CN109055292A - 一种生产l-木糖的重组变形假单胞菌及其应用 - Google Patents
一种生产l-木糖的重组变形假单胞菌及其应用 Download PDFInfo
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- 230000006798 recombination Effects 0.000 title claims abstract description 9
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- 239000008103 glucose Substances 0.000 claims abstract description 25
- 108090000854 Oxidoreductases Proteins 0.000 claims abstract description 17
- 108010011939 Pyruvate Decarboxylase Proteins 0.000 claims abstract description 17
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- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 claims abstract description 6
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 claims abstract description 6
- 239000000174 gluconic acid Substances 0.000 claims abstract description 6
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- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 claims description 16
- 229940050410 gluconate Drugs 0.000 claims description 16
- RKFLKNFLAJISPF-OGXRZFKVSA-L calcium;(3s,4s)-3,4,6-trihydroxy-2,5-dioxohexanoate Chemical compound [Ca+2].OCC(=O)[C@@H](O)[C@H](O)C(=O)C([O-])=O.OCC(=O)[C@@H](O)[C@H](O)C(=O)C([O-])=O RKFLKNFLAJISPF-OGXRZFKVSA-L 0.000 claims description 13
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- 239000002609 medium Substances 0.000 claims description 10
- 239000007836 KH2PO4 Substances 0.000 claims description 8
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 8
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 5
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- NAOLWIGVYRIGTP-UHFFFAOYSA-N 1,3,5-trihydroxyanthracene-9,10-dione Chemical compound C1=CC(O)=C2C(=O)C3=CC(O)=CC(O)=C3C(=O)C2=C1 NAOLWIGVYRIGTP-UHFFFAOYSA-N 0.000 claims description 3
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- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
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- ZAQJHHRNXZUBTE-WVZVXSGGSA-N L-xylulose Chemical compound OC[C@H](O)[C@@H](O)C(=O)CO ZAQJHHRNXZUBTE-WVZVXSGGSA-N 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 2
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- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 2
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- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 2
