本發(fā)明屬于代謝工程領(lǐng)域，具體涉及一種重組大腸桿菌及其構(gòu)建方法與通過代謝工程生產(chǎn)葡萄糖二酸的方法。
背景技術(shù)：
：葡萄糖二酸(glucuricacid,saccharicacid)又名葡萄糖質(zhì)酸，是含有4個手性碳原子的化合物，通常以手性化合物d-葡萄糖二酸的形式存在，在水溶液中自發(fā)氧化，形成單內(nèi)酯d-葡萄糖二酸-1,4內(nèi)酯和d-葡萄糖二酸-3,6-內(nèi)酯以及少量的雙內(nèi)酯d-葡萄糖二酸-1,4；3,6-內(nèi)酯。葡萄糖二酸是一種天然、無毒的化合物，少量生產(chǎn)于包括人類的哺乳動物和一些植物中，如番茄、葡萄柚等。葡萄糖二酸具有重要的生物功能，被美國能源部確定為“最有價值的生物煉制產(chǎn)品”。它和許多營養(yǎng)物質(zhì)一樣，可以幫助和增強免疫及身體排毒，進而減少人類疾病，研究稱葡萄糖二酸有藥理作用，它能特異性的強烈抑制一些與癌癥發(fā)生有關(guān)的酶如β-葡萄糖醛酸苷酶的活性。它還可以降低膽固醇，同時其鈣鹽也是一種食品添加劑。葡萄糖二酸及其衍生物能明顯抑制凝血酶誘導花生四烯酸過氧化反應(yīng)，可以起到明顯的抗炎癥作用；葡萄糖二酸及其衍生物影響血小板在氧化應(yīng)激條件的活化，通過抗氧化機制有助于預(yù)防過度血小板激活，因此葡萄糖二酸及其衍生物可能是預(yù)防心血管疾病風險的頂尖保健品，而一些水果蔬菜中含有大量的葡萄糖二酸及其衍生物，可作為膳食預(yù)防心血管疾病風險。葡萄糖二酸由于對人體無毒，其結(jié)構(gòu)與單糖結(jié)構(gòu)相似，可以通過人體的糖轉(zhuǎn)運系統(tǒng)進入細胞內(nèi)部，且能夠聚集在病灶和少量被缺血組織快速吸收等優(yōu)點，因此可以作為顯像劑應(yīng)用于心肌梗死和腫瘤的研究中。另外，葡萄糖二酸也可應(yīng)用于生活生產(chǎn)中普遍用到的聚合物單體的生產(chǎn)中，且這些聚合物在自然界中水解為小分子單體可以被植物和微生物吸收利用，所以它作為合成多種高效環(huán)保的新興生物質(zhì)能源的原料，具有巨大的潛在經(jīng)濟價值。目前葡萄糖二酸的制備方法主要是化學法，以硝酸氧化法和tempo為催化劑的氧化法等。硝酸氧化法主要將葡萄糖氧化為葡萄糖及小分子物質(zhì)。此種方法生產(chǎn)的副產(chǎn)物多且復雜，同時有大量no和no2等有害氣體排放，對環(huán)境產(chǎn)生污染，所以逐漸被新方法替代。tempo氧化法是指利用2,2,6,6-四甲基-1-哌啶自由基(tempo)介導的電化學氧化葡萄糖合成葡萄糖二酸的方法。由于它摧毀的氧化反應(yīng)物金屬參與反應(yīng)且反應(yīng)條件溫和，對產(chǎn)物具有選擇性并能夠限制不可回收的副產(chǎn)物的產(chǎn)物，便于操作，是目前常用的一種方法；但是由于在反應(yīng)過程中要控制反應(yīng)溫度和ph值以達到最佳，還有昂貴的催化劑也是需要改進的方面。長期以來，微生物發(fā)酵法來實現(xiàn)有機酸高效環(huán)保生產(chǎn)的常用方法。同化學法相比，微生物發(fā)酵法在原料損耗、產(chǎn)品純度等方面有很大改善，成為一種更優(yōu)的合成方法。葡萄糖二酸是存在于哺乳動物體內(nèi)作為代謝終產(chǎn)物被發(fā)現(xiàn)，在體內(nèi)，從葡萄糖到葡萄糖二酸，至少需要10步反應(yīng)。prather等從釀酒酵母(saccaromycescerevisiae)和小鼠獲取肌醇-1-磷酸合成酶(ino1)和肌醇氧化酶(miox)，及丁香假單胞菌(pseudomonassyringae)獲得醛酸脫氫酶(udh)，克隆到大腸桿菌中，實現(xiàn)了葡萄糖二酸的生物合成。目前尚未發(fā)現(xiàn)有通過重組植物來源的肌醇氧化酶的大腸桿菌來發(fā)酵生產(chǎn)葡萄糖二酸的報道，植物中葡萄糖二酸含量少，且提取分離純化較難，鑒于此，本發(fā)明中構(gòu)建的植物來源基因異構(gòu)重組在大腸桿菌，在微生物發(fā)酵生產(chǎn)葡萄糖二酸方面，提供了新的方法與思路。技術(shù)實現(xiàn)要素：本發(fā)明通過將不同植物來源的肌醇-1-磷酸合成酶基因(ino1)、肌醇氧化酶基因(miox)及來源于根癌農(nóng)桿菌的醛酸脫氫酶基因(udh)克隆表達至大腸菌株中，構(gòu)建重組菌，該重組菌能以葡萄糖、甘油、蔗糖或者肌醇為底物合成葡萄糖二酸。本發(fā)明通過以下技術(shù)方案實現(xiàn)。一種重組大腸桿菌，該重組大腸桿菌同時表達肌醇-1-磷酸合成酶基因ino1、肌醇氧化酶基因miox、醛酸脫氫酶基因udh。