CN112899314A - 一种促进重组解脂亚罗酵母菌合成根皮素的方法 - Google Patents
一种促进重组解脂亚罗酵母菌合成根皮素的方法 Download PDFInfo
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Abstract
本发明公开了一种促进重组解脂亚罗酵母菌合成根皮素的方法，所述方法以酪氨酸为原料，重组解脂亚罗酵母菌为宿主菌，经过宿主菌体内若干酶的催化反应获得根皮素。本发明利用解脂亚罗酵母菌细胞内乙酰辅酶A的高积累量，通过引入POX2启动子的有益突变体片段，驱动乙酰辅酶A转化为丙二酰辅酶A的关键基因ACC1和对羟基苯丙酰辅酶A合成关键基因4CL高效过表达，提高合成根皮素的两种重要前体物质丙二酰辅酶A和对羟基苯丙酰辅酶A积累量，以最终提升宿主菌内根皮素产量。本发明通过在微生物体内构建根皮素合成代谢通路，有效避免了大规模的提取、分离过程，环境友好、无污染，符合当今绿色化生产的新需求。
Description
技术领域
本发明属于根皮素的生物合成技术领域，具体涉及一种促进重组解脂亚罗酵母菌合成根皮素的方法。
背景技术
根皮素(Phloretin)是国外新近研究开发出来的一种新型天然皮肤美白剂，主要分布于苹果、梨等多汁水果的果皮及根皮。根皮素为珍珠白结晶粉末，能溶于乙醇和丙酮，几乎不溶于水，其保湿作用非常强，能够促进配方中功能因子的吸收利用，使其发挥出良好功效，可应用于面膜、护肤膏霜、乳液和精华素中。此外，根皮素还具有改善记忆力的功能以及抗癌、抗氧化、抗肿瘤等多种重要的生物活性，在新型药物和天然保健食品开发中具有广泛的应用前景。
目前，现有根皮素的提取方法，主要有酸水解、酶解和直接提取等。在实际生产中，常以柚皮苷为原料，兰尼镍为催化剂，在氢氧化钠溶液中，催化氢化合成柚皮苷二氢查尔酮，再利用盐酸溶液水解柚皮苷二氢查尔酮，得到根皮素。但这种方法存在化学废液排放的问题，会造成一定程度的环境污染。而酶解、直接提取等方法也普遍存在提取效率低、污染严重等多种问题。因此，利用微生物异源生物合成根皮素，条件温和且污染较小，成为了一种理想的合成方法。根皮素的生物合成途径是以对羟基苯丙酰辅酶A和丙二酰辅酶A为前体物质。专利CN107805646A报道的是以苯丙氨酸为原料，大肠杆菌为宿主菌合成根皮素。专利CN103571892A是以柚皮苷或其糖苷配基为原料，大肠杆菌、酿酒酵母或巴斯德毕赤酵母为宿主菌；CN107586795A和CN109913508A均以对羟基苯丙酸为原料，分别以酿酒酵母和蓝藻细胞为宿主菌。以上所述专利，大多仅限于根皮素合成通路的构建，只有CN107586795A，通过在酿酒酵母中整合强化乙醇到丙二酰辅酶A途径的关键基因，以及引入丙二酸转化为丙二酰辅酶A路径，提高了根皮素合成重要前体物质丙二酰辅酶A积累量，从而提升了根皮素产量。
发明内容
本发明的目的是提供一种以解脂亚罗酵母为宿主菌，微生物异源合成根皮素的方法，从满足根皮素生物合成的对羟基苯丙酰辅酶A和丙二酰辅酶A两个前体物质出发，通过提高这两种前体物质的积累量，最终提升宿主菌内根皮素产量，以解决现有提取方法存在的提取效率低、分离纯化步骤繁琐、化学污染严重等问题。
针对上述目的，本发明所采用的技术方案是：以外源酪氨酸为原料，重组解脂亚罗酵母为宿主菌，经过宿主菌体内若干酶的催化反应获得根皮素，其合成路线如下所示：
根皮素生物合成关键基因确定：以外源酪氨酸为底物，酪氨酸在酪氨酸解氨酶(TAL)作用下生成4-香豆酸，4-香豆酸在烯酸还原酶(2-ER)作用下生成对羟基苯丙酸，进而在4-香豆酰辅酶A连接酶(4CL)作用下生成对羟基苯丙酰辅酶A，乙酰辅酶A在乙酰辅酶A羧化酶(ACC1)作用下生成丙二酰辅酶A，然后在查尔酮合成酶(CHS)的催化作用下，1分子的对羟基苯丙酰辅酶A和3分子的丙二酰辅酶A发生作用，最终合成根皮素。
上述重组解脂亚罗酵母菌的构建方法如下：
1、将酪氨酸解氨酶基因TAL、烯酸还原酶基因2-ER、4-香豆酰辅酶A连接酶基因4CL、查尔酮合成酶基因CHS、乙酰辅酶A羧化酶基因ACC1分别连接至载体pJN44，构建含有TAL表达盒的质粒pJN44-TAL、含有2-ER表达盒的质粒pJN44-2-ER、含有4CL表达盒的质粒pJN44-4CL、含有CHS表达盒的质粒pJN44-CHS和含有ACC1表达盒的质粒pJN44-ACC1；
