CN112062821B - 一种碳分解代谢调控蛋白CcpA突变体K31A - Google Patents
一种碳分解代谢调控蛋白CcpA突变体K31A Download PDFInfo
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- CN112062821B CN112062821B CN202011015122.XA CN202011015122A CN112062821B CN 112062821 B CN112062821 B CN 112062821B CN 202011015122 A CN202011015122 A CN 202011015122A CN 112062821 B CN112062821 B CN 112062821B
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Abstract
本发明公开了一种碳分解代谢调控蛋白CcpA突变体K31A，属于蛋白质工程技术领域。本发明对从地衣芽孢杆菌基因组中克隆的CcpA基因进行突变，得到一种碳分解代谢调控蛋白CcpA突变体K31A，其氨基酸序列如SEQ ID NO.2所示，编码其的基因核苷酸序列如SEQ ID NO.1所示。本发明构建了包含编码上述CcpA突变体K31A基因的重组表达载体，并将构建的表达载体转化到地衣芽孢杆菌CcpA基因缺陷株中获得重组地衣芽孢杆菌。所获得的重组地衣芽孢杆菌能够明显改变由于葡萄糖存在而产生的碳分解代谢阻遏现象，能够降低地衣芽孢杆菌对木糖的偏好性。
Description
技术领域
本发明属于蛋白质工程领域，涉及一种碳分解代谢调控蛋白CcpA突变体K31A，还涉及表达该突变体的重组表达载体及重组微生物细胞，进一步涉及该突变体在地衣芽孢杆菌发酵过程中对当木糖与葡萄糖共存时对木糖利用的影响。
背景技术
地衣芽孢杆菌(Bacillus licheniformis)是一种革兰氏阳性菌，具有耐热、酶系丰富、产酶量高、生长速率适中、蛋白折叠、全基因组信息公开等优点。与大肠杆菌相比，地衣芽孢杆菌具有耐热性高、pH耐受性较低、生物量高等优势，因此，不仅广泛地被用于外源基因的表达宿主，在发酵工业上也具有巨大的潜力。
木糖作为一种优质碳源，可以通过木质纤维素水解而得到，随着目前环境与能源的压力，木质纤维素等可再生的碳越来越受到人们的青睐。地衣芽孢杆菌可以利用木糖，但是其对木糖的利用会受到葡萄糖的抑制。而木质纤维素在水解过程中产生大量木糖的同时，也会产生大量的葡萄糖。由于碳分解代谢阻遏效应，葡萄糖的存在会抑制地衣芽孢杆菌对木糖的利用，为了消除葡萄糖存在而产生的碳分解代谢阻遏效应，许多解除或者降低碳分解代谢阻遏效应的尝试已经在其他的微生物中进行，主要分为两类：一是通过人为构建代谢途径的方式增加木糖的利用；二是通过对细菌本身的代谢途径进行干预，来构建碳分解代谢阻遏降低的工程菌。但是通过敲除碳分解代谢调控蛋白CcpA蛋白的方式来抑制碳分解代谢阻遏，降低葡萄糖对木糖利用的抑制，增加木糖的利用效率，菌体生长会受到明显的抑制作用，其他的代谢途径也受到明显的影响。因此通过基因敲除构建的工程菌未能投入商业化生产。
因而，目前本领域普遍认为单纯敲除碳分解代谢调控蛋白会导致菌体生长受限，破坏菌体的代谢途径，往往得不到人们预期的结果，随着蛋白质工程的进步，对蛋白的理性改造越来越受到人们的青睐。因此通过对碳分解代谢调控蛋白的活性位点的定向改造来降低由于葡萄糖的存在而引起的木糖利用抑制可能是一个有效的手段。
发明内容
针对现有技术存在的上述问题，本发明提供了一种碳分解代谢调控蛋白CcpA突变体K31A(Lys31Ala)及其应用。本发明从地衣芽孢杆菌ATCC 9945A基因组中，克隆得到编码碳分解代谢调控蛋白CcpA蛋白的基因序列，将其编码负责DNA结合结构域的氨基酸进行定点突变，并在大肠杆菌中进行异源表达与纯化；体外测定CcpA蛋白与调控位点cre位点的结合能力，初步筛选出与cre位点结合能力有明显差距的CcpA突变体K31A蛋白；将该突变体蛋白在地衣芽孢杆菌CcpA缺陷株中进行表达，验证该突变体在葡萄糖存在下对木糖利用的影响，得到一种可以改善地衣芽孢杆菌对木糖利用偏好性的CcpA突变体K31A。
本发明的技术方案如下：
本发明提供了一种碳分解代谢调控蛋白CcpA突变体K31A，氨基酸序列如SEQ IDNO.2所示。
本发明还提供了编码上述碳分解代谢调控蛋白CcpA突变体K31A的基因，所述基因的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了含上述基因的重组表达载体。
本发明还提供了表达上述碳分解代谢调控蛋白CcpA突变体K31A的重组微生物细胞。
进一步地，所述重组微生物细胞以地衣芽孢杆菌(Bacillus Licheniformis)为宿主。