- 229960002675 xylitol Drugs 0.000 description 2
- HLLSOEKIMZEGFV-UHFFFAOYSA-N 4-(dibutylsulfamoyl)benzoic acid Chemical compound CCCCN(CCCC)S(=O)(=O)C1=CC=C(C(O)=O)C=C1 HLLSOEKIMZEGFV-UHFFFAOYSA-N 0.000 description 1
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- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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- 150000002972 pentoses Chemical class 0.000 description 1
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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Abstract
本发明公开了一种生产L‑木糖的重组变形假单胞菌及其应用,属于生物工程技术领域。本发明将合成的2‑酮基葡萄糖酸还原酶基因和来源于Corynebaterium ATCC 31090的2,5‑二酮基葡萄糖酸还原酶基因和Saccharomyces cerevisiae的丙酮酸脱羧酶基因,以双质粒系统在宿主变形假单胞菌中成功表达,获得的基因工程菌株在摇瓶上发酵56h,L‑木糖的产量可达16.2g/L,转化率达到了20.3%,在3L和15L发酵罐上分别发酵48h和44h,L‑木糖的产量分别可达37.6g/L和45.8g/L,葡萄糖转化率分别为47.0%和57.3%。该方法原料成本低,对环境无污染,且操作简单,具有重要的经济效益和社会效益。
Description
技术领域
本发明涉及一种生产L-木糖的重组变形假单胞菌及其应用,属于生物工程技术领域。
背景技术
木糖是一种五碳糖,L-木糖主要存在于植物和动物体内。木糖属于无热量甜味剂,广泛应用于食品、医药、化工等领域。食用木糖能改善人体的微生物环境,提高机体免疫能力,是糖尿病人理想的甜味剂。
目前制备木糖均采用酸法提取,酸法生产木糖多采用硫酸进行水解,但酸法生产木糖有如下缺点:①生产过程中采用大量硫酸,硫酸使原料中的纤维素和半纤维素及其它糖类物质水解,产生多种副产物,影响后提取工艺和产品质量。②酸法提取木糖由于生产过程中加入大量强酸强碱液体,生产过程中产生大量生产废液,对环境造成了巨大污染。③由于酸水解的糖液杂质多,使得后处理效率低,化工程序多,对设备要求高,增加环保成本。
目前,L-木糖的生产主要是由化学合成的方法,存在上述的不利因素。L-木糖也可由L-木酮糖转化生成,但是L-木酮糖属于稀有糖,价格较高,不宜作为生产L-木糖的原料。
变形假单胞菌属于假单胞菌属,为革兰氏阴性杆菌。假单胞菌中葡萄糖的代谢是通过ED途径(Entner-Doudoroff pathway)完成的,在好氧条件下,葡萄糖在周质空间被氧化为葡萄糖酸,研究表明,在假单胞菌中大多数由葡萄糖产生的葡萄糖酸(约90%)被转化为2-酮基葡萄糖酸(2KGA)。目前,变形假单胞菌(Pseudomonas plecoglossicida)是国内2KGA生产的主要工业用菌。
发明内容
本发明的第一个目的是提供一种生产L-木糖的重组菌,是以变形假单胞菌为宿主,利用双质粒表达系统表达2-酮基葡萄糖酸脱氢酶基因、2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因。
在本发明的一种实施方式中,所述双质粒表达系统包括pME6032质粒和pBBR1MCS-2质粒。
在本发明的一种实施方式中,所述pME6032质粒用于表达2-酮基葡萄糖酸脱氢酶基因。
在本发明的一种实施方式中,所述pBBR1MCS-2质粒用于表达2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因。
在本发明的一种实施方式中,所述2-酮基葡萄糖酸脱氢酶基因2GADH由三个亚基组成,分别表示为2GADH-1,2GADH-2,2GADH-3,其序列分别以SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示,以pME6032质粒为载体,在变形假单胞菌中表达SEQ ID NO.1、SEQ IDNO.2、SEQ ID NO.3所示基因2GADH的三个亚基。
在本发明的一种实施方式中,所述2,5-二酮基葡萄糖酸还原酶基因2,5DKG来自Corynebaterium ATCC 31090。
在本发明的一种实施方式中,所述丙酮酸脱羧酶基因PDC来自Saccharomycescerevisiae。
在本发明的一种实施方式中,所述变形假单胞菌宿主包括Pseudomonasplecoglossicida CGMCC 7150、Pseudomonas plecoglossicida CGMCC 1.16111、Pseudomonas plecoglossicida CGMCC 1.12685、Pseudomonas plecoglossicida CGMCC1.761中的任一种。
