CN112877349B - 一种重组表达载体、包含其的基因工程菌及其应用 - Google Patents
一种重组表达载体、包含其的基因工程菌及其应用 Download PDFInfo
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Abstract
本发明公开一种重组表达载体,所述重组表达载体包含特定的对香豆酰辅酶A连接酶的基因和芪合成酶的基因。包含该重组表达载体的重组表达转化体应用于赤松素生物合成过程中可以有效减少中间代谢物肉桂酰辅酶A及赤松素对肉桂酸转化的抑制,增加底物肉桂酸的消耗,且大幅度提高肉桂酸至赤松素的转化效率。
Description
技术领域
本发明涉及生物技术及生物催化领域,尤其涉及对香豆酰辅酶A、芪合成酶及其编码基因及应用。
背景技术
赤松素,又名银松素、反式-3,5-二羟基二苯乙烯等,属于芪类化合物。在植物属于低分子量的次生代谢产物,主要存在于松木心材中的一种植物多酚,保护植物免受细菌和真菌的侵蚀。近年来,许多研究已证实赤松素有众多的生物活性,如预防心血管疾病、抗氧化、防癌和治疗关节炎等生物活性。据报道,赤松素能够保护动物细胞免受氧化应激,这种作用已在人的视网膜细胞中观察到,添加一定浓度的赤松素可以改善氧化应激后的存活率。也有报道称赤松素可以防止牛主动脉内皮细胞坏死。因此,在保健食品和药品应用方面有着广阔的市场前景。
现阶段获得赤松素的途径主要以植物提取和化学合成为主,赤松素大多数是从松树树心中进行提取,其含量低下,只有1-40 mg/g松木,另外因许多结构类似物难以分离,使得提取工艺复杂和成本昂贵。化学合成则面临反应底物昂贵、有毒副产品、副产物分离纯化困难及环境污染的难题。因此,另一种获得赤松素的方法是利用微生物制备,显得十分环境友好,生产周期短,培养成本低,产物提纯渐变,避免了大量的有机溶剂、重金属和强酸强碱的使用。
赤松素的基本骨架是由一分子肉桂酰辅酶A来源的B环和三分子丙二酰辅酶A环化形成的A环组成,其生物合成途径是以L-苯丙氨酸为起点,经苯丙氨酸解氨酶(PAL)转化为肉桂酸,对香豆酰辅酶A连接酶(4CL)催化肉桂酸生成肉桂酰辅酶A。丙二酰辅酶A是由乙酰辅酶A或丙二酸在丙二酰辅酶A合成酶(ACC)的作用下形成,其在芪合成酶(STS)的催化作用下,与肉桂酰辅酶A缩合生成赤松素。在微生物合成赤松素的研究中,途径设计和构建是成功制备该产物的前提。2003年,研究者(Becker J V W, Armstrong G O, Van d M M J, etal. Metabolic engineering ofSaccharomyces cerevisiaefor the synthesis of thewine-related antioxidant resveratrol [J]. FEMS Yeast Research, 2003,4: 79-85.)采用胡杨来源的4CL和葡萄来源的STS在酵母中组装了芪类物质的生物合成途径,首次实现了芪类物质的异源合成。Wang等(Wang S Y, Zhang S W, Xiao A F, et al.Metabolic engineering ofEscherichia colifor the biosynthesis of variousphenylpropanoid derivatives [J]. Metabolic Engineering, 2015, 29:153-159.)在大肠杆菌中组装来自三叶草的PAL、拟南芥的4CL和花生的STS,合成了13 mg/L赤松素。Salas-Navarrete(Salas-Navarrete C, Hernández-Chávez G, Flores N, et al.Increasing pinosylvin production in,Escherichia coli, by reducing theexpression level of the gene, fabI-encoded enoyl-acyl carrier proteinreductase [J]. Electronic Journal of Biotechnology, 2018, 33:11-16)等过表达天蓝色链霉菌4CL和葡萄STS,成功合成34.89 mg/L赤松素。
丙二酰辅酶A是芪类物质和黄酮类物质合成的重要前体,也是微生物脂肪酸合成的代谢节点。内源性代谢途径对胞内的丙二酰辅酶A极具竞争性和支配性,只有微量丙二酰辅酶A参与赤松素的合成。因此,强化赤松素合成前体丙二酰辅酶A的供给受到广泛关注。目前有如下两种方法:(1)生物胞内的乙酰辅酶A羧化酶(ACC)将葡萄糖代谢合成的乙酰辅酶A转化成丙二酰辅酶A,研究者通常在大肠杆菌中过表达谷氨酸棒状杆菌(Corynebacterium glutamicum)或发光光杆状菌(Photorhabdus luminescens)来源的ACC以提高胞内丙二酰辅酶A水平;(2)丙二酸盐载体蛋白(MatC)将外源添加的丙二酸盐运输至胞内,然后在丙二酸辅酶A合酶(MatB)的催化作用下合成丙二酸辅酶A。研究者在重组菌株中过表达三叶草根瘤菌的matB和matC基因后,胞内丙二酰辅酶A含量显著提高;(3)丙二酰辅酶A是脂肪酸合成的底物,在胞内的丙二酰辅酶A:ACP转酰基酶(FabD)的催化作用下合成丙二酰-ACP,流入脂肪酸合成途径。
发明内容
发明目的:为解决现有技术中存在的技术问题,本发明提出了一种重组表达载体,所述重组表达载体包含表达特定的对香豆酰辅酶A连接酶的基因和表达芪合成酶的基因,通过表达对香豆酰辅酶A连接酶和芪合成酶,得到的基因工程菌在赤松素生物合成中可减少了肉桂酰辅酶A及赤松素的下游反馈抑制,增加了底物肉桂酸的消耗,提高了肉桂酸至赤松素的转化率。
为实现上述目的,本发明提出了一种重组表达载体,所述重组表达载体包含表达对香豆酰辅酶A连接酶的基因和表达芪合成酶的基因,其中,
