CN103966217B - A kind of Gluconobacter oxvdans gradient intensity promotor - Google Patents
A kind of Gluconobacter oxvdans gradient intensity promotor Download PDFInfo
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Abstract
The invention discloses a kind of Gluconobacter oxvdans gradient intensity promotor, belong to gene and metabolic engineering field.Does the present invention select Gluconobacter? constitutive expression gene and a upper intergenic sequence in oxydans, promoter prediction software is used to carry out preliminary screening, then the potential strong promoter of the height scoring obtained is strengthened green fluorescent protein (EGFP) as reporter gene, does quantitative and qualitative analysis respectively by fluorescent microscope and fluorescence microplate reader detect, finally from G.oxydans? picking out intensity sequence in WSH-003 is: P
tuf-WSH003aMP.AMp.Amp gt; P
tuf-621HaMP.AMp.Amp gt; P
sldh≈ P
psldh≈ P
sodaMP.AMp.Amp gt; P
aldhpromotor.Does the present invention filter out graded series promotor and a kind of new strong promoter, is industrial producing strain G.oxydans? the gene of WSH-003 and metabolic engineering, particularly in the adjustment of gene transcription level, provide very large help.
Description
Technical field
The present invention relates to a kind of Gluconobacter oxvdans gradient intensity promotor, belong to gene and metabolic engineering field.
Background technology
L-sorbose (L-sorbose) superior strain that Gluconobacter oxvdans (Gluconobacteroxydans) WSH-003 can be substrate with D-glucitol (D-sorbitol), and quite tolerant D-glucitol and L-sorbose, be widely used in industrial ascorbic production.Along with the announcement in succession of the gene order-checking of KetogulonicigeniumvulgareWSH-001 (taking sorbose as the high yield 2-KLG production bacterial strain of substrate) and G.oxydansWSH-003, the integrated use of genetically engineered and information biology etc. makes the structure of vitamins C one step bacterium be promoted greatly.Wherein, by building a step genetic engineering bacterium, the direct production from D-glucitol to 2-KLG can be realized in shake flask fermentation, but output has much room for improvement.Except improve the production performance of a step bacterium from fermentation condition optimization aspect except, also comprise and screen the strong promoter (P in Goxydans621H more conventional than bibliographical information from GoxydansWSH-003
tuf) stronger promotor and graded series promotor be used for metabolic engineering.
The present invention is according to the gene order-checking result of GoxydansWSH-003, in conjunction with genomics, proteomics and the metabonomic analysis of its gene order-checking annotation and high homology bacterial strain Goxydans621H etc., search possible strong constitutive gene promotor and carry out software Preliminary detection by information biology, and then use reporter gene to identify in GoxydansWSH-003, then finally obtain strong promoter and graded series promotor.
The research of the domestic transcriptional control level in Gluconobacter oxvdans promotor is less at present, and mostly concentrate on thalline improvement and mutagenesis, important dehydrogenation zymologic property research on.Therefore, the present invention from the gene expression regulation of mRNA level in-site research thalline, screening controlling element, and then provide strong backing for the metabolic regulation of Gluconobacter oxvdans.
Summary of the invention
First technical problem that the present invention will solve is to provide the strong promoter that a group derives from Gluconobacter oxvdans: P
tuf-WSH003, P
sldh, P
psldh, P
sod, P
aldh.
Described promotor P
tuf-WSH003, P
sldh, P
psldh, P
sod, P
aldhnucleotide sequence respectively as shown in SEQIDNO.1, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5, SEQIDNO.6.
Described strong promoter P
tuf-WSH003, be the P in the strongest Gluconobacteroxydans621H of reported in literature than ever
tuf-621Halso strong strong promoter (Merfort, M., etal., High-yield5-keto-D-gluconicacidformationismediatedbysolu bleandmembrane-boundgluconate-5-dehydrogenasesofGluconob acteroxydans, inApplMicrobiolBiotechnol2006.p.443-51).By promotor method for truncating, determining its core sequence, is the 450-599bp of sequence shown in SEQIDNO.1.
Second technical problem that the present invention will solve is to provide a kind of intensity gradient strong promoter deriving from Gluconobacter oxvdans, by P
tuf-WSH003, P
tuf-621H, P
sldh, P
psldh, P
sod, P
aldhin the different promotor of two or more intensity form.Described promotor P
tuf-WSH003, P
tuf-621H, P
sldh, P
psldh, P
sod, P
aldhnucleotide sequence respectively as shown in SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5, SEQIDNO.6.
It is P that described 6 kinds of promotors constitute intensity gradient
tuf-WSH003>P
tuf-621H>P
sldh≈ P
psldh≈ P
sod>P
aldhintensity gradient promotor.
Described intensity gradient promotor can be used for gene or the metabolic engineering of Gluconobacter oxvdans.
