CN104263739A - Nucleic acid molecule for encoding xylose translocator and xylose translocator encoded by nucleic acid molecule - Google Patents
Nucleic acid molecule for encoding xylose translocator and xylose translocator encoded by nucleic acid molecule Download PDFInfo
- Publication number
- CN104263739A CN104263739A CN201410526356.9A CN201410526356A CN104263739A CN 104263739 A CN104263739 A CN 104263739A CN 201410526356 A CN201410526356 A CN 201410526356A CN 104263739 A CN104263739 A CN 104263739A
- Authority
- CN
- China
- Prior art keywords
- xylose
- translocator
- sequence
- nucleic acid
- acid molecule
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a nucleic acid molecule for encoding a xylose translocator, the xylose translocator encoded by the nucleic acid molecule and a host cell expressing xylose-specific translocator. An experiment shows that the transport capacity of the xylose translocator provided by the invention over xylose is comparable to that of an efficient endogenous translocator of saccharomyces cerevisiae. By the translocator, xylose rather than glucose is specifically transported to the saccharomyces cerevisiae, and the transportation of the xylose is free of the limitation of the glucose. The translocator is favorable for improving the xylose transport capacity during the co-fermentation of the glucose and the xylose, and a space is developed for the application of the xylose translocator to the technical fields of enzyme engineering, genetic engineering, metabolic engineering and synthetic biology.
Description
Technical field
The present invention relates to a kind of nucleic acid molecule of encodes xylose translocator and the xylose transport albumen of coding thereof, and described xylose transport albumen is in the application of enzyme engineering, genetically engineered, metabolic engineering and synthetic biology technical field.The invention still further relates to the host cell and the application aborning of this host cell of expressing this xylose transport albumen.
Background technology
Miscellaneous lignocellulosic sources annual production is huge, and comprising a large amount of agricultural industrial wood waste, its comprehensive utilization both can bring economic benefit, can reduce again the environmental pollution because the improper process such as burning cause.In lignocellulosic material, wood sugar component concentration is up to about 30%, is only second to glucose component, and carrying out comprehensive utilization to it can significantly improve raw material availability.Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is widely used in food and industrial microorganism, and it has the characteristics such as the vigorous and grade-safe of strong robustness, metabolism.Wild type Saccharomyces cerevisiae can not utilize wood sugar, but can utilize its isomer---xylulose.Pass through genetic engineering means, express Xylose reductase and xylitol dehydrogenase in yeast saccharomyces cerevisiae simultaneously, or it is the ability of xylulose that single expression xylose isomerase can give yeast saccharomyces cerevisiae by xylose, and then the optimization expression of the enzyme to be related to by relevant metabolic pathway, can obtain can be cell factory (the Van Vleet J H and Jeffries T W.Yeast metabolic engineering for hemicellulosic ethanol production.Curr Opin Biotech of various target product by xylose, 2009, 20:300-306.).
Wood sugar is the first step that wood sugar is utilized to the transport in brewing yeast cell.High level expression HUCEP-8 can promote that yeast saccharomyces cerevisiae is to the absorption of wood sugar.Yeast saccharomyces cerevisiae does not have special xylose transport albumen, it depends on hexose transport albumen to the absorption of wood sugar, but these nonspecific translocator transports to wood sugar are subject to the strong competitive inhibition of glucose, thus have impact on bacterial strain and having (the Subtil T and Boles E.Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae.Biotechnol Biofuels of the xylose utilization efficiency under glucose environment, 2012,5:14).Although Farwick etc. devise the screening system based on growth phenomenon in the recent period, screen the wood sugar transport protein that multiple release glucose suppresses, utilize this system, although screening obtains a transport xylose and does not transport glucose, and there is not the yeast saccharomyces cerevisiae Inner source HUCEP-8 Gal2p muton Gal2p-N376F suppressed by glucose in wood sugar transport, (Farwick A about but the wood sugar transportcapacity of this muton is only the half of wild-type Gal2p wood sugar transportcapacity, et al.Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose.P Natl Acad Sci USA, 2014, 111:5159-5164.).And on the other hand, occurring in nature exists the multiple natural microorganism utilizing wood sugar, from these microorganisms, clone xylose transport albumen is also the direction that researcher is made great efforts.Such as, the Gxf1p expressing Candida intermedia improves xylose metabolism ability (the Leandro M J that Wine brewing yeast strain improves bacterial strain under lower concentration wood sugar condition, et al.Two glucose/xylose transporter genes from the yeast Candida intermedia:first molecular characterization of a yeast xylose-H+symporter.Biochem J, 2006,395:543-549; Runquist D, et al.Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae.Appl Microbiol Biot, 2009,82:123-130.); The Sut1p expressing Scheffersomyces (Pichia) stipitis improves bacterial strain is total to fermentation stage ethanol production (Katahira S at glucose and xylose, et al.Improvement of ethanol productivity during xylose and glucose co-fermentation by xylose-assimilating S.cerevisiae via expression of glucose transporter Sut1.Enzyme Microb Tech, 2008,43:115-119.); At5g59250p and At5g17010p expressing Arabidopsis thaliana makes the transport velocity of bacterial strain wood sugar and wear rate improve 46% and 40% (Hector R E respectively, et al.Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption.Appl Microbiol Biot, 2008,80:675-684.).But these albumen can both transport glucose simultaneously, wood sugar transport is still affected.In addition, there is research display, Trichosporon cutaneum (Hu C, et al.Simultaneous utilization of glucose and xylose for lipid production by Trichosporon cutaneum.Biotechnol Biofuels, 2011,4:25.) present the synchronous utilization level of good glucose and xylose during the fermentation with Meyerozyma guilliermondii bacterial strain, be expected to become efficient xylose transport dietary protein origin.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the problem to be solved in the present invention is to provide a kind of nucleic acid molecule of encodes xylose translocator and the xylose transport albumen of coding thereof, and expresses the host cell of this wood sugar specificity translocator.
The nucleic acid molecule of encodes xylose translocator of the present invention, is characterized in that, its nucleotides sequence is classified as one of following sequence:
(1) nucleotide sequence shown in SEQ ID NO.1, this sequence is the mutant of GeneID:5124182 in Genebank, and its 1078-1080 position Nucleotide sports TTC by AAT;
(2) nucleotide sequence of the nucleotide sequence of (1) is different from due to the degeneracy of genetic codon;
(3) with the sequence of nucleotide sequence at least 80% homology shown in (1), and its Nucleotide corresponding to (1) 1078-1080 position is TTC;
(4) because the degeneracy of genetic codon is different from the nucleotide sequence of the nucleotide sequence described in (3).
The nucleic acid molecule of above-mentioned encodes xylose translocator, the nucleotide sequence shown in its nucleotide sequence preferred SEQ ID NO.1, this nucleic acid molecule called after MGT05196 (N360F).
Xylose transport albumen of the present invention, is characterized in that, its aminoacid sequence is one of following sequence:
(1) aminoacid sequence shown in SEQ ID NO.2, this sequence is the Point mutont of albumen protein_id=" XP_001482176.1 " in Genebank, and its amino-acid residue of 360 sports phenylalanine (F) by the glutamine (N) of protein_id=" XP_001482176.1 ".
(2) have the homology of 80% at least with the aminoacid sequence shown in (1), and the corresponding amino-acid residue of the aminoacid sequence shown in its to SEQ ID NO.2 360 is phenylalanine residue.
The nucleotide sequence of encodes xylose translocator of the present invention and the aminoacid sequence of coding thereof, its sequence does not comprise the sequence of published coding protein_id=" XP_001482176.1 ", does not comprise coding and the aminoacid sequence shown in SEQ ID NO.2 has the homology of at least 80% but is equivalent to the sequence that 360 of the aminoacid sequence shown in SEQ ID NO.2 are not phenylalanine (F) yet.
Above-mentioned xylose transport albumen, the aminoacid sequence shown in its aminoacid sequence preferred SEQ ID NO.2, this aminoacid sequence called after Mgt05196p (N360F).
A kind of recombinant vectors, it contains the complete coding reading frame sequence of the nucleic acid molecule of above-mentioned encodes xylose translocator.
Wherein: described recombinant vectors is preferably recombinant plasmid pJFE3-MGT05196 (N360F).
Express a host cell for xylose transport albumen, it is bacterium, yeast or filamentous fungus, the complete coding reading frame sequence of the nucleic acid molecule containing above-mentioned encodes xylose translocator in this host cell or recombinant vectors.
Wherein: described host cell preferably saccharomyces cerevisiae BSW4M51X (N360F).
Xylose transport albumen of the present invention is utilizing the application in the raw material production Xylitol containing wood sugar, ethanol, polyvalent alcohol, organic acid, hydro carbons or terpenoid.