2、将构建的质粒pJN44-2-ER、pJN44-4CL、pJN44-CHS和pJN44-ACC1中含目的基因的表达盒进行酶切，并依次连接至质粒pJN44-TAL，得到重组质粒pJN44-TAL/2-ER/4CL/CHS/ACC1；
3、提取解脂亚罗酵母基因组，通过PCR获得其基因组上整合位点GUT2的上下游同源臂，进而构建载体pURA-GUT2 L&R；将重组质粒pJN44-TAL/2-ER/4CL/CHS/ACC1中含目的基因的表达盒酶切并连接至载体pURA-GUT2 L&R上，得到重组质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1。
4、以POX2启动子基因序列为模板，通过易错PCR反应筛选得到优化的POX2启动子突变体，将POX2启动子突变体片段连接至质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，分别替换掉ACC1和4CL表达盒的原有启动子，然后将所得质粒整合至解脂亚罗酵母菌染色体，得到重组解脂亚罗酵母菌；其中，所述POX2启动子突变体的序列为SEQ ID No.1、SEQ IDNo.2、SEQ ID No.3和SEQ ID No.4中任意一种。
上述步骤4中，优选易错PCR反应体系为：100μL，所含8mmol/L MgCl2、0.6mmol/LMnCl2、50mmol/L KCl、10mol/L Tris-HCl，25℃pH 8.3，6U的Taq DNA聚合酶，dATP、dGTP、dCTP、dTTP浓度分别为0.2、0.2、1和1mmol/L；易错PCR反应条件为：96℃预变性5min，96℃变性2min，58℃退火2min，72℃延伸1min，进行30个循环。
上述步骤4中，利用限制性内切酶BamHI和HindⅢ对质粒pURA-GUT2L&R-TAL/2-ER/4CL/CHS/ACC1上ACC1和4CL表达盒的原有启动子进行双酶切，并在POX2启动子突变体片段两端分别添加BamHI和HindⅢ酶切位点，然后将POX2启动子突变体片段分别连接至质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，替换ACC1和4CL表达盒的原有启动子。
上述步骤4中，优选的将序列为SEQ ID No.1的POX2启动子突变体连接至质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，替换掉ACC1表达盒的原有启动子，将序列为SEQID No.3的POX2启动子突变体连接至质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，替换掉4CL表达盒的原有启动子。
本发明的有益效果如下：
1、本发明通过在微生物体内构建根皮素合成代谢通路，发酵产生了根皮素，有效避免了大规模的提取、分离过程，环境友好、无污染，符合当今绿色化生产的新需求。
2、本发明以解脂亚罗酵母工程菌为宿主菌，利用其细胞内乙酰辅酶A的高积累量，过表达乙酰辅酶A转化为丙二酰辅酶A的关键基因ACC1，以提高宿主菌内丙二酰辅酶A含量。同时，过表达对羟基苯丙酰辅酶A合成关键基因4CL，提高根皮素合成另一重要前体物质对羟基苯丙酰辅酶A积累量，最终提升了宿主菌内根皮素产量。
3、本发明在过表达ACC1和4CL这两个关键基因时，分别引入了POX2启动子的有益突变体片段，以驱动基因高效过表达。
具体实施方式
下面结合实施例对本发明进一步详细说明，但本发明的保护范围不仅限于这些实施例。
实施例1