本发明的第二个目的是提供一种生产L-木糖的重组变形假单胞菌的构建方法,所述构建方法的具体步骤为:
(1)通过化学合成SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示基因,将上述序列同时与载体pME6032连接形成重组质粒pME6032-2gadh;
(2)将上述重组质粒pME6032-2gadh转化至变形假单胞菌中得到重组变形假单胞菌;
(3)分别扩增2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因,将上述序列同时与载体pBBR1MCS-2连接形成重组质粒pBBR1MCS-2-25dkg-pdc;
(4)再将步骤(3)所得的重组质粒pBBR1MCS-2-25dkg-pdc转化至含有重组质粒pME6032-2gadh的变形假单胞菌中得到含有双质粒的重组菌。
本发明的第三个目的是提供所述变形假单胞菌在生产L-木糖中的应用,将所述重组菌培养至OD650为0.6-0.8,加入IPTG诱导2-酮基葡萄糖酸脱氢酶基因、2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因的表达,以葡萄糖为底物合成L-木糖。
进一步得,所述应用中的种子培养基:葡萄糖14.0-15.0g/L,玉米浆3.5-4.0g/L,尿素1.5-2.5g/L,KH2PO4 1.5-2.5g/L,MgS04·7H2O 0.4-0.6g/L,CaCO3 0.8-1.2g/L,pH值7.0;所述应用中的发酵培养基:葡萄糖75.0-85.0g/L,玉米浆3.0-4.0g/L,尿素1.5-2.5g/L,KH2PO4 1.5-2.5g/L,MgSO4·7H2O 0.4-0.6g/L,CaCO3 9.0-11.0g/L,pH值6.8。
本发明取得有益效果:
(1)变形假单胞菌是国内2-酮基葡萄糖酸(2KGA)生产的主要工业用菌,在工业生产中可达到较高的糖酸转化率,采用本发明的方法,可以将2-酮基葡萄糖酸在2-酮基葡萄糖酸脱氢酶、2,5-二酮基葡萄糖酸还原酶和丙酮酸脱羧酶的催化下生成L-木糖,为L-木糖的生产提供了新的原料和方法。
(2)本发明使用发酵法生产L-木糖的过程工艺操作简单,不会给环境带来污染,以葡萄糖为底物,摇瓶上发酵56h,L-木糖的产量可达16.2g/L,转化率达到了20.3%,在3L和15L发酵罐上分别发酵48h和44h,L-木糖的产量分别可达37.6g/L和45.8g/L,葡萄糖转化率分别为47.0%和57.3%。
(3)本发明方法可直接用于酵母菌的生物转化实验,生产木糖醇,此种方法不需要脱毒,发酵性能好,利于木糖转化为木糖醇。因此,本发明制备L-木糖的方法为以后绿色工业化生产奠定基础。
附图说明
图1所示为重组菌株P.plecoglossicida-2gadh-25dkg-pdc的摇瓶发酵结果。
图2所示为重组菌株P.plecoglossicida-2gadh-25dkg-pdc的3L罐分批发酵结果。
图3所示为重组菌株P.plecoglossicida-2gadh-25dkg-pdc的15L罐分批发酵结果。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细的说明。
L-木糖浓度通过高效液相色谱法测定。高效液相色谱条件:色谱柱为HPX-87H(Bio-Rad Hercules),柱温35℃;检测器为示差折光检测器;流动相为5mM H2SO4,流速为0.6mL/min。发酵上清液通过0.22μm微孔过滤除去杂质后直接用于L-木糖的检测。
葡萄糖浓度通过国产SBA-40C生物传感分析仪测定。
葡萄糖转化率的计算方法:氨糖总量/总耗糖量×100%。
2-酮基葡萄糖酸脱氢酶基因2GADH亚基1序列如SEQ ID NO.1所示;
2-酮基葡萄糖酸脱氢酶基因2GADH亚基2序列如SEQ ID NO.2所示;
2-酮基葡萄糖酸脱氢酶基因2GADH亚基3序列如SEQ ID NO.3所示;
引物如表1所示,
表1相关引物
实施例1
(1)重组质粒pME6032-2gadh的构建及鉴定
通过化学合成获得序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示序列,设计引物(2GADH-1、2GADH-2、2GADH-3,见表1),将以上2-酮基葡萄糖酸脱氢酶基因(2GADH)的3个亚基的基因以酶切位点EcoRI、SacI、KpnI、NcoI依次在pME6032质粒16℃过夜连接,连接产物pME6032-gadh化学法转化至大肠杆菌JM109感受态细胞。将转化液涂布于含50mg/L四环素的LB平板,提取质粒测序验证构建的重组质粒。
(2)重组菌株P.plecoglossicida-2gadh的构建
将(1)中构建完成的质粒pME6032-2gadh通过电转化的方法转入出发菌株Pseudomonas.plecoglossicida CGMCC 7150中,通过四环素筛选得到阳性菌株P.plecoglossicida-2gadh。
(3)重组质粒pBBR1MCS-2-25dkg-pdc的构建及鉴定
