CN103923869A - Bacillus subtilis gene engineering bacterial producing Neu5Ac, construction method and application thereof - Google Patents
Bacillus subtilis gene engineering bacterial producing Neu5Ac, construction method and application thereof Download PDFInfo
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Abstract
The present invention discloses Bacillus subtilis gene engineering bacterial producing Neu5Ac, a construction method and an application thereof. According to the present invention, according to the gene engineering bacterial, genes for coding UDP-N-acetyl glucosamine epimerase and Neu5Ac synthetase are introduced into Bacillus subtilis so as to be expressed, and expression of glucosamine 6-phosphate synthetase of the Bacillus subtilis is enhanced to construct the complete metabolic pathway from glucose or glycerol to Neu5Ac in the Bacillus subtilis so as to utilize glucose or glycerol as a substrate to carry out fermentation culture so as to produce Neu5Ac; and the adopted Bacillus subtilis is widely used in the food industry, is safe and harmless, and is suitable for production of Neu5Ac required by food and health products.
Description
Technical field
The present invention relates to a kind of subtilis genetic engineering bacterium and construction process and application of producing N-acetyl-neuraminate, belong to technical field of bioengineering.
Background technology
Sialic acid, claims again Nidus collocaliae acid, is extensively present in natural various organism, and sialic acid is a series of general designations that contain 9 carbon atoms and have the acid aminosugar of pyranose structure, and wherein topmost component is N-acetyl-neuraminate (Neu5Ac).
The current application of N-acetyl-neuraminate mainly contains two fields, and one is as medicine intermediate, for the synthesis of Tamiflu zanamivir; Another is as foodstuff additive, for baby milk powder, so that it approaches breast milk, promotes the neural growth of baby.
As the N-acetyl-neuraminate of medicine intermediate, can use biological catalysis and fermentative Production, wherein biological catalysis is taking 2-Acetamido-2-deoxy-D-glucose and Sodium.alpha.-ketopropionate as raw material, taking the coli somatic of high expression level neuraminic acid zymohexase and 2-Acetamido-2-deoxy-D-glucose epimerase or bacteria breaking liquid as catalyzer, transform and generate N-acetyl-neuraminate.Fermentative Production N-acetyl-neuraminate is that intestinal bacteria are carried out to metabolic engineering, can be from glucose or directly synthetic N-acetyl-neuraminate of glycerine.
As the N-acetyl-neuraminate main or directly extraction from the natural matter (as egg, whey, bird's nest etc.) that contains N-acetyl-neuraminate at present of foodstuff additive, the product that the method is produced is natural origin, safe and reliable, but because content is too low and complicated component causes extraction process complexity, with high costs.And current biotransformation method and fermentation method are all to produce with intestinal bacteria, intestinal bacteria produce owing to having intracellular toxin in fermenting process, the finished product of producing for it may have adverse influence, therefore the N-acetyl-neuraminate that, intestinal bacteria are produced not yet obtains authority's accreditation in the application in foodstuff additive field.
Subtilis and metabolism product thereof are widely used in human food prods and healthcare products, not yet find at present toxic side effect, by (the Food and Drug Administration of FDA (Food and Drug Adminstration), FDA) certification is for it is generally acknowledged safety (Generally Recognized as Safe, GRAS) quasi-microorganism, have advantages of safe and harmlessly, can be used for the production of human food prods's field of health care products.But compare intestinal bacteria, the genetic modification more complicated of subtilis, metabolism background is unintelligible, there is not yet after transformation for the synthetic report of producing of N-acetyl-neuraminate.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of subtilis genetic engineering bacterium and construction process and application of producing N-acetyl-neuraminate.
The present invention selects subtilis as the starting strain of producing N-acetyl-neuraminate, but the pathways metabolism of analyzing subtilis is known, it starts to be synthesized to UDP-N-acetylglucosamine from glucose, but lack the approach that is synthesized to N-acetyl-neuraminate from UDP-N-acetylglucosamine, cannot reach taking glucose as substrate, fermentation method is the object of synthetic N-acetyl-neuraminate directly.