1、根据目的基因的氨基酸序列和解脂亚罗酵母密码子偏好性，优化出基因的碱基序列，送至商业基因合成公司合成目的基因：酪氨酸解氨酶基因(TAL，Gene ID：54319287)、烯酸还原酶基因(2-ER，Gene ID:44999865)、4-香豆酰辅酶A连接酶基因(4CL，Gene ID:110880015)、查尔酮合成酶基因(CHS，Gene ID:110908971)、乙酰辅酶A羧化酶基因(ACC1，Gene ID:2909424)。合成后，将经密码子优化的酪氨酸解氨酶基因(TAL)和表达载体pJN44(含启动子PTEF和终止子Txpr2)分别用限制性内切酶SmaI在37℃下处理2h，纯化并胶回收目的基因和载体的DNA片段；将回收的两个DNA片段在NEB DNA连接酶(NEB公司，产品编号：M0367S)作用下进行连接反应，得到含TAL表达盒的质粒pJN44-TAL。同理，构建得到含有2-ER表达盒的质粒pJN44-2-ER、含有4CL表达盒的质粒pJN44-4CL、含有CHS表达盒的质粒pJN44-CHS和含有ACC1表达盒的质粒pJN44-ACC1。
2、将含2-ER表达盒的质粒pJN44-2-ER和含TAL表达盒的质粒pJN44-TAL分别用限制性内切酶XbaI/SpeI、XbaI在37℃下处理2h，纯化并胶回收2-ER表达盒和质粒pJN44-TAL的DNA片段；将回收的两个DNA片段在NEB DNA连接酶(NEB公司，产品编号：M0367S)作用下进行连接反应，得到质粒pJN44-TAL/2-ER。同理，将4CL表达盒酶切并连接至质粒pJN44-TAL/2-ER，得到质粒pJN44-TAL/2-ER/4CL，将CHS表达盒酶切并连接至质粒pJN44-TAL/2-ER/4CL，得到质粒pJN44-TAL/2-ER/4CL/CHS，将ACC1表达盒酶切并连接至质粒pJN44-TAL/2-ER/4CL/CHS，最终得到重组质粒pJN44-TAL/2-ER/4CL/CHS/ACC1。按照酵母转化试剂盒说明书(Zymo Research corporation，USA)操作，将重组质粒pJN44-TAL/2-ER/4CL/CHS/ACC1转入解脂亚罗酵母菌进行游离表达，待长出转化子，挑选单克隆，提取质粒进行验证，对验证正确的菌株进行培养，并在培养后的菌液中添加酪氨酸底物，在30℃、200rpm/min下反应120h。采用高效液相色谱(HPLC)进行检测，结果表明，菌液中产生了根皮素，其产量为363.5mg/L。
3、提取解脂亚罗酵母基因组，通过PCR获得其基因组上整合位点GUT2的上下游同源臂，进而构建载体pURA-GUT2 L&R。将步骤2构建的质粒pJN44-TAL/2ER/4CL/CHS/ACC1和载体pURA-GUT2 L&R分别用限制性内切酶XbaI/SpeI、XbaI在37℃下处理2h，纯化并胶回收得到含五个目的基因(TAL、2-ER、4CL、CHS、ACC1)表达盒和载体pURA-GUT2 L&R的DNA片段，将回收的两个DNA片段在NEB DNA连接酶(NEB公司，产品编号：M0367S)作用下进行连接反应，得到重组质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1。
4、首先以POX2启动子基因序列为模板，设计引物POX2-EPf/POX2-EPr(POX2-EPf碱基序列：cgcggatcctttcccttatacttttcccca；POX2-Epr碱基序列：cccaagcttggcgtcgttgc)，经易错PCR得到突变的POX2启动子。易错PCR反应体系为：100μL，含8mmol/L MgCl2、0.6mmol/L MnCl2、50mmol/L KCl、10mol/L Tris-Cl，pH 8.3(25℃)，6U的Taq DNA聚合酶，dATP、dGTP、dCTP、dTTP浓度分别为0.2、0.2、1和1mmol/L。易错PCR反应条件为：96℃预变性5min，96℃变性2min，58℃退火2min，72℃延伸1min，进行30个循环。酶切突变过后的启动子，然后将这些突变的启动子混合片段与经相同酶切后的质粒pJN44连接，获得重组质粒库，并将这些重组质粒按照酵母转化试剂盒说明书(Zymo Research corporation，USA)操作，转化进入解脂亚罗酵母菌，进而获得突变POX2启动子重组解脂亚罗酵母细胞库。转化过后，涂布于相应的营养缺陷型培养基，30℃倒置培养3～4天，直到单克隆出现，采用菌落PCR鉴定转化子，挑选阳性克隆。