分别以Corynebaterium ATCC 31090和Saccharomyces cerevisiae基因组为模板,设计引物(2,5DKG、PDC,见表1),扩增2,5-二酮基葡萄糖酸还原酶基因(2,5DKG)和丙酮酸脱羧酶基因(PDC),将2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因以酶切位点KpnI、EcoRI、BamHI依次在pBBR1MCS-2质粒16℃过夜连接,连接产物pBBR1MCS-2-25dkg-pdc化学法转化至大肠杆菌JM109感受态细胞。将转化液涂布于含50mg/L卡纳霉素的LB平板,提取质粒测序验证构建的重组质粒。
(4)重组菌株P.plecoglossicida-2gadh-25dkg-pdc的构建
将(3)中构建完成的质粒pBBR1MCS-2-25dkg-pdc通过电转化的方法转入(2)中得到的菌株P.plecoglossicida-2gadh中,通过氨苄青霉素筛选得到阳性菌株P.plecoglossicida-2gadh-25dkg-pdc。
实施例2
出发菌株为Pseudomonas plecoglossicida CGMCC 1.12685,其余步骤与实施例1相同,筛选得到阳性重组菌株P.plecoglossicida1-2gadh-25dkg-pdc。
实施例3重组菌株P.plecoglossicida-2gadh-25dkg-pdc的培养及L-木糖发酵
种子培养基:葡萄糖15.0g/L,玉米浆4.0g/L,尿素2.0g/L,KH2PO4 2.0g/L,MgS04·7H2O 0.5g/L,CaCO3 1.0g/L,pH值7.0。
发酵培养基:葡萄糖80.0g/L,玉米浆4.0g/L,尿素2.0g/L,KH2PO4 2.0g/L,MgSO4·7H2O 0.5g/L,CaCO3 10.0g/L,pH值6.8。
摇瓶培养:取适量菌(实施例1中所得)悬液接种于装有50ml种子培养基的500ml摇瓶中,于30℃,220r/min条件下培养16-20h,以10%的接种量转移至加有50ml发酵培养基的500ml摇瓶中,于30℃、220r/min条件下培养至OD650为0.6时,采用IPTG进行诱导,终浓度为0.5mM。诱导温度为25℃,发酵至64h,定期取样。
测定L-木糖的产量,结果如图1所示,发酵56h时,重组菌株P.plecoglossicida-2gadh-25dkg-pdc生产L-木糖的总量达到最大值,为16.2g/L,葡萄糖转化率为20.3%。
实施例4重组菌株P.plecoglossicida-2gadh-25dkg-pdc的3L发酵罐发酵
3L罐上发酵培养基及条件如下:
发酵培养基:葡萄糖80.0g/L,玉米浆4.0g/L,尿素2.0g/L,KH2PO4 2.0g/L,MgSO4·7H2O 0.5g/L,CaCO3 10.0g/L,pH值6.8。
发酵条件:初始装液量为2.0L,种子接种量为10%,初始发酵条件为:培养温度为30℃,pH为6.8,搅拌速度为400rpm,通气量为1.5vvm,当菌体OD650为0.6时,采用IPTG进行诱导,终浓度为0.5mM,诱导温度为25℃,发酵至64h,定期取样。
测定L-木糖的产量,结果如图2所示,发酵48h时,重组菌株P.plecoglossicida-2gadh-25dkg-pdc生产L-木糖的总量达到最大值,为37.6g/L,葡萄糖转化率为47.0%。
实施例5重组菌株P.plecoglossicida-2gadh-25dkg-pdc的15L发酵罐发酵
15L罐上发酵培养基及条件如下:
发酵培养基:葡萄糖80.0g/L,玉米浆4.0g/L,尿素2.0g/L,KH2PO4 2.0g/L,MgSO4·7H2O 0.5g/L,CaCO3 10.0g/L,pH值6.8。
发酵条件:初始装液量为10.0L,种子接种量为10%,初始发酵条件为:培养温度为30℃,pH为6.8,搅拌速度为400rpm,通气量为0.8vvm,罐压0.5bar,当菌体OD650为0.6时,采用IPTG进行诱导,终浓度为0.5mM,诱导温度为25℃,发酵至64h,定期取样。
测定L-木糖的产量,结果如图3所示,发酵44h时,重组菌株P.plecoglossicida-2gadh-25dkg-pdc生产L-木糖的总量达到最大值,为45.8g/L,葡萄糖转化率为57.3%。
对比例1采用pME6032单质粒表达构建重组菌
采用实施例1的策略,将2-酮基葡萄糖酸脱氢酶基因(2GADH)的3个亚基的基因、2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因以酶切位点EcoRI、SacI、KpnI、NcoI、BgiII、XhoI依次与pME6032质粒连接,构建完成的质粒pME6032-2gadh-25dkg-pdc表达在P.plecoglossicida CGMCC 7150中,结果显示,无法成功表达。
对比例2采用pBBR1MCS单质粒表达构建重组菌
采用实施例1的策略,将2-酮基葡萄糖酸脱氢酶基因(2GADH)的3个亚基的基因、2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因以酶切位点KpnI、XhoI、HindIII、EcoRI、BamHI、SacI依次与pBBR1MCS质粒连接,构建完成的质粒pBBR1MCS-2gadh-25dkg-pdc表达在P.plecoglossicida CGMCC 7150中,结果显示,无法成功表达。
对比例3:采用来源于欧文氏菌的2-酮基葡萄糖酸脱氢酶基因(2GADH)