For solving the problems of the technologies described above, the present invention is heterogenous expression coding UDP-N-acetylglucosamine epimerization enzyme coding gene neuC and N-acetyl-neuraminate synthetase-coding gene neuB in subtilis, supply the synthetic required enzyme from UDP-N-acetylglucosamine to N-acetyl-neuraminate, framework plays the complete pathways metabolism from glucose to N-acetyl-neuraminate, thereby realizes the object of utilizing the synthetic N-acetyl-neuraminate of glucose direct fermentation.
For further improving the output of N-acetyl-neuraminate, the glmS gene of high expression level subtilis of the present invention self coding 6-phosphorylated amino glucose synthetic enzyme, get through from glucose or glycerine to the synthetic bottleneck of N-acetyl-neuraminate, increase metabolic flux, thereby increase the output of N-acetyl-neuraminate.
For this reason, the invention provides a kind of N-acetyl-neuraminate subtilis (Bacillus subtilis) genetic engineering bacterium that produces, it is characterized in that described genetic engineering bacterium carries out heterogenous expression and builds and obtain by UDP-N-acetylglucosamine epimerase gene, N-acetyl-neuraminate synthase gene and 6-phosphorylated amino glucose synthase gene are imported in subtilis.
UDP-N-acetylglucosamine epimerase gene neuC of the present invention and N-acetyl-neuraminate synthase gene neuB derive from campylobacter jejuni (Campylobacter jejuni) maybe can express the microorganism of identical function enzyme, the acquisition of gene can be synthetic according to neuB in GenBank No.AF400048 and the full gene of neuC gene order, or the genomic dna that utilizes campylobacter jejuni (as strains A TCC43438) is that template obtains by pcr amplification, or adopted similar means to obtain from other biological body.Because the UDP-N-acetylglucosamine epimerase gene neuC and the N-acetyl-neuraminate synthase gene neuB that derive from campylobacter jejuni are chained together, therefore heterogenous expression is two genes can be expressed together, also two genes separately can be expressed.
Described 6-phosphorylated amino glucose synthase gene derives from subtilis maybe can express the microorganism of identical function enzyme, the acquisition of gene can be synthetic according to the full gene of SD sequence of the glmS gene in subtilis 168 strain gene group sequence GenBank No.AL009126.3 and upstream thereof, or the genomic dna that utilizes Bacillus subtilus 168 bacterial strains is that template pcr amplification obtains, or adopted similar means to obtain from other biological body.
In one embodiment, described UDP-N-acetylglucosamine epimerase gene, N-acetyl-neuraminate synthase gene and 6-phosphorylated amino glucose synthase gene in same carrier together with clonal expression.As the scientific research personnel of this area, utilize conventional molecule clone technology, also three genes can be separated in different carriers to clonal expression or complete in a similar manner heterogenous expression, thereby reach same object.
In one embodiment, described subtilis host is subtilis 164 or subtilis 168, and the genetic engineering bacterium obtaining has been preserved in Chinese Typical Representative culture collection center, address is in Wuhan, China university, preserving number is respectively CCTCC NO:M2013731 and CCTCC NO:M2014038, preserving number is that the classification number of CCTCC NO:M2013731 is: subtilis CASOV-3Bacillus subtilis CASOV-3, the classification number of CCTCC NO:M2014038 is: subtilis CASOV-4Bacillus subtilis CASOV-4.The present embodiment also can be selected other bacillus subtilis strains, reaches similar experiment purpose.
