CN116875475A - 一株高产大麻素合成前体的酵母菌株及其构建方法和应用 - Google Patents
一株高产大麻素合成前体的酵母菌株及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一株高产大麻素合成前体的酵母菌株及其构建方法和应用,属于合成生物学技术及微生物领域。本发明以酿酒酵母为宿主,筛选了参与蛋白质折叠蛋白的基因以获得提高OA/OLO产量的工程菌株,进一步的下调FAS1和MvaE酶的表达,随后在酿酒酵母基因组rDNA位点多拷贝插入基因表达盒CsAAE1‑TKS‑OACcassette,获得OA/OLO产量提高的基因工程菌株;本发明通过优化发酵体系中的底物,以己酸钠与己酸乙酯混合底物,进一步提高了OA/OLO的产量,10L发酵罐产量约10mMOA及10mM OLO,折合产量分别为2.2g/L及1.8g/L。
Description
技术领域
本发明涉及一株高产大麻素合成前体的酵母菌株及其构建方法和应用,具体涉及对酵母菌株进行了特定改造及相应的底物配比优化,使其适用于高产大麻素生物合成途径中间产物橄榄酸及橄榄醇,属于合成生物学技术及微生物领域。
背景技术
在大麻素的生物合成途径中有一个共同的前体:大麻萜酚酸(cannabigerolicacid,CBGA)。CBGA的合成来源于两个前体:牻牛儿基二磷酸(geranyl diphosphate,GPP)与橄榄醇酸(olivetolic acid,OA)。GPP来源于2-甲基-D-赤藓糖醇-4-磷酸生物合成途径(MEP途径)或者甲戊二羟酸生物合成途径(mevalonate pathway,MVA途径),OA的合成从己酰辅酶A(hexanoyl CoA)开始经聚酮合酶(PKS)生物合成途径引入3分子丙二酰辅酶A(malonyl CoA)得到;己酰辅酶A的来源是脂肪酸从头合成的或外源添加的己酸(hexanoicacid)。CBGA可以转化为大麻二酚酸CBDA、四氢大麻酚酸THCA等大麻素,这些羧酸形式的大麻素通过加热可以脱羧基转化为大麻萜酚(CBG)、大麻二酚(CBD)、四氢大麻酚(THC)等常见大麻素。
橄榄醇酸(olivetolic acid,OA)、橄榄醇(olivetol,OLO)是来源于植物的一种III型聚酮类化合物,具有抗菌、抗肿瘤和抗紫外线等活性,在大麻素生物合成过程中提供大麻素聚酮核部分,也是大麻素生物合成过程中的第一个关键节点。研究发现大麻萜酚酸合成酶(CBGASynthase,CBGAS)以GPP为供体,并以OA为专一异戊烯基受体,故OA的产量影响到CBGA的产量,进而影响大麻素的产量。OL为OA的脱羧产物,虽不是CBGAS的底物,但可被异戊二烯基转移酶NphB识别,用于生物催化直接合成CBG;或应用于化学合成中,以橙花醛为初始底物合成CBD。
目前,很少有报道从植物天然提取橄榄醇和橄榄醇酸。且从植物中提取制备橄榄醇(酸)受多种因素的影响,例如植物对气候和疾病的敏感性、没有GAP标准化、含量较低、占用耕地面积大且周期较长等。然而化学合成工艺复杂、成本高并且产率很低,并且存在环境污染和条件苛刻等问题。
生物合成法主要是以大肠杆菌和酿酒酵母为底盘菌,2022年,公开号为CN114703171A的专利报道了以大肠杆菌为底盘菌,发酵的OA产量为349.87mg/L。2022年,公开号为CN114262695A的专利报道了以酿酒酵母为底盘菌,通过敲除三个硫酯水解酶抑制己酰辅酶A的水解以促进OA的产生,但发酵的OA产量仍较低,离工业化生产还有差距。现有菌株改造对OA/OLO的产量提升仍十分有限,对推进OA/OLO的工业化生产仍存在局限,因此,仍需要开发新的基因、酶和菌株资源和底物以促进OA/OLO的产量提升。
