CN106754598B - 一种高产肝素前体的重组枯草芽孢杆菌及其应用 - Google Patents

一种高产肝素前体的重组枯草芽孢杆菌及其应用 Download PDF

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CN106754598B
CN106754598B CN201611149201.3A CN201611149201A CN106754598B CN 106754598 B CN106754598 B CN 106754598B CN 201611149201 A CN201611149201 A CN 201611149201A CN 106754598 B CN106754598 B CN 106754598B
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康振
陈坚
堵国成
张琳培
王浩
周正雄
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Abstract

本发明公开了一种高产肝素前体的重组枯草芽孢杆菌及其应用,属于生物工程技术领域。本发明在产肝素前体的枯草芽孢杆菌中,共表达了UDP‑GlcUA途径和UDP‑GlcNAc途径中的关键酶基因,获得了肝素前体产量及分子量均增大的重组菌株。当共表达tuaD,gtaB,glmU,glmM和glmS基因,摇瓶中肝素前体产量提高至2.89g/L,分子量为75.90kDa。通过在3L发酵罐中进行补料分批发酵,肝素前体的产量最终积累到7.25g/L,分子量为46.66kDa。本发明为工业化生产肝素奠定了基础。

Description

一种高产肝素前体的重组枯草芽孢杆菌及其应用
技术领域
本发明涉及一种高产肝素前体的重组枯草芽孢杆菌及其应用,属于生物工程技术领域。
背景技术
肝素(Heparin)和硫酸乙酰肝素(HS)属于一类高度硫酸化的糖胺聚糖(GAGs),糖胺聚糖是一类由重复的二糖单元组成的不分支的带负电荷的多糖,由于其独特的生理功能,糖胺聚糖组成了一类具有巨大治疗应用潜力的化合物。肝素作为一种抗凝血和抗血栓药物,在深部静脉血栓形成、肾透析和留置导管分流术、及术后血栓控制等医疗措施中使用。同时还具有抗血脂,抗炎、抗肿瘤、抑制细菌粘附等作用,自1918年被发现后被广泛应用于临床作为抗凝血药物。其结构复杂、生物活性多样等特点,研发以肝素和硫酸乙酰肝素为骨架的新药已成为近年来多糖药物研究的热点。
肝素主要通过动物提取法获得,然而该方法存在种间疾病感染和过硫酸软骨素污染等风险,且提取过程往往造成环境污染。另外,通过化学法也可从头合成低分子量的肝素,但因原料昂贵、步骤繁多和产量极低等缺点难以进行大规模制备。近年来,以肝素前体为合成起点进行硫酸化的化学酶法逐渐受到研究者的重视,因此,在微生物中实现肝素前体的高产成为了重要环节。
发明内容
本发明提供了一种高产肝素前体的重组枯草芽孢杆菌,是在枯草芽孢杆菌B.subtilis 168基因组整合表达KfiC和KfiA基因的基础上,以pP43NMK为表达载体共表达肝素前体合成途径的关键酶基因。
在本发明的一种实施方式中,所述共表达是指共表达UDP-GlcUA途径的关键酶基因pgcA、gtaB、tuaD,或共表达UDP-GlcNAc途径的关键酶基因glmS、glmM、glmU。
在本发明的一种实施方式中,所述共表达是指组合共表达,所述组合共表达是共表达tuaD和glmU,或共表达tuaD、glmU、gtaB、glmM、glmS。。
本发明的第二个目的是提供所述重组枯草芽孢杆菌的构建方法,是以整合共表达KfiC和KfiA基因的B.subtilis 168为宿主,以pP43NMK为表达载体共表达肝素前体合成途径的关键酶基因;所述共表达是指共表达UDP-GlcUA途径的关键酶基因pgcA、gtaB、tuaD,或共表达UDP-GlcNAc途径的关键酶基因glmS、glmM、glmU。
在本发明的一种实施方式中,所述共表达是指组合共表达,所述组合共表达是共表达tuaD和glmU,或共表达tuaD,gtaB,glmU,glmM和glmS。
本发明的第三个目的是提供一种发酵生产肝素前体的方法,所述方法是将所述重组菌接种至发酵培养基中,在37℃下培养24~60h。
在本发明的一种实施方式中,所述发酵培养基以蔗糖为碳源。
