CN114934061A - 工程菌及其在全细胞催化酮基泛解酸内酯生产d-泛解酸内酯中的应用 - Google Patents
工程菌及其在全细胞催化酮基泛解酸内酯生产d-泛解酸内酯中的应用 Download PDFInfo
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- CN114934061A CN114934061A CN202210556247.6A CN202210556247A CN114934061A CN 114934061 A CN114934061 A CN 114934061A CN 202210556247 A CN202210556247 A CN 202210556247A CN 114934061 A CN114934061 A CN 114934061A
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Abstract
本发明属于生物催化技术领域。本发明公开了工程菌及其在全细胞催化酮基泛解酸内酯生产D‑泛解酸内酯中的应用。本发明的工程菌实现酮基泛解酸内酯还原酶突变体在辅酶再生***中催化合成D‑泛解酸内酯,底物浓度高达100g/L,转化率≥99.0%,重量收率高达95%。
Description
技术领域
本发明属于生物催化技术领域。
背景技术
泛酸,又称遍多酸、维生素B5,广泛存在于生物界内,是动物体内合成辅酶A的前体,其主要作用是参与动物体内蛋白质、脂肪和糖的代谢。泛酸的活性成分为D型,但D-泛酸在高温、酸碱下不稳定,因此相比于D-泛酸,其稳定性更好的衍生物在食品、医药、饲料等行业得到了更广泛的应用。目前被广泛应用的泛酸衍生物主要有D-泛酸钙、D-泛醇以及D-泛硫乙胺等,其中主要以D-泛酸钙为主。
D-泛酸及其衍生物的工业化生产以D-泛解酸内酯(D-Pantolactone,D-PL)作为前体物质。泛解酸内酯因具有手性碳原子而有三种存在形式:DL-泛解酸内酯(外消旋泛解酸内酯)、D-泛解酸内酯和L-泛解酸内酯,其中只有D-泛解酸内酯具有生物活性,可以作为D-泛酸及其衍生物合成的重要前体。
D-泛解酸内酯的合成主要有两种方法:1)化学合成法,以异丁醛—甲醛—氢氰酸经过一系列步骤得到DL-泛解酸内酯,后通过对映体过量结晶法或手性拆分剂获得D-泛解酸内酯,而L-泛解酸内酯经萃取外消旋后再次拆分。该方法在合成路线中涉及有毒化合物氢氰酸的使用,导致生产企业环保压力提高;2)微生物水解酶法,是目前获得D-泛解酸内酯的主要渠道。该方法以化学合成的外消旋泛解酸内酯为原料,利用内酯水解酶选择性水解消旋体中的D-泛解酸内酯,接着分离D-泛解酸和L-泛解酸内酯,分离后的D-泛解酸经酸化成环形成D-泛解酸内酯,而L-泛解酸内酯经消旋后重复参与水解。该工艺不涉及有毒物质的使用,但也存在反复萃取消旋、酸碱耗用高等问题。因此,开发工艺简单、高效环保的不对称还原路线具有重要的研究意义。
发明内容
有鉴于此,本发明提供了一种质粒,所述质粒pBAD-DCR由DCR基因***到表达载体pBAD获得;所述DCR基因的氨基酸序列如SEQ ID NO.2所示;以及用于辅因子循环的质粒pBAD-GDH,所述质粒pBAD-GDH由GDH基因***到表达载体pBAD获得;所述GDH基因的氨基酸序列如SEQ ID NO.8所示。
本发明还提供了一种重组质粒,包括:质粒pBAD-DCR-GDH、质粒pBAD-E224G-GDH和质粒pBAD-E224Q-GDH;所述质粒pBAD-DCR-GDH由T4连接酶连接酶切质粒pBAD-DCR后的DCR片段、酶切权利要求1所述质粒pBAD-GDH后的GDH片段和酶切pBAD后的线性载体片段获得;所述质粒pBAD-E224G-GDH由T4连接酶连接酶切质粒pBAD-E224G后的E224G片段、酶切权利要求1所述质粒pBAD-GDH后的GDH片段和酶切pBAD后的线性载体片段获得;所述质粒pBAD-E224G由E224G基因***到表达载体pBAD获得;所述E224G基因的氨基酸序列如SEQ ID NO.4所示;所述质粒pBAD-E224Q-GDH由T4连接酶连接酶切质粒pBAD-E224Q的后的E224Q片段、酶切切权利要求1所述质粒pBAD-GDH后的GDH片段和酶切pBAD后的线性载体片段获得;所述质粒pBAD-E224Q由E224Q基因***到表达载体pBAD获得;所述E224Q基因的氨基酸序列如SEQID NO.6所示。
