CN114410664A - 四氢嘧啶生物合成基因及其应用 - Google Patents
四氢嘧啶生物合成基因及其应用 Download PDFInfo
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Abstract
四氢嘧啶生物合成基因及其应用，涉及四氢嘧啶合成基因簇。四氢嘧啶合成基因ectABC发现于一株深海盐单胞菌Halomonas sedimenti QX‑2T，其核苷酸序列如SEQ ID NO.1的2244个碱基序列所示。包括3个基因区别于目前已知的四氢嘧啶合成基因簇序列，属于新的基因序列。通过对其进行基因工程技术克隆表达，发现进行基础发酵后，具有减少难以分离的次级代谢产物以及高产四氢嘧啶的特性。满足作为科学研究以及对四氢嘧啶应用于化妆品行业作为防晒剂、保湿剂、抗氧化剂。
Description
技术领域
本发明涉及基因工程技术领域，尤其是涉及一株深海来源的盐单胞菌Halomonassedimenti QX-2T产四氢嘧啶生物合成基因簇及其应用。
背景技术
四氢嘧啶(Ectoine)，1,4,5,6-四氢-2-甲基-4-嘧啶羧酸，首先发现于外硫红螺菌属(Ectothiorhodospira)，并因此而被命名。四氢嘧啶是一种环化的氨基酸，是一类广泛存在于细菌中的相容性溶质，尤其是在极端微生物嗜盐菌或耐盐菌中存在。极端微生物由于其特殊的生存环境，形成独特的环境适应机制；因此为了适应环境而会产生多种次级代谢产物，极端的高盐环境具有较高的环境渗透压，一般细胞由于难以承受极端的渗透压而会裂解死亡。而耐嗜盐菌由于会产生次级代谢产物四氢嘧啶等物质，可以维持渗透压以及细胞的稳定性，所以耐嗜盐微生物可以在极端的高浓度盐分中得以生存。
四氢嘧啶是细胞中主要的渗透压调节剂，除了调节细胞渗透压，对大分子也有一定稳定作用，此外四氢嘧啶具有多种生物学功能，因此是高档化妆品采用的生物工程制剂之一：1)作为水分结合剂，四氢嘧啶能够长效保湿(7天)，帮助滋润皮肤、重组皮肤细胞膜；2)抗氧化(预防衰老、改善皱纹)，延缓皮肤的过早老化等；3)四氢嘧啶能对抗紫外线对皮肤的伤害，修复紫外线导致的细胞DNA损伤，减少皮肤因紫外线照射形成的晒斑，抑制黑色素的生成；4)抗逆保护作用，缓解皮肤所受的各种压力以及干燥环境所致皮肤老化，表面活性剂引起的皮肤劣化等。四氢嘧啶应用于化妆品领域的优势在于它与细胞内的新陈代谢相容，并不影响细胞的生物大分子功能或生理过程。其不仅是一种重要的渗透压补偿溶质，而且对处于高温、高盐、冷冻、干燥、辐射等不良环境刺激下的细胞和生物大分子有着很好的保护作用。因此，对于四氢嘧啶的基因保护、挖掘具有重要作用。
已经有多项研究结果表明。中度嗜盐菌中四氢嘧啶的合成途径以天冬氨酸半醛(ASA)为前体，共分3步；第一步由L-二氨基丁酸转氨酶(L-Diaminobutyric acidtransaminase，DAT)催化合成L-二氨基丁酸(DABA)，第二步由L-二氨基丁酸乙酰转移酶(L-Diaminobutyric acid transferase，DAA)催化合成N-乙酰-L-2，4二氨基丁酸(ADABA)，第三步由四氢嘧啶合酶(Ectoine synthase，ES)催化合成四氢嘧啶，相关酶系DAT、DAA和ES的合成基因分别为ectB、ectA、ectC。由于四氢嘧啶有一个手性分子，很难化学合成，目前还没有研究结果表明ectoine的化学合成，所以只能进行生物合成。
发明内容
本发明的第一目的在于提供一种四氢嘧啶生物合成基因簇。
本发明的另一目的在于提供一种四氢嘧啶生物合成基因簇的应用。
本发明在深海环境样本中分离到一株Halomonas sp.微生物，经过鉴定菌株属于Halomonas sedimenti QX-2T。该株菌已公开发表，作为一株模式菌株保藏于海洋微生物菌种管理保藏中心(MCCC)以及韩国菌种保藏中心(KCTC)，保藏编号分别为MCCC 1A17876T，KCTC 82199T。
所述四氢嘧啶生物合成基因簇为基因ectABC，其来源于Halomonas sedimentiQX-2T，用大肠杆菌重导的菌株能够大量的表达ectABC，表达水平高，该生物合成基因簇的核苷酸序列如SEQ ID NO.1的2244个碱基序列所示，包括3个基因，具体为：
