CN116622532A - 合成阿魏酸的酵母菌株和构建方法及其在制备阿魏酸及胡椒代谢物中的应用 - Google Patents
合成阿魏酸的酵母菌株和构建方法及其在制备阿魏酸及胡椒代谢物中的应用 Download PDFInfo
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Abstract
本发明涉及合成阿魏酸的酵母菌株和构建方法及其在制备阿魏酸及胡椒代谢物中的应用。该菌株以酿酒酵母为底盘菌株,并过表达如下基因:PahpaB、SehpaC和AtCOMT。在酿酒酵母体内整合咖啡酸合成关键酶基因(PahpaB和SehpaC)和阿魏酸合成关键酶基因(AtCOMT),获得阿魏酸产量较高的重组菌株。本研究构建全新的阿魏酸合成重组酵母细胞,通过代谢改造提高了阿魏酸生物合成产量。借助该酿酒酵母细胞工厂挖掘了阿魏酸衍生的胡椒次生代谢产物合成的关键酶基因(PnPKS,PnOMT,PnNAT)功能,构建了能够生物合成具有药理活性产物的工程菌株,同时提供了类似化合物生物法生产的方法与技术。
Description
技术领域
本发明属于合成生物学技术领域,涉及合成阿魏酸的酵母菌株和构建方法及其在制备阿魏酸及胡椒代谢物中的应用,所述酵母工程菌株具有阿魏酸生物合成途径的酵母菌株。尤其涉及高效合成阿魏酸的酿酒酵母菌株的制备,以及衍生的胡椒次生代谢产物合成酶功能挖掘与提供重组酵母细胞中异源合成类似化合物的方法。首次公开了阿魏酸衍生的胡椒次生代谢产物生物合成关键酶基因-氮酰基转移酶(PnNAT)、聚酮合成酶(PnPKS)、氧甲基转移酶(PnOMT)三类酶基因的功能和序列。
背景技术
阿魏酸,又称为3-甲氧基-4-羟基肉桂酸是一种酚酸类有机化合物,是由苯丙烷类合成途径的重要植物次生代谢产物。阿魏酸具有较强的抗氧化、抗菌和抗病毒生物活性。阿魏酸常见于植物细胞壁,与木质素和多糖交联形成复合物来支撑细胞的形状。传统方法获得阿魏酸需要在碱性溶液中阿魏酸酯酶和木聚糖酶分解细胞壁,或者利用超声波处理破解细胞壁成分。此类方法受限于产率低、耗能高、污染严重等工业生产上的实际问题。因此通过微生物发酵合成不需要繁琐的分离纯化工艺,既环保又能廉价的成本获得大量阿魏酸纯品。同时,以高产阿魏酸酵母菌株为宿主细胞,挖掘阿魏酸衍生的代谢产物生物合成途径中关键酶,构建新颖化合物的生物合成重组酵母菌株。
发明内容
基于此,有必要提供一种高产阿魏酸的重组酵母菌株构建方法,在制备高产阿魏酸及阿魏酸衍生的胡椒代谢物生产中的应用。
一种重组酵母菌株,以酿酒酵母为底盘菌株,并过表达如下基因:PahpaB、SehpaC和AtCOMT。
研究发现,在酿酒酵母体内整合咖啡酸合成关键酶基因(PahpaB和SehpaC)和阿魏酸合成关键酶基因(AtCOMT),能够获得阿魏酸产量较高的重组酵母菌株。经试验验证,过表达了PahpaB、SehpaC和AtCOMT的重组酵母菌株的咖啡酸和阿魏酸的产量分别为203.6mg/L和108.1mg/L。
其中一个实施例中,所述酿酒酵母的基因型为:MATa ura3-52can1Δ::CAS9-natNT2 TRP1 LEU2 HIS3 gpp1ΔXII-2::(GPM1p-AtPAL2-FBA1t)+(TDH3p-AtC4H-CYC1t)+(tHXT7p-AtATR2-pYX212t)+(PGK1p-CYB5-ADH1t)X-3::(TEF1p-ARO4-CYC1t)XII-5::(ADH1t-Bbxfpk-TDH3p)+(tHXT7p-Ckpta-CYC1t)XII-1::(TEF1p-SehpaC-FBA1t)+(TPS1t-PahpaB-CCW12p)+(TDH3p-AtCOMT-CYC1t)。
其中一个实施例中,进一步敲除了如下基因中的至少一种:aro8、aro9、aro10、pdc5和aro80。
其中一个实施例中,进一步敲除aro9基因且过表达aro8基因。
其中一个实施例中,aro8基因与Pgk1p启动子及Tps1t终止子融合后过表达于aro9基因的敲除位点上。
其中一个实施例中,进一步敲除了如下基因中的至少一种:pad1和fdc1。
其中一个实施例中,进一步过表达如下基因中的至少一种:Sam1、Sam2、Sah1和Met6。
其中一个实施例中,进一步敲除了pdc5基因和aro10基因。