发明内容
为了解决上述技术问题,本发明提供了一种高产橄榄醇酸和/或橄榄醇的工程菌株,可以进一步用于大麻素的生产。
本发明的第一个目的是提供一种高产橄榄醇酸和/或橄榄醇的工程菌株,以生产大麻萜酚酸的酿酒酵母为宿主,整合表达蛋白质折叠蛋白相关基因,基因表达盒CsAAE1-TKS-OAC cassette;所述基因表达盒CsAAE1-TKS-OAC cassette包括串联表达的酰基活化酶基因CsAAE1、聚酮合酶基因TKS、橄榄酸环化酶基因OAC和橄榄油酸酯香叶基转移酶基因CsPT。
在一种实施方式中,所述蛋白质折叠蛋白相关基因选自PDI1、SEC22、KAR2、ERO1、SED5或BET1中的任意两个。
在一种实施方式中,所述蛋白质折叠蛋白相关基因为PDI1和SEC22。
在一种实施方式中,所述基因PDI1和SEC22分别整合至酿酒酵母基因组的1414a或106a位点。
在一种实施方式中,所述工程菌还过表达Fad1、Fmn1、Hac1和Gal4,敲除Pep4和Gal80、下调FAS1基因和MvaE酶的表达,多拷贝表达OAC。
在一种实施方式中,所述下调FAS1基因和MvaE酶的表达为利用启动子pHXT1启动表达基因FAS1、MvaE酶。
在一种实施方式中,所述基因表达盒CsAAE1-TKS-OAC cassette多拷贝整合至酿酒酵母基因组的rDNA位点。
在一种实施方式中,所述多拷贝表达OAC为在1622b位点过表达TKS和OAC、在YPRCd15c位点过表达OAC、在HO位点过表达OAC、OAC3、OAC5、OAC4。
在一种实施方式中,所述基因PDI1、SEC22、KAR2、SED5、ERO1或BET1编码的氨基酸序列分别如SEQ ID NO.1~6所示。
在一种实施方式中,所述基因FAS1编码的氨基酸序列SEQ ID NO.7所示。
在一种实施方式中,所述启动子pHXT1的核苷酸序列如SEQ ID NO.8所示。
在一种实施方式中,所述酰基活化酶基因CsAAE1、聚酮合酶基因TKS、橄榄酸环化酶基因OAC编码和橄榄油酸酯香叶基转移酶基因CsPT的氨基酸序列如SEQ ID NO.9~12所示。
在一种实施方式中,所述Fad1核苷酸序列可以是NC_001136.10(372688..373608,complement)。
在一种实施方式中,所述Fmn1核苷酸序列可以是NC_001136.10(935236..935892,complement)。
在一种实施方式中,所述Hac1核苷酸序列可以是NC_001138.5(75179..76147)。
在一种实施方式中,所述Gal4核苷酸序列可以是NC_001148.4(79711..82356,complement)。
在一种实施方式中,所述Pep4核苷酸序列可以是NC_001148.4(259714..260931,complement)。
在一种实施方式中,所述Gal80核苷酸序列可以是NC_001145.3(171594..172901)。
在一种实施方式中,所述OAC3、OAC5、OAC4的氨基酸序列如SEQ ID NO.18~20所示。
本发明的第二个目的是一种生产大麻素合成前体的方法,所述方法为将上述工程菌株接种于反应体系中发酵制备大麻素合成前体,所述大麻素合成前体为橄榄醇酸和/或橄榄醇。
在一种实施方式中,所述方法以己酸乙酯和己酸钠的混合物为底物。
在一种实施方式中,所述己酸乙酯和己酸钠的混合比例为(1:1)~(50:1)。
在一种实施方式中,所述己酸乙酯和己酸钠的混合比例为3:1。
