CN110003316A - A kind of amino acid sequence, mutant and application encoding saccharide transporter - Google Patents

Abstract

The disclosure belongs to saccharomyces cerevisiae saccharide transporter technical field, and in particular to a kind of amino acid sequence, mutant and application for encoding saccharide transporter.The turn-over capacity of saccharide transporter and yeast cells fermentative activity are closely related in saccharomyces cerevisiae, provide the fermentation efficiency that the hexose transport albumen with good transport ability is conducive to improve saccharomyces cerevisiae；On the other hand, so that saccharomyces cerevisiae is obtained wood-sugar fermentation ability by genetic modification, be conducive to improve resource utilization, improve productivity effect.In order to achieve the above technical purposes, present disclose provides the saccharide transporter amino acid sequences and mutant that can be applied to saccharomyces cerevisiae, brewing yeast cell after genetic modification can realize good sugar utilization and the selection index system to carbon source, be expected to provide more saccharomyces cerevisiae application modes.

Description

A kind of amino acid sequence, mutant and application encoding saccharide transporter

Technical field

This disclosure relates to saccharomyces cerevisiae saccharide transporter technical field, and in particular to a kind of amino for encoding saccharide transporter Acid sequence, coding mutation body, the recombinant plasmid comprising the sequence and recipient cell are improving saccharomyces cerevisiae sugar transport ability side The application in face.

Background technique

The information for disclosing the background technology part is merely intended to increase the understanding to the general background of the disclosure, without certainty It is considered as recognizing or implying in any form that information composition has become existing skill well known to persons skilled in the art Art.

Saccharomyces cerevisiae (Saccharomyces cerevisiae) is widely used in food and industrial microorganism, extensively It is general to be applied to brewing industry and alcohol fuel manufacturing industry, with strong robustness, it is metabolized the characteristics such as vigorous and grade-safe. The growth and fermentation of saccharomyces cerevisiae are closely related with hexose transport protein family, and the saccharomyces cerevisiae in fermentation process can be aerobic Energy is obtained with zymohexose under conditions of anaerobic, the fermentation efficiency of the yeast cells activity white with sugared turning egg(s) is closely bound up.

Lignocellulosic sources are many kinds of, and annual output is high, and the comprehensive utilization for improving xylose component can significantly improve money Source utilization rate reduces food production cost.Natural saccharomyces cerevisiae cannot utilize xylose, be only capable of utilizing xylulose.Pass through gene Engineering means express Xylose reductase and xylitol dehydrogenase or single expression xylose isomerase simultaneously in saccharomyces cerevisiae Ability of the saccharomyces cerevisiae by xylose for xylulose, and then the optimization table for the enzyme being related to by relevant metabolic pathway can be assigned Reach, can obtain can by xylose be various target products cell factory.

Transport of the xylose into brewing yeast cell is the first step that xylose is utilized.High level expression saccharide transporter can To promote absorption of the saccharomyces cerevisiae to xylose.Saccharomyces cerevisiae does not have special xylose transport albumen, relies on the absorption of xylose In hexose transport albumen.Although it has been recognised by the inventors that having been reported that the Gal2 albumen of saccharomyces cerevisiae origin is that an effect is extensive, has Saccharide transporter (Young et al.2011, the Applied and Environmental of a variety of hexoses and pentose turn-over capacity Microbiology 77 (10): 3311-3319), but these nonspecific transport proteins to the transport of xylose by grape The strong Reverse transcriptase of sugar, to affect xylose utilization efficiency (Subtil and of the bacterial strain in the case where there is glucose environment Boles 2012, Biotechnology for Biofuels 5:14), the life for related microorganisms fermented food and feed Production brings limitation.

Summary of the invention

In view of the above technical problems, it has been recognised by the inventors that on the one hand may be used to further increase the production efficiency of saccharomyces cerevisiae The transfer efficiency of hexose is improved in a manner of through genetic modification；On the other hand, promoted nature in there are it is a variety of naturally can benefit With the microorganism of xylose, excellent xylose transport albumen is found from these microorganisms, passes through genetic modification and imports wine brewing ferment Mother can eliminate substrate to the Reverse transcriptase of xylose transport, improve saccharomyces cerevisiae to the transfer efficiency of xylose, reduction is produced into This is expected to realize the control to saccharomyces cerevisiae culture medium by the transformation to saccharide transporter, and obtaining has carbon source tendentious Saccharomyces cerevisiae provides more convenience for food production and the preparation of alcohol fuel.