The invention also discloses a kind of application of producing N-acetyl-neuraminate subtilis, utilize above-mentioned engineering bacteria to ferment with glucose or glycerine, produce N-acetyl-neuraminate, described application comprises the following steps:
1) activation culture engineering bacteria seed;
2) cultured seed is amplified step by step, and carry out aerobic cultivation in corresponding fermentor tank; And supplementary suitable carbon nitrogen source;
3) add sec.-propyl-β-D-sulfo-galactopyranoside, the expression of induction associated protein.The present invention transforms subtilis by genetic engineering technique, makes it with glucose or directly synthetic N-acetyl-neuraminate of glycerine, have production technique simple, eco-friendly advantage.The subtilis that the present invention simultaneously adopts is as the host who produces N-acetyl-neuraminate, and the N-acetyl-neuraminate of producing does not have the threat of contaminated with endotoxins, safe and reliable, can be used for health care of food product field, has the value of suitability for industrialized production.
Brief description of the drawings
Below in conjunction with accompanying drawing and embodiment, the present invention is further detailed explanation:
Fig. 1 be in subtilis glucose or glycerine to the synthetic rebuilding approach of N-acetyl-neuraminate.
Embodiment
Plasmid, the PCR reagent etc. using in following examples adopts commerical prod, and concrete operations are carried out to specifications.Other not marked experimental implementation are carried out according to conventional molecule manipulation method.
The structure of embodiment 1:neuBC and glmS gene tandem expression carrier
One, the structure of neuBC expression vector pHT01-neuBC
1) according to the synthetic neuBC gene of the full gene of the gene order of neuBC (GenBank No.AF400048), two ends add BamHI and XbaI site, concrete sequence (SEQ ID NO.1) as follows.
GGATCCATGAAAGAAATAAAAATACAAAATATAATCATAAGTGAAGAAAAAGCACCCTTAGTCGTACCTGAAATAGGCATTAATCATAATGGCAGTTTAGAACTAGCTAAAATTATGGTAGATGCAGCCTTTAGCGCAGGTGCTAAGATTATAAAGCATCAAACTCATATTGTTGAAGATGAGATGAGTAAGGCCGCTAAAAAAGTAATTCCTGGTAATGCAAAAATAAGCATTTATGAGATTATGCAAAAATGTGCTTTGGATTATAAAGATGAGCTAGCACTTAAAGAATACACAGAAAAATTAGGTCTTGTTTATCTTAGCACACCTTTTTCTCGTGCAGGTGCGAACCGCTTAGAAGATATGGGAGTTAGTGCTTTTAAGATTGGTTCAGGTGAGTGTAATAATTATCCGCTTATTAAACACATAGCAGCCTTTAAAAAGCCTATGATAGTTAGCACAGGAATGAATAGTATTGAAAGTATAAAACCAACTGTAAAAATCTTATTAGACAATGAAATTCCTTTTGTTTTAATGCACACGACCAATCTTTACCCAACCCCGCATAATCTTGTAAGATTAAACGCTATGCTTGAGTTAAAAAAAGAATTTTCTTGTATGGTAGGCTTAAGCGACCACACAACAGATAATCTTGCGTGTTTAGGTGCAGTTGTACTTGGAGCTTGTGTGCTTGAAAGACATTTTACTGATAGTATGCATAGAAGTGGCCCTGATATAGTTTGTTCTATGGATACAAAGGCTTTAAAAGAGCTAATTATACAAAGTGAGCAAATGGCTATAATAAGAGGAAATAATGAAAGTAAAAAAGCGGCTAAACAAGAACAAGTTACAATTGATTTTGCCTTTGCAAGTGTAGTTAGCATTAAAGATATTAAAAAAGGCGAAGTTTTATCTATGGATAATATTTGGGTTAAAAGACCTGGACTTGGTGGAATTAGTGCGGCTGAATTTGAAAATATTTTAGGCAAAAAAGCATTAAGAGATATAGAAAATGATGCTCAGTTAAGCTATGAGGATTTTGCGTGAAAAAAATCCTTTTTATAACAGGCTCTAGGGCTGATTATTCTAAGATTAAATCTTTAATGTACAGGGTGCAAAACTCAAGCGAATTTGAACTTTACATCTTTGCAACAGGAATGCACTTAAGTAAAAATTTTGGCTATACAGTTAAAGAACTTTATAAAAATGGCTTTAAAAATATTTATGAATTTATAAATTATGATAAATATTATCAAACTGATAAGGCTTTAGCTACTACAATTGATGGATTTTCAAGGTATGCAAATGAGCTAAAACCTGATTTAATCGTAGTACATGGAGATAGAATTGAGCCTTTAGCAGCAGCTATTGTTGGAGCATTAAATAATATCTTAGTAGCGCATATTGAAGGCGGAGAGATTTCAGGAACTATTGACGATAGCTTACGCCACGCTATATCAAAACTAGCTCATATTCATTTAGTAAATGATGAGTTTGCAAAAAGGCGTTTAATGCAGCTTGGAGAAGATGAAAAATCTATTTTTATCATAGGTTCGCCTGATTTAGAACTTTTAAACGATAATAAAATTTCACTTAGCGAAGCAAAAAAATATTATGATATAAATTATGAAAACTACGCTTTGCTTATGTTTCATCCTGTTACAACTGAAATTACTAGCATTAAAAATCAAGCAGACAATTTAGTAAAAGCACTGATACAAAGTAATAAAAATTATATTGTTATTTATCCAAATAATGATTTAGGTTTTGAATTAATCTTGCAAAGCTATGAAGAGTTTAAAAATAACCCTAGATTTAAGCTTTTTCCATCGCTTAGATTTGAGTATTTTATAACTTTGT TAAAAAATGCTGATTTTATAATAGGTAATTCAAGTTGTATTTTAAAAGAGGCCTTATACTTAAAAACAGCAGGGATTTTAGTTGGCTCAAGACAAAATGGAAGACTTGGCAATGAAAATACACTAAAAGTTAATGCAAATAGTGATGAAATACTAAAAGCTATTAACACTATTCATAAAAAACAAGATTTATTTAGCGCTAAGTTAGAGATTTTAGATAGCTCAAAATTATTTTTTGAATATTTACAAAGCGGAGATTTTTTTAAACTCAGCACACAAAAAGTTTTTAAGGATATAAAATGA
TCTAGA
2) by BamHI and XbaI enzyme cutting for synthetic gene fragment, reclaim for subsequent use.
3) extracting expression vector pHT01(MOBITEC company), with BamHI and XbaI double digestion, reclaim for subsequent use.
4) connect above-mentioned enzyme and cut carrier and the fragment that purifying is good, with the connection of T4DNA ligase enzyme, and proceed to bacillus coli DH 5 alpha (purchased from TAKARA company) competent cell, obtain expression vector pHT01-neuBC.
Two, the structure of neuBC and glmS gene tandem expression carrier
1) according to the glmS gene in subtilis 168 strain gene group sequence GenBank No.AL009126.3 and the primers of upstream thereof: forward primer F-glmS-XbaI:CGC
tCTAGAgGAGGAAGAAAAATATGTGTG(SEQ ID NO.2) and reverse primer R-glmS-AatII:GTA
gACGTCtTACTCCACAGTAACACTCTTCGC(SEQ ID NO.3), XbaI and AatII restriction enzyme site sequence have been added in primer two ends, so that follow-up genetic manipulation.
2) taking total DNA of subtilis 168 bacterial strains as template, obtain glmS gene and upstream fragment following (SEQ ID NO.4) thereof with above-mentioned primer (shown in SEQ ID NO.2-3) pcr amplification, and cut with XbaI and AatII enzyme, reclaim for subsequent use.