優(yōu)選的，所述肌醇-1-磷酸合成酶基因ino1來源于以下任意一種：釀酒酵母(saccaromycescerevisiae)、畢赤酵母(pichiapastoris)、番茄(solanaceaelycopersicon1706)、玉米(zeamays)、大豆(glycinemax)。進一步優(yōu)選的，所述肌醇-1-磷酸合成酶基因ino1，在本發(fā)明的一種實施方式中，來源于番茄(solanaceaelycopersicon1706)。優(yōu)選的，所述肌醇氧化酶基因miox來源于以下任意一種：畢赤酵母(pichiapastorisgs115)、番茄(solanaceaelycopersicon1706)、玉米(zeamays)、大豆(glycinemax)。進一步優(yōu)選的，所述肌醇氧化酶基因miox，在本發(fā)明的一種實施方式中，來源于番茄(solanaceaelycopersicon1706)。優(yōu)選的，所述醛酸脫氫酶基因udh來源于根癌農(nóng)桿菌(agrobacteriumtumefaciensgv3103)。優(yōu)選的，所述肌醇-1-磷酸合成酶基因ino1、肌醇氧化酶基因miox、醛酸脫氫酶基因udh，在本發(fā)明的一種實施方式中，其核苷酸序列分別是seqidno.1、seqidno.2、seqidno.3所示的序列。優(yōu)選的，所述肌醇-1-磷酸合成酶基因ino1、肌醇氧化酶基因miox、醛酸脫氫酶基因udh表達的蛋白質(zhì)，在本發(fā)明的一種實施方式中，其氨基酸序列分別是seqidno.4、seqidno.5、seqidno.6所示的序列。優(yōu)選的，所述重組大腸桿菌的出發(fā)宿主菌為大腸桿菌。優(yōu)選的，所述肌醇-1-磷酸合成酶基因ino1、肌醇氧化酶基因miox及醛酸脫氫酶基因udh為整合表達。以上所述的重組大腸桿菌的構(gòu)建方法，所述方法包括如下步驟：(1)將肌醇-1-磷酸合成酶基因ino1和肌醇氧化酶基因miox連接到雙元表達載體上轉(zhuǎn)化到大腸桿宿主菌種，得到重組大腸桿菌1；(2)將醛酸脫氫酶基因udh連接到表達載體后轉(zhuǎn)化到步驟(1)得到的重組大腸桿菌1中，得重組大腸桿菌。優(yōu)選的，該方法具體包括如下步驟：(1)以番茄(solanaceaelycopersicon1706)基因組為模板擴增miox基因、ino1基因，并分別加上合適酶切位點，將片段連接到雙元表達載體petduet1后，用化學法轉(zhuǎn)化dh5α中，篩選獲得正確的重組質(zhì)粒，命名為petduet1-ino1-miox，將重組質(zhì)粒轉(zhuǎn)化到宿主菌e.colibl21(de3)中既得到e.colibl21(de3)/ino1-miox；(2)以根癌農(nóng)桿菌(agrobacteriumtumefaciensgv3103)基因組為模板擴增udh基因，連接到pet28a后，用化學法轉(zhuǎn)化步驟(1)構(gòu)建的表達宿主e.colibl21(de3)/ino1-miox中，篩選正確的重組大腸桿菌，命名為e.colibl21(de3)/ino1-miox-udh。一種利用所述以上重組大腸桿菌生產(chǎn)葡萄糖二酸的方法，該方法是以葡萄糖、甘油、蔗糖或者肌醇為底物催化合成葡萄糖二酸。優(yōu)選的，所述方法具體是將重組大腸桿菌種子液以2％-5％的接種量接種到發(fā)酵培養(yǎng)基中，于od600約為0.6，加入終濃度為0.1-1mmiptg和10g/l葡萄糖于25℃-30℃，160rpm-200rpm，培養(yǎng)60-100h。進一步優(yōu)選的，所述發(fā)酵培養(yǎng)基，在本發(fā)明的一種實施方式中，其碳源為葡萄糖和/或肌醇。進一步優(yōu)選的，所述方法，在本發(fā)明的一種實施方式中，具體是將種子培養(yǎng)液按2％接種量接種到含有50ml發(fā)酵培養(yǎng)基的量為250ml搖瓶中，于od600約為0.6，加入終濃度為0.1-1mmiptg和10g/l葡萄糖，溫度為30℃，搖床轉(zhuǎn)速為180rpm，培養(yǎng)時間為100小時。與現(xiàn)有技術(shù)相比，本發(fā)明具有如下優(yōu)點：(1)本發(fā)明提供了一種通過異構(gòu)植物來源關(guān)鍵酶基因重組大腸桿菌發(fā)酵生產(chǎn)葡萄糖二酸的方法，重組大腸桿菌在lb培養(yǎng)基中生產(chǎn)葡萄糖二酸2.53g/l，對照大腸桿菌bl21(de3)中，沒有檢測到葡萄糖二酸。