CN108913610A - The building and application of the engineered strain of xylitol are generated using glucose and xylose altogether - Google Patents

Abstract

The present invention is to generate the building and application of the engineered strain of xylitol using glucose and xylose altogether, it was found that glucose depression effect is significantly eliminated in the heat-resistant yeast that hexokinase KmHXK is knocked out, a variety of sugar are realized to utilize altogether with glucose, it can restore the influence that glucose metabolism is subject to after KmHXK is knocked out by being overexpressed glucokinase KmGLK, it finds that the bacterium still maintains the feature of glucose depression effect releasing in the present invention, can be used as platform bacterial strain；The present invention is successfully obtained with the platform bacterial strain can be efficiently altogether using the heat-resistant yeast bacterial strain YHY013 of glucose and xylose production xylitol, and deposit number is CGMCC No.15347.The bacterial strain can efficiently use the glucose and xylose xylitol zymolysis production in the natural fermented substrate such as xylose mother liquid or Corncob hydrolysate under conditions of 42 DEG C, and be more than other reported bacterial strains using the yield that Corncob hydrolysate produces xylitol.

Description

The building and application of the engineered strain of xylitol are generated using glucose and xylose altogether

Technical field

The present invention relates to field of biotechnology.Specifically, present invention discover that the heat-resisting ferment that hexokinase KmHXK is knocked out Glucose depression effect is significantly eliminated in mother, is realized a variety of sugar and is utilized altogether with glucose, by being overexpressed Portugal Glucokinase KmGLK can restore the influence that glucose metabolism is subject to after KmHXK is knocked out, and find that the bacterium still maintains in the present invention The feature that glucose depression effect releases, can be used as platform bacterial strain；The present invention is on the basis of the platform bacterial strain, to the bacterium Xylose metabolism path is transformed, and the Thermotolerant yeast of glucose and xylose production xylitol can efficiently be utilized altogether by successfully obtaining Strain YHY013, deposit number are CGMCC 15347.The bacterial strain can efficiently use xylose mother liquid or corncob under the high temperature conditions Glucose and xylose xylitol zymolysis production in the natural fermented substrate such as hydrolyzate, and xylose is produced using Corncob hydrolysate The yield of alcohol is more than reported bacterial strain.

Background technique

Agricultural wastes including corncob are a kind of biomass class energy for having very much development potentiality, pass through hydrolysis Pretreatment can be classified as the corncob residue (CCR) and supernatant hydrolyzate two parts of solid.CCR is due to being wherein rich in glucose So it is generally used to production bio-ethanol, and xylose accounts for the 90% of total reducing sugar in hydrolyzate, therefore is generally used to crystallization and extracts xylose, The pure xylose extracted can be used as present industrial production xylitol raw material (Balan, 2014；Behera et al., 2014； Isikgor&Becer, 2015).Remaining thick liquid, that is, xylose mother liquid after extracting xylose in Corncob hydrolysate, total reducing sugar For content between 60-75%, xylose accounts for the 50-70% of total reducing sugar, and glucose accounts for total reducing sugar about 8-10%, but due in xylose mother liquid Complicated component and miscellaneous sugar content is very high, make wherein xylose be difficult to be utilized, generally as liquid waste processing, not only cause environment dirt Dye is also a kind of energy waste.

Different from industrial chemical catalysis, bioanalysis production xylitol does not need pure xylose as raw material (Mohamad Et al., 2014), therefore xylose mother liquid perhaps can be directly used to produce xylitol by bioanalysis, and directly benefit The crystallization purifying process that xylose can be reduced with Corncob hydrolysate, further reduces the cost.Resource is both saved in this way, simultaneously Pollution is decreased, and has implemented the thought of scientific development and sustainable development.

Heat-resistant yeast Kluyveromyces marxianus (kluyveromyces marxianus) produces xylose for industrial fermentation Alcohol has following several advantages：1. be a kind of yeast of GRAS (general regarding as safe) rank, therefore can be with It is produced for food and medicine；2. can include xylose using various saccharides growth fermentation；3. can fast-growth and hair at high temperature Ferment, thus reduce the refrigeration costs in industrial fermentation and meanwhile greatly reduce bacterial strain pollution a possibility that (Kumar et al., 2009).Since kluyveromyces marxianus has quality much more outstanding than saccharomyces cerevisiae, kluyveromyces marxianus is by increasingly More productions (Fonseca et al., 2008) for bioenergy.So developing into wood using kluyveromyces marxianus Production of sugar polyol bacterial strain has very important application value.

Although kluyveromyces marxianus itself can use xylose as carbon source, works as in carbon source and be mixed with glucose When kluyveromyces marxianus can contain in xylose mother liquid and Corncob hydrolysate preferentially using glucose and inhibit xylose metabolism The glucose depression effect for also all containing glucose while abundant xylose, therefore how releasing heat-resistant yeast realize glucose and The total of xylose utilizes the key for becoming the biomass economies such as xylose mother liquid and Corncob hydrolysate.Have some about to biology Glucose and xylose realizes the research utilized altogether in matter, is studied due to the genomic information and metabolic pathway of saccharomyces cerevisiae Compare it is clear, therefore significant portion research all be around saccharomyces cerevisiae (Saccharomyces cerevisiae) be unfolded, mainly Research Thinking is by some key genes expression in transformation glucose signals access, while some non-glucose of heterogenous expression Transport protein and metabolism related gene, realize glucose and non-glucose carbon source total utilization (Kogje&Ghosalkar, 2016；Oh et al., 2013；Santangelo, 2006).But about heat-resistant yeast, the work of this respect is just opened Begin.Research before the present inventor is realized by expressing external source xylose specific transporters in kluyveromyces marxianus Glucose and xylose utilizes altogether, and obtained final bacterial strain YZJ119 highest can use corncob (including corncob residue (CCR) With hydrolyzate two parts) ethyl alcohol of 44.58g/L and the xylitol (Zhang et al., 2016) of 32.03g/L are generated simultaneously, But the glucose and xylose of the bacterial strain, altogether using only realizing in sugar transport level, there are no give full play to heat-resistant yeast Glucose and xylose be total to Utilization ability.

Therefore, there remain a need in the art of to develop with higher xylitol production capacity and can be hydrolyzed using corncob The heat-resisting bacterial strain of the biological raw materials such as liquid generation xylitol.

Summary of the invention

The purpose of the present invention is construct a kind of heat-resisting works yeast that can produce xylitol using glucose and xylose altogether Bacterial strain.Specifically, the present invention, which constructs one kind, to be had higher xylitol production capacity and can utilize Corncob hydrolysate Equal biological raw materials generate the heat-resisting works yeast strain of xylitol.

