CN106967159A - Application of the iolT1 and iolT2 albumen in xylose transport - Google Patents

Application of the iolT1 and iolT2 albumen in xylose transport Download PDF

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CN106967159A
CN106967159A CN201710149680.7A CN201710149680A CN106967159A CN 106967159 A CN106967159 A CN 106967159A CN 201710149680 A CN201710149680 A CN 201710149680A CN 106967159 A CN106967159 A CN 106967159A
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xylose
glucose
iolt2
transport
iolt1
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CN106967159B (en
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李燕军
陈宁
韩洪军
袁启发
高立栋
张顺棠
赵岩
黄龙辉
王婷
谢希贤
张成林
徐庆阳
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Lotus Health Industry Group Ltd By Share Ltd
Tianjin University of Science and Technology
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Tianjin University of Science and Technology
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    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • C12Y503/01005—Xylose isomerase (5.3.1.5)

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Abstract

The invention belongs to gene engineering technology field, and in particular to the patent application of the new opplication of inositol transport protein iolT1 and iolT2 in xylose transport.In corynebacterium glutamicum, inositol transport protein iolT1 and iolT2 can transport flesh alcohol and glucose, while having the ability of transhipment xylose；The turn-over capacity and the PTS system in corynebacterium glutamicum（PtsG）Xylose transport ability quite, and the transhipment of xylose is not suppressed with complementary effect, and its transhipment to xylose by glucose transport with PTS system.The discovery is beneficial to the structure of corynebacterium glutamicum engineered strain efficiently using xylose, is particularly advantageous for building the synchronous engineered strain using glucose and xylose.This will efficiently be had laid a good foundation for exploitation using the engineering strain of the raw materials such as ligno-cellulose hydrolysate, with preferable exploitation prospect.

Description

Application of the iolT1 and iolT2 albumen in xylose transport
Technical field
The invention belongs to gene engineering technology field, and in particular to inositol transport protein IolT1 and IolT2 are in xylose transport In new opplication patent application.
Background technology
Miscellaneous lignocellulosic sources annual production is huge, and including a large amount of agricultural industrial wood wastes, it is integrated Using can both bring economic benefit, the environmental pollution caused by the improper processing such as burning can be reduced again.It is former in lignocellulosic Xylose constituent content is up to 18 ~ 30% in material, is only second to glucose component, therefore carries out comprehensive utilization to it that original can be significantly improved Expect utilization rate.
It is existing for one of lignocellulosic material Land use systems, be using microbial fermentation technology to xylose therein Or glucose component carries out fermenting and producing.Its main technical principle is：In bacterium, xylose passes through xylose isomerase （Xylose isomerase, XI）Catalysis forms xylulose, then in Xylulokinase（Xylulokinase, XK）In the presence of Xylulose -5- phosphoric acid is formed, into pentose phosphate pathway.
Corynebacterium glutamicum is the production bacterial strain of one kind of multiple amino acid, its have strong robustness, be metabolized it is vigorous and The characteristics such as food security.Have in microbial fermentation production and have been widely used, but lignocellulosic material is utilized, paddy Propylhomoserin corynebacteria itself can not directly utilize xylose, but due to there is XK encoding genes on its genome, thus pass through base Because of engineering means, XI is overexpressed in corynebacterium glutamicum, so that it is wooden ketone to assign corynebacterium glutamicum by xylose Sugar ability, and then by relevant metabolic pathway and enzyme Optimal Expression, acquisition can by xylose be various targets production The cell factory of product.
Corynebacterium glutamicum fermentation is using during xylose, and transport of the xylose into C. glutamicum cells is Its first step being utilized, thus high level expression saccharide transporter can promote absorption of the corynebacterium glutamicum to xylose. But there are some researches show corynebacterium glutamicum does not have special xylose transport albumen, thus speculates its absorption master to xylose To depend on hexose transport albumen.
Have been reported proposition：The ability that the PTS system of glucose has transhipment xylose is transported in corynebacterium glutamicum, and When PTS system has glucose and xylose simultaneously in the environment, the transhipment of xylose substantially can be by the competing of glucose transport Striving property suppresses（Wang etc., Investigation ofptsG gene in response to xylose utilization in Corynebacterium glutamicum, 2014, Journal of Industrial Microbiology and Biotechnology）.Therefore to realize corynebacterium glutamicum simultaneously using two kinds of carbon sources of glucose and xylose, it is necessary to find Other can transport xylose and not by the albumen of grape Glyco inhabiting.For a long time, researcher speculates and thought：It is bar-shaped in glutamic acid In bacillus, the transport of xylose is sugar/proton symporter albumen by MFS families to complete.However, the party so far The research in face, which does not have, to make substantial progress.