In order to realize above-mentioned technical effect, the disclosure the following technical schemes are provided:

For the disclosure in a first aspect, providing a kind of amino acid sequence of saccharide transporter, the amino acid sequence is following sequence One of column:

(1) in Genebank albumen protein_id=" XP_006966515.1 " amino acid sequence, the amino acid sequence Column are named as Xltr1；

(2) mutant of amino acid sequence Xltr1, the mutant are as follows: Xltr1 in (1) described in^F300A、Xltr1^N326A、 Xltr1^D39A、Xltr1^R132A、Xltr1^Q288A、Xltr1^Q291A、Xltr1^Q292A、Xltr1^F298A、Xltr1^I299A、Xltr1^Y301A、 Xltr1^Y302A、Xltr1^W407A、Xltr1^N434A、Xltr1^Q292S、Xltr1^Q292Y、Xltr1^Q292W、Xltr1^Q292F、Xltr1^F300S、 Xltr1^F300Y、Xltr1F^300W、Xltr1^F300K、Xltr1^N326S、Xltr1^N326F、Xltr1^N326Y、Xltr1^N326W；

(3) there is the amino acid sequence of 80% and the above similitude with (1) or (2).

Amino acid sequence shown in sequence (1) i.e. SEQ ID NO:1, wherein preferred mutational site is Xltr1^F300A、 Xltr1^N326F、Xltr1^Q288A、Xltr1^Q291A、Xltr1^Q292A、Xltr1^F300A、Xltr1^N326A、Xltr1^N434A、Xltr1^Q292S、 Xltr1^Q292F、Xltr1^F300S、Xltr1^F300Y、Xltr1^F300W、Xltr1^F300K、Xltr1^N326S。

Extracellular metabolin of the Xltr1 albumen as trichoderma reesei, has a saccharide transporter activity, and the research of the disclosure is into one Step discovery, which can be used as the saccharide transporter of saccharomyces cerevisiae, can not only effectively improve saccharomyces cerevisiae to glucose, sweet The utilization rate of dew sugar and fructose, additionally it is possible to which increase dramatically increases saccharomyces cerevisiae to the utilization rate of xylose, is saccharomyces cerevisiae origin egg 8.25 times of white Gal2p can effectively improve saccharomyces cerevisiae to the utilization rate of xylose.

Above-mentioned mutant Xltr1^F300AIndicate protein_id=" XP_006966515.1 " indicated amino acid sequence Its exon the 300th sports alanine A by phenylalanine F；

Similarly, Xltr1^N326AIndicating protein_id=" XP_006966515.1 " indicated amino acid sequence, it is outer aobvious Son the 326th sports alanine A by asparagine N；Other mutant indicate meaning can with and so on.

Above-mentioned mutant all has certain technical effect, such as Xltr1 compared to this source protein of saccharomyces cerevisiae^Q288A、 Xltr1^Q291A、Xltr1^Q292A、Xltr1^F300A、Xltr1^N326A、Xltr1^Q292S、Xltr1^Q292F、Xltr1^F300S、Xltr1^F300Y、 Xltr1^F300W、Xltr1^F300K、Xltr1^N326SThere is good utilizing status Deng to carbon source；Wherein, Xltr1^F300AAlthough transporting Portugal The ability of grape sugar only has the 67.5% of Gal2p, but its xylose transport ability almost disappears.On the other hand, Xltr1^N326FGlucose transport ability completely disappears, and xylose transport ability is significantly larger than Gal2p.Above-mentioned mutant is applied to The control to carbon source in substrate may be implemented in saccharomyces cerevisiae, realizes that more productions may.

Further preferred mutant is Xltr1^F300A、Xltr1^N326F、Xltr1^F300S、Xltr1^N434A。

Xltr1^F300SUtilization of the mutant strain under the conditions of mixing hexose (glucose, mannose, fructose) utilizes, to glucose Rate is accelerated, and reduces to the utilization speed of mannose and fructose, can be applied to the separation of mixed sugar, removes the Portugal in mixed sugar Grape sugar ingredient, extends the application of the bacterial strain.

Xltr1^N326FUnder the conditions of mixed sugar (glucose, xylose) utilization, glucose transport ability completely disappears mutant strain, And xylose transport ability is significantly larger than Gal2p, is its 2.37 times.

Xltr1^N434AFor the xylose utilization critical sites that the disclosure is found for the first time, the amino after the mutation is not disclosed before this Acid sequence can be applied to glycoprotein transport.

The disclosure is studies have shown that Xltr1^F300AThere is no xylose transport ability, but remains the turn-over capacity of hexose.The characteristic It can be applied equally to the separation of mixed sugar, in addition, the mutation result is it may be said that the transhipment of hexose is extremely closed in this bright site Key, for further exploitation, the related transport protein of sugar has valuable reference function.

Disclosure second aspect, provide it is a kind of encode saccharide transporter nucleotide sequence, the nucleotides sequence be classified as with One of lower sequence:

(1) GeneID:18487109 in Genebank；

(2) since Codon degeneracy can be translated to obtain the nucleotide sequence of first aspect (1) the part sequence；

(3) since Codon degeneracy can be translated to obtain the nucleotide sequence of first aspect (2) the part mutant.

(4) there is the core of 80% and the above similitude with second aspect (1) or (2) or (3) the part nucleotide sequence Nucleotide sequence.

The disclosure third aspect, provides a kind of recombinant plasmid, and the recombinant plasmid has nucleotides sequence described in second aspect The complete coding reading frame sequence of column.