CGC
TCTAGAggaggaagaaaaatatgtgtggaatcgtaggttatatcggtcagcttgatgcgaaggaaattttattaaaagggttagagaagcttgagtatcgcggttatgactctgctggtattgctgttgccaacgaacagggaatccatgtgttcaaagaaaaaggacgcattgcagatcttcgtgaagttgtggatgccaatgtagaagcgaaagccggaattgggcatactcgctgggcgacacacggcgaaccaagctatctgaacgctcacccgcatcaaagcgcactgggccgctttacacttgttcacaacggcgtgatcgagaactatgttcagctgaagcaagagtatttgcaagatgtagagctcaaaagtgacaccgatacagaagtagtcgttcaagtaatcgagcaattcgtcaatggaggacttgagacagaagaagcgttccgcaaaacacttacactgt taaaaggctcttatgcaattgctttattcgataacgacaacagagaaacgatttttgtagcgaaaaacaaaagccctctattagtaggtcttggagatacattcaacgtcgtagcatctgatgcgatggcgatgcttcaagtaaccaacgaatacgtagagctgatggataaagaaatggttatcgtcactgatgaccaagttgtcatcaaaaaccttgatggtgacgtgattacacgtgcgtcttatattgctgagcttgatgccagtgatatcgaaaaaggcacgtaccctcactacatgttgaaagaaacggatgagcagcctgttgttatgcgcaaaatcatccaaacgtatcaagatgaaaacggcaagctgtctgtgcctggcgatatcgctgccgctgtagcggaagcggaccgcatctatatcattggctgcggaacaagctaccatgcaggacttgtcggtaaacaatatattgaaatgtgggcaaacgtgccggttgaagtgcatgtagcgagtgaattctcctacaacatgccgcttctgtctaagaaaccgctcttcattttcctttctcaaagcggagaaacagcagacagccgcgcggtactcgttcaagtcaaagcgctcggacacaaagccctgacaatcacaaacgtacctggatcaacgctttctcgtgaagctgactatacattgctgcttcatgcaggccctgagatcgctgttgcgtcaacgaaagcatacactgcacaaatcgcagttctggcggttcttgcttctgtggctgctgacaaaaatggcatcaatatcggatttgacctcgtcaaagaactcggtatcgctgcaaacgcaatggaagctctatgcgaccagaaagacgaaatggaaatgatcgctcgtgaatacctgactgtatccagaaatgctttcttcatcggacgcggccttgactacttcgtatgtgtcgaaggcgcactgaagctgaaagagatttcttacatccaggcagaaggttttgccggcggtgagctaaagcacggaacgattgccttgatcgaacaaggaacaccagtattcgcactggcaactcaagagcatgtaaacctaagcatccgcggaaacgtcaaagaagttgctgctcgcggagcaaacacatgc atcatctcactgaaaggcctagacgatgcggatgacagattcgtattgccggaagtaaacccagcgcttgctccgttggtatctgttgttccattgcagctgatcgcttactatgctgcactgcatcgcggctgtgatgtggataaacctcgtaaccttgcgaagagtgttactgtggagtaa
GACGTCTAC
3) extracting and purifying expression vector pHT01-neuBC, with XbaI and AatII double digestion, reclaims for subsequent use.
4) connect above-mentioned enzyme and cut carrier and the fragment that purifying is good, with the connection of T4DNA ligase enzyme, and proceed to bacillus coli DH 5 alpha (purchased from TAKARA company) competent cell, obtain expression vector pHT01 – neuBC-glmS.
The structure of embodiment 2:N-n acetylneuraminic acid n subtilis genetic engineering bacterium
1) e.colistraindh5α that contains carrier pHT01-neuBC-glmS by liquid LB substratum incubated overnight, extracting plasmid pHT01-neuBC-glmS.
2) cultivate respectively subtilis 164 and 168 bacterial strains, prepare competent cell, and electric shock conversion plasmid vector pHT01-neuBC-glmS enters, obtain engineering strain 164/pHT01-neuBC-glmS and 168/pHT01-neuBC-glmS, can obtain and produce N-acetyl-neuraminate genetic engineering bacterium, called after CASOV-3 and CASOV-4, these two bacterial strains are preserved in Chinese Typical Representative culture collection center respectively on December 31st, 2013 and on February 18th, 2014, and preserving number is respectively CCTCC NO:M2013731 and CCTCC NO:M2014038.