所述对香豆酰辅酶A连接酶是如下(a)或(b)蛋白质:
(a)具有SEQ ID NO.1所示的氨基酸序列的蛋白质,具有该氨基酸序列的香豆酰辅酶A,命名为Ptr4CL4,来源于毛果杨(Populus trichocarpa Torr.&Gray)
(b)由SEQ ID NO.1所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有对香豆酰辅酶A活性的由(a)衍生的蛋白质;
所述芪合成酶是如下(c)或(d)的蛋白质:
(c)具有SEQ ID NO .2所示的氨基酸序列的蛋白质,命名为PpSTS,来源于意大利松(Pinus pinea L);
(d)由SEQ ID NO.2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有芪合成酶活性的由(c)衍生的蛋白质。
所述表达对香豆酰辅酶A连接酶的基因的核苷酸序列为如下(1)或(2)所述的核苷酸序列:
(1)具有SEQ ID NO .3所示的核苷酸序列;
(2)编码(a)或(b)所述的蛋白质的核苷酸序列;
所述表达芪合成酶的基因的核苷酸序列为如下(3)或(4)所述的核苷酸序列:
(3)具有SEQ ID NO .4所示的核苷酸序列;
(4)编码(c)或(d)所述的蛋白质的核苷酸序列。
在一种优选的实施方案中,本发明所述的重组表达载体包含具有SEQ ID NO .3所示的核苷酸序列的基因和具有SEQ ID NO .4所示的核苷酸序列的基因,其中,具有SEQ IDNO .3所示的核苷酸序列的基因编码的对香豆酰辅酶A命名为Ptr4CL4,全长1713 bp。其编码序列从第1个碱基起至第1713个碱基止,起始密码子为ATG,终止密码子为TAA。具有SEQID NO .4所示的核苷酸序列的基因编码的芪合成酶命名为PpSTS,全长1182 bp。其编码序列从第1个碱基起至第1182个碱基止,起始密码子为ATG,终止密码子为TAA。该序列无内含子。
如本领域技术人员所知,由于密码子的简并性,编码氨基酸序列的核苷酸序列不仅仅局限于上述序列。本发明的对香豆酰辅酶A基因及芪合成酶基因的核苷酸序列也可以是编码(b)或(d)所示蛋白质的氨基酸序列的其他任何核苷酸序列。另外,还可以通过适当引入替换、缺失或插入来提供一个多聚核苷酸的同系物。本发明中多聚核苷酸的同系物可以通过对序列表中序列的一个或多个碱基在保持酶活性范围内进行替换、缺失或增加来制得。
其中,可通过本领域常规方法将本发明所述核苷酸序列连接于各种载体上构建而成。所述的载体可为本领域常规的各种载体,如市售的质粒、粘粒、噬菌体或病毒载体等。
优选第,选用质粒pRSFDuet-1。较佳的,可通过下述方法制得本发明的重组表达载体:将通过PCR扩增所得的核酸产物用Promega纯化试剂盒纯化,同时将载体pRSFDuet-1用分别限制性内切酶NdeI和XhoI、BamHI和HindIII双酶切胶回收,经全式金无缝拼接试剂盒Assembly Mix连接,形成含有本发明的对香豆酰辅酶A、芪合成酶基因的重组表达质粒pRSFDuet-Ppsts-Ptr4cl4。
扩增上述核苷酸序列全长或其任意片段的引物对也是本发明保护的范围。
本发明进一步提出了上述重组表达载体的重组表达转化体,通过将上述重组表达载转化至宿主细胞中得到。
所述的宿主细胞可为本领域常规的各种宿主细胞,只要能满足重组表达载体可稳定地自行复制,且所携带的本发明的对香豆酰辅酶A、芪合成酶基因可被有效表达即可。本发明优选大肠杆菌,更优选大肠杆菌(Escherichia coli)BL21(DE3)。将前述重组表达质粒pRSFDuet-Ppsts-Ptr4cl4转化至E.coli BL21(DE3)中,即可得本发明优选的基因工程菌株,即E.coliBL21(pRSFDuet-Ppsts-Ptr4cl4)。
上述核苷酸序列或上述重组表达载体或重组表达转化体在制备对香豆酰辅酶A、芪合成酶中的应用也是本发明保护的范围。
本发明进一步提出了上述重组表达转化体在赤松素合成上的应用。
本发明将来源于毛果杨及意大利松中克隆得到的对香豆酰辅酶A基因Ptr4cl4、芪合成酶基因Ppsts,可在宿主细胞中表达该基因以生产对香豆酰辅酶APtr4cl4,芪合成酶Ppsts,用于赤松素的合成。
具体地发酵方法为:按照1%接种量的重组表达转换体接种到LB培养基中,摇管活化,然后按1%接种量转接至装有LB的锥形瓶中,继续培养,取种子培养液,离心弃上清,将菌泥按初始OD600 0.6~0.8接种至发酵培养基中,并在发酵培养基中加入肉桂酸,同时加入终浓度0.5~1.5 mM IPTG诱导培养。
优选地,在发酵同时添加20-100 μM浅蓝菌素。
具体地,发酵培养基包含M9盐11.3 g/L,酵母提取物5~10 g/L,3-(N-吗啉基)丙磺酸(MOPS)35~45 g/L,甘油3~8% (v/v),大肠杆菌的培养温度为28~45ºC,转速150~300 rpm,培养时间为8~15 h,发酵时间12~72 h。
有益效果:本发明通过将表达对香豆酰辅酶A连接酶的基因和表达芪合成酶的基因重组并转化表达,解决了目前下游反馈抑制、STS活力低以及丙二酰辅酶A供应不足的问题,使用Ptr4CL4减少了肉桂酰辅酶A及赤松素的抑制,增加了底物肉桂酸的消耗,大幅度提高了肉桂酸至赤松素转化率。
附图说明
图1为pETDuet-Ptr4cl4质粒双酶切结果图,其中,M:DL 5,000 DNA Marker;1:pETDuet-Ptr4cl4;2:pETDuet-Ptr4cl4质粒双酶切产物;
图2为提取质粒双酶切验证实验结果,1:pRSFDuet-Ppsts质粒;2:pRSFDuet-Ptps质粒;3:pRSFDuet-PspsQ361R质粒;4:pRSFDuet-Ppsts质粒双酶切产物;5:pRSFDuet-Ptps质粒双酶切产物;6:pRSFDuet-PspsQ361R质粒双酶切产物;M:5000 Maker;
