CN107699533A - A kind of recombined bacillus subtilis of acetylglucosamine output increased - Google Patents

A kind of recombined bacillus subtilis of acetylglucosamine output increased Download PDF

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CN107699533A
CN107699533A CN201710948044.0A CN201710948044A CN107699533A CN 107699533 A CN107699533 A CN 107699533A CN 201710948044 A CN201710948044 A CN 201710948044A CN 107699533 A CN107699533 A CN 107699533A
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bacillus subtilis
glms
acetylglucosamine
recombined
recombined bacillus
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刘龙
陈坚
堵国成
李江华
武耀康
陈泰驰
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Jiangnan University
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Abstract

The invention discloses a kind of recombined bacillus subtilis of acetylglucosamine output increased, belong to field of genetic engineering.It is with bacillus subtilis BSGNKAP PxylA‑glmS‑P43GNA1 is starting strain, and strong constitutive promoter P is integrated on genomevegThe phosphatase yqaB genes of control, and the promoter of 6 phosphorylated amino glucose synthase is replaced for strong constitutive promoter Pveg, obtained accumulation acetylglucosamine Bacillus subtilis genes engineering bacteria BSGNY Pveg‑glmS‑P43‑GNA1.3L fermentation tank acetylglucosamine yield reaches 60.5g/L, acetylglucosamine yield is 0.311g/g glucose, production intensity is 0.983g/L/h, raising of the acetylglucosamine in the extracellular yield of recombined bacillus subtilis is realized, and produces Glucosamine for further metabolic engineering bacillus subtilis and lays a good foundation.

Description

A kind of recombined bacillus subtilis of acetylglucosamine output increased
Technical field
The present invention relates to a kind of recombined bacillus subtilis of acetylglucosamine output increased, belong to genetic engineering Field.
Background technology
Acetylglucosamine is a kind of monose in organism, be widely present in bacterium, yeast, mould, plant and In animal body.In human body, acetylglucosamine is the synthesis precursor of glycosaminoglycan disaccharide unit, and it is to repairing and maintaining soft Bone and joint tissue function play an important roll.Therefore, acetylglucosamine is widely used as medicine and nutritious food addition To treat and repair joint injury.In addition, acetylglucosamine also has many applications in cosmetic field.At present, acetyl Glucosamine is mainly produced using chitin in acidolysis shrimp shell or crab shell, and waste liquid environmental pollution is more tight caused by the method Weight, and obtained product easily causes allergic reaction, and the crowd for being not suitable for seafood allergy takes.
Bacillus subtilis (Bacillus subtilis) is that one kind is widely used as Food enzyme and important nutrient laden The production host of product, its product are " generally regarded as safe " (GRAS) level of security by FDA certifications.Cause This, is the effective way for producing aliment security level acetylglucosamine with metabolic engineering means structure recombined bacillus subtilis Footpath.However, recombined bacillus subtilis (the BSGNKAP-PxylA-glmS- built in patent application 201510761678.6 P43-GNA1 the shortcomings that yield is not high, product yield is relatively low) be present.Therefore, acetylglucosamine route of synthesis generation is improved Thank to flux, be the problem urgently to be resolved hurrily for improving acetylglucosamine yield.Therefore, acetylglucosamine route of synthesis is improved Metabolic flux, it is to improve acetylglucosamine yield urgent problem to be solved.Acetyl ammonia is catalyzed in bacillus subtilis at present The final step reaction of base glucose route of synthesis, i.e., 6- phosphates Glucosamine is converted into the enzyme of acetylglucosamine It is unclear.It is reported that to second after three phosphatases of the HAD-like families that expression comes from Escherichia coli in saccharomyces cerevisiae The synthesis of acylamino- glucose has obvious facilitation；And it need to only strengthen 6- phosphoric acid ammonia during using Escherichia coli as Host Strains Base glucosylceramide synthase glmS and 6- phosphorylated amino glucose acetylase genes GNA1 can reach very high acetamido glucose Candy output, it is likely to turn because abundant phosphatase in its periplasmic space be present and can be catalyzed 6- phosphates Glucosamine Turn to acetylglucosamine.And bacillus subtilis is gram-positive bacteria, it is empty that pericentral siphon as Escherichia coli is not present in it Between structure, therefore speculate in recombined bacillus subtilis BSGNKAP-PxylA-glmS-P43-GNA1 6- phosphates amino The reaction that glucose is converted into acetylglucosamine may be the rate-limiting step of its synthesis of acetyl Glucosamine.Withered grass gemma Have its expression intensity of some constitutive promoters will not be effected by environmental factors in bacillus, wherein promoter Pveg intensity compared with By force, referring specifically to the .A part toolbox to tune genetic expression such as document GUIZIOU S in Bacillus subtilis[J].Nucleic Acids Research,2016,44(15):gkw624。
The content of the invention
In order to solve the above problems, the invention provides a kind of recombinant bacillus gemma for efficiently synthesizing acetylglucosamine Bacillus.