The invention discloses a kind of nucleic acid molecule of encodes xylose translocator and the xylose transport albumen of coding thereof, and express the host cell of this wood sugar specificity translocator.Experiment proves xylose transport albumen provided by the invention being on close level to the transportcapacity of wood sugar and yeast saccharomyces cerevisiae efficient Inner source translocator Gal2p.This albumen is narrow spectrum does not transport glucose to transport xylose in yeast saccharomyces cerevisiae, and it is not subject to the suppression of glucose to the transport of wood sugar.Prompting expresses this translocator by being conducive to improving when co-fermentation of glucose and xylose the ability of wood sugar transport, for xylose transport albumen has opened up space in the application of enzyme engineering, genetically engineered, metabolic engineering and synthetic biology technical field.
Accompanying drawing explanation
Fig. 1: recombinant plasmid pJFE3-MGT05196 (N360F) physical map.This recombinant plasmid is yeast saccharomyces cerevisiae 2 μ plasmid, and MGT05196 (N360) gene is expressed under yeast saccharomyces cerevisiae constitutive promoter TEF1 promotor controls.
Fig. 2: the bacterial strain of expressing different translocator hatches wood sugar accumulation volume analysis in body in wood sugar.
Fig. 3: the growth of recombinant bacterial strain on maltose and glucose.
Fig. 4: bacterial strain BSW4M51X (N360F) growth characteristics analysis on different carbon source.The growth characteristics of bacterial strain BSW4M51X (N360F) in the SC-URA auxotroph substratum adding different carbon source.
Wherein, ● for the addition of 20g/L wood sugar, ◆ for the addition of 20g/L glucose, ■ adds 10g/L glucose for the addition of 20g/L wood sugar, ▲ add 20g/L glucose for the addition of 20g/L wood sugar.
Embodiment
Wine brewing yeast strain EBY.VW4000 (MAT α leu2-3,112 ura3-52 trp1-289 his3-Δ 1 Mal2-8c SUC2hxt17 Δ hxt13 Δs:: loxP hxt15 Δ:: loxP hxt16 Δ:: loxP hxt14 Δ:: loxP hxt12 Δ:: loxP hxt9 Δ:: loxP hxt11 Δ:: loxP hxt10 Δ:: loxP hxt8 Δ:: loxP hxt514::loxP hxt2 Δ:: loxP hxt367 Δ:: loxP gal2 Δ stl1 Δ:: loxP agt1 Δ:: loxP ydl247w Δ:: loxP yjr160c Δ:: loxP) (Wieczorke R, et al.Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae.FEBS Letters, 1999,464:123-128.) be the bacterial strain (hxt null) that a strain has lacked all Inner sources hexose transport albumen, it for want of can not at grown on glucose to the ability of transmitter loss hexose, and can only grow on this carbon substrate with autospecific transport protein of maltose.In this bacterial strain, express HUCEP-8 can make bacterial strain again at grown on glucose, and its energy for growth can reflect the glucose transport capacity of expressed translocator.The EBY.VW4000 expressing HUCEP-8 is hatched certain hour in wood sugar, and then measures the gross accumulation amount of wood sugar and Xylitol in cell, the transportcapacity of expressed translocator to wood sugar can be reflected.After introduce xylose utilization approach in EBY.VW4000, bacterial strain is that energy for growth on sole carbon source can reflect the transportcapacity of translocator to wood sugar at wood sugar.
Below in conjunction with embodiment, the present invention is further illustrated.
The clone of embodiment 1 translocator encoding gene
Extract M.guilliermondii ATCC 6260 chromosomal DNA as template, pcr amplification is carried out with the Fast Pfu polysaccharase that Quan Shi King Company produces, overlapping extension is adopted to introduce rite-directed mutagenesis (Urban A, et al.A rapid and efficient method for site-directed mutagenesis using one-step overlap extension PCR.Nucleic acids research, 1997,25:2227-2228.).Be primer with primer MGT05196-F and MGT05196-N360F-R, pcr amplified dna fragment MGT05196-up, the annealing temperature selected during its pcr amplification is preferably 56 DEG C; Be primer PCR amplification of DNA fragments MGT05196-down with primer MGT05196p-N360F-F and MGT05196-R, the annealing temperature selected during its pcr amplification is preferably 55 DEG C.Then, with DNA fragmentation MGT05196-up and MGT05196-down primer and template each other, carry out the overlap-extension PCR without primer, its annealing temperature is preferably 56 DEG C.Obtain final product through DNA sequencing, turn out to be two ends with extra contacts SEQ ID NO.1 shown in nucleotide sequence, i.e. gene M GT05196 (N360F).The extra contacts sequence that its 5 ' end band has is 5 '-GCAATCTAATCTAAGTTTTAATTACAAAGGATCC-3 ', and the extra contacts sequence that its 3 ' end band has is 5 '-CTCGAGGTCGACCTGCAGGATTGAATTGAATTGAAAT-3 '.
