalysis 3-hydroxy propanal is converted into 1, and the efficient of ammediol obviously improves.Carrier pYX212 is a kind of fabric shuttle-type plasmid, it is the free type expression vector of yeast saccharomyces cerevisiae, copy number is 50～200, the elements such as selection type marker gene URA3,2 μ that have the URA defective type, host's yeast saccharomyces cerevisiae W303-1A that the present invention selects is the URA defective type, so the preferential free type expression vector of selecting pYX212 as yeast saccharomyces cerevisiae.
Zeocin is a kind of glycoprotein microbiotic that belongs to bleomycin family, can act on most of bacteriums in vivo and (comprise: E.coli) and fungi (as yeast).In order efficiently to select positive transformant, the present invention inserts carrier pYX212 with resistant gene zeocin, has made up carrier pYX212-zeocin.
The present invention does not select the promotor on the carrier pYX212 for use, but has selected the promotor that derives from zymic glyceraldehyde 3-phosphate dehydro-genase gene (GAP) for use, and this promotor is the strong promoter of composing type, can start the expression of goal gene efficiently.
The free type expression vector that has heterologous gene can transform host system by transforming lithium salts method, PEG method, protoplasm body or electroporation.The success cell transformed promptly contains the cell of the free type expression vector that the present invention makes up, and can be identified by method well known in the art, as collecting cell, extracts plasmid after the cracking, carries out enzyme then and cuts with PCR and identify.
The host system that the present invention selects is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) W303-1A, yeast saccharomyces cerevisiae is a kind of model animals, genetic background is clear, in biology field, brought into play important effect, characteristics such as easy to operate, the growth that the zymic expression system has a prokaryotic expression system rapidly, nutritional requirement is simple.Yeast saccharomyces cerevisiae itself can be that fermenting substrate is produced glycerine with glucose, so the present invention selects yeast saccharomyces cerevisiae synthetic 1 as host's heterogenous expression glycerine, two key gene dhaB of ammediol and yqhD.
Beneficial effect of the present invention: gene dhaB and yqhD are placed the downstream of GAP respectively, thereby guaranteed the effective expression of gene; Because 1, the ammediol oxydo-reductase is to metal ion and NAD
+Concentration ratio responsive, with by 1 of yqhD coding, the ammediol oxidoreductase isozyme is compared, 1 of dhaT coding, and the ammediol oxydo-reductase is very low to 3-hydroxy propanal catalytic efficiency, substitute gene dhaT with gene yqhD, avoided 1, the inactivation of ammediol oxydo-reductase has reduced the accumulation of intermediate product 3-hydroxy propanal, help 1, the generation of ammediol.The present invention at home and abroad first will dhaB and yqhD series connection back in yeast saccharomyces cerevisiae W303-1A, express, first dhaB and yqhD are placed respectively to be connected on yeast saccharomyces cerevisiae W303-1A behind the promotor downstream of source zymic glyceraldehyde 3-phosphate dehydro-genase gene (GAP) and to express.
Utilize the Zeocin resistant panel to select to have the yeast saccharomyces cerevisiae W303-1A of the carrier pYX212-zeocin-pGAP-yqhD-pGAP-dhaB that the present invention makes up, i.e. recombinant Saccharomyces cerevisiae.By cultivating recombinant Saccharomyces cerevisiae, detect in the fermented liquid 1, the content of ammediol.Cultivation is chosen in 250mL and shakes on the bottle and carry out, and cultivates and divides two stages, and the fs is mainly used in the zymic growth, and it is synthetic that subordinate phase is mainly used in product.Can detect 1 with gas phase, liquid phase, ammediol; Can adopt the 5% discontinuous slab-electrophoresis that concentrates glue and 12% separation gel to carry out albumen sepn, and detect glycerol dehydratase and 1, the expression of heterologous protein (DHAB and YQHD) is analyzed in the work of ammediol oxidoreductase isozyme enzyme.