Preferably, the carrier of the recombinant plasmid is pJFE3.

Disclosure fourth aspect provides a kind of recipient cell for expressing saccharide transporter, comprising the in the recipient cell On the one hand the nucleotide sequence of saccharide transporter is encoded described in the amino acid sequence of the saccharide transporter and/or second aspect And/or recombinant plasmid described in the third aspect.

Preferably, the recipient cell is bacterium or fungi；It is further preferred that being saccharomyces cerevisiae.

The 5th aspect of the disclosure, provides the method for a kind of raising saccharomyces cerevisiae sugar absorption and Utilization ability, and this method includes Following steps: recombinant plasmid described in the third aspect is imported in brewing yeast cell.

The aspect of the disclosure the 6th provides sugar described in the amino acid sequence of saccharide transporter described in first aspect, second aspect Recipient cell described in recombinant plasmid described in the nucleotide sequence of transport protein, the third aspect or fourth aspect is improving saccharomyces cerevisiae The application of sugared use aspects.

Preferably, the application is to produce xylitol, ethyl alcohol, polyalcohol, organic acid, hydro carbons or terpene using saccharomyces cerevisiae Application in compound.

Compared with prior art, the disclosure the utility model has the advantages that

1. the application present disclose provides Xltr1 albumen as saccharomyces cerevisiae saccharide transporter, for natural saccharomyces cerevisiae Cell cannot utilize the status of xylose, and the saccharide transporter present disclose provides the albumen as saccharomyces cerevisiae can be mentioned effectively Utilization rate of the high saccharomyces cerevisiae to carbon source and the transport capacity to xylose.Nucleotide sequence is imported by way of genetic modification In brewing yeast cell, make cell that can ferment using xylose as carbon source, and under same condition of culture, imports disclosure sequence It obtains saccharomyces cerevisiae significantly to increase the utilization rate of maltose, considerably beyond the utilization rate to glucose.By the sequence in the application Saccharomyces cerevisiae is imported, can effectively eliminate the Reverse transcriptase of other carbon sources in culture medium, improves cell to the utilization rate of xylose, It is expected to bring good economic benefit.

2. the disclosure additionally provides the mutant of Xltr1 albumen, as the saccharide transporter of saccharomyces cerevisiae, equally have good Good Transport Activity is expected to realize control saccharomyces cerevisiae to single carbon source in addition to improving saccharomyces cerevisiae to the utilization rate of carbon source Intake, realize more production methods.

Detailed description of the invention

The Figure of description for constituting a part of this disclosure is used to provide further understanding of the disclosure, and the disclosure is shown Meaning property embodiment and its explanation do not constitute the improper restriction to the disclosure for explaining the disclosure.

Fig. 1 is recombinant plasmid pJFE3-Xltr1 physical map in embodiment 2；

Fig. 2 is that Xltr1 difference mutain bacterial strain is expressed in embodiment 4 in culture medium upgrowth situation figure.

Specific embodiment

It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the disclosure.Unless another It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field The identical meanings of understanding.

It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.

As background technique is introduced, the turn-over capacity of saccharide transporter and yeast cells fermentative activity in saccharomyces cerevisiae It is closely related, the fermentation efficiency that the hexose transport albumen with good transport ability is conducive to improve saccharomyces cerevisiae is provided；It is another Aspect makes saccharomyces cerevisiae obtain wood-sugar fermentation ability by genetic modification, is conducive to improve resource utilization, improves production effect Benefit.In order to achieve the above technical purposes, present disclose provides Xltr1 albumen as saccharomyces cerevisiae saccharide transporter application and its He equally has the active mutant of sugar transport.

In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool The technical solution of the disclosure is described in detail in the embodiment and comparative example of body.

In following embodiment:

(1) saccharomyces cerevisiae: Wine brewing yeast strain EBY.VW4000 (MAT α leu2-3,112 ura3-52 trp1-289 his3-Δ1 Mal2-8c SUC2hxt17 Δhxt13Δ::loxP hxt15Δ::loxP hxt16Δ::loxP hxt14 Δ::loxP hxt12Δ::loxPhxt9Δ::loxP hxt11Δ::loxP hxt10Δ::loxP hxt8Δ::loxP hxt514::loxP hxt2Δ::loxP hxt367Δ::loxP Gal2pΔstl1Δ::loxP agt1Δ::loxP ydl247wΔ::loxP yjr160cΔ::loxP)(Wieczorke et al.1999,Febs Letters 464(3): 123-128) be one plant of bacterial strain (hxt null) for having lacked all endogenous hexose transport albumen, due to a lack of to transmitter loss oneself The ability of sugar cannot be in grown on glucose, and can only be in this carbon substrate with autospecific transport protein of maltose Upper growth.Saccharide transporter is expressed in the bacterial strain can make training of the bacterial strain again in glucose and the independent carbon source of other hexoses It supports and is grown on base, growth ability can reflect out the glucose or other hexose transport capacities of expressed transport protein.It will The EBY.VW4000 of expression saccharide transporter is incubated for certain time in xylose, and then measures the total of intracellular xylose and xylitol Accumulation can reflect expressed transport protein to the transport capacity of xylose.When the introducing xylose utilization way in EBY.VW4000 After diameter, bacterial strain can reflect transport protein to the transport capacity of xylose for the growth ability on sole carbon source in xylose.