Using M.guilliermondii ATCC 6260 chromosomal DNA as template, MGT05196-F and MGT05196-R is primer, pcr amplification is the nucleotide sequence in Genebank shown in GeneID:5124182 with the concrete sequence of extra contacts, i.e. Mgt05196p encoding gene MGT05196, the annealing temperature selected during its pcr amplification is preferably 55 DEG C.The extra contacts sequence that its 5 ' end band has is 5 '-GCAATCTAATCTAAGTTTTAATTACAAAGGATCC-3 ', and the extra contacts sequence that its 3 ' end band has is 5 '-CTCGAGGTCGACCTGCAGGATTGAATTGAATTGAAAT-3 '.
Wherein, above-mentioned primer MGT05196-F, MGT05196-R, MGT05196-N360F-R and MGT05196p-N360F-F primer sequence is:
MGT05196-F:5’-GCAATCTAATCTAAGTTTTAATTACAAAGGATCCATGTCGTCGAATGAGCAGGTTACTC-3’
MGT05196-R:5’-ATTTCAATTCAATTCAATCCTGCAGGTCGACCTCGAGTCAAACCCTTTCGGCTTCGTCC-3’
MGT05196p-N360F-F:5’-GTTCTTGGTATAGTG
TTCTTTGCATCCACTTTTG-3’
MGT05196-N360F-R:5’-CAAAAGTGGATGCAAA
GAACACTATACCAAGAAC-3’
Embodiment 2 builds Mgt05196p (N360F) expression plasmid
With the MGT05196 with extra contacts (N360F) gene DNA fragment obtained in BamH I and Xho I double digestion embodiment 1, and be connected to yeast saccharomyces cerevisiae 2 μ plasmid vector pJFE3 (the Shen Y cut through BamH I and Sal I enzyme, et al.An efficient xylose-fermenting recombinant Saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile.Appl Microbiol Biot, 2012,96:1079-1091.) on.Wherein, in linked system, the concentration ratio of the plasmid vector pJFE3 that (N360F) gene DNA fragment of the MGT05196 with extra contacts of BamH I and Xho I double digestion and BamH I and Sal I enzyme are cut is 10:1 ~ 3:1.Its preferred concentration is than being 5:1.Use T4 ligase enzyme catalysis ligation, reaction conditions is 16 ~ 25 DEG C, 1 ~ 4 hour.Its preferred reaction conditions is 16 DEG C, 4 hours.Ligation liquid direct transformation of E. coli Trans5 α Chemically Competent Cell (purchased from Beijing Quanshijin Biotechnology Co., Ltd), in containing the LB substratum of 50 μ g/ml penbritins, screen transformant, the cell be not converted can not at this grow on plates.The transformant of the single clone of picking, the plasmid in transformant cells is extracted with plasmid extraction kit Plasmid Mini Kit I (purchased from Omega Bio-tek company), the plasmid that electrophoresis detection is extracted from each transformant, wherein size is the plasmid of 7765bp is correct recombinant plasmid, this recombinant plasmid called after pJFE3-MGT05196 (N360F), on this plasmid, MGT05196 (N360) gene expresses (Fig. 1) under yeast saccharomyces cerevisiae constitutive promoter TEF1 promotor controls.
Wherein intestinal bacteria transform and operate according to Trans5 α Chemically Competent Cell (purchased from Beijing Quanshijin Biotechnology Co., Ltd) specification sheets.
Wherein, plasmid extraction operates according to plasmid extraction kit Plasmid Mini Kit I (purchased from Omega Bio-tek company) specification sheets.
Use the same method, the MGT05196 gene DNA fragment with extra contacts also uses BamH I and Xho I double digestion, and is connected on the plasmid pJFE3 that cuts through BamH I and Sal I enzyme.Correct recombinant plasmid size is also 7765bp, this plasmid called after pJFE3-MGT05196.