出发菌株为P.plecoglossicida CGMCC 7150,步骤与实施例1相同,发现来源于欧文氏菌的2-酮基葡萄糖酸脱氢酶基因(2GADH)在宿主菌株中不能成功表达。
对比例4:重组菌株P.plecoglossicida-2gadh-25dkg-pdc发酵培养基调整
具体实施方式同实施例3,区别在于发酵培养基中葡萄糖、玉米浆他尿素的浓度分别如下表1所示,结果显示当葡萄糖、玉米浆和尿素浓度分别为80.0g/L、4.0g/L和2.0g/L时,L-木糖产量和转化率可达到相对较高,分别为16.2g/L和20.3%。
表1发酵培养基的成分调整及对应产量和转化率
编号 | 葡萄糖(g/L) | 玉米浆(g/L) | 尿素(g/L) | L-木糖(g/L) | 转化率(100%) |
1 | 70.0 | 4.0 | 2.0 | 12.0 | 17.1 |
2 | 80.0 | 4.0 | 2.0 | 16.2 | 20.3 |
3 | 90.0 | 4.0 | 2.0 | 16.4 | 18.2 |
4 | 80.0 | 2.0 | 2.0 | 13.5 | 15.9 |
5 | 80.0 | 5.0 | 2.0 | 14.0 | 17.5 |
6 | 80.0 | 4.0 | 1.0 | 13.4 | 16.8 |
7 | 80.0 | 4.0 | 3.0 | 13.0 | 16.3 |
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> 上海凌凯医药科技有限公司
<120> 一种生产L-木糖的重组变形假单胞菌及其应用
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gctctgtctc gtactatcac cgaacacaaa cagattgatt ctgttttagc agcgcgtttc 240
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ctgcagtctc cggatgattc tgcgcagcag ctgctgaaca aagcgaccca ggctggtctg 360
catgattttc tgtatcagat cgttgttgct tggtataccg gtaccgttgg tgatgattac 420
cacggtaccc tggttgctta taaacaggct ttaatgtatc agaccgtttc tgatggcctg 480
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gttctggaag cgggtctgcg tatcgatcgc gcccaggccg ttgaaaactg gcgtaacatg 180
ccgtttgcga accgtgcagg cagcgatttc cagggtctgt atccgcagtc caaattcgcg 240
ccggcgccgc tgtacttccc acgtaacaac tacgttaacg ttaccggccc gaacgcggat 300
tccttccagc agggttacct gcgtaccgtt ggtggtacca cgtggcactg ggcggcgagc 360
tgctggcgtc accacccgag cgatttcgtg atgcagtcca aatacggtgt tggtcgtgat 420
tggccaatcg gctatgatga actggaaccg tggtactgca aagcagaaaa cgaaatcggt 480
gttgctggcc cgaacgaccc ggcgcgtcag agcccgaccg aacgtagcca gccgtatccg 540
atggatatgg tgccgttcgc acacggtgat aactacttcg cgtccgttgt taacccgcat 600
ggctacaacc tggttccgat tccgcagggc cgttccaccc gtccgtggga aggtcgtccg 660
acctgctgcg gcaacaacaa ctgtcagccg atctgtccga tcggtgcgat gtacaacggt 720
atccaccacg ttgaacgtgc ggaacgtaac ggcgcggtgg tgctggcaga agctgttgtt 780
tacaagatgg ataccgattc caacaaccgt attaccgctg tacactggct ggacaccagt 840
ggcgcaagcc acaaagccac cgctaaagcg tttgcgctgg cgtgtaacgg tatcgaaacc 900
ccgcgtctgc tgctgatggc tgcgaacgat gcgaacccga acggcattgc caacgcgagc 960
gatatggttg gccgtaatat gatggaccat agcggttttc actgtagctt cctgaccaaa 1020
gagccggttt ggctgggtaa aggtccggcg cagtcttcct gcatggtggg ctaccgtgac 1080
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gttaccgcga cccaacaggc catgaaaaaa ggtctggttg gcaaagctct ggatgaagaa 1200