(1)负责催化L-二氨基丁酸(DABA)合成N-乙酰-L-2，4二氨基丁酸(ADABA)的二氨基丁酸乙酰转移酶基因(L-2,4-diaminobutyric acid acetyltransferase)，即ectA，位于核苷酸序列SEQ ID NO.1的第1-579位，共579个碱基，编码192个氨基酸。
(2)负责将合成四氢嘧啶的前体物质天冬氨酸半醛(ASA)催化生成L-二氨基丁酸(DABA)的二氨基丁酸-2-氧戊二酸氨基转移酶基因(diaminobutyrate--2-oxoglutarateaminotransferase)，即ectB，位于核苷酸序列SEQ ID NO.1的第580-1851位，共1272个碱基，编码423个氨基酸。
(3)负责将N-乙酰-L-2，4二氨基丁酸(ADABA)催化生成目标产物四氢嘧啶(ectoine)的四氢嘧啶合成酶基因(ectoine synthase)，即ectC，位于苷酸序列SEQ IDNO.1的第1852-2244位，共393个碱基，编码130个氨基酸。
所述四氢嘧啶生物合成基因簇的应用，包括但不限于：
1)基因ectABC的合成产物在制备化妆品抗氧化剂等中应用。
2)基因ectABC通过基因工程技术获得其重组蛋白，该序列编码的蛋白质在制备四氢嘧啶中的应用。
3)基因ectA在制备N-乙酰-L-2，4二氨基丁酸(ADABA)中的应用。
4)基因ectB在制备L-二氨基丁酸(DABA)中的应用。
5)基因ectC在制备四氢嘧啶(ectoine)中的应用。
6)所述四氢嘧啶生物合成基因簇在制备抗氧化的生物制剂中的应用。
实验表明，四氢嘧啶能有效清除DPPH自由基活性，减弱由氧化带来的氧化损伤作用，从而可以作为一种抗氧化剂应用在化妆品中，具有很大的应用价值和广泛的应用前景。
本发明研究发现，Halomonas sedimenti QX-2T通过简单的发酵，检测其胞内四氢嘧啶的产量，就具有较高的产量，这可能与其作为一株深海中度嗜盐菌、具有一定的新基因、新结构有关，本发明的基因ectABC编码Halomonas sedimenti QX-2T菌株的ectione的生物合成，用大肠杆菌重导的菌株能够大量的表达ectABC，表达水平高，由于产物较单一，便于纯化，减少次级代谢产物纯化的难度。具有高产的化合物的特征，基因合成产物在制药、食品、化妆品、生物制剂、酶制剂、农业和化学合成药物及有机电子材料等领域具有很大的应用价值和广泛的应用前景。
附图说明
图1为盐单胞菌次级代谢产物HPLC图。
图2为重组菌株次级代谢产物HPLC图。
图3为四氢嘧啶对DPPH自由基清除率图。
具体实施方式
下面结合实施例对发明做进一步说明。下列实施例中未注明具体条件的实验方法，通常可按照常规条件和方法进行，如J.萨姆布鲁克(Sambrook)等编写的《分子克隆实验指南》中所述的条件，或按照制造厂商所建议的条件运行。
所述四氢嘧啶生物合成基因簇为ectABC，是编码Halomonas sedimenti QX-2T的ectoine合成基因簇，其DNA序列、氨基酸序列如序列表SEQ ID NO.1所示。
经过测序比对发现基因ectABC的序列与目前已知公开发表的ectoine合成基因簇基因序列无一致序列，相似度低，为新基因序列。
实施例1:ectABC基因的克隆分离
通过对Halomonas sedimenti QX-2T进行全基因组测序得到ectione合成基因及其基因序列，包括ectABC基因，全长2452bp。利用生物学软件Primer5.0设计上游引物(5’-3’)和下游引物(5’-3’)以基因组DNA为模板，进行PCR反应，PCR反应组成如下(50μL反应体系)：上游和下游引物各2μL，DNA模板1μL，10×Buffer 5μL，dNTP Mix 5μL，Ex Taq 0.5μL，ddH2O 34.5μL。反应条件为：94℃预变性5min，94℃变性30s，52.4℃退火30s，72℃延伸1min20s，72℃终延伸5min，共30个循环。反应结束后，将PCR产物回收得到ectoine合成基因簇ectABC。琼脂糖凝胶电泳，大小符合ectABC片段长度。