Embodiment 3 expresses translocator in yeast saccharomyces cerevisiae
With empty carrier plasmid pJFE3 transformed saccharomyces cerevisiae bacterial strain EBY.VW4000, the SC-URA auxotroph culture medium flat plate adding 20g/L maltose screens transformant, and the cell be not converted can not at this grow on plates.The transformant called after BSW4PP obtained.
By recombinant plasmid pJFE3-GAL2 (Wang C, et al.Improvement of L-arabinose fermentation by modifying the metabolic pathway and transport in Saccharomyces cerevisiae.BioMed Research International, 2013,461204.) transform bacterial strain EBY.VW4000, the SC-URA auxotroph culture medium flat plate adding 20g/L maltose screens transformant, and the cell be not converted can not at this grow on plates.The transformant called after BSW4PG obtained.
Recombinant plasmid pJFE3-MGT05196 is transformed bacterial strain EBY.VW4000, and the SC-URA auxotroph culture medium flat plate adding 20g/L maltose screens transformant, and the cell be not converted can not at this grow on plates.The transformant called after BSW4M51 obtained.
Recombinant plasmid pJFE3-MGT05196 (N360F) is transformed bacterial strain EBY.VW4000, and the SC-URA auxotroph culture medium flat plate adding 20g/L maltose screens transformant, and the cell be not converted can not at this grow on plates.The transformant called after BSW4M51 (N360F) obtained.
Wherein, Saccharomyces cerevisiae transformant use PEG/LiAc induction chemical conversion process (Fang Zhijia, etc.The new exploration of Saccharomyces cerevisiae transformant method." laboratory study and exploration " 04 phase in 2012).
Wherein, containing 1.7g/L YNB (yeast basic nitrogen source, purchased from Shanghai Sheng Gong biotechnology limited-liability company) in SC-URA auxotrophy substratum, 5g/L ammonium sulfate, 0.77g/L SC-URA (purchased from MP Biomedicals); Solid medium adds 20g/L agar powder in addition, and pH is adjusted to 6.0 ~ 7.0; Sterilising conditions: 115 DEG C, 30min; The maltose that final concentration is 20g/L is added as carbon source during use.
Embodiment 4 wood sugar accumulation volume method compares the wood sugar transportcapacity of transport protein
At 30 DEG C, bacterial strain BSWPP, BSW4PG, BSW4M51 and BSW4M51 (N360F) is cultivated in the SC-URA auxotroph substratum adding 20g/L maltose, culture transfer after 12 hours in the SC-URA auxotroph substratum of fresh interpolation 20g/L maltose, and switching amount is 10%.At 30 DEG C, continue cultivation after 10 hours, 8000 leave the heart collects thalline in 3 minutes.Then cell is resuspended in the SC-URA auxotroph substratum adding 20g/L wood sugar.Regulate the bacteria suspension OD of bacterial strain BSWPP, BSW4PG, BSW4M51 and BSW4M51 (N360F)
600unanimously, preferred OD
600be equal to 5, hatch as 30 DEG C.Hatching 30min, during 60min and 120min, get each bacterial strain 10mL bacteria suspension respectively, 8000 leave the heart 3 minutes, abandon supernatant.With twice, the aseptic washing bacterium mud that 10mL temperature is 4 DEG C.The method of washing is the sterilized water re-suspended cell of 4 DEG C by 10mL temperature, then 8000 leaves the heart 3 minutes, abandon supernatant.Washed cell is resuspended in 3mL deionized water, spend the night in 37 DEG C of incubators centrifugal after placing, with the wood sugar in HPLC mensuration supernatant liquor and Determination of Xylitol, according to the mole number of wherein wood sugar and Xylitol total content, calculate wood sugar total amount (the Du J that somatic cells absorbs, et al.Discovery and characterization of novel D-xylose-specific transporters from Neurospora crassa and Pichia stipitis.Mol Biosyst, 2010,6:2150-2156.).The OD of bacteria suspension is measured with spectrophotometer BioPhotometer plus (purchased from Eppendorf, Germany)
600, and according to formula: dry cell weight (mg/mL)=0.2365 × OD
600+ 0.1149 (Wang C, et al.Improvement of L-arabinose fermentation by modifying the metabolic pathway and transport in Saccharomyces cerevisiae.BioMed Research International, 2013,461204.), cell concentration is scaled dry cell weight.The turn-over capacity of sugar represents by the wood sugar total amount that unit somatic cells absorbs, and its measure unit is mg/g DCW.Result shows, and the wood sugar that bacterial strain BSWPP sample only measures minute quantity absorbs, and the wood sugar amount that bacterial strain BSW4PG, BSW4M51 and BSW4M51 (N360F) absorb does not have notable difference (Fig. 2).This result shows, translocator Mgt05196p and Mgt05196p (N360F) has the wood sugar transportcapacity suitable with Gal2p.