其中一个实施例中,进一步过表达AtCOMT基因。
其中一个实施例中,AtCOMT基因过表达于pdc5基因的敲除位点和aro10基因的敲除位点中的至少一个上;及/或,AtCOMT基因为多拷贝。
其中一个实施例中,进一步敲除了bna7基因。
其中一个实施例中,进一步过表达AtCOMT基因,AtCOMT基因过表达于bna7基因的敲除位点上;进一步地,AtCOMT基因为多拷贝。
其中一个实施例中,进一步过表达Pn4CL3基因,且过表达PnNAT4基因和PnNAT5基因中的一种。
其中一个实施例中,PnNAT4基因的核苷酸序列包括如下序列中的一种:如SEQ IDNo.5所示的序列,或与如SEQ ID No.5所示的序列具有至少70%同源性的序列。
其中一个实施例中,PnNAT5基因的核苷酸序列包括如下序列中的一种:如SEQ IDNo.6所示的序列,或与如SEQ ID No.6所示的序列具有至少70%同源性的序列。
其中一个实施例中,进一步过表达Pn4CL3基因,且过表达PnPKS1基因和PnPKS2基因中的一种。
其中一个实施例中,PnPKS1基因的核苷酸序列包括如下序列中的一种:如SEQ IDNo.1所示的序列,或与如SEQ ID No.1所示的序列具有至少70%同源性的序列。
其中一个实施例中,PnPKS2基因的核苷酸序列包括如下序列中的一种:如SEQ IDNo.2所示的序列,或与如SEQ ID No.2所示的序列具有至少70%同源性的序列。
其中一个实施例中,进一步过表达PnOMT1基因和PnOMT2基因中的一种。
其中一个实施例中,PnOMT1基因的核苷酸序列包括如下序列中的一种:如SEQ IDNo.3所示的序列,或与如SEQ ID No.3所示的序列具有至少70%同源性的序列。
其中一个实施例中,PnOMT2基因的核苷酸序列包括如下序列中的一种:如SEQ IDNo.4所示的序列,或与如SEQ ID No.4所示的序列具有至少70%同源性的序列。
一种重组酵母菌株的构建方法,包括如下步骤:以酿酒酵母为底盘菌株,过表达所述酿酒酵母的如下基因:PahpaB、SehpaC和AtCOMT。
上述重组酵母菌株在制备阿魏酸、阿魏酸的衍生物中的应用。
上述重组酵母菌株在制备胡椒生物碱类代谢产物中的应用。
在其中一个实施例中,所述胡椒生物碱类代谢产物包括阿魏酰哌啶。
上述重组酵母菌株在11-甲氧基-去甲甲氧基醉椒素生物合成中的应用。
上述重组酵母菌株在11-甲氧基-12羟基-脱氢卡瓦因生物合成中的应用。
附图说明
图1为重组酵母菌株构建示意图;
图2为实施例1中重组酿酒酵母菌株阿魏酸合成途径示意图与产量统计结果图;
图3为实施例2中重组酿酒酵母菌株优化苯丙烷代谢途径示意图与产量统计结果图;
图4为实施例3中重组酿酒酵母菌株阿魏酸合成SAM辅因子循环示意图与产量统计结果图;
图5为实施例4中重组酿酒酵母菌株过表达多拷贝AtCOMT基因产量统计结果图;
图6为实施例5中胡椒代谢产物合成路径示意图与阿魏哌啶合成酶PnNAT功能验证结果图;
图7为实施例6中胡椒内酯合成酶PnPKS与PnOMT功能验证结果图。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例及附图对本发明的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本发明。但是本发明能够以很多不同于在此描述的其它方式来实施,本领域技术人员可以在不违背本发明内涵的情况下做类似改进,因此本发明不受下面公开的具体实施的限制。
本申请实施方式提供一种重组酵母菌株,以酿酒酵母为底盘菌株,并过表达如下基因:PahpaB(GenBank登录号:PKG21040)、SehpaC(GenBank登录号:GAR62209)和AtCOMT(GenBank登录号:AT5G54160)。PahpaB、SehpaC为咖啡酸合成关键酶基因,AtCOMT为阿魏酸合成关键酶基因。研究发现,在酿酒酵母体内整合咖啡酸合成关键酶基因(PahpaB和SehpaC)和阿魏酸合成关键酶基因(AtCOMT),能够获得阿魏酸产量较高的重组酵母菌株。经试验验证,过表达了PahpaB、SehpaC和AtCOMT的重组酵母菌株的咖啡酸和阿魏酸的产量分别为203.6mg/L和108.1mg/L。
其中,酿酒酵母的基因型如下:
MATa ura3-52 can1Δ::CAS9-natNT2 TRP1 LEU2 HIS3 gpp1ΔXII-2::(GPM1p-AtPAL2-FBA1t)+(TDH3p-AtC4H-CYC1t)+(tHXT7p-AtATR2-pYX212t)+(PGK1p-CYB5-ADH1t)X-3::(TEF1p-ARO4-CYC1t)XII-5::(ADH1t-Bbxfpk-TDH3p)+(tHXT7p-Ckpta-CYC1t)XII-1::(TEF1p-SehpaC-FBA1t)+(TPS1t-PahpaB-CCW12p)+(TDH3p-AtCOMT-CYC1t)。
其中一些实施例中,进一步敲除了如下基因中的至少一种:aro8(GenBank登录号:YGL202W)、aro9(GenBank登录号:YHR137W)、aro10(GenBank登录号:YDR380W)、pdc5(GenBank登录号:YLR134W)和aro80(GenBank登录号:YDR421W)。敲除了酿酒酵母内源的阿魏酸合成阻遏相关的酶aro8、aro9、aro10、pdc5和aro80基因中的至少一个有利于提高阿魏酸的产量,减少副产物的合成。
aro8、aro9为苯丙酮酸与苯丙烷可逆反应的合成酶基因。敲除aro8、aro9基因有利于提高阿魏酸的产量。敲除苯丙酮酸起始的分子途径上aro10和pdc5,有利于提高阿魏酸的产量。被苯丙氨酸、酪氨酸和色氨酸芳香类氨基酸反馈抑制的转录调控因子aro80的敲除,有利于提高阿魏酸的产量。经试验验证,以20g/L葡萄糖为碳源进行发酵,在aro80敲除的重组酿酒酵母合成阿魏酸的产量为147.8mg/L,咖啡酸的产量为232.6mg/L,检测出副产物2-苯乙醇(2-phenylethanol,2PE)比敲除aro80前减少约221.9mg/L。
其中一个实施例中,进一步敲除aro9基因且过表达aro8基因。通过敲除aro9的同时过表达酵母内源的aro8,更有利于合成苯丙氨酸。进一步地,aro8基因与Pgk1p启动子及Tps1t终止子融合后过表达于aro9基因的敲除位点上。
在其中一些实施例中,进一步敲除了如下基因中的至少一种:pad1和fdc1。pad1和fdc1为分解阿魏酸的酶基因。敲除这两种基因中的至少一种能够抑制阿魏酸的分解,提高阿魏酸的产量。进一步过表达如下基因中的至少一种:Sam1(GenBank登录号:YLR180W)、Sam2(GenBank登录号:YDR502C)、Sah1(GenBank登录号:YER043C)和Met6(GenBank登录号:YER091C)。Sam1、Sam2、Sah1和Met6为阿魏酸合成的必要辅因子SAM合成循环途径上酶基因,过表达上述基因有利于提高阿魏酸的合成。
在其中一些实施例中,进一步敲除了pdc5基因和aro10基因。pdc5基因和aro10基因为苯丙氨酸合成分支途径中的酶基因,敲除该两种基因有利于提高阿魏酸的产量。进一步过表达AtCOMT基因。此设置有利于提高阿魏酸的产量。更进一步地,AtCOMT基因过表达于pdc5基因的敲除位点和aro10基因的敲除位点中的至少一个上。AtCOMT基因为多拷贝。在一个具体示例中,2个拷贝AtCOMT基因分别过表达于pdc5基因的敲除位点和aro10基因的敲除位点上。
在其中一些实施例中,进一步敲除了bna7基因。bna7基因为受高浓度阿魏酸负反馈调控,敲除该基因有利于提高阿魏酸的产量。进一步多拷贝过表达AtCOMT基因,AtCOMT基因过表达于bna7基因的敲除位点上。在一个具体示例中,2个拷贝AtCOMT基因过表达于bna7基因的敲除位点上。
上述重组酵母菌株在制备阿魏酸中的应用。
在其中一些实施例中,进一步过表达Pn4CL3(GenBank登录号:MH078050)基因,且过表达PnNAT4基因和PnNAT5基因中的一种。Pn4CL3基因为阿魏酰辅酶A合成所需酶基因。PnNAT4基因和PnNAT5基因为胡椒的氮酰基转移酶基因。进一步过表达Pn4CL3基因,且过表达PnNAT4基因和PnNAT5基因中的一种,有利于胡椒生物碱类代谢产物的合成。其中,胡椒生物碱类代谢产物例如为阿魏酰哌啶。该重组酵母菌株能够应用于制备胡椒生物碱类代谢产物。
其中,PnNAT4基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.5所示的序列,或与如SEQ ID No.5所示的序列具有至少70%同源性的序列。