(2) Xltr1 is amino acid sequence shown in SEQ ID NO:1.

(3) bioinformatics means are utilized, by homologous modeling and molecular docking, predict the protein structure of Xltr1 albumen, And by the analysis to conservative amino acid residues, and according to Xltr1 albumen and glucose, fructose, mannose and xylose molecule Docking as a result, preferred amino acid site is mutated.

Below with reference to embodiment, the present invention is further illustrated.

The clone of 1 transport protein encoding gene of embodiment

Saccharomyces cerevisiae laboratory strains genome is extracted as template, using Gal2p-F and Gal2p-R as primer, is carried out The amplification in the area Gal2CDS, annealing temperature are equally chosen as 56 DEG C, use the In-Fusion HD Cloning of Clontech company Gal2 segment is connect by high-efficient cloning kit with pJFE3 plasmid.Extract Trichoderma reesei QM6a (ATCC 13631) RNA carries out reverse transcription and obtains cDNA as template, carries out PCR amplification with the KOD high fidelity enzyme of TOYOBO company, leads to Xltr1 segment is connect by the method for crossing infusion with pJFE3 plasmid.Reuse point mutation kit KOD-PLUS- The rite-directed mutagenesis of MUTAGENISIS introducing table 1.Pcr amplified DNA segment Xltr1, the annealing temperature that when PCR amplification selects for 56℃.List of primers such as Fig. 1.

Table 1 tests the primer list

Remarks:

1. " F " indicates that forward primer, " R " indicate reverse primer.

2.F300X-R/N326X-R the amino acid residue before and after waiting the mutation for representing Xltr1, F300X represent F300A/S/ W/Y；N326X represents N326A/S/F.

Embodiment 2 constructs pJFE3_Xltr1p expression plasmid

As shown in Figure 1, the recombinant plasmid is 2 μ plasmid of saccharomyces cerevisiae, Gal2p and Xltr1 gene is in saccharomyces cerevisiae composing type The lower expression of promoter TEF1 promoter control；The DNA fragmentation of Gal2p and Xltr1 gene is connected in using the method for GB-dir On pJFE3 plasmid.Correct monoclonal transformant is screened by the sequencing to segment, wherein size is respectively 7817bp, The plasmid of 7720bp is correct recombinant plasmid, which is named as pJFE3-Gal2p and pJFE3-Xltr1p, in the matter On grain, Gal2p and Xltr1p gene is expressed under the control of saccharomyces cerevisiae constitutive promoter TEF1 promoter.

Embodiment 3 expresses transport protein in saccharomyces cerevisiae

With empty carrier plasmid pJFE3 transformed saccharomyces cerevisiae bacterial strain EBY.VW4000, in the SC-URA of addition 20g/L maltose Transformant is screened on auxotroph culture medium flat plate, the cell not being converted cannot be grown on the plate.The conversion of acquisition Son is named as BSW4PP.

Recombinant plasmid pJFE3-Gal2p and pJFE3-Xltr1p are converted into bacterial strain EBY.VW4000, in addition 20g/L malt Transformant is screened on the SC-URA auxotroph culture medium flat plate of sugar, the cell not being converted cannot be grown on the plate. The transformant of acquisition is named as FJTR3_Xltr1.

By the different loci mutation conversion bacterial strain EBY.VW4000 of recombinant plasmid pJFE3-Xltr1, in addition 20g/L malt Transformant is screened on the SC-URA auxotroph culture medium flat plate of sugar, the cell not being converted cannot be grown on the plate. Transformant after mutation is respectively designated as FJTR3_Xltr1^F300AAnd FJTR3_Xltr1^N326FDeng (being shown in Table 2 and table 3).

Wherein, Saccharomyces cerevisiae transformant uses method (Costaglioli the et al.1994, Current of electrotransformation Genetics 27(1):26-30)。

Wherein, 1.7g/L YNB (yeast basic nitrogen source, purchased from the raw work life in Shanghai is contained in SC-URA auxotrophy culture medium Object Engineering stock Co., Ltd), 5g/L ammonium sulfate, 0.77g/L SC-URA (is purchased from MP Biomedicals)；Solid medium In addition 20g/L agar powder is added, pH is adjusted to 6.0~7.0；Sterilising conditions: 115 DEG C, 30min；It is added when use final concentration of The maltose of 20g/L is as carbon source.

The growth change of the yeast in different carbon sources of embodiment 4

As shown in Fig. 2, conversion bacterial strain BSW4PP and FJTR3_Xltr1 addition 20g/L mannose or glucose or Xylose or fructose be sole carbon source culture medium flat plate on cultivate.The results show that control zero load bacterial strain BSW4PP is trained in xylose Supporting cannot grow on base, be only capable of faint growth on mannose and fructose culture medium, and bacterial strain FJTR3_Xltr1 is in xylose, Grow very well on mannose and fructose culture medium (Fig. 2).