The present inventor knocks out the grape to heat-resistant yeast (Kluyveromyces marxianus) by comparison different genes Glyco inhabiting effects have found the important function of hexokinase (hexokinase, KmHXK) in the path, find hexose Kinases KmHXK knocks out the glucose depression effect for largely eliminating heat-resistant yeast, which can be used as platform bacterial strain Realize that a variety of sugar including xylose utilize altogether with glucose；The transformation in platform bacterial strain combination xylose metabolism path further mentions High glucose and xylose utilizes the ability of xylitol zymolysis production altogether；Final transformation bacterial strain is with bean cake powder and corn pulp mixing Object as can be efficiently used in the culture medium of nitrogen source xylose mother liquid or Corncob hydrolysate production xylitol；This is current glucose It is total to the best heat-resistant yeast engineering bacteria (table 2) of utilizing status with xylose, is that ferment is tieed up by heat-resistant yeast Marx Crewe for the first time Mother carries out the trial using production xylitol to xylose mother liquid, and effect is preferably (table 3), while being also currently with corncob water Solve the liquid production highest yeast of xylitol yield (table 3)；Final bacterial strain of the invention is demonstrated in xylose mother liquid and corncob water Solve the advantage on the industrial utilizations of biomass such as liquid.

The present invention knocks out the influence to heat-resistant yeast kluyveromyces marxianus glucose effect by comparison different genes, It was found that the knockout of hexokinase gene (KmHXK) can release the depression effect that glucose utilizes a variety of sugar；It was found that passing through Expression glucokinase KmGLK restores to maintain glucose depression effect while KmHXK knocks out bacterium to glucose metabolism ability The feature of releasing；Such bacterial strain can be used as platform bacterial strain, play a role in the development and utilization of many biomass；And it is making It is considered controlling the knockout of the correspondence gene KmMIG1 of the gene ScMIG1 of glucose depression effect in brewer yeast, cannot but solves Inhibition except glucose to xylose utilization.

The Xylose reductase base of present invention constitutive expression Neurospora sp (Neurospora crassa) in the platform bacterial strain Because of (xylose reductase genes, NcXYL1), glucose effect, which releases, combines external source Xylose reductase constitutive expression, The ability that heat-resistant yeast kluyveromyces marxianus utilizes glucose and xylose production xylitol altogether is greatly improved；Pass through again It is overexpressed the total utilization that xylose specific transporters (ScGal2N376F) further increases glucose and xylose, constructs Portugal Grape sugar and xylose are total to Utilization ability and greatly improve, and can utilize xylose mother liquid or jade using bean cake powder and corn pulp mixture as nitrogen source The heat-resisting works yeast strain of rice core hydrolyzate production xylitol.Building process is shown in Fig. 1.

The present invention is eventually used for YHY013.This bacterial strain efficiently can produce xylitol using glucose and xylose altogether, can be with bean cake powder and corn pulp mixture Nitrogen source directly utilizes xylose mother liquid or Corncob hydrolysate xylitol zymolysis production.Above-mentioned heat-resisting works yeast strain is in 2018 It is stored within 7 days 2 months China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC, city of BeiJing, China southern exposure year The institute 3 of area North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode：100101), corresponding deposit number is CGMCC No.15347 is named as kluyveromyces marxianus (Kluyveromyces marxianus) YHY013 bacterial strain.

Generally speaking, the application releases grape Glyco inhabiting to obtain platform bacterial strain, in platform bacterium from xylose metabolism level In conjunction with the other related transformations of xylose metabolism to obtain YHY013 bacterial strain on the basis of strain, before the present inventor The YZJ119 bacterial strain of building, glucose depression effect further release, and glucose and xylose is big using production xylitol ability altogether Amplitude improves, while demonstrating the exploitability of platform bacterial strain.

Specifically, the present invention includes the following contents：

In a first aspect, the present invention provides a kind of heat-resisting works ferment that can produce xylitol using glucose and xylose altogether Mother strains, it is especially a kind of directly to utilize xylose mother liquid or the heat-resisting works ferment of Corncob hydrolysate xylitol zymolysis production Mother strains.The bacterial strain is obtained by following methods：By comparing the glucose depression effect of different genes knock-out bacterial strain, obtain The key gene KmHXK, KmHXK of the approach knock out fungi degradation in addition to glucose is to the depression effect of a variety of sugar, after expression KmGLK is restored glucose metabolism ability caused by being knocked out due to KmHXK and declined, and the verified bacterial strain still maintains grape Glyco inhabiting Effect releases feature, therefore can be used as the platform bacterial strain built altogether using the K. marxianus bacterial strain of mixed sugar；Meanwhile it sending out It is considered controlling the knockout of the correspondence gene KmMIG1 of the gene ScMIG1 of glucose depression effect in present saccharomyces cerevisiae, but not Inhibition of the glucose to xylose utilization can be released；Related gene in xylose utilization path is changed on the basis of platform bacterial strain It makes, screening obtains efficiently utilizing the heat-resisting works yeast strain of glucose and xylose production xylitol altogether；The bacterial strain can be with Bean cake powder and corn pulp, which are fermented at a temperature of 42 DEG C for nitrogen source using xylose mother liquid and Corncob hydrolysate, generates xylitol, this bacterium Strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.15347 is named as YHY013 bacterial strain.

Plasmid used in the present invention is as follows：PZJ011 (Zhang et al., 2014), pZJ012 (Zhang et al., 2014), pZJ061 (Zhang et al., 2016), pKmMIG1-ScURA3-T and pHY008.

The construction method of plasmid is as follows in the present invention：

(1) with heat-resistant yeast NBRC1777 (purchased from the state-run biology money of Japanese independent administrative corporation's product assessment technique mechanism Source center (NBRC)) genome is template, use PrimeSTAR HS archaeal dna polymerase (Dalian precious biology) and primer KMMIG1- F (SEQ ID No.1), KMMIG1-R (SEQ ID No.2) carry out PCR amplification, obtain gene KmMIG1 (GenBank： BAP70066.1 (117191 to 119413)).Then gene KmMIG1 is inserted into pGEM-T Easy (Promega company of the U.S.) Carrier, to obtain pKmMIG1-T carrier.Then with saccharomyces cerevisiae genome (Saccharomyces cerevisiae W303) it is template, uses PrimeSTAR HS archaeal dna polymerase (the precious biology in Dalian) and primer SCURA3-SMAI-FULL-F (SEQ ID No.3), SCURA3-SMAI-FULL-R (SEQ ID No.4) carry out PCR amplification, obtain ScURA3 expressed intact Frame (GenBank：AM697670.1 (2062 to 3158)), phosphatizing treatment is carried out to the segment by T4PNK kinases.With Carrier dephosphorylation is carried out with FastAP dephosphorylation enzyme again after BamHI digestion pKmMIG1-T carrier, then by dephosphorylation Segment carry out flat end processing, finally by the ScURA3 complete genome segment of the carrier segments of flat end and phosphorylation connect, To obtain plasmid pKmMIG1-ScURA3-T (Fig. 2A)

(2) it is template with plasmid pZJ061, pZJ011, uses PrimeSTAR HS archaeal dna polymerase (the precious biology in Dalian) With TER-NOTI-F (SEQ ID No.5), KMTDH3-R (SEQ ID No.6)；KMTDH3-SCGAL2-F (SEQ ID No.7), SCGAL2-NOTI-R (SEQ ID No.8) primer carries out PCR amplification and obtains product, and obtained product is respectively T_ScTDH3- P_KmTDH3With ScGAL2-N376F gene.Amplified production is merged to obtain segment T_ScTDH3-P_KmTDH3-ScGAL2-N376F.It will melt Close segment T_ScTDH3-P_KmTDH3- ScGAL2-N376F and carrier pZJ061 carries out Not I single endonuclease digestion, then connects, to obtain The ScGAL2-N376F plasmids comprising two copies are obtained, are named pHY008 (Fig. 2 B).