图3为pRSFDuet-sts-Ptr4cl4质粒的双酶切产物;其中,pRSFDuet-Ppsts-Ptr4cl4质粒双酶切产物;5-8:pRSFDuet-Ptps-Ptr4cl4质粒双酶切产物; 9-12:pRSFDuet-Psps-Ptr4cl4质粒双酶切产物;M:5000 Maker;
图4为赤松素和肉桂酸混合样品的色谱图及标准曲线。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。给出了详细的实施方式和具体的操作过程,实施例将有助于理解本发明,但是本发明的保护范围不限于下述的实施例。
实施例1:对香豆酰辅酶A基因的优化及重组表达载体的构建
原始的对香豆酰辅酶A(4-coumarate-CoA ligase family protein [Populustrichocarpa],GenBank: EEF00197.1)的氨基酸序列来源于从NCBI数据库注释信息中筛选而来。在不改变编码的氨基酸序列的前提下,按照大肠杆菌密码子偏好性,将Ptr4CL4基因进行密码子优化,全基因合成方法合成了对香豆酰辅酶A的全基因序列Ptr4CL4。人工合成的对香豆酰辅酶A基因Ptr4CL4的核苷酸序列如SEQ ID NO.3中所示。合成的Ptr4CL4基因两端还带有NdeⅠ和XhoⅠ酶切位点,以便和表达载体进行连接。连接结果如图1所示。采用Ptr4CL4基因序列为模板扩增芪合成酶Ptr4CL4基因,所用引物为:
Ptr4CL4-up:5’-ACATATGATGAGTGTTGCCACCGTGG-3’
Ptr4CL4-dn:5’-AGACTCGAGTTAACTCATGGTGGTT-3’
下划线处分别为NdeⅠ和XhoⅠ酶切位点。
实施例2:芪合成酶基因的优化及重组表达载体的构建
原始的芪合成酶(stilbene synthase [Pinus pinea],GenBank: ALN42233.1)的氨基酸序列来源于从NCBI数据库注释信息中筛选而来。在不改变编码的氨基酸序列的前提下,按照大肠杆菌密码子偏好性,将编码该芪合成酶的基因进行密码子优化,全基因合成方法合成了芪合成酶全基因序列Ppsts,其核苷酸序列如SEQ ID NO.4所示。合成的Ppsts基因两端还带有BamHⅠ和HindⅠⅠⅠ酶切位点,以便和表达载体进行连接。采用Ppsts基因的核苷酸序列为模板扩增芪合成酶Ppsts基因,所用引物为:
PpSTS-up:5’-CAGGATCCGATGGGCGCTGTTGACT-3’
PpSTS-dn:5’-CGCAAGCTTTTATTGCAGCGGAACA-3’
下划线处分别为BamHⅠ和HindⅠⅠⅠ酶切位点。
委托滁州通用合成的Ppsts(SEQ ID NO. 4)、Ptps(SEQ ID NO. 5)和Psps Q361R 基因(SEQ ID NO. 6)的核苷酸序列长度分别为1182 bp、1179 bp和1194 bp,其中,Ptps和Psps Q361R 基因为另外两种已知的芪合成酶基因,Ptps基因的Genebank 登录号为KF998274.1,Psps Q361R 基因的Genebank 登录号为P48407.1,其均被分别插在pRSFDuet-1(3829 bp)的BamH Ⅰ和Hind Ⅲ酶切位点。提取质粒双酶切验证实验结果如图2所示。以pETDuet-Ptr4cl4质粒(该质粒委托商业公司制备)为模板进行扩增目的基因Ptr4cl4,所用引物为:
Ptr4CL4-up:5’-ACATATGATGAGTGTTGCCACCGTGG-3’
Ptr4CL4-dn:5’-AGACTCGAGTTAACTCATGGTGGTT-3’
下划线处分别为NdeⅠ和XhoⅠ酶切位点。
采用BglⅡ和KpnⅠ双酶切pRSFDuet-Ppsts、pRSFDuet-Ptps和pRSFDuet-Psps Q361R 质粒,胶回收后用pEASY-Uni定向重组试剂盒连接目的片段与Ptr4cl4基因,并转化入E. coliTrans T1。挑取转化子质粒,并采用ApaⅠ和EcoR Ⅰ双酶切,产物大小在4400 bp和2200bp左右,如图3所示。将测序正确的质粒导入表达菌株E. coli BL21 (DE3)中,构建重组大肠杆菌E. coliBLPS(pRSFDuet-Psps Q361R -Ptr4cl4)、E. coliBLPT(pRSFDuet-Ptps-Ptr4cl4)和E. coliBLPP-2(pRSFDuet-Ppsts-Ptr4cl4),不同STS合成赤松素能力,在含有80 mg/L肉桂酸的M9CA培养基中,菌泥按初始OD6000.6~0.8接种至发酵培养基发酵48 h后均能合成赤松素,E. coliBLPS在48 h内合成了0.048 mg/L 赤松素,E. coliBLPT和E. coliBLPP-2的赤松素均具有较强的赤松素合成能力,且E. coliBLPP-2的赤松素产量最高(0.444 mg/L),因此本专利涉及的pRSFDuet-Ppsts-Ptr4cl4合成赤松素的能力最强。
实施例5:重组菌株发酵合成赤松素。
赤松素采用如下方法检测:
色谱柱:Eclipse XDB-C18反相柱(250 mm×4.6 mm,5 μm),柱温:30 ℃,流动相A为100%乙腈、B为1.5%乙酸,采用40%的流动相A等梯度洗脱,流速:1mL/min,紫外检测波长:294 nm,进样量:10 μL。
赤松素和肉桂酸混合样品的色谱图及标准曲线如图4所示。