进一步地，所述地衣芽孢杆菌为CcpA基因缺陷菌株，所述CcpA基因缺陷菌株的构建方法是：地衣芽孢杆菌中原始CcpA基因由于被SEQ ID NO.3所示的外源基因插入而失活；CcpA基因经外源基因插入后基因序列变为SEQ ID NO.4。
本发明还提供了上述的碳分解代谢调控蛋白CcpA突变体K31A在改善地衣芽孢杆菌对木糖利用偏好性中的应用。
本发明还提供了上述的基因在改善地衣芽孢杆菌对木糖利用偏好性中的应用。
本发明还提供了上述的重组表达载体在改善地衣芽孢杆菌对木糖利用偏好性中的应用。
本发明还提供了上述的重组微生物细胞在改善地衣芽孢杆菌对木糖利用偏好性中的应用。
本发明的主要思路如下：
(1)根据蛋白的同源建模预CcpA蛋白的关键氨基酸，对氨基酸进行定点突变；
(2)将得到的突变体蛋白在大肠杆菌中进行异源表达与纯化；
(3)在体外对突变体与核酸的结合能力进行筛选；
(4)将筛选得到的突变体蛋白构建CcpA蛋白重组表达质粒；
(5)CcpA突变体K31A蛋白体内影响验证，验证其在菌体内对木糖与葡萄糖利用的影响。
本发明的有益效果：
本发明对从地衣芽孢杆菌中克隆的CcpA基因进行突变，得到的一种碳分解代谢调控蛋白CcpA突变体K31A，其氨基酸序列如SEQ ID NO.2所示，编码其的基因核苷酸序列如SEQ ID NO.1所示。本发明构建了包含编码上述CcpA突变体K31A基因的重组表达载体，并将构建的表达载体转化到地衣芽孢杆菌CcpA基因缺陷菌株中获得重组地衣芽孢杆菌；所获得的重组地衣芽孢杆菌能够明显改变由于葡萄糖存在而产生的碳分解代谢阻遏现象，能够降低地衣芽孢杆菌对木糖的偏好性。
附图说明
图1是地衣芽孢杆菌CcpA蛋白突变体K31A的定点突变菌落PCR扩增验证产物，其中，泳道1是Marker，泳道2-8是挑取的转化后的菌落阳性验证，目的条带长度1002bp。
图2是CcpA突变体K31A蛋白纯化后SDS-PAGE凝胶验证，其中，泳道1是Marker，泳道2是细胞破碎液，泳道3-5分别是蛋白纯化的洗脱液1、洗脱液2与洗脱液3，目的条带长度1002bp。
图3是地衣芽孢杆菌CcpA蛋白敲除流程图。
图4是地衣芽孢杆菌CcpA基因敲除验证图，其中，泳道1是Marker(2000bp)；泳道5是敲除菌株左交换验证(1460bp)，泳道6是右交换验证(1460bp)，泳道7是双交换验证(2098bp)。
图5是地衣芽孢杆菌CcpA缺陷株生长曲线。
图6是CcpA突变体K31A在CcpA基因缺陷株中的过表达重组质粒图谱。
图7是重组菌株K31A-BL21-ΔCcpA在木糖葡萄糖混合碳源培的养基中的生长曲线。
图8是重组菌K31A-BL21-ΔCcpA在以木糖葡萄糖混合碳源的培养基中对木糖与葡萄糖的消耗曲线。
具体实施方式
下面结合实施例和附图进一步来描述本发明。本发明中使用的地衣芽孢杆菌ATCC9945A是商用菌株，可通过购买获得。本发明使用的构建表达载体、转化、PCR等手段均为常规的分子生物学方法。
(一)培养基：CcpA蛋白突变体体内功能验证培养基为添加了30g/L葡萄糖、30g/L木糖的LB培养基。
(二)地衣芽孢杆菌CcpA基因缺陷菌株的构建方法
从地衣芽孢杆菌基因组上CcpA基因上游299bp处开始选取554bp作为同源臂一，在地衣芽孢杆菌CcpA基因下游991bp处开始选取500bp作为同源臂二，将含有FRT位点的Kan基因插入至两个同源臂中间，构成CcpA基因的敲除盒，将该敲除盒连接至pNZT1-Tet粒上，构建CcpA基因敲除质粒。将该质粒转化至地衣芽孢杆菌中构建含有CcpA基因敲除盒的重组菌。将该重组菌在30℃下活化培养，将活化培养后的种子液在42℃下培养，将种子液在含Kan的LB平板上进行培养，挑取单双交换菌株，将得到的单交换菌株在30℃下培养，将培养后的种子液在37℃下划线培养，挑取双交换菌株，作为地衣芽孢杆菌CcpA基因缺陷菌株。
(三)高效液相色谱测定培养基中的木糖与葡萄糖的含量的方法
地衣芽孢杆菌CcpA突变体K31A过表达菌株在LB培养基中培养活化，之后取1mL菌液接种至250mL摇瓶中并在LB培养基中添加30g/L葡萄糖、30g/L木糖。每隔3h取样测定培养基中的碳源的消耗，将培养基在12000rpm下离心5min，将得到的上清取出加入等体积的10％三氯乙酸去除培养基中的杂质。然后利用氨基柱进行检测，流动相为乙腈与水(3：7)作为流动相，进行检测。
实施例1地衣芽孢杆菌CcpA蛋白的关键氨基酸的选取
地衣芽孢杆菌CcpA基因作为一个全局型调控因子主要通过蛋白与核酸位点进行结合，然后发挥其调控功能，CcpA蛋白与核酸结合后其相应的结合部位的亚结构的相对位置会发生一定的变化，在这一变换过程中起支点作用的氨基酸有可能位于CcpA蛋白亚结构中α螺旋的两端。同时CcpA蛋白与核酸结合需要蛋白与cre位点的识别，在蛋白与cre位点识别中其主要作用的氨基酸有可能位于α螺旋的中间，因此我们在选取氨基酸位点时主要分布在CcpA蛋白α螺旋的两端以及中间。