Embodiment 5 growth method compares the ability of translocator transport glucose
At 30 DEG C, bacterial strain BSWPP, BSW4PG, BSW4M51 and BSW4M51 (N360F) is cultivated in the SC-URA auxotroph substratum adding 20g/L maltose, culture transfer after 12 hours in the SC-URA auxotroph substratum of fresh interpolation 20g/L maltose, and switching amount is 10%.At 30 DEG C, continue cultivation after 12 hours 8000 to leave the heart 3 minutes and collect thalline.Then be resuspended in aseptic double-distilled water after washing 2 times with aseptic double-distilled water, cultivate 9 hours with carbon source residual in consumer for 30 DEG C.Afterwards, again collect thalline resuspended with distilled water, regulate bacteria suspension OD
600be 1, and ten times of gradient dilutions are carried out to this bacteria suspension.Selected by the cell of dilution on the SC-URA auxotroph culture medium flat plate that drops in and add 20g/L maltose and glucose respectively, each point is 4 μ l bacteria suspensions, cultivates after 5 days and observe at 30 DEG C.Result shows, and bacterial strain BSW4PG and BSW4M51 can bacterial strain BSWPP and BSW4M51 (N360F) can not at grown on glucose (Fig. 3) at grown on glucose.This result shows that translocator Gal2p and Mgt05196p can transport glucose, and Mgt05196p (N360F) can not transport glucose.
Embodiment 6 builds xylose metabolism approach in the yeast saccharomyces cerevisiae of expressing translocator
Integrative plasmid pYMIK-xy127 (the Wang Y with Xylose reductase, xylitol dehydrogenase and xylulokinase gene utilizing previous work to build, et al.Establishment of a xylose metabolic pathway in an industrial strain of Saccharomyces cerevisiae.Biotechnology letters, 2004,26,885-890.), in yeast saccharomyces cerevisiae, xylose metabolism approach is built.With restriction enzyme A paI digested plasmid pYMIK-xy127, obtain linearizing pYMIK-xy127.With linearizing pYMIK-xy127 conversion bacterial strain BSW4M51 (N360F).At the interpolation final concentration containing 200mg/L microbiotic G418 (purchased from Promega) be 20g/L maltose SC-URA auxotroph culture medium flat plate on screen transformant, the cell be not converted can not at this grow on plates.The recombinant bacterial strain obtained is with xylose metabolism approach, and this Strain Designation is BSW4M51X (N360F).
Wherein, Saccharomyces cerevisiae transformant use PEG/LiAc induction chemical conversion process (Fang Zhijia, etc.The new exploration of Saccharomyces cerevisiae transformant method." laboratory study and exploration " 04 phase in 2012).
Wherein, containing 1.7g/L YNB (yeast basic nitrogen source, purchased from Shanghai Sheng Gong biotechnology limited-liability company) in SC-URA auxotrophy substratum, 5g/L ammonium sulfate, 0.77g/L SC-URA (purchased from MP Biomedicals); Solid medium adds 20g/L agar powder in addition, and pH is adjusted to 6.0 ~ 7.0; Sterilising conditions: 115 DEG C, 30min; Add the maltose that final concentration is 20g/L during use as carbon source, adding final concentration is that the microbiotic G418 of 200mg/L is as screening pressure.
Embodiment 7 expresses the growth characteristics analysis of recombinant bacterial strain on different carbon source of translocator Mgt05196p (N360F) and xylose metabolism approach
In the SC-URA auxotroph substratum adding 20g/L maltose, cultivate bacterial strain BSW4M51 (N360F) cell of 12 hours at 30 DEG C, be transferred in the SC-URA auxotroph substratum of 20g/L maltose, switching amount is 10%.BSW4M51 (N360F) cell after switching, cultivate 12 hours again at 30 DEG C after, 8000 leave the heart collects thalline for 3 minutes as seed.Be inoculated into respectively by this seed and with the addition of 20g/L wood sugar, 20g/L glucose, 20g/L wood sugar add 10g/L glucose, and 20g/L wood sugar adds in the SC-URA auxotroph substratum of 20g/L glucose, initial inoculum is OD
600equal 1.Cultivate at 30 DEG C and timing mensuration culture OD
600growth.Result display (Fig. 4), the recombinant bacterial strain BSW4M51X (N360F) expressing Mgt05196p (N360F) can utilize on wood sugar and grow, but can not grown on glucose be utilized, and the growth of this bacterial strain on wood sugar be not by the suppression that glucose exists.This result shows, translocator Mgt05196p (N360F) only transhipment wood sugar does not transport glucose, and it is not subject to the suppression of glucose to the transhipment of wood sugar.