在本发明的一种实施方式中,所述发酵培养基的组成成为分:20g/L酵母粉,50g/L蔗糖,3.9g/L硫酸钾,1.5g/L硫酸镁,50mM磷酸盐缓冲液,pH 6.5-7.5。
在本发明的一种实施方式中,所述发酵培养基的组成成为分:20g/L酵母粉,15g/L蔗糖,3.9g/L硫酸钾,1.5g/L硫酸镁,50mM磷酸盐缓冲液,pH 6.5-7.5。
本发明还提供所述重组枯草芽孢杆菌在医药、保健品领域的应用,所述应用具体是制备含肝素的药品、医药配制品。
有益效果:本发明通过在产肝素前体的枯草芽孢杆菌中进行合成途径优化,即共表达UDP-GlcUA途径和UDP-GlcNAc途径中的关键酶基因,提高了肝素前体的产量,且重组质粒pP43-DU-PBMS的转入并没有成为菌体生长的负担,菌体量反而比对照菌提高了17.3%,具有较大的应用价值。首先,本发明过程中使用的宿主为食品级,可满足医疗卫生和食品安全的要求,无内毒素和病原感染的风险;其次,共表达tuaD,gtaB,glmU,glmM和glmS基因导致两前体物供应充足且比例平衡,并进一步通过3L罐补料分批培养,使肝素前体的积累达到最高,为7.25g/L,分子量为46.66kD。本研究为高产肝素前体提供了新策略,一方面可应用于其它糖胺聚糖,另一方面为工业化大规模生产肝素前体奠定了基础。
附图说明
图1为共表达途径基因的重组质粒构建示意图;
图2为PCR验证重组枯草芽孢杆菌的构建,M为5000bp的Marker,1-4分别表示含有质粒pP43-DBA,pP43-UMS,pP43-DU和pP43-DU-PBMS的重组菌;
图3为共表达途径基因后重组菌的肝素前体产量;
图4为重组菌B.subtilis E168H/pP43-DU-PBMS与对照株B.subtilis E168H在摇瓶培养中的细胞生长与产量曲线;
图5为重组菌B.subtilis E168H/pP43-DU-PBMS在3L罐中的补料分批培养。
具体实施方式
肝素前体发酵产物分子量检测分析方法:采用高效体积排阻色谱-多角度激光散射进行分析,选择示差折光检测器RI,使用凝胶色谱柱Ultrahydrogel Linear进行分析。流动相选择0.1M硝酸钠进行洗脱,流速为0.5mL/min,柱子温度设定为50℃,进样量为20μL,每个样品洗脱时间为20min,利用软件计算平均分子质量和多分散性系数。
实施例涉及的核苷酸序列信息:
(1)SEQ ID NO.1序列信息为枯草芽孢杆菌来源的葡萄糖磷酸变位酶的基因pgcA编码序列;
(2)SEQ ID NO.2序列信息为枯草芽孢杆菌来源的UDP-葡萄糖焦磷酸化酶的基因gtaB编码序列;
(3)SEQ ID NO.3序列信息为枯草芽孢杆菌来源的UDP-葡萄糖脱氢酶基因tuaD编码序列;
(4)SEQ ID NO.4序列信息为枯草芽孢杆菌来源的酰胺转移酶的基因glmS编码序列;
(5)SEQ ID NO.5序列信息为枯草芽孢杆菌来源的氨基葡糖磷酸变位酶的基因glmM的编码序列;
(6)SEQ ID NO.6序列信息为枯草芽孢杆菌来源的UDP-N-乙酰氨基葡糖焦磷酸化酶的基因glmU的编码序列;
实施例1共表达途径基因的重组枯草芽孢杆菌的构建
首先制备B.subtilis E168H感受态细胞,所需试剂为无机盐母液(g/L):K2HPO4140,KH2PO4 60,(NH4)2SO4 20,(Na3C6H5O7·2H2O)10,MgSO4·7H2O 2;GMI溶液(每100mL):无机盐母液9.6mL,20%葡萄糖2.5mL,5%水解酪蛋白0.4mL,10%酵母汁1mL;GMII溶液(每100mL):无机盐母液9.7mL,20%葡萄糖2.5mL,5%水解酪蛋白0.08mL,10%酵母汁0.04mL,1M MgCl20.25mL,1M CaCl20.05mL。将B.subtilis E168H单菌落接种于5mL GMI溶液,30℃、125rpm培养16h,取2mL培养液转接到18mL GMI溶液,37℃、200rpm培养3.5h,再取10mL培养液转接到90mL GMII溶液,37℃、200rpm培养90min,在4℃、5000g离心10min收集菌体,仅保留10mL液体重悬菌体,以500μL为单位分装。
