CN112680389B - 一种提高微生物对甲醇的耐受性和利用率的方法 - Google Patents
一种提高微生物对甲醇的耐受性和利用率的方法 Download PDFInfo
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Abstract
本发明公开了通过弱化菌株中cgl0833基因的表达来构建甲醇耐受性提高的菌株的方法。本发明还公开了所构建的甲醇耐受性提高的甲醇生物转化菌株以及利用所述菌株进行甲醇生物转化的方法。本发明构建的菌株在利用甲醇和其它辅助碳源组成的混合碳源时，甲醇的利用比例显著提高，从而实质性提高了菌株对于甲醇的利用效率。
Description
技术领域
本发明涉及生物技术领域。具体地说，本发明涉及提高菌株对甲醇的耐受性和利用率的方法、这种方法获得的菌株以及这种方法和获得的菌株在生物转化甲醇中的应用。
背景技术
甲醇是煤炭化工、页岩气化工和工农业废弃物气化的初级平台产品，我国甲醇产能已达8000万吨/年，占国际总量50％以上，呈现过剩趋势，甲醇经济的发展急需配套的甲醇转化利用技术【陈继军,陈光达,2016；张世新等,2013】。生物转化具有产品多样、产品选择性高和过程绿色环保等特点。因此，改造常用的平台菌株，开发甲醇生物转化方法，发展基于甲醇的生物制造产业，具有重要意义。目前已经发现多条甲醇生物转化途径，例如核酮糖单磷酸途径(RuMP途径)、木酮糖单磷酸途径、核酮糖二磷酸途径和丝氨酸循环等。其中，RuMP途径是目前研究和应用较多的甲醇生物转化途径。在RuMP途径中，甲醇首先被甲醇脱氢酶(Mdh)氧化成甲醛，甲醛与核酮糖-5-磷酸(Ru5P)在3-己酮糖-6-磷酸合成酶(Hps)的催化下生成己酮糖-6-磷酸(H6P)，H6P被6-磷酸-3-己酮糖异构酶(Phi)催化生成果糖-6-磷酸(F6P)，F6P进入糖酵解途径被利用，或通过戊糖磷酸途径进行碳重排再生Ru5P(Whitaker,W.B.,Sandoval,N.R.,Bennett,R.K.,Fast,A.G.,Papoutsakis,E.T.,2015.Syntheticmethylotrophy:engineering the production of biofuels and chemicals based onthe biology of aerobic methanol utilization.Curr.Opin.Biotechnol.33,165–175.)。
现有技术主要是通过在平台菌株如大肠杆菌(Escherichia coli)和谷氨酸棒杆菌(Corynebacterium glutamicum)中阻断甲醛氧化生成甲酸和二氧化碳的途径，减少碳损失，同时表达Mdh、Hps和Phi，构建RuMP途径，赋予菌株生物转化甲醇的能力。例如，Witthoff等研究者在C.glutamicum中失活了负责氧化甲醛生成甲酸的关键酶AdhE和Ald，同时表达了Mdh、Hps和Phi。所获得的基因工程菌株在甲醇作为唯一碳源的无机盐培养基中无法生长，在含有葡萄糖和甲醇的无机盐培养基中可以生长，最终消耗55mM葡萄糖和约80mM甲醇，甲醇与葡萄糖利用量的比例约为1.45:1(Witthoff,S.,Schmitz,K.,Niedenfuhr,S.,Noh,K.,Noack,S.,Bott,M.,Marienhagen,J.,2015.Metabolic engineering ofCorynebacterium glutamicum for methanol metabolism.Appl.Environ.Microbiol.81,2215-2225)。Bennett等研究者在E.coli中失活了负责氧化甲醛生成甲酸的关键酶FrmA，同时表达了Mdh、Hps和Phi，以及来源于天然甲醇利用菌中的戊糖磷酸途径关键酶。所获得的基因工程菌株在甲醇作为唯一碳源的无机盐培养基中无法生长，在含有葡萄糖和甲醇的无机盐培养基中可以生长，最终消耗约275mM葡萄糖和38.3mM甲醇，甲醇与葡萄糖利用量的比例约为0.14:1(Bennett,R.K.,Gonzalez,J.E.,Whitaker,W.B.,Antoniewicz,M.R.,Papoutsakis,E.T.,2018.Expression of heterologous non-oxidative pentosephosphate pathway from Bacillus methanolicus and phosphoglucose isomerasedeletion improves methanol assimilation and metabolite production by asynthetic Escherichia coli methylotroph.Metab.Eng.45,75-85)。Tuyishime等研究者在C.glutamicum中失活了负责氧化甲醛生成甲酸的关键酶AdhE和Ald，失活了戊糖磷酸途径的关键酶核糖磷酸异构酶RpiB，同时表达了Mdh、Hps和Phi，所获得的基因工程菌株在甲醇作为唯一碳源的无机盐培养基中无法生长，在含有木糖和甲醇的无机盐培养基中可以生长，进一步通过适应性进化筛选得到生长速度和甲醇利用速度大幅提升的突变株MX-11，该突变株生长过程中消耗甲醇96.9mM和木糖25.32mM，甲醇和木糖的利用比例为3.83:1(Tuyishime,P.,Wang,Y.,Fan,L.,Zhang,Q.,Li,Q.,Zheng,P.,Sun,J.,Ma,Y.,2018.Engineering Corynebacterium glutamicum for methanol-dependent growth andglutamate production.Metab.Eng.49,220-231)。
现有技术虽然实现了甲醇与另一种辅助碳源的共利用，但是甲醇相对辅助碳源的利用比例仍较低。由于甲醇的价格低于常用碳源如葡萄糖的价格，因此提高菌株利用混合碳源时甲醇的利用比例，对于降低原料成本十分重要。此外，甲醇作为有机溶剂，对微生物细胞具有一定的毒性，因此亟需提高菌株对高浓度甲醇的耐受性。
发明内容
本发明的目的在于提供一种能够提高菌株对甲醇的耐受性和利用率的方法；
本发明的另一目的在于提供所述方法获得的菌株以及本发明的方法和获得的菌株在甲醇的生物转化中的应用。
本发明的另一目的在于提供酶的突变体，该突变体的引入使得所述菌株的甲醇生物转化能力提高。
在第一方面，本发明提供一种甲醇的耐受性和利用率提高的菌株的构建方法，所述方法包括：弱化所述菌株中cgl0833基因的表达。
在优选的实施方式中，所述弱化是将所述cgl0833基因的表达降低但不使得该基因失活或完全敲除。
在优选的实施方式中，所述弱化是指弱化所述菌株中本身包含的野生型cgl0833基因的表达，或者在本身不含cgl0833基因的菌株中外源性地导入表达弱化的突变型cgl0833基因。
在优选的实施方式中，所述cgl0833基因是野生型cgl0833基因，其核苷酸序列如SEQ ID NO:5所示，编码的氨基酸序列如SEQ ID NO:6所示；或者，所述cgl0833基因编码氨基酸序列与SEQ ID NO:6具有84％以上同源性(优选85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同源性)且来源于棒杆菌属具有cgl0833蛋白活性的多肽。
在进一步的优选的实施方式中，所述cgl0833基因的核苷酸序列如SEQ ID NO:5所示，编码的氨基酸序列如SEQ ID NO:6所示；或者，所述cgl0833基因编码的氨基酸序列如SEQ ID NO:20至SEQ ID NO:29任一序列所示。
SEQ ID NO:20(Corynebacterium glutamicum strain USDA-ARS-USMARC-56828)
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLDIHEFKWQALVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNDAVEKHAASDYAATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFLAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH
SEQ ID NO:21(Corynebacterium glutamicum strain B253)
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLNIHEFKWQAVVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFIKVSGGLTTLLNDAVEKHAASDYAATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFIAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH
SEQ ID NO:22(Corynebacterium glutamicum strain CICC10064)
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLDIHEFKWQALVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNKAVETHAASDYAATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARQSVTWAIVLIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFIAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH
SEQ ID NO:23(Corynebacterium glutamicum strain WM001)
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLNIHEFKWQALVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNKAVETHAASDYAATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFIAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH
SEQ ID NO:24(Corynebacterium glutamicum ZL-6)
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMVDVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLNIHEFKWQALVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNKAVETHAASDYAATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFIAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH
SEQ ID NO:25(Corynebacterium glutamicum strain AR1)
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLNIHEFKWQALVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNKAVETHAASDYAATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFLAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH
SEQ ID NO:26(Corynebacterium glutamicum strain XV)
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLNIHEFKWQALVVGIVGIVMIAYVLLSGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNKAVETHAASDYAATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFIAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH
SEQ ID NO:27(Corynebacterium glutamicum ATCC 14067)
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLNIHEFKWQAVVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNKAVDTHAASEYATTKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIILIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFIAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH
SEQ ID NO:28(Corynebacterium glutamicum SCgG2)
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLDIHEFKWQAVVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNKAVDTHAASEYATTKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLILGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGESTEAEQVRVSRITVVVIGLVSIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALVLIFLSPAVSGSDSAMVPGADWAIFPLKNPGIVSIPLAFIAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH
SEQ ID NO:29(Corynebacterium glutamicum strain N24)
MNSTIVLAQEDTGNPILNISVFVVFIVVTMTVVMRAGKTTKEASDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAIALNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAAFGTLFVSLFYLIAQMAGAGSLVSVLLDIHEFHWQAVVIGVVGVVMIAYVLLGGMKGTTYVQMIKACLLVGGVLIMTIWTFVAVKGGVGTLLEDAVAMHASSERAATLGYEATAILEPGLQYGATVTKQLDFVSLGLALVLGTAGLPHVLMRFYTVPTATEARKSVTWAIILIGSFYLMTLILGYGAAALVGPDRIVNAPGAANAAAPLLALELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYDAVLRDGKSSEEEQVRVSRITIVVIGVLSIVLGILAMSQNVAFLVALAFAIAASANLPCILYSLYWKKFNTTGAVAAIYTGLVSALVLIVFSPAVSGTPTSMVPSDGGGWDIFPLSNPGIISIPLAFIAGWIGTMVGKPDNLDDLQAEMEVRSLTGVGVEGAVEH
在具体的实施方式中，所述弱化是将所述cgl0833基因的表达降低至野生型cgl0833基因表达的10％-90％；优选降低至野生型cgl0833基因表达的15％-85％；最优选降低至野生型cgl0833基因表达的18％-80％。
在优选的实施方式中，所述弱化通过以下方法之一或组合实现：野生型cgl0833基因的错义突变、野生型cgl0833基因的同义突变、采用CRISPRi方法对野生型cgl0833基因转录进行抑制、基因启动子或翻译调控区改变令其转录或翻译弱化、改变基因序列使其mRNA稳定性减弱或酶结构不稳定等。
在优选的实施方式中，所述错义突变是将野生型cgl0833基因的核苷酸序列中第1439位的C突变为T，或将cgl0833基因所编码蛋白的第480位丝氨酸突变为苯丙氨酸。
在优选的实施方式中，所述方法构建的菌株的甲醇耐受性为300mM以上；优选400mM以上；更优选500mM以上、600mM以上、700mM以上、800mM以上、900mM以上；最优选1000mM以上。在一优选例中，所述方法构建的菌株利用甲醇和木糖组成的混合碳源时，甲醇和木糖的消耗量之比大于4:1；优选大于5:1；更优选大于6:1；最优选大于7:1。
在优选的实施方式中，所述方法构建的菌株可以利用甲醇为唯一碳源生长。
在优选的实施方式中，所述菌株包括但不限于肠杆菌属家族(Escherichia)、棒杆菌属家族(Corynebacterium)、芽孢杆菌属家族(Bacillus)；优选棒杆菌属家族(Corynebacterium)。
在优选的实施方式中，所述菌株包括但不限于大肠杆菌(Escherichia coli)、谷氨酸棒杆菌(Corynebacterium glutamicum)、枯草芽孢杆菌(Bacillus subtilis)；优选谷氨酸棒杆菌(Corynebacterium glutamicum)。
在第二方面，本发明提供一种甲醇的耐受性和利用率提高的菌株，所述菌株中cgl0833基因的表达被弱化。
在具体的实施方式中，所述菌株采用第一方面所述的方法构建。
在优选的实施方式中，所述菌株包括但不限于肠杆菌属家族(Escherichia)、棒杆菌属家族(Corynebacterium)、芽孢杆菌属家族(Bacillus)；优选棒杆菌属家族(Corynebacterium)。
在优选的实施方式中，所述菌株包括但不限于谷氨酸棒杆菌(Corynebacteriumglutamicum)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)；优选谷氨酸棒杆菌(Corynebacterium glutamicum)。
在优选的实施方式中，所述菌株的甲醇耐受性为300mM以上；优选400mM以上；更优选500mM以上、600mM以上、700mM以上、800mM以上、900mM以上；最优选1000mM以上。在一优选例中，所述方法构建的菌株利用甲醇和木糖组成的混合碳源时，甲醇和木糖的消耗量之比大于4:1；优选大于5:1；更优选大于6:1；最优选大于7:1。
在优选的实施方式中，所述方法构建的菌株可以利用甲醇为唯一碳源生长。
在第三方面，本发明提供一种突变型cgl0833基因或其编码的蛋白，所述突变型cgl0833基因的相应野生型cgl0833基因的核苷酸序列如SEQ ID NO:5所示，相应野生型cgl0833基因编码的氨基酸序列如SEQ ID NO:6所示；或者，所述cgl0833基因编码氨基酸序列与SEQ ID NO:6具有84％以上同源性(优选85％以上、90％以上、95％以上、96％以上、97％以上、98％以上、99％以上同源性)且来源于棒杆菌属具有cgl0833蛋白活性的多肽。
在进一步的优选的实施方式中，所述cgl0833基因的核苷酸序列如SEQ ID NO:5所示，编码的氨基酸序列如SEQ ID NO:6所示；或者，所述cgl0833基因编码的氨基酸序列如SEQ ID NO:20至SEQ ID NO:29任一序列所示。
在优选的实施方式中，相比于野生型cgl0833基因，所述突变型cgl0833基因的表达降低但不完全丧失。
在具体的实施方式中，所述突变型cgl0833基因的表达降低至野生型cgl0833基因表达的10％-90％；优选降低至野生型cgl0833基因表达的15％-85％；最优选降低至野生型cgl0833基因表达的18％-80％。
在具体的实施方式中，与野生型cgl0833基因的核苷酸序列相比，所述突变型cgl0833基因的核苷酸序列中相应的第1439位的C突变为T；或者，与野生型cgl0833基因编码的氨基酸序列相比，突变型cgl0833基因所编码氨基酸序列中相应的第480位丝氨酸突变为苯丙氨酸。
在第四方面，本发明提供第三方面所述的突变型cgl0833基因或其编码的蛋白在提高菌株的甲醇耐受性或构建甲醇生物转化菌株中的用途。
在第五方面，本发明提供一种表达载体，所述表达载体包含第三方面所述的突变型cgl0833基因。
在第六方面，本发明提供一种宿主细胞，所述宿主细胞包含第五方面所述的表达载体或基因组上整合有第三方面所述的突变型cgl0833基因。
在第七方面，本发明提供一种生物转化甲醇的方法，包括利用第一方面所述构建方法构建的菌株或第二方面所述菌株或第三方面所述的基因或编码蛋白或第五方面所述的表达载体或第六方面所述的宿主细胞进行甲醇的生物转化。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了cgl0833基因错义突变对谷氨酸棒杆菌甲醇耐受性的影响；
图2显示了cgl0833基因错义突变对cgl0833表达水平的影响。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现弱化菌株中的cgl0833基因，能够显著提高该菌株的甲醇耐受性以及该菌株利用甲醇和其它辅助碳源组成的混合碳源时的甲醇利用比例，从而实质性提高了菌株对于甲醇的利用效率。在此基础上完成了本发明。
术语定义
本文所用的术语“外源性”是指某体系中包含了原来不存在的物质。例如，通过转化等方式在某菌株中引入该菌株中原本不存在的基因，则该基因对于该菌株是“外源性”的。
本文所用的术语“弱化”是指降低、削弱、减少某种基因的表达，使得该菌株的表达低于野生型菌株的表达水平，或其编码的蛋白具有减弱的活性。为实现本发明的目的，可以采用本领域已知的各种技术手段，只要能够降低基因的表达即可。所述技术手段可以在任何水平，例如在转录水平，基因启动子区域改变、CRISPRi抑制基因转录，使转录水平降低；如在翻译水平，改变基因序列使其mRNA稳定性减弱，使用sRNA技术加快mRNA降解，使基因翻译水平降低；如在蛋白水平，基因突变使蛋白失活或部分功能失活，或降低蛋白的活性或稳定性；以上方法可单独使用或组合实现。
cgl0833基因及其突变体
本文所述的cgl0833基因编码的蛋白具有一元羧酸转运活性、Na+/脯氨酸转运活性、Na+/泛磷酸转运活性，也被称为NCgl0799，cg0953，在棒杆菌属微生物中天然存在。
在具体的实施方式中，野生型cgl0833基因可以来源于谷氨酸棒杆菌ATCC13032，其核苷酸序列如SEQ ID NO:5所示，其编码的氨基酸序列如SEQ ID NO:6、SEQ ID NO:20至SEQ ID NO:29的任一序列所示。也可以是来源于谷氨酸棒杆菌JH41、谷氨酸棒杆菌B253、谷氨酸棒杆菌AR1、谷氨酸棒杆菌ATCC21831、谷氨酸棒杆菌CICC10064、谷氨酸棒杆菌B414、谷氨酸棒杆菌WM001、谷氨酸棒杆菌AJ1511、谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌CP、谷氨酸棒杆菌XV、谷氨酸棒杆菌C1、谷氨酸棒杆菌TQ2223、谷氨酸棒杆菌MB001、谷氨酸棒杆菌K051、谷氨酸棒杆菌USDA-ARS-USMARC-56828、谷氨酸棒杆菌N24、谷氨酸棒杆菌ZL-6、谷氨酸棒杆菌ATCC14067、谷氨酸棒杆菌Y1、谷氨酸棒杆菌ATCC15168、谷氨酸棒杆菌SCgG2、谷氨酸棒杆菌SCgG1、谷氨酸棒杆菌R、Corynebacterium crudilactis JZ16、Corynebacteriumefficiens YS-314、Corynebacterium deserti GIMN1.010，但不限于以上这些菌株。
同样地，属于棒杆菌属且具有弱化的cgl0833基因的微生物可以是谷氨酸棒杆菌ATCC13032，也可以是谷氨酸棒杆菌JH41、谷氨酸棒杆菌B253、谷氨酸棒杆菌AR1、谷氨酸棒杆菌ATCC21831、谷氨酸棒杆菌CICC10064、谷氨酸棒杆菌B414、谷氨酸棒杆菌WM001、谷氨酸棒杆菌AJ1511、谷氨酸棒杆菌ATCC13869、谷氨酸棒杆菌CP、谷氨酸棒杆菌XV、谷氨酸棒杆菌C1、谷氨酸棒杆菌TQ2223、谷氨酸棒杆菌MB001、谷氨酸棒杆菌K051、谷氨酸棒杆菌USDA-ARS-USMARC-56828、谷氨酸棒杆菌N24、谷氨酸棒杆菌ZL-6、谷氨酸棒杆菌ATCC14067、谷氨酸棒杆菌Y1、谷氨酸棒杆菌ATCC15168、谷氨酸棒杆菌SCgG2、谷氨酸棒杆菌SCgG1、谷氨酸棒杆菌R、Corynebacterium crudilactis JZ16、Corynebacterium efficiens YS-314、Corynebacterium deserti GIMN1.010，但不限于以上这些菌株。经本发明的教导，以上这些具有弱化的cgl0833基因的棒杆菌也是甲醇耐受性和利用率提高的菌株，也应该在本发明的保护范围内。