For heat-resisting works yeast strain described in first aspect, starting strain is K.marxianus YHJ010 (ginseng See Hong et al., 2007)；Different genes, which knock out bacterium, to be had：Hexokinase gene KmHXK knocks out bacterium YLM001 (Zhang et Al., 2017), glucokinase gene KmGLK knocks out bacterium YLM002 (Zhang et al., 2017), and transcription factor KmMIG1 strikes Degerming YLM012；Heterogenous expression ScURA3 obtains control strain YWD016 (Zhang et al., 2017) in YHJ010； After expressing K mGLK gene in YLM001 (compensating for leads to the decline of bacterial strain metabolizable glucose ability since KmHXK is knocked out) The verified platform bacterium that can be used as the releasing of glucose depression effect of obtained bacterial strain YLM005 (Zhang et al., 2017) Strain.

Heat-resisting works yeast strain described in first aspect, wherein the Xylose reductase and xylose specificity that recombinantly express turn Fortune albumen is respectively the xylose specific transporter (grape of Neurospora sp Xylose reductase gene (NcXYL1) and Saccharomyces cerevisiae The mutated gene (ScGAL2-N376F) of saccharide transporter).

In second aspect, the present invention provides the method for the heat-resisting works yeast strain of building first aspect, the method Include the following steps：

KmMIG1-ScURA3 segment in pKmMIG1-ScURA3-T is transferred in YHJ010, to knock out in YHJ010 KmMIG1 gene, be named as YLM012；

Containing ScURA3 label is introduced due to transformation early period in K.marxianus YLM005, which is knocked out to obtain Bacterial strain YHY003.Then it is ScURA3 by label and copies containing there are two (respectively by the promoter (P in heat-resistant yeast source_KmTDH3) With the promoter (P of Saccharomyces cerevisiae_ScTDH3) control) and Neurospora sp Xylose reductase gene (NcXYL1) plasmid pZJ011, It is transformed into YHY003.Obtained conversion bacterial strain is named as：YHY006；

It is ScLEU2 by label and copies containing there are two (respectively by the promoter (P in heat-resistant yeast source_KmTDH3) and wine brewing Promoter (the P of yeast sources_ScTDH3) control Neurospora sp Xylose reductase gene (NcXYLl) plasmid pZJ012, be transformed into In YHY006, obtained conversion bacterial strain is named as：YHY008；

Then the ScURA3 that pZJ011 plasmid is introduced into is knocked out from YHY008 and obtains YHY009；It is again ScURA3 by label Containing there are two copies (respectively by the promoter (P in heat-resistant yeast source_KmTDH3) and Saccharomyces cerevisiae promoter (P_ScTDH3) S.cerevisiae xylose transport protein mutant (ScGal2N376F) the plasmid pHY008 of control is transferred in YHY009, is obtained Bacterial strain YHY013.

In a third aspect, the present invention provides a kind of method for producing xylitol, and the method includes by bacterial strain YHY013 It is seeded in the culture medium comprising glucose and xylose and carries out fermented and cultured, extracted from fermentation liquid and obtain xylitol.

In one embodiment, the method for the production xylitol includes that bacterial strain YHY013 is directly seeded in xylose mother Fermented and cultured is carried out in liquid or Corncob hydrolysate, is extracted from fermentation liquid and is obtained xylitol.

The method of the production xylitol is can to generate wood using glucose and xylose fermentation altogether using bacterial strain YHY013 The ability of sugar alcohol.

About the culture medium of fermentation, example may include the pure glucose and xylose compound of addition pure sugar culture-medium, The xylose mother liquid of Corncob hydrolysate or Corncob hydrolysate after extracting xylose.Corncob hydrolysate or xylose mother liquid include wood Sugar and glucose, can be used as the natural fermented substrate of bacterial strain YHY013.Furthermore it is possible to add cheap dregs of beans in the medium Powder and corn pulp are as nitrogen source,, can be by bean cake powder and corn pulp concentration all by adding nitrogen source for example, during the fermentation Control is in 10g/L.

When for this paper, term " pure sugar culture-medium ", which refers to, adds pure glucose and xylose compound and other cultures The culture medium that based component is formulated, it is this kind of in itself comprising glucose and xylose etc. to distinguish Corncob hydrolysate or xylose mother liquid Carbohydrate and the culture medium that can be used as natural fermented substrate.Pure glucose and xylose compound is commercially available herein.

It, can be by Starting glucose and xylose weight concentration in the culture medium for adding pure glucose and xylose compound Ratio control is 1: 4.Specifically, Initial sugar concentration can be 30g/L glucose and 120g/L xylose in pure sugar culture-medium, or first Beginning sugared concentration can be with 50g/L glucose and 200g/L xylose.

Include a large amount of xylose and glucose in Corncob hydrolysate, can be used as the natural fermented bottom of YHY013 bacterial strain Object.Generally, in Corncob hydrolysate, initial glucose concentration is about 11.68g/L, and xylose concentration is about 165.29g/L, It can satisfy the fermentation needs of bacterial strain YHY013.

Also, as the xylose mother liquid of the remaining thick liquid after extracting xylose in Corncob hydrolysate, total reducing sugar contains Amount is between 60-75%, and wherein xylose accounts for the 50-70% of total reducing sugar, and glucose accounts for total reducing sugar about 8-10%, is also used as the present invention Bacterial strain YHY013 natural fermented substrate.When using xylose mother liquid as fermentation medium, when being originated in the culture medium Concentration of glucose is usually 13.88g/L, and xylose concentration is usually 53.90g/L.It during the fermentation, can be according to practical need Supplement material xylose mother liquid, for example, can be with feed supplement two or more times.

Preferably, fermentation carries out in the fermenter, and fermentation temperature is 42 DEG C, and preceding 12 hours mixing speeds are 300rpm, Mixing speed control later is 350rpm, and oxygen-supply quantity is controlled always in 0.5vvm.