以pP43NMK为表达载体,共表达肝素前体合成途径基因的4个重组型质粒pP43-DBA,pP43-UMS,pP43-DU和pP43-DU-PBMS(图1)为先前构建(公开于Production ofspecific-molecular-weight hyaluronan by metabolically engineered Bacillussubtilis 168,Metabolic Engineering,2016,Jinpeng),通过化转方式转化入B.subtilisE168H感受态细胞中,并涂布于含50μg/mL卡那霉素的筛选平板,挑选转化子进行PCR验证。转化pP43-DBA,pP43-UMS,pP43-DU和pP43-DU-PBMS质粒的重组菌分别用引物gtaB-F/pgcA-R,glmM-F/glmS-R,tuaD-F/glmU-R和glmU-F/gtaB-R验证,结果如图2所示,并命名重组菌为B.subtilis E168H/pP43-DBA,B.subtilis E168H/pP43-UMS,B.subtilis E168H/pP43-DU,B.subtilis E168H/pP43-DU-PBMS。
引物序列信息:5’-3’方向
gtaB-F:ATGAAAAAAGTACGTAAAGCCATAA
pgcA-R:TTATTTTGCTGTTGACTCAACAA
glmM-F:ATGGGCAAGTATTTTGGAACAGACG
glmS-R:TTACTCCACAGTAACACTCTTCGCA
tuaD-F:GTGAAAAAAATAGCTGTCATTGGAAC
glmU-R:TTATTTTTTATGAATATTTTTCACATAATC
glmU-F:ATGGATAAGCGGTTTGCAGTTG
gtaB-R:TTAGATTTCTTCTTTGTTTAGTAAAC
实施例2重组枯草芽孢杆菌的摇瓶发酵
分别挑取上述构建的4株重组枯草芽孢杆菌及对照菌B.subtilis E168H单菌落,接种于种子培养基,置于200rpm 37℃过夜培养16h。按体积比为10%的接种量转接于25mL发酵培养基中(250mL摇瓶),发酵培养基为:20g/L酵母粉,50g/L蔗糖,硫酸钾3.9g/L,硫酸镁1.5g/L,50mM磷酸盐缓冲液,pH 7.0,其中重组菌培养液中需添加50μg/mL卡那霉素。置于200rpm 37℃培养54h,并于接种后第2h添加木糖母液使其终浓度为20g/L进行诱导。
摇瓶培养中取样的发酵液经10000g离心5min后,向上清液中加入3倍体积的无水乙醇,混匀并于4℃放置1h,再次经10000g离心5min,收集沉淀,加入蒸馏水重新溶解沉淀,该纯化步骤重复三次。肝素前体的含量采用Bitter-Muir硫酸咔唑比色法测定,在比色管中加入1mL硼砂硫酸试剂和200μL经一定倍数稀释后的肝素前体样品,混匀并在沸水中煮15min,冷却至室温,再加入50μL咔唑试剂,混匀并再在沸水中煮15min,冷却后测定530nm处的吸光值,根据标准曲线计算产量。
根据附图3中第48h肝素前体产量,较对照菌株B.subtilis E168H(1.71g/L),含重组质粒的4株重组菌在摇瓶上产量均有提高,其中B.subtilis E168H/pP43-DBA产量为2.37g/L,B.subtilis E168H/pP43-UMS产量为2.20g/L,B.subtilis E168H/pP43-DU产量为2.52g/L,E168H/pP43-DU-PBMS产量最高,为2.89g/L,提高幅度最大,为69.01%。说明两前体物UDP-GlcUA和UDP-GlcNAc的比例平衡对加快肝素前体的聚合速度非常有利。并且,如图4所示,重组菌B.subtilis E168H/pP43-DU-PBMS在第12-24h进入菌体生长稳定期,OD600最高为22.4,而对照菌OD600最高为19.1,提高17.3%。与对照株相比,重组质粒pP43-DU-PBMS的转入并没有成为菌体生长的负担,菌体量反而稍有提高,另外,根据MALLS-SEC结果(表1),上述4株重组菌所产肝素前体的Mw分别为53.65kDa,73.83kDa,58.79kDa和75.90kDa,较对照菌(39.72kDa)也均增大。同时,产物的多分散性系数Ip在1.09-1.42之间,尤其是当前体物平衡时,产物分子量分布范围更集中,说明可通过途径优化实现更统一分子量的肝素前体的生产。
表1摇瓶中重组菌与对照菌在第48h时所产肝素前体的分子量比较
Figure BDA0001179447920000051
a数量平均分子量(Mn);b质量平均分子量(Mw);c聚合度(Ip=Mw/Mn).