其中,PnNAT5基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.6所示的序列,或与如SEQ ID No.6所示的序列具有至少70%同源性的序列。
在其中一些实施例中,进一步过表达辅酶A连接酶Pn4CL3基因,且过表达PnPKS1基因和PnPKS2基因中的一种。PnPKS1和PnPKS2基因为胡椒来源聚酮合成酶,是胡椒内酯合成酶基因。进一步过表达Pn4CL3基因,且过表达PnPKS1基因和PnPKS2基因中的一种有利于合成得11-甲氧基-去甲甲氧基醉椒素。该重组酵母菌株能够应用于制备11-甲氧基-去甲甲氧基醉椒素。
其中,PnPKS1基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.1所示的序列,或与如SEQ ID No.1所示的序列具有至少70%同源性的序列。
其中,PnPKS2基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.2所示的序列,或与如SEQ ID No.2所示的序列具有至少70%同源性的序列。
在其中一些实施例中,进一步过表达PnOMT1基因和PnOMT2基因中的一种。PnOMT1基因和PnOMT2基因为合成11-甲氧基-去甲甲氧基醉椒素酵母菌株内整合胡椒中克隆的一种氧甲基转移酶。进一步过表达PnOMT1基因和PnOMT2基因中的一种有利于11-甲氧基-12羟基-脱氢卡瓦因的生物合成。该重组酵母菌株能够应用于制备11-甲氧基-12羟基-脱氢卡瓦因。
其中,PnOMT1基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.3所示的序列,或与如SEQ ID No.3所示的序列具有至少70%同源性的序列。
其中,PnOMT2基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.4所示的序列,或与如SEQ ID No.4所示的序列具有至少70%同源性的序列。
本研究一实施方式还提供上述重组酵母菌株的构建方法,包括如下步骤:以酿酒酵母为底盘菌株,过表达酿酒酵母的如下基因:PahpaB、SehpaC和AtCOMT。
重组酵母菌株的具体描述详见上文,此处不再赘述。
其中,重组酵母菌株的构建方法采用CRISPR/Cas9方法,参照文献(MANS,Robert,et al.CRISPR/Cas9:a molecular Swiss army knife for simultaneous introductionof multiple genetic modifications in Saccharomyces cerevisiae.FEMS YeastResearch,2015,15.1)。酿酒酵母转化方法参照文献(GIETZ,R.Daniel;SCHIESTL,RobertH.High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEGmethod.Nature protocols,2007,2.1:31-34.)。
在其中一个实施例中,首先整合咖啡酸和阿魏酸生物合成酶PahpaB,SehpaC和AtCOMT,获得一株阿魏酸生物合成功能的重组酿酒酵母菌株,其阿魏酸合成产量达到108.1mg/L和203.6mg/L的咖啡酸。其次敲除苯丙酮酸与苯丙烷可逆反应的合成酶基因aro8和aro9,aro9敲除同时过表达酵母内源的Aro8(该酶更趋向于合成苯丙氨酸)。敲除苯丙酮酸起始的分子途径上aro10和pdc5和转录调控因子aro80。以20g/L葡萄糖为碳源进行发酵,在aro80敲除的重组酿酒酵母合成阿魏酸产量达147.8mg/L和232.6mg/L咖啡酸,检测出副产物2-苯乙醇(2-phenylethanol,2PE)比敲除aro80前减少约221.9mg/L。
需要说明的是,本研究所采用的敲除手段,不限于为CRISPR/CAS9方法,也可以是其他方法,例如RNA干扰、酶活性降低、低强度启动子置换或敲除等能达到相同效果的其他技术。