atccgttatc gtgcggttca cagcgtggat ctgtccatct ctctggaacc gctgccggac 1260
ccggaaaacc gtctgaccct gtctaaaacc cgtaaagatc cgcacggcct gccgtgcccg 1320
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caggcatgcc acaccgcgcc gggtagcgcg accgcgttct ccggtggcta cgcgatcgcg 180
tccccgatgg gcgtgatcta ctctaccaat atcaccccga gcgcggatgg tatcggcccg 240
tactctgaag cggaattttc ccaggcggtg cgtcacggcg tgcgtgcgga tggcgcgcag 300
ctgtacccgg cgatgccgta cacctcttat agcaaaatca ctgacgaaga tctgcatgct 360
ctgtactact acttcatgca cggcgttaaa ccggtggaac agaaaaaccg ccagaccagc 420
ctgccgttcc cgttcaacct gcgtttctcc atgttcttct ggaacctgat gttcgcggat 480
gataaaccgt acctgagcga tgatagccag tctgctgaat ggaaccgtgg taactacctg 540
gtgaacggcc tggcgcactg caacacctgc cacaccccgc gtggcgttct gatgcaggaa 600
gcaggtaacc gcccgctggc gggtgcgccg attggtagct ggtacgctcc gaacattacc 660
tccgatgcga tcagcggcat cggtggttgg cgtaacgatg aactggtgca gtacctgaaa 720
accggccgtg cggaaggtaa aaaccaggcg gcaggtggta tggcggaagc ggttgaacac 780
tctctgcagt acctgagcga tagcgatctg aaagcgattg cggtttatct gaaaagcacc 840
accccgattc gtgatgaagg tgatacccag ccggcgtact ccttcggtaa accggcggat 900
gttgaaaaca gcatccgcgg tcgtaacgcc aacaacgcta accactctct gaccaacggt 960
gctgcgctgt tctctggcaa ctgcgcgtct tgccaccagc cggatggcgc gggctctgca 1020
aatcaggcgt atccgtctct gttccacaac accgccaccg gcatgcataa cccggcgaac 1080
ctgattgcgg cgatcctgtt cggcgttcag cgtaacaccg ctgcgggcca ggtcctgatg 1140
ccgggattta gtagcccatc ttacgtagat aaattgagtg acgcacaggt agcagacatt 1200
agtaatttcg ttctggcgca ctatggtaac ccggaagtta ccgtttctgc aggtgatgtt 1260
gcgtgggttc gtcagggcgg ccacccgccg ctgctggcgc gtgcgcagcc gtggatcatg 1320
ccgggcatcg tggcgctgat cgtgatcctg ctggcggttt gcgcgggtct gacctggcgt 1380
cgtcgtcgtc gtatcgcgcg tgataacggt taa 1413
<210> 4
<211> 33
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<213> 人工序列
<400> 4
cgcggatccg atgaacctga aaatcgaacc gga 33
<210> 5
<211> 33
<212> DNA
<213> 人工序列
<400> 5
cccaagcttt tacaggtttt caatcagaga cgg 33
<210> 6
<211> 34
<212> DNA
<213> 人工序列
<400> 6
cgcggatccg atgaaaaaac cggttttcac cgcg 34
<210> 7
<211> 35
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<213> 人工序列
<400> 7
cccaagcttt catgcatcac ctttcatacg caggc 35
<210> 8
<211> 32
<212> DNA
<213> 人工序列
<400> 8
cgcggatccg atgaaacagc tgctgatggc aa 32
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<400> 10