In addition, when fermenting in the fermenter, additionally it is possible to add in xylose mother liquid culture medium or Corncob hydrolysate culture medium Adding cheap bean cake powder and corn pulp mixture is that nitrogen source carries out xylitol zymolysis production.During the fermentation, benefit can be passed through Adding bean cake powder and corn pulp and keeping bean cake powder concentration is 10g/L, and holding corn pulp concentration is 10g/L.

When fermenting in the fermenter, the initial inoculum of bacterial strain YHY013 is OD₆₀₀It can be 1.

Specifically, the present invention provides following technical proposals：

1. one kind can be altogether using the heat-resisting works yeast strain of glucose and xylose production xylitol, deposit number is CGMCC No.15347。

2. heat-resisting works yeast strain described in the 1st can directly be sent out using xylose mother liquid or Corncob hydrolysate Ferment generates xylitol.

3. a kind of method for producing xylitol, the method includes connecing the bacterial strain that deposit number is CGMCC No. 15347 Kind carries out fermented and cultured in the culture medium comprising glucose and xylose, extracts from fermentation liquid and obtains xylitol.

4. the method according to the 3rd, wherein the fermentation carries out in the fermenter, fermentation temperature is 42 DEG C.

5. the method according to the 4th, wherein during the fermentation, preceding 12 hours mixing speeds are 300rpm, it Mixing speed control afterwards is 350rpm, and oxygen-supply quantity is controlled always in 0.5vvm.

6. the method according to the 3rd, wherein the culture medium is using bean cake powder and corn pulp mixture as nitrogen source, preferably Ground, the two content all control as 10g/L.

7. according to the method described in claim 3, wherein the inoculum concentration of the bacterial strain is initial OD₆₀₀It is 1.

8. the method according to the 3rd, wherein in the culture medium comprising glucose and xylose Starting glucose and The weight concentration ratio of xylose is 1: 4.

9. the method according to the 3rd, wherein the culture medium is Corncob hydrolysate, it is preferable that in the corn In core hydrolyzate, initial glucose concentration 11.68g/L, xylose concentration 165.29g/L.

10. the method according to the 3rd, wherein the culture medium is what Corncob hydrolysate obtained after extracting xylose Xylose mother liquid, it is preferable that in the xylose mother liquid initial glucose concentration be 13.88g/L, xylose concentration 53.90g/L, Feed supplement xylose mother liquid is two or more times in fermentation process.

Advantage and good effect

Influence in the present invention by comparison different genes knockout to heat-resistant yeast glucose depression effect, it was found that the line Key gene KmHXK in road, the knockout of the gene largely relieve kluyveromyces marxianus (K.marxianus) Glucose depression effect；Glucose generation caused by verified overexpression glucokinase KmGLK is compensated for due to KmHXK knockout Thank ability decline while still maintain grape Glyco inhabiting release characteristic (Fig. 4), therefore can be used as polysaccharide mixing utilize it is resistance to The platform bacterial strain of hot saccharomycete building；And the knockout of the MIG1 of control glucose depression effect is believed in saccharomyces cerevisiae Glucose depression effect can not be released, illustrates may there there be the inhibition of xylose different glucose in kluyveromyces marxianus Path (Fig. 3).Obtained bacterium after being further transformed to the related gene of xylose utilization approach on the platform base YHY013 is under the conditions of 42 DEG C of high temperature for strain, glucose and xylose can be utilized altogether with very effective, although involved in YHY013 The transformation of xylose utilization approach it has been reported that be not still completed in the case where glucose effect releases such a background, so It is also that YZJ119 (Zhang et al., 2016) cannot reach that YHY013, which can reach glucose and xylose to be total to utilizing status, 's；Just because of glucose and xylose can be utilized effectively altogether, YHY013 can more effectively be hydrolyzed xylose mother liquid and corncob The biomass such as liquid are used xylitol zymolysis production.

The KmHXK of K.marxianus knocks out bacterium YLM001 and YLM005 (after expression in YLM001 in the present invention KmGLK) the knockout bacterium compared to KmGLK and KmMIG1 is containing 2- deoxyglucose (2-deoxy-D-glucose, 2-DG) Various non-glucose carbon source plates on have obviously growth vigor, illustrate that the knockout of KmHXK largely relieves The glucose depression effect of K.marxianus.YHY013 in the present invention can be utilized at 42 DEG C, under shake flask culture conditions The xylitol of 20g/L glucose (initial concentration) and 80g/L xylose (initial concentration) fermenting and producing 60.05g/L, throughput rate For 1.77g/L/h (Fig. 5)；When fermentation tank culture, 30g/L glucose (initial concentration) and 120g/L xylose (initial concentration) are utilized It can be up to 3.11g/L/h with the xylitol of fermenting and producing 93.33g/L, rate, when concentration is increased to 50g/ glucose and 200g/L Xylitol yield is up to 158.55g/L (Fig. 6) when xylose.Although YZJ119 can be generated effectively using glucose and xylose altogether in document Xylitol (Zhang et al., 2016), but the accumulation of xylitol here must have a large amount of glucose metabolism and provide energy Amount and coenzyme, when glucose deficiency, the accumulation efficiency of xylitol is not just considerable, 20g/L glucose and 80g/L xylose shaking flask Fermentation only generates the xylitol of 24.9g/L, throughput rate 0.73g/L/h；And grape in xylose mother liquid and Corncob hydrolysate The ratio of sugar is all relatively low compared to xylose, therefore YHY013 is utilizing xylose mother liquid or corncob water compared to YZJ119 Advantage is had more in terms of solving liquid production xylitol.YHY013 in the present invention can be using bean cake powder and corn pulp mixture as nitrogen source Culture medium in, in the fermenter, by xylose mother liquid and 82.34g/ may finally be produced in the way of feed supplement xylose mother liquid L xylitol, rate 1.25g/L/h；It can be produced in the fermenter using Corncob hydrolysate (xylose containing 165.29g/L) 118.63g/L xylose, rate are 1.98g/L/h (Fig. 7).

Detailed description of the invention

From detailed description with reference to the accompanying drawing, features described above of the invention and advantage be will be apparent from, wherein：

The building flow chart of Fig. 1 YHY013 bacterial strain of the present invention.

PKmMIG1-ScURA3-T plasmid (A) and pHY008 plasmid (B) map in Fig. 2 present invention.

Fig. 3 different genes knock out bacterium and control strain YWD016 without or with 1,5-anhydroglucitol (2-DG) no With the cultured on solid medium situation of carbon source.

Cultured on solid medium situation of Fig. 4 platform bacterial strain YLM005 in the different carbon source without or with 2-DG (YLM001 and the YWD016 respectively positive and negative control).