Description of drawings
Fig. 1 plasmid PGAPZB-yqhD
The enzyme of Fig. 2 plasmid PGAPZB-yqhD is cut checking Lane1 carrier PGAPZB EcoR I enzyme and is cut; Lane2 yqhD EcoR I enzyme is cut; Lane3 PGAPZB-yqhD EcoR I enzyme is cut; Lane4PGAPZB-yqhD Bgl I enzyme is cut; Lane5 dna molecular amount standard.
Fig. 3 plasmid PGAPZB-dhaB
Fig. 4 plasmid PGAPZB-dhaB enzyme is cut checking Lanel dna molecular amount standard; Lane2 carrier PGAPZB Xba I enzyme is cut; Lane3 dhaB Xba I enzyme is cut; Lane4 PGAPZB-dhaB Xba I enzyme is cut; Lane5 PGAPZB-dhaB BamH I enzyme is cut Lane6 dna molecular amount standard.
Fig. 5 plasmid pYX212-zeocin
The enzyme of Fig. 6 plasmid pYX212-zeocin is cut checking Lanel dna molecular amount standard; Lane2 carrier pYX212 EcoR I enzyme is cut; Lane3 zeocin EcoR I enzyme is cut; Lane4 pYX212-zeocinEcoR I enzyme is cut; Lane5 pYX212-zeocin EcoR I enzyme is cut; Lane6 dna molecular amount standard.
Fig. 7 plasmid pYX212-zeocin-pGAP-yqhD
The enzyme of Fig. 8 plasmid pYX212-zeocin-pGAP-yqhD is cut checking Lane 1DNA molecular weight standard; Lane2 pYX212-zeocin/Xba I enzyme is cut; Lane3 pYX212-zeocin-pGAP-yqhD/Xba I enzyme is cut.
Fig. 9 plasmid pYX212-zeocin-pGAP-yqhD-pGAP-dhaB
The enzyme of Figure 10 plasmid pYX212-zeocin-pGAP-yqhD-pGAP-dhaB is cut checking Lane1DNA molecular weight standard; Lane2 pYX212-zeocin-pGAP-yqhD-pGAP-dhaB Xba I enzyme is cut.
The proteic SDS-PAGE of Figure 11 recombinant Saccharomyces cerevisiae analyzes Lane1 yeast saccharomyces cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB; Lane2 yeast saccharomyces cerevisiae W303-1A; Lane3 molecular weight of albumen standard.
In Figure 12 recombinant Saccharomyces cerevisiae and the control fermentation characteristic S.cerevisiaeW303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB fermented liquid 1, the accumulation of ammediol (▲) and glycerine (■); The accumulation of glycerine in S.cerevisiae W303-1A (◆) fermented liquid.
Growth curve S.cerevisiaeW303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB (◆) the and S.cerevisiaeW303-1A (■) of Figure 13 recombinant Saccharomyces cerevisiae.
Specific implementation method
Embodiment 1: the structure of plasmid PGAPZB-yqhD:
EcoR I enzyme is cut carrier pHsh-dhaB-yqhD,, discharge and be about 1 of 1.2kb size, ammediol oxidoreductase isozyme encoding gene yqhD reclaims the test kit purifying with PCR fragment glue; Extract plasmid PGAPZB, cut carrier PGAPZB with EcoR I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 2.9kb with the PCR fragment, uses T
4Ligase enzyme connects yqhD/EcoR I and PGAPZB/EcoR I, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 25 μ g/mLZeocin resistances, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 25 μ g/mL Zeocin resistances, extract plasmid, carrying out enzyme with EcoR I and Bgl I respectively cuts, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after PGAPZB-yqhD, positive recombinant called after JM109/PGAPZB-yqhD.
Embodiment 2: the structure of plasmid PGAPZB-dhaB:
Xba I enzyme is cut carrier pHsh-dhaB-yqhD, discharges the dehydrating glycerin enzyme coding gene dhaB that is about the 2.7kb size, reclaims the test kit purifying with PCR fragment glue; Extract plasmid PGAPZB, cut carrier PGAPZB with Xba I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 2.9kb with the PCR fragment, uses T
4Ligase enzyme connects dhaB/Xba I and PGAPZB/Xba I, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 25 μ g/mL Zeocin resistances, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 25 μ g/mL Zeocin resistances, extract plasmid, carrying out enzyme with Xba I and BamH I respectively cuts, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after PGAPZB-dhaB, positive recombinant called after JM109/PGAPZB-dhaB.