In other relevant researchs, Japanese Scientists Ikeda etc.（Identification and application of a different glucose uptake system that functions as an alternative to the phosphotransferase system in Corynebacterium glutamicum, 2011, Applied Microbiology and Biotechnology）Find first：Corynebacterium glutamicum mysoinositol transport protein IolT1 and IolT2 possesses glucose transport function, but its expression needs frameshift mutation inactivation aporepressor IolR encoding gene.Then, Germany scientist Lindner etc.（Phosphotransferase system-independent glucose utilization in Corynebacterium glutamicumBy inositol permeases and glucokinases, 2011, Applied and Environmental Microbiology）Also similar result is reported.But transport egg on inositol Whether white IolT1 and IolT2 has xylose transport ability, and whether can improve corynebacterium glutamicum for lignocellulosic The utilization ratio of raw material, still lacks further application report.
The content of the invention
The present invention is knocked out by specific gene in corynebacterium glutamicum and PTS Xi System deactivated strains are built, it is therefore intended that The relation between inositol transport protein IolT1 and IolT2 and xylose transport is verified, so as to be the exploitation profit of lignocellulosic material With establishing theoretical and application foundation.
Details are as follows for the technical scheme of the application.
Application of the IolT1 and IolT2 albumen in xylose transport, in corynebacterium glutamicum, inositol transport protein IolT1 and IolT2, can transport flesh alcohol and glucose, while having the ability of transhipment xylose；The turn-over capacity and glutamic acid rod PTS system in shape bacillus（PtsG）Xylose transport ability quite, and with PTS system to the transhipment of xylose with complementary work With, and its transhipment to xylose do not suppressed by glucose transport.
A kind of synchronous microbial fermentation processes using xylose and glucose, specifically include following steps：
（1）It is basic by bacterial strain of common corynebacterium glutamicum, in its genomeiolRGene is knocked out, and obtains paddy ammonia The gene knock-out bacterial strain Δ of sour corynebacteriaiolR；
The corynebacterium glutamicum, specifically for exampleC. glutamicumThe bacterial strains of ATCC 13032；
（2）To step（1）Middle gene knock-out bacterial strain, imports and is overexpressed recombinant plasmid pXMJ19-xylATo be overexpressed xylose isomerase Enzyme XI, so that gene knock-out bacterial strain has the ability using xylose；
（3）Microbial fermentation is carried out using the fermentation medium containing xylose and glucose, you can made full use of wooden in culture medium Sugared substrate, so as to realize the exploitation to lignocellulosic material.
On the basis of early-stage Study, compared by protein sequence, inventor has found, IolT1/IolT2 and Escherichia coli xylose Transport protein XylE has compared with high homology.Therefore, whether there is xylose transport capacity to the two albumen, inventor is carried out Further experimental study.
In the application, by ΔptsG（PTS system is inactivated）、ΔptsGΔiolR（PTS system inactivate withiolRGene Knock out）And ΔptsGΔiolRΔiolT1ΔiolT2（PTS system inactivation,iolRGene withiolT1WithiolT2Gene is simultaneously Knock out）Deng bacterial strain system constructing, the relation between inositol transport protein IolT1 and IolT2 and xylose transport is carried out fully to grind Study carefully.Result of study shows, in corynebacterium glutamicum, IolT1 and IolT2 albumen has the ability of transhipment xylose, Neng Goushi The synchronous transhipment and metabolism of existing glucose and xylose, and PtsG is required to low concentration xylose transport.
In general, the application is demonstrated by technique for gene engineering means：In corynebacterium glutamicum, inositol transhipment egg White IolT1 and IolT2 has the function of transhipment xylose, and the discovery is beneficial to corynebacterium glutamicum work efficiently using xylose The structure of journey bacterial strain, is particularly advantageous for building the synchronous engineered strain using glucose and xylose.In terms of concrete operations, base is utilized Because of engineering technology, the PtsG of corynebacterium glutamicum is lacked by knocking out, and utilizes the two albumen（IolT1 and IolT2）Together When transport glucose and xylose, the synchronous utilization of xylose and glucose can be achieved.This efficiently will utilize lignocellulosic for exploitation The engineering strain of the raw materials such as hydrolyzate is had laid a good foundation, with preferable exploitation prospect.