本研究通过在酿酒酵母体内整合咖啡酸合成关键酶基因(PahpaB和SehpaC)和阿魏酸合成关键酶基因(AtCOMT),能够获得阿魏酸产量较高的重组酵母菌株。经试验验证,过表达了PahpaB、SehpaC和AtCOMT的重组酵母菌株的咖啡酸和阿魏酸的产量分别为203.6mg/L和108.1mg/L。
本研究构建全新的阿魏酸合成重组酵母细胞,通过代谢改造提高了阿魏酸生物合成产量。借助该工程酵母菌株挖掘了阿魏酸衍生的胡椒次生代谢产物合成的关键酶基因功能,构建了能够合成具有药理活性产物的工程菌株,同时提供了此类化合物工程化生产的方法与技术。
以下为具体实施例部分。
实施例中采用试剂和仪器如非特别说明,均为本领域常规选择。实施例中未注明具体条件的实验方法,通常按照常规条件,例如文献、书本中所述的条件或者试剂盒生产厂家推荐的方法实现。实施例中所使用的试剂均为市售。
如无特别说明:
本申请实施例以酿酒酵母为例来详细说明重组酿酒酵母生物合成阿魏酸与及其衍生产物的合成方法,所用酿酒酵母菌株信息为:MATa ura3-52can1Δ::CAS9-natNT2TRP1 LEU2 HIS3 gpp1ΔXII-2::(GPM1p-AtPAL2-FBA1t)+(TDH3p-AtC4H-CYC1t)+(tHXT7p-AtATR2-pYX212t)+(PGK1p-CYB5-ADH1t)X-3::(TEF1p-ARO4-CYC1t)XII-5::(ADH1t-Bbxfpk-TDH3p)+(tHXT7p-Ckpta-CYC1t)。参考文献为(LIU,Quanli,et al.Rewiringcarbon metabolism in yeast for high level production of aromaticchemicals.Nature communications,2019,10.1:1-13)。
本申请实施例所构建菌株均采用CRISPR/CAS9方法,参照文献(MANS,Robert,etal.CRISPR/Cas9:a molecular Swiss army knife for simultaneous introduction ofmultiple genetic modifications in Saccharomyces cerevisiae.FEMS YeastResearch,2015,15.1)。酿酒酵母转化方法参照文献(GIETZ,R.Daniel;SCHIESTL,RobertH.High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEGmethod.Nature protocols,2007,2.1:31-34.)。
以下实施例的重组酵母菌株的构建示意图如图1所示。
本申请所涉及的序列信息如下表4所示。
表4本申请所涉及的序列信息
实施例1阿魏酸生物合成的重组酿酒酵母菌株的制备
构建阿魏酸合成菌株,具体步骤如下:
按照参考文献(MIKKELSEN,Michael Dalgaard,et al.Microbial production ofindolylglucosinolate through engineering of a multi-gene pathway in aversatile yeast expression platform.Metabolic engineering,2012,14.2:104-111.)中叙述的方法设计酵母XII-1位点切割的gRNA引物(XII-1sgRNA F&R,5’-gaaagataaatgatcggtatgt gcagttgattcacgttttagagctagaaatagcaagt-3’,下划线表示在酵母基因组中点特异性识别位点),该引物用于扩增2μm片段。反应体系如下表1:
表1扩增2μm片段的反应体系
以引物CRISPR plasmid back F&R(5’-gatcatttatctttcactgcggagaag-3’)扩增质粒骨架。反应体系如下表2:
表2扩增质粒骨架的反应体系