本文所述的“突变型cgl0833基因”是指，与相应野生型cgl0833基因相比，所述突变型cgl0833基因的表达降低。例如，所述突变型cgl0833基因的表达降低至野生型cgl0833基因表达的10％-90％；优选降低至野生型cgl0833基因表达的15％-85％；最优选降低至野生型cgl0833基因表达的18％-80％。在具体的实施方式中，突变型cgl0833基因或其编码的蛋白是指，与野生型cgl0833基因的核苷酸序列相比，所述突变型cgl0833基因的核苷酸序列中相应的第1439位的C突变为T；或者，与野生型cgl0833基因编码的氨基酸序列相比，突变型cgl0833基因所编码氨基酸序列中相应的第480位丝氨酸突变为苯丙氨酸。
本发明的甲醇耐受性的菌株和甲醇生物转化菌株
本文所述的“甲醇耐受性提高的菌株”是指该菌株对甲醇的耐受性较出发菌株(即未使用本发明方法改造前的菌株)有所提高，或能够在高甲醇环境下生长，进而对于甲醇的利用效率显著提高。
具体地说，本发明通过弱化cgl0833基因的表达来显著提高菌株对甲醇的耐受性。然而，本领域技术人员应该明白，本发明的方法并不限于弱化菌株中本身包含的野生型cgl0833基因的表达。当在本身不含cgl0833基因的菌株中外源性地导入表达弱化的突变型cgl0833基因时，该菌株对甲醇的耐受性同样得到显著提高。换言之，本发明的方法可以适用于本身含有野生型cgl0833基因的菌株，例如本身具备一定程度甲醇耐受性的菌株，从而进一步提高其甲醇耐受性；本发明的方法也可以适用于本身不具备甲醇耐受性的菌株，例如在该菌株中外源性导入表达弱化的突变型cgl0833基因，从而使得其具备甲醇耐受性。
对于弱化cgl0833基因表达的具体技术手段，本领域技术人员可以采取本领域熟知的各种方法，只要能够降低所述cgl0833基因的表达即可。基于本发明的教导，本领域技术人员应该知晓，本文所述的弱化cgl0833基因的表达并不是使得该基因完全失活或被完全敲除。
本文所用的术语“生物转化”具备本领域技术人员常规理解的意义，即，使用酶、休止细胞或活细胞，将甲醇转化为对环境无害的产物(如二氧化碳)，或菌体和各种化学品等产品。
本文所述的“甲醇生物转化菌株”、“生物转化甲醇的菌株”或“本发明菌株”具备相同的含义，均是指通过生物学途径转化甲醇的微生物，从而能够借助该甲醇生物转化菌株将甲醇转化为对环境无害的产物(如二氧化碳)，或将甲醇转化成菌体、氨基酸、有机酸、多元醇等产品。
本发明的菌株或者本发明方法构建的菌株对甲醇表现出优异的耐受性，例如甲醇耐受性达到300mM以上；优选400mM以上；更优选500mM以上、600mM以上、700mM以上、800mM以上、900mM以上；最优选1000mM以上。然而，基于本发明的教导，本领域技术人员会知晓，本发明菌株的甲醇耐受性提高的实质在于其在利用甲醇和其它辅助碳源组成的混合碳源时，对甲醇的利用率大大提高。例如，本发明的菌株利用甲醇和木糖组成的混合碳源时，甲醇和木糖的消耗量之比大于4:1；优选大于5:1；更优选大于6:1；最优选大于7:1；甚至可以以甲醇为唯一碳源进行生长。因此，本发明的菌株或者本发明方法构建的菌株是优异的甲醇生物转化菌株。
采用本发明的方法构建的菌株可以是任何可通过上述方法构建的甲醇生物转化菌株，包括但不限于肠杆菌属家族(Escherichia)、棒杆菌属家族(Corynebacterium)、芽孢杆菌属家族(Bacillus)；优选棒杆菌属家族(Corynebacterium)。在进一步的实施例中，本发明构建的甲醇耐受性菌株或甲醇生物转化菌株可以是谷氨酸棒杆菌(Corynebacteriumglutamicum)、大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)，优选谷氨酸棒杆菌(Corynebacterium glutamicum)。
本发明的优点：
1.本发明为构建甲醇耐受性提高的菌株，进而构建甲醇生物转化效率提高的菌株提供了全新的思路；和
2.本发明的菌株在利用甲醇和其它辅助碳源组成的混合碳源时，甲醇的利用比例显著提高；和
3.本发明的方法能够进一步提高本身已经具有相当甲醇耐受性的菌株的甲醇生物转化效率，从而能够具备显著的经济价值和社会价值，为实现甲醇生物转化和甲醇化工奠定了基础，使甲醇真正有望成为生物发酵和生物制造产业的新原料平台。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring HarborLaboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
本发明所用试剂和原料均市售可得。
实施例
实施例1.在谷氨酸棒杆菌MX-11中引入cgl0833基因错义突变
菌株C.glutamicum MX-11出自文献(Tuyishime,P.,Wang,Y.,Fan,L.,Zhang,Q.,Li,Q.,Zheng,P.,Sun,J.,Ma,Y.,2018.Engineering Corynebacterium glutamicum formethanol-dependent growth and glutamate production.Metab.Eng.49,220-231)。
(1)构建pK18mobsacB-tet-cgl0833C1439T质粒
a.使用BamHI内切酶线性化pK18mobsacB-tet空载体。
b.以C.glutamicum ATCC 13032的基因组DNA为模板，使用单链核苷酸cgl0833-F1(TATGACATGATTACGAATTCTACTTGATCGCTCAGATGGCTGG；SEQ ID NO:1)和cgl0833-R1(CTGCTGGGGACAGGAAGATCAGCAGCAGC；SEQ ID NO:2)作为引物，扩增cgl0833的上半部分片段，同时引入cgl0833第1439位碱基C到T的突变。
c.以C.glutamicum ATCC 13032的基因组DNA为模板，使用单链核苷酸cgl0833-F2(ATCTTCCTGTCCCCAGCAGTCTCCGGTAATG；SEQ ID NO:3)和cgl0833-R2(CGACGGCCAGTGCCAAGCTTAGAGGAGTGCAGCATGAGATCAT C；SEQ ID NO:4)作为引物，扩增cgl0833的下半部分片段，同时引入cgl0833第1439位碱基C到T的突变。
d.使用ClonExpress II One Step Cloning Kit(南京诺唯赞生物科技有限公司)将上述cgl0833上半部分片段、cgl0833下半部分片段和线性化的pK18mobsacB-tet质粒连接，构建pK18mobsacB-tet-cgl0833C1439T质粒。
(2)在C.glutamicum MX-11中引入cgl0833C1439T突变
a.参照文献(Ruan,Y.,Zhu,L.,Li,Q.,2015.Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cellwall and increasing the cytoplasmic membrane fluidity.Biotechnol.Lett.37,2445-2452)的方法，制备C.glutamicum MX-11的感受态细胞。在感受态制备过程中，在文献中所述培养基中额外加入木糖和核糖作为C.glutamicum MX-11的碳源。
b.将上述pK18mobsacB-tet-cgl0833C1439T质粒转化至C.glutamicum MX-11的感受态细胞中，参照文献(
A.,Tauch,A.,
W.,Kalinowski,J.,Thierbach,G.,Pühler,A.,1994.Small mobilizable multi-purpose cloning vectors derived from theEscherichia coli plasmids pK18 and pK19:selection of defined deletions in thechromosome of Corynebacterium glutamicum.Gene 145,69-73)的方法，在C.glutamicumMX-11中引入cgl0833C1439T突变，对应cgl0833编码蛋白质的第480位丝氨酸突变为苯丙氨酸，构建的突变株命名为MX-11-cgl0833C1439T。
cgl0833野生型基因序列如下：
ATGAATTCCACTATTCTCCTTGCACAAGACGCTGTTTCTGAGGGCGTCGGTAATCCGATTCTTAACATCAGTGTCTTCGTCGTCTTCATTATTGTGACGATGACCGTGGTGCTTCGCGTGGGCAAGAGCACCAGCGAATCCACCGACTTCTACACCGGTGGTGCTTCCTTCTCCGGAACCCAGAACGGTCTGGCTATCGCAGGTGACTACCTGTCTGCAGCGTCCTTCCTCGGAATCGTTGGTGCAATTTCACTCAACGGTTACGACGGATTCCTTTACTCCATCGGCTTCTTCGTCGCATGGCTTGTTGCACTGCTGCTCGTGGCAGAGCCACTTCGTAACGTGGGCCGCTTCACCATGGCTGACGTGCTGTCCTTCCGACTGCGTCAGAAACCAGTCCGCGTCGCTGCGGCCTGCGGTACCCTCGCGGTTACCCTCTTTTACTTGATCGCTCAGATGGCTGGTGCAGGTTCGCTTGTGTCCGTTCTGCTGGACATCCACGAGTTCAAGTGGCAGGCAGTTGTTGTCGGTATCGTTGGCATTGTCATGATCGCCTACGTTCTTCTTGGCGGTATGAAGGGCACCACATACGTTCAGATGATTAAGGCAGTTCTGCTGGTCGGTGGCGTTGCCATTATGACCGTTCTGACCTTCGTCAAGGTGTCTGGTGGCCTGACCACCCTTTTAAATGACGCTGTTGAGAAGCACGCCGCTTCAGATTACGCTGCCACCAAGGGGTACGATCCAACCCAGATCCTGGAGCCTGGTCTGCAGTACGGTGCAACTCTGACCACTCAGCTGGACTTCATTTCCTTGGCTCTCGCTCTGTGTCTTGGAACCGCTGGTCTGCCACACGTTCTGATGCGCTTCTACACCGTTCCTACCGCCAAGGAAGCACGTAAGTCTGTGACCTGGGCTATCGTCCTCATTGGTGCGTTCTACCTGATGACCCTGGTCCTTGGTTACGGCGCTGCGGCACTGGTCGGTCCAGACCGCGTCATTGCCGCACCAGGTGCTGCTAATGCTGCTGCTCCTCTGCTGGCCTTCGAGCTTGGTGGTTCCATCTTCATGGCGCTGATTTCCGCAGTTGCGTTCGCTACCGTTCTCGCCGTGGTCGCAGGTCTTGCAATTACCGCATCCGCTGCTGTTGGTCACGACATCTACAACGCTGTTATCCGCAACGGTCAGTCCACCGAAGCGGAGCAGGTCCGAGTATCCCGCATCACCGTTGTCGTCATTGGCCTGATTTCCATTGTCCTGGGAATTCTTGCAATGACCCAGAACGTTGCGTTCCTCGTGGCCCTGGCCTTCGCAGTTGCAGCATCCGCTAACCTGCCAACCATCCTGTACTCCCTGTACTGGAAGAAGTTCAACACCACCGGCGCTGTGGCCGCTATCTACACCGGTCTCATCTCCGCGCTGCTGCTGATCTTCCTGTCCCCAGCAGTCTCCGGTAATGACAGCGCAATGGTTCCAGGTGCAGACTGGGCAATCTTCCCACTGAAGAACCCAGGCCTCGTCTCCATCCCACTGGCATTCATCGCTGGTTGGATCGGCACTTTGGTTGGCAAGCCAGACAACATGGATGATCTTGCTGCCGAAATGGAAGTTCGTTCCCTCACCGGTGTCGGTGTTGAAAAGGCTGTTGATCACTAA(SEQ IDNO:5)