实施例3重组枯草芽孢杆菌B.subtilis E168H/pP43-DU-PBMS的3L罐补料分批培养
将重组菌单菌落接种至150mL种子培养基中,37℃、200r/min培养16h,将种子培养液按体积比为10%的接种量转接至含1.35L发酵培养基的3L发酵罐中,并添加50μg/mL卡那霉素。在接种后第2h添加木糖溶液使终浓度为20g/L进行诱导。使用5M NaOH溶液控制pH为7.0,温度为37℃,搅拌转速在接种后8h内为600r/min,8h后为800r/min,通气量为2.0vvm。补料料液为800g/L的蔗糖母液,当发酵液中蔗糖浓度低于5g/L时开始补料,维持残糖浓度在0-5g/L。在8-12h期间流加速度分别为7.5、7.5、15、10g/L/h,此后保持5g/L/h的流速至发酵结束。同时采用HPSEC-MALLS测定发酵终点肝素前体的质量平均分子量(Mw),数量平均分子量(Mn)和分散系数Ip
如图5所示,可以看出大量的肝素前体的积累主要集中于菌体生长的稳定期,即细胞生长与产物生成非偶联。在发酵64h时,肝素前体的产量达到最高,为7.25g/L,是摇瓶水平的2.51倍,此后产量保持稳定。产物的Mw为46.66kDa,较摇瓶培养时降低,可能因为搅拌桨机械剪切力的作用使糖链切断,但Ip值1.28却显示3L罐发酵仍较好地保持了相对集中的分子量分布。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 江南大学
<120> 一种高产肝素前体的重组枯草芽孢杆菌及其应用
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 1746
<212> DNA
<213> 人工序列
<400> 1
atgacttgga gaaagagcta tgaacgctgg aaacagacag aacatttaga tctggaatta 60
aaagagcgcc ttattgaatt agagggagat gaacaggccc ttgaggactg tttctataaa 120
gaccttgaat tcggtaccgg cggaatgcgc ggggaaatcg gcgccgggac aaatcggatg 180
aatatttaca ctgtgcgcaa agcatcggcc gggtttgcgg catacatctc gaagcaaggt 240
gaggaagcga aaaaacgggg cgttgtcatt gcttatgatt cccgccataa gtctccggag 300
ttcgcgatgg aagcggcaaa aacacttgcg acacaaggca tccaaacata cgtgtttgat 360
gagcttcgcc cgacgccaga gctgtcattc gctgttagac agctgaacgc ttatggtgga 420
attgtggtaa cggcaagcca taacccgcct gaatataacg gctacaaagt atacggggat 480
gatggcggcc agctgcctcc aaaggaagcg gacatcgtca ttgagcaggt aaacgcgatt 540
gaaaatgagc tgacgatcac agtggacgaa gaaaataagt taaaagaaaa aggcttaatc 600
aaaatcatcg gtgaagatat tgataaagtt tatacagaaa aactgacgtc catttctgta 660
catcctgaat tatcggaaga agtagatgta aaggttgttt tcacaccgct gcatggaact 720
gcaaataaac cggtcagacg cggtcttgaa gcactcggct acaaaaatgt aacggttgtc 780
aaagaacagg aactgccgga ttcaaacttc tccactgtta catcgccgaa cccggaagag 840
catgcggcat tcgaatatgc cattaagctt ggggaggagc agaatgcaga tattctcatc 900
gcgacagatc ctgatgctga ccgcctcggc atcgcggtga aaaacgatca aggcaaatat 960
acagtgctga caggaaacca aaccggagcg ttgctgcttc attacctgct ttctgaaaag 1020
aaaaaacaag gcatcctgcc tgataacggt gttgttctca aaacgatcgt cacaagcgaa 1080
atcggccgtg ctgtagcttc ttcattcggc cttgatacga ttgatacgct gacaggcttt 1140
aagtttatcg gtgaaaagat taaggaatac gaagcatcag gccagtatac cttccaattc 1200
ggttatgaag agagctacgg ttatttaatc ggggattttg cccgcgataa ggacgccatt 1260
caggctgcgc ttttggcagt tgaagtttgc gcgttctata aaaaacaggg aatgtcattg 1320
tatgaggcgc tcatcaatct ctttaacgaa tatggttttt atcgtgaagg gctgaaatcc 1380
ctgacgctga aaggcaaaca aggagcagag caaattgaag cgattcttgc ttccttcaga 1440