将重组菌株按1%接种量接种到5 mL LB培养基中,37 ℃、200 rpm摇管活化12 h,然后按1%接种量转接至装有20 mL LB的锥形瓶中,继续培养11~12 h。取种子培养液,离心弃上清,将菌泥按初始D600 0.6~0.8接种至发酵培养基中,其中,发酵培养基成分为: M9盐11.3 g/L;酵母提取物 10 g/L;MOPS 42 g/L;甘油 5%(v/v),含80 mg/L肉桂酸,加入终浓度1 mM IPTG诱导,在发酵同时添加60 μM浅蓝菌素抑制脂肪酸代谢提高胞内丙二酰辅酶A的浓度。30 ℃、200 rpm培养48 h后取上清检测赤松素产量,含有Ptr4CL4的重组菌株E. coli BLPP-2的肉桂酸转化率达到93.2%,合成了68.68 mg/L的赤松素,相比于含有Ptr4CL5的重组菌株E. coli BLPP仅合成了9.61 mg/L的赤松素,产量提高了615.4%。
序列表
<110> 南京林业大学
<120> 一种重组表达载体、包含其的基因工程菌及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 570
<212> PRT
<213> 对香豆酰辅酶A连接酶Ptr4CL4的氨基酸序列(Artificial Sequence)
<400> 1
Met Met Ser Val Ala Thr Val Glu Pro Pro Lys Pro Glu Leu Ser Pro
1 5 10 15
Pro Gln Asn Gln Asn Ala Pro Ser Ser His Glu Thr Asp His Ile Phe
20 25 30
Arg Ser Lys Leu Pro Asp Ile Thr Ile Ser Asn His Leu Pro Leu His
35 40 45
Ala Tyr Cys Phe Glu Asn Leu Ser Asp Phe Ser Asp Arg Pro Cys Leu
50 55 60
Ile Ser Gly Ser Thr Gly Lys Thr Tyr Ser Phe Ala Glu Thr His Leu
65 70 75 80
Ile Ser Arg Lys Val Ala Ala Gly Leu Ser Asn Leu Gly Ile Lys Lys
85 90 95
Gly Asp Val Ile Met Thr Leu Leu Gln Asn Cys Pro Glu Phe Val Phe
100 105 110
Ser Phe Met Gly Ala Ser Met Ile Gly Ala Val Thr Thr Thr Val Asn
115 120 125
Pro Phe Tyr Thr Pro Gly Glu Ile Phe Lys Gln Phe Ser Ala Ser Arg
130 135 140
Ala Lys Leu Ile Ile Thr Gln Ser Gln His Val Asn Lys Leu Arg Asp
145 150 155 160
Ser Asp Cys His Glu Asn Asn Gln Lys Pro Glu Glu Asp Phe Ile Val
165 170 175
Ile Thr Ile Asp Asp Pro Pro Glu Asn Cys Leu His Phe Asn Val Leu
180 185 190
Val Glu Ala Asn Glu Ser Glu Met Pro Thr Val Ser Ile His Pro Asp
195 200 205
Asp Pro Val Ala Leu Pro Phe Ser Ser Gly Thr Thr Gly Leu Pro Lys
210 215 220
Gly Val Ile Leu Thr His Lys Ser Leu Ile Thr Ser Val Ala Gln Gln
225 230 235 240
Val Asp Gly Glu Ile Pro Asn Leu Tyr Leu Lys Gln Asp Asp Val Val
245 250 255
Leu Cys Val Leu Pro Leu Phe His Ile Phe Ser Leu Asn Ser Val Leu
260 265 270
Leu Cys Ser Leu Arg Ala Gly Ser Ala Val Leu Leu Met Gln Lys Phe
275 280 285
Glu Ile Gly Ser Leu Leu Glu Leu Ile Gln Lys His Asn Val Ser Val
290 295 300
Ala Ala Val Val Pro Pro Leu Val Leu Ala Leu Ala Lys Asn Pro Met
305 310 315 320
Val Ala Asn Phe Asp Leu Ser Ser Ile Arg Val Val Leu Ser Gly Ala
325 330 335
Ala Pro Leu Gly Lys Glu Leu Glu Glu Ala Leu Arg Ser Arg Val Pro
340 345 350
Gln Ala Ile Leu Gly Gln Gly Tyr Gly Met Thr Glu Ala Gly Pro Val
355 360 365
Leu Ser Met Cys Leu Ala Phe Ser Lys Gln Pro Leu Pro Thr Lys Ser
370 375 380
Gly Ser Cys Gly Thr Val Val Arg Asn Ala Glu Leu Lys Val Ile Asp
385 390 395 400
Pro Glu Thr Gly Ser Ser Leu Gly Arg Asn Gln Pro Gly Glu Ile Cys
405 410 415
Ile Arg Gly Ser Gln Ile Met Lys Gly Tyr Leu Asn Asp Ala Glu Ala
420 425 430