在一种实施方式中,所述反应体系以葡萄糖为碳源。
在一种实施方式中,所述方法为将上述工程菌株活化得到种子液,将种子液接种于含有己酸乙酯和己酸钠反应体系中,发酵培养大麻素合成前体。
本发明还提供了上述工程菌株或上述方法在制备大麻素前体和/或大麻素中的应用。
有益效果:
本发明以生产大麻萜酚酸的酿酒酵母为宿主,在过表达Fad1、Fmn1、Hac1和Gal4,敲除Pep4和Gal80,多拷贝OAC的基础上,筛选了参与蛋白质折叠蛋白的基因以获得提高OA/OLO产量的工程菌株,进一步的将FAS1和MvaE酶原来的启动子替换为pHXT1启动子,随后在酿酒酵母基因组rDNA位点多拷贝插入基因表达盒CsAAE1-TKS-OAC cassette,获得OA/OLO产量提高的基因工程菌株;本发明通过优化发酵体系中的底物,以己酸钠与己酸乙酯混合底物,进一步提高了OA/OLO的产量,10L发酵罐产量约10mM OA及10mM OLO,折合产量分别为2.2g/L及1.8g/L。
附图说明
图1为MG36质粒CsAAE1-TKS-OAC表达盒示意图;
图2改造蛋白重叠相关重组酵母菌株孔板水平发酵测试;
图3多拷贝插入重组酵母菌株摇瓶水平发酵测试;
图4重组酵母菌株摇瓶水平底物发酵测试;
图5重组酿酒酵母在生产OA/OLO。
具体实施方式
术语
CBGA:大麻萜酚酸;
CsAAE1:酰基活化酶,将己酸或己酸钠转化为己酰基-CoA;
TKS:聚酮合酶;
OAC:橄榄酸环化酶;
GFP:绿色荧光蛋白;
Leu2:亮氨酸;
pHXT1:启动子,响应葡萄糖浓度的启动子;
PDI1:蛋白质二硫异构酶;
KAR2:参与蛋白质导入ER的ATP酶;
ERO1:ER中氧化蛋白折叠所需的硫醇氧化酶;
SED5:顺式-高尔基体t-SNARE合成蛋白;
BET1:囊泡运输所需的II型膜蛋白;
SEC22:R-SNARE蛋白;
FAS1:内源脂肪酸合成酶;
Pep4:编码空泡蛋白酶;
Fad1:编码黄素腺嘌呤二核苷酸合成酶;
Fmn1:编码核黄素激酶;
pGal1:启动子,是编码半乳糖激酶的Gal1基因上游的600bp部分,Gal1基因的核苷酸序列是NC_001134.8(279021..280607)。
Gal80:参与抑制Gal基因的转录调节因子;
Gal4:编码转录激活蛋白GAL4
实验方法:
下述实施例所用的PCR扩增方法、不同片段的融合方法、基因敲除及过表达方法可以采用本领域常见的技术手段,例如融合PCR、同源重组、CRISPR-Cas9技术。所用的酶、试剂盒均是可以购买的已经商业化的产品。
过表达,是指把基因上调表达,即该基因被过度的转录、翻译,终基因表达产物超过正常水平。
转化,是采用醋酸锂法,先将宿主菌株在1×YPD培养基中活化,30℃,200rpm培养过夜。然后接种到新的2×YPD培养基中使起始OD值为0.2,30℃继续培养4-4.5h后,取5OD菌液,常温3000rcf,离心5min,弃上清,并用灭菌超纯水洗涤两次,获得酵母细胞;配制DNA混合物,每个构建取5OD的菌液获得细胞,并与50μL DNA混合物混合,使细胞重悬,50μLDNA混合物由2μg所述插入片段、250ng工具质粒以及足量ddH2O混合而成。在悬浮的细胞中加入醋酸锂转化混合物,经培养后获取细胞,涂布到筛选平板上,获取单菌落,即为重组酿酒酵母,通过蓝光板,观看菌株是否存在荧光现象,选取阳性菌落。