caaaatccgc cgcagaaaat ggcgggcaaa caggttgtca cagcagaaga ttacgctgta 1500
agcaaacgga cgcttctgac tgaaagcaaa gaagaagcca tcgacttgcc aaaatcaaat 1560
gtattgaaat acttcctgga agacgggtct tggttctgtc tccgtccttc tggaactgag 1620
ccgaaggtta aattttattt cgccgtaaaa gggtcatctt tggaagacag tgaaaagcga 1680
cttgccgtcc tttctgaaga tgtaatgaag acggtggatg aaattgttga gtcaacagca 1740
aaataa 1746
<210> 2
<211> 879
<212> DNA
<213> 人工序列
<400> 2
atgaaaaaag tacgtaaagc cataattcca gcagcaggct taggaacacg ttttcttccg 60
gctacgaaag caatgccgaa agaaatgctt cctatcgttg ataaacctac cattcaatac 120
ataattgaag aagctgttga agccggtatt gaagatatta ttatcgtaac aggaaaaagc 180
aagcgtgcga ttgaggatca ttttgattac tctcctgagc ttgaaagaaa cctagaagaa 240
aaaggaaaaa ctgagctgct tgaaaaagtg aaaaaggctt ctaacctggc tgacattcac 300
tatatccgcc aaaaagaacc taaaggtctc ggacatgctg tctggtgcgc acgcaacttt 360
atcggcgatg agccgtttgc ggtactgctt ggtgacgata ttgttcaggc tgaaactcca 420
gggttgcgcc aattaatgga tgaatatgaa aaaacacttt cttctattat cggtgttcag 480
caggtgcccg aagaagaaac acaccgctac ggcattattg acccgctgac aagtgaaggc 540
cgccgttatc aggtgaaaaa cttcgttgaa aaaccgccta aaggcacagc accttctaat 600
cttgccatct taggccgtta cgtattcacg cctgagatct tcatgtattt agaagagcag 660
caggttggcg ccggcggaga aattcagctc acagacgcca ttcaaaagct gaatgaaatt 720
caaagagtgt ttgcttacga ttttgaaggc aagcgttatg atgttggtga aaagctcggc 780
tttatcacaa caactcttga atttgcgatg caggataaag agcttcgcga tcagctcgtt 840
ccatttatgg aaggtttact aaacaaagaa gaaatctaa 879
<210> 3
<211> 1386
<212> DNA
<213> 人工序列
<400> 3
gtgaaaaaaa tagctgtcat tggaacaggt tatgtaggac tcgtatcagg cacttgcttt 60
gcggagatcg gcaataaagt tgtttgctgt gatatcgatg aatcaaaaat cagaagcctg 120
aaaaatgggg taatcccaat ctatgaacca gggcttgcag acttagttga aaaaaatgtg 180
ctggatcagc gcctgacctt tacgaacgat atcccgtctg ccattcgggc ctcagatatt 240
atttatattg cagtcggaac gcctatgtcc aaaacaggtg aagctgattt aacgtacgtc 300
aaagcggcgg cgaaaacaat cggtgagcat cttaacggct acaaagtgat cgtaaataaa 360
agcacagtcc cggttggaac agggaaactg gtgcaatcta tcgttcaaaa agcctcaaag 420
gggagatact catttgatgt tgtatctaac cctgaattcc ttcgggaagg gtcagcgatt 480
catgacacga tgaatatgga gcgtgccgtg attggttcaa caagtcataa agccgctgcc 540
atcattgagg aacttcatca gccattccat gctcctgtca ttaaaacaaa cctagaaagt 600
gcagaaatga ttaaatacgc cgcgaatgca tttctggcga caaagatttc ctttatcaac 660
gatatcgcaa acatttgtga gcgagtcggc gcagacgttt caaaagttgc tgatggtgtt 720
ggtcttgaca gccgtatcgg cagaaagttc cttaaagctg gtattggatt cggcggttca 780
tgttttccaa aggatacaac cgcgctgctt caaatcgcaa aatcggcagg ctatccattc 840