Brief description of the drawings
Fig. 1 is that different genes lack corynebacterium glutamicum bacterial strain in minimal medium CGXII（Xylose is sole carbon source） In growth curve；
Fig. 2 is 13032（Scheme A）And ΔptsGΔiolR（Scheme B）Bacterial strain is in minimal medium CGXII（Xylose and glucose is Carbon source）In glucose and xylose growth and the sugared curve of consumption, in figure：▲ OD values are represented, ■ represents concentration of glucose, ● represent wood Sugared concentration.
Embodiment
Explanation is further explained to the application with reference to embodiment, before specific embodiment is introduced, with regard to following realities Part biological material context in example is applied to be briefly discussed below.
Biomaterial：
Corynebacterium glutamicum（C. glutamicum）ATCC 13032, from American Type Culture Collection center （American Type Culture Collection, ATCC）；
Primer sequence is synthesized and related examining order, is completed to provide by Jin Weizhi companies.
Embodiment 1
It is mostly important operating procedure in the present invention because related strain is built, thus the present embodiment is with regard to corynebacterium glutamicum （C. glutamicum）13032/pXT01-xylABacterial strain（Hereinafter referred to as PTSG/pXT01-xylA, normal strains, which are converted, to be had pXT01-xylAPlasmid）、ΔptsG/pXT01-xylABacterial strain（PTSG systems are inactivated, and conversion has pXT01-xylAPlasmid）、ΔptsGΔiolR/pXT01-xylABacterial strain（PTSG systems are inactivated,iolRGene knockout, and conversion has pXT01-xylAPlasmid）、 ΔptsGΔiolRΔiolT1ΔiolT2/pXT01-xylABacterial strain（PTSG systems are inactivated,iolRGene knockout,iolT1WithiolT2Gene is also knocked, and conversion has pXT01-xylA plasmids）Building process be briefly discussed below.
First,ptsG、iolR、iolT1WithiolT2The knockout of gene
（1）C. glutamicumThe extraction of 13032 genomic DNAs,
Single bacterium colony is inoculated in into LB to shake in pipe, after 32 DEG C of h of incubated overnight 12,30 mL LB culture mediums are accessed by 2% inoculum concentration Middle progress Shaking culture is stayed overnight；CTAB methods are extracted, and specific extraction process refers to following operating process：
A, by the rpm of bacterium solution 13000 centrifuge 2 min, remove supernatant, be collected into 1.5 mL EP pipes；
Plus 600 μ L pH=8.0 TE buffer suspension thallines, 13000 rpm centrifuge 2 min, abandon supernatant；
B plus 600 μ L TE suspension thallines again, add 6 μ L RNAase（10 μ g/mL), 30 μ L lysozyme（50 μ g/mL）Mix, 37 DEG C of 30 min of insulation；
Plus 30 μ L 10% SDS and 10 μ L Proteinase K, mix, 65 DEG C insulation 15 min；
After the 5 mol/L NaCl solutions mixing for adding 100 μ L, 80 μ L CTAB/NaCl solution are added, overturns and mixes 50 It is secondary, 65 DEG C of min of water-bath 10；
C, the phenol for adding 750 μ L：Chloroform：Isoamyl alcohol（25：24：1）Extracting, overturns and mixes 50 times, 13000 rpm centrifugations 15 min；
Supernatant is sucked in 1.5 new mL EP pipes, add isometric phenol：Chloroform：Isoamyl alcohol（25：24：1）Extracting, top Mix 50 times, 13000 rpm centrifuge 15 min；
In the EP pipes that supernatant is sucked to 1.5 new mL, plus 1 times of volume isopropanol, overturn and mix, -80 DEG C of min of cold shock 15, 13000 rpm centrifuge 10 min, abandon supernatant；
D, 70% ethanol of addition, flick resuspension, stand 6 min, and 13000 rpm centrifuge 5 min, remove supernatant；
Oven drying（37℃）Afterwards, 50 μ L deionized waters are added, dissolving DNA surveys DNA concentration, -20 DEG C save backup.