将2μm片段和质粒骨架通过Gibson Assembly方法组装构建本申请中指定的指定位点切割质粒。以aro8基因敲除为实例,酿酒酵母基因组为模板,引物Aro8up F(5’-ccggttcatacactatttttccaag-3’)和引物Aro8 up-(aro8dw)R(5’-tgtatttacctctctatttggatagtaacgatcggttgtcctattc-3’)扩增aro8敲除修复上游片段;引物Aro8 dw-(aro8up)F(5’-gacaaccgatcgttactatccaaatagagaggtaaatacattggaag-3’)和引物Aro8 dw R(5’-ggaagagagaagattagcgtttaaag-3’)扩增aro8敲除修复下游片段;获得片段后,以引物Aro8up F和引物Aro8 dw R进行融合PCR,获得修复片段Aro8up-dw。将获得异源合成途径整合或者修复片段和精准切割质粒转入酿酒酵母细胞。
具体步骤如下:挑取新鲜酵母克隆至1mL YPD培养基(Yeast Extract,Peptone,Dextrose),培养过夜,取适量菌液转接至20mL YPD中,使起始OD600=0.1,30℃,200rpm培养至OD600=0.6,3000g离心去除培养基,用1mL无菌水重悬细胞沉淀,3000g离心去除上清后加入1mL 0.1M醋酸锂重悬,3000g离心去除上清,加入200μL 0.1M醋酸锂制得酿酒细胞感受态细胞,将获得酵母感受态通过醋酸锂/聚乙二醇转化法进行重组酿酒酵母构造,转化体系如下表3:
表3重组酿酒酵母的转化体系
聚乙二醇3500(50%w/v) | 120μL |
醋酸锂(1.0M) | 18μL |
鲑鱼精DNA(2.0mg/mL) | 25μL |
修复片段 | 1~2μg |
敲除质粒 | 1~2μg |
总体积(加水) | 至180μL |
为了评价改造菌株合成阿魏酸的能力,分别将出发菌株和改造菌株摇瓶发酵。具体步骤如下:挑取新鲜酵母单克隆至1mL DelftD培养基(酵母无机盐培养基以2%Dextrose为碳源)中(参考文献见VERDUYN C,Postma E,Scheffers W A,etal.Effect of benzoicacid on metabolic fluxes in yeasts:a continuous-culture study on theregulation of respiration and alcoholic fermentation.Yeast,1992,8.7:501-517),培养过夜,取适量菌液转接至20mL DelftD,使起始OD600=0.2,30℃,200rpm培养,发酵4天后取0.5mL的发酵液并添加等量的100%乙醇,在振荡器中2000rpm充分振荡混合10min,4℃,12000rpm离心10min,取上清经0.22μm滤膜过滤,进行液相质谱分析。
液相质谱分析方法如下:使用Phenomenex Kinetex C18 column(100×2.1mm,粒径为2.6μm)色谱柱,柱温30℃,进样量5μL。流动相A含有0.1%甲酸的去离子水,B相是含有0.1%甲酸的乙腈,流速为0.2ml/min。质谱仪电喷雾电压为3.0kV,载气为N2(纯度大于99%)流速为120L/h,干燥气温度为400℃。采用ESI负离子(-)模式检测目标产物(咖啡酸,[M-H]-m/z 179;阿魏酸,[M-H]-m/z 193)。
如图2所示,过表达了PahpaB,SehpaC和AtCOMT的Fe01菌株分别检测出咖啡酸和阿魏酸,含量分别为203.6mg/L和108.1mg/L。
实施例2阿魏酸合成途径优化方法提高产量的重组酿酒酵母菌株的制备
在网站(http://yeastriction.tnw.tudelft.nl/#!/)设计aro8,aro9,aro80,aro10和pdc5敲除gRNA引物,获得引物用于扩增2μm片段,参照实施例1构建精准切割质粒。以酿酒酵母基因组为模板,分别构建aro8,aro9,aro80,aro10和pdc5的修复片段;过表达Aro8是以酿酒酵母内源基因与Pgk1p酿酒酵母启动子,Tps1t酿酒酵母终止子融合后过表达于aro9敲除位点。参考实施案例1,以Fe01重组酵母菌株为出发菌株,分别构建获得重组酿酒酵母菌株Fe02、Fe03、Fe04、Fe05和Fe06。菌株基因型如图1所示;对获得酿酒酵母菌株进行发酵分析,结果如图3所示,在敲除转录调控因子aro80基因的酿酒酵母阿魏酸明显提升,产量达到148.8mg/L的同时副产物有显著减少。aro10和pdc5的敲除显著减少副产物的生成,后续改造中叠加敲除该位点。