本发明另外还提供了一种工程菌,所述工程菌pBAD-DCR-GDH BW25113由权利要求2所述质粒pBAD-DCR-GDH化转入感受态细胞大肠杆菌BW25113获得工程菌。
在本发明的具体实施例中,所述工程菌pBAD-E224G-GDH BW25113由权利要求2所述质粒pBAD-E224G-GDH化转入感受态细胞大肠杆菌BW25113获得工程菌pBAD-E224G-GDHBW25113。
在本发明的具体实施例中,所述工程菌pBAD-E224Q-GDH BW25113由权利要求2所述质粒pBAD-E224Q-GDH化转入感受态细胞大肠杆菌BW25113获得工程菌pBAD-E224Q-GDHBW25113。
本发明最后提供了工程菌在全细胞催化酮基泛解酸内酯生产D-泛解酸内酯中的应用。
本发明具有以下有益效果:
1、使用高效的酮基泛解酸内酯还原酶,以及辅酶再生***来催化合成D-泛解酸内酯,底物浓度高达100g/L,转化率为≥99.0%,重量收率高达95%。生产成本和产品质量优于化学合成和微生物水解酶法,适合于工业化生产;
2、酶促反应选择性高,无副产物,产品后提取和精制简单。
附图说明
图1.利用酮基泛解酸内酯还原酶突变体高效催化生产D-泛解酸内酯的路线图。
图2.D-泛解酸内酯的气相分析图。
图3.D-泛解酸内酯的气相分析图。
具体实施方式
以下结合实施例对本发明作进一步详细说明,但并不因此将本发明限制在所述的实施例范围之中。
酮基泛解酸内酯购自北京伊诺凯科技有限公司
大肠杆菌BW25113菌株购自上海泽叶生物科技有限公司。
pBAD载体购自北京庄盟国际生物基因科技有限公司。
未特别说明的实验步骤均按照试剂说明书操作。
实施例1.
工程菌pBAD-DCR-GDH BW25113高效催化酮基泛解酸内酯生产D-泛解酸内酯
在50mM、pH7.0、10mL的PB缓冲溶液中加入制备的工程菌pBAD-DCR-GDH BW25113湿菌体1.0g,加入酮基泛解酸内酯(KPL)0.5g,后加入1倍于KPL投料量的葡萄糖(摩尔比)并通过GDH促使辅因子循环再生,控制pH在7.0、30℃的条件下反应,气相色谱监控进程至KPL反应完全。后用6M HCl酸化至pH=3.5左右,80℃加热反应溶液15min,加入0.5%的H2O2脱色30min,后用等体积乙酸乙酯萃取三次,合并有机相,蒸馏浓缩得到D-泛解酸内酯粗品0.526g。用乙酸乙酯/石油醚(60-90℃)重结晶得到纯品共0.483g。
D-泛解酸内酯的检测分析条件:取上述样品溶液300ul,加入1/3体积量的6M HCl酸化后于80℃加热15分钟,冷却至室温后加入同等体积的色谱纯乙酸乙酯混匀,12000rpm高速离心2min后取有机相溶液,无水Na2SO4干燥后使用Agilent 7890气相色谱仪分析,所用色谱柱为Astec CHIRALDEX B-PN气相毛细管色谱柱,氮气流速为6.5ml/min,进样口和检测器温度均为200℃,程序升温为100℃(保持2min),0.5℃/min至110℃,共运行12.6min。
实施例2.
工程菌pBAD-E224G-GDH BW25113全细胞催化酮基泛解酸内酯生产D-泛解酸内酯
在50mM、pH7.0、10mL的PB缓冲溶液中加入制备的工程菌pBAD-E224G-GDH BW25113湿菌体1.0g,加入酮基泛解酸内酯(KPL)0.70g,后加入1倍于KPL投料量的葡萄糖(摩尔比)并通过GDH促使辅因子循环再生,控制pH在7.0、30℃的条件下反应,气相色谱监控进程至KPL反应完全。后用6M HCl酸化至pH=3.5左右,80℃加热反应溶液15min,加入0.5%的H2O2脱色30min,后用等体积乙酸乙酯萃取三次,合并有机相,蒸馏浓缩得到D-泛解酸内酯粗品0.73g。用乙酸乙酯/石油醚(60-90℃)重结晶得到纯品共0.68g。
实施例3.
工程菌pBAD-E224G-GDH BW25113全细胞催化酮基泛解酸内酯生产D-泛解酸内酯
在50mM、pH6.5、10mL的PB缓冲溶液中加入制备的工程菌pBAD-E224G-GDH BW25113湿菌体1.5g,加入酮基泛解酸内酯(KPL)1g,后加入1倍于KPL投料量的葡萄糖(摩尔比)并通过GDH促使辅因子循环再生,控制pH在6.5、30℃的条件下反应,气相色谱监控进程至KPL反应完全。后用6M HCl酸化至pH=3.5左右,80℃加热反应溶液15min,加入0.5%的H2O2脱色30min,后用等体积乙酸乙酯萃取三次,合并有机相,蒸馏浓缩得到D-泛解酸内酯粗品1.05g。用乙酸乙酯/石油醚(60-90℃)重结晶得到纯品共0.97g。
实施例4.