aagctcatcg aagctgtcat tgaaacgaac gaaaagcagc gtgttcatat tgtagataaa 900
cttttgactg ttatgggaag cgtcaaaggg agaaccattt cagtcctggg attagccttc 960
aaaccgaata cgaacgatgt gagatccgct ccagcgcttg atattatccc aatgctgcag 1020
cagctgggcg cccatgtaaa agcatacgat ccgattgcta ttcctgaagc ttcagcgatc 1080
cttggcgaac aggtcgagta ttacacagat gtgtatgctg cgatggaaga cactgatgca 1140
tgcctgattt taacggattg gccggaagtg aaagaaatgg agcttgtaaa agtgaaaacc 1200
ctcttaaaac agccagtcat cattgacggc agaaatttat tttcacttga agagatgcag 1260
gcagccggat acatttatca ctctatcggc cgtcccgctg ttcggggaac ggaaccctct 1320
gacaagtatt ttccgggctt gccgcttgaa gaattggcta aagacttggg aagcgtcaat 1380
ttataa 1386
<210> 4
<211> 1803
<212> DNA
<213> 人工序列
<400> 4
atgtgtggaa tcgtaggtta tatcggtcag cttgatgcga aggaaatttt attaaaaggg 60
ttagagaagc ttgagtatcg cggttatgac tctgctggta ttgctgttgc caacgaacag 120
ggaatccatg tgttcaaaga aaaaggacgc attgcagatc ttcgtgaagt tgtggatgcc 180
aatgtagaag cgaaagccgg aattgggcat actcgctggg cgacacacgg cgaaccaagc 240
tatctgaacg ctcacccgca tcaaagcgca ctgggccgct ttacacttgt tcacaacggc 300
gtgatcgaga actatgttca gctgaagcaa gagtatttgc aagatgtaga gctcaaaagt 360
gacaccgata cagaagtagt cgttcaagta atcgagcaat tcgtcaatgg aggacttgag 420
acagaagaag cgttccgcaa aacacttaca ctgttaaaag gctcttatgc aattgcttta 480
ttcgataacg acaacagaga aacgattttt gtagcgaaaa acaaaagccc tctattagta 540
ggtcttggag atacattcaa cgtcgtagca tctgatgcga tggcgatgct tcaagtaacc 600
aacgaatacg tagagctgat ggataaagaa atggttatcg tcactgatga ccaagttgtc 660
atcaaaaacc ttgatggtga cgtgattaca cgtgcgtctt atattgctga gcttgatgcc 720
agtgatatcg aaaaaggcac gtaccctcac tacatgttga aagaaacgga tgagcagcct 780
gttgttatgc gcaaaatcat ccaaacgtat caagatgaaa acggcaagct gtctgtgcct 840
ggcgatatcg ctgccgctgt agcggaagcg gaccgcatct atatcattgg ctgcggaaca 900
agctaccatg caggacttgt cggtaaacaa tatattgaaa tgtgggcaaa cgtgccggtt 960
gaagtgcatg tagcgagtga attctcctac aacatgccgc ttctgtctaa gaaaccgctc 1020
ttcattttcc tttctcaaag cggagaaaca gcagacagcc gcgcggtact cgttcaagtc 1080
aaagcgctcg gacacaaagc cctgacaatc acaaacgtac ctggatcaac gctttctcgt 1140
gaagctgact atacattgct gcttcatgca ggccctgaga tcgctgttgc gtcaacgaaa 1200
gcatacactg cacaaatcgc agttctggcg gttcttgctt ctgtggctgc tgacaaaaat 1260
ggcatcaata tcggatttga cctcgtcaaa gaactcggta tcgctgcaaa cgcaatggaa 1320
gctctatgcg accagaaaga cgaaatggaa atgatcgctc gtgaatacct gactgtatcc 1380
agaaatgctt tcttcatcgg acgcggcctt gactacttcg tatgtgtcga aggcgcactg 1440
aagctgaaag agatttctta catccaggca gaaggttttg ccggcggtga gctaaagcac 1500
