CN116478841B - 一株产油脂的酿酒酵母突变株mu310及其应用 - Google Patents
一株产油脂的酿酒酵母突变株mu310及其应用 Download PDFInfo
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Abstract
本发明属于生物工程技术领域,具体涉及一株高产油脂的酿酒酵母突变株MU310及其应用。本发明提供了一株高产油脂的酿酒酵母突变株MU310,其分类命名为Saccharomyces cerevisiae MU310,已于2023年3月23日保藏于中国普通微生物菌种保藏中心,保藏号为CGMCC No.26859,保藏地址为北京市朝阳区北辰西路1号院3号。本发明以野生型酿酒酵母Saccharomyces cerevisiae SC018作为出发菌株经过诱变获得高产油脂的酿酒酵母突变株MU310,该突变株在限氮培养基中培养4天后,使细胞内的油脂含量由出发株的31.12%提高到44.07%。该突变株经过20次的连续接种传代,细胞内的油脂含量没有发生明显的变化,说明该突变株的遗传性状相对稳定,可作为规模化生产酿酒酵母油脂和棕榈油酸的优良菌株,为推动微生物油脂的产业化发展提供了种质资源。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一株高产油脂的酿酒酵母突变株MU310及其应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
棕榈油酸是一种珍稀的十六碳单不饱和脂肪酸,不饱和双键位于甲基端的第七个碳原子上,具有抗炎抗氧化、调节胆固醇水平、预防糖尿病和心血管疾病、控制体重、美白等保健作用,还是一种良好的生物柴油原材料,所以其在医药、保健品、水产饵料、化工产品以及生物能源等方面具有非常重要的应用价值。
目前,天然的棕榈油酸主要来源于野生动植物资源。然而,野生动植物资源非常有限,并且严重受制于季节、地域等因素,难以规模化持续性供应,无法满足迅速增长的棕榈油酸产品的市场需求。为解决动植物来源脂肪酸的原料限制,已有很多研究致力于开发脂肪酸生产的微生物技术方法,这些研究通过藻类或酵母等产油微生物筛选获得高产油脂的菌株,并进行油脂的工业化发酵生产,以克服传统农业模式的地域、季节以及占用大量土地的限制。目前在以酵母发酵生产脂肪酸方面的研究,大都以圆红冬孢酵母、产油油脂酵母、浅白色隐球酵母等传统的产油酵母为底盘微生物,鲜有非产油酵母酿酒酵母的报道。
研究发现,酿酒酵母中的棕榈油酸的含量要远远高于其它种类的酵母,约占其总脂肪酸含量的50%。酿酒酵母具有繁殖快、生长周期短、代谢旺盛和非致病性等特点,并且其培养过程不受季节、地域等因素的限制,不占耕地,可实现全年大规模培养和生产。此外,酿酒酵母油脂品质较好、气味佳,更易于作为棕榈油酸的新的生产资源。
然而,酿酒酵母油脂含量极低,被普遍认为不属于产油微生物,缺乏作为油脂生产平台的潜力。本发明申请人前期已经通过大量的筛选评价获得了一株产油能力较强的酿酒酵母菌株,经缺氮诱导后油含量在30%左右,通过发酵工艺优化还可将其含油量提高至40%左右,显示酿酒酵母具有较强的产油能力。然而,单纯的依靠野生株的筛选来获得更高油脂含量的酿酒酵母菌株,无论是从工作量还是可行性上都存在巨大的问题。
发明内容
为了解决现有技术的不足,本发明的目的是提供一株高产油脂的酿酒酵母突变株MU310及其应用。本发明旨在通过博来霉素化学诱变对野生型酿酒酵母进行诱变育种,结合高通量筛选方法获得油脂含量高的酿酒酵母突变株,为酿酒酵母生产棕榈油技术的建立提供高效产油菌种,丰富产油微生物的种质资源。本发明提供的酿酒酵母突变株MU310是以野生型酿酒酵母SC018为出发菌株进行诱变和筛选获得的高产油脂的突变株,该突变株细胞能积累更高含量的油脂,为利用酿酒酵母生产油脂提供了优良的种质资源,并为推动微生物油脂产业提供了技术支撑。