The bacterial strain and control strain YZJ119 that Fig. 5 present invention constructs are using 20g/L glucose and 80g/L xylose 42 The result of xylitol zymolysis production under the conditions of DEG C shaking flask.

The YHY013 that Fig. 6 present invention constructs utilizes different Initial sugar concentrations fermenting and producing xylose under fermentation condition Alcohol result.(A) 30g/L glucose and 120g/L xylose；(B) 50g/L glucose and 200g/L xylose.

The bacterial strain YHY013 constructed in Fig. 7 present invention utilizes wood in the culture medium using bean cake powder and corn pulp as nitrogen source The result of sugared mother liquor (A) and Corncob hydrolysate (B) production xylitol.

Preservation explanation

Energy of the invention utilizes the heat-resisting works yeast strain kluyveromyces marxianus of wood-sugar fermentation (Kluyveromyces marxianus) YHY013 was stored in Chinese microorganism strain preservation management on 2 7th, 2018 Common micro-organisms center (CGMCC, city of BeiJing, China Chaoyang District North Star West Road 1 institute 3, the micro- life of the Chinese Academy of Sciences of the committee Object research institute, postcode：100101), corresponding deposit number is CGMCC No.15347, is named as kluyveromyces marxianus (Kluyveromyces marxianus) YHY013 bacterial strain.

Specific embodiment

The present invention is further described referring to specific embodiment, it will be appreciated by those skilled in the art that this hair It is bright to be not limited to these specific embodiments.

Reagent and bacterial strain：All reagents in the present invention are the reagents of the SILVER REAGENT of market purchase or more.Wherein, wooden Sugar, glucose, the basic nitrogen source of yeast, plastic recovery kit, T4 ligase with T4 PNK kinases, FastAP dephosphorylation enzyme and All restriction enzymes derive from Shanghai Sheng Gong bio-engineering corporation.Yeast powder and peptone have Oxoid company and peace Two kinds of sources of fine jade company, bean cake powder derive from Shandong En Mu company, and corn pulp is from Shanghai Fang Qi company, xylose mother liquid source In Shandong Longli Biology Science and Technology Co., Ltd, corncob is purchased from the Yantai, Shandong Province locality market of farm produce；E. coli jm109 (DE3) host strain (Promega company) that uses when bacterial strain is as molecular cloning, includes 100 μ g/ml ampicillins Luria-Bertani (LB) culture medium is for cultivating E.coli.Synthetic media (glucose 20g/L, the basic nitrogen source of yeast 6.7g/L, leucine 30mg/ml, uracil 20mg/ml, tyrosine 20mg/ml) it is mainly used for converting.YPD culture medium (10g/L Yeast extract, 20g/L peptone, 20g/L glucose) it is used for the preceding culture of yeast.

The preparation of 1. bacterial strain of embodiment：

1. the building that glucose and xylose utilizes the heat-resistant yeast bacterial strain of xylitol zymolysis production altogether：

It is starting strain with heat-resistant yeast YHJ010 (referring to Hong et al., 2007), carries out related gene transformation.

1) yeast chemical conversion steps：

1. bacterium to be rebuilt lines on YPD plate, in 37 DEG C of overnight incubations.

2. is inoculated into the fresh YPD fluid nutrient medium of 5ml, 37 DEG C from picking monoclonal on the YPD plate being incubated overnight, 250rpm, overnight incubation.

3. takes 500 μ l cultures to be transferred to equipped in the fresh YPD fluid nutrient medium test tube of 5ml, 37 DEG C, 250rpm is shaken Bed culture 5h.

4. takes out culture, it is centrifuged 5000rpm under room temperature, 3 minutes, abandons supernatant, retains thallus.

5. prepares 500 μ l and converts buffer：400 μ l 50%PEG4000；50 μ l 2M lithium acetates；50 μl 1M DTT (being dissolved in 10mM sodium acetate, pH 5.2).

6. thallus is resuspended using 150 μ l conversion buffer in, 5000rpm is centrifuged 3 minutes, removes supernatant.

7. converts buffer resuspension thallus with 50 μ l, the plasmid of 5 μ l (1-10 μ g) linearisation is added, it is slight to shake 30sec。

8. is water-bath 15 minutes under the conditions of 47 DEG C.

9. thallus is coated on the synthesis culture containing leucine (Leu) or tryptophan (Trp) or uracil (Ura) by Base, 37 DEG C are cultivated 2 days.

It is cultivated 10. being cloned in liquid YPD on picking plate, extracts genome, and conversion results are identified by PCR and are fermented Verification the verifying results.

2) detailed process that heat-resistant yeast of the present invention respectively expresses bacterial strain is constructed：

1. the plasmid in the building present invention：

1) building of plasmid pMIG1-ScURA3-T：

PCR amplification is carried out by template of heat-resistant yeast YHJ010 genome, obtains gene KmMIG1 (GenBank： BAP70066.1 (117191 bases to 119413 bases)).Then gene KmMIG1 is inserted into the (U.S. pGEM-T Easy Promega company) carrier, to obtain pKmMIG1-T carrier.With saccharomyces cerevisiae genome (Saccharomyces Cerevisiae W303) it is template, PCR amplification is carried out, ScURA3 expressed intact frame (GenBank is obtained： AM697670.1 (the 2062nd base to 3158 bases)), phosphatizing treatment is carried out to the segment by T4PNK kinases.With BamHI digestion Carrier dephosphorylation is carried out with FastAP dephosphorylation enzyme again after pKmMIG1-T carrier, then carries out dephosphorylized segment Flat end processing, finally connects the ScURA3 complete genome segment of the carrier segments of flat end and phosphorylation, to obtain matter Grain pKmMIG1-ScURA3-T (Fig. 2A)

Concrete operation step is：

(1) with heat-resistant yeast K.marxianus NBRC1777 (purchased from Japanese independent administrative corporation's product assessment technique machine The state-run Biological Resource Center of structure (NBRC)) genome is template, use PrimeSTAR HS archaeal dna polymerase (Dalian precious biology) PCR amplification is carried out, gene KmMIG1 is obtained with primer KMMIG1-F, KMMIG1-R.Then gene KmMIG1 is inserted into pGEM-T Easy (Promega company of the U.S.) carrier, to obtain pKmMIG1-T carrier.

1. the PCR system of KmMIG1：

PCR program

2. after obtaining KmMIG1, after the end DNA adds " A " base respectively, insertion pGEM-T easy carrier (is purchased from Promega in).