实施例2：大肠杆菌克隆载体pUC-ectABC的构建
1、用EcoRⅠ以及NotⅠ酶将ectABC与pUC载体分别进行双酶切，反应体系为：pUC载体/ectABC 8μL，EcoRⅠ1μL，NotⅠ1μL，缓冲液3.5μL，ddH2O 6.5μL
2、37℃下酶切3h，电泳后分别回收两个目的片段2.2kb和5.6kb，两者与ectABC/pUC载体片段大小相同
3、采用T4连接酶进行连接，连接体系(10μL)如下：2×Buffer 5μL，2.2kbDNA片段3μL，5.6kb pUC载体1μL，T4连接酶1μL，20℃过夜连接。此即得到连接液，之后并转化大肠杆菌DH5α。
实施例3：大肠杆菌重组株DH5α-PUC-ectABC的构建
在1.5mL Eppendorf管中加入100μL解冻的感受态大肠杆菌DH5α和10μL连接液，冰浴30min，42℃热激90s，冰浴30min，加入LB培养基900μL，37℃振荡培养1h，将菌液涂布于含4μL IPTG、100μg/mL Amp及40μg/mL X-gal的LB平板上，37℃过夜培养。挑白色单菌落做PCR检测，琼脂糖凝胶电泳中呈现2.2kb特异带者为阳性转化菌落，即含有pUC-ectABC的大肠杆菌菌落。
实施例6：重组ectoine的制备
挑取大肠杆菌重组菌株的单克隆于含有浓度为100μg/mL氨苄青霉素的5mL LB液体培养基中，37℃振荡培养12h，取菌液1mL接种于100mL同样LB液体培养基中扩大培养，37℃180rpm振荡培养24h后测OD600，然后抽取10mL于离心管中，12000r/min离心5min，弃上清；加人纯水10mL，用超声细胞破碎仪破碎7min，12 000r/min离心5min，取其上清经0.22um水系微孔过滤器过滤,即重组菌株四氢嘧啶提取液。
野生型菌株Halomonas sedimenti QX-2T四氢嘧啶提取液的制备方法同上。
实施例7：高效液相色谱(HPIC)检测四氢嘧啶含量
配制1.0mg/ml的四氢嘧啶标准品母液，并建立标准曲线(峰面积与浓度)。HPLC检测条件：流动相为水︰乙腈(V/V,20︰80)，检测波长210nm，流速为1.0mL/min，柱压3.486-4.761MPa，柱温30℃，上样量10μL。按照此条件与方法检查样品中四氢嘧啶的浓度，将实例6得到的野生型以及重组菌株的四氢嘧啶提取液分别取10μL，与四氢嘧啶标准品按照相同条件检测。结果表明，野生型菌株的HPLC图谱(图1)显示，野生型菌株的次级代谢产物种类较多，相比于重组菌株的HPLC图谱(图2)，重组菌株的次级代谢产物的种类明显减少，且发现显著ectione产量显著提升。这提示在下一步分离纯化四氢嘧啶化合物会减少其他难以分离的次级代谢产物的难度，并且得到产量更高、纯度更好的四氢嘧啶化合物。以便进一步用于后续的科学研究以及产业化研究开发。
实施例8：四氢嘧啶对DPPH自由基清除活性测定
(1)DPPH溶液的配制：称取DPPH4mg，配制浓度为0.004％的DPPH无水乙醇溶液。
(2)DPPH自由基清除能力:精密称取128mg四氢嘧啶溶于10mL蒸馏水中，得浓度为12.8mg/mL的样品液，然后梯度稀释，分别得0.2、0.4、0.8、1.6、3.2、6.4mg/mL的样品液，(蒸馏水做空白对照，每组样品设三个平行)避光条件下加入1.0mL DPPH(0.004％)溶液，摇匀，避光反应30min，离心，取上层清液。BHT为阳性对照在测定吸光度值，计算百分清除率:
百分清除率(％)＝[(A0-A1)/A0]×100％
其中A1表示样品(或阳性对照品)的吸光度值；
A0表示空白组的吸光度值。清除率越大，抗氧化能力越强。
实验结果显示，相比于阳性对照组BHT的清除率，当样品浓度为2mg/mL时，其DPPH自由基的清除能力较阳性对照组能力弱，而当样品浓度大于等于4mg/ml时，其自由基清除能力强于阳性对照组的清除能力，且随着浓度的降低，四氢嘧啶的自由基清除能力在逐渐减弱。(图3)。这表明在一定浓度范围内四氢嘧啶具有良好的清除DPPH自由基的能力，而由此说明四氢嘧啶具有良好的抗氧化作用与功效。