（2）Gene in the amplification of target gene, the corynebacterium glutamicum ATCC 13032 announced according to GenBankptsG （Gene ID：1021001）、iolR（Gene ID：3342760）、iolT1（Gene ID：3342760）WithiolT2Sequence （Gene ID：1021001）, and their upstream and downstream sequence, design specific primer（5’-3’）It is as shown in the table：
。
With step（1）It is middle extractedC. glutamicum13032 genomic DNAs are template, withptsGUpstream is homologous Arm sense primer（iolT1-up-S）And anti-sense primer（iolT1-up-A）For primer, enter performing PCR amplification, obtainptsGUpstream The bp fragments of homology arm about 500, while withptsGDownstream homology arm sense primer（iolT1-down-S）And anti-sense primer（ptsG- down-A）For primer, PCR amplifications are obtainedptsGThe bp fragments of downstream homology arm about 500；
PCR reaction conditions are：95 DEG C of min of pre-degeneration 5；98 DEG C of denaturation 10 s, 55 DEG C of 5 s of annealing, 72 DEG C extend 30 s, 30 Circulation；72 DEG C of 10 min of last extension（High-fidelity Prime STAR HS polymerases）.
Reaction takes 3 μ L amplified productions after terminating, carry out 1% agarose gel electrophoresis detection amplified fragments size.
Reclaim（Using Cycle Pure Kit, OMEGA）Pcr amplification product, the upstream and downstream homology arm using recovery is template （Addition is mol ratio 1：1）, while withptsG- up-S andptsG- down-A is primer, carries out over-lap PCR, expands upstream and downstream The overlapping fragmentses of homology arm.
iolT2、iolRWithptsGUpstream and downstream homology arm overlapping fragmentses amplification method ibid.
（3）Reclaim PCR primer, and with carrier pK18 mobsacBIt is attached, to step（2）In pcr amplification product Reclaimed, and carry out double digestion, while to pK18 mobsacBCarrier carries out corresponding double digestion, reclaims digestion products, and It is attached；
During double digestion：
iolT1Upstream and downstream overlapping fragmentses double enzyme site is：HindIII andSphI；
iolT2Upstream and downstream overlapping fragmentses double enzyme site is：EcoRI andSphI；
iolRUpstream and downstream overlapping fragmentses double enzyme site is：SphI andPstI；
ptsGUpstream and downstream overlapping fragmentses double enzyme site is：XbaI andEcoRI；
50 μ L double digestion system Reference Design is as follows：
Plasmid（pK18 mobsacBCarrier）Or purpose fragment（Pcr amplification product）, 3000 ~ 5000 ng；
Q.Cut 1（Restriction enzyme site enzyme）, 1.5 μ L；
Q.Cut 2（Restriction enzyme site enzyme）, 1.5 μ L；
10 × Q.Cut Buffer, 5 μ L；
Deionized water, adds to 50 μ L.
After digestion products are reclaimed, the design of 10 μ L linked systems is as follows：Purpose fragment（PCR primer fragment after digestion）With load Body（Carrier segments after digestion）Mol ratio is 3 ~ 9：Between 1, the μ L of Solution I ligases 5, remaining uses deionized water polishing； 16 DEG C of 4 h of connection.
（4）Conversion and identification recombinant plasmid, utilize CaCl2Method, by step（3）In recombinant plasmid（Connection product）Conversion Enter bacillus coli DH 5 alpha competent cell；Then LB solid mediums are coated with, positive transformant is selected and shake after pipe culture, adopt With alkaline lysis, DNA is extracted, the PCR identifications and digestion identification of recombinant plasmid is carried out.
It will identify that correct plasmid is sent into row sequencing, sequencing result sequence in DNAMAN softwares and GenBank of each fragment Row are compared, to verify geneptsG、iolR、iolT1WithiolT2The correctness that overlapping fragmentses are built, that is, recombinant plasmid Build correct.