实施例2地衣芽孢杆菌CcpA蛋白表达载体的构建
以地衣芽孢杆菌CcpA蛋白基因组ATCC 9945A为模板，扩增CcpA基因，将得到的基因连接在pET28a载体上，构建地衣芽孢杆菌CcpA蛋白表达载体。将其转化至大肠杆菌BL21(DE3)中构建重组菌株。
实施例3地衣芽孢杆菌CcpA蛋白定点突变及表达与纯化
以实施例2中构建的地衣芽孢杆菌CcpA蛋白表达载体为模板对地衣芽孢杆菌CcpA蛋白进行定点突变。
定点突变引物序列如下：
K31A-F(SEQ ID NO.5)：5’gaaatccgaacgtcgcgccgacgacgagaaagaaggtgcttgaagccatc3’；
K31A-R(SEQ ID NO.6)：5’ttctcgtcgtcggcgcgacgttcggatttccgttcacaaccctggaaacg3’。
将定点突变后的CcpA蛋白(氨基酸序列如SEQ ID NO.2所示，编码其的基因核苷酸序列如SEQ ID NO.1所示)在大肠杆菌中进行异源表达与纯化，菌落PCR扩增验证图如图1所示，SDS-PAGE凝胶验证图如图2所示，表明地衣芽孢杆菌CcpA蛋白突变体成功导入大肠杆菌并在大肠杆菌中成功表达。
实施例4突变体蛋白与cre位点结合和能力的测定
将实施例3得到的突变体蛋白在体外利用荧光偏振与EMSA测定其与cre位点的结合能力。该结果表明，突变体K31A与对照组相比，较对照组与cre位点的结合能力有明显的降低。
实施例5地衣芽孢杆菌CcpA基因的敲除
在地衣芽孢杆菌CcpA蛋白两端设计同源臂，在同源臂种间插入Kan基因，构成基因敲除盒，将该敲除盒连接在pNZT1-Tet质粒上，利用同源重组原理进行敲除。敲除流程如图3所示。敲除后的验证图如图4所示，结果显示在敲除菌株中成功扩增出左交换条带、右交换条带以及双交换条带。
实施例6地衣芽孢杆菌CcpA突变体K31A蛋白的过表达
将得到的与cre位点结合能力有明显差距的突变体K31A连接在P43启动子后，与pHY300连接，构建重组表达载体，如图6所示。在地衣芽孢杆菌CcpA缺陷株(生长曲线如图5所示)中进行表达，得到重组表达CcpA蛋白突变体的菌株K31A-BL21-ΔCcpA。
实施例7CcpA突变体K31A蛋白对地衣芽孢杆菌对木糖及葡萄糖利用的影响
将实施例6得到的重组表达CcpA蛋白突变体的菌株K31A-BL21-ΔCcpA在以木糖与葡萄糖混合碳源的培养基(添加了30g/L葡萄糖、30g/L木糖的LB培养基)中在37℃，250rpm下进行培养，取样利用高效液相色谱测定培养基中的木糖与葡萄糖的含量。
结果如图7和图8所示，培养21h后，木糖残糖量从29.2g/L下降至22.8g/L。重组表达CcpA蛋白突变体的菌株K31A-BL21-ΔCcpA，较原始菌株而言，其对葡萄糖的利用速率没有较大差别，但是在葡萄糖存在的情况下对木糖的利用速率有明显的提升。
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> 一种碳分解代谢调控蛋白CcpA突变体K31A
<160> 4
<170> PatentIn version 3.3
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atgagtaatg tgacaatata tgatgtagca cgcgaagcaa atgtaagtat ggcaaccgtt 60
tccagggttg tgaacggaaa tccgaacgtc gcgccgacga cgagaaagaa ggtgcttgaa 120
gccatcgagc gtcttggcta tcgtccaaat gccgtggcaa ggggccttgc aagcaaaaag 180
acgacgactg tcggcgtgat cattcccgat atttccagca tcttttattc agagctggcg 240
aggggaatcg aagatatcgc aacgatgtac aagtacaata ttattttgag caactccgac 300
cagaatatgg acaaagaact tcatcttttg aatacgatgc tgggaaaaca agttgacggt 360
atcgtcttta tgagcggaaa tgtcactgaa gagcatgtgg aggagtttaa gcggtcacca 420
gttccgatcg tgcttgcggc atctgttgaa gaaaaagggg aaacgccgtc ggttgcgatc 480
gattatgaac aggcgattta tgatgcggct accatgctga ttgaaaaagg ccataagcgc 540