序列表
<110> 自然资源部第三海洋研究所；中国大洋矿产资源研究开发协会（中国大洋事务管理局）
<120> 四氢嘧啶生物合成基因及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2244
<212> DNA
<213> Halomonas sedimenti QX-2T(2 Ambystoma laterale x Ambystomajeffersonianum)
<400> 1
atgagtacgc cgacacaacc ttttaccccc tctgctgacc ttgctaggcc aacggtggct 60
gatgctgttg tgggtcatgc ggagatgccg ctgtttattc gcaaacccaa tgcagatgat 120
ggctggggag tttacgagct tatcaaagcc tgtccgccgc tggatgtgaa ttcagcctat 180
gcctacctac tgcttgctac tcagtttcgt gacacctgcg cagtggcgac caatgaagac 240
ggcgaagtgg tggggtttgt gtctggctat gttaaggaca atgcccccga cacctatttt 300
ctgtggcaag tggctgtcgg tgaaaaggca cgcggcacag gcctagcgcg tcgcttggtt 360
gaggcgatca tgtcgcgtcc cgagcttgat gatgtgcatc accttgaaac taccatcacg 420
cctgacaatc tagcctcttg gggcttattt cgtcgcctgg ctgcgcgctg gcatgcgccg 480
ctaaacagcc gcgaatactt ctctaccgaa cagctcggag gagagcatga cccggaaaac 540
ctggttcgta tcggcccgtt ccaaacagac cgcatctaaa tgcagaccca gacgcttgaa 600
cgcatggaat ccaatgtacg tacttattct cgttcgtttc cggtggtgtt taccaaagcg 660
caaaatgcgc gcctaaccga tgagaatggc cgtgagtaca ttgatttcct cgccggcgcg 720
ggcacgctga actacggcca caataatccg cacatgaagc aggcgatgat tgattacctg 780
tcgactgacg gtgttgttca cggtttggat atgtggacca atgccaagcg cgattatctt 840
gaaacgctgg aagaagttat tttcaagcca cgcgggctgg attataaagt gcatctgcct 900
ggcccaaccg gtaccaatgc cgtagaggcc gctatccgct tggctcgtgt ggctaaaggc 960
cgtcacaaca ttgtcacctt caccaatggt ttccacggcg tcaccatggg ggctttggcg 1020
accacaggta atcgtaaatt ccgtgaagcc acgggtggca tacccaccca gggcgctagc 1080
tttctgcctt acgatggtta catgggtgag cataccgata ctctggatta ttttgaaaag 1140
ctgctcagcg acaaatctgg cggtttggat attccagcgg gcgttatcat tgaaaccgtg 1200
caaggcgagg gcggtattaa cgttgcaggc ttggagtggt tgaagcgtct cgaagggatt 1260
tgccgtgccc acgatattct gctgattatc gatgatattc aggcaggctg tggccgtaca 1320