The 3rd technical problem that the present invention will solve is to provide a kind of method of screening gradient promotor and strong promoter in Goxydans.
Said method comprising the steps of:
(1) according to G.oxydans genome sequence, all nucleotide fragments between the ORF selecting G.oxydans central metabolic pathways enzyme gene and a upper gene;
(2) adopt promotor Online Judge software, possible promoter sequence is marked;
(3) by the potential strong promoter of the high score value of acquisition, be connected with EGFP by fusion DNA vaccine, be then connected to wide host's type carrier pBBR1MCS2, being built into restructuring and carrying a pBBR1MCS2-promoter-egfp;
(4) recombinant vectors is imported G.oxydans cell by the method for triparental mating, utilize fluorescent microscope and fluorescence microplate reader to carry out quantitative and qualitative analysis detection, obtain the promotor of varying strength.
The present invention filters out a series of gradient promotor, by the combination between the promotor of varying strength, can regulate and control the expression intensity of different enzyme.For gene and the metabolic engineering of Gluconobacter oxydans sensation, particularly in the adjustment of gene transcription level, provide new thinking.
Accompanying drawing explanation
Fig. 1 is the screening process of the strong promoter in GoxydansWSH-003.
Fig. 2 is the fluorescence microscopy figure of fluoroscopic examination bacterial strain when each autofluorescence maximum value of the promotor of 4 different gradient type powers.
Fig. 3 is the detection by quantitative of the fluorescence microplate reader of the promotor fluoroscopic examination bacterial strain of 4 different gradient type in Fig. 2.
Fig. 4 is the fluorescence intensity of each detection bacterial strain utilizing promoter sequence method for truncating to build.
Embodiment
Selecting of embodiment 1 promotor
Containing the multiple important desaturase relevant with the metabolism such as glucose, glycerine, sorbyl alcohol in Goxydans, these enzymes may have potential strong composition type expression promoter, as the promotor of sorbito dehy drogenase (sorbitoldehydrogenase), membrane-bound aldehyde dehydrogenase (aldehydedehydrogenase), membrane-bound Hexose phosphate dehydrogenase (membrane-boundglucosedehydrogenase) etc.In addition, the P in the strongest Gluconobacteroxydans621H of reported in literature is chosen
tuf-621h in contrast.According to G.oxydansWSH-003 Genomic sequence information (GenBankaccessionNO.AHKI00000000), all nucleotide fragments between the ORF (open reading frame) choosing goal gene and a upper gene, then according to promotor software (
http:// www.softberry.com) score of promoter region evaluated and tested out, select the promotor that score value is higher, use EGFP to identify as reporter gene.
The structure of embodiment 2 expression vector pBBR1MCS2-promter-egfp
According to G.oxydansWSH-003 Genomic sequence information (GenBankaccessionNO.AHKI00000000), design fusion DNA vaccine primer, (egfp gene order comprises terminator for the high promotor nucleotide sequence of marking of amplification acquisition and the gene order strengthening green fluorescent protein egfp respectively, sequence is shown in SEQIDNO.7), then connected by fusion DNA vaccine, obtain the promoter-egfp of two ends with restriction enzyme site, carry out T-A clone and check order, positive colony correct for order-checking is connected the wide host's type carrier pBBR1MCS2 cutting site with same enzyme, be built into expression vector pBBR1MCS2-promter-egfp (Fig. 1).
The restructuring of embodiment 3 fluorescin detects the structure of bacterial strain
By the method for triparental mating, each cloning vector pBBR1MCS2-promoter-egfp correct for sequence verification is imported in G.oxydansWSH-003 (be preserved in Southern Yangtze University's Chinese Universities ' industrial microorganism resource and information center, be numbered CICIM-CUB7004).E.coliJM109 containing recombinant expression vector is as donor bacterium, and the E.coliHB101 containing joint helper plasmid pRK2013 is as auxiliary bacterium.G.oxydansWSH-003 grows into OD
600=0.9, E.coli grows into OD
600=0.8.Get each 1ml mixing of E.coliJM109 and E.coliHB101, centrifugal, with the resuspended thalline of 2mlLB mix with 4mlG.oxydansWSH-003, centrifugal, with 0.8mlY-S substratum (yeast extract 20g/L, sorbyl alcohol 80g/L) resuspended thalline, bacterium liquid is coated on containing filter paper but not containing on antibiotic Y-S solid medium flat board, cultivates 24h for 30 DEG C.By the bacterium that filter paper grows with on the flat board be coated on after sterilized water wash-out containing Ampicillin Trihydrate and cephalo western spit of fland mycin, cultivate 2-4 days for 30 DEG C, picking resistant strain carries out PCR checking.