Thr Ala Asn Ile Ile Asp Val Glu Gly Trp Leu His Thr Gly Asp Ile
435 440 445
Gly Tyr Val Asp Asp Asp Asp Glu Ile Phe Ile Val Asp Arg Val Lys
450 455 460
Glu Ile Ile Lys Phe Lys Gly Phe Gln Val Pro Pro Ala Glu Leu Glu
465 470 475 480
Ala Leu Leu Val Asn His Pro Ser Ile Ala Asp Ala Ala Val Val Pro
485 490 495
Gln Lys Asp Glu Val Ala Gly Glu Val Pro Val Ala Phe Val Val Arg
500 505 510
Ser Asn Asp Leu Asp Leu Asn Glu Glu Ala Val Lys Asp Tyr Ile Ala
515 520 525
Lys Gln Val Val Phe Tyr Lys Lys Leu His Lys Val Phe Phe Val His
530 535 540
Ser Ile Pro Lys Ser Ala Ala Gly Lys Ile Leu Arg Lys Asp Leu Arg
545 550 555 560
Ala Lys Leu Ala Thr Ala Thr Thr Met Ser
565 570
<210> 2
<211> 393
<212> PRT
<213> 芪合成酶PpSTS的氨基酸序列(Artificial Sequence)
<400> 2
Met Gly Ala Val Asp Phe Glu Gly Phe Arg Lys Leu Gln Arg Ala
1 5 10 15
Asp Gly Phe Ala Ser Ile Leu Ala Ile Gly Thr Ala Asn Pro Pro
20 25 30
Asn Ala Val Asp Gln Ser Thr Tyr Pro Asp Phe Tyr Phe Arg Ile
35 40 45
Thr Gly Asn Glu His Asn Thr Glu Leu Lys Asp Lys Phe Lys Arg
50 55 60
Ile Cys Glu Arg Ser Ala Ile Lys Gln Arg Tyr Met Tyr Leu Thr
65 70 75
Glu Glu Ile Leu Lys Lys Asn Pro Asp Val Cys Ala Phe Val Glu Val Pro Ser Leu Asp
80 85 90 95
Ala Arg Gln Ala Met Leu Ala Thr Glu Val Pro Arg Leu Ala Lys Glu Ala Ala Glu Lys
100 105 110 115
Ala Ile Gln Glu Trp Gly Gln Ser Lys Ser Arg Ile Thr His Leu Ile Phe Cys Ser Thr
120 125 130 135
Thr Thr Pro Asp Leu Pro Gly Ala Asp Phe Glu Val Ala Lys Met Leu Gly Leu His Pro
140 145 150 155
Ser Val Lys Arg Val Gly Val Phe Gln His Gly Cys Phe Ala Gly Gly Thr Val Leu Arg
160 165 170 175
Met Ala Lys Asp Leu Ala Glu Asn Asn Arg Gly Ala Arg Val Leu Val Ile Cys Ser Glu
180 185 190 195
Thr Thr Ala Val Thr Phe Arg Gly Pro Ser Glu Thr His Leu Asp Ser Leu Val Gly Gln
200 205 210 215
Ala Leu Phe Gly Asp Gly Ala Ser Ala Leu Ile Val Gly Ala Asp Pro Ile Pro Gln Val
220 225 230 235
Glu Lys Ala Cys Phe Glu Ile Val Trp Thr Ala Gln Thr Val Val Pro Asn Ser Glu Gly
240 245 250 255
Ala Ile Gly Gly Lys Val Arg Glu Val Gly Leu Thr Phe Gln Leu Lys Gly Ala Val Pro
260 265 270 275
Asp Leu Ile Ser Ala Asn Ile Glu Asn Cys Leu Val Glu Ala Phe Ser Gln Phe Lys Ile Ser
280 285 290 295
Asp Trp Asn Lys Leu Phe Trp Val Val His Pro Gly Gly Arg Ala Ile Leu Asp Arg Val
300 305 310 315
Glu Ala Lys Leu Asn Leu Asp Pro Thr Lys Leu Ile Pro Thr Arg His Val Met Ser Glu
320 325 330 335
Tyr Gly Asn Met Ser Ser Ala Cys Val His Phe Ile Leu Asp Gln Thr Arg Lys Ala Ser
340 345 350 355
Leu Gln Asn Gly Cys Ser Thr Ser Gly Glu Gly Leu Glu Met Gly Val Leu Phe Gly Phe