工程菌pBAD-E224Q-GDH BW25113全细胞催化酮基泛解酸内酯生产D-泛解酸内酯
在50mM,pH7.0,10mL的PB缓冲溶液中加入制备的pBAD-E224Q-GDH BW25113湿菌体1.5g,加入酮基泛解酸内酯(KPL)0.75g,后加入1倍于底物KPL投料量的葡萄糖(摩尔比)并通过GDH促使辅因子循环再生,控制pH在7.0、30℃的条件下反应,气相色谱监控进程至KPL反应完全。后用6M HCl酸化至pH=3.5左右,80℃加热反应溶液15min,加入0.5%的H2O2脱色30min,后用等体积乙酸乙酯萃取三次,合并有机相,蒸馏浓缩得到D-泛解酸内酯粗品0.78g。用乙酸乙酯/石油醚(60-90℃)重结晶得到纯品共0.73g。
实施例5.
工程菌pBAD-E224Q-GDH BW25113全细胞催化酮基泛解酸内酯生产D-泛解酸内酯
在50mM,pH6.5,10mL的PB缓冲溶液中加入制备的pBAD-E224Q-GDH BW25113湿菌体2.0g,加入酮基泛解酸内酯(KPL)0.95g,后加入1倍于底物KPL投料量的葡萄糖(摩尔比)并通过GDH促使辅因子循环再生,控制pH在6.5、30℃的条件下反应,气相色谱监控进程至KPL反应完全。后用6M HCl酸化至pH=3.5左右,80℃加热反应溶液15min,加入0.5%的H2O2脱色30min,后用等体积乙酸乙酯萃取三次,合并有机相,蒸馏浓缩得到D-泛解酸内酯粗品0.98g。用乙酸乙酯/石油醚(60-90℃)重结晶得到纯品共0.92g。
实施例6
1、来源于平滑假丝酵母Candida parapsilosis CDC31的新的重组酮基泛解酸内酯还原酶pBAD-DCR-BW25113的构建
合成来源于平滑假丝酵母Candida parapsilosis CDC31的酮基泛解酸内酯还原酶基因,简称:DCR,Genebank ID:HE605209.1,DCR基因的核苷酸序列如SEQ ID NO.1所示和氨基酸序列如SEQ ID NO.2,DCR基因***到表达载体pBAD的NdeI和EcoRI位点得到质粒pBAD-DCR。质粒pBAD-DCR随后转入大肠杆菌宿主BW25113,得到pBAD-DCR-BW25113菌株。37℃过夜培养后挑单菌落至装有5mL LB的试管培养(100μg/mL氨苄抗性),以甘油管形式保存菌种。剩余种子液按2%比例接种到800mL,含100μg/mL氨苄抗性的LB培养基,37℃、200rpm培养至OD600=0.6左右,30℃、200rpm下加入终浓度为1%的L-***糖进行诱导培养8-10h,离心收集细胞。细胞悬浮于1/20发酵液体积的50mM的磷酸缓冲液(pH 7.0)并超声破碎,离心后得到重组的野生型DCR粗酶液。
2、构建和获得高活性酮基泛解酸内酯还原酶突变体
利用分子对接软件LeDock(https://lephar.com),将底物分子酮基泛解酸内酯KPL***到DCR的活性中心进行了分析;蛋白结构比对以及图像的分析、处理等生物信息学操作均通过PyMOL软件完成。分子对接表明酮基泛解酸内酯还原酶DCR的Thr27、Lys28、Glu224以及Thr225残基可能对其还原活力具有潜在影响。
以pBAD-DCR质粒为模板,应用QuickChange定点饱和突变方法构建了上述氨基酸残基位点的饱和突变体,各突变体基因序列经测序验证正确后,将其转入BW25113。后接入50ml含有100μg/ml氨苄抗性的LB液体培养基,在37℃,200rpm/min过夜培养后,用甘油管保存菌种。
以野生型pBAD-DCR-BW25113菌株作为对照,将剩余各突变体种子液按照2%的接种量接入800ml LB培养基(100μg/ml氨苄抗性)继续在37℃下振荡培养至OD600=0.6左右,后加入终浓度为1%的L-***糖后,在30℃下诱导培养8-10h后离心收集细胞。细胞悬浮于1/20发酵液体积的50mM的磷酸缓冲液(pH 7.0)并超声破碎,离心后得到重组的野生型DCR粗酶液。
在EP管中加入300μL上述裂解酶液和0.078mol酮基泛解酸内酯,在pH7.0,30℃条件下反应5h后,用6M HCl酸化至pH3.5后在80℃加热15min,等体积乙酸乙酯萃取后进气相色谱仪检测。
通过四轮筛选后获得第224位点残基突变后的活性明显提高的两个突变体E224G和E224Q,所述突变体E224G的核苷酸序列如SEQ ID NO.3所示和氨基酸序列如SEQ ID NO.4所示,所述突变体E224Q的核苷酸序列如SEQ ID NO.5所示和氨基酸序列如SEQ ID NO.6。
表1.DCR高效突变体的氨基酸残基变化及转化率比较
菌株名称 | 氨基酸残基变化 | 底物 | 转化率 |
野生型(Seq ID NO:1 and 2) | 无 | KPL | 25 |
E224G(Seq ID NO:3 and 4) | Glu→Gly | KPL | 32.4 |
E224Q(Seq ID NO:5 and 6) | Glu→Gln | KPL | 31.5 |
3、酮基泛解酸内酯还原酶DCR与葡萄糖脱氢酶GDH共表达工程菌pBAD-DCR-GDHBW25113的构建
平滑假丝酵母Candida parapsilosis CDC31的酮基泛解酸内酯还原酶DCR,所述DCR基因的核苷酸序列如SEQ ID NO.1所示和氨基酸序列如SEQ ID NO.2所示。
Bacillus megateriu(ATCC 14581)的GDH基因,所述GDH基因的核苷酸序列如SEQID NO.7所示和氨基酸序列如SEQ ID NO.8所示。
设计DCR基因引物对:5’-ctagccatggccactcaaagtaacttactaccaaa-3’和5’-ccctcgagctacaaatctttaaattgctcatggaa-3’。
GDH基因引物对:5’-ccctcgagtctagagaaagaggggacaaactagatgtatacagatttaaaagataaagta-3’和5’-ggggtaccttagcctcttcctgcttggaaagaagggtacagcgtcataccaccatcagca-3’。