为了实现上述目的,本发明是通过如下的技术方案来实现:
第一方面,本发明提供了一株高产油脂的酿酒酵母突变株MU310,其分类命名为Saccharomyces cerevisiae MU310,已于2023年3月23日保藏于中国普通微生物菌种保藏中心,保藏号为CGMCCNo.26859,保藏地址为北京市朝阳区北辰西路1号院3号。
第二方面,本发明提供了一种微生物菌剂,所述微生物菌剂含有第一方面所述的酿酒酵母突变株MU310或其发酵产物或其代谢产物。
第三方面,本发明提供了一种酿酒酵母油脂的生产方法,将第一方面所述的酿酒酵母突变株MU310加入到培养基中,培养生产酿酒酵母油脂。
第四方面,本发明提供了第一方面所述的酿酒酵母突变株MU310和/或第二方面所述的微生物菌剂在生产油脂中的应用。
上述本发明的一种或多种技术方案取得的有益效果如下:
本发明以野生型酿酒酵母Saccharomyces cerevisiae SC018作为出发菌株经过诱变获得高产油脂的酿酒酵母突变株MU310,该突变株在限氮培养基中培养4天后,其细胞内的油脂含量显著高于野生型出发菌株,使细胞内的油脂含量由31.12%提高到44.07%。该突变株经过20次的连续接种传代,细胞内的油脂含量没有发生明显的变化,说明该突变株的遗传性状相对稳定,可作为规模化生产酿酒酵母油脂和棕榈油酸的优良菌株,为推动微生物油脂的产业化发展提供了种质资源。同时,突变株油脂含量的大幅提升可以使微生物油脂的提取效率和提取成本发幅度降低,有利于微生物油脂的工业化生产。
保藏信息
酿酒酵母(Saccharomyces cerevisiae)MU310已于2023年3月23日保藏于中国普通微生物菌种保藏中心,保藏号为CGMCC No.26859,保藏地址为北京市朝阳区北辰西路1号院3号。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例1中博来霉素浓度对酿酒酵母致死率的影响曲线;
图2为实施例1中连续三轮博来霉素诱变与高通量筛选获的菌株的生物量与油脂含量,其中SC018为野生型出发菌株,2H/45为第一轮诱变获得突变株,MU230为第二轮诱变获得的突变株,其余为第三轮诱变获得突变株;
图3为实施例1中连续三轮诱变获得菌株的油脂产量。
具体实施方式
本发明的第一种典型实施方式,一株高产油脂的酿酒酵母突变株MU310,其分类命名为Saccharomyces cerevisiae MU310,已于2023年3月23日保藏于中国普通微生物菌种保藏中心,保藏号为CGMCCNo.26859,保藏地址为北京市朝阳区北辰西路1号院3号。
该实施方式的一种或多种实施例中,所述酿酒酵母突变株MU310的ITS序列具有如SEQ ID No.1所示的核苷酸序列。
本发明的第二种典型实施方式,一种微生物菌剂,所述微生物菌剂含有第一种典型实施方式所述的酿酒酵母突变株MU310或其发酵产物或其代谢产物。
本发明的第三种典型实施方式,一种酿酒酵母油脂的生产方法,其特征在于,将第一种典型实施方式所述的酿酒酵母突变株MU310加入到培养基中,培养生产酿酒酵母油脂。
该实施方式的一种或多种实施例中,所述培养基包括种子培养基和油脂诱导培养基。
该实施方式的一种或多种实施例中,所述种子培养基为YPD培养基,所述YPD培养基的组分包括酵母浸粉9-11g/L、蛋白胨19-21g/L和葡萄糖19-21g/L。