cttgcgtttg tatcaggacc tatgactgag ccggtcaatc aagcgaaaaa acttcaaggc 600
tttaaaagag cgcttgagga taaggggctg acatttaaag aagagtatgt cgcagaaggc 660
gattatacgt acgattcagg aatggaagcg ctggaggcgc taatgaagct ggatgaaaaa 720
ccgacggccg tcctgtcagc gacagacgaa atggcactcg gcgttattca cgcagcacag 780
gataaaggac tggctattcc ggatgacctt gaagtgatcg gctttgacaa tacaaggctt 840
tcattaatgg ttcgaccgca gctgtcgact gtcgtccagc cgacgtatga tatcggtgcc 900
gtagcgatga gacttctgac aaagctgatg aacaaagagg aagtcgaaga gcatattgtc 960
cagctgccgc atcgcattga actcagacaa tcaacaaaat ga 1002
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Met Leu Gly Lys Gln Val Asp Gly Ile Val Phe Met Ser Gly Asn Val
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Thr Glu Glu His Val Glu Glu Phe Lys Arg Ser Pro Val Pro Ile Val
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Leu Ala Ala Ser Val Glu Glu Lys Gly Glu Thr Pro Ser Val Ala Ile
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Asp Tyr Glu Gln Ala Ile Tyr Asp Ala Ala Thr Met Leu Ile Glu Lys
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Gly His Lys Arg Leu Ala Phe Val Ser Gly Pro Met Thr Glu Pro Val
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Asn Gln Ala Lys Lys Leu Gln Gly Phe Lys Arg Ala Leu Glu Asp Lys
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atcgaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt cggccagttt 60
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ttattaatag ctgaataaga acggtgctct ccaaatattc ttatttagaa aagcaaatct 180
aaaattatct gaaaagggaa tgagaatagt gaatggacca ataataatga ctagagaaga 240
aagaatgaag attgttcatg aaattaagga acgaatattg gataaatatg gggatgatgt 300
taaggctatt ggtgtttatg gctctcttgg tcgtcagact gatgggccct attcggatat 360
tgagatgatg tgtgtcatgt caacagagga agcagagttc agccatgaat ggacaaccgg 420
tgagtggaag gtggaagtga attttgatag cgaagagatt ctactagatt atgcatctca 480
ggtggaatca gattggccgc ttacacatgg tcaatttttc tctattttgc cgatttatga 540
ttcaggtgga tacttagaga aagtgtatca aactgctaaa tcggtagaag cccaaacgtt 600
ccacgatgcg atttgtgccc ttatcgtaga agagctgttt gaatatgcag gcaaatggcg 660