利用96深孔板，通过高通量筛选方法筛选构建的突变体，并用荧光酶标仪检测重组菌的酵母增强型绿色荧光蛋白yEGFP荧光强度。随机挑取200个突变子进行筛选，检测到荧光强度较对照重组菌(野生型POX2启动子调控yEGFP表达菌株)显著提高的4个重组菌，进而得到4个启动子强度高于野生型POX2启动子的有益突变体POX2-1、POX2-2、POX2-3和POX2-4。为了验证以上重组菌的yEGFP荧光强度改变确实是因为启动子序列的突变引起，将突变POX2启动子序列克隆并测序，POX2-1、POX2-2、POX2-3和POX2-4的基因序列依次见SEQ ID No.1、SEQ ID No.2、SEQ ID No.3和SEQ ID No.4。
利用限制性内切酶BamHI和HindⅢ对质粒pJN44-TAL/2-ER/4CL/CHS/ACC1上ACC1表达盒的原有启动子进行双酶切，并在POX2-1、POX2-2、POX2-3和POX2-4启动子突变体片段两端分别添加BamHI和HindⅢ酶切位点，然后将POX2-1、POX2-2、POX2-3和POX2-4启动子突变体片段分别连接至pJN44-TAL/2-ER/4CL/CHS/ACC1载体，替换ACC1表达盒的原有启动子。再按照酵母转化试剂盒说明书(Zymo Research corporation，USA)操作，分别转化进入解脂亚罗酵母菌进行游离表达，对应得到重组菌，将重组菌进行培养，在培养后的菌液中添加酪氨酸底物，30℃、200rpm/min下反应120h。采用HPLC测定菌液中根皮素的含量，其产量分别可达663.4mg/L、632.8mg/L、612.9mg/L和642.6mg/L，经比较可得，引入突变过后的POX2-1启动子来驱动ACC1基因表达，效果最佳。
同理，将POX2-1、POX2-2、POX2-3和POX2-4启动子突变体片段分别连接至pJN44-TAL/2-ER/4CL/CHS/ACC1载体，替换4CL表达盒的原有启动子，转化得到对应重组菌，经相同条件发酵培养过后，测得其产量分别可达528.9mg/L、566.7mg/L、587.6mg/L、532.7mg/L，经比较可得，引入突变过后的POX2-3启动子来驱动4CL基因表达，效果最佳。
利用限制性内切酶BamHI和HindⅢ对质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1上ACC1表达盒和4CL表达盒的原有启动子进行双酶切，在筛选得到的POX2-1和POX2-3启动子突变体片段两端分别添加BamHI和HindⅢ酶切位点，然后将POX2-1和POX2-3启动子突变体片段连接至pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1载体，分别替换ACC1和4CL表达盒的原有启动子，再按照酵母转化试剂盒说明书(Zymo Research corporation，USA)操作，将该重组质粒转化进入解脂亚罗酵母菌，利用同源重组作用，将含五个目的基因的表达盒整合至解脂亚罗酵母菌染色体，进而得到能够稳定合成根皮素的重组解脂亚罗酵母菌。将所得重组解脂亚罗酵母菌进行培养，在培养后的菌液中添加酪氨酸底物，30℃、200rpm/min下反应120h。经HPLC检测，根皮素最终产量可达783.5mg/L。
实施例2
本实施例中，按照实施例1的方法构建质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，然后利用限制性内切酶BamHI和HindⅢ对质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1上ACC1表达盒和4CL表达盒的原有启动子进行双酶切，在实施例1筛选得到的POX2-1和POX2-2启动子突变体片段两端分别添加BamHI和HindⅢ酶切位点，然后将POX2-1和POX2-2启动子突变体片段连接至pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1载体，分别替换ACC1和4CL表达盒的原有启动子，再按照酵母转化试剂盒说明书(Zymo Research corporation，USA)操作，将该重组质粒转化进入解脂亚罗酵母菌，利用同源重组作用，将含五个目的基因的表达盒整合至解脂亚罗酵母菌染色体，进而得到能够稳定合成根皮素的重组解脂亚罗酵母菌。将所得重组解脂亚罗酵母菌进行培养，在培养后的菌液中添加酪氨酸底物，30℃、200rpm/min下反应120h。经HPLC检测，根皮素最终产量可达763.1mg/L。