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<210> 11
<211> 33
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<400> 11
acgcgtcgac tcatccattg tctcgggcta tcc 33
<210> 12
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<400> 12
catgccatgg attcaattac tttgggtaaa tatttgttcg 40
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<212> DNA
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Claims (10)
1.一种生产L-木糖的重组菌,其特征在于,所述重组菌是以变形假单胞菌为宿主,表达2-酮基葡萄糖酸脱氢酶基因、2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因的重组菌。
2.根据权利要求1所述的一种生产L-木糖的重组菌,其特征在于,以双质粒表达系统表达2-酮基葡萄糖酸脱氢酶基因、2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因,所述双质粒表达系统包括pME6032质粒和pBBR1MCS-2质粒。
3.根据权利要求2所述的一种生产L-木糖的重组菌,其特征在于,所述pME6032质粒用于表达2-酮基葡萄糖酸脱氢酶基因,所述pBBR1MCS-2质粒用于表达2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因。
4.根据权利要求1-3任一所述的一种生产L-木糖的重组菌,其特征在于,所述2-酮基葡萄糖酸脱氢酶2GADH由三个亚基组成,分别如序列SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示;所述2,5-二酮基葡萄糖酸还原酶基因2,5DKG来自Corynebaterium ATCC 31090;所述丙酮酸脱羧酶基因PDC来自Saccharomyces cerevisiae。
5.根据权利要求1所述的一种生产L-木糖的重组菌,其特征在于,所述变形假单胞菌宿主包括Pseudomonas plecoglossicida CGMCC 7150、Pseudomonas plecoglossicidaCGMCC 1.16111、Pseudomonas plecoglossicida CGMCC 1.12685、Pseudomonasplecoglossicida CGMCC 1.761中任一种。
6.权利要求1-5所述的一种生产L-木糖的重组菌的构建方法,其特征在于,包括以下步骤:
(1)将SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3所示基因同时与载体pME6032质粒连接形成重组质粒pME6032-2gadh;
(2)将上述重组质粒pME6032-2gadh转化至变形假单胞菌中得到重组变形假单胞菌;
(3)分别扩增2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因,将二者序列同时与载体pBBR1MCS-2连接形成重组质粒pBBR1MCS-2-25dkg-pdc;
(4)将步骤(3)所得的重组质粒pBBR1MCS-2-25dkg-pdc转化至含有重组质粒pME6032-2gadh的变形假单胞菌中得到含有双质粒的重组菌。
7.应用权利要求1-5任一所述的重组菌生产L-木糖的方法,其特征在于,将所述重组菌在发酵培养基中培养至OD650为0.6-0.8,加入IPTG诱导2-酮基葡萄糖酸脱氢酶基因、2,5-二酮基葡萄糖酸还原酶基因和丙酮酸脱羧酶基因的表达,以葡萄糖为底物合成L-木糖。
8.根据权利要求7所述的方法,其特征在于,所述发酵培养基:葡萄糖75.0-85.0g/L,玉米浆3.0-4.0g/L,尿素1.5-2.5g/L,KH2PO4 1.5-2.5g/L,MgSO4·7H2O 0.4-0.6g/L,CaCO39.0-11.0g/L,pH值6.8。
9.根据权利要求7或8所述的方法,其特征在于,所述重组菌在发酵前需在种子培养基上培养,所述种子培养基:葡萄糖14.0-15.0g/L,玉米浆3.5-4.0g/L,尿素1.5-2.5g/L,KH2PO4 1.5-2.5g/L,MgS04·7H2O 0.4-0.6g/L,CaCO3 0.8-1.2g/L,pH值7.0。
10.权利要求1~5任一所述的一种生产L-木糖的重组菌在食品、医药、化工领域的应用。
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CN114134057A (zh) * | 2021-12-03 | 2022-03-04 | 河北省科学院生物研究所 | 一株酿酒酵母swgcjm001及其培养方法和应用 |
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