taatattcgt gtgcaaggac cgacaacatt tctaccatcc ttgactgtac aggtagcaat 720
ggcaggtgcc atgttgattg gtctgcatca tcgcatctgt tatacgacga gcgcttcggt 780
cttaactgaa gcagttaagc aatcagatct tccttcaggt tatgaccatc tgtgccagtt 840
cgtaatgtct ggtcaacttt ccgactctga gaaacttctg gaatcgctag agaatttctg 900
gaatgggatt caggagtgga cagaacgaca cggatatata gtggatgtgt caaaacgcat 960
accattttga acgatgacct ctaataattg ttaatcatgt tggttacgta tttattaact 1020
tctcctagta ttagtaatta tcatggctgt catggcgcat taacggaata aagggtgtgc 1080
ttaaatcggg ccattttgcg taataagaaa aaggattaat tatgagcgaa ttgaattaat 1140
aataaggtaa tagatttaca ttagaaaatg aaaggggatt ttatgcgtgg aagttcctat 1200
tccgaagttc ctattctcta gaaagtatag gaacttcgat 1240
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gctttcgagc ggtccttttt ttagttgtca tcaaaaattg tcgaaaaaat gtcttatttt 60
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tattaggtaa aaccgtgtat aatttcagag agcccctttt gttttggcag gttatgaaaa 180
ataatgtaaa cttgagttct gtttatttta cataaagttt tacattttga catttctcta 240
catgaaaatg tttatgctat aggaaaagaa aagtgtaccc agttgaagga gtggtaaaaa 300
tgagtaatgt gacaatatat gatgtagcac gcgaagcaaa tgtaagtatg gcaaccgttt 360
ccagggttgt gaacggaaat ccgaacgtca agccgacgac gagaaagaag gtgcttgaag 420
ccatcgagcg tcttggctat cgtccaaatg ccgtggcaag gggccttgca agcaaaaaga 480
cgacgactgt cggcgtgatc attcccgata tttccagcat cttttattca gagctggcga 540
ggggaatcga agatatcgaa gttcctattc cgaagttcct attctctaga aagtatagga 600
acttcggcca gtttgttgaa gattagatgc tataattgtt attaaaagga ttgaaggatg 660
cttaggaaga cgagttatta atagctgaat aagaacggtg ctctccaaat attcttattt 720
agaaaagcaa atctaaaatt atctgaaaag ggaatgagaa tagtgaatgg accaataata 780
atgactagag aagaaagaat gaagattgtt catgaaatta aggaacgaat attggataaa 840
tatggggatg atgttaaggc tattggtgtt tatggctctc ttggtcgtca gactgatggg 900
ccctattcgg atattgagat gatgtgtgtc atgtcaacag aggaagcaga gttcagccat 960
gaatggacaa ccggtgagtg gaaggtggaa gtgaattttg atagcgaaga gattctacta 1020
gattatgcat ctcaggtgga atcagattgg ccgcttacac atggtcaatt tttctctatt 1080
ttgccgattt atgattcagg tggatactta gagaaagtgt atcaaactgc taaatcggta 1140