First purpose of the present invention is to provide a kind of recombined bacillus subtilis of acetylglucosamine output increased, Recombined bacillus subtilis integrant expression phosphatase gene yqaB, and overexpression 6- phosphorylated amino glucose synthase bases Because of glmS.
In one embodiment of the invention, the integrant expression phosphatase gene yqaB is with strong constitutive promoter PvegPhosphatase gene yqaB expression is controlled, is replaced and is integrated on genome by homologous recombination.
In one embodiment of the invention, the amino acid sequence such as SEQ ID of the phosphatase gene yqaB expression Shown in NO.3.
In one embodiment of the invention, overexpression 6- phosphorylated amino glucoses synthase gene glmS is by 6- phosphorus Sour Glucosamine synthase gene glmS promoter PxylAReplace with strong constitutive promoter Pveg。
In one embodiment of the invention, the recombined bacillus subtilis is with bacillus subtilis BSGNKAP- PxylA-glmS-P43- GNA1 is starting strain.
In one embodiment of the invention, the bacillus subtilis BSGNKAP-PxylA-glmS-P43- GNA1 is Based on bacillus subtilis 168, genotype makees what following transformation obtained：ΔnagPΔgamPΔgamAΔnagAΔnagB ΔldhΔpt aΔglcKΔpckAΔpyk::Lox72, and with xylose evoked promoter PxylARegulating and expressing glmS, in plasmid On with promoter P43Regulating and expressing GNA1 recombination expression.
In one embodiment of the invention, the bacillus subtilis BSGNKAP, is Application No. 201510761678.6 patent application in the recombined bacillus subtilis BSGNKAP that builds.
In one embodiment of the invention, it is with BSGNKAP-PxylA-glmS-P43- GNA1 is starting strain, is passed through Homologous recombination is in genome nagAB sites with strong promoter PvegControl phosphatase gene yqaB expression；And by 6- phosphorylated aminos Portugal Grape sugar synthase gene glmS promoter replaces with strong constitutive promoter Pveg。
Second object of the present invention is to provide above-mentioned recombined bacillus subtilis in medicine, nutrient and healthcare products or cosmetic Application in product.
In one embodiment of the invention, the application is to utilize recombined bacillus subtilis production acetyl ammonia Base glucose.
In one embodiment of the invention, the application is after production bacterial strain is activated, with 5-10% inoculum concentration It is transferred to fermentation medium, 35-38 DEG C, cultivate 50-72h under the conditions of 500-900rpm.
Beneficial effects of the present invention：
The present invention strengthens the sugar synthesis of 6- phosphorylated amino glucoses by being overexpressed 6- phosphorylated amino glucose sugar synthase glmS Reaction, cell growth is restored, and the yield of acetylglucosamine and yield improve a lot, solve because strengthen The emulative precursor substance 6- phosphorylated amino glucoses using intracellular Cell wall synthesis of meeting after reaction, to cell caused by Adversely affected caused by growth.