5 xylose accumulation method of embodiment compares the xylose transport capacity of transport protein

Bacterial strain FJTR3_ is cultivated at 30 DEG C in the SC-URA auxotroph culture medium of addition 20g/L maltose Xltr1 and its mutant strain, SC-URA auxotroph of the switching culture to fresh addition 20g/L maltose after 12 hours In culture medium, switching amount is 10%.After continuing culture at 30 DEG C 10 hours, 8000 revs/min of centrifugations, 3 minutes collection thallus.So Cell is resuspended in afterwards in the SC-URA auxotroph culture medium of addition 20g/L xylose.Adjust bacterial strain FJTR3_Xltr1 and its The bacteria suspension OD600 of mutant strain is consistent, and preferably OD600 is equal to 20, is incubated for as 30 DEG C.Be incubated for 30min, 60min and When 120min, each bacterial strain 10mL bacteria suspension is taken respectively, and 8000 revs/min are centrifuged 3 minutes, abandon supernatant.The nothing for being 4 DEG C with 10mL temperature Bacterium washes bacterium mud twice.The method washed is that cell is resuspended in the sterile water for being 4 DEG C with 10mL temperature, is then centrifuged 3 points for 8000 revs/min Clock abandons supernatant.Washed cell is resuspended in 3mL deionized water, is centrifuged after placing overnight in 37 DEG C of incubators, is surveyed with HPLC Determine the xylose and Determination of Xylitol in supernatant, according to the molal quantity of wherein xylose and xylitol total content, calculates somatic cells The xylose total amount (Du et al.2010, Mol Biosyst 6 (11): 2150-2156.) of absorption.Use spectrophotometer The OD600 of BioPhotometer plus (being purchased from Eppendorf, Germany) measurement bacteria suspension, and according to formula: dry cell weight DCW (mg/mL)=0.2154*OD600+0.1358, is scaled dry cell weight for cell concentration.The turn-over capacity unit thallus of sugar The xylose total amount that cell absorbs indicates that measurement unit is mg/g DCW.It the results are shown in Table 2.

Embodiment 6 compares the ability of transport protein transhipment glucose in the method that growth rate compares

Bacterial strain FJTR3_ is cultivated at 30 DEG C in the SC-URA auxotroph culture medium of addition 20g/L maltose Gal2p, FJTR3_Xltr1 and its mutant strain, SC- of the switching culture to fresh addition 20g/L maltose after 12 hours In URA auxotroph culture medium, switching amount is 10%.It is received within centrifugation 3 minutes for 8000 turns after continuing at 30 DEG C culture 12 hours Collect thallus.Then it is resuspended in aseptic double-distilled water after washing 2 times with aseptic double-distilled water, 10% biomass of switching to fresh addition In the SC-URA auxotroph culture medium of 20g/L glucose.Adjusting bacteria suspension initial OD 600 is 1,30 DEG C of 200rpm cultures 5 It.It the results are shown in Table 2.

Table 2 expresses the bacterial strain glucose of Xltr1 and its mutain and the comparison of transhipment xylose ability

It is compared as seen from Table 2 with the origin protein Gal-9 2p of saccharomyces cerevisiae, the yeast of Xltr1 expression, xylose transport ability It is stronger, it is 8.25 times of Gal2p, and the mutant Xltr1 of Xltr1^F300AAlthough the ability of transhipment glucose only has Gal2p's 67.5%, but its xylose transport ability almost disappears.On the other hand, the mutant Xltr1 of Xltr1^N326F, its Portugal Grape sugar transport ability completely disappears, and xylose transport ability is significantly larger than Gal2p, is its 2.37 times.

Embodiment 7

In the present embodiment, inventor screens for the transport protein of following mutational site coding, referring to embodiment 1-3 Method construction recombination plasmid, and import in Wine brewing yeast strain EBY.VW4000, investigate with glucose, mannose and fructose and be Sugared utilization efficiency when carbon source

1. acquisition modes:

Using bioinformatics means, by homologous modeling and molecular docking, according to Xltr1 albumen and glucose, fructose With mannose molecules docking as a result, and conserved amino acid sequence analysis, preferred amino acid site is mutated.

2. activity verifying specific steps:

A activated strains: first plate activate, rear picking single colonie SC-URA addition maltose fluid nutrient medium in into Row is incubated overnight, and is forwarded within second day fresh culture, and initial OD 600 adjusts to 0.1-0.2, cultivates 16-18h, OD600 is made to exist 2.0 left and right；

Thallus is centrifuged by b, and is washed repeatedly using sterile water 3 times, until noresidue culture medium；

C is forwarded to containing carrying out in different carbon source (glucose, mannose, fructose) culture medium；

D is sampled in different time points: 0h, 6h, 12h, for 24 hours, 36h, 48h, 72h；

E detects sugared content remaining in sample using high performance liquid chromatography, as a result as shown in table 3 below:

Table 3 expresses Xltr1 and its bacterial strain of mutain is sugared using fast in mixed sugar (glucose, mannose, fructose) The comparison of rate

Annotation: t₃₀, t₅₀: the time consumed by sugar consumption 30% and 50% is respectively represented, the consumption speed of sugar can be characterized Rate；NQ representative can not calculate the time, and the time consumed by sugar consumption 30% and 50% is about infinity.