cgl0833野生型氨基酸序列如下：
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLDIHEFKWQAVVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNDAVEKHAASDYAATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFIAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDH*(SEQ ID NO:6)
实施例2.甲醇生物转化
(1)培养基
在不含葡萄糖的CGXII培养基中，添加甲醇和木糖作为碳源，额外添加1mM异丙基硫代半乳糖苷(IPTG)、5mg/L氯霉素和25mg/L卡那霉素。CGXII培养基配方参照文献(Keilhauer,C.,Eggeling,L.,Sahm,H.,1993.Isoleucine synthesis inCorynebacterium glutamicum:molecular analysis of the ilvB-ilvN-ilvCoperon.J.Bacteriol.175,5595-5603)。
(2)培养条件
将C.glutamicum菌株MX-11和MX-11-cgl0833C1439T分别接种于含有上述培养基的摇瓶中，初始OD600 nm约为0.5(初始细胞干重约为0.153gCDW/L)。将摇瓶置于摇床中培养，温度为30℃，转速为220rpm，摇瓶为250mL，装液量为50mL，使用不透气封口膜封闭摇瓶以防止甲醇挥发。
(3)取样检测
定时取样，检测培养液中剩余的甲醇和木糖浓度。甲醇和木糖浓度检测方法为：将培养液样品在12,000×g离心10分钟，使用上清进行检测。使用岛津公司的高速液相色谱仪(Shimadzu Prominence UFLC)和Bio-Rad公司的色谱柱Aminex HPX-87H column(300×7.8mm)，色谱柱柱温为55℃，检测器为示差折光检测器，检测器温度为55℃，流动相为5mMH2SO4，流速为0.5mL/min，进样量为10μL。当生物量不再升高时，停止培养。计算菌株对甲醇和木糖的消耗量，结果如表1所示。
当培养基中的初始甲醇浓度由125mM提高至460mM时，菌株MX-11的生长和甲醇利用受到明显抑制。采用本发明的方法构建的菌株MX-11-cgl0833C1439T，对高浓度甲醇的耐受性明显提高，在含有460mM甲醇的培养基中，甲醇和辅助碳源的利用比例提高至7.04:1，显著高于MX-11的甲醇和辅助碳源利用比例3.83:1。本发明的方法可显著提高菌株对高浓度甲醇的耐受性和甲醇利用效率，具有重要的应用价值。
表1.不同菌株的甲醇和木糖消耗量a
a数据为三次独立实验的平均值
实施例3.构建cgl0833和绿色荧光蛋白egfp融合表达的谷氨酸棒杆菌ATCC13032突变株
(1)构建pK18mobsacB-cgl0833-egfp质粒
a.使用BamHI内切酶线性化pK18mobsacB空载体。
b.以pTRCmob-egfp质粒(Wang,Y.,Cao,G.,Xu,D.,Fan,L.,Wu,X.,Ni,X.,Zhao,S.,Zheng,P.,Sun,J.,Ma,Y.,2018.A novel Corynebacterium glutamicum L-glutamateexporter.Applied and environmental microbiology 84,e02691-17.)为模板，使用单链核苷酸egfp-F(GTGAGCAAGGGCGAGGAGC；SEQ ID NO:7)和egfp-R(TTACTTGTACAGCTCGTCCATGC；SEQ ID NO:8)作为引物，扩增egfp基因片段。
c.以C.glutamicum ATCC 13032的基因组DNA为模板，使用单链核苷酸cgl0833-F3(GAGCTCGGTACCCGGGGATCCATTATGACCGTTCTGACCTTCGT；SEQ ID NO:9)和cgl0833-R3(AGCTCCTCGCCCTTGCTCACGTGATCAACAGCCTTTTCAACA；SEQ ID NO:10)作为引物，扩增cgl0833在染色体上游的部分片段。
d.以C.glutamicum ATCC 13032的基因组DNA为模板，使用单链核苷酸cgl0833-F4(TGGACGAGCTGTACAAGTAAATCTAGTTTCTGAAGTTATTTAAACCG；SEQ ID NO:11)和cgl0833-R4(CAGGTCGACTCTAGAGGATCCCCCACCATTCCTGGAAACTC；SEQ ID NO:12)作为引物，扩增cgl0833在染色体下游的部分片段。
e.使用ClonExpress II One Step Cloning Kit(南京诺唯赞生物科技有限公司)将上述egfp基因片段、cgl0833在染色体上游的部分片段、cgl0833在染色体下游的部分片段和线性化的pK18mobsacB质粒连接，构建pK18mobsacB-cgl0833-egfp质粒。
(2)在C.glutamicum ATCC 13032中引入cgl0833-egfp融合基因
a.参照文献(Ruan,Y.,Zhu,L.,Li,Q.,2015.Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cellwall and increasing the cytoplasmic membrane fluidity.Biotechnol.Lett.37,2445-2452)的方法，制备C.glutamicum ATCC 13032的感受态细胞。
b.将上述pK18mobsacB-cgl0833-egfp质粒转化至C.glutamicum ATCC 13032的感受态细胞中，参照文献(
A.,Tauch,A.,
W.,Kalinowski,J.,Thierbach,G.,Pühler,A.,1994.Small mobilizable multi-purpose cloning vectors derived from theEscherichia coli plasmids pK18 and pK19:selection of defined deletions in thechromosome of Corynebacterium glutamicum.Gene 145,69-73)的方法，在C.glutamicumATCC 13032中引入cgl0833-egfp融合基因，构建的突变株命名为ATCC13032-cgl0833-egfp。
cgl0833-egfp融合基因的核苷酸序列如下：
ATGAATTCCACTATTCTCCTTGCACAAGACGCTGTTTCTGAGGGCGTCGGTAATCCGATTCTTAACATCAGTGTCTTCGTCGTCTTCATTATTGTGACGATGACCGTGGTGCTTCGCGTGGGCAAGAGCACCAGCGAATCCACCGACTTCTACACCGGTGGTGCTTCCTTCTCCGGAACCCAGAACGGTCTGGCTATCGCAGGTGACTACCTGTCTGCAGCGTCCTTCCTCGGAATCGTTGGTGCAATTTCACTCAACGGTTACGACGGATTCCTTTACTCCATCGGCTTCTTCGTCGCATGGCTTGTTGCACTGCTGCTCGTGGCAGAGCCACTTCGTAACGTGGGCCGCTTCACCATGGCTGACGTGCTGTCCTTCCGACTGCGTCAGAAACCAGTCCGCGTCGCTGCGGCCTGCGGTACCCTCGCGGTTACCCTCTTTTACTTGATCGCTCAGATGGCTGGTGCAGGTTCGCTTGTGTCCGTTCTGCTGGACATCCACGAGTTCAAGTGGCAGGCAGTTGTTGTCGGTATCGTTGGCATTGTCATGATCGCCTACGTTCTTCTTGGCGGTATGAAGGGCACCACATACGTTCAGATGATTAAGGCAGTTCTGCTGGTCGGTGGCGTTGCCATTATGACCGTTCTGACCTTCGTCAAGGTGTCTGGTGGCCTGACCACCCTTTTAAATGACGCTGTTGAGAAGCACGCCGCTTCAGATTACGCTGCCACCAAGGGGTACGATCCAACCCAGATCCTGGAGCCTGGTCTGCAGTACGGTGCAACTCTGACCACTCAGCTGGACTTCATTTCCTTGGCTCTCGCTCTGTGTCTTGGAACCGCTGGTCTGCCACACGTTCTGATGCGCTTCTACACCGTTCCTACCGCCAAGGAAGCACGTAAGTCTGTGACCTGGGCTATCGTCCTCATTGGTGCGTTCTACCTGATGACCCTGGTCCTTGGTTACGGCGCTGCGGCACTGGTCGGTCCAGACCGCGTCATTGCCGCACCAGGTGCTGCTAATGCTGCTGCTCCTCTGCTGGCCTTCGAGCTTGGTGGTTCCATCTTCATGGCGCTGATTTCCGCAGTTGCGTTCGCTACCGTTCTCGCCGTGGTCGCAGGTCTTGCAATTACCGCATCCGCTGCTGTTGGTCACGACATCTACAACGCTGTTATCCGCAACGGTCAGTCCACCGAAGCGGAGCAGGTCCGAGTATCCCGCATCACCGTTGTCGTCATTGGCCTGATTTCCATTGTCCTGGGAATTCTTGCAATGACCCAGAACGTTGCGTTCCTCGTGGCCCTGGCCTTCGCAGTTGCAGCATCCGCTAACCTGCCAACCATCCTGTACTCCCTGTACTGGAAGAAGTTCAACACCACCGGCGCTGTGGCCGCTATCTACACCGGTCTCATCTCCGCGCTGCTGCTGATCTTCCTGTCCCCAGCAGTCTCCGGTAATGACAGCGCAATGGTTCCAGGTGCAGACTGGGCAATCTTCCCACTGAAGAACCCAGGCCTCGTCTCCATCCCACTGGCATTCATCGCTGGTTGGATCGGCACTTTGGTTGGCAAGCCAGACAACATGGATGATCTTGCTGCCGAAATGGAAGTTCGTTCCCTCACCGGTGTCGGTGTTGAAAAGGCTGTTGATCACGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA(SEQ ID NO:13)
cgl0833-egfp融合基因的氨基酸序列如下：
MNSTILLAQDAVSEGVGNPILNISVFVVFIIVTMTVVLRVGKSTSESTDFYTGGASFSGTQNGLAIAGDYLSAASFLGIVGAISLNGYDGFLYSIGFFVAWLVALLLVAEPLRNVGRFTMADVLSFRLRQKPVRVAAACGTLAVTLFYLIAQMAGAGSLVSVLLDIHEFKWQAVVVGIVGIVMIAYVLLGGMKGTTYVQMIKAVLLVGGVAIMTVLTFVKVSGGLTTLLNDAVEKHAASDYAATKGYDPTQILEPGLQYGATLTTQLDFISLALALCLGTAGLPHVLMRFYTVPTAKEARKSVTWAIVLIGAFYLMTLVLGYGAAALVGPDRVIAAPGAANAAAPLLAFELGGSIFMALISAVAFATVLAVVAGLAITASAAVGHDIYNAVIRNGQSTEAEQVRVSRITVVVIGLISIVLGILAMTQNVAFLVALAFAVAASANLPTILYSLYWKKFNTTGAVAAIYTGLISALLLIFLSPAVSGNDSAMVPGADWAIFPLKNPGLVSIPLAFIAGWIGTLVGKPDNMDDLAAEMEVRSLTGVGVEKAVDHVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*(SEQ ID NO:14)
实施例4.在谷氨酸棒杆菌ATCC 13032和ATCC 13032-cgl0833-egfp菌株中引入cgl0833基因错义突变