360 365 370 375
Gly Pro Gly Leu Thr Ile Glu Thr Val Val Leu Lys Ser Val Pro Leu Gln
380 385 390 393
<210> 3
<211> 1713
<212> DNA
<213> 对香豆酰辅酶A连接酶基因的核苷酸序列(Artificial Sequence)
<400> 3
atgatgagtg ttgccaccgt ggaaccgccg aaaccggaac tgagcccgcc gcagaatcag 60
aatgccccga gtagtcatga aaccgatcat atttttcgca gtaaactgcc ggatattacc 120
attagtaatc atctgccgct gcatgcatat tgttttgaaa atctgagcga ttttagcgat 180
cgtccgtgtc tgattagtgg tagtaccggc aaaacctata gctttgcaga aacccatctg 240
attagtcgta aagtggccgc aggtctgagt aatctgggta ttaagaaagg cgatgtgatt 300
atgaccctgc tgcagaattg cccggaattt gtttttagtt ttatgggcgc cagcatgatt 360
ggtgcagtta ccaccaccgt taatccgttt tataccccgg gcgaaatttt taaacagttt 420
agcgcaagtc gtgcaaaact gattattacc cagagccagc atgtgaataa gctgcgtgat 480
agcgattgcc atgaaaataa tcagaaaccg gaagaagatt ttatcgttat taccattgac 540
gatccgccgg aaaattgcct gcattttaat gtgctggttg aagcaaatga aagcgaaatg 600
ccgaccgtga gcattcatcc ggatgatccg gtggcactgc cgtttagtag tggcaccacc 660
ggtctgccga aaggcgttat tctgacccat aaaagcctga ttaccagtgt ggcacagcag 720
gtggatggtg aaattccgaa tctgtatctg aaacaggatg atgtggtgct gtgcgtgctg 780
ccgctgtttc atatttttag tctgaatagt gtgctgctgt gcagtctgcg tgccggtagt 840
gccgtgctgc tgatgcagaa atttgaaatt ggcagtctgc tggaactgat tcagaaacat 900
aatgttagtg ttgccgccgt tgttccgccg ctggttctgg ccctggcaaa aaatccgatg 960
gtggcaaatt ttgatctgag cagcattcgt gtggtgctga gcggtgcagc cccgctgggt 1020
aaagaactgg aagaagccct gcgcagccgc gttccgcagg ctattctggg ccagggttat 1080
ggcatgaccg aagccggtcc ggttctgagc atgtgtctgg cattttctaa acagccgctg 1140
ccgaccaaaa gtggcagctg cggcaccgtt gttcgcaatg ccgaactgaa agttattgat 1200
ccggaaaccg gtagcagcct gggtcgtaat cagccgggcg aaatctgtat tcgtggcagc 1260
cagattatga aaggttatct gaatgatgca gaagcaaccg caaatattat tgatgttgaa 1320
ggttggctgc ataccggtga cattggctat gttgatgatg atgatgaaat ttttatcgtg 1380
gatcgtgtga aagaaattat taagtttaag ggcttccagg tgccgccggc cgaactggaa 1440
gccctgctgg tgaatcatcc gagtattgca gatgcagcag ttgttccgca gaaagatgaa 1500
gtggccggtg aagtgccggt tgcatttgtt gttcgcagta atgatctgga tctgaatgaa 1560
gaagccgtga aagattatat tgccaaacag gttgttttct ataagaaact gcataaagtg 1620
ttcttcgtgc atagtattcc gaaaagcgcc gccggcaaaa ttctgcgcaa agatctgcgc 1680
gccaaactgg caaccgcaac caccatgagt taa 1713
<210> 4
<211> 1182
<212> DNA
<213> 芪合成酶的基因的核苷酸序列(Artificial Sequence)
<400> 4
atgggcgctg ttgacttcga aggcttccgc aaactgcaac gtgccgatgg cttcgcaagc 60
attctggcca ttggtacagc aaatccgccg aatgcagtgg atcagagtac ctatccggac 120
ttctacttcc gtattaccgg taatgaacat aataccgaac tgaaagataa gttcaaacgc 180