Mutant Xltr1^Q291A、Xltr1^F300S、Xltr1^F300Y、Xltr1^N326A、Xltr1^N326SIt is right in mixed sugar utilization Apparent quickening, mutant Xltr1 has occurred in the utilization rate of glucose^N326A、Xltr1^N326SThe utilization rate of mannose is become Slowly, mutant Xltr1^Q291A、Xltr1^N326SIt then obviously becomes faster to the utilization rate of mannose, mutant Xltr1^Q288S、 Xltr1^Q291A、Xltr1^F300Y、Xltr1^N326A、Xltr1^N326SIt obviously becomes faster to the utilization rate of fructose, mutant Xltr1^Q292A、 Xltr1^Q292S、Xltr1^F300S、Xltr1^F300WIt is slack-off to the utilization rate of fructose.It therefore, can be by the way that above-mentioned mutant be imported The mode of saccharomyces cerevisiae realizes the separation of selective use and mixed sugar to carbon source.

The foregoing is merely preferred embodiment of the present disclosure, are not limited to the disclosure, for the skill of this field For art personnel, the disclosure can have various modifications and variations.It is all within the spirit and principle of the disclosure, it is made any to repair Change, equivalent replacement, improvement etc., should be included within the protection scope of the disclosure.

SEQUENCE LISTING

<110>the intelligent SeaBird in Rongcheng City reaches Biotechnology Co., Ltd

<120>a kind of amino acid sequence, mutant and application for encoding saccharide transporter

<130> 2010

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 543

<212> PRT

<213>artificial sequence

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Met Arg Phe Ser Glu Lys Leu Gly Phe Lys Arg Pro Asp Asp Glu Ala

1 5 10 15

Gly Ala Ser Trp Val Ala Ile Leu Met Gly Phe Phe Val Ala Phe Gly

20 25 30

Gly Val Leu Tyr Gly Tyr Asp Thr Gly Thr Ile Ser Gly Ile Met Ala

35 40 45

Met Pro Tyr Phe Lys Asp Leu Phe Ser Thr Gly Tyr Arg Asn Pro Asn

50 55 60

Gly Glu Leu Asp Ile Thr Ala Thr Gln Glu Ser Ala Ile Val Ser Ile

65 70 75 80

Leu Ser Ala Gly Thr Phe Phe Gly Ala Leu Ala Ser Pro Leu Leu Ala

85 90 95

Asp Phe Leu Gly Arg Arg Pro Ala Leu Met Ile Ser Thr Trp Val Phe

100 105 110

Asn Leu Gly Val Val Leu Gln Thr Ile Ala Thr Ala Ile Pro Met Phe

115 120 125

Leu Ala Gly Arg Phe Phe Ala Gly Phe Gly Val Gly Leu Ile Ser Ala

130 135 140

Leu Ile Pro Leu Tyr Gln Ser Glu Thr Ala Pro Lys Trp Ile Arg Gly

145 150 155 160

Ala Ile Val Gly Ala Tyr Gln Leu Ala Ile Thr Ile Gly Leu Leu Leu

165 170 175

Ala Ala Val Val Asn Asn Ala Thr Ala Lys Arg His Asp Ser Gly Ser

180 185 190

Tyr Arg Ile Pro Ile Ala Val Gln Phe Ala Trp Ser Leu Val Leu Phe

195 200 205

Val Gly Met Ile Phe Leu Pro Glu Thr Pro Arg Phe Leu Val Arg Ser

210 215 220

Gly Lys Leu Glu Lys Ala Arg Ala Ala Leu Ser Arg Ile Arg Arg Leu

225 230 235 240

Ser Pro Glu His Glu Ala Leu Ala Ala Glu Leu Gly Gln Ile Gln Ala

245 250 255

Asn Leu Glu Ala Glu Ser Ser Val Arg Lys Ala Thr Tyr Ala Asp Cys

260 265 270

Phe Arg Arg Pro Met Leu Lys Arg Gln Phe Thr Gly Met Ala Leu Gln

275 280 285

Ala Leu Gln Gln Leu Thr Gly Ile Asn Phe Ile Phe Tyr Tyr Gly Thr

290 295 300

Arg Tyr Phe Gln Asn Ser Gly Val Ser Ser Gly Phe Thr Ile Gly Met

305 310 315 320

Ile Thr Ala Gly Ile Asn Val Ala Ser Thr Ile Pro Gly Leu Leu Ala

325 330 335

Ile Asp Arg Trp Gly Arg Arg Pro Leu Leu Leu Leu Gly Ala Val Gly

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Met Cys Val Ser Gln Leu Ile Val Ala Val Val Gly Thr Val Ser Thr