该实施方式的一种或多种实施例中,所述油脂诱导培养基为限氮培养基,所述限氮培养基的组分包括酵母浸粉3-5g/L,(NH4)2SO40.3-0.5g/L,KH2PO4 0.9-1.1g/L,Na2HPO40.2-0.3g/L,MgSO4·7H2O1.4-1.6g/L,FeCl3·6H2O 0.14-0.16g/L,CaCl2·2H2O 0.14-0.16g/L,ZnSO4·7H2O 0.01-0.03g/L,MnSO4·H2O 0.05-0.07g/L,葡萄糖19-21g/L。
该实施方式的一种或多种实施例中,所述培养包括种子培养和油脂诱导培养,所述种子培养的条件为置于种子培养基28℃培养2天,所述油脂诱导培养的条件为置于油脂诱导培养基在28℃下180rpm摇床培养4天并每天补加30g/L的葡萄糖。
本发明的第四种典型实施方式,第一种典型实施方式所述的酿酒酵母突变株MU310和/或第二种典型实施方式所述的微生物菌剂在生产油脂中的应用。
该实施方式的一种或多种实施例中,所述油脂包括棕榈油酸。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例与对比例详细说明本发明的技术方案。
实施例1
酿酒酵母(Saccharomyces cerevisiae)突变株MU310及其筛选培养的具体过程如下:
(1)配制酿酒酵母YPD种子培养基。酿酒酵母种子培养基YPD成分包括20g/L蛋白胨,10g/L酵母浸粉,20g/L葡萄糖,115℃灭菌30min。
(2)配制酿酒酵母限氮培养基。酿酒酵母限氮培养基成分包括酵母浸粉4g/L,(NH4)2SO4 0.4g/L,KH2PO4 1g/L,Na2HPO4 0.25g/L,MgSO4·7H2O 1.5g/L,FeCl3·6H2O0.15g/L,CaCl2·2H2O 0.15g/L,ZnSO4·7H2O 0.02g/L,MnSO4·H2O 0.06g/L,葡萄糖20g/L,115℃灭菌30min。
(3)博来霉素诱变浓度的确定。挑取出发菌株酿酒酵母SC018单菌落接种至YPD种子培养基,28℃,180rpm培养24h;稀释菌液OD600至8~9,加入不同体积的博来霉素,使其终浓度分别为0μg/mL、30μg/mL、50μg/mL、70μg/mL、100μg/mL、120μg/mL、150μg/mL,置于28℃,180rpm摇床震荡2h,取1mL反应混合物4000rpm离心1min,用无菌水重悬,倒去上清,再次离心重悬;将重悬后的菌液稀释不同的倍数,取20μL涂布于YPD平板,28℃培养2d。以添加0μg/mL博来霉素的处理组为对照,计算致死率,致死率%=(对照菌落数-诱变后菌落数)/对照菌落数×100。博来霉素致死率曲线如图1所示,随着诱变剂浓度增加,酿酒酵母的致死率也逐渐增加,当博来霉素诱变浓度为50μg/mL时,致死率在96%左右,选择该浓度为博来霉素的诱变处理浓度。
(4)诱变与初筛。挑取出发菌株酿酒酵母SC018单菌落接种至YPD种子培养基,28℃,180rpm培养24h;稀释菌液至OD600 8~9,加入一定体积的博来霉素,使其终浓度为50μg/mL,将反应物置于28℃,180rpm摇床震荡2h,取1mL反应混合物4000rpm离心1min,用无菌水重悬,倒去上清,再次离心重悬;将重悬后的菌液接种至限氮培养基,置于28℃摇床,180rpm培养4天,每天补加30g/L葡萄糖。
采用流式细胞仪分选诱变后高油含量的细胞。用无菌水重悬诱导后的菌体,将菌液浓度稀释至107/mL,取1mL稀释的菌液加入0.2μLBODIPY 505/515荧光染料,置于流式上样管中,通过BD公司流式细胞仪鞘液,利用流式细胞仪在488nm激光激发下捕捉荧光信号,选择FITC通道,530±15nm带通滤片。根据FSC-A、SSC-A(表征细胞颗粒度)和FITC-A(表征荧光强度)信号设置分选门收集目标细胞,选择细胞荧光强度频数分布的最高荧光值的0.1~1%的区域进行收集,以1000evt/s的速度对目的细胞进行分选。将分选后的目的细胞涂布于YPD平板上,置于28℃培养2天。