菌落PCR及测序验证:待筛选平板上长出单克隆菌落后,进行菌落PCR及测序验证,具体步骤是:用枪头挑取少量细胞分别置于20μL 20mmol/LNaOH溶液,涡旋混匀,于金属浴95℃孵育20min,涡旋混匀,取1μL菌液作为模板进行菌落PCR反应,反应引物根据验证序列不同而有所不同,比对克隆条带与阴性克隆条带大小,挑选菌落PCR阳性克隆的菌液送致金唯智公司进行测序验证。测序正确的菌株进行划线保存和甘油冻存。
对酵母的基因组改造,包括基因的插入、敲除、启动子替换等,其原理为CRISPR/Cas9的酵母同源重组技术,相关技术基于本公司前期已开发并运用的SOP,可参考专利CN114657078A;CN114591923B。
2×YPD培养基配方:酵母浸膏20.0g/L,蛋白胨40.0g/L,葡萄糖40.0g/L。
半合成培养基:(NH)2SO4:5g/L,KH2PO4:3g/L,MgSO4:0.32g/L,Leu:0.2g/L,Ura:0.2g/L,Trp:0.2g/L,琥珀酸:5.9g/L,酵母浸膏10.0g/L,葡萄糖20.0g/L。
醋酸锂转化混合物:50%W/V PEG3350260μL,1mol/L LiOAc 36μL,变性鲑鱼精DNA10μL(变性鲑鱼精DNA使用前先置于95℃金属浴变性5min),ddH2O 4μL。
缺少尿嘧啶的筛选平板:酵母氮源母液1.7g/L,硫酸铵5g/L,各种氨基酸如表1所示,琼脂20g/L,葡萄糖20g/L,注:葡萄糖分开灭菌。
表1筛选平板中各种氨基酸的含量
氨基酸 | (mg/L) | 氨基酸 | (mg/L) |
腺嘌呤半硫酸盐 | 18 | L-苯丙氨酸 | 76 |
L-丙氨酸 | 76 | L-脯氨酸 | 76 |
L-精氨酸 | 76 | L-苏氨酸 | 76 |
L-天冬氨酸 | 76 | L-丝氨酸 | 76 |
L-天冬酰胺 | 76 | L-色氨酸 | 76 |
L-半胱氨酸 | 76 | L-酪氨酸 | 76 |
L-谷氨酸 | 76 | L-缬氨酸 | 76 |
L-甘氨酸 | 76 | L-蛋氨酸 | 76 |
L-异亮氨酸 | 76 | L-赖氨酸 | 76 |
L-谷氨酰胺 | 76 | L-亮氨酸 | 360 |
L-组氨酸 | 76 |
重组酿酒酵母产OA或OLO的量的检测方法:样品收集后,根据样品OD600,先用破壁酶2U/OD于30℃,200rpm摇床温育处理60min,然后加入0.2mL体积0.5mm玻璃珠和0.4ml的乙酸乙酯:甲酸(0.05%),在高速组织研磨仪中,以65Hz处理180s,间隔30s,重复三次,每次处理后将研磨托盘置于冰上冷却1min,震荡15-30s,瞬时离心后,取上层有机层0.28mL至1.5mL离心管中,重复两次,并将收集的上层有机相合并。三次提取的有机层,Evaporation,mode V-AL,45℃1h挥干至无溶剂残留,用重悬液AHF(乙腈:H2O:甲酸=80:20:0.05%,含内标PHB(对羟基苯甲酸丙酯溶液标准物质,15μM)140μL重悬,0.22μm PVDF滤膜过滤至液相检测瓶内插管中,作为检测样品。每种样品2个平行。样品准备好之后,用HPLC进行检测,检测条件见表2。
表2 HPLC检测条件
表3下述实施例中涉及到的菌株
实施例1替换基因组中的MvaE酶的启动子菌株的构建
(1)ySC461菌株构建
通过2×Phanta Max Master Mix(Phanta DNA聚合酶)进行PCR扩增整合片段,以酿酒酵母yG278(公开于公开号为CN115927029A的中国专利申请文件)为模板,以表4中的引物1和引物2扩增1622b-up-TKS片段,以表4中的引物3和引物4扩增OAC-1622b-dn片段,将上述片段转化到宿主酿酒酵母ySC340,获得菌株ySC461,以表4中的引物5和引物6进行PCR验证,获得PCR阳性克隆的菌液,用于基因测序。实现在1622b位点过表达TKS和OAC。