a.参照文献(Ruan,Y.,Zhu,L.,Li,Q.,2015.Improving the electro-transformation efficiency of Corynebacterium glutamicum by weakening its cellwall and increasing the cytoplasmic membrane fluidity.Biotechnol.Lett.37,2445-2452)的方法，制备C.glutamicum ATCC 13032和C.glutamicum ATCC 13032-cgl0833-egfp的感受态细胞。
b.将上述pK18mobsacB-tet-cgl0833C1439T质粒转化至C.glutamicum ATCC 13032和C.glutamicum ATCC 13032-cgl0833-egfp的感受态细胞中，参照文献(
A.,Tauch,A.,
W.,Kalinowski,J.,Thierbach,G.,Pühler,A.,1994.Small mobilizablemulti-purpose cloning vectors derived from the Escherichia coli plasmids pK18and pK19:selection of defined deletions in the chromosome of Corynebacteriumglutamicum.Gene 145,69-73)的方法，在C.glutamicum ATCC13032和C.glutamicum ATCC13032-cgl0833-egfp中引入cgl0833C1439T突变，对应cgl0833编码蛋白质的第480位丝氨酸突变为苯丙氨酸，构建的突变株分别命名为ATCC 13032-cgl0833C1439T和ATCC 13032-cgl0833C1439T-egfp。
实施例5.cgl0833基因错义突变对cgl0833表达水平和谷氨酸棒杆菌甲醇耐受性的影响
(1)培养基
在CGXII培养基中，添加5g/L葡萄糖作为碳源，额外添加1000mM甲醇(32g/L)。CGXII培养基配方参照文献(Keilhauer,C.,Eggeling,L.,Sahm,H.,1993.Isoleucinesynthesis in Corynebacterium glutamicum:molecular analysis of the ilvB-ilvN-ilvC operon.J.Bacteriol.175,5595-5603)。
(2)培养条件
将C.glutamicum菌株ATCC 13032、ATCC 13032-cgl0833C1439T、ATCC 13032-cgl0833-egfp和ATCC 13032-cgl0833C1439T-egfp分别接种于含有上述培养基的摇瓶中，初始OD600nm约为0.1(初始细胞干重约为0.03gCDW/L)。将摇瓶置于摇床中培养，温度为30℃，转速为220rpm，摇瓶为250mL，装液量为50mL，使用不透气封口膜封闭摇瓶以防止甲醇挥发。
(3)取样检测
定时取样，检测培养液的生物量和荧光强度。荧光强度检测方法如下：将培养液进行适当稀释，取100微升稀释后的菌悬液至96孔板中，使用酶标仪(SpectraMax M5,Molecular Devices)检测菌悬液在600nm处的吸光值，以及荧光强度，荧光强度检测所用激发光波长为488nm，发射光波长为520nm。
第12小时的菌株生长情况如图1所示，首先，在菌株ATCC13032中引入cgl0833C1439T突变明显提高了菌株在高浓度甲醇下的生长；其次，cgl0833与egfp融合表达不影响菌株在高浓度甲醇下的生长；第三，在cgl0833与egfp融合表达的菌株中引入cgl0833C1439T突变仍明显提高了菌株在高浓度甲醇下的生长。
第12小时的菌株荧光强度如图2所示，在cgl0833与egfp融合表达的菌株ATCC13032-cgl0833-egfp中引入cgl0833C1439T突变，使得cgl0833-egfp融合蛋白的表达水平下降了65％，说明cgl0833C1439T突变对cgl0833基因表达有弱化作用。
实施例6.cgl0833基因表达弱化对谷氨酸棒杆菌ATCC 13032的甲醇耐受性的影响
使用成熟的CRISPRi方法对C.glutamicum ATCC 13032的cgl0833基因转录进行抑制，从而对cgl0833的基因表达水平进行弱化。CRISPRi技术是通过在细胞内表达dCas9蛋白与引导RNA(gRNA)，dCas9蛋白与gRNA形成复合体，在gRNA的引导下与基因组特定基因靶点进行结合，从而抑制靶基因的转录，对靶基因的表达水平进行弱化(Cleto,S.,Jensen,J.V.,Wendisch,V.F.,Lu,T.K.,2016.Corynebacterium glutamicum metabolicengineering with CRISPR interference(CRISPRi).ACS Synth.Biol.5,375-385)。通过调节gRNA的序列和长度，可实现不同效率的靶基因的弱化。
(1)所使用的gRNA
所使用的的gRNA序列如表2所示，其中对照不具有靶向cgl0833的gRNA，因此对cgl0833的表达无弱化作用。将如下gRNA序列构建至CRISPRi工具质粒上，转化进入C.glutamicum ATCC 13032-cgl0833-egfp菌株。
表2.gRNA序列、cgl0833表达弱化效率和cgl0833表达弱化对菌株甲醇耐受性的影响a
a数据为三次独立实验的平均值
bcgl0833表达弱化效率＝(1-实验组菌株的egfp荧光强度/对照组的egfp荧光强度)*100％
(2)培养基
在CGXII培养基中，添加5g/L葡萄糖作为碳源，额外添加1000mM甲醇(32g/L)，额外添加5mg/L氯霉素维持CRISPRi工具质粒的稳定性，在培养至4小时时，向培养基中添加0.1mM IPTG诱导dCas9蛋白和gRNA的表达。CGXII培养基配方参照文献(Keilhauer,C.,Eggeling,L.,Sahm,H.,1993.Isoleucine synthesis in Corynebacterium glutamicum:molecular analysis of the ilvB-ilvN-ilvC operon.J.Bacteriol.175,5595-5603)。
(3)培养条件
将对照菌株，以及表达dCas9和gRNA的实验组菌株分别接种于含有上述培养基的摇瓶中，初始OD600nm约为0.1(初始细胞干重约为0.03gCDW/L)。将摇瓶置于摇床中培养，温度为30℃，转速为220rpm，摇瓶为250mL，装液量为50mL，使用不透气封口膜封闭摇瓶以防止甲醇挥发。
(4)取样检测
定时取样，检测培养液的生物量和荧光强度。荧光强度检测方法如下：将培养液进行适当稀释，取100微升稀释后的菌悬液至96孔板中，使用酶标仪(SpectraMax M5,Molecular Devices)检测菌悬液在600nm处的吸光值，以及荧光强度，荧光强度检测所用激发光波长为488nm，发射光波长为520nm。
不同程度弱化cgl0833对菌株甲醇耐受性的影响如表2所示，对cgl0833的表达进行弱化，可有效提高菌株对甲醇的耐受性。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 一种提高微生物对甲醇的耐受性和利用率的方法
<130> P2019-1553
<160> 29
<170> SIPOSequenceListing 1.0
<210> 1
<211> 43
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
tatgacatga ttacgaattc tacttgatcg ctcagatggc tgg 43
<210> 2
<211> 29
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 2
ctgctgggga caggaagatc agcagcagc 29
<210> 3
<211> 31
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
atcttcctgt ccccagcagt ctccggtaat g 31
<210> 4
<211> 44
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 4
cgacggccag tgccaagctt agaggagtgc agcatgagat catc 44
<210> 5
<211> 1656
<212> DNA
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 5
atgaattcca ctattctcct tgcacaagac gctgtttctg agggcgtcgg taatccgatt 60
cttaacatca gtgtcttcgt cgtcttcatt attgtgacga tgaccgtggt gcttcgcgtg 120
ggcaagagca ccagcgaatc caccgacttc tacaccggtg gtgcttcctt ctccggaacc 180
cagaacggtc tggctatcgc aggtgactac ctgtctgcag cgtccttcct cggaatcgtt 240
ggtgcaattt cactcaacgg ttacgacgga ttcctttact ccatcggctt cttcgtcgca 300
tggcttgttg cactgctgct cgtggcagag ccacttcgta acgtgggccg cttcaccatg 360
gctgacgtgc tgtccttccg actgcgtcag aaaccagtcc gcgtcgctgc ggcctgcggt 420
accctcgcgg ttaccctctt ttacttgatc gctcagatgg ctggtgcagg ttcgcttgtg 480
tccgttctgc tggacatcca cgagttcaag tggcaggcag ttgttgtcgg tatcgttggc 540
attgtcatga tcgcctacgt tcttcttggc ggtatgaagg gcaccacata cgttcagatg 600
attaaggcag ttctgctggt cggtggcgtt gccattatga ccgttctgac cttcgtcaag 660
gtgtctggtg gcctgaccac ccttttaaat gacgctgttg agaagcacgc cgcttcagat 720
tacgctgcca ccaaggggta cgatccaacc cagatcctgg agcctggtct gcagtacggt 780
gcaactctga ccactcagct ggacttcatt tccttggctc tcgctctgtg tcttggaacc 840
gctggtctgc cacacgttct gatgcgcttc tacaccgttc ctaccgccaa ggaagcacgt 900
aagtctgtga cctgggctat cgtcctcatt ggtgcgttct acctgatgac cctggtcctt 960
ggttacggcg ctgcggcact ggtcggtcca gaccgcgtca ttgccgcacc aggtgctgct 1020
aatgctgctg ctcctctgct ggccttcgag cttggtggtt ccatcttcat ggcgctgatt 1080
tccgcagttg cgttcgctac cgttctcgcc gtggtcgcag gtcttgcaat taccgcatcc 1140
gctgctgttg gtcacgacat ctacaacgct gttatccgca acggtcagtc caccgaagcg 1200
gagcaggtcc gagtatcccg catcaccgtt gtcgtcattg gcctgatttc cattgtcctg 1260
ggaattcttg caatgaccca gaacgttgcg ttcctcgtgg ccctggcctt cgcagttgca 1320
gcatccgcta acctgccaac catcctgtac tccctgtact ggaagaagtt caacaccacc 1380
ggcgctgtgg ccgctatcta caccggtctc atctccgcgc tgctgctgat cttcctgtcc 1440