实施例3
本实施例中，按照实施例1的方法构建质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，然后利用限制性内切酶BamHI和HindⅢ对质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1上ACC1表达盒和4CL表达盒的原有启动子进行双酶切，在实施例1筛选得到的POX2-4和POX2-3启动子突变体片段两端分别添加BamHI和HindⅢ酶切位点，然后将POX2-4和POX2-3启动子突变体片段连接至pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1载体，分别替换ACC1和4CL表达盒的原有启动子，再按照酵母转化试剂盒说明书(Zymo Research corporation，USA)操作，将该重组质粒转化进入解脂亚罗酵母菌，利用同源重组作用，将含五个目的基因的表达盒整合至解脂亚罗酵母菌染色体，进而得到能够稳定合成根皮素的重组解脂亚罗酵母菌。将所得重组解脂亚罗酵母菌进行培养，在培养后的菌液中添加酪氨酸底物，30℃、200rpm/min下反应120h。经HPLC检测，根皮素最终产量可达754.2mg/L。
实施例4
本实施例中，按照实施例1的方法构建质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，然后利用限制性内切酶BamHI和HindⅢ对质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1上ACC1表达盒和4CL表达盒的原有启动子进行双酶切，在实施例1筛选得到的POX2-4和POX2-2启动子突变体片段两端分别添加BamHI和HindⅢ酶切位点，然后将POX2-4和POX2-2启动子突变体片段连接至pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1载体，分别替换ACC1和4CL表达盒的原有启动子，再按照酵母转化试剂盒说明书(Zymo Research corporation，USA)操作，将该重组质粒转化进入解脂亚罗酵母菌，利用同源重组作用，将含五个目的基因的表达盒整合至解脂亚罗酵母菌染色体，进而得到能够稳定合成根皮素的重组解脂亚罗酵母菌。将所得重组解脂亚罗酵母菌进行培养，在培养后的菌液中添加酪氨酸底物，30℃、200rpm/min下反应120h。经HPLC检测，根皮素最终产量可达739.5mg/L。
序列表
<110> 陕西师范大学
<120> 一种促进重组解脂亚罗酵母菌合成根皮素的方法
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ccagagtata cttatatacc aaagggatgg gtcctcagga atcacacaag caacgacgcc 300
Claims (4)
1.一种促进重组解脂亚罗酵母菌合成根皮素的方法，其特征在于：所述方法是以酪氨酸为原料，重组解脂亚罗酵母菌为宿主菌，经过宿主菌体内若干酶的催化反应获得根皮素；
上述重组解脂亚罗酵母菌的构建方法如下：
(1)将酪氨酸解氨酶基因TAL、烯酸还原酶基因2-ER、4-香豆酰辅酶A连接酶基因4CL、查尔酮合成酶基因CHS、乙酰辅酶A羧化酶基因ACC1分别连接至载体pJN44，构建含有TAL表达盒的质粒pJN44-TAL、含有2-ER表达盒的质粒pJN44-2-ER、含有4CL表达盒的质粒pJN44-4CL、含有CHS表达盒的质粒pJN44-CHS和含有ACC1表达盒的质粒pJN44-ACC1；
(2)将构建的质粒pJN44-2-ER、pJN44-4CL、pJN44-CHS和pJN44-ACC1中含目的基因的表达盒进行酶切，并依次连接至质粒pJN44-TAL，得到重组质粒pJN44-TAL/2-ER/4CL/CHS/ACC1；