Bacillus megateriu(ATCC 14581)的GDH基因,***到表达载体pBAD的NdeI和EcoRI位点得到质粒pBAD-GDH。所述GDH基因的核苷酸序列如SEQ ID NO.7所示和氨基酸序列如SEQ ID NO.8所示。
分别以质粒pBAD-DCR和pBAD-GDH为模板进行PCR扩增,用NcoI、XhoI和XhoI、KpnI进行酶切,PCR得到的基因序列两端分别带有NcoI、XhoI和XhoI、KpnI两个酶切位点,PCR产物经琼脂糖凝胶电泳纯化,利用琼脂糖凝胶DNA回收试剂盒回收目的片段。NcoI、KpnI酶切pBAD载体,用T4连接酶连接DCR片段、GDH片段和pBAD载体片段,连接产物转化DH5α后涂氨苄抗性平板,过夜培养后挑单菌落至装有5mL LB的试管培养,用质粒提取试剂盒提取质粒,PCR扩增确定***基因条带大小正确的质粒送测序,保存测序正确的质粒并命名为pBAD-DCR-GDH,按照化转操作流程,转入感受态细胞大肠杆菌BW25113,涂氨苄抗性LB固体平板,37度静置培养过夜,挑单菌落至装有5mL LB的试管培养5h后保存甘油菌种,获得共表达工程菌pBAD-DCR-GDH BW25113。
4、突变体E224G与葡萄糖脱氢酶GDH共表达工程菌pBAD-E224G-GDH的构建
分别以质粒pBAD-E224G和pBAD-GDH为模板进行PCR扩增,所用引物、酶切位点以及操作流程均同共表达重组菌株pBAD-DCR-GDH BW25113的构建一致,从而获得工程菌pBAD-E224G-GDH BW25113。
5、突变体E224Q与葡萄糖脱氢酶GDH共表达工程菌pBAD-E224Q-GDH的构建
分别以质粒pBAD-E224Q和pBAD-GDH为模板进行PCR扩增,所用引物、酶切位点以及操作流程均同共表达重组菌株pBAD-DCR-GDH BW25113的构建一致,从而获得工程菌pBAD-E224Q-GDH BW25113。
6、工程菌的培养
1)工程菌pBAD-DCR-GDH BW25113:
吸取甘油菌种管保存的菌种pBAD-DCR-GDH BW25113于5mL,含100μg/mL氨苄霉素的LB液体培养基中,37℃、200rpm培养5h。后按2%比例转接入800mL,含100μg/mL卡那霉素的LB培养基,37℃、200rpm培养至OD600约0.6左右。25℃、200rpm条件下加入终浓度为1%的L-***糖进行诱导培养8-10h,离心收集细胞。
2)工程菌pBAD-E224G-GDH BW25113:
吸取甘油菌种管保存的菌种pBAD-E224G-GDH BW25113于5mL,含100μg/mL氨苄霉素的LB液体培养基中,37℃、200rpm培养5h。后按2%比例接种到800mL,含100μg/mL卡那霉素的LB培养基,37℃、200rpm培养至OD600约0.6左右。25℃、200rpm条件下加入终浓度为1%的L-***糖进行诱导培养8-10h,离心收集细胞。
3)工程菌pBAD-E224Q-GDH BW25113:
吸取甘油菌种管保存的菌种pBAD-E224Q-GDH BW25113于5mL,含100μg/mL氨苄霉素的LB液体培养基中,37℃、200rpm培养5h。后按2%比例接种到800mL,含100μg/mL卡那霉素的LB发酵培养基,37℃、200rpm培养至OD600约0.6左右。25℃、200rpm条件下加入终浓度为1%的L-***糖进行诱导培养8-10h,离心收集细胞。
以上所述仅是本发明的优选实施方式,本发明的保护范围并不仅局限于上述实施例,凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理前提下的若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国科学院微生物研究所
<120> 工程菌及其在全细胞催化酮基泛解酸内酯生产D-泛解酸内酯中的应用
<130> IM2022057I
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 924
<212> DNA
<213> 平滑假丝酵母(Candida parapsilosis)
<400> 1
atgactcaaa gtaacttact accaaaaaca tttcgtacca aatctgggaa ggagatatca 60
attgcacttg gaacagggac caaatggaaa caagcccaaa caattaacga tgttagtact 120
gagttggtgg acaatatcct tttggggtta aagttgggtt ttagacacat tgatactgct 180
gaagcttaca acacgcaaaa ggaagttggt gaagccctca aaagaaccga tgttccaaga 240
gaggatattt gggttaccac aaaatatagt ccaggttggg gttcaatcaa ggcatacagt 300
aaatcgccaa gtgattcaat tgataaagct ttggcacagc ttggtgttga ctacgttgat 360
ttatttttga ttcactcccc attcttcacc actgagcaaa ctcatggata tacattagag 420
caagcttggg aagctttggt tgaagcaaag aaggcgggaa aggttagaga aattggtatc 480
tcaaatgctg ctattccaca cttggaaaaa ctctttgctg cctccccgag tcctgagtac 540
taccctgttg tcaaccaaat tgaattccat ccattcttgc aaaaccaatc taaaaacatt 600
gttagatttt gtcaagagca tgggatcttg gtcgaagctt tttcgccatt ggcgccattg 660