实施例3优化重组酿酒酵母的SAM辅因子供给提高阿魏酸产量的方法
将实施例2的Fe04重组酵母菌株为起始,按照实施例2中精准切割质粒制备方法,构建了分解阿魏酸的pad1(GenBank登录号:YDR538W)和fdc1(GenBank登录号:YDR539W)的敲除质粒。Pad1和fdc1基因是相邻的两个基因,可以使用一个修复片段和整合片段。在该位点分别构建了敲除和过表达SAM辅因子循环途径基因Sam1,Sam2,Sah1和Met6。构建的重组酵母菌株Fe07、Fe08、Fe09、Fe10和Fe11按实施例1的酿酒酵母产量评价方法,检测出阿魏酸的产量第一次超越了咖啡酸的产量达到了267.0mg/L。
实施例4重组酵母菌株内过表达多拷贝AtCOMT提高阿魏酸产量的方法
实施例2中pdc5和aro10的敲除也有显著减少副产物的生成,因此在此位点敲除的同时过表达多拷贝的AtCOMT。在实施例3中构建的Fe10菌株的基础上过分别表达了1个拷贝和2个拷贝的AtCOMT。重组酵母菌株Fe18、Fe19、Fe20和Fe21产物合成能力评价结果显示,2个拷贝的AtCOMT有显著提高阿魏酸的产量,其达到390.6mg/L。
紧接着敲除感应阿魏酸含量起负调控作用的基因bna7(GenBank登录号:YDR428C),敲除同时按实施例4的方法过表达1个和2个拷贝的AtCOMT,构建了Fe22、Fe23和Fe24菌株。构建菌株评价结果显示有显著提高阿魏酸产量的效果。重组酿酒酵母改造菌株的产量达到了533.1mg/L。
实施例5利用高产阿魏酸的重组酵母菌株挖掘未知功能基因的方法
如图6中模式图所示,利用阿魏酸高产重组酵母菌株Fe21为起始过表达Pn4CL3和PnNAT4(SEQ ID No.5所示)和PnNAT5(SEQ ID No.6所示)基因。验证出PnNAT4和PnNAT5可以合成出阿魏酰哌啶,但PnNAT5展现出转化效率更高。此物质是阿魏酸衍生的胡椒次生代谢产物,是新发掘的酶基因。与此同时,在Fe21菌株内过表达Pn4CL3和PnPKS1(SEQ ID No.1所示)和PnPKS2(SEQ ID No.2所示)基因,代谢产物检测分析发现11-甲氧基-去甲甲氧基醉椒素,挖掘全新功能的PnPKS1和PnPKS2基因,展现出PnPKS1活性更高。在该重组酵母菌内过表达了PnOMT1(SEQ ID No.3所示)和PnOMT2(SEQ ID No.4所示)基因,代谢产物检测出11-甲氧基-12羟基-脱氢卡瓦因胡椒代谢产物,鉴定PnOMT1和PnOMT2的功能,展现出PnOMT2的活性更高。
本研究通过提供一种高效合成阿魏酸的酿酒酵母菌株的改造方法,通过敲除酵母细胞内源的阿魏酸分解途径、敲除代谢途径的调控因子、解除阿魏酸合成的抑制机制和提高阿魏酸生物合成所必需的辅因子供给能力,构建了高效合成阿魏酸的酵母细胞菌株。同时利用高产阿魏酸的平台菌株挖掘了3种胡椒次生代谢产物合成相关的关键6种酶基因。实现了异源高效生物合成胡椒代谢产物和为阿魏酸衍生的化合物生物合成提供了方法和技术。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (27)
1.一种重组酵母菌株,其特征在于,以酿酒酵母为底盘菌株,并过表达如下基因:PahpaB、SehpaC和AtCOMT。
2.根据权利要求1所述的重组酵母菌株,其特征在于,所述酿酒酵母的基因型为:MATaura3-52 can1Δ::CAS9-natNT2 TRP1 LEU2 HIS3 gpp1ΔXII-2::(GPM1p-AtPAL2-FBA1t)+(TDH3p-AtC4H-CYC1t)+(tHXT7p-AtATR2-pYX212t)+(PGK1p-CYB5-ADH1t)X-3::(TEF1p-ARO4-CYC1t)XII-5::(ADH1t-Bbxfpk-TDH3p)+(tHXT7p-Ckpta-CYC1t)XII-1::(TEF1p-SehpaC-FBA1t)+(TPS1t-PahpaB-CCW12p)+(TDH3p-AtCOMT-CYC1t)。
3.根据权利要求1所述的重组酵母菌株,其特征在于,进一步敲除了如下基因中的至少一种:aro8、aro9、aro10、pdc5和aro80。