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Gly Gln Arg Pro Asn Gly Glu Ile Phe Val Lys Ser Leu Ala Gly Gln

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Gln Ala Ala Val Ala Phe Val Cys Ile Phe Ile Ala Phe Phe Ala Ser

385 390 395 400

Thr Trp Gly Pro Leu Ala Trp Val Val Thr Gly Glu Ile Tyr Pro Leu

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Ala Thr Arg Ala Lys Ala Leu Ser Met Thr Thr Ala Thr Asn Trp Leu

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Phe Asn Trp Ala Ile Ala Tyr Ser Thr Pro Tyr Leu Val Asn Tyr Gly

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Pro Gly Tyr Ala Asn Leu Gln Ser Lys Ile Phe Phe Val Trp Phe Gly

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Ala Cys Phe Leu Cys Ile Ala Leu Val Trp Phe Phe Ile Tyr Glu Thr

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Lys Gly Leu Ser Leu Glu Glu Val Asp Glu Leu Tyr Ala Glu Val Lys

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Val Ala Arg Lys Ser Thr Thr Trp Lys Pro Thr Pro Arg Leu Glu Ala

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Ala Gly Ser Thr Thr Ser Glu Glu Ser Lys Asp Glu Ser Gly Pro Lys

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Glu Ala Ser Pro His Val Met Asp Gln Gly Val Glu Leu Gln Val

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ctgtcggctg gtactttctt cggcgctctt gcgtcccctc ttctggccga cttcctgggc 300