ggaacgattg ccttgatcga acaaggaaca ccagtattcg cactggcaac tcaagagcat 1560
gtaaacctaa gcatccgcgg aaacgtcaaa gaagttgctg ctcgcggagc aaacacatgc 1620
atcatctcac tgaaaggcct agacgatgcg gatgacagat tcgtattgcc ggaagtaaac 1680
ccagcgcttg ctccgttggt atctgttgtt ccattgcagc tgatcgctta ctatgctgca 1740
ctgcatcgcg gctgtgatgt ggataaacct cgtaaccttg cgaagagtgt tactgtggag 1800
taa 1803
<210> 5
<211> 1347
<212> DNA
<213> 人工序列
<400> 5
atgggcaagt attttggaac agacggtgta agaggtgtcg ccaatagtga gcttacacct 60
gagctggcct ttaaagtcgg acgtttcggc ggttatgtgc tgacaaaaga caaacaacgt 120
ccaaaagtgc tgataggccg cgatacacgc atctccggcc atatgctgga gggagccctt 180
gtcgccggac ttttatccat tggcgcagaa gtcatgcgcc tgggtgtcat ttctacacca 240
ggtgtatctt atttgacaaa agcgatggat gcagaggcgg gcgtcatgat ttccgcttct 300
cataacccag tgcaggataa cggcatcaaa ttctttgggg gagatggatt taagctttct 360
gatgaacagg aggctgaaat tgagcgcctg atggacgaac ctgaggataa gctgccaaga 420
cctgtcggag cagaccttgg acttgtaaac gattattttg aaggcggaca aaaatatctg 480
caattcttaa aacagacagc tgatgaagat ttcacaggca ttcatgtggc attggactgt 540
gccaatggcg caacgtcatc cttggcgaca cacctgtttg ctgatttaga tgcagatgtt 600
tctacaatgg ggacttcccc gaacggatta aacattaatg acggcgtcgg ttcgactcat 660
cccgaagcgc tcagcgcgtt tgtcaaagag aaaaacgcgg atctcggtct tgcgttcgac 720
ggtgacggcg accgcctgat tgctgtcgat gaaaaaggaa atattgtaga cggcgaccaa 780
atcatgtaca tatgctcaaa acatctgaaa tcagagggcc gtttaaagga tgatacagtg 840
gtttcaaccg tgatgagcaa cctcggcttc tataaggcgc tcgaaaaaga aggcatcaaa 900
agcgtgcaga cagctgtcgg cgaccgctac gtagtagaag caatgaaaaa agacggctac 960
aacgtcggcg gagagcagtc aggacatctt attttccttg attacaacac gacaggggac 1020
ggattattgt ctgctattat gctgatgaac actttaaaag caacaggcaa gccgctgtca 1080
gagcttgcag ctgaaatgca gaagttcccg cagctgttag tcaatgtgag agtgactgat 1140
aaatataaag ttgaagaaaa tgaaaaagta aaagcagtta tttctgaagt tgaaaaagaa 1200
atgaacggcg acggccggat tttggtgcgc ccttcaggaa ctgaaccgct cgtccgtgtc 1260
atggctgaag cgaagacgaa agagctgtgc gatgagtatg tcaatcgcat tgttgaagtc 1320
gtccggtcag aaatgggatt agagtaa 1347
<210> 6
<211> 1371
<212> DNA
<213> 人工序列
<400> 6
atggataagc ggtttgcagt tgttttagcg gctggacaag gaacgagaat gaaatcgaag 60
ctttataaag tccttcatcc agtttgcggt aagcctatgg tagagcacgt cgtggacgaa 120
gccttaaaat tatctttatc aaagcttgtc acgattgtcg gacatggtgc ggaagaagtg 180
aaaaagcagc ttggtgataa aagcgagtac gcgcttcaag caaaacagct tggcactgct 240
catgctgtaa aacaggcaca gccatttctt gctgacgaaa aaggcgtcac aattgtcatt 300