表4引物序列
(2)ySC471菌株构建
通过2×Phanta Max Master Mix(Phanta DNA聚合酶)进行PCR扩增整合片段,以酿酒酵母CEN.PK2-1C为模板,以表5中的引物1和引物2扩增YPRcd15c-up片段,以表5中的引物3和引物4扩增YPRcd15c-dn片段,以酿酒酵母yG278为模板,以表5中的引物5和6扩增OAC片段,将上述片段转化到步骤(1)构建的宿主酿酒酵母ySC461,获得菌株ySC471,以表5中的引物1和引物4进行PCR验证,获得PCR阳性克隆的菌液,用于基因测序。实现在YPRCd15c位点过表达OAC。
表5引物序列
(3)ySC476菌株构建
通过2×Phanta Max Master Mix(Phanta DNA聚合酶)进行PCR扩增整合片段,以酿酒酵母CEN.PK2-1C为模板,以表6中的引物1和引物2扩增HO-up片段,以表6中的引物3和引物4扩增HO-dn片段,以酿酒酵母yG278为模板,以表6中的引物5和6扩增OAC-OAC3-OAC5-OAC4片段,将上述片段转化到步骤(2)构建的宿主酿酒酵母ySC471,获得菌株ySC476,以表6中的引物7和引物8进行PCR验证,获得PCR阳性克隆的菌液,用于基因测序。实现在HO位点过表达OAC、OAC3、OAC5、OAC4。
表6引物序列
(4)ySC481菌株构建
通过2×Phanta Max Master Mix(Phanta DNA聚合酶)进行PCR扩增整合片段,以yG024(公开于公开号为CN114369541A的中国专利申请文件)为模板,以表7中的引物1和引物2扩增MvaE-up-pHXK1片段,以表7中的引物3和引物4扩增MvaE-dn片段,将上述片段转化到步骤(3)构建的宿主酿酒酵母ySC476,获得菌株ySC481,以表7中的引物5和引物6进行PCR验证,获得PCR阳性克隆的菌液,用于基因测序。获得启动子pHXK1调控MvaE的重组酿酒酵母ySC481。
表7引物序列
实施例2过表达参与蛋白质折叠蛋白的酿酒酵母菌株构建
通过2×Phanta Max Master Mix(PhantaDNA聚合酶)进行PCR扩增整合片段,以CEN.PK2-1C的基因组为模板,以表8中的引物1和引物2扩增获得1414a-up片段,以ySC222的基因组为模板,以表8中的引物3和引物4扩增获得启动子pPGK1片段,以CEN.PK2-1C的基因组为模板,以表8中的引物5和引物6扩增获得1414a-dn片段,以CEN.PK2-1C的基因组为模板,以表8中的引物7和引物8扩增获得106a-up片段,以ySC222的基因组为模板,以表8中的引物9和引物10扩增获得启动子pPGK1片段,以CEN.PK2-1C的基因组为模板,以表8中的引物11和引物12扩增获得106a-dn片段,以CEN.PK2-1C的基因组为模板,以表8中的引物13和引物14扩增获得PDI1-tPDI1片段,以表8中的引物15和引物16扩增获得KAR2-tKAR2片段,以表8中的引物17和引物18扩增获得ERO1-tERO1片段,以表8中的引物19和引物20扩增获得SED5-tSED5片段,以表8中的引物21和引物22扩增获得BET1-tBET1片段,以表8中的引物23和引物24扩增获得SEC22-tSEC22片段,然后将上述片段分别组合转化到宿主酿酒酵母ySC481的1414a位点和106a位点,获得菌株yAW1、yAW2、yAW3、yAW4、yAW5、yAW6、yAW7、yAW8、ySC812(基因信息见表3)。表8中的引物25和引物26及引物27和引物28用于对菌株yAW1、yAW2、yAW3、yAW4、yAW5、yAW6、yAW7、yAW8、ySC812进行PCR反应,获得菌落PCR阳性克隆的菌液,用于基因测序。