atctgtgaac gtagtgccat taaacagcgt tatatgtatc tgaccgaaga aattctgaaa 240
aaaaatcctg atgtgtgtgc attcgttgaa gttccgagtc tggatgcacg tcaggcaatg 300
ctggcaaccg aagttccgcg cctggcaaaa gaagccgccg aaaaagccat tcaggaatgg 360
ggccagagta aaagccgcat tacccatctg atcttctgta gcaccaccac cccggatctg 420
ccgggtgccg acttcgaagt tgccaaaatg ctgggcctgc atccgagtgt taaacgcgtg 480
ggtgtgttcc agcatggttg cttcgcaggc ggtacagttc tgcgtatggc aaaagactta 540
gcagaaaata atcgtggtgc ccgtgtgctg gttatctgta gtgaaaccac cgcagtgacc 600
ttccgtggtc cgagcgaaac acatctggat agcctggttg gtcaggccct gttcggcgat 660
ggtgccagcg cactgattgt gggcgcagat ccgattccgc aggttgaaaa agcatgcttc 720
gaaattgtgt ggaccgcaca gaccgttgtt ccgaatagtg aaggtgccat tggcggtaaa 780
gttcgtgaag tgggtctgac cttccagctg aaaggtgcag ttccggatct gattagcgca 840
aatattgaaa attgtctggt tgaagcattc agccagttca aaattagtga ttggaataaa 900
ctgttctggg tggttcatcc gggtggtcgc gcaattctgg atcgtgttga agcaaaactg 960
aatctggacc ctaccaaact gattccgacc cgtcatgtta tgagcgaata tggtaatatg 1020
agcagcgcat gcgtgcactt cattctggat cagacccgca aagccagtct gcaaaatggt 1080
tgcagcacca gtggtgaagg cctggaaatg ggcgttctgt tcggcttcgg tccgggcctg 1140
accattgaaa ccgtggtgct gaaaagtgtt ccgctgcaa taa 1182
<210> 5
<211> 1179
<212> DNA
<213> Ptps的核苷酸序列(Artificial Sequence)
<400> 5
atggggggcg ttgattttga aggtttcagg aagttgcaga gggcagatgg cttcgcttcg 60
atccttgcta tcggcactgc caatccaccc aatgctgtgg atcagagcac atatccagat 120
tactacttcc gaatcaccgg taacgagcat aacacagagc tcaaggataa gttcaagcga 180
atatgtgaaa ggtcggcaat aagacaaaga tacatgtacc tgacggagga gattctcaag 240
aagaatcccg atgtgtgcgc gtttgtggag gtgccatcgt tggacgcacg gcaggcgatg 300
ttggctatgg aggtgccccg actggcaaaa gaggctgctg aaaaggccat tcacgagtgg 360
gggcagtgca agtctgggat cactcatctc atattttgca gcacaacgac tccggatcta 420
cccggagcag actttgaggt agccaagttg ctcgggctgc acccgagtgt gaagagagtg 480
ggcgtgttcc aacatggctg cttcgccgga ggcaccgttc ttcgactggc gaaagacctt 540
gccgaaaaca atcgaggagc tcgggtgctg gtcatctgca gtgaaaccac cgccgttacc 600
ttccgtggac cctccgagac tcacctggac agcctggtgg ggcaagctct attcggcgac 660
ggtgcgtctg ccctcatcgt gggagctgat cccatccctc aagtggagaa ggcatgtttc 720
gaaatcgttc ggacatccca gacagttgtt cccaacagcg acggagccat cggtgggaag 780
gtgagagaag tcggacttac cttccaactc aaaggggcgg ttccggatct tatctctgcc 840
aacattgaaa actgtctcgt ggaggcgttc agtcaattca aaatatccga ctggaacaag 900
ttgttctggg ttgttcatcc cggaggacgt gccatccttg atcgggtgga ggccaagctc 960
aatctggatc ccacaaaact gatacccacc aggcacgtta tgagcgagta cggaaacatg 1020
tcgagtgcat gcgtccactt catattggat gagacgagga aagcgtctct acgaaacgat 1080