gcaagagttg aaactaatgc tctcgctgag acattaaaga gattggcgga aaagtacaaa 720
aagaccgaag ctcaagtttt attgaggtat actttgcaaa gaggtatttt gccagtgaca 780
acatcgtcaa aggaaagcag attaaaagag tctttgaatt tgtttgactt tgaattgact 840
gacgaggagg ttaatgaaat caacaagatt ggcgatgcta atccctatag agctttcttc 900
catgagcaat ttaaagattt gtag 924
<210> 2
<211> 307
<212> PRT
<213> 平滑假丝酵母(Candida parapsilosis)
<400> 2
Met Thr Gln Ser Asn Leu Leu Pro Lys Thr Phe Arg Thr Lys Ser Gly
1 5 10 15
Lys Glu Ile Ser Ile Ala Leu Gly Thr Gly Thr Lys Trp Lys Gln Ala
20 25 30
Gln Thr Ile Asn Asp Val Ser Thr Glu Leu Val Asp Asn Ile Leu Leu
35 40 45
Gly Leu Lys Leu Gly Phe Arg His Ile Asp Thr Ala Glu Ala Tyr Asn
50 55 60
Thr Gln Lys Glu Val Gly Glu Ala Leu Lys Arg Thr Asp Val Pro Arg
65 70 75 80
Glu Asp Ile Trp Val Thr Thr Lys Tyr Ser Pro Gly Trp Gly Ser Ile
85 90 95
Lys Ala Tyr Ser Lys Ser Pro Ser Asp Ser Ile Asp Lys Ala Leu Ala
100 105 110
Gln Leu Gly Val Asp Tyr Val Asp Leu Phe Leu Ile His Ser Pro Phe
115 120 125
Phe Thr Thr Glu Gln Thr His Gly Tyr Thr Leu Glu Gln Ala Trp Glu
130 135 140
Ala Leu Val Glu Ala Lys Lys Ala Gly Lys Val Arg Glu Ile Gly Ile
145 150 155 160
Ser Asn Ala Ala Ile Pro His Leu Glu Lys Leu Phe Ala Ala Ser Pro
165 170 175
Ser Pro Glu Tyr Tyr Pro Val Val Asn Gln Ile Glu Phe His Pro Phe
180 185 190
Leu Gln Asn Gln Ser Lys Asn Ile Val Arg Phe Cys Gln Glu His Gly
195 200 205
Ile Leu Val Glu Ala Phe Ser Pro Leu Ala Pro Leu Ala Arg Val Glu
210 215 220
Thr Asn Ala Leu Ala Glu Thr Leu Lys Arg Leu Ala Glu Lys Tyr Lys
225 230 235 240
Lys Thr Glu Ala Gln Val Leu Leu Arg Tyr Thr Leu Gln Arg Gly Ile
245 250 255
Leu Pro Val Thr Thr Ser Ser Lys Glu Ser Arg Leu Lys Glu Ser Leu
260 265 270
Asn Leu Phe Asp Phe Glu Leu Thr Asp Glu Glu Val Asn Glu Ile Asn
275 280 285
Lys Ile Gly Asp Ala Asn Pro Tyr Arg Ala Phe Phe His Glu Gln Phe
290 295 300
Lys Asp Leu
305
<210> 3
<211> 924
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgactcaaa gtaacttact accaaaaaca tttcgtacca aatctgggaa ggagatatca 60
attgcacttg gaacagggac caaatggaaa caagcccaaa caattaacga tgttagtact 120
gagttggtgg acaatatcct tttggggtta aagttgggtt ttagacacat tgatactgct 180
gaagcttaca acacgcaaaa ggaagttggt gaagccctca aaagaaccga tgttccaaga 240
gaggatattt gggttaccac aaaatatagt ccaggttggg gttcaatcaa ggcatacagt 300
aaatcgccaa gtgattcaat tgataaagct ttggcacagc ttggtgttga ctacgttgat 360
ttatttttga ttcactcccc attcttcacc actgagcaaa ctcatggata tacattagag 420
caagcttggg aagctttggt tgaagcaaag aaggcgggaa aggttagaga aattggtatc 480
tcaaatgctg ctattccaca cttggaaaaa ctctttgctg cctccccgag tcctgagtac 540
taccctgttg tcaaccaaat tgaattccat ccattcttgc aaaaccaatc taaaaacatt 600