Claims (9)
1. a nucleic acid molecule for encodes xylose translocator, is characterized in that, its nucleotides sequence is classified as one of following sequence:
(1) nucleotide sequence shown in SEQ ID NO.1, this sequence is the mutant of GeneID:5124182 in Genebank, and its 1078-1080 position Nucleotide sports TTC by AAT;
(2) nucleotide sequence of the nucleotide sequence of (1) is different from due to the degeneracy of genetic codon;
(3) with the sequence of nucleotide sequence at least 80% homology shown in (1), and its Nucleotide corresponding to (1) 1078-1080 position is TTC;
(4) because the degeneracy of genetic codon is different from the nucleotide sequence of the nucleotide sequence described in (3).
2. the nucleic acid molecule of encodes xylose translocator as claimed in claim 1, it is characterized in that, the nucleotides sequence of described encodes xylose translocator is classified as the nucleotide sequence shown in SEQ ID NO.1, this nucleic acid molecule called after MGT05196 (N360F).
3. an xylose transport albumen, is characterized in that, its aminoacid sequence is one of following sequence:
(1) aminoacid sequence shown in SEQ ID NO.2, this sequence is the Point mutont of albumen protein_id=" XP_001482176.1 " in Genebank, and its amino-acid residue of 360 sports phenylalanine (F) by the glutamine (N) of protein_id=" XP_001482176.1 ".
(2) have the homology of 80% at least with the aminoacid sequence shown in (1), and the corresponding amino-acid residue of the aminoacid sequence shown in its to SEQ ID NO.2 360 is phenylalanine residue.
4. xylose transport albumen as claimed in claim 3, it is characterized in that, the aminoacid sequence of described xylose transport albumen is the aminoacid sequence shown in SEQ ID NO.2, this aminoacid sequence called after Mgt05196p (N360F).
5. a recombinant vectors, is characterized in that: described recombinant vectors contains the complete coding reading frame sequence of the nucleic acid molecule of the encodes xylose translocator described in claim 1 or 2.
6. recombinant vectors as claimed in claim 5, is characterized in that: described recombinant vectors is recombinant plasmid pJFE3-MGT05196 (N360F).
7. express a host cell for xylose transport albumen, it is characterized in that: described host cell is bacterium, yeast or filamentous fungus, this host cell includes have the right nucleotide sequence described in requirement 1 or recombinant vectors according to claim 5.
8. host cell of expressing xylose transport albumen as claimed in claim 7, is characterized in that: described host cell is yeast saccharomyces cerevisiae BSW4M51X (N360F).
9. the xylose transport albumen described in claim 3 or 4 is utilizing the application in the raw material production Xylitol containing wood sugar, ethanol, polyvalent alcohol, organic acid, hydro carbons or terpenoid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410526356.9A CN104263739A (en) | 2014-10-08 | 2014-10-08 | Nucleic acid molecule for encoding xylose translocator and xylose translocator encoded by nucleic acid molecule |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410526356.9A CN104263739A (en) | 2014-10-08 | 2014-10-08 | Nucleic acid molecule for encoding xylose translocator and xylose translocator encoded by nucleic acid molecule |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104263739A true CN104263739A (en) | 2015-01-07 |
Family
ID=52155320
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410526356.9A Pending CN104263739A (en) | 2014-10-08 | 2014-10-08 | Nucleic acid molecule for encoding xylose translocator and xylose translocator encoded by nucleic acid molecule |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104263739A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105199976A (en) * | 2015-11-05 | 2015-12-30 | 山东大学 | Recombinant saccharomyces cerevisiae strain for efficiently co-fermenting glucose and xylose and application of recombinant saccharomyces cerevisiae strain |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559527A (en) * | 2012-01-11 | 2012-07-11 | 山东大学 | Saccharomyces cerevisiae bacterial strain with high xylose transport capacity and application thereof |
| WO2014033018A1 (en) * | 2012-08-28 | 2014-03-06 | Dsm Ip Assets B.V. | Yeast strains engineered to produce ethanol from acetate |