tgttcaacca ccggagaggg attggaaatg ggagtcctgt ttggattcgg gccgggcctc 1140
accatcgaaa cagtggttct caagagcgtt cctctttag 1179
<210> 6
<211> 1189
<212> DNA
<213> PspsQ361R基因的核苷酸序列(Artificial Sequence)
<400> 6
atgtctgtag gaatgggcgt tgatttggaa gctttcagga aatcccagag ggcagatggc 60
ttcgcttcaa tccttgctat cggtacggcc aatcccccca atgttgtgga tcagagcaca 120
tatccagatt actactttcg aaacaccaat aacgaggata acacagacct caaggataag 180
ttcaagcgaa tatgtgaaag gtcggcaata aaaaagagac acatgtacct cacggaggag 240
attctgaaga agaatccgga attgtgcgca ttcttggagg tgccatcact ggacacacgg 300
caggcgatgt tggcggtgga ggtgccccgg ctaggaaaag aggccgctga aaaggcgatt 360
gaggagtggg gacaacccaa gtcgaggatc actcatctca tcttttgcac cacaaccact 420
ccagatttac ccggagccga ctttgaggta gccaagttgc tggggctgca ccccagtgtg 480
aagagagtgg gcgtgttcca acatggctgc ttcgccggag gcaccgttct tcgcctggcg 540
aaagaccttg ccgaaaacaa tcgaggagct cgggtgctgg tcgtgtgcag tgaaaacacc 600
gccgttacct tccgcggacc ctccgagact cacctggatg gcctagtggg cctagctctg 660
ttcggcgatg gtgcggctgc cctcatcgtg ggagctgatc ccatccctca agtggagaag 720
ccctgtttcg aaatcgtttg gacagcccag acagttgttc ccaacagcga cggagcaatc 780
agtgggaagc tgagagaggt gggattgacc ttccaactca aaggcgcggt tccggatctc 840
atctctacca acattgaaaa gtgtctggtg gaggcgttca gtcagttcaa tatctccgac 900
tggaaccagt tgttctggat tgctcatccc ggaggacgtg ccatccttga ccaggtggag 960
gcaagcctca atttggatcc cacaaaactc agagccacca ggcacgttat gagcgagtac 1020
ggaaacatgt ccagtgcgtg cgtccacttc atattggatg agaccaggaa ggcgtctcga 1080
caaaacggat gttcaaccag cggaggggga ttccaaatgg gagtcctctt tggattcggg 1140
ccgggcctca ccgtcgaaac agtcgttctc aagagcattc ctttccctt 1189
<210> 7
<211> 26
<212> DNA
<213> Ptr4CL4上游引物(Artificial Sequence)
<400> 7
acatatgatg agtgttgcca ccgtgg 26
<210> 8
<211> 25
<212> DNA
<213> Ptr4CL4下游引物(Artificial Sequence)
<400> 8
agactcgagt taactcatgg tggtt 25
<210> 9
<211> 25
<212> DNA
<213> PpSTS上游引物(Artificial Sequence)
<400> 9
caggatccga tgggcgctgt tgact 25
<210> 10
<211> 25
<212> DNA
<213> PpSTS下游引物(Artificial Sequence)
<400> 10
cgcaagcttt tattgcagcg gaaca 25
Claims (1)
1.一种赤松素的合成方法,其特征在于,按照1%接种量的重组表达转化体接种到LB培养基中,摇管活化,然后按1%接种量转接至装有LB的锥形瓶中,继续培养,取种子培养液,离心弃上清,将菌泥按初始OD600 0.6~0.8接种至发酵培养基中,并在发酵培养基中加入肉桂酸,同时加入终浓度0.5~1.5 mM IPTG诱导培养;其中,在发酵同时添加20-100 μM浅蓝菌素;发酵培养基包含M9盐11.3 g/L,酵母提取物5~10 g/L,3-(N-吗啉基)丙磺酸(MOPS)35~45 g/L,甘油3~8% (v/v),大肠杆菌的培养温度为28~45ºC,转速150~300 rpm,培养时间为8~15 h,发酵时间12~72 h;其中,重组表达转化体通过将重组表达载体转化至大肠杆菌得到,所述重组表达载体包含表达对香豆酰辅酶A连接酶的基因和表达芪合成酶的基因,所述表达对香豆酰辅酶A连接酶的基因编码GenBank: EEF00197.1对应的氨基酸序列;所述表达芪合成酶的基因编码GenBank: ALN42233.1对应的氨基酸的序列。
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