gttagatttt gtcaagagca tgggatcttg gtcgaagctt tttcgccatt ggcgccattg 660
gcaagagttg gtactaatgc tctcgctgag acattaaaga gattggcgga aaagtacaaa 720
aagaccgaag ctcaagtttt attgaggtat actttgcaaa gaggtatttt gccagtgaca 780
acatcgtcaa aggaaagcag attaaaagag tctttgaatt tgtttgactt tgaattgact 840
gacgaggagg ttaatgaaat caacaagatt ggcgatgcta atccctatag agctttcttc 900
catgagcaat ttaaagattt gtag 924
<210> 4
<211> 307
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Thr Gln Ser Asn Leu Leu Pro Lys Thr Phe Arg Thr Lys Ser Gly
1 5 10 15
Lys Glu Ile Ser Ile Ala Leu Gly Thr Gly Thr Lys Trp Lys Gln Ala
20 25 30
Gln Thr Ile Asn Asp Val Ser Thr Glu Leu Val Asp Asn Ile Leu Leu
35 40 45
Gly Leu Lys Leu Gly Phe Arg His Ile Asp Thr Ala Glu Ala Tyr Asn
50 55 60
Thr Gln Lys Glu Val Gly Glu Ala Leu Lys Arg Thr Asp Val Pro Arg
65 70 75 80
Glu Asp Ile Trp Val Thr Thr Lys Tyr Ser Pro Gly Trp Gly Ser Ile
85 90 95
Lys Ala Tyr Ser Lys Ser Pro Ser Asp Ser Ile Asp Lys Ala Leu Ala
100 105 110
Gln Leu Gly Val Asp Tyr Val Asp Leu Phe Leu Ile His Ser Pro Phe
115 120 125
Phe Thr Thr Glu Gln Thr His Gly Tyr Thr Leu Glu Gln Ala Trp Glu
130 135 140
Ala Leu Val Glu Ala Lys Lys Ala Gly Lys Val Arg Glu Ile Gly Ile
145 150 155 160
Ser Asn Ala Ala Ile Pro His Leu Glu Lys Leu Phe Ala Ala Ser Pro
165 170 175
Ser Pro Glu Tyr Tyr Pro Val Val Asn Gln Ile Glu Phe His Pro Phe
180 185 190
Leu Gln Asn Gln Ser Lys Asn Ile Val Arg Phe Cys Gln Glu His Gly
195 200 205
Ile Leu Val Glu Ala Phe Ser Pro Leu Ala Pro Leu Ala Arg Val Gly
210 215 220
Thr Asn Ala Leu Ala Glu Thr Leu Lys Arg Leu Ala Glu Lys Tyr Lys
225 230 235 240
Lys Thr Glu Ala Gln Val Leu Leu Arg Tyr Thr Leu Gln Arg Gly Ile
245 250 255
Leu Pro Val Thr Thr Ser Ser Lys Glu Ser Arg Leu Lys Glu Ser Leu
260 265 270
Asn Leu Phe Asp Phe Glu Leu Thr Asp Glu Glu Val Asn Glu Ile Asn
275 280 285
Lys Ile Gly Asp Ala Asn Pro Tyr Arg Ala Phe Phe His Glu Gln Phe
290 295 300
Lys Asp Leu
305
<210> 5
<211> 924
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgactcaaa gtaacttact accaaaaaca tttcgtacca aatctgggaa ggagatatca 60
attgcacttg gaacagggac caaatggaaa caagcccaaa caattaacga tgttagtact 120
gagttggtgg acaatatcct tttggggtta aagttgggtt ttagacacat tgatactgct 180
gaagcttaca acacgcaaaa ggaagttggt gaagccctca aaagaaccga tgttccaaga 240
gaggatattt gggttaccac aaaatatagt ccaggttggg gttcaatcaa ggcatacagt 300
aaatcgccaa gtgattcaat tgataaagct ttggcacagc ttggtgttga ctacgttgat 360
ttatttttga ttcactcccc attcttcacc actgagcaaa ctcatggata tacattagag 420
caagcttggg aagctttggt tgaagcaaag aaggcgggaa aggttagaga aattggtatc 480
tcaaatgctg ctattccaca cttggaaaaa ctctttgctg cctccccgag tcctgagtac 540