Embodiment 3: the structure of plasmid pYX212-zeocin:
With carrier PGAPZB is template, and pcr amplification goes out the resistant gene zeocin of 1.2kb size, reclaims the test kit purifying with PCR fragment glue; Extract plasmid pYX212, cut carrier pYX212 with EcoR I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 8.3kb with the PCR fragment, uses T
4Ligase enzyme connects zeocin/EcoR I and pYX212/EcoR I, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 25 μ g/mL Zeocin resistances, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 25 μ g/mL Zeocin resistances, extract plasmid, carrying out enzyme with EcoR I respectively cuts, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after pYX212-zeocin, positive recombinant called after JM109/pYX212-zeocin.
Embodiment 4: the structure of plasmid pYX212-zeocin-pGAP-yqhD and pYX212-zeocin-pGAP-yqhD-pGAP-dhaB:
The structure of plasmid pYX212-zeocin-pGAP-yqhD:
Extract pYX212-zeocin, cut carrier pYX212-zeocin with BamH I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 8.3kb with the PCR fragment; Extract plasmid PGAPZB-yqhD, PGAPZB-yqhD by agarose gel electrophoresis analytical reaction product, target stripe occur at the application of sample swimming lane through Bgl II and BamH I double digestion, reclaims the target fragment that test kit reclaims out 2.1kb with the PCR fragment; Use T
4Ligase enzyme connects two enzymes and cuts the good fragment of purifying, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose are inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/mL penbritins, extract plasmid, Xba I enzyme is cut, agarose gel electrophoresis is identified positive colony, and positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after pYX212-zeocin-pGAP-yqhD, positive recombinant called after JM109/pYX212-zeocin-pGAP-yqhD.
The structure of pYX212-zeocin-pGAP-yqhD-pGAP-dhaB:
Extracting plasmid PGAPZB-dhaB, is that template is carried out pcr amplification with it, and the primer is as follows:
P1：5’-CGC
AGATCTTTTTTGTAGAAATG-3’
P2：5’-CGC
AGATCTGCACAAACGAAGGTCTCAC-3’
PCR method is as follows: add in the 50 μ L reaction systems: each 1.5 μ L of the primer P1 of 10mol/L and P2, the dNTP 5 μ L of 2mmol/L, 10 * ExTaq Buffer, 5 μ L, the ExTaq archaeal dna polymerase 0.5 μ L of 5U/ μ L, template 1 μ g adds distilled water polishing 50 μ L; The PCR condition is: 95 ℃ of sex change 5 minutes, 52 ℃ 3 minutes, 72 ℃ were extended 5 minutes, 94 ℃ of sex change 1 minute circulate 35 times.
Analyze the purifying reaction product by agarose gel electrophoresis, target stripe occurs, reclaim the target fragment that test kit reclaims out 3.6kb with the PCR fragment at the application of sample swimming lane; The target fragment that purifying is good is cut through the BglII enzyme, reclaims test kit with the PCR fragment and reclaims; PYX212-zeocin-pGAP-yqhD cuts through BamH I enzyme, reclaims test kit with the PCR fragment and reclaims; Use T
4Ligase enzyme connects two enzymes and cuts the good fragment of purifying, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose are inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/mL penbritins, extract plasmid, cut by Xba I enzyme, agarose gel electrophoresis is identified positive colony, and positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after pYX212-zeocin-pGAP-yqhD-pGAP-dhaB, positive recombinant called after JM109/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB.