ccagcagtct ccggtaatga cagcgcaatg gttccaggtg cagactgggc aatcttccca 1500
ctgaagaacc caggcctcgt ctccatccca ctggcattca tcgctggttg gatcggcact 1560
ttggttggca agccagacaa catggatgat cttgctgccg aaatggaagt tcgttccctc 1620
accggtgtcg gtgttgaaaa ggctgttgat cactaa 1656
<210> 6
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 6
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asp Ile His Glu Phe Lys Trp Gln Ala Val Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Asp Ala Val Glu Lys His Ala Ala Ser Asp
225 230 235 240
Tyr Ala Ala Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Ile Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 7
<211> 19
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 7
gtgagcaagg gcgaggagc 19
<210> 8
<211> 23
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 8
ttacttgtac agctcgtcca tgc 23
<210> 9
<211> 44
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 9
gagctcggta cccggggatc cattatgacc gttctgacct tcgt 44
<210> 10
<211> 42
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 10
agctcctcgc ccttgctcac gtgatcaaca gccttttcaa ca 42
<210> 11
<211> 47
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 11
tggacgagct gtacaagtaa atctagtttc tgaagttatt taaaccg 47
<210> 12
<211> 41
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 12
caggtcgact ctagaggatc ccccaccatt cctggaaact c 41
<210> 13
<211> 2370
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 13
atgaattcca ctattctcct tgcacaagac gctgtttctg agggcgtcgg taatccgatt 60
cttaacatca gtgtcttcgt cgtcttcatt attgtgacga tgaccgtggt gcttcgcgtg 120
ggcaagagca ccagcgaatc caccgacttc tacaccggtg gtgcttcctt ctccggaacc 180
cagaacggtc tggctatcgc aggtgactac ctgtctgcag cgtccttcct cggaatcgtt 240
ggtgcaattt cactcaacgg ttacgacgga ttcctttact ccatcggctt cttcgtcgca 300
tggcttgttg cactgctgct cgtggcagag ccacttcgta acgtgggccg cttcaccatg 360
gctgacgtgc tgtccttccg actgcgtcag aaaccagtcc gcgtcgctgc ggcctgcggt 420
accctcgcgg ttaccctctt ttacttgatc gctcagatgg ctggtgcagg ttcgcttgtg 480
tccgttctgc tggacatcca cgagttcaag tggcaggcag ttgttgtcgg tatcgttggc 540
attgtcatga tcgcctacgt tcttcttggc ggtatgaagg gcaccacata cgttcagatg 600
attaaggcag ttctgctggt cggtggcgtt gccattatga ccgttctgac cttcgtcaag 660
gtgtctggtg gcctgaccac ccttttaaat gacgctgttg agaagcacgc cgcttcagat 720
tacgctgcca ccaaggggta cgatccaacc cagatcctgg agcctggtct gcagtacggt 780
gcaactctga ccactcagct ggacttcatt tccttggctc tcgctctgtg tcttggaacc 840
gctggtctgc cacacgttct gatgcgcttc tacaccgttc ctaccgccaa ggaagcacgt 900
aagtctgtga cctgggctat cgtcctcatt ggtgcgttct acctgatgac cctggtcctt 960
ggttacggcg ctgcggcact ggtcggtcca gaccgcgtca ttgccgcacc aggtgctgct 1020
aatgctgctg ctcctctgct ggccttcgag cttggtggtt ccatcttcat ggcgctgatt 1080
tccgcagttg cgttcgctac cgttctcgcc gtggtcgcag gtcttgcaat taccgcatcc 1140
gctgctgttg gtcacgacat ctacaacgct gttatccgca acggtcagtc caccgaagcg 1200
gagcaggtcc gagtatcccg catcaccgtt gtcgtcattg gcctgatttc cattgtcctg 1260
ggaattcttg caatgaccca gaacgttgcg ttcctcgtgg ccctggcctt cgcagttgca 1320
gcatccgcta acctgccaac catcctgtac tccctgtact ggaagaagtt caacaccacc 1380
ggcgctgtgg ccgctatcta caccggtctc atctccgcgc tgctgctgat cttcctgtcc 1440
ccagcagtct ccggtaatga cagcgcaatg gttccaggtg cagactgggc aatcttccca 1500
ctgaagaacc caggcctcgt ctccatccca ctggcattca tcgctggttg gatcggcact 1560
ttggttggca agccagacaa catggatgat cttgctgccg aaatggaagt tcgttccctc 1620
accggtgtcg gtgttgaaaa ggctgttgat cacgtgagca agggcgagga gctgttcacc 1680
ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg 1740
tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc 1800
accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag 1860
tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc 1920
gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc 1980
gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 2040
ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac 2100
gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac 2160
aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 2220
gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa gctgagcaaa 2280
gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc 2340
actctcggca tggacgagct gtacaagtaa 2370
<210> 14
<211> 789
<212> PRT
<213> 人工序列(Artificial sequence)
<400> 14
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asp Ile His Glu Phe Lys Trp Gln Ala Val Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Asp Ala Val Glu Lys His Ala Ala Ser Asp
225 230 235 240
Tyr Ala Ala Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Ile Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His Val Ser Lys Gly Glu Glu Leu Phe Thr
545 550 555 560
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
565 570 575
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
580 585 590
Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
595 600 605
Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
610 615 620
Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro
625 630 635 640
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn
645 650 655
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
660 665 670
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu
675 680 685
Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met
690 695 700
Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His
705 710 715 720
Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
725 730 735
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
740 745 750
Ser Thr Gln Ser Lys Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
755 760 765
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