表8引物序列
实施例3以己酸钠为底物合成橄榄酸(醇)
以己酸钠为底物,利用实施例2制备得到的工程酵母菌yAW1、yAW2、yAW3、yAW4、yAW5、yAW6、yAW7、yAW8、ySC812进行摇瓶培养,分别挑取2-3个单菌落,接种到1×YPD培养基中,培养16h,按0.2OD/mL转接到20mL半合成培养基,30℃培养24h开始补加1M己酸钠,早晚各一次,共补4次,使发酵液中己酸钠的终浓度为3mM,接种后,每隔24h补加2%葡萄糖,直至发酵结束,从接种开始计算发酵时间,在发酵72h和96h取样检测。结果显示,ySC812的产量最高,ySC812 OA的产量为0.26mM;OD为14.87(图2)。可见,相比其他组合,过表达PDI1和SEC22组合的工程菌在制备生产OA和OLO上都具有一定的优势,可用于下一步的改造。
实施例4构建整合表达FAS1基因的菌株ySC815
以酿酒酵母yG011基因组为模板,通过2×Phanta Max Master Mix(Phanta DNA聚合酶),使用表9中的引物1和引物2扩增PHXT1启动子,将扩增的片段转化至实施例2构建的酿酒酵母宿主菌ySC812,通过表9中的引物3和引物4进行PCR验证,获得PCR阳性克隆的菌液,用于基因测序。经测序,成功的构建替换了启动子的菌株ySC815。
表9引物序列
实施例5构建整合表达CsAAE1-TKS-OAC cassette的菌株ySC1821
(1)质粒MG36的构建
通过2×Phanta Max Master Mix(Phanta DNA聚合酶)进行PCR扩增整合片段,以pUASR(为公开于文献DOI:10.1038/s41467-020-15977-4中的PZEVAR,在本申请中命名为pUASR,核苷酸序列如SEQ ID NO.13)为模板,以表10中的引物1和引物2扩增获得启动子pUASR,以yCAN31的基因组为模板,以表10中的引物3和引物4扩增获得CsAAE1片段,以tSyn1(为公开于文献DOI:10.1038/s41467-020-15977-4中的Tsynth2,在本申请中命名为tSyn1,核苷酸序列如SEQ ID NO.14)为模板以表10中的引物5和引物6扩增获得tSyn1片段,以pGPD(公开于文献DOI:10.1038/s41467-020-15977-4中,核苷酸序列如SEQ ID NO.15)为模板,以表10中的引物7和引物8扩增获得pGPD片段,以yCAN31的基因组为模板,以表10中的引物9和引物10扩增获得TKS-OAC片段,以tSyn3(为公开于文献DOI:10.1038/s41467-020-15977-4中的Tsynth9,在本申请中命名为tSyn3,核苷酸序列如SEQ ID NO.16)为模板,以表10中的引物11和引物12扩增获得tSyn3片段,以yCAN31的基因组为模板,以表10中的引物13和引物14扩增获得pGal1-CsPT片段,以tSyn5(为公开于文献DOI:10.1038/s41467-020-15977-4中的Tsynth18,在本申请中命名为tSyn5,核苷酸序列如SEQ ID NO.17)为模板,以表10中的引物15和16扩增获得tSyn5片段,将质粒backbome(公开于公开号CN113999783A的中国专利申请文件)用限制性内切酶PacI和KpnI进行双酶切,将上述片段和双酶切后的线性化载体混合转化至大肠杆菌DH5α(来源全式金),涂布于含氨苄(50ug/ml)LB平板进行转化子的筛选,用表10中的引物17和引物18采用菌落PCR鉴定转化子,对鉴定正确的转化子提取质粒,并将质粒送测序,测序正确的质粒命名为MG36(图1)。