(3)提取解脂亚罗酵母基因组，通过PCR获得其基因组上整合位点GUT2的上下游同源臂，进而构建载体pURA-GUT2 L&R；将重组质粒pJN44-TAL/2-ER/4CL/CHS/ACC1中含目的基因的表达盒酶切并连接至载体pURA-GUT2 L&R上，得到重组质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1；
(4)以POX2启动子基因序列为模板，通过易错PCR反应筛选得到优化的POX2启动子突变体，将POX2启动子突变体片段连接至质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，分别替换掉ACC1和4CL表达盒的原有启动子，然后将所得质粒整合至解脂亚罗酵母菌染色体，得到重组解脂亚罗酵母菌；
上述POX2启动子突变体的序列为SEQ ID No.1、SEQ ID No.2、SEQ ID No.3和SEQ IDNo.4中任意一种。
2.根据权利要求1所述的促进重组解脂亚罗酵母菌合成根皮素的方法，其特征在于：步骤(4)中，易错PCR反应体系为：100μL，所含8mmol/L MgCl2、0.6mmol/L MnCl2、50mmol/LKCl、10mol/L Tris-HCl，25℃pH 8.3，6U的Taq DNA聚合酶，dATP、dGTP、dCTP、dTTP浓度分别为0.2、0.2、1和1mmol/L；易错PCR反应条件为：96℃预变性5min，96℃变性2min，58℃退火2min，72℃延伸1min，进行30个循环。
3.根据权利要求1所述的促进重组解脂亚罗酵母菌合成根皮素的方法，其特征在于：步骤(4)中，利用限制性内切酶BamHI和HindⅢ对质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1上ACC1和4CL表达盒的原有启动子进行双酶切，并在POX2启动子突变体片段两端分别添加BamHI和HindⅢ酶切位点，然后将POX2启动子突变体片段分别连接至质粒pURA-GUT2L&R-TAL/2-ER/4CL/CHS/ACC1，替换ACC1和4CL表达盒的原有启动子。
4.根据权利要求1所述的促进重组解脂亚罗酵母菌合成根皮素的方法，其特征在于：步骤(4)中，将序列为SEQ ID No.1的POX2启动子突变体连接至质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，替换掉ACC1表达盒的原有启动子，将序列为SEQ ID No.3的POX2启动子突变体连接至质粒pURA-GUT2 L&R-TAL/2-ER/4CL/CHS/ACC1，替换掉4CL表达盒的原有启动子。
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748532A (zh) * | 2020-05-25 | 2020-10-09 | 天津大学 | 新的p-香豆酰-CoA连接酶在生物合成根皮素中的应用 |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103484420A (zh) * | 2013-10-15 | 2014-01-01 | 江南大学 | 一株以酪氨酸为底物合成柚皮素的基因工程菌及其构建方法 |
| DE102012213492A1 (de) * | 2012-07-31 | 2014-02-06 | Symrise Ag | Verfahren zur biotechnologischen Herstellung von Dihydrochalkonen |
| CN107586795A (zh) * | 2017-10-10 | 2018-01-16 | 嘉兴欣贝莱生物科技有限公司 | 一种酿酒酵母发酵生产根皮素的方法 |
| US20180155733A1 (en) * | 2015-05-12 | 2018-06-07 | Evolva Sa | A method for producing resveratrol |