gaagcccaaa cgttccacga tgcgatttgt gcccttatcg tagaagagct gtttgaatat 1200
gcaggcaaat ggcgtaatat tcgtgtgcaa ggaccgacaa catttctacc atccttgact 1260
gtacaggtag caatggcagg tgccatgttg attggtctgc atcatcgcat ctgttatacg 1320
acgagcgctt cggtcttaac tgaagcagtt aagcaatcag atcttccttc aggttatgac 1380
catctgtgcc agttcgtaat gtctggtcaa ctttccgact ctgagaaact tctggaatcg 1440
ctagagaatt tctggaatgg gattcaggag tggacagaac gacacggata tatagtggat 1500
gtgtcaaaac gcataccatt ttgaacgatg acctctaata attgttaatc atgttggtta 1560
cgtatttatt aacttctcct agtattagta attatcatgg ctgtcatggc gcattaacgg 1620
aataaagggt gtgcttaaat cgggccattt tgcgtaataa gaaaaaggat taattatgag 1680
cgaattgaat taataataag gtaatagatt tacattagaa aatgaaaggg gattttatgc 1740
gtggaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt cgatatcggt 1800
gccgtagcga tgagacttct gacaaagctg atgaacaaag aggaagtcga agagcatatt 1860
gtccagctgc cgcatcgcat tgaactcaga caatcaacaa aatgatcatg attaaataaa 1920
cgagaaagaa agcaagtgtt cacagctttt ttcgtgaatt gcttgctttc ttttccttga 1980
gctggagaga ttatgaaaaa atgaaacgat ttgattatct gacaccggtc ggtttgattt 2040
tgggtatttt cattttagta atcggcgttg tctctggagc ggggctttcc ggtttttatt 2100
cgtttatcga tttgacgtct ttctttatcg tgacgggcgg attgtgtgca gctgttttca 2160
tcagcttttc cccgaaagat ttaaaaagag cgccggctgt gctgaagcag gtctttattt 2220
cggaagaaga cgatgtacgc gacttggtca aaacctttgt cagcctgtct gaacaggcgc 2280
gcaaacaagg aatt 2294
Claims (10)
1.一种碳分解代谢调控蛋白CcpA突变体K31A，其特征在于，所述碳分解代谢调控蛋白CcpA突变体K31A的氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述碳分解代谢调控蛋白CcpA突变体K31A的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.1所示。
3.含权利要求2所述基因的重组表达载体。
4.表达权利要求1所述碳分解代谢调控蛋白CcpA突变体K31A的重组微生物细胞。
5.根据权利要求4所述的重组微生物细胞，其特征在于，所述重组微生物细胞以地衣芽孢杆菌(Bacillus Licheniformis)为宿主。
6.根据权利要求5所述的重组微生物细胞，其特征在于，所述地衣芽孢杆菌为CcpA基因缺陷菌株，所述CcpA基因缺陷菌株的构建方法是：地衣芽孢杆菌中原始CcpA基因被SEQ IDNO.3所示的外源基因插入而失活；CcpA基因经外源基因插入后基因序列变为SEQ ID NO.4。
7.权利要求1所述的碳分解代谢调控蛋白CcpA突变体K31A在改善地衣芽孢杆菌对木糖利用偏好性中的应用。
8.权利要求2所述的基因在改善地衣芽孢杆菌对木糖利用偏好性中的应用。
9.权利要求3所述的重组表达载体在改善地衣芽孢杆菌对木糖利用偏好性中的应用。
10.权利要求4-6任一所述的重组微生物细胞在改善地衣芽孢杆菌对木糖利用偏好性中的应用。
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