Add A system：

TA clones linked system：

(2) it with saccharomyces cerevisiae genome (Saccharomyces cerevisiae W303) for template, uses PrimeSTAR HS archaeal dna polymerase (the precious biology in Dalian) and primer SCURA3-SMAI-FULL-F (SEQ ID No.3), SCURA3-SMAI-FULL-R (SEQ ID No.4) carries out PCR amplification, obtains ScURA3 expressed intact frame, passes through T4PNK enzyme pair ScURA3 complete genome carries out phosphatizing treatment；With after BamHI digestion pKmMIG1-T carrier again with FastAP dephosphorylation enzyme into Then dephosphorylized segment is carried out flat end processing by row carrier dephosphorylation；Finally by the carrier segments of flat end and phosphorus The ScURA3 complete genome of acidification connects, to obtain plasmid pKmMIG1-ScURA3-T (Fig. 2A)

1. the PCR system of ScURA3 expressed intact frame：

PCR program

2. the phosphorylation system of ScURA3 expressed intact frame segment：

3. the digestion and phosphorylation system of pKmMIG1-T carrier：

4. the pKmMIG1-T flat end system of phosphorylation：

5. the linked system of ScURA3 expressed intact frame and pKmMIG1-T carrier：

2) building of plasmid pHY008

Be template with plasmid pZJ061, pZJ011, using PrimeSTAR HS archaeal dna polymerase (Dalian precious biology) and TER-NOTI-F (SEQ ID No.5), KMTDH3-R (SEQ ID No.6)；KMTDH3-SCGAL2-F (SEQ ID No.7), SCGAL2-NOTI-R (SEQ ID No.8) primer carries out PCR amplification and obtains product, and obtained product is respectively T_ScTDH3- P_KmTDH3With ScGAL2-N376F gene.Amplified production is merged to obtain as T_ScTDH3-P_KmTDH3-ScGAL2-N376F.It will fusion Segment T_ScTDH3-P_KmTDH3- ScGAL2-N376F and carrier pZJ061 carries out Not I single endonuclease digestion, then connects, to obtain Comprising two ScGAL2-N376F plasmids, name pHY008 (Fig. 2 B).

Concrete operation step is：

(1) using plasmid pZJ011 or pZJ061 as template, PCR amplification obtains product, and obtained product is respectively T_ScTDH3- P_KmTDH3With ScGAL2-N376F gene.

①T_ScTDH3-P_KmTDH3The PCR system of segment：

PCR program

2. the PCR system of ScGAL2-N376F gene：

PCR program

(2) by amplified production T_ScTDH-P_KmTDH3It is merged with ScGAL2-N376F, fusion product T_ScTDH3-P_KmTDH3- ScGAL2-N376F。

T_ScTDH3-P_KmTDH3- ScGAL2-N376F Gene Fusion PCR system：

PCR program

(3) segment T will be merged_ScTDH3-P_KmTDH3- ScGAL2-N376F is inserted into plasmid pZJ061 and obtains plasmid pHY008 (i.e. double copy ScGAL2-N376F expression plasmids).

①T_ScTDH3-P_KmTDH3The digestion system of-ScGAL2-N376F gene：

2. the digestion system of pZJ061 carrier：

③T_ScTDH3-P_KmTDH3The linked system of-ScGAL2-N376F gene and pZJ002 carrier：

4. acquisition is inserted T_ScTDH3-P_KmTDH3The pZJ061 plasmid of-ScGAL2-N376F gene order, is named as pHY008。

2) building of the Engineering Yeast bacterium in the present invention：

Using pKmMIG1-ScURA3-T as plasmid template.PCR amplification goes out the knockout frame KmMIG-1-ScURA3 piece of KmMIG1 Section, which is transferred in YHJ010, after homologous recombination, is knocked the KmMIG1 gene of bacterial strain YHJ010, while making bacterial strain Restore URA3 gene function.In the synthetic media (formula containing Leu and Trp：Glucose 20g/L, the basic nitrogen source 6.7 of yeast G/L, uracil 2mg/ml, agar 15g/L) on screening positive clone, be named as YLM012.

Using pMD18T- Δ ScURA3 plasmid as template, PCR amplification ScURA3 knocks out segment.ScURA3 is knocked out segment to turn It dissolves into hexokinase (KmHXK) the gene knockout YLM005 that glucokinase (KmGLK) is overexpressed simultaneously, after homologous recombination, It is knocked the ScURA3 gene in bacterial strain YLM005, loses the ability of the synthesis of uracil.Containing leucine, uracil With the synthetic media (formula of 5 '-FOA：Glucose 20g/L, yeast basic nitrogen source 6.7g/L, uracil 2mg/ml, agar ScURA3 knock-out bacterial strain is screened on plate 15g/L), the Strain Designation of acquisition is YHY003.

With Sma I digestion pZJ011 carrier.Digestion products are converted to YHY003, in the synthetic media for containing only leucine (formula：Glucose 20g/L, yeast basic nitrogen source 6.7g/L, 15 g/L of agar) on screening ScURA3 gene function restore sun Property clone, at the same time increase newly two parts of NcXYL1 genes function, be named as YHY006.

With Sma I digestion pZJ012 carrier.Digestion products are converted to YHY006, ScLEU2 is screened on synthetic media The positive colony that gene function restores, strain contains four parts of NcXYL1 gene copies at this time, is named as YHY008.

Using pMD18T-ScURA3 as template, PCR amplification ScURA3 knocks out segment.ScURA3 knockout segment is transformed into In YHY008, after homologous recombination, it is knocked the ScURA3 gene in bacterial strain YHY008, loses the ability of the synthesis of uracil. ScURA3 knock-out bacterial strain is screened on the synthetic media plate containing uracil and 5 '-FOA, the Strain Designation of acquisition is YHY009。

With Sma I digestion pHY008 carrier.Digestion products are converted to YHY009, screens and restores on synthetic media The positive colony of ScURA3 gene function, and the function of newly-increased 2 parts of ScGAL2-N376F genes, are named as YHY013.

3) genome is extracted, the positive strain of yeast conversion is identified by PCR.

(1) heat-resistant yeast genome extraction step：

1. picking monoclonal accesses in 5ml liquid YPD, 37 DEG C, 250rpm, culture is for 24 hours.

2. 12000rpm under room temperature, bacterium is received in centrifugation in 5 seconds, abandons supernatant.

3. thallus, 12000rpm is resuspended in .500 μ l distilled water, bacterium is received in centrifugation in 5 seconds, abandons supernatant.

4. takes 200 μ l laboratory autogamy 1xbreaking buffers (TritonX-100 (2% (w/v)), SDS (1% (w/v)), NaCl (100mM), Tris-Cl (10mM, pH8.0), EDTA (1mM)) be resuspended thallus, and by bacterium solution be transferred to containing In the EP pipe of 0.3g bead (425-600 μm, Sigma, the U.S.).

After 5. 200 μ l phenol chloroformic solutions are added in, high speed concussion 3 minutes, 200 μ l 1x TE of addition (10 mM Tris-Cl, PH 8.0,1mM EDTA).Slight concussion.