（5）Corynebacterium glutamicum competent cell is prepared, following operating procedure is specifically referred to：
A, from -80 DEG C of glycerine protect tube in use transfer needle picking Pidolidone corynebacteria, three zonings on LB plating mediums Overnight incubation in line, 32 DEG C of incubators；
B, the picking single bacterium colony on step streak plate culture medium 1., are inoculated into 5 mL BHIS culture medium test tubes, 30 DEG C, 200 r/min cultivate 12 h；
The BHIS culture mediums that liquid amount is 100 mL are transferred to by 1% inoculum concentration（Containing the 150 sweet ammonia of μ L Tween 80 and 2.5 g Acid）500 mL round-bottomed bottles in, 18 DEG C, 160 rpm cultivate 18 h, determine OD600Value；
BHIS culture mediums（g/L）：Brain heart extract（Brain-Heart Infusion）37, sorbierite（Sorbitol）91, pH 7.0 ~ 7.2,115 DEG C of min of high pressure steam sterilization 15 of culture medium；
C, by OD in step B600=0.6 or so nutrient solution stands 20 min on ice；
Then in super-clean bench, in the centrifuge tube that bacterium solution is collected into 50 mL, 4 DEG C, 6500 rpm, 10 min of centrifugation are abandoned Clearly；
D, be resuspended with 10% glycerine of 50 mL precoolings and wash bacterium, 4 DEG C, 6500 rpm centrifuge 10 min, abandon supernatant, repeat this step 3 It is secondary（That is, repeat to wash three times）；
Thalline is resuspended with 10% glycerine of 2 mL precoolings, managed with the EP of precooling, the packing of 100 μ L/ pipes, -80 DEG C save backup.
（6）Electricity conversion, by step（4）The middle electric step of converting of the correct recombinant plasmid of sequencing identification（5）In prepared competence Cell, specific step of converting is with reference to as follows：
A, in superclean bench, with absolute ethyl alcohol by electric revolving cup purge it is clean after, be inverted on clean filter paper, about After 20 min, the min of about precooling 20 is placed on ice；
B, the corynebacterium glutamicum competent cell preserved from -80 DEG C of refrigerators taking-ups（Step（5）In it is prepared）, it is placed on ice Melt 5 min or so；
By the recombinant plasmid successfully constructed（Step（4）In it is prepared）Competent cell is added, is placed in step A's after flicking mixing After precooling in electric revolving cup；Place min of precooling 20 or so in ice；
C, electric revolving cup both sides are dried with filter paper, be put into electroporation rapidly, 1 mL preheatings are rapidly added after being shocked by electricity under the conditions of 2500V BHIS resuscitation fluids, gently blow and beat it is uniform after, be transferred in centrifuge tube, be placed in the min of thermal shock 6 in 46 DEG C of water-baths；
After thermal shock terminates, then 32 DEG C, 200 r/min about 2 h of recovery carry out further bacterial strain screening and culture.
（7）Screening and the corynebacterium glutamicum recombinant bacterial strain of identification gene knockout, to step（6）After middle electricity conversion recovery Thalline, bacterial strain screening is carried out with reference to following incubation step：
After A, recovery are completed, 8000 rpm/min centrifuge 2 min to be enriched with thalline, and the coating of be enriched with thalline is mould containing that is blocked The LB resistant panels of element（Kmr, 10 μ g/mL）, 32 DEG C of 48 h of culture；
By the single bacterium colony grown on flat board respectively to selecting the LB flat boards of the sucrose of mass content 15% and LB resistances containing kanamycins Flat board（Kmr, 10 μ g/mL）On, 32 DEG C of 18 h of culture；
B, pick out and grown in LB resistant panels, but the bacterium colony not grown in 15% sucrose LB flat boards, transfer LB culture mediums, 32 DEG C, 200 rpm/min cultivate 18 h, extract bacterium colony genome, using Kana-N and Kana-C as primer（Primer sequence such as SEQ Shown in ID NO.1, SEQ ID NO.2, it is specially：Kana-N:AAGGGACTGGCTGCTATTGGG, Kana-C: GCCCATTCGCCGCCAAGCTC；The bp of amplification length 447）, the PCR detections of Kana resistant genes are carried out, checking occurs for the first time The recon of restructuring, to verifying that correct recon protects bacterium.