表10引物序列
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(2)菌株ySC1821的构建
利用Leu2基因为筛选标记,在rDNA位点过表达CsAAE1-TKS-OAC cassette。重组酿酒酵母菌株在多拷贝rDNA位点过表达优化的酰基活化酶基因、聚酮合酶基因、橄榄酸环化酶基因和橄榄油酸酯香叶基转移酶基因。为方便rDNA位点多拷贝插入,前期先构建了rDNA位点整合插入表达盒(rDNAintegration cassette),包含1)rDNA位点上下游同源臂;2)用作筛选标记的leu2基因或loxL-Ura3-Cre-LoxR cassette;3)以单独启动子及终止子串联表达的CsAAE1/TKS/OAC;4)组成型表达的GFP;5)单独启动子及终止子表达的CsPT。其中,loxL-Ura3-Cre-LoxR cassette设计为在诱导条件下可通过Cre介导的LoxL/R间的同源重组将cassette自身消除,GFP用于通过荧光强度判断不同单菌落间表达盒表达强度的相对强弱。
通过2×Phanta Max Master Mix(Phanta DNA聚合酶)进行PCR扩增整合片段。以酿酒酵母CEN.PK2-1C的基因组为模板,使用表11中的引物1和引物2扩增rDNA-up片段,以步骤(1)构建的质粒MG36为模板,使用表11中的引物3和引物4扩增CsAAE1-TKS片段,以质粒MG36为模板,使用表11中的引物5和引物6扩增OAC片段,以GFP基因为模板,使用表11中的引物7和引物8扩增GFP片段,以Leu2为模板,使用表11中的引物9和引物10扩增Leu2片段,CEN.PK2-1C的基因组为模板,使用表11中的引物11和引物12扩增rDNA-dn片段,将上述片段混合转化实施例4构建的宿主酵母菌株ySC815。挑转化子单菌落培养后通过酶标仪检测各菌株GFP荧光强度。选取不同荧光强度范围的菌株进行孔板发酵初筛,结果如图3所示,菌株ySC1821表现出最高的OA和OLO产量。经测序,菌株ySC1821整合了5个拷贝的表达框CsAAE1-TKS-OAC cassette。
表11引物序列
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实施例6以不同底物合成橄榄酸(醇)
将实施例5获得的工程酵母菌ySC1821为发酵菌株,设置底物溶液中(己酸乙酯:己酸钠)的混合比例为3:1、只添加己酸乙酯或只添加己酸钠,以探究不同底物溶液对合成橄榄酸(醇)的影响。
挑取2-3个单菌落,接种到1×YPD培养基中,培养16h,按0.2OD/mL转接到20mL半合成培养基,接种后,30℃培养24h开始补加不同比例的己酸乙酯/己酸钠混合底物,底物补加使用1M己酸乙酯(溶于肉豆蔻酸异丙酯,IPM)/1M己酸钠,早晚各一次,共补4次,使发酵液中己酸钠的终浓度为3mM,己酸乙酯的终浓度为9mM,接种后,每隔24h补加2%葡萄糖,直至发酵结束,从接种开始计算发酵时间,在发酵72h和96h取样检测。结果显示,当底物含有己酸乙酯时,ySC1821的OA产量为1.501mM,OD为8.73(图4),当底物中不含有己酸钠时,ySC1821的OA产量为0.846mM,OD为6.43。