Embodiment 5: the structure of recombinant Saccharomyces cerevisiae:
Get the JM109/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB of a freezing preservation, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/mL penbritins, extract plasmid, with efficient Lithium Acetate conversion method transformed saccharomyces cerevisiae, conversion product is coated SM (YPD+150 μ g/mL Zeocin) flat board, in 30 ℃, cultivated 2 days.The bacterium colony that grows is rule again to the SM flat board, the bacterium colony that grows tentatively is defined as positive transformant, extract the plasmid of positive transformant according to the extracting method of yeast plasmid, direct then transformed into escherichia coli JM109, extract plasmid again, cut by PCR and Xba I enzyme, the proof plasmid has changed yeast saccharomyces cerevisiae W303-1A really over to, the positive transformant that obtains there be not continuous passage cultivation on the flat board of selective pressure, per ten substitute the SM plate screening once, the bacterial classification that deletion can not be grown is promptly thought the bacterial classification of genetic stability through the bacterial classification that still can grow after 30 generations on the SM flat board.
Embodiment 6: the expression of recombinant Saccharomyces cerevisiae W303-1A fermentation character and heterologous protein:
Recombinant Saccharomyces cerevisiae W303-1A fermentation character:
The recombinant Saccharomyces cerevisiae of genetic stability is inoculated in the triangular flask of 250mL of 30mL seed culture medium (peptone 2%, yeast extract paste 1%, glucose 2%), after the incubated overnight, be inoculated in 40mL fermention medium (peptone 2%, yeast extract paste 1% with 10%, glucose 10%, VB
120.001%) in the 500mL triangular flask, cultivate 70h, centrifugal collection thalline is used to survey dry cell weight; Supernatant liquor is used to detect 1, the content of 3-PD.
In the fermented liquid 1, the content of ammediol and glycerine adopts gas Chromatographic Determination.Agilent 1490 type gas chromatographs, the stainless steel column of 2m (
3mm), homemade polymer microsphere GDX-401 (110 order) is a stationary phase.Column temperature: 250 ℃, the sample introduction temperature: 260 ℃, detected temperatures: 260 ℃, nitrogen is as carrier gas, uses hydrogen flame detector, sample introduction 5 μ L, and 1, the retention time of ammediol is 2.6min, calculates in the fermented liquid 1 with external standard method, the content of ammediol.
After screening strain recombinant Saccharomyces cerevisiae fermentation 70h, glycerol content is about 3.5g/L in the fermented liquid, 1, and ammediol content is about 1.5g/L.And glycerol content is about 6.0g/L in the contrast S.cerevisiae W303-1A fermented liquid, but does not produce 1, ammediol.Find the biomass and contrast basically identical of S.cerevisiaeW303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB simultaneously.
The expression of heterologous protein:
With above-mentioned product 1, the recombinant Saccharomyces cerevisiae of 3-PD is inoculated in 30mL seed culture medium (peptone 2%, yeast extract paste 1%, glucose 2%) in the triangular flask of 250mL, after the incubated overnight, be inoculated in 40mL fermention medium (peptone 2% with 10%, yeast extract paste 1%, glucose 10%, VB
120.001%) in the 500mL triangular flask, cultivate 70h, centrifugal collection thalline, adopt the ultrasonic disruption cell, carry out the SDS-PAGE checking, detect recombinant Saccharomyces cerevisiae glycerol dehydratase enzyme and live (DHAB) and 1, ammediol oxidoreductase isozyme (YQHD) enzyme is lived, simultaneously with yeast saccharomyces cerevisiae W303-1A in contrast.