The fluoroscopic examination of embodiment 4 recombinant bacterial strain
The correct each positive strain G.oxydansWSH-003pBBR1MCS2-promoter-egfp of checking is inoculated in Y-S substratum, get each fluoroscopic examination bacterial strain 1.25ml seed liquor in 250ml shaking flask (kana, 20ug/ml cephalo 50ug/ml) the 30 DEG C cultivation containing 25ml sorbyl alcohol substratum.Get 2ml bacterium liquid respectively at 4h, 16h, 24h, 32h, use PBS to wash 3 times, remove substratum impurity and dead cell.Carry out fluoroscopic examination with NUNC96 hole black enzyme plate, use 96 hole white Tissue Culture Plates to carry out OD
600detect (fluorescence and OD
600detect and all do 4 multiple holes, every hole adds 200ul and detects liquid), instrument is the multi-functional microplate reader of BioTekSynergy4, and function software is Gen5.Fluorescence intensity optimum configurations is, excites: 488/9nm launches: 509/9nm, and top is luminous, and tritium gas lamp, gain is for automatically to adjust gain.OD
600optimum configurations is, absorb light wavelength 600nm, and selects adjustment light path.Finally, fluorescent microscope is used to carry out fluorescence microscopy (Fig. 2).
According to the fluorescent value (Fig. 3) that fluorescence microplate reader detects, and the strong promoter gradient promotor different with series of intensity in combined with fluorescent microscopy figure and then screening G.oxydansWSH-003.
The promotor P of final acquisition transcriptional elongation factor Tu (elongationfactorTu)
tuf, the promotor P of sorbito dehy drogenase (sorbitoldehydrogenase)
sldh, the promotor P of sorbitol dehydrogenase enzyme precursor (sorbitoldehydrogenaseprecursor)
psldh, the promotor P of membrane-bound aldehyde dehydrogenase (aldehydedehydrogenase)
aldh, the promotor P of superoxide-dismutase (superoxidedismutase)
sod, and the P in strong promoter Gluconobacteroxydans621H conventional in document
tuf-621H.The intensity sequence of above-mentioned promotor is: P
tuf-WSH003>P
tuf-621H>P
sldh≈ P
psldh≈ P
sod>P
aldh.
Embodiment 5 strong promoter P
tuf-WSH003core sequence qualification
P
tuf-WSH003promotor place nucleotide sequence total length be 599bp, utilize promotor segmentation brachymemma strategy, PCR obtains the nucleotide fragments of upstream from start codon 100bp, 150bp, 200bp, 250bp, 300bp and 400bp respectively, and with the nucleotide fragments of total length 599bp as positive control.Above each fragment is connected egfp gene, and construction of expression vector pBBR1MCS2-promter-egfp and fluorescin restructuring detect bacterial strain, construction process is identical with embodiment 3 with embodiment 2 respectively.Then, use the method for embodiment 4 to detect the fluorescence intensity of recombinant bacterium, be all unified in the logarithmic growth later stage (the 16th hour) detection time, and be negative control bacterial strain with G.oxydansWSH-003.Qualification result display (see Fig. 4), P
tuf-WSH003promotor just has the activity starting egfp genetic transcription at its upstream from start codon 150bp place, and this and previous promoter prediction software (softberry software is conventional promoter prediction software, it can reach 80% to the accuracy of the promoter prediction of bacterium and specificity) closely (-35th district are present in upstream from start codon 147bp place to acquired results, sequence is CTGAAG,-10th district are present in 127bp place, upstream, and sequence is AGTCACTAT).Therefore, in conjunction with result and the software prediction result of promotor brachymemma, the nucleus of this promotor is at its initiator codon 150bp place (in Seq1 450-599bp).
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (5)
1. deriving from a strong promoter for Gluconobacter oxvdans, it is characterized in that, is the P of nucleotide sequence as SEQIDNO.1
tuf-WSH003.
2. derive from a gradient intensity promotor for Gluconobacter oxvdans, it is characterized in that, by P
tuf-WSH003with P
tuf-621H, P
sldh, P
psldh, P
sod, P
aldhin the different promotor of one or more intensity form; Described promotor P
tuf-WSH003, P
tuf-621H, P
sldh, P
psldh, P
sod, P
aldhnucleotide sequence respectively as shown in SEQIDNO.1, SEQIDNO.2, SEQIDNO.3, SEQIDNO.4, SEQIDNO.5, SEQIDNO.6.
3. gradient intensity promotor according to claim 2, is characterized in that, be made up of described 6 kinds of promotors, promotor intensity gradient is P
tuf-WSH003>P
tuf-621H>P
sldh≈ P
psldh≈ P
sod>P
aldh.
4. strong promoter according to claim 1 or the arbitrary described application of gradient intensity promotor in Gluconobacter oxvdans gene or metabolic engineering of claim 2-3.
5. carry carrier or the genetic engineering bacterium of promotor described in claim 1.
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