taccctgttg tcaaccaaat tgaattccat ccattcttgc aaaaccaatc taaaaacatt 600
gttagatttt gtcaagagca tgggatcttg gtcgaagctt tttcgccatt ggcgccattg 660
gcaagagttc agactaatgc tctcgctgag acattaaaga gattggcgga aaagtacaaa 720
aagaccgaag ctcaagtttt attgaggtat actttgcaaa gaggtatttt gccagtgaca 780
acatcgtcaa aggaaagcag attaaaagag tctttgaatt tgtttgactt tgaattgact 840
gacgaggagg ttaatgaaat caacaagatt ggcgatgcta atccctatag agctttcttc 900
catgagcaat ttaaagattt gtag 924
<210> 6
<211> 307
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Thr Gln Ser Asn Leu Leu Pro Lys Thr Phe Arg Thr Lys Ser Gly
1 5 10 15
Lys Glu Ile Ser Ile Ala Leu Gly Thr Gly Thr Lys Trp Lys Gln Ala
20 25 30
Gln Thr Ile Asn Asp Val Ser Thr Glu Leu Val Asp Asn Ile Leu Leu
35 40 45
Gly Leu Lys Leu Gly Phe Arg His Ile Asp Thr Ala Glu Ala Tyr Asn
50 55 60
Thr Gln Lys Glu Val Gly Glu Ala Leu Lys Arg Thr Asp Val Pro Arg
65 70 75 80
Glu Asp Ile Trp Val Thr Thr Lys Tyr Ser Pro Gly Trp Gly Ser Ile
85 90 95
Lys Ala Tyr Ser Lys Ser Pro Ser Asp Ser Ile Asp Lys Ala Leu Ala
100 105 110
Gln Leu Gly Val Asp Tyr Val Asp Leu Phe Leu Ile His Ser Pro Phe
115 120 125
Phe Thr Thr Glu Gln Thr His Gly Tyr Thr Leu Glu Gln Ala Trp Glu
130 135 140
Ala Leu Val Glu Ala Lys Lys Ala Gly Lys Val Arg Glu Ile Gly Ile
145 150 155 160
Ser Asn Ala Ala Ile Pro His Leu Glu Lys Leu Phe Ala Ala Ser Pro
165 170 175
Ser Pro Glu Tyr Tyr Pro Val Val Asn Gln Ile Glu Phe His Pro Phe
180 185 190
Leu Gln Asn Gln Ser Lys Asn Ile Val Arg Phe Cys Gln Glu His Gly
195 200 205
Ile Leu Val Glu Ala Phe Ser Pro Leu Ala Pro Leu Ala Arg Val Gln
210 215 220
Thr Asn Ala Leu Ala Glu Thr Leu Lys Arg Leu Ala Glu Lys Tyr Lys
225 230 235 240
Lys Thr Glu Ala Gln Val Leu Leu Arg Tyr Thr Leu Gln Arg Gly Ile
245 250 255
Leu Pro Val Thr Thr Ser Ser Lys Glu Ser Arg Leu Lys Glu Ser Leu
260 265 270
Asn Leu Phe Asp Phe Glu Leu Thr Asp Glu Glu Val Asn Glu Ile Asn
275 280 285
Lys Ile Gly Asp Ala Asn Pro Tyr Arg Ala Phe Phe His Glu Gln Phe
290 295 300
Lys Asp Leu
305
<210> 7
<211> 786
<212> DNA
<213> Bacillus megateriu
<400> 7
atgtatacag atttaaaaga taaagtagtt gtaattacag gtggatcaac aggtttagga 60
cgcgcaatgg ctgttcgttt cggtcaagaa gaagcaaaag ttgttattaa ctattacaac 120
aatgaagaag aagctttaga tgcgaaaaaa gaagtagaag aagcaggcgg acaagcaatc 180
atcgttcaag gcgacgtaac aaaagaagaa gatgttgtaa accttgttca aacagctatt 240
aaagaattcg gtacattaga cgttatgatt aataacgctg gtgttgaaaa cccagttcct 300
tctcatgagt tatctttaga caactggaat aaagtaatcg atacaaactt aacgggcgca 360
tttttaggaa gccgcgaagc gattaaatat tttgttgaaa acgacattaa aggaaacgtt 420
attaacatgt ctagtgttca tgaaatgatt ccttggccat tatttgttca ttacgcagca 480