-
2014
- 2014-10-08 CN CN201410526356.9A patent/CN104263739A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559527A (en) * | 2012-01-11 | 2012-07-11 | 山东大学 | Saccharomyces cerevisiae bacterial strain with high xylose transport capacity and application thereof |
| WO2014033018A1 (en) * | 2012-08-28 | 2014-03-06 | Dsm Ip Assets B.V. | Yeast strains engineered to produce ethanol from acetate |
Non-Patent Citations (5)
| Title |
|---|
| ALEXANDER FARWICK ET AL.: "Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose", 《PNAS》 * |
| LANDER,E. ET AL.: "NCBI：GenBank：XM_001482126.1", 《NCBI》 * |
| LANDER,E. ET AL.: "NCBI：GenBank：XP_001482176.1", 《NCBI》 * |
| YU SHEN ET AL.: "An efficient xylose-fermenting recombinant Saccharomyces cerevisiae strain obtained through adaptive evolution and its global transcription profile", 《APPL MICROBIOL BIOTECHNOL》 * |
| 王娜等: "酵母菌木糖转运蛋白研究进展", 《中国农业科技导报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105199976A (en) * | 2015-11-05 | 2015-12-30 | 山东大学 | Recombinant saccharomyces cerevisiae strain for efficiently co-fermenting glucose and xylose and application of recombinant saccharomyces cerevisiae strain |
| CN105199976B (en) * | 2015-11-05 | 2020-12-18 | 山东大学 | Recombinant saccharomyces cerevisiae strain for co-fermenting glucose and xylose and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200255871A1 (en) | Recombinant yeast cell | |
| JP2013524797A (en) | Method for producing cells capable of converting arabinose | |
| US8993301B2 (en) | Vector with codon-optimised genes for an arabinose metabolic pathway for arabinose conversion in yeast for ethanol production | |
| BR112019025777A2 (en) | recombinant yeast cell | |
| EP2495306B1 (en) | Specific arabinose transporter of the plant arabidosis thaliana for the construction of pentose-fermenting yeasts | |
| Huisjes et al. | Toward pectin fermentation by Saccharomyces cerevisiae: expression of the first two steps of a bacterial pathway for D-galacturonate metabolism | |
| EP3158075A2 (en) | Method for fermenting sugars using genetically engineered yeast | |
| CN102725306A (en) | Novel pentose transporters and uses thereof | |
| CN112195145A (en) | Cells with improved pentose conversion | |
| JP5813977B2 (en) | Mutant yeast belonging to the genus Kluyveromyces and method for producing ethanol using the same | |
| CN106754979B (en) | Gene for regulating and controlling long-chain fatty acid transport of candida tropicalis and application of gene | |
| CN107937296A (en) | One kind has acetic acid furfural vanillic aldehyde tolerance recombinant Saccharomyces cerevisiae and preparation method, application | |
| CN104263739A (en) | Nucleic acid molecule for encoding xylose translocator and xylose translocator encoded by nucleic acid molecule | |
| CN111088177A (en) | Construction and application of heat-resistant yeast engineering bacteria for producing glycerol under high-temperature aerobic condition | |
| CN113373073B (en) | Method for improving transport capacity and utilization capacity of levoglucosan of saccharomyces cerevisiae strain | |
| CN103421698B (en) | Construction and application of high-temperature high-yield xylitol engineering strains | |
| CN104844698A (en) | Method for promoting microbial cells to transport glucose, xylose and arabinose and application thereof in fermentation of biobased products | |
| CN104403956B (en) | The structure of xylitol high temperature high-yielding engineering bacterial strain and application | |
| CN106929530A (en) | A kind of method for improving yeast cells to composite inhibitor tolerance | |
| CN105462867B (en) | The saccharomyces cerevisiae of one plant of enduring high-concentration furfural and its application | |
| CA2877141A1 (en) | Fungal cells that produce decreased amounts of peptaibols | |
| CN111378691B (en) | Corn hydrolysate culture medium for producing beta-farnesene through fermentation and application thereof | |
| CN109415716A (en) | Arabinose isomerase for yeast | |
| WO2023093794A1 (en) | Acid-resistant yeast strain for high-yield production of l-malic acid, and construction method therefor and use thereof | |
| KR101828580B1 (en) | Methods of utilizing xylose as a carbon source using secretory expression of xylose isomerase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150107 |