cgccgccctg ccttgatgat ctcgacttgg gtcttcaacc tgggcgtcgt cctccaaaca 360

atcgcaacgg caatccccat gttcctggct ggtcgcttct ttgccggctt cggcgtcggt 420

ctcatctccg ccttgatccc cctgtaccag tccgaaacag cacccaaatg gatccgcggc 480

gccatcgtcg gcgcctacca actggccatc acaatcggcc tcctcctcgc cgccgtcgtc 540

aacaacgcca cggccaagcg ccacgactcg ggcagctacc gcatccccat cgccgtccag 600

ttcgcctggt ccctcgtcct cttcgtcggc atgatcttcc tgcccgagac gccgcgcttc 660

ctcgtccgct ccggcaagct cgagaaagcc agggccgccc tctcgcgcat ccgccgcctg 720

tccccggagc acgaggctct cgccgccgag ctcggccaga tccaggccaa tctcgaggcc 780

gagagcagcg tccgcaaggc cacgtacgca gactgcttcc gccgccccat gctcaagcgc 840

cagttcaccg gcatggccct gcaggcgctg cagcagctga cgggcatcaa cttcatcttc 900

tactacggga cgcggtattt ccagaactcg ggcgtctcca gcggcttcac cattggcatg 960

atcacggcgg gcatcaacgt cgcctccacc atccccggtc tcttggccat tgaccgctgg 1020

ggccgccgcc ctctgctcct cctcggcgcc gtcggcatgt gcgtctccca gctcatcgtc 1080

gccgtggtcg gcacagtttc caccggccag cgccccaacg gagaaatctt tgtaaagtcc 1140

ctggccggcc agcaggcggc cgtcgccttc gtctgcatct tcatcgcctt cttcgccagc 1200

acctggggcc ccctcgcgtg ggtcgtcacc ggcgaaatct acccgctggc cacgcgggca 1260

aaggccctga gcatgaccac ggccacaaac tggctcttca actgggccat tgcctactcc 1320

accccttacc tcgtcaacta cggcccgggc tacgccaacc tgcagtccaa gatcttcttc 1380

gtctggttcg gcgcctgctt cctctgcatc gcccttgtct ggttcttcat ctacgagacc 1440

aagggcctta gcctggagga ggtcgacgag ctctatgccg aggtcaaggt cgcccgcaag 1500

tctaccacct ggaagcccac cccgcgcctg gaggctgctg gaagtaccac cagcgaggag 1560

tcaaaggatg agtctggtcc caaggaggcg agtcctcatg ttatggacca aggagttgaa 1620

cttcaagtgt aa 1632

<210> 3

<211> 25

<212> DNA

<213>artificial sequence

<400> 3

atggcagttg aggagaacaa tatgc 25

<210> 4

<211> 23

<212> DNA

<213>artificial sequence

<400> 4

cttattctag catggccttg tac 23

<210> 5

<211> 19

<212> DNA

<213>artificial sequence

<400> 5

ttcgtcgcct ccaccatcc 19

<210> 6

<211> 19

<212> DNA

<213>artificial sequence

<400> 6

agagtcgcct ccaccatcc 19

<210> 7

<211> 19

<212> DNA

<213>artificial sequence

<400> 7

gcggcgctgc agcagctga 19

<210> 8

<211> 19

<212> DNA

<213>artificial sequence

<400> 8

cagggccatg ccggtgaac 19

<210> 9

<211> 21

<212> DNA

<213>artificial sequence

<400> 9

gcgcagctga cgggcatcaa c 21

<210> 10

<211> 19

<212> DNA

<213>artificial sequence

<400> 10

cagcgcctgc agggccatg 19

<210> 11

<211> 21

<212> DNA

<213>artificial sequence

<400> 11

caggcgctga cgggcatcaa c 21

<210> 12

<211> 19

<212> DNA

<213>artificial sequence

<400> 12

cagcgcctgc agggccatg 19

<210> 13

<211> 20

<212> DNA

<213>artificial sequence

<400> 13

gcctactacg ggacgcggta 20

<210> 14

<211> 20

<212> DNA

<213>artificial sequence

<400> 14

tggtactacg ggacgcggta 20

<210> 15

<211> 20

<212> DNA

<213>artificial sequence

<400> 15

tactactacg ggacgcggta 20

<210> 16

<211> 21

<212> DNA

<213>artificial sequence

<400> 16

gatgaagttg atgcccgtca g 21

<210> 17

<211> 19

<212> DNA

<213>artificial sequence

<400> 17

gccgtcgcct ccaccatcc 19

<210> 18

<211> 19

<212> DNA

<213>artificial sequence

<400> 18

gatgcccgcc gtgatcatg 19

<210> 19

<211> 19

<212> DNA

<213>artificial sequence

<400> 19

ttcgtcgcct ccaccatcc 19

<210> 20

<211> 19

<212> DNA

<213>artificial sequence

<400> 20

tgggtcgcct ccaccatcc 19

<210> 21

<211> 19

<212> DNA

<213>artificial sequence

<400> 21

tacgtcgcct ccaccatcc 19

<210> 22

<211> 19

<212> DNA

<213>artificial sequence

<400> 22

gatgcccgcc gtgatcatg 19

Claims (10)

1. a kind of amino acid sequence of saccharide transporter, which is characterized in that the amino acid sequence is one of following sequence:

(1) in Genebank albumen protein_id=" XP_006966515.1 " amino acid sequence, the amino acid sequence life Entitled Xltr1；

(2) mutant of amino acid sequence Xltr1, the mutant in (1) described in are as follows: Xltr1F300A, Xltr1N326A, Xltr1D39A、Xltr1R132A、Xltr1Q288A、Xltr1Q291A、Xltr1Q292A、Xltr1F298A、Xltr1I299A、 Xltr1Y301A、Xltr1Y302A、Xltr1W407A、Xltr1N434A、Xltr1Q292S、Xltr1Q292Y、Xltr1Q292W、 Xltr1Q292F、Xltr1F300S、Xltr1F300Y、Xltr1F300W、Xltr1F300K、Xltr1N326S、Xltr1N326F、 Xltr1N326Y,Xltr1N326W；

(3) there is the amino acid sequence of 80% and the above similitude with (1) or (2) amino acid sequence in claim 1.

2. a kind of nucleotide sequence for encoding saccharide transporter, which is characterized in that the nucleotides sequence is classified as one of following sequence:

(1) in Genebank GeneID:18487109 nucleotide sequence；

(2) since Codon degeneracy can translate to obtain in claim 1 nucleotide sequence of (1) part sequence；

(3) since Codon degeneracy can translate to obtain in claim 1 nucleotide sequence of (2) part mutant.

(4) there is the nucleosides of 80% and the above similitude with (1) or (2) in claim 2 or (3) part nucleotide sequence Acid sequence.

3. a kind of recombinant plasmid, which is characterized in that the recombinant plasmid has the complete of nucleotide sequence as claimed in claim 2 Encode reading frame sequence.

4. recombinant plasmid as claimed in claim 3, which is characterized in that the carrier of the recombinant plasmid is pJFE3.

5. a kind of recipient cell for expressing saccharide transporter, which is characterized in that comprising described in claim 1 in the recipient cell Amino acid sequence and/or claim 2 described in nucleotide sequence and/or recombinant plasmid as claimed in claim 3.

6. recipient cell as claimed in claim 5, which is characterized in that the recipient cell is bacterium or filamentous fungi.

7. recipient cell as claimed in claim 6, which is characterized in that the recipient cell is saccharomyces cerevisiae.

8. a kind of method for improving saccharomyces cerevisiae sugar and absorbing with Utilization ability, which is characterized in that the method includes wanting right Recombinant plasmid described in asking 3 imports in brewing yeast cell.

9. the nucleotides sequence of saccharide transporter described in the amino acid sequence of saccharide transporter described in claim 1, claim 2 Recipient cell described in column, recombinant plasmid as claimed in claim 3 or claim 4 is improving answering for saccharomyces cerevisiae sugar use aspects With.

10. application as claimed in claim 9, which is characterized in that the application is to produce tyrosol, hydroxyl including the use of saccharomyces cerevisiae The polyalcohols such as base tyrosol, rhodioside, benzyl carbinol, lactic acid, shikimic acid, xylitol, ethyl alcohol, organic acid and hydro carbons or terpene Close the application in object.