Aldehyde material that glycerine generates under the effect of glycerol dehydratase and 3-methyl-2-[4-morpholinodithio ketone hydrazone MBTH forms the compound of brown or pink colour, at 305nm maximum absorption is arranged, by measuring the absorbancy of reaction solution, measure the amount of product with cessation method, because glycerine has stronger restraining effect to glycerol dehydratase, so adopt 1, the 2-propylene glycol is that substrate is analyzed the dehydrating glycerin enzyme activity.Reaction system: 2.5mL: wherein, 0.1mL KCl (0.05mol/L), 0.1mL 1,2-PD (0.2mol/L), 0.1mL VB
12(15 μ mol/L), 0.7mL enzyme liquid adds the timing of enzyme liquid, and 37 ℃, 10min adds 1mL 0.1mol/L Tripotassium Citrate damping fluid (pH3.6) termination reaction, adds 0.5mL0.1%MBTH colour developing 15min, 37 ℃.Measure the 305nm absorbancy, with 1, the 2-propylene glycol is that to multiply by that coefficient 1.41 is converted to glycerine be that the enzyme of glycerol dehydratase of substrate is lived in the enzyme work of the glycerol dehydratase that records of substrate.Enzyme activity unit (U/mL)=(VT * Δ A * K)/(ε * VS), wherein, VT: reaction solution cumulative volume; VS: extract sample volume; Δ A: per minute absorbancy changing value; K: diluted sample multiple; ε: molar absorptivity.
Enzyme work unit definition is under the standard reaction condition, and 1min catalysis generates 1 μ mol propionic aldehyde and is defined as the enzyme work of 1 unit.
1, the mensuration that ammediol oxidoreductase isozyme (YQHD) enzyme is lived: because 3-hydroxy propanal instability does not have commercialization, so enzyme activity determination comes quantitatively according to the speed that NADP+ under 25 ℃ of conditions is reduced into NADPH.Reaction system: 3mL:0.3mL 1,3-PD (0.1mol/L), 0.25mL NADP (2mmol/L), 0.3mL 30mmol/L (NH
4)
2SO
4, 2.15mL K
2CO
3-KHCO
3Damping fluid (pH 9.0) adds the crude enzyme liquid initial action, and 340nm measures the variation of 3min light absorption value.Other mensuration and method of calculation are the same, wherein ε
340nm=6.3mmol
-1Cm
-1L, reversed reaction 1, the enzyme work that coefficient 3.95 is converted to positive reaction is multiply by in the enzyme work of ammediol oxidoreductase isozyme.The enzyme unit definition of living is that 1 international unit (1U) equals the per minute kind and reduces 1 μ mol substrate or generate the enzyme amount of 1 μ mol product.The dehydrating glycerin enzyme activity that the result records this strain recombinant Saccharomyces cerevisiae is the 24U/mg total protein, 1, and ammediol oxidoreductase isozyme vigor is the 15U/mg total protein, lives and detect in control strain less than the enzyme of these two kinds of enzymes.
Protein content determination: the crude enzyme liquid protein content adopts the Bradford method to measure, and is standard protein with BSA.
Find this strain recombination yeast 61,43,21, the protein characteristic band appears in the 16kD place, conform to the target protein size of bibliographical information.It is that substrate produces 1, ammediol genes of brewing yeast engineering bacteria that the present invention has successfully made up with glucose at home first.
Claims (4)
1. be that substrate produces 1 with glucose, the construction process of ammediol recombinant Saccharomyces cerevisiae, it is characterized in that the glycerol dehydrase gene dhaB that derives from klebsiella that to obtain and derive from colibacillary 1, ammediol oxidoreductase isozyme gene yqhD, be connected with carrier PGAPZB respectively, i.e. carrier construction PGAPZB-dhaB and PGAPZB-yqhD; Cut with the method for PCR with enzyme and to obtain expression casette PGAP-dhaB-AOX1TT and PGAP-yqhD-AOX1TT; The zeocin resistant gene is inserted yeast saccharomyces cerevisiae expression vector pYX212, carrier construction pYX212-zeocin; PGAP-yqhD-AOX1TT and PGAP-dhaB-AOX1TT are connected into carrier pYX212-zeocin successively, carrier construction pYX212-zeocin-pGAP-yqhD-pGAP-dhaB; Change it over to yeast saccharomyces cerevisiae W303-1A with the Lithium Acetate conversion method, obtain recombinant Saccharomyces cerevisiae;
(1) structure of plasmid PGAPZB-yqhD:
EcoR I enzyme is cut carrier pHsh-dhaB-yqhD, discharges 1 of 1.2kb size, and ammediol oxidoreductase isozyme encoding gene yqhD reclaims the test kit purifying with PCR fragment glue; Cut carrier PGAPZB with EcoR I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 2.9kb with the PCR fragment; Use T
4Ligase enzyme connects yqhD/EcoR I and PGAPZB/EcoR I, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 25 μ g/mL Zeocin resistances, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 25 μ g/mL Zeocin resistances, extract plasmid, carrying out enzyme with EcoR I and Bgl I cuts, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after PGAPZB-yqhD, positive recombinant called after JM109/PGAPZB-yqhD;
(2) structure of plasmid PGAPZB-dhaB:
Cut carrier pHsh-dhaB-yqhD with Xba I enzyme, discharge the dehydrating glycerin enzyme coding gene dhaB of 2.7kb size, reclaim the test kit purifying with PCR fragment glue; Cut carrier PGAPZB with Xba I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 2.9kb with the PCR fragment; Use T
4Ligase enzyme connects dhaB/Xba I and PGAPZB/Xba I, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 25 μ g/mL Zeocin resistances, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 25 μ g/mL Zeocin resistances, extract plasmid, carrying out enzyme with Xba I and BamH I cuts, agarose gel electrophoresis is identified positive colony, positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after PGAPZB-dhaB, positive recombinant called after JM109/PGAPZB-dhaB;
(3) structure of plasmid pYX212-zeocin:
With carrier PGAPZB is template, and pcr amplification goes out the resistant gene zeocin of 1.2kb size, reclaims the test kit purifying with PCR fragment glue; Extract plasmid pYX212, cut carrier pYX212 with EcoR I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 8.3kb with the PCR fragment; Use T
4Ligase enzyme connects zeocin/EcoR I and pYX212/EcoR I, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 25 μ g/mL Zeocin resistances, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose are inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 25 μ g/mL Zeocin resistances, extract plasmid, carrying out enzyme with EcoR I cuts, agarose gel electrophoresis is identified positive colony, and positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after pYX212-zeocin, positive recombinant called after JM109/pYX212-zeocin;
(4) structure of plasmid pYX212-zeocin-pGAP-yqhD and pYX212-zeocin-pGAP-yqhD-pGAP-dhaB:
The structure of plasmid pYX212-zeocin-pGAP-yqhD:
Extract pYX212-zeocin, cut carrier pYX212-zeocin with BamH I enzyme, the agarose gel electrophoresis purifying reclaims the target fragment that test kit reclaims out 8.3kb with the PCR fragment; Plasmid PGAPZB-yqhD by agarose gel electrophoresis analytical reaction product, target stripe occur at the application of sample swimming lane through Bgl II and BamH I double digestion, reclaims the target fragment that test kit reclaims out 2.1kb with the PCR fragment; Use T
4Ligase enzyme connects two enzymes and cuts the good fragment of purifying, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose are inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/mL penbritins, extract plasmid, Xba I enzyme is cut, agarose gel electrophoresis is identified positive colony, and positive recombinant is kept among the LB that contains 20% glycerine, freezing being stored in-80 ℃; Recombinant plasmid called after pYX212-zeocin-pGAP-yqhD, positive recombinant called after JM109/pYX212-zeocin-pGAP-yqhD;
The structure of pYX212-zeocin-pGAP-yqhD-pGAP-dhaB:
Extracting plasmid PGAPZB-dhaB, is that template is carried out pcr amplification with it, and the primer is as follows:
P1：5’-CGC
AGATCTTTTTTGTAGAAATG-3’
P2：5’-CGC
AGATCTGCACAAACGAAGGTCTCAC-3’
PCR method is as follows: add in the 50 μ L reaction systems: each 1.5 μ L of the primer P1 of 10mol/L and P2, the dNTP 5 μ L of 2mmol/L, 10 * ExTaq Buffer, 5 μ L, the ExTaq archaeal dna polymerase 0.5 μ L of 5U/ μ L, template 1 μ g adds distilled water polishing 50 μ L; The PCR condition is: 95 ℃ of sex change 5 minutes, 52 ℃ 3 minutes, 72 ℃ were extended 5 minutes, 94 ℃ of sex change 1 minute circulate 35 times;