tgcggagata cgccgctttt gacagcagag acgatggaac agatgctgaa agaacataca 360
caaagagaag cgaaagctac gattttaact gcggttgcag aagatccaac tggatacggc 420
cgcattattc gcagcgaaaa cggagcggtt caaaaaatag ttgagcataa ggacgcctct 480
gaagaagaac gtcttgtaac tgagatcaac accggtacgt attgttttga caatgaagcg 540
ctatttcggg ctattgatca ggtgtctaat gataatgcac aaggcgagta ttatttgccg 600
gatgtcatag agattcttaa aaatgaaggc gaaactgttg ccgcttacca gactggtaat 660
ttccaagaaa cgctcggagt taatgataga gttgctcttt ctcaggcaga acaatttatg 720
aaagagcgca ttaataaacg gcatatgcaa aatggcgtga cgttgattga cccgatgaat 780
acgtatattt ctcctgacgc tgttatcgga agcgatactg tgatttaccc tggaactgtg 840
attaaaggtg aggtgcaaat cggagaagat acgattattg gccctcatac ggagattatg 900
aatagtgcca ttggcagccg tacggttatt aaacaatcgg tagtcaatca cagtaaagtg 960
gggaatgatg taaacatagg accttttgct cacatcagac ctgattctgt catcgggaat 1020
gaagtgaaga tcgggaattt tgtagaaatt aaaaagactc aattcggaga ccgaagcaag 1080
gcatctcatc taagctatgt cggcgatgct gaggtaggca ctgatgtaaa cctgggctgc 1140
ggttcaatta ctgtcaatta tgatggaaag aataagtatt tgacaaaaat tgaagatggc 1200
gcgtttatcg gctgcaattc caacttggtt gcccctgtca cagtcggaga aggcgcttat 1260
gtggcggcag gttcaactgt tacggaagat gtacctggaa aagcacttgc tattgccaga 1320
gcgagacaag taaataaaga cgattatgtg aaaaatattc ataaaaaata a 1371
<210> 7
<211> 25
<212> DNA
<213> 人工序列
<400> 7
atgaaaaaag tacgtaaagc cataa 25
<210> 8
<211> 23
<212> DNA
<213> 人工序列
<400> 8
ttattttgct gttgactcaa caa 23
<210> 9
<211> 25
<212> DNA
<213> 人工序列
<400> 9
atgggcaagt attttggaac agacg 25
<210> 10
<211> 25
<212> DNA
<213> 人工序列
<400> 10
ttactccaca gtaacactct tcgca 25
<210> 11
<211> 26
<212> DNA
<213> 人工序列
<400> 11
gtgaaaaaaa tagctgtcat tggaac 26
<210> 12
<211> 30
<212> DNA
<213> 人工序列
<400> 12
ttatttttta tgaatatttt tcacataatc 30
<210> 13
<211> 22
<212> DNA
<213> 人工序列
<400> 13
atggataagc ggtttgcagt tg 22
<210> 14
<211> 26
<212> DNA
<213> 人工序列
<400> 14
ttagatttct tctttgttta gtaaac 26

Claims (6)

1.一种高产肝素前体的重组枯草芽孢杆菌,其特征在于,是在枯草芽孢杆菌B.subtilis 168基因组整合表达KfiC和KfiA基因的基础上,以pP43NMK为表达载体共表达肝素前体合成途径的关键酶基因,所述共表达是指共表达tuaD、glmU、gtaB、glmM、glmS。
2.权利要求1所述重组枯草芽孢杆菌的构建方法,其特征在于,以整合共表达KfiC和KfiA基因的B.subtilis 168为宿主,以pP43NMK为表达载体共表达肝素前体合成途径的关键酶基因;所述共表达是指共表达tuaD、gtaB、glmU、glmM、glmS。
3.一种发酵生产肝素前体的方法,其特征在于,所述方法是将权利要求1所述的重组枯草芽孢杆菌接种至发酵培养基中,在37℃下培养24~60h。
4.根据权利要求3所述的方法,其特征在于,所述发酵培养基以蔗糖为碳源。
5.根据权利要求3所述的方法,其特征在于,所述发酵培养基的组成成为分:20g/L酵母粉,50g/L蔗糖,3.9g/L硫酸钾,1.5g/L硫酸镁,50mM磷酸盐缓冲液,pH 6.5-7.5。
6.权利要求1所述的重组枯草芽孢杆菌在医药领域制备含肝素的药品、医药配制品方面的应用。
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