(2)本發(fā)明所用的來源于番茄中的肌醇-1-磷酸合成酶基因和肌醇氧化酶基因與現(xiàn)有報道的來源于小鼠的miox氨基酸差異較大，并且目前沒有來源于番茄中的ino1基因和miox基因能夠?qū)⑵咸烟寝D(zhuǎn)化為醛酸的相關(guān)報道。(3)本發(fā)明的重組大腸桿菌合成葡萄糖二酸，還具有培養(yǎng)時營養(yǎng)要求低、生產(chǎn)快、培養(yǎng)廉價、遺傳穩(wěn)定、表達水平高等許多優(yōu)點。(4)本發(fā)明采用代謝工程的策略，改造微生物菌株來合成目的產(chǎn)物葡萄糖二酸，為生物法高效生產(chǎn)葡萄糖二酸打下了堅實的基礎(chǔ)。附圖說明圖1為重組大腸桿菌搖瓶發(fā)酵葡萄糖二酸生成量隨發(fā)酵時間增加的曲線圖；圖2a為葡萄糖二酸標樣的hplc譜圖；圖2b為重組大腸桿菌發(fā)酵液的hplc譜圖。具體實施方式以下結(jié)合實例與附圖對本發(fā)明的具體實施作進一步的說明，但本發(fā)明的實施方式不限于此，對未特別說明的工藝參數(shù)，可參照常規(guī)技術(shù)進行。葡萄糖二酸的檢測：waser1525(binaryhplcpump)，2414時差檢測器和2487紫外檢測器。葡萄糖二酸標樣的配制：準確稱取50mg葡萄糖二酸溶于5mmh2so4中，將溶解后的溶液轉(zhuǎn)移至10ml容量瓶中定容，其濃度為100mg/l。再用5mmh2so4分別稀釋至4mg/ml、3mg/ml、2mg/ml、1mg/ml。樣品制備：1ml發(fā)酵液在12000rpm下離心5min，取上清經(jīng)0.22um濾膜過濾，濾液經(jīng)液相分析。分析條件：流動相：5mmh2so4，等度洗脫色譜柱：hpx-87h檢測器：2414時差檢測器和2487紫外檢測器實施例1重組大腸桿菌e.colibl21(de3)/ino1-miox的構(gòu)建以番茄(solanaceaelycopersicon1706)基因組為模板擴增ino1基因、miox基因，用ino1-f(序列如seqidno.7所示)、ino1-r(序列如seqidno.8所示)為引物擴增ino1基因，用miox-f(序列如seqidno.9所示)、miox-r(序列如seqidno.10所示)為引物擴增miox基因。將獲得的ino1基因、miox基因，通過引物兩端的酶切位點，經(jīng)雙酶切，連接到具有相應(yīng)切口的表達載體petduet1(購于novegen公司)，轉(zhuǎn)化到dh5α(購于全式金公司)中，在確保閱讀框正確的前體下，鑒定出重組表達質(zhì)粒petduet1-ino1-miox，經(jīng)測序比對，序列正確。重組質(zhì)粒經(jīng)化學法轉(zhuǎn)入表達宿主e.colibl21(de3)(購于全式金公司)中，重組克隆子經(jīng)pcr驗證正確，命名為e.colibl21(de3)/ino1-miox。實施例2重組大腸桿菌e.colibl21(de3)/ino1-miox-udh的構(gòu)建以根癌農(nóng)桿菌(agrobacteriumtumefaciensgv3103)基因組為模板擴增udh基因，udh-f(序列如seqidno.11所示)、udh-r(序列如seqidno.12所示)為引物擴增udh基因，通過引物上的雙酶切位點，連接至具有相應(yīng)切口的表達載體pet28a(購于biovector質(zhì)粒載體菌種細胞基因保藏中心)上，轉(zhuǎn)化至e.colidh5α(購于全式金公司)中，在確保閱讀框正確的前提下鑒定出重組表達質(zhì)粒pet28a-udh，經(jīng)dna測序比對，重組序列正確。重組質(zhì)粒經(jīng)化學法轉(zhuǎn)入實施例1構(gòu)建的表達宿主e.colibl21(de3)/ino1-miox中，篩選正確的重組大腸桿菌，命名為e.colibl21(de3)/ino1-miox-udh。表1為實施例1、2中用到的引物。表1引物序列udh-fcgcaagcttatgaaacggcttcttgttaccudh-rcgctcgagcggtgtcgtctcggttatatino1-fcgcggatccgatgtttattgaaaattttaaggtino1-rgcgctgcaggatttgtattccaaaatcatgmiox-fcggatatcgatgactattctcattgagcagcctmiox-rccgctcgagaccacctcagctttgttggaaaat實施例3重組大腸桿菌發(fā)酵生產(chǎn)葡萄糖二酸將重組大腸桿菌e.colibl21(de3)/ino1-miox-udh進行發(fā)酵培養(yǎng)。