Recombined bacillus subtilis provided by the invention can efficiently utilize glucose synthesis of acetyl Glucosamine, and it is in 3L Yield can reach 60.5g/L, while recombined bacillus subtilis 3L provided by the invention fermentation acetamido glucoses on fermentation tank Sugared yield is 0.311g/g glucose, and production intensity is 0.983/L/h, realizes acetylglucosamine in recombinant bacillus gemma The raising of the extracellular yield of bacillus, and produce Glucosamine for further metabolic engineering bacillus subtilis and established base Plinth.
Brief description of the drawings
Fig. 1 is influence of the different constitutive promoters to regulation and control yqaB expression；
Fig. 2 is influence of the different constitutive promoters to regulation and control glmS expression.
Embodiment
Seed culture medium (g/L)：Tryptone 10, dusty yeast 5, NaCl 10.
Medium of shaking flask fermentation (g/L)：Tryptone 6, dusty yeast 12, (NH4) SO46, K2HPO4·3H2O 12.5, KH2PO42.5, CaCO35, micro- 10ml/L；Trace element solution contains based on g/L：MnSO4·5H2O 1.0, CoCl2· 6H2O 0.4, NaMoO4·2H2O 0.2, ZnSO4·7H2O 0.2, AlCl3·6H2O 0.1, CuCl2·H2O 0.1, H3BO40.05, HCl containing 5M.
Upper tank fermentation medium (g/L)：Tryptone 20, dusty yeast 20, K2HPO4·3H2O 12.5, KH2PO42.5 CaCO35, micro- 10ml/L；Trace element solution contains based on g/L：MnSO4·5H2O 1.0, CoCl2·6H2O 0.4, NaMoO4·2H2O 0.2, ZnSO4·7H2O 0.2, AlCl3·6H2O 0.1, CuCl2·H2O 0.1, H3BO40.05, containing 5M HCl。
The assay method of acetylglucosamine：
High performance liquid chromatography (HPLC) detection method：Agilent 1260, RID detector, HPX-87H posts (Bio- RadHercules, CA), mobile phase：5mM H2SO4, flow velocity 0.6mL/min, 35 DEG C of column temperature, sampling volume is 10 μ L.
The calculating of acetylglucosamine yield：
Acetylglucosamine yield (YGlcNAc/GlcGlucose (the g/ of)=acetylglucosamine yield (g/L)/consumption L)。
Embodiment 1：Build recombined bacillus subtilis BSGNY-PxylA-glmS-P43-GNA1
According to the bacillus subtilis announced on NCBI, (Bacillus subtilis 168 are purchased from American Type Culture Collection, ATCC No.27370) nagAB genes upstream and downstream sequence and phosphoric acid enzyme coding gene yqaB (nucleotides sequences Row such as NCBI-Gene ID:945776), terminator λ T0, strong constitutive promoter PvegAnd the sequence of chloramphenicol resistance gene Row, sequence expression cassette as shown in SEQ ID NO.1 is built, phosphatase yqaB is incorporated into the nagAB sites of genome and used Strong constitutive promoter PvegExpressed.
The expression cassette built is converted into BSGNKAP-PxylA-glmS-P43- GNA1 (i.e. patent applications The recombined bacillus subtilis BSGNKAP built in 201510761678.6), pass through chlorampenicol resistant plate screening, bacterium colony PCR Checking, confirmation obtain recombined bacillus subtilis BSGNY-PxylA-glmS-P43-GNA1.By plasmid pTSC (Agricultural University Of Nanjing, Doctor Yan Xin give, NCBI accession no.EU864234) conversion recombined bacillus subtilis BSGNY, eliminate chloramphenicol Resistance.