agtaaaggcg gtatgaaact aatgacgaaa acattggctc ttgaatatgc gccaaaaggt 540
atccgcgtaa ataacattgg accaggtgcg atgaacacac caattaacgc agagaaattt 600
gcagatcctg tacaacgtgc agacgtagaa agcatgattc caatgggtta catcggtaaa 660
ccagaagaag tagcagcagt tgcagcattc ttagcatcat cacaagcaag ctatgtaaca 720
ggtattacat tatttgctga tggtggtatg acgctgtacc cttctttcca agcaggaaga 780
ggctaa 786
<210> 8
<211> 261
<212> PRT
<213> Bacillus megateriu
<400> 8
Met Tyr Thr Asp Leu Lys Asp Lys Val Val Val Ile Thr Gly Gly Ser
1 5 10 15
Thr Gly Leu Gly Arg Ala Met Ala Val Arg Phe Gly Gln Glu Glu Ala
20 25 30
Lys Val Val Ile Asn Tyr Tyr Asn Asn Glu Glu Glu Ala Leu Asp Ala
35 40 45
Lys Lys Glu Val Glu Glu Ala Gly Gly Gln Ala Ile Ile Val Gln Gly
50 55 60
Asp Val Thr Lys Glu Glu Asp Val Val Asn Leu Val Gln Thr Ala Ile
65 70 75 80
Lys Glu Phe Gly Thr Leu Asp Val Met Ile Asn Asn Ala Gly Val Glu
85 90 95
Asn Pro Val Pro Ser His Glu Leu Ser Leu Asp Asn Trp Asn Lys Val
100 105 110
Ile Asp Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
115 120 125
Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Met Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Met Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr
165 170 175
Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Met Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Val Gln Arg Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Lys Pro Glu Glu Val
210 215 220
Ala Ala Val Ala Ala Phe Leu Ala Ser Ser Gln Ala Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260
Claims (7)
1.质粒pBAD-GDH,其特征在于,所述质粒pBAD-GDH由GDH基因***到表达载体pBAD获得;所述GDH基因的氨基酸序列如SEQ ID NO.8所示。
2.重组质粒,其特征在于,包括:质粒pBAD-DCR-GDH、质粒pBAD-E224G-GDH和质粒pBAD-E224Q-GDH;
所述质粒pBAD-DCR-GDH由T4连接酶连接酶切质粒pBAD-DCR后的DCR片段、酶切权利要求1所述质粒pBAD-GDH后的GDH片段和酶切pBAD后的线性载体片段获得;所述质粒pBAD-DCR由DCR基因***到表达载体pBAD获得;所述DCR基因的氨基酸序列如SEQ ID NO.2所示;
所述质粒pBAD-E224G-GDH由T4连接酶连接酶切质粒pBAD-E224G后的E224G片段、酶切权利要求1所述质粒pBAD-GDH后的GDH片段和酶切pBAD后的线性载体片段获得;所述质粒pBAD-E224G由E224G基因***到表达载体pBAD获得;所述E224G基因的氨基酸序列如SEQ IDNO.4所示;
所述质粒pBAD-E224Q-GDH由T4连接酶连接酶切质粒pBAD-E224Q后的E224Q片段、酶切切权利要求1所述质粒pBAD-GDH后的GDH片段和酶切pBAD后的线性载体片段获得;所述质粒pBAD-E224Q由E224Q基因***到表达载体pBAD获得;所述E224Q基因的氨基酸序列如SEQ IDNO.6所示。
3.工程菌,其特征在于,所述工程菌pBAD-DCR-GDH BW25113由权利要求2所述质粒pBAD-DCR-GDH化转入感受态细胞大肠杆菌BW25113获得工程菌。
4.工程菌,其特征在于,所述工程菌pBAD-E224G-GDH BW25113由权利要求2所述质粒pBAD-E224G-GDH化转入感受态细胞大肠杆菌BW25113获得工程菌pBAD-E224G-GDHBW25113。
5.工程菌,其特征在于,所述工程菌pBAD-E224Q-GDH BW25113由权利要求2所述质粒pBAD-E224Q-GDH化转入感受态细胞大肠杆菌BW25113获得工程菌pBAD-E224Q-GDHBW25113。
6.权利要求3至5任一工程菌在全细胞催化酮基泛解酸内酯生产D-泛解酸内酯中的应用。
7.依据权利要求6所述工程菌在全细胞催化酮基泛解酸内酯生产D-泛解酸内酯中的应用,其特征在于,所述工程菌与酮基泛解酸内酯加入量的质量比为2-4:1-2。
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