ggtaagttct ttagcttcga acatgcgggt attacacctg acattattac caactcgaaa 1380
tcgctctccg gttttggcct gccatttgcc catgtgttga tgcgccctga gctggacaaa 1440
tggaaaccgg gtcaatataa cggcactttc cgtggtttca gcctggcgat ggtgaccgcc 1500
acagcggcgt tgaaaaaata ctggactaat gacacctttg agcgtgatgt tcagcgcaaa 1560
gggcgtattg tagaagagcg ctttcagaag ttagcagcgt tgttaacgga acacggcatg 1620
cctgccactg aacgtggtcg tggtttgatg cgcggtattg acgtcgtgtc gggtgatatt 1680
gccgataaaa ttaccagtaa agcctttgag catggtctgg taattgaaac cagtggtcag 1740
gatggtgaag tggttaagtg tctatgcccg ctaaccatta gcgatgatga tctgctggaa 1800
ggtttggata ttctcgaaat gtgcgttaaa gctgttgttg ctagcgaata aatgatcgtt 1860
cgtaatcttg atgaagcgcg taaaacagac cgtctagtga ccgctgaaaa tggtaactgg 1920
gacagcacgc gccttgttct ggccaacgat aatgcaggtt tttcgttcca tattacccgc 1980
atttttccag gcactgaaac gcatatccac tacaaaaatc actacgaagc agtgttttgc 2040
tacgaaggtg aaggcgaagt tgaaaccctg gctgacggta aaatttggcc gatcaaagca 2100
ggcgacattt atctactcga ccagcatgat gaacacttgc tgcgcggcaa agagaagggc 2160
atgacggttg cctgtgtatt tactccgccg atcacaggta atgaggttca ccaggaagat 2220
ggctcgtacg cagccccaga gtaa 2244
Claims (8)
1.四氢嘧啶生物合成基因簇，其特征在于其核苷酸序列如SEQ ID NO.1的2244个碱基序列所示。
2.如权利要求1所述四氢嘧啶生物合成基因簇，其特征在于其包括以下3个基因：
(1)负责催化L-二氨基丁酸合成N-乙酰-L-2，4二氨基丁酸的二氨基丁酸乙酰转移酶基因，即ectA，位于核苷酸序列SEQ ID NO.1的第1-579位，共579个碱基，编码192个氨基酸；
(2)负责将合成四氢嘧啶的前体物质天冬氨酸半醛催化生成L-二氨基丁酸的二氨基丁酸-2-氧戊二酸氨基转移酶基因，即ectB，位于核苷酸序列SEQ ID NO.1的第580-1851位，共1272个碱基，编码423个氨基酸；
(3)负责将N-乙酰-L-2，4二氨基丁酸催化生成目标产物四氢嘧啶的四氢嘧啶合成酶基因，即ectC，位于苷酸序列SEQ ID NO.1的第1852-2244位，共393个碱基，编码130个氨基酸。
3.如权利要求1所述四氢嘧啶生物合成基因簇在作为抗氧化剂在化妆品制备中应用。
4.如权利要求1所述四氢嘧啶生物合成基因簇的应用，其编码蛋白催化制备四氢嘧啶。
5.如权利要求2所述基因ectA在催化制备N-乙酰-L-2，4二氨基丁酸中的应用。
6.如权利要求2所述基因ectB在催化制备L-二氨基丁酸中的应用。
7.如权利要求2所述基因ectC在催化制备四氢嘧啶中的应用。
8.如权利要求1所述四氢嘧啶生物合成基因簇在制备抗氧化的生物制剂中的应用。
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