6. .12000rpm, 5 minutes, centrifugation took top layer's clear liquid to be transferred in new EP pipe, the anhydrous of 1ml pre-cooling is added Ethyl alcohol.

7. .12000rpm, is centrifuged 10 minutes by 4 DEG C, supernatant is abandoned, it is drying precipitated at room temperature, and be resuspended with 400 μ l 1x TE Precipitating.

8. is added in 2 μ l RNase (RNA hydrolase, 2mg/ml) to EP pipe, mix, 37 DEG C, digestion 1h.

9. takes 40 μ l 3M sodium acetates (pH 5.2) to be added in pipe, the dehydrated alcohol of 1ml pre-cooling is mixed and is added.

10. .12000rpm, 4 DEG C, 30 minutes, centrifugation was abandoned supernatant and is dried at room temperature.It is resuspended and is precipitated with suitable volumes, this is Pastoris genomic dna.

(2) PCR system of the positive strain of yeast conversion is identified：

1. the PCR system of NcXYL1 gene in the genome of YHY006：

Wherein primer N_CXYL1-F1 is as shown in SEQ ID No.9, N_CXYL1-R1 is as shown in SEQ ID No.10.

PCR program

2. the genome detection of the positive strain of other yeast conversions is same as above system.As described above, respectively with the spy of gene Specific primer is as primer, using genome as template, after PCR amplification, can specificity amplify corresponding mrna length band Bacterial strain, as positive strain, and carry out next step experiment.

2. heat-resistant yeast bacterial strain glucose of embodiment inhibits to release situation

Whether the engineered strain that the embodiment is used to verify in the present invention releases grape Glyco inhabiting compared to wild-type strain Effect.The result shows that the glucose depression effect of YLM001 is largely released really, 2- deoxidation Portugal can contained A variety of non-glucose carbon source solid medium tablets growth of grape sugar (2-DG)；The knockout of KmGLK is for glucose depression effect Do not generate any influence；And KmMIG1 is only in the sucrose containing 2-DG, gossypose, maltose, mannose and gala sugar culture-medium On compared to control YWD016 have growth vigor slightly, and on the xylose and glycerin medium containing 2-DG growth conditions with It compares almost the same, illustrates that the knockout of KmMIG1 does not release the inhibition that xylose and glycerol utilize in glucose；It verifies simultaneously Glucose has been still maintained while being overexpressed the YLM005 that KmGLK is obtained on YLM001 and having restored metabolizable glucose ability The releasing feature of depression effect.

1. the recovery bacterial strain on YPD culture medium flat plate：Control strain：YWD016, experimental strain：YLM001, YLM002, YLM012, YLM005,37 DEG C are cultivated 1 day.

2. picking them separately monoclonal, it is connected in 5ml liquid YPG culture medium, 37 DEG C, 250rpm, overnight incubation.

3. thalline were collected by centrifugation, then be resuspended with sterile water, ultraviolet specrophotometer surveys cell concentration, obtains bacteria concentration OD₆₀₀ For 4 bacterium solution, then obtaining concentration by sterile water gradient dilution is 1,10^-1, 10^-2, 10^-3, 10^-4, 10^-5Bacterium solution.

4. the YP culture medium for drawing different carbon source of the bacterium solution drop in being free of or containing 2-DG of 2ul difference dilution is flat On plate, 42 DEG C of cultures are for 24 hours.(Fig. 3 and Fig. 4)

5. YLM001 and YLM005 can be in the non-glucose containing 2-DG from Fig. 3 and 4 it is found that compared to control strain Normal growth on carbon source culture medium flat plate shows to relieve glucose in YLM001 and YLM005 bacterial strain to other a variety of sugar The inhibition utilized；YLM012 is only in the sucrose containing 2-DG, gossypose, maltose, on mannose and gala sugar culture-medium compared to Control YWD016 have growth vigor slightly, and on the xylose and glycerin medium containing 2-DG growth conditions with compare substantially Unanimously, illustrate that the knockout of KmMIG1 does not release the inhibition that xylose and glycerol utilize in glucose；And YLM002 is having 2-DG Non-glucose carbon source culture medium flat plate on growth with to compare YWD106 almost the same, illustrate KmGLK on grape Glyco inhabiting road It is not key gene in diameter.

Fermentation situation of bacterial strain under the conditions of 42 DEG C is transformed in embodiment 3.

It is poor that the different engineered strains that the embodiment is used to compare acquisition are total to utilizing status utilizing status to glucose and xylose It is different.The result shows that the bacterial strain YHY013 that all cydorge genes are all transferred to utilizes the effect of glucose and xylose production xylitol most altogether It is good.

1. the recovery bacterial strain on YPD culture medium flat plate：Compare strain：YZJ119 tests strain：YLM005, YHY006, YHY008 and YHY013,37 DEG C are cultivated 1 day.

2. picking them separately monoclonal, it is connected to 5ml liquid YPD medium.37 DEG C, 250rpm, overnight.

3. preparing 30ml fermentation medium to be sub-packed in 250ml conical flask.Formula：20g/L glucose, 80g/L xylose, 10g/L yeast extract, 20g/L bacteriological peptone, sterilizing are stand-by.

4. taking in appropriate overnight culture access 30ml wood-sugar fermentation culture medium, make their initial OD₆₀₀It is 2 DEG C of Isosorbide-5-Nitrae, 250rpm culture.

5. in 0h, 6h, 12h, 22h, 28h, 34h, 46h sampling, and supernatant is taken to test and analyze (Fig. 4) by HPLC.

6. as can be seen from Figure 4, at 42 DEG C, using xylose and glucose as under the culture medium condition of culture of carbon source：

The result shows that the bacterial strain YHY013 that all cydorge genes are all transferred to utilizes glucose and xylose production xylitol altogether Effect is best, and the xylitol of 60.05g/L, throughput rate 1.77g/ are generated using 20g/L glucose and 80g/L wood-sugar fermentation L/h (Fig. 5) has very big advantage compared to YZJ119 (xylitol yield is 24.9g/L, throughput rate 0.73g/L/h).

Cultivation and fermentation produces xylitol to embodiment 4.YHY013 bacterial strain in the fermenter

The example is used to illustrate that final bacterial strain YHY013 to have better xylitol in the amplification culture closer to industry Production capacity.The result shows that it is more preferable really than shaking flask culture effect to produce xylitol by ferment tank.

1. the recovery bacterial strain YHY013 on YPD culture medium flat plate.

2. picking monoclonal is connected in 30mL liquid YPD medium, 37 DEG C, 250rpm, culture is for 24 hours.

3. preparing the fermentation medium 500mL of different sugar concentration in 1L fermentor, sterilizing is stand-by.

4. taking in appropriate seed culture medium access fermentation medium, control fermentation starting OD₆₀₀It is 2 DEG C of Isosorbide-5-Nitrae, first 12 hours Speed of agitator be 300rpm, mixing speed control later is 350rpm, and oxygen-supply quantity controls always in 0.5vvm.