Embodiment 2：Build recombined bacillus subtilis BSGNY-Pveg-glmS-P43-GNA1
According to the bacillus subtilis announced on NCBI, (Bacillus subtilis 168 are purchased from American Type Culture Collection, ATCC No.27370) glmS genes and its upstream sequence, terminator λ T0, strong constitutive promoter Pveg、 And the sequence of chloramphenicol resistance gene, glmS promoter of the structure sequence as shown in SEQ ID NO.2 replaces frame, and glmS is former Some promoters replace with strong constitutive promoter Pveg.The recombinant bacillus bud replacement frame built being transferred in embodiment one Spore bacillus BSGNY-PxylA-glmS-P43- GNA1, verify that confirmation is recombinated by chlorampenicol resistant plate screening, bacterium colony PCR Bacillus subtilis BSGNY-Pveg-glmS-P43-GNA1.By plasmid pTSC (Agricultural University Of Nanjing, doctor Yan Xin give, NCBI accession no.EU864234) conversion recombined bacillus subtilis BSGNY-Pveg-glmS-P43- GNA1, eliminate chlorine Chloramphenicol resistance.
Embodiment 3：Recombined bacillus subtilis BSGNY-Pveg-glmS-P43-GNA1 fermenting and producing acetamido glucoses Sugar
The checking that obtained recombinated bacillus carry out 3L fermentation tank levels is built using embodiment 2, by 37 DEG C, 220rpm Lower culture 12h seed is transferred to upper tank fermentation medium with 5% inoculum concentration, in 3L fermentation tanks, 35-37 DEG C, 500-900rpm Under the conditions of cultivate 52-72h.Acetylglucosamine content reaches 60.5g/L in final fermented supernatant fluid, while the present invention provides Recombined bacillus subtilis 3L fermentation acetylglucosamine yield be 0.311g/g glucose, production intensity is 0.983g/ L/h (result is as shown in table 1), raising of the acetylglucosamine in the extracellular yield of recombined bacillus subtilis is realized, and be Further metabolic engineering bacillus subtilis production Glucosamine is laid a good foundation.
Table 1 is overexpressed yqaB and synthesizes situation with cell growth before and after glmS and acetylglucosamine
Reference examples 1：Using different constitutive promoters regulation and control yqaB expression
Because phosphatase yqaB expression can produce certain inhibitory action to thalli growth, therefore utilize the side of embodiment 1 Formula structure integrates frame, and respectively using .Apart toolbox to tune genetic such as document GUIZIOU S expression in Bacillus subtilis[J].Nucleic Acids Research,2016,44(15):gkw624. The constitutive promoter P of the varying strength of reportymdA、PfbaA、PlepA、PrelARegulate and control yqaB expression, convert bacillus subtilis Recombined bacillus subtilis genome nagAB sites are integrated into by way of homologous recombination afterwards, build obtained restructuring Bacillus carries out shake flask fermentation.Training method is as follows：The seed that 12h is cultivated under 37 DEG C, 220rpm is turned with 5% inoculum concentration Enter Medium of shaking flask fermentation, xylose is added after two hours and is induced, 48h is cultivated under the conditions of 37 DEG C, 220rpm.Fermentation results See Fig. 1, when the remitted its fury of promoter, the influence of cell growth can also reduce, but the yield of acetylglucosamine Decrease, so the promoter P that selection is strongervegTo regulate and control yqaB expression.
Reference examples 2：GlmS promoter is replaced using different constitutive promoters
The replacement frame of glmS promoters is built by the way of embodiment 2, utilizes promoter PymdA、PfbaA、PlepAReplace weight Group bacillus subtilis BSGNY-PxylA-glmS-P43GlmS promoter in-GNA1, the recombinated bacillus for building to obtain enter Row shake flask fermentation.For training method with reference examples 1, fermentation results are as shown in Figure 2.When glmS promoters replace with PvegWhen, thalline life Length is restored, and yield of the acetylglucosamine in shaking flask reaches 15.4g/L, and the yield to glucose is 0.312g/ G, and replace with promoter PymdAWhen yield be slightly below Pveg, replace with PfbaAAnd PlepAWhen yield decreased compared with control.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, it can all do various change and modification, therefore the protection model of the present invention Enclose being defined of being defined by claims.