Embodiment 3 glucose are that carbon source through fermentation is produced N-acetyl-neuraminate
1, seed and fermention medium:
(NH4)
2sO
44g/L; KH
2pO
46g/L; K
2hPO
43H
2o8g/L; Yeast extract (purchased from OXOID company) 5g/L; Peptone 10g/L; Glucose 5g/L.
Feed supplement liquid: 500g/L glucose.
2, fermenting process:
1) the mono-bacterium colony of picking CASOV-3 or CASOV-4, in liquid amount 4ml LB test tube, is cultivated 8-12 hour for 37 DEG C.
2) 2ml first order seed is inoculated in 200ml seed culture medium, cultivates 8～10 hours for 37 DEG C.
3) secondary seed is inoculated in the fermentor tank of liquid amount 3.5L, 37 DEG C, 300～800 revs/min of stirring velocitys, dissolved oxygen remains on more than 30%, with ammoniacal liquor control pH 6.9.
4), after glucose has consumed, start to add glucose with the speed of 5g/L.h.
5) fermented liquid thalline OD
600, add IPTG(IPTG final concentration is 0.2mM when=10 the left and right), 37 DEG C of cultivations, the feed supplement speed that can slow down after 70 hours, to finishing fermentation.
After testing, when 80 hours fermentation ends, in CASOV-3 bacterial strain, the concentration of product N-acetyl-neuraminate can reach 25.40g/L, and in CASOV-4 bacterial strain, the concentration of product N-acetyl-neuraminate can reach 22.80g/L.
Embodiment 4 glycerine are that carbon source through fermentation is produced N-acetyl-neuraminate
Glycerine with 20g/L replaces glucose, and other compositions of fermention medium are with embodiment 2, and supplemented medium is 600g/L glycerine;
Zymotechnique is with embodiment 2.
After testing, when 80 hours fermentation ends, the concentration of CASOV-3 product N-acetyl-neuraminate can reach 29.20g/L, and the concentration of CASOV-4 product N-acetyl-neuraminate can reach 25.80g/L.Comparing glucose is raw material, and output increases.
Above cultivation results shows to have the ability of high yield N-acetyl-neuraminate through the subtilis of metabolic engineering technique construction, has possessed industrialized potentiality.
Sequence table
<110> Wuhan Zhongke Guanggu Green Biological Technology Co., Ltd.
<120> subtilis genetic engineering bacterium and construction process and application of producing N-acetyl-neuraminate
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caaagtgagc aaatggctat aataagagga aataatgaaa gtaaaaaagc ggctaaacaa 840
gaacaagtta caattgattt tgcctttgca agtgtagtta gcattaaaga tattaaaaaa 900
ggcgaagttt tatctatgga taatatttgg gttaaaagac ctggacttgg tggaattagt 960
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ataaggcttt agctactaca attgatggat tttcaaggta tgcaaatgag ctaaaacctg 1320
atttaatcgt agtacatgga gatagaattg agcctttagc agcagctatt gttggagcat 1380
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gcttacgcca cgctatatca aaactagctc atattcattt agtaaatgat gagtttgcaa 1500
aaaggcgttt aatgcagctt ggagaagatg aaaaatctat ttttatcata ggttcgcctg 1560
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gtattgctgt tgccaacgaa cagggaatcc atgtgttcaa agaaaaagga cgcattgcag 180
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gctttacact tgttcacaac ggcgtgatcg agaactatgt tcagctgaag caagagtatt 360