| CN108138151A (zh) * | 2015-06-05 | 2018-06-08 | 埃沃尔瓦公司 | 苯丙素类和二氢苯丙素类衍生物的生物合成 |
| CN109913508A (zh) * | 2018-06-05 | 2019-06-21 | 嘉兴欣贝莱生物科技有限公司 | 一种利用蓝藻合成根皮素的方法 |
| CN110117550A (zh) * | 2019-01-09 | 2019-08-13 | 嘉兴欣贝莱生物科技有限公司 | 基于酿酒酵母发酵生产根皮素的工艺及酿酒酵母 |
-
2021
- 2021-02-04 CN CN202110157119.XA patent/CN112899314B/zh not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102012213492A1 (de) * | 2012-07-31 | 2014-02-06 | Symrise Ag | Verfahren zur biotechnologischen Herstellung von Dihydrochalkonen |
| CN103484420A (zh) * | 2013-10-15 | 2014-01-01 | 江南大学 | 一株以酪氨酸为底物合成柚皮素的基因工程菌及其构建方法 |
| US20180155733A1 (en) * | 2015-05-12 | 2018-06-07 | Evolva Sa | A method for producing resveratrol |
| CN108138151A (zh) * | 2015-06-05 | 2018-06-08 | 埃沃尔瓦公司 | 苯丙素类和二氢苯丙素类衍生物的生物合成 |
| CN107586795A (zh) * | 2017-10-10 | 2018-01-16 | 嘉兴欣贝莱生物科技有限公司 | 一种酿酒酵母发酵生产根皮素的方法 |
| CN109913508A (zh) * | 2018-06-05 | 2019-06-21 | 嘉兴欣贝莱生物科技有限公司 | 一种利用蓝藻合成根皮素的方法 |
| CN110117550A (zh) * | 2019-01-09 | 2019-08-13 | 嘉兴欣贝莱生物科技有限公司 | 基于酿酒酵母发酵生产根皮素的工艺及酿酒酵母 |
Non-Patent Citations (3)
| Title |
|---|
| CHUNMEI JIANG ET AL.: ""Raising the production of phloretin by alleviation of by-product of chalcone synthase in the engineered yeast"", 《SCI CHINA LIFE SCI》 * |
| MICHAEL EICHENBERGER ET AL.: ""Metabolic engineering of Saccharomyces cerevisiae for de novo production of dihydrochalcones with known antioxidant, antidiabetic, and sweet tasting properties"", 《METABOLIC ENGINEERING 》 * |
| 刘金丛 等: ""微生物合成根皮素及其糖苷研究进展"", 《中国生物工程杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111748532A (zh) * | 2020-05-25 | 2020-10-09 | 天津大学 | 新的p-香豆酰-CoA连接酶在生物合成根皮素中的应用 |
| CN111748532B (zh) * | 2020-05-25 | 2022-04-15 | 天津大学 | 新的p-香豆酰-CoA连接酶在生物合成根皮素中的应用 |
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