（C, it will verify that correct recombinant bacterium is passed on twice in pipe in shaking containing 15% sucrose in step B（Due in 15% sugarcane In the case of sugar is present,sacBThe expression of gene has lethal effect to Host Strains, therefore it is homologous to can be used to screen second of generation The bacterial strain of restructuring）；
D, by thalline after Secondary Culture in step C, on the flat board that 15% sucrose is applied to after dilution, 32 DEG C culture 36h, will grow Single bacterium colony respectively to o'clock to 15% sucrose plate and the LB resistant panels containing kanamycins（Kmr, 10 μ g/mL）On, 32 DEG C of trainings 18 h are supported, are filtered out in the single bacterium colony that 15% sucrose plate grows but is not grown on the LB flat boards containing kanamycins；
The correct single bacterium colony of phenotype is transferred into LB to shake in pipe, 32 DEG C, 200 rpm/min incubated overnights；
Bacterium colony genome after incubated overnight is extracted, using genomic DNA as template, performing PCR is entered with the homologous recombination primer of design and expanded Increase（Primer sequence refers to abovementioned steps（2））, in PCR amplifications, after 72 DEG C of last extensions, add 0.5 μ L Ex Taq enzymes to add Poly A tails.
Pcr amplification product is reclaimed, and reclaimed DNA fragmentation is connected on pMD18-T-simple carriers, 10 μ L company Junctor system Reference Design is as follows：
The pcr amplification product reclaimed, 4.5 μ L；
PMD18-T-simple carriers, 0.5 μ L；
Solution I, 5 μ L；
16 DEG C of connections are stayed overnight.
Using CaCl2Method, bacillus coli DH 5 alpha competent cell is transformed into by connection product；Using alkaline lysis, extract DNA, enters performing PCR identification and digestion identification；It will identify that correct plasmid carries out sequencing analysis, and determine that each gene will successfully strike Remove, i.e. gene deletion strains build correct.
By screening, identification, final build obtains ΔptsGBacterial strain（PTSG systems are inactivated）、ΔptsGΔiolRBacterial strain （PTSG systems are inactivated,iolRGene knockout）、ΔptsGΔiolRΔiolT1ΔiolT2Bacterial strain（PTSG systems are inactivated,iolR Gene knockout,iolT1WithiolT2Gene is also knocked）.
2nd, xylose isomerase expression vector pXT01- is builtxylA, with reference to prior art, build xylose isomerase expression of enzymes and carry Body pXT01-xylA。
3rd, electricity conversion, by gene deletion strains constructed in step 1（ΔptsGBacterial strain, ΔptsGΔiolRBacterial strain, ΔptsGΔiolRΔiolT1ΔiolT2Bacterial strain）, andC. glutamicumIt is thin that 13032 bacterial strains prepare competence respectively Born of the same parents, using electrotransformation, by xylose isomerase expression vector pXT01- constructed in step 2xylAAbove-mentioned impression is converted respectively State cell, it is final to obtain the different genes knockout recombinant bacterial strain that xylose isomerase is overexpressed by screening, that is, obtain：
Corynebacterium glutamicum（C. glutamicum）13032/pXT01-xylABacterial strain（Hereinafter referred to as PTSG/pXT01-xylA, normal strains, which are converted, pXT01-xylAPlasmid）,
ΔptsG/pXT01-xylABacterial strain（PTSG systems are inactivated, and conversion has pXT01-xylAPlasmid）,
ΔptsGΔiolR/ pXT01-xylABacterial strain（PTSG systems are inactivated,iolRGene knockout, and conversion has pXT01-xylA Plasmid）,
ΔptsGΔiolRΔiolT1ΔiolT2/pXT01-xylABacterial strain（PTSG systems are inactivated,iolRGene knockout,iolT1 WithiolT2Gene is also knocked, and conversion has pXT01-xylAPlasmid）.
It is to be understood that experimental implementation is not described in detail in aforesaid operations with reference to the relevant step of other in prior art or embodiment Rapid description can be carried out, text brief period, no longer excessively repeated description.
Embodiment 2
On the basis of the recombinant bacterial strain constructed by embodiment 1, inventor is wooden to different corynebacterium glutamicum recombinant bacterial strains The growth utilization power of sugar and glucose is tested and analyzed, and related experiment process is briefly discussed below.