tgcaagatgt agagctcaaa agtgacaccg atacagaagt agtcgttcaa gtaatcgagc 420
aattcgtcaa tggaggactt gagacagaag aagcgttccg caaaacactt acactgttaa 480
aaggctctta tgcaattgct ttattcgata acgacaacag agaaacgatt tttgtagcga 540
aaaacaaaag ccctctatta gtaggtcttg gagatacatt caacgtcgta gcatctgatg 600
cgatggcgat gcttcaagta accaacgaat acgtagagct gatggataaa gaaatggtta 660
tcgtcactga tgaccaagtt gtcatcaaaa accttgatgg tgacgtgatt acacgtgcgt 720
cttatattgc tgagcttgat gccagtgata tcgaaaaagg cacgtaccct cactacatgt 780
tgaaagaaac ggatgagcag cctgttgtta tgcgcaaaat catccaaacg tatcaagatg 840
aaaacggcaa gctgtctgtg cctggcgata tcgctgccgc tgtagcggaa gcggaccgca 900
tctatatcat tggctgcgga acaagctacc atgcaggact tgtcggtaaa caatatattg 960
aaatgtgggc aaacgtgccg gttgaagtgc atgtagcgag tgaattctcc tacaacatgc 1020
cgcttctgtc taagaaaccg ctcttcattt tcctttctca aagcggagaa acagcagaca 1080
gccgcgcggt actcgttcaa gtcaaagcgc tcggacacaa agccctgaca atcacaaacg 1140
tacctggatc aacgctttct cgtgaagctg actatacatt gctgcttcat gcaggccctg 1200
agatcgctgt tgcgtcaacg aaagcataca ctgcacaaat cgcagttctg gcggttcttg 1260
cttctgtggc tgctgacaaa aatggcatca atatcggatt tgacctcgtc aaagaactcg 1320
gtatcgctgc aaacgcaatg gaagctctat gcgaccagaa agacgaaatg gaaatgatcg 1380
ctcgtgaata cctgactgta tccagaaatg ctttcttcat cggacgcggc cttgactact 1440
tcgtatgtgt cgaaggcgca ctgaagctga aagagatttc ttacatccag gcagaaggtt 1500
ttgccggcgg tgagctaaag cacggaacga ttgccttgat cgaacaagga acaccagtat 1560
tcgcactggc aactcaagag catgtaaacc taagcatccg cggaaacgtc aaagaagttg 1620
ctgctcgcgg agcaaacaca tgcatcatct cactgaaagg cctagacgat gcggatgaca 1680
gattcgtatt gccggaagta aacccagcgc ttgctccgtt ggtatctgtt gttccattgc 1740
agctgatcgc ttactatgct gcactgcatc gcggctgtga tgtggataaa cctcgtaacc 1800
ttgcgaagag tgttactgtg gagtaagacg tctac 1835
Claims (8)
1. produce subtilis (Bacillus subtilis) genetic engineering bacterium for N-acetyl-neuraminate, it is characterized in that described genetic engineering bacterium carries out heterogenous expression and builds and obtain by UDP-N-acetylglucosamine epimerase gene, N-acetyl-neuraminate synthase gene and 6-phosphorylated amino glucose synthase gene are imported in subtilis.
2. the subtilis genetic engineering bacterium of product N-acetyl-neuraminate as claimed in claim 1, is characterized in that described UDP-N-acetylglucosamine epimerase gene and N-acetyl-neuraminate synthase gene derive from campylobacter jejuni (Campylobacter jejuni) and maybe can express the microorganism of identical function enzyme.
3. the subtilis genetic engineering bacterium of product N-acetyl-neuraminate as claimed in claim 1, is characterized in that described 6-phosphorylated amino glucose synthase gene derives from subtilis and maybe can express the microorganism of identical function enzyme.
4. the subtilis genetic engineering bacterium of the product N-acetyl-neuraminate as described in claims 1 to 3 any one, it is characterized in that clonal expression together with described UDP-N-acetylglucosamine epimerase gene, N-acetyl-neuraminate synthase gene and 6-phosphorylated amino glucose synthase gene are in same carrier, or be cloned in proceeding to after different carriers after same bacterial strain and separately express.