4.根据权利要求1所述的重组酵母菌株,其特征在于,进一步敲除aro9基因且过表达aro8基因。
5.根据权利要求4所述的重组酵母菌株,其特征在于,aro8基因与Pgk1p启动子及Tps1t终止子融合后过表达于aro9基因的敲除位点上。
6.根据权利要求3-5任一项所述的重组酵母菌株,其特征在于,进一步敲除了如下基因中的至少一种:pad1和fdc1。
7.根据权利要求6所述的重组酵母菌株,其特征在于,进一步过表达如下基因中的至少一种:Sam1、Sam2、Sah1和Met6。
8.根据权利要求7所述的重组酵母菌株,其特征在于,进一步敲除了pdc5基因和aro10基因。
9.根据权利要求8所述的重组酵母菌株,其特征在于,进一步过表达AtCOMT基因。
10.根据权利要求9所述的重组酵母菌株,其特征在于,AtCOMT基因过表达于pdc5基因的敲除位点和aro10基因的敲除位点中的至少一个上;及/或,AtCOMT基因为多拷贝。
11.根据权利要求9-10任一项所述的重组酵母菌株,其特征在于,进一步敲除了bna7基因。
12.根据权利要求11所述的重组酵母菌株,其特征在于,进一步过表达AtCOMT基因,AtCOMT基因过表达于bna7基因的敲除位点上;进一步地,AtCOMT基因为多拷贝。
13.根据权利要求9-10任一项所述的重组酵母菌株,其特征在于,进一步过表达Pn4CL3基因,且过表达PnNAT4基因和PnNAT5基因中的一种。
14.根据权利要求13所述的重组酵母菌株,其特征在于,PnNAT4基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.5所示的序列,或与如SEQ IDNo.5所示的序列具有至少70%同源性的序列。
15.根据权利要求13所述的重组酵母菌株,其特征在于,PnNAT5基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.6所示的序列,或与如SEQ IDNo.6所示的序列具有至少70%同源性的序列。
16.根据权利要求9-10任一项所述的重组酵母菌株,其特征在于,进一步过表达Pn4CL3基因,且过表达PnPKS1基因和PnPKS2基因中的一种。
17.根据权利要求16所述的重组酵母菌株,其特征在于,PnPKS1基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.1所示的序列,或与如SEQ IDNo.1所示的序列具有至少70%同源性的序列。
18.根据权利要求16所述的重组酵母菌株,其特征在于,PnPKS2基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.2所示的序列,或与如SEQ IDNo.2所示的序列具有至少70%同源性的序列。
19.根据权利要求16所述的重组酵母菌株,其特征在于,进一步过表达PnOMT1基因和PnOMT2基因中的一种。
20.根据权利要求19所述的重组酵母菌株,其特征在于,PnOMT1基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.3所示的序列,或与如SEQ IDNo.3所示的序列具有至少70%同源性的序列。
21.根据权利要求19所述的重组酵母菌株,其特征在于,PnOMT2基因的核苷酸序列包括如下序列中的一种:如SEQ ID No.4所示的序列,或与如SEQ IDNo.4所示的序列具有至少70%同源性的序列。
22.一种重组酵母菌株的构建方法,其特征在于,包括如下步骤:以酿酒酵母为底盘菌株,过表达所述酿酒酵母的如下基因:PahpaB、SehpaC和AtCOMT。
23.权利要求1-12及22任一项所述的重组酵母菌株在制备阿魏酸、阿魏酸的衍生物中的应用。
24.权利要求13-15任一项所述的重组酵母菌株在制备胡椒生物碱类代谢产物中的应用。
25.根据权利要求23所述的应用,其特征在于,所述胡椒生物碱类代谢产物包括阿魏酰哌啶。
26.权利要求16-18任一项所述的重组酵母菌株在制备11-甲氧基-去甲甲氧基醉椒素中的应用。
27.权利要求19-21任一项所述的重组酵母菌株在制备11-甲氧基-12羟基-脱氢卡瓦因生物合成中的应用。
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