將單克隆重組大腸桿菌e.colibl21(de3)/ino1-miox-udh接種于25ml的lb培養(yǎng)基(胰蛋白胨10g/l，酵母提取物5g/l，氯化鈉10g/l，ph7)中，在37℃、180rpm培養(yǎng)16h。然后再按2％接種量接種至50ml(搖瓶容量為500ml)發(fā)酵培養(yǎng)基(胰蛋白胨10g/l，酵母提取物5g/l，氯化鈉10g/l)中發(fā)酵，于od600為0.6時加入誘導劑iptg使其濃度為0.2mm，同時加入葡萄糖使其濃度為10g/l，在溫度為30℃，搖床轉(zhuǎn)速為180rpm下，培養(yǎng)100小時。培養(yǎng)結(jié)束后，取1ml發(fā)酵液在12000rpm下離心5min，取上清經(jīng)0.22um濾膜過濾，通過hplc檢測產(chǎn)物。圖2a為葡萄糖二酸標樣的hplc譜圖，圖2b為重組大腸桿菌發(fā)酵液的hplc譜圖。由標樣可以看出葡萄糖二酸的出峰時間為10.058min，在重組菌株發(fā)酵液中可以檢測到具有同樣出峰時間的葡萄糖二酸。圖1為重組大腸桿菌搖瓶發(fā)酵葡萄糖二酸生成量隨發(fā)酵時間增加的曲線圖；由圖1可知，隨著搖瓶發(fā)酵時間的增加，葡萄糖的量減少，葡萄糖二酸的生成量增加，達到2.53g/l。sequencelisting<110>華南理工大學<120>一種重組大腸桿菌及其構(gòu)建方法與通過代謝工程生產(chǎn)葡萄糖二酸的方法<130><160>12<170>patentinversion3.5<210>1<211>1533<212>prt<213>人工序列<400>1alathrglythrthrthralathrthrglyalaalaalaalathrthr151015thrthralaalaglyglythrglyglyalaalaalaglycyscyscys202530alaalaalathrglythrglyalaalaglythralathralathrthr354045glyalaglyalaalathrglyalaalaalathrthrcysalathrthr505560cysthrglythrglythralathrglyalathrthralathrglyala65707580alaalacyscysalacysalaglyalaglycysthrthrglythrthr859095cysalathrglyalaalaglyalaglyalaglyalaalaalathrgly100105110glyalaalacysthrthralathrcysalaalathrglyglyalathr115120125thrglythrthralaalaglycyscysthralaalaalaalacysthr130135140glythrcysalaalaalathralathrglyalaalathrthrthrala145150155160alaalaalacysthrglyalathralacyscyscysalathrglythr165170175glycyscysalaalaalaalathrthrglyglyglyglyglythrthr180185190alathrglycysthrthrglythrthrglyglyalathrglyglygly195200205glyalaglyglyalaalaalacysalaalathrglyglythrthrcys210215220alaalacysalathrthrglyalacysthrglyglyalaglyglythr225230235240glythrthralathrthrglycysglyalaalathrcysglyalagly245250255alaalaglyglyalaalathrthrthrcysalathrglyglyglycys260265270alaalacysglyalaalaalaglyalaalaalaalaalaglythrgly275280285cysalaalacysalaalaglycyscysalaalathrthralathrthr290295300thrthrglyglyglythrcysthrcysthrthralacysthrcysala305310315320glyglycysalathrcysalaalacyscysalathrthrcysglyala325330335glythrthrglyglyglythrcysthrthrthrcysalaalathrgly340345350glycysglyalaalaglyalaglyalathrcysthralathrglycys355360365alacyscyscysthrthrcysalaalaalaalaglycyscysthrcys370375380cysthrthrcyscyscysalathrglyglythrcysalaalacyscys385390395400cysalaglyalacysglyalathrglythralaglythralathrthr405410415thrglyglyalaglyglyalathrglyglyglyalacysalathrthr420425430alaglycysalaalacysalathrglyalaalathrthrthrglygly435440445cysalaglyalathrglycysthralathrglyglythrcysalagly450455460glyglycysthralaalaglyglythrthrthrthrcysglyalaala465470475480glythrthrglyalathrcysthrglycysalaalaalaalaglycys485490495alaglycysthrglyalaglyglycyscyscysthralacysalathr500505510glyglyalaalathrcyscysalathrglyglythrthrcyscyscys515520525cysthrthrcyscysthrglyglythralathrcysthralathrgly530535540alacyscyscysthrglyalacysthrthrcysalathrthrglycys545550555560glyglycysthralaalacyscysalaalaglyalaalaglycysala565570575cysglythrglycyscysalaalacysalaalacysglythrglyala580585590thrcysalaalaalaglyglyalaalacyscysalaalaglyalaala595600605alaglyalaalacysalaalaglythrthrglyalaalacysalaala610615620alathrthrglythrthralaalaalaglyalathralathrthrala625630635640glyglyglyalaglythrthrcysalaalaglyglyalaglyalaala645650655glyalaalacysalaalaglyglythralaglyalacysalaalagly660665670alathralaglythrglyglythrthrcysthralathrglyglyala675680685cysthrglycyscysalaalacysalacyscysglyalaalaalagly690695700glythralacysalaglythralaalathrglythrglyglythrthr705710715720glythrthrglyglycyscysthrthralaalathrglyalacysala725730735cyscysalathrglyglyalaalaalaalacyscysthrthrthrthr740745750alaglycysthrglycysthrglythrglyglyalathralaglyala755760765alaalathrglyalaglyglycysthrglyalaalaalathralathr770775780cysthrcyscysthrthrcysthralacysalathrthrglycysala785790795800thrglycysthralathrthrglycysthrthrglythralathrthr805810815alathrglyglyalaalaalaalathrglythrglycyscysthrthr820825830thrcysalathrcysalaalacysglyglyalaalaglycyscyscys835840845thrcysalaalaalaalacysalacysthrthrthrthrglythrthr850855860cyscysalaglyglythrcysthrthralathrthrglyalathrthr865870875880thrglyglycyscysalathralaalaalaglyalaglyalaalaala885890895cysalacysthrthrthralaalathrthrglyglythrglyglythr900905910glyalathrglyalacysthrthrth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