5. the subtilis genetic engineering bacterium of product N-acetyl-neuraminate as claimed in claim 1, is characterized in that described subtilis host is for subtilis 164 or subtilis 168 or other similar bacterial strains.
6. the subtilis genetic engineering bacterium of product N-acetyl-neuraminate as claimed in claim 5, is characterized in that described genetic engineering bacterium is that preserving number is the bacterial strain of CCTCC NO:M2013731 or CCTCC NO:M2014038.
7. the purposes of the subtilis engineering bacteria of product N-acetyl-neuraminate as claimed in claim 1, is characterized in that utilizing described engineering bacteria to produce N-acetyl-neuraminate, and its step comprises:
1) activation culture engineering bacteria seed;
2) cultured seed is amplified step by step, and carry out aerobic cultivation in corresponding fermentor tank; In culturing process, supplement suitable carbon nitrogen source; And
3) add sec.-propyl-β-D-sulfo-galactopyranoside, the expression of induction associated protein.
8. the purposes of the subtilis engineering bacteria of the product N-acetyl-neuraminate of claim 7, is characterized in that utilizing glucose or glycerine to ferment as carbon source.
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| CN104996986A (en) * | 2015-07-10 | 2015-10-28 | 武汉中科光谷绿色生物技术有限公司 | Food application of N-acetylneuraminic acid fermented from E. coli |
| CN105255802A (en) * | 2015-10-14 | 2016-01-20 | 江南大学 | Method for increasing yield of recombinant bacillus subtilis acetylglucosamine through expression of NAD(P)H oxidases |
| WO2016015469A1 (en) * | 2014-08-01 | 2016-02-04 | 张帆 | Bacillus subtilis for producing n-acetylglucosamine, construction method therefor and uses thereof |
| WO2016119293A1 (en) * | 2015-01-27 | 2016-08-04 | 邹季虹 | Strain for producing glucosamine by microbial fermentation and method therefor |
| CN108424868A (en) * | 2018-03-22 | 2018-08-21 | 江南大学 | A kind of recombinant bacterium using natural double carbon source high yield N-acetyl-neuraminates |
| CN109161576A (en) * | 2018-09-26 | 2019-01-08 | 武汉中科光谷绿色生物技术有限公司 | Promote the method for fermentation of bacillus subtilis production N-acetyl-neuraminate |
| CN111394292A (en) * | 2020-03-30 | 2020-07-10 | 江南大学 | Multi-way composite neuraminic acid-producing bacillus subtilis and application thereof |
| CN111411065A (en) * | 2020-03-30 | 2020-07-14 | 江南大学 | Recombinant bacterium for producing N-acetylneuraminic acid based on artificial double carbon sources |
| CN113249285A (en) * | 2021-05-13 | 2021-08-13 | 浙江工业大学 | Recombinant bacillus subtilis for high yield of N-acetylneuraminic acid and construction method and application thereof |
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| WO2016015469A1 (en) * | 2014-08-01 | 2016-02-04 | 张帆 | Bacillus subtilis for producing n-acetylglucosamine, construction method therefor and uses thereof |
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| CN111411065A (en) * | 2020-03-30 | 2020-07-14 | 江南大学 | Recombinant bacterium for producing N-acetylneuraminic acid based on artificial double carbon sources |
| CN111411065B (en) * | 2020-03-30 | 2022-07-05 | 江南大学 | Recombinant bacterium for producing N-acetylneuraminic acid based on artificial double carbon sources |
| CN111394292B (en) * | 2020-03-30 | 2022-08-09 | 江南大学 | Multi-way composite neuraminic acid-producing bacillus subtilis and application thereof |
| CN113249285A (en) * | 2021-05-13 | 2021-08-13 | 浙江工业大学 | Recombinant bacillus subtilis for high yield of N-acetylneuraminic acid and construction method and application thereof |
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