Substitutions of the protein I olT1 and IolT2 to PTS system is tested
Study and thought：Ability of the PTS system with transhipment xylose of transhipment glucose in corynebacterium glutamicum, and PTS In the environment simultaneously when there is glucose and xylose in system, the transhipment of xylose substantially can by glucose transport competitiveness Suppress.Therefore, inventor is with Δ constructed in embodiment 1ptsGΔiolR/pXT01-xylABacterial strain（ptsGGene knockout, Therefore PTS system is lacked,iolRGene knockout, relieve to iolT1 and iolT2 genes suppress so that IolT1 and IolT2 albumen is expressed）As experimental strain, with normal（C. glutamicum）13032/pXT01-xylABacterial strain is made For control, when having investigated with IolT1 and IolT2 albumen substitution PTS system, IolT1 and IolT2 albumen is to xylose and glucose Turn-over capacity（Appreciation gist is used as using the sugared situation of the consumption of bacterial strain）.Specific incubation is described below.
（1）Actication of culture
Constructed bacterial strain in embodiment 1 is subjected to activation culture, to recover bacterial strain vigor, during activation culture, cultivation temperature is 32 DEG C, culture medium refers to use activation medium, and specific formula is：
Activation medium（g/L）：Peptone 10, beef extract 10, dusty yeast 5, NaCl 2.5, the mL/L of corn steep liquor 15, agar 25, PH 7.0 ~ 7.2, sterilising conditions：121℃、20 min.
（2）Seed culture
By step（1）After middle activation in bacterial strain access seed culture medium, 32 DEG C, the h of 200 rpm incubation times 12 or so, culture is extremely OD600=10 ± 2 or so；Seed culture medium Reference Design is as follows：
Seed culture medium（g/L）：Glucose 10, brain heart infusion 37, pH 7.0 ~ 7.2, sterilising conditions：121℃、20 min.
（3）Shake flask fermentation culture
By the inoculum concentration of 4% volume ratio, by step（2）In seed culture fluid inoculation fermentation culture medium, 32 DEG C, 200 rpm conditions It is lower to be cultivated；Fermentation medium design is as follows：
Fermentation medium（CGXII）（g/L）：Xylose 20（Or xylose 12, glucose 12）, (NH4)2SO420, K2HPO41, KH2PO41, MgSO40.25, urea 5, the M of MOPS 42,0.1 CaCl21 mL, 0.2 g/L biotin 200 μ L, pH 7.0 ~ 7.2, sterilising conditions：121 DEG C, 20 min.
Experimental result：
Experimental result is included to xylose, two aspects of glucose utilization in the velocity determination of different strains Fermentative growth and zymotic fluid Measure situation, be specifically described as follows.
The measure of cell concentration
In fermentation process, timing sampling, after bacteria liquid sample is diluted, at 600 nm（V1200 ultraviolet specrophotometers）Determine sample Product absorbance, to have judged thalli growth speed in fermentation process.
The OD of strain growth process by sole carbon source of xylose600Growth curve is as shown in figure 1, in Fig. 1, ATCC 13032 be starting strain；ΔptsGForptsGKnock-out bacterial strain；ΔptsGΔiolRTo knock out simultaneouslyptsGAnd repressor protein geneiolRBacterial strain；ΔptsGΔiolRΔiolT1ΔiolT2For 4 gene knock-out bacterial strains.
It will be seen from figure 1 that knocking out after PTS system, the OD values of recombinant bacterial strain have dropped 80% or so, show that PTS system is gone After removing, it is impossible to which effectively using sugar in culture medium, thus growth result there occurs obvious reduction；This result is to specialize in Transhipments of the IolT1/IolT2 of MFS families to xylose is made to be used as best reference pair photograph.
Glucose, the measure of xylose consumption in zymotic fluid
In fermentation process, the rpm of bacteria liquid sample 12000 is centrifuged 5 min by timing sampling, takes supernatant to dilute after 10 times, mistake 0.22 μm of film, to sugared in filtrate（Xylose, glucose）Determined using HPLC, during measure：
It is measured using the high performance liquid chromatographs of Agilent 1200,
Chromatographic column：Aminex HPX-87H（300 × 7.8 mm, Bio-Rad）；
Detector：Composition distribution；
Mobile phase：5 mM sulfuric acid；
The mL/min of flow velocity 0.6.
Glucose and xylose consumption curve is drawn according to HPLC testing results, drawing result is as shown in Figure 2（Incubation In, contain same concentrations in culture medium simultaneously（It is 12 g/L）Xylose and glucose, i.e., simultaneously using xylose and glucose as Carbon source）.