770 775 780
Asp Glu Leu Tyr Lys
785
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 15
cagaaacagc gtcttgtgca 20
<210> 16
<211> 17
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 16
aaacagcgtc ttgtgca 17
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 17
cagaaacagc gtcttctgca 20
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 18
cagaaagacc gtcttgtgca 20
<210> 19
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 19
cagaaacagc gtcttgtcca 20
<210> 20
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 20
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asp Ile His Glu Phe Lys Trp Gln Ala Leu Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Asp Ala Val Glu Lys His Ala Ala Ser Asp
225 230 235 240
Tyr Ala Ala Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Leu Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 21
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 21
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asn Ile His Glu Phe Lys Trp Gln Ala Val Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Ile Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Asp Ala Val Glu Lys His Ala Ala Ser Asp
225 230 235 240
Tyr Ala Ala Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Ile Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 22
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 22
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asp Ile His Glu Phe Lys Trp Gln Ala Leu Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Lys Ala Val Glu Thr His Ala Ala Ser Asp
225 230 235 240
Tyr Ala Ala Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Gln Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Ile Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 23
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 23
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asn Ile His Glu Phe Lys Trp Gln Ala Leu Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Lys Ala Val Glu Thr His Ala Ala Ser Asp
225 230 235 240
Tyr Ala Ala Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Ile Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 24
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 24
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Val Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asn Ile His Glu Phe Lys Trp Gln Ala Leu Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Lys Ala Val Glu Thr His Ala Ala Ser Asp
225 230 235 240
Tyr Ala Ala Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Ile Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 25
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 25
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asn Ile His Glu Phe Lys Trp Gln Ala Leu Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Lys Ala Val Glu Thr His Ala Ala Ser Asp
225 230 235 240
Tyr Ala Ala Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Leu Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 26
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 26
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asn Ile His Glu Phe Lys Trp Gln Ala Leu Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Ser Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Lys Ala Val Glu Thr His Ala Ala Ser Asp
225 230 235 240
Tyr Ala Ala Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Ile Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 27
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 27
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asn Ile His Glu Phe Lys Trp Gln Ala Val Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Lys Ala Val Asp Thr His Ala Ala Ser Glu
225 230 235 240
Tyr Ala Thr Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Ile Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Val Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Gln Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Ile
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Leu Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Asn Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Leu Val Ser Ile Pro Leu Ala
500 505 510
Phe Ile Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 28
<211> 551
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 28
Met Asn Ser Thr Ile Leu Leu Ala Gln Asp Ala Val Ser Glu Gly Val
1 5 10 15
Gly Asn Pro Ile Leu Asn Ile Ser Val Phe Val Val Phe Ile Ile Val
20 25 30
Thr Met Thr Val Val Leu Arg Val Gly Lys Ser Thr Ser Glu Ser Thr
35 40 45
Asp Phe Tyr Thr Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu
50 55 60
Ala Ile Ala Gly Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val
65 70 75 80
Gly Ala Ile Ser Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly
85 90 95
Phe Phe Val Ala Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu
100 105 110
Arg Asn Val Gly Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu
115 120 125
Arg Gln Lys Pro Val Arg Val Ala Ala Ala Cys Gly Thr Leu Ala Val
130 135 140
Thr Leu Phe Tyr Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val
145 150 155 160
Ser Val Leu Leu Asp Ile His Glu Phe Lys Trp Gln Ala Val Val Val
165 170 175
Gly Ile Val Gly Ile Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met
180 185 190
Lys Gly Thr Thr Tyr Val Gln Met Ile Lys Ala Val Leu Leu Val Gly
195 200 205
Gly Val Ala Ile Met Thr Val Leu Thr Phe Val Lys Val Ser Gly Gly
210 215 220
Leu Thr Thr Leu Leu Asn Lys Ala Val Asp Thr His Ala Ala Ser Glu
225 230 235 240
Tyr Ala Thr Thr Lys Gly Tyr Asp Pro Thr Gln Ile Leu Glu Pro Gly