并且从图4可以看出,实施例3获得的工程酵母菌ySC812和实施例4获得的工程酵母菌ySC815在橄榄酸(醇)合成反应中,虽然OD值高于工程菌株ySC1821,但产量显著降低。
实施例7摇瓶发酵测试ySC1821等菌株以不同底物合成橄榄酸(醇)
实施例使用百伦生物4联10L不锈钢发酵罐。取实施例5获得的ySC1821甘油管接种至装有20mL1×YPD培养基的250ml摇瓶中制备一级种子液,于30℃/200rpm过夜培养后转接装有500mL 1×YPD培养基的2000ml摇瓶中制备二级种子液,于30℃/200rpm培养后接种发酵罐。控制发酵罐中的初始OD600约0.2,发酵培养基使用半合成培养基,发酵参数使用:1.5vvm/0.5Mpa/DO≥50%/pH 5.0。发酵过程采用流加补料方式补加葡萄糖(碳源),控制残糖<5g/L,乙醇浓度<10g/L。自发酵起始18h开始,采用分批补加的方式分6次加入底物,其中,底物己酸乙酯补料使用己酸乙酯含量2.5%的IPM母液(己酸乙酯/IPM(V/V)=2.5%),底物己酸钠补料使用浓度1M己酸钠的水溶液,其终浓度分别为18mM己酸乙酯及6mM己酸钠。发酵过程中每24h取样检测发酵液OD及产物。发酵结束后发酵液经在位高温灭菌处理后按发酵废液处理。结果显示,发酵144小时菌株可积累约10mM OA及10mM OLO,折合产量分别为2.2g/L及1.8g/L(图5)。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
Claims (10)
1.一种高产橄榄醇酸和/或橄榄醇的工程菌株,以酿酒酵母为宿主,整合表达蛋白质折叠蛋白相关基因,基因表达盒CsAAE1-TKS-OACcassette;所述基因表达盒CsAAE1-TKS-OACcassette包括串联表达的CsAAE1、TKS和OAC。
2.根据权利要求1所述的工程菌株,其特征在于,所述蛋白质折叠蛋白相关基因选自PDI1、SEC22、KAR2、ERO1、SED5或BET1中的任意两个。
3.根据权利要求1或2所述的工程菌株,其特征在于,所述工程菌还过表达Fad1、Fmn1、Hac1和Gal4,敲除Pep4和Gal80、下调FAS1基因和MvaE酶的表达,多拷贝表达OAC。
4.根据权利要求3所述的工程菌株,其特征在于,所述下调FAS1基因和MvaE酶的表达为利用启动子pHXT1启动表达基因FAS1、MvaE酶。
5.根据权利要求1所述的工程菌株,其特征在于,所述基因表达盒CsAAE1-TKS-OACcassette多拷贝整合至酿酒酵母基因组的rDNA位点。
6.根据权利要求3所述的工程菌株,其特征在于,所述多拷贝表达OAC为在1622b位点过表达TKS和OAC、在YPRCd15c位点过表达OAC、在HO位点过表达OAC、OAC3、OAC5、OAC4。
7.一种生产大麻素合成前体的方法,其特征在于,所述大麻素合成前体为橄榄醇酸和/或橄榄醇,所述方法为将权利要求1~6任一所述工程菌株接种于反应体系中发酵制备大麻素合成前体。
8.根据权利要求7所述的方法,其特征在于,所述方法以己酸乙酯和己酸钠的混合物为底物。
9.根据权利要求8所述的方法,其特征在于,所述己酸乙酯和己酸钠的混合比例为(1:1)~(50:1)。
10.权利要求1~6任一所述工程菌株或权利要求7~9任一所述方法在制备大麻素前体和/或大麻素中的应用,所述大麻素合成前体为橄榄醇酸和/或橄榄醇。
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