Sequence table
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gtcggttcga ctcatcccga agcgctcagc gcgtttgtca aagagaaaaa cgcggatctc 360
ggtcttgcgt tcgacggtga cggcgaccgc ctgattgctg tcgatgaaaa aggaaatatt 420
gtagacggcg accaaatcat gtacatatgc tcaaaacatc tgaaatcaga gggccgttta 480
aaggatgata cagtggtttc aaccgtgatg agcaacctcg gcttctataa ggcgctcgaa 540
aaagaaggca tcaaaagcgt gcagacagct gtcggcgacc gctacgtagt agaagcaatg 600
aaaaaagacg gctacaacgt cggcggagag cagtcaggac atcttatttt ccttgattac 660
aacacgacag gggacggatt attgtctgct attatgctga tgaacacttt aaaagcaaca 720
ggcaagccgc tgtcagagct tgcagctgaa atgcagaagt tcccgcagct gttagtcaat 780
gtgagagtga ctgataaata taaagttgaa gaaaatgaaa aagtaaaagc agttatttct 840
gaagttgaaa aagaaatgaa cggcgacggc cggattttgg tgcgcccttc aggaactgaa 900
ccgctcgtcc gtgtcatggc tgaagcgaag acgaaagagc tgtgcgatga gtatgtcaat 960
cgcattgttg aagtcgtccg gtcagaaatg ggattagagt aacaccggat ttgatcccgt 1020
ataccgttcg tatagcatac attatacgaa gttatgccat agtgactggc gatgctgtcg 1080
gaatggacga cggcaatagt tacccttatt atcaagataa gaaagaaaag gatttttcgc 1140
tacgctcaaa tcctttaaaa aaacacaaaa gaccacattt tttaatgtgg tcttttattc 1200
ttcaactaaa gcacccatta gttcaacaaa cgaaaattgg ataaagtggg atatttttaa 1260
aatatatatt tatgttacag taatattgac ttttaaaaaa ggattgattc taatgaagaa 1320
agcagacaag taagcctcct aaattcactt tagataaaaa tttaggaggc atatcaaatg 1380
aactttaata aaattgattt agacaattgg aagagaaaag agatatttaa tcattatttg 1440
aaccaacaaa cgacttttag tataaccaca gaaattgata ttagtgtttt ataccgaaac 1500
ataaaacaag aaggatataa attttaccct gcatttattt tcttagtgac aagggtgata 1560
aactcaaata cagcttttag aactggttac aatagcgacg gagagttagg ttattgggat 1620
aagttagagc cactttatac aatttttgat ggtgtatcta aaacattctc tggtatttgg 1680
actcctgtaa agaatgactt caaagagttt tatgatttat acctttctga tgtagagaaa 1740
tataatggtt cggggaaatt gtttcccaaa acacctatac ctgaaaatgc tttttctctt 1800
tctattattc catggacttc atttactggg tttaacttaa atatcaataa taatagtaat 1860
taccttctac ccattattac agcaggaaaa ttcattaata aaggtaattc aatatattta 1920
ccgctatctt tacaggtaca tcattctgtt tgtgatggtt atcatgcagg attgtttatg 1980
aactctattc aggaattgtc agataggcct aatgactggc ttttataata tgagataatg 2040
ccgactgtac tttttacagt cggttttcta acgatacatt aataggtacg aaaaagcaac 2100
tttttttgcg cttaaaacca gtcataccaa taaataactt cgtatagcat acattatacg 2160
aacggtacgg aattccgctg ataggtggta ttcagaacgc tcggttgccg ccgggcgttt 2220
tttatttttg tcaaaataat tttattgaca acgtcttatt aacgttgata taatttaaat 2280
tttatttgac aaaaatgggc tcgtgttgta caataaatgt ggagaaaaac tagtagtgat 2340