(5)复筛。随机从平板上挑取50个单克隆分别置于装有30mL限氮培养基的三角瓶中,置于28℃摇床,180rpm培养4天,每天补加30g/L葡萄糖,分析每个样品的生物量和油脂含量,取油脂含量较高的几株酵母再次培养验证。实验结果如图2和图3所示,取每一轮的一株油脂含量最高的菌株重复诱变和筛选,经过连续三轮博来霉素诱变与筛选后得到一株生物量为10.5g/L、油脂含量为44.07%的酿酒酵母突变株MU310,其油脂含量较出发菌株相对提高了41.61%。
对突变株进行菌种鉴定。以MU310菌株基因组为模板,通过真菌鉴定通用测序引物ITS1和ITS4进行PCR扩增,ITS1引物序列为:TCCGTAGGTGAACCTGCGG(SEQ ID No.2),ITS4引物序列为:TCCTCCGCTTATTGATATGC(SEQ ID No.3)。扩增产物由南京擎科生物科技有限公司进行测序,将测序结果在NCBI数据库中与已知的核酸序列进行同源性比对,结果显示为酿酒酵母。酿酒酵母突变株MU310的ITS序列具有如SEQ ID No.1所示的核苷酸序列。
(6)遗传稳定性检测。将筛获的突变株MU310在YPD培养基中进行连续20次的转接传代培养,每次传代的接种量为4%,培养时间为2天。转接20次后,将MU310接入含有50mL限氮培养基的三角瓶中,培养4天,每天补加30g/L的葡萄糖,培养结束后收集菌体,测定其油脂含量。结果表明,筛获的酿酒酵母突变株在经过连续20次的传代后油脂含量仍可达到细胞干重的44%,说明其具有良好的遗传稳定性,可被稳定地应用于酿酒酵母油脂的工业化生产中。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一株产油脂的酿酒酵母突变株MU310,其分类命名为Saccharomyces cerevisiaeMU310,已于2023年3月23日保藏于中国普通微生物菌种保藏中心,保藏号为CGMCCNo.26859,保藏地址为北京市朝阳区北辰西路1号院3号。
2.一种微生物菌剂,其特征在于,所述微生物菌剂含有权利要求1所述的酿酒酵母突变株MU310。
3.一种酿酒酵母油脂的生产方法,其特征在于,将权利要求1所述的酿酒酵母突变株MU310加入到培养基中,培养生产酿酒酵母油脂。
4.如权利要求3所述的生产方法,其特征在于,所述培养基包括种子培养基和油脂诱导培养基。
5.如权利要求4所述的生产方法,其特征在于,所述种子培养基为YPD培养基,所述YPD培养基的组分包括酵母浸粉9-11g/L、蛋白胨19-21g/L和葡萄糖19-21g/L。
6.如权利要求4所述的生产方法,其特征在于,所述油脂诱导培养基为限氮培养基,所述限氮培养基的组分包括酵母浸粉3-5g/L,(NH4)2SO4 0.3-0.5g/L,KH2PO4 0.9-1.1g/L,Na2HPO4 0.2-0.3g/L,MgSO4·7H2O 1.4-1.6g/L,FeCl3·6H2O 0.14-0.16g/L,CaCl2·2H2O0.14-0.16g/L,ZnSO4·7H2O 0.01-0.03g/L,MnSO4·H2O 0.05-0.07g/L,葡萄糖19-21g/L。
7.如权利要求4所述的生产方法,其特征在于,所述培养包括种子培养和油脂诱导培养,所述种子培养的条件为置于种子培养基28℃培养2天,所述油脂诱导培养的条件为置于油脂诱导培养基在28℃下180rpm摇床培养4天并每天补加30g/L的葡萄糖。
8.权利要求1所述的酿酒酵母突变株MU310和/或权利要求2所述的微生物菌剂在生产油脂中的应用。
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