From Fig. 2 (A),C. glutamicumDuring 13032 10 h of fermentation, glucose consumption to 1 g/L or so, xylose Just start quickly to be consumed, be exhausted to xylose during 42 h.As can be seen here, original starting strain bacterial strain is preferentially to utilize grape Sugar.And from Fig. 2 (B), ΔptsGΔiolRBacterial strain possesses the ability of synchronous fermenting xylose and glucose, but cultivates 40 H to remain about 3.6 g/L xyloses all the time in wild Oryza species, it is seen that PtsG is required to low concentration xylose transport.
With reference to Fig. 1, Fig. 2, analysis as can be seen that knocking outptsGOn the basis of again pass through knock outiolRActivationiolT1WithiolT2Expression after, the final OD that bacterial strain grows on xylose minimal medium adds 2.5 times.This explanation IolT1 and IolT2 inositols transport protein has the ability of transhipment xylose.
Further, with reference to above-mentioned incubation, inventor is to ΔptsGΔiolRΔiolT1ΔiolT2Recombinant bacterial strain Cultivated, its final OD is with only knocking outptsGAnd un-activationiolT1WithiolT2The bacterial strain of expression is suitable（OD600Growth is bent Line is as shown in Figure 1）.
To sum up result can be seen that：In corynebacterium glutamicum, IolT1 and IolT2 albumen has the energy of transhipment xylose Power.Inactivate after aporepressor IolR, IolT1 and IolT2 albumen is expressed, turn-over capacity and paddy of the two albumen to xylose PTS system in propylhomoserin corynebacteria（PtsG）Xylose transport ability be suitable, when PTS system is inactivated, alternative PTS Performance of the system to the transport function of xylose., can be in lignocellulosic material when being fermented using recombinant bacterial strain based on this characteristic Xylose, synchronous utilize of dextrose components have established necessary application foundation.
SEQUENCE LISTING
<110>Lotus flower health industry Group Plc
University Of Science and Technology Of Tianjin
<120>Application of the iolT1 and iolT2 albumen in xylose transport
<130> none
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Engineer
<400> 1
aagggactgg ctgctattgg g 21
<210> 2
<211> 20
<212> DNA
<213>Engineer
<400> 2
gcccattcgc cgccaagctc 20

Claims (3)

  1. Application of the 1.IolT1 and IolT2 albumen in xylose transport, it is characterised in that in corynebacterium glutamicum, inositol turns Protein I olT1 and IolT2 are transported, the ability with transhipment xylose；The turn-over capacity is to the transhipment of xylose not by glucose transport Suppress.
  2. 2. a kind of synchronous microbial fermentation processes using xylose and glucose, it is characterised in that this method specifically includes as follows Step：
    （1）It is basic by bacterial strain of common corynebacterium glutamicum, in its genomeiolRGene is knocked out, and obtains paddy ammonia The gene knock-out bacterial strain Δ of sour corynebacteriaiolR；
    （2）To step（1）Middle gene knock-out bacterial strain, imports and is overexpressed recombinant plasmid pXMJ19-xylATo be overexpressed xylose isomerase Enzyme XI；
    Correct inoculation will be built to be cultivated in using xylose and glucose as the culture medium of carbon source.
  3. 3. the microbial fermentation processes of xylose and glucose are synchronously utilized as claimed in claim 2, it is characterised in that step（1） Described in corynebacterium glutamicum, be speciallyC. glutamicumThe bacterial strains of ATCC 13032.
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CN112585263A (en) * 2018-07-11 2021-03-30 于利奇研究中心有限公司 D-xylose-dehydrogenase from coryneform bacteria and method for producing D-xylonic acid
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CN112779201A (en) * 2021-01-14 2021-05-11 宜昌东阳光生化制药有限公司 Recombinant microorganism, application thereof and method for preparing shikimic acid and oseltamivir
CN112779201B (en) * 2021-01-14 2023-05-09 宜昌东阳光生化制药有限公司 Recombinant microorganism and application thereof, and method for preparing shikimic acid and oseltamivir
CN114107149A (en) * 2021-11-12 2022-03-01 清华大学 Method for high-yield production of hyaluronic acid, recombinant bacterium construction and application thereof
CN114107149B (en) * 2021-11-12 2023-11-28 清华大学 Method for high-yield hyaluronic acid, recombinant bacterium construction and application thereof
WO2023142853A1 (en) * 2022-01-29 2023-08-03 廊坊梅花生物技术开发有限公司 Method for constructing genetically engineered bacterium for highly producing threonine

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