5. as shown in fig. 6, compartment time sampling, and supernatant is taken to detect sugared content by HPLC.

6. as can be seen from Figure 6, under the conditions of fermentation tank culture, can be fermented life using 30g/L glucose and 120g/L xylose The xylitol of 93.33g/L is produced, rate is up to 3.11g/L/h, wooden when concentration is increased to 50g/ glucose and 200g/L xylose Sugar alcohol yield is up to 158.55g/L (Fig. 6), than the production that shaking flask culture is more conducive to xylitol.

Specifically, " fermentation medium of different sugar concentration " used in the present embodiment refers to xylose and grape in culture medium The initial content of sugar is different, for example, comprising 30g/L glucose and 120g/L xylose, or include 50g/L glucose and 200g/L wood Sugar.But all comprising 10g/L bean cake powder and 10g/L corn pulp as nitrogen source in the culture medium.

The acquisition of 5. Corncob hydrolysate of embodiment

The glucose and xylose that the example is used to discharge in corncob carrys out xylitol zymolysis production, and method is referring generally to document Method in (Zhang et al., 2016).The result shows that the method for acid processing can discharge the glucose in corncob really And xylose.

1. 0.5% (w/w) H of corncob₂SO₄With 1.5% (w/w) H₃PO₄Mixed acid handles 1h under the conditions of 127 DEG C, Gu Liquor ratio is 1: 4.

2. residue is removed with filtered through gauze and obtains hydrolyzate.

3. passing through Ca (OH)₂It is 10.0 that hydrolyzate, which is adjusted to pH, and the precipitating of generation is filtered to remove.

4. then handling 3h under the conditions of 250rpm at 50 DEG C with activated carbon again by HCl neutralizing hydrolysis liquid pH to 6.0.

5. processed hydrolyzate is finally concentrated into xylose concentration about 200g/L or so by Rotary Evaporators at 70 DEG C.

Generally, industrial Corncob hydrolysate is also to be obtained by acid-hydrolyzed mode, above-mentioned laboratory simulation industry The ingredient for the Corncob hydrolysate that process obtains and glucose and xylose content therein substantially with industrial corn core hydrolyzate It is similar, it can be used for the experiment that subsequent authentication YHY013 bacterial strain of the invention utilizes Corncob hydrolysate fermentation.

Utilization power of the embodiment 6.YHY013 bacterial strain to xylose mother liquid and Corncob hydrolysate

Whether the example is for verifying final bacterial strain YHY013 can be using bean cake powder and corn pulp mixture as the training of nitrogen source Support base effective use xylose mother liquid and Corncob hydrolysate xylitol zymolysis production.The result shows that YHY013 really can be with bean cake powder It is nitrogen source with corn pulp mixture and efficiently uses xylose mother liquid or Corncob hydrolysate xylitol zymolysis production.

1. the recovery bacterial strain on YPD culture medium flat plate.Test strain：YHY013.37 DEG C are cultivated 1 day.

2. picking monoclonal is connected to 5ml liquid YPD medium.37 DEG C, 250rpm, overnight.

3. preparing 30ml fermentation medium to be sub-packed in 250ml conical flask.Formula：10g/L bean cake powder, 10g/L corn Slurry, the xylose mother liquid or Corncob hydrolysate of certain dilution, sterilizing are stand-by.

4. taking in appropriate overnight culture access 30ml wood-sugar fermentation culture medium, make their initial OD₆₀₀It is 2 DEG C of Isosorbide-5-Nitrae, 250rpm culture.

5. in 0h, 6h, 12h, 18h, for 24 hours, 30h sampling, and supernatant is taken to test and analyze (Fig. 7) by HPLC.

6. from Fig. 7 and table 3 it is found that YHY013 effectively can produce wood using xylose mother liquid or Corncob hydrolysate really Sugar alcohol may finally produce 82.33g/L xylitol, rate 1.25g/L/h by feed supplement in the fermenter；Utilize corncob Hydrolyzate (xylose containing 165.29g/L) can produce 118.63g/L xylose in the fermenter, and rate is 1.98g/L/h (Fig. 7).

The result of Fig. 7 and table 3 explanation, YHY013 effectively can produce xylitol using glucose and xylose altogether, and can Using using xylose mother liquid or Corncob hydrolysate as carbon source, bean cake powder and corn pulp mixture are that nitrogen source effectively produces xylitol.

Therefore, YHY013 bacterial strain was preserved in Chinese microorganism strain preservation management committee on 2 7th, 2018 by the present inventor Member's meeting common micro-organisms center (CGMCC, city of BeiJing, China Chaoyang District North Star West Road 1 institute 3, grind by Chinese Academy of Sciences microorganism Study carefully institute, postcode：100101), corresponding deposit number is CGMCC No.15347, is named as kluyveromyces marxianus (Kluyveromyces marxianus) YHY013 bacterial strain).

It should be understood that although carrying out particularly shown and description to the present invention with reference to its illustrative embodiment, It should be understood by those skilled in the art that without departing substantially from spirit of the invention as defined in appended claims Under conditions of range, any of various embodiments can be carried out in the variation for wherein carrying out various forms and details Combination.

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Claims (10)

1. one kind can utilize the heat-resisting works yeast strain of glucose and xylose production xylitol, deposit number CGMCC altogether No.15347。

2. heat-resisting works yeast strain described in claim 1 can directly be sent out using xylose mother liquid or Corncob hydrolysate Ferment generates xylitol.

3. a kind of method for producing xylitol, the method includes wrapping the strain inoculated that deposit number is CGMCC No.15347 Fermented and cultured is carried out in culture medium containing glucose and xylose, is extracted from fermentation liquid and is obtained xylitol.

4. fermentation temperature is 42 DEG C according to the method described in claim 3, wherein the fermentation carries out in the fermenter.

5. preceding 12 hours mixing speeds are 300rpm according to the method described in claim 4, wherein during the fermentation, it Mixing speed control afterwards is 350rpm, and oxygen-supply quantity is controlled always in 0.5vvm.

6. according to the method described in claim 3, wherein the culture medium is using bean cake powder and corn pulp mixture as nitrogen source, preferably Ground, the two content all control as 10g/L.

7. according to the method described in claim 3, wherein the inoculum concentration of the bacterial strain is initial OD₆₀₀It is 1.

8. according to the method described in claim 3, wherein in the culture medium comprising glucose and xylose Starting glucose and The weight concentration ratio of xylose is 1: 4.

9. according to the method described in claim 3, wherein the culture medium is Corncob hydrolysate.

10. according to the method described in claim 3, wherein the culture medium is what Corncob hydrolysate obtained after extracting xylose Xylose mother liquid, feed supplement xylose mother liquid is two or more times in fermentation process.