agcgccaaaa aacatgtagg aggggacgat tgaaagtccc cttgaaattt gactttcttc 2400
gtctcctttt acaatcttag gaggaagaaa aatatgtgtg gaatcgtagg ttatatcggt 2460
cagcttgatg cgaaggaaat tttattaaaa gggttagaga agcttgagta tcgcggttat 2520
gactctgctg gtattgctgt tgccaacgaa cagggaatcc atgtgttcaa agaaaaagga 2580
cgcattgcag atcttcgtga agttgtggat gccaatgtag aagcgaaagc cggaattggg 2640
catactcgct gggcgacaca cggcgaacca agctatctga acgctcaccc gcatcaaagc 2700
gcactgggcc gctttacact tgttcacaac ggcgtgatcg agaactatgt tcagctgaag 2760
caagagtatt tgcaagatgt agagctcaaa agtgacaccg atacagaagt agtcgttcaa 2820
gtaatcgagc aattcgtcaa tggaggactt gagacagaag aagcgttccg caaaacactt 2880
acactgttaa aaggctctta tgcaattgct ttattcgata acgacaacag agaaacgatt 2940
tttgtagcga aaaacaaaag ccctctatta gtaggtcttg gagatacatt caacgtcgta 3000
gcatctgatg cgatggcgat gcttcaagta accaacgaat acgtagagct gatggataaa 3060
gaaatggtta tcgtcactga tgaccaagtt gtcatcaaaa accttgatgg tgacgtgatt 3120
acacgtgcgt cttatattgc tgagcttgat gccagtgata tcgaaaaagg cacgtaccct 3180
cactacatgt tgaaagaaac ggatgagcag cctgttgtta tgcgcaaaat catccaaacg 3240
tatcaagatg aaaacggcaa gctgtctgtg cctggcgata tcgctgccgc tgtagcggaa 3300
gcggaccgca tctatatcat tggctgcgga acaagctacc atgcaggact tgtcggtaaa 3360
caatatattg aaatgtgggc aaacgtgccg gttgaagtgc atgtagcgag tgaattctcc 3420
tacaacatgc cgc 3433
<210> 3
<211> 188
<212> PRT
<213>Artificial sequence
<400> 3
Met Tyr Glu Arg Tyr Ala Gly Leu Ile Phe Asp Met Asp Gly Thr Ile
1 5 10 15
Leu Asp Thr Glu Pro Thr His Arg Lys Ala Trp Arg Glu Val Leu Gly
20 25 30
His Tyr Gly Leu Gln Tyr Asp Ile Gln Ala Met Ile Ala Leu Asn Gly
35 40 45
Ser Pro Thr Trp Arg Ile Ala Gln Ala Ile Ile Glu Leu Asn Gln Ala
50 55 60
Asp Leu Asp Pro His Ala Leu Ala Arg Glu Lys Thr Glu Ala Val Arg
65 70 75 80
Ser Met Leu Leu Asp Ser Val Glu Pro Leu Pro Leu Val Asp Val Val
85 90 95
Lys Ser Trp His Gly Arg Arg Pro Met Ala Val Gly Thr Gly Ser Glu
100 105 110
Ser Ala Ile Ala Glu Ala Leu Leu Ala His Leu Gly Leu Arg His Tyr
115 120 125
Phe Asp Ala Val Val Ala Ala Asp His Val Lys His His Lys Pro Ala
130 135 140
Pro Asp Thr Phe Leu Leu Cys Ala Gln Arg Met Gly Val Gln Pro Thr
145 150 155 160
Gln Cys Val Val Phe Glu Asp Ala Asp Phe Gly Ile Gln Ala Ala Arg
165 170 175
Ala Ala Gly Met Asp Ala Val Asp Val Arg Leu Leu
180 185

Claims (9)

  1. A kind of 1. recombined bacillus subtilis of acetylglucosamine output increased, it is characterised in that the recombinant bacillus bud Spore bacillus integrant expression phosphatase gene yqaB, and overexpression 6- phosphorylated amino glucose synthase genes glmS.