245 250 255
Leu Gln Tyr Gly Ala Thr Leu Thr Thr Gln Leu Asp Phe Ile Ser Leu
260 265 270
Ala Leu Ala Leu Cys Leu Gly Thr Ala Gly Leu Pro His Val Leu Met
275 280 285
Arg Phe Tyr Thr Val Pro Thr Ala Lys Glu Ala Arg Lys Ser Val Thr
290 295 300
Trp Ala Ile Val Leu Ile Gly Ala Phe Tyr Leu Met Thr Leu Ile Leu
305 310 315 320
Gly Tyr Gly Ala Ala Ala Leu Val Gly Pro Asp Arg Val Ile Ala Ala
325 330 335
Pro Gly Ala Ala Asn Ala Ala Ala Pro Leu Leu Ala Phe Glu Leu Gly
340 345 350
Gly Ser Ile Phe Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val
355 360 365
Leu Ala Val Val Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly
370 375 380
His Asp Ile Tyr Asn Ala Val Ile Arg Asn Gly Glu Ser Thr Glu Ala
385 390 395 400
Glu Gln Val Arg Val Ser Arg Ile Thr Val Val Val Ile Gly Leu Val
405 410 415
Ser Ile Val Leu Gly Ile Leu Ala Met Thr Gln Asn Val Ala Phe Leu
420 425 430
Val Ala Leu Ala Phe Ala Val Ala Ala Ser Ala Asn Leu Pro Thr Ile
435 440 445
Leu Tyr Ser Leu Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala
450 455 460
Ala Ile Tyr Thr Gly Leu Ile Ser Ala Leu Val Leu Ile Phe Leu Ser
465 470 475 480
Pro Ala Val Ser Gly Ser Asp Ser Ala Met Val Pro Gly Ala Asp Trp
485 490 495
Ala Ile Phe Pro Leu Lys Asn Pro Gly Ile Val Ser Ile Pro Leu Ala
500 505 510
Phe Ile Ala Gly Trp Ile Gly Thr Leu Val Gly Lys Pro Asp Asn Met
515 520 525
Asp Asp Leu Ala Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly
530 535 540
Val Glu Lys Ala Val Asp His
545 550
<210> 29
<211> 549
<212> PRT
<213> 谷氨酸棒杆菌(Corynebacterium glutamicum)
<400> 29
Met Asn Ser Thr Ile Val Leu Ala Gln Glu Asp Thr Gly Asn Pro Ile
1 5 10 15
Leu Asn Ile Ser Val Phe Val Val Phe Ile Val Val Thr Met Thr Val
20 25 30
Val Met Arg Ala Gly Lys Thr Thr Lys Glu Ala Ser Asp Phe Tyr Thr
35 40 45
Gly Gly Ala Ser Phe Ser Gly Thr Gln Asn Gly Leu Ala Ile Ala Gly
50 55 60
Asp Tyr Leu Ser Ala Ala Ser Phe Leu Gly Ile Val Gly Ala Ile Ala
65 70 75 80
Leu Asn Gly Tyr Asp Gly Phe Leu Tyr Ser Ile Gly Phe Phe Val Ala
85 90 95
Trp Leu Val Ala Leu Leu Leu Val Ala Glu Pro Leu Arg Asn Val Gly
100 105 110
Arg Phe Thr Met Ala Asp Val Leu Ser Phe Arg Leu Arg Gln Lys Pro
115 120 125
Val Arg Val Ala Ala Ala Phe Gly Thr Leu Phe Val Ser Leu Phe Tyr
130 135 140
Leu Ile Ala Gln Met Ala Gly Ala Gly Ser Leu Val Ser Val Leu Leu
145 150 155 160
Asp Ile His Glu Phe His Trp Gln Ala Val Val Ile Gly Val Val Gly
165 170 175
Val Val Met Ile Ala Tyr Val Leu Leu Gly Gly Met Lys Gly Thr Thr
180 185 190
Tyr Val Gln Met Ile Lys Ala Cys Leu Leu Val Gly Gly Val Leu Ile
195 200 205
Met Thr Ile Trp Thr Phe Val Ala Val Lys Gly Gly Val Gly Thr Leu
210 215 220
Leu Glu Asp Ala Val Ala Met His Ala Ser Ser Glu Arg Ala Ala Thr
225 230 235 240
Leu Gly Tyr Glu Ala Thr Ala Ile Leu Glu Pro Gly Leu Gln Tyr Gly
245 250 255
Ala Thr Val Thr Lys Gln Leu Asp Phe Val Ser Leu Gly Leu Ala Leu
260 265 270
Val Leu Gly Thr Ala Gly Leu Pro His Val Leu Met Arg Phe Tyr Thr
275 280 285
Val Pro Thr Ala Thr Glu Ala Arg Lys Ser Val Thr Trp Ala Ile Ile
290 295 300
Leu Ile Gly Ser Phe Tyr Leu Met Thr Leu Ile Leu Gly Tyr Gly Ala
305 310 315 320
Ala Ala Leu Val Gly Pro Asp Arg Ile Val Asn Ala Pro Gly Ala Ala
325 330 335
Asn Ala Ala Ala Pro Leu Leu Ala Leu Glu Leu Gly Gly Ser Ile Phe
340 345 350
Met Ala Leu Ile Ser Ala Val Ala Phe Ala Thr Val Leu Ala Val Val
355 360 365
Ala Gly Leu Ala Ile Thr Ala Ser Ala Ala Val Gly His Asp Ile Tyr
370 375 380
Asp Ala Val Leu Arg Asp Gly Lys Ser Ser Glu Glu Glu Gln Val Arg
385 390 395 400
Val Ser Arg Ile Thr Ile Val Val Ile Gly Val Leu Ser Ile Val Leu
405 410 415
Gly Ile Leu Ala Met Ser Gln Asn Val Ala Phe Leu Val Ala Leu Ala
420 425 430
Phe Ala Ile Ala Ala Ser Ala Asn Leu Pro Cys Ile Leu Tyr Ser Leu
435 440 445
Tyr Trp Lys Lys Phe Asn Thr Thr Gly Ala Val Ala Ala Ile Tyr Thr
450 455 460
Gly Leu Val Ser Ala Leu Val Leu Ile Val Phe Ser Pro Ala Val Ser
465 470 475 480
Gly Thr Pro Thr Ser Met Val Pro Ser Asp Gly Gly Gly Trp Asp Ile
485 490 495
Phe Pro Leu Ser Asn Pro Gly Ile Ile Ser Ile Pro Leu Ala Phe Ile
500 505 510
Ala Gly Trp Ile Gly Thr Met Val Gly Lys Pro Asp Asn Leu Asp Asp
515 520 525
Leu Gln Ala Glu Met Glu Val Arg Ser Leu Thr Gly Val Gly Val Glu
530 535 540
Gly Ala Val Glu His
545
Claims (14)
1.一种甲醇的耐受性和利用率提高的菌株的构建方法，所述方法包括：弱化所述菌株中cgl0833基因的表达；所述菌株是通过生物学途径转化甲醇的谷氨酸棒杆菌(Corynebacterium glutamicum)。
2.如权利要求1所述的方法，其特征在于，所述弱化是将所述cgl0833基因的表达降低至野生型cgl0833基因表达的10%-90%。
3.如权利要求2所述的方法，其特征在于，所述弱化是将所述cgl0833基因的表达降低至野生型cgl0833基因表达的15%-85%。
4.如权利要求3所述的方法，其特征在于，所述弱化是将所述cgl0833基因的表达降低至野生型cgl0833基因表达的18%-80%。
5. 一种甲醇的耐受性和利用率提高的菌株，所述菌株中cgl0833基因的表达被弱化；所述菌株是通过生物学途径转化甲醇的谷氨酸棒杆菌 (Corynebacterium glutamicum)。
6.如权利要求5所述的甲醇的耐受性和利用率提高的菌株，其特征在于，所述菌株采用权利要求1-4中任一项所述的方法构建。
7.一种突变型cgl0833基因或其编码的蛋白，所述突变型cgl0833基因是指与相应野生型cgl0833基因相比，其表达降低的cgl0833基因；
与野生型cgl0833基因的核苷酸序列相比，所述突变型cgl0833基因的核苷酸序列中相应的第1439位的C突变为T；或者，与野生型cgl0833基因编码的氨基酸序列相比，突变型cgl0833基因所编码氨基酸序列中相应的第480位丝氨酸突变为苯丙氨酸；
所述野生型cgl0833基因编码的氨基酸序列如SEQ ID NO: 6所示。
8.如权利要求7所述的突变型cgl0833基因或其编码的蛋白，其特征在于，所述突变型cgl0833基因的表达降低至野生型cgl0833基因表达的10%-90%。
9.如权利要求8所述的突变型cgl0833基因或其编码的蛋白，其特征在于，所述突变型cgl0833基因的表达降低至野生型cgl0833基因表达的15%-85%。
10.如权利要求9所述的突变型cgl0833基因或其编码的蛋白，其特征在于，所述突变型cgl0833基因的表达降低至野生型cgl0833基因表达的18%-80%。
11.权利要求7-10中任一项所述的突变型cgl0833基因或其编码的蛋白在提高菌株的甲醇耐受性或构建甲醇生物转化菌株中的用途。
12.一种表达载体，所述表达载体包含权利要求7-10中任一项所述的突变型cgl0833基因。
13.一种谷氨酸棒杆菌宿主细胞，所述宿主细胞包含权利要求12所述的表达载体或基因组上整合有权利要求7-10中任一项所述的突变型cgl0833基因。
14.一种生物转化甲醇的方法，包括利用权利要求1-4中任一项所述构建方法构建的菌株或权利要求5或6所述菌株或权利要求7-10中任一项所述的基因或编码的蛋白或权利要求12所述的表达载体或权利要求13所述的宿主细胞进行甲醇的生物转化。
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