Analyze the purifying reaction product by agarose gel electrophoresis, target stripe occurs, reclaim the target fragment that test kit reclaims out 3.6kb with the PCR fragment at the application of sample swimming lane; The target fragment that purifying is good is cut through the BglII enzyme, reclaims test kit with the PCR fragment and reclaims; PYX212-zeocin-pGAP-yqhD cuts through BamH I enzyme, reclaims test kit with the PCR fragment and reclaims; Use T
4Ligase enzyme connects two enzymes and cuts the good fragment of purifying, connects product transformed into escherichia coli JM109 competent cell, and conversion product is coated the LB agar plate that contains 100 μ g/mL penbritins, 37 ℃ of overnight incubation; 5 bacterium colonies of random choose are inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/mL penbritins, extract plasmid, cut by Xba I enzyme, agarose gel electrophoresis is identified positive colony, and positive recombinant is kept among the LB that contains 20% glycerine, freezingly is stored in one 80 ℃; Recombinant plasmid called after pYX212-zeocin-pGAP-yqhD-pGAP-dhaB, positive recombinant called after JM109/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;
(5) structure of recombinant Saccharomyces cerevisiae:
Get the JM109/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB of a freezing preservation, be inoculated in 20mL and contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/mL penbritins, extract plasmid, with efficient Lithium Acetate conversion method transformed saccharomyces cerevisiae W303-1A, conversion product is coated the SM flat board of YPD+150 μ g/mL Zeocin, in 30 ℃, cultivated 2 days; The bacterium colony that grows is rule again to the SM flat board, the bacterium colony that grows tentatively is defined as positive transformant, extract the plasmid of positive transformant according to the extracting method of yeast plasmid, direct then transformed into escherichia coli JM109, extract plasmid again, cut by PCR and Xba I enzyme, the checking plasmid has changed yeast saccharomyces cerevisiae W303-1A over to, the positive transformant that obtains there be not continuous passage cultivation on the YPD flat board of microbiotic Zeocin, per ten substitute the SM plate screening once, the bacterial classification that deletion can not be grown promptly is the recombinant Saccharomyces cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB of genetic stability through the bacterial classification that still can grow on the SM flat board after 30 generations.
2. the construction process of recombinant Saccharomyces cerevisiae according to claim 1, it is characterized in that dhaB among the free type expression vector pYX212-zeocin-pGAP-yqhD-pGAP-dhaB and yqhD do not utilize the promotor on the carrier pYX212, but place the promotor downstream of source zymic glyceraldehyde 3-phosphate dehydro-genase gene GAP respectively, expressing after the series connection on the carrier pYX212-zeocin then.
3. the recombinant Saccharomyces cerevisiae of using the described method of claim 1 to make up, it is characterized in that to detect glycerol dehydratase DHAB and 1, the enzyme of two kinds of enzymes of ammediol oxidoreductase isozyme YQHD is lived, success is expressed gene dhaB and yqhD series connection back in yeast saccharomyces cerevisiae, and expression of heterologous genes is to not influence of zymic growth, can be that substrate ferments with glucose, fermentation 70h detects 1, ammediol in the fermented liquid.
4. use the application of the recombinant Saccharomyces cerevisiae of the described method structure of claim 1, it is characterized in that with its fermentation produce 1, ammediol: recombinant Saccharomyces cerevisiae W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB is inoculated in the triangular flask of 250mL of the 30mL seed culture medium that contains peptone 2%, yeast extract paste 1% and glucose 2%, behind 30 ℃, 150rpm incubated overnight, be inoculated in 10% and contain peptone 2%, yeast extract paste 1%, glucose 10% and VB
12In the 500mL triangular flask of 0.001% 40mL fermention medium, 30 ℃, 150rpm are cultivated 70h, and centrifugal collection thalline is surveyed dry cell weight; With supernatant liquor detect 1, the content of ammediol, record 1, the content of ammediol is 1.5g/L.
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