  2. 2. recombined bacillus subtilis according to claim 1, it is characterised in that the integrant expression phosphatase gene yqaB It is with strong constitutive promoter PvegPhosphatase gene yqaB expression is controlled, it is withered to be integrated into restructuring by homologous recombination replacement In careless bacillus gene group.
  3. 3. recombined bacillus subtilis according to claim 2, it is characterised in that the ammonia of the phosphatase gene yqaB expression Base acid sequence is as shown in SEQ ID NO.3.
  4. 4. recombined bacillus subtilis according to claim 1, it is characterised in that overexpression 6- phosphorylated amino glucoses are closed Enzyme gene glmS is that 6- phosphorylated amino glucose synthase genes glmS promoter is replaced with into strong constitutive promoter Pveg。
  5. 5. recombined bacillus subtilis according to claim 1, it is characterised in that the recombined bacillus subtilis is with withered Careless bacillus BSGNKAP-PxylA-glmS-P43- GNA1 is starting strain.
  6. 6. recombined bacillus subtilis according to claim 5, it is characterised in that the bacillus subtilis BSGNKAP- PxylA-glmS-P43- GNA1 is based on bacillus subtilis 168, and genotype makees what following transformation obtained：ΔnagPΔ gamPΔgamAΔnagAΔnagBΔldhΔptaΔglcKΔpckAΔpyk::Lox72, and with xylose evoked promoter PxylARegulating and expressing glmS, on plasmid with promoter P43Regulate and control GNA1 recombination expression.
  7. 7. application of the recombined bacillus subtilis described in claim 1 in medicine, nutrient and healthcare products or cosmetics.
  8. 8. apply according to claim 7, it is characterised in that the application is produced using the recombined bacillus subtilis Acetylglucosamine.
  9. 9. apply according to claim 7, it is characterised in that the application is after production bacterial strain is activated, with 5-10%'s Inoculum concentration is transferred to fermentation medium, 35-38 DEG C, cultivate 50-72h under the conditions of 500-900rpm.
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CN108330095A (en) * 2018-03-01 2018-07-27 江南大学 It is a kind of accumulation N-acetyl-neuraminate recombination Corynebacterium glutamicum and its application
CN108330095B (en) * 2018-03-01 2020-12-29 江南大学 Recombinant corynebacterium glutamicum for accumulating N-acetylneuraminic acid and application thereof
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
CN108441461A (en) * 2018-03-22 2018-08-24 江南大学 A kind of recombinant bacterium using artificial double carbon source high yield N-acetyl-neuraminates
CN108424868B (en) * 2018-03-22 2020-11-03 江南大学 Recombinant bacterium for high yield of N-acetylneuraminic acid by utilizing natural dual-carbon source
CN108486025A (en) * 2018-04-02 2018-09-04 山东润德生物科技有限公司 Recombined bacillus subtilis and application
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CN108504678A (en) * 2018-04-12 2018-09-07 江南大学 A method of improving recombined bacillus subtilis chitin oligo saccharide yield
CN108517330A (en) * 2018-04-12 2018-09-11 江南大学 A method of it knocking out ybbD and improves recombined bacillus subtilis chitin oligo saccharide
CN108531436A (en) * 2018-04-12 2018-09-14 江南大学 A kind of accumulation chitin oligo saccharide recombined bacillus subtilis and its application
WO2021175759A1 (en) * 2020-03-04 2021-09-10 Basf Se Method for the production of constitutive bacterial promoters conferring low to medium expression
CN112877272A (en) * 2021-04-28 2021-06-01 中国农业科学院北京畜牧兽医研究所 Escherichia coli engineering bacteria of N-acetylglucosamine and fermentation production method

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