CN106987531B - 一株葡萄酒复膜孢酵母及其在单萜生产中的应用 - Google Patents
一株葡萄酒复膜孢酵母及其在单萜生产中的应用 Download PDFInfo
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Abstract
本发明公开了一株葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24，保藏号为CGMCC No.13676。该酵母可用于单萜类生物发酵的生产及与酿酒酵母混合发酵酿造葡萄酒。与模式酿酒酵母Saccharomyces cerevisiae菌株相比，以YPD培养基为基质，该菌发酵能够获得香茅醇、香叶醇、芳樟醇和α‑松油醇4种单萜而酿酒酵母极少或完全不产生单萜。该菌是我国首次从葡萄园中获得分离与鉴定，并首次确定葡萄酒复膜孢酵母中的株系具有从头合成单萜类的能力。该菌的获得为高产单萜工程菌的开发提供重要材料与技术支撑。
Description
技术领域
本发明涉及一株葡萄酒复膜孢酵母及其在单萜生产中的应用，特别涉及一株葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24及其在单萜生产和其他发酵产物中增香的应用。
背景技术
单萜是由两个异戊二稀单元组成具10个碳原子的萜类化合物，单萜及其含氧衍生物具有较强的生物活性和香气，是构成水果、花卉、香精油、葡萄酒等香气的关键组分，如香叶醇、香茅醇、芳樟醇等能提供优雅的花香和果香（http://www.flavornet.org/）。这些单萜既可作为化妆品工业的重要原料，同时也是重要的可食用功能或药用成分，不同的单萜分别具有抗菌、抗氧化、镇痛、缓解焦虑、降血压等多种功能（Breitmaier E. 2006.Terpenes: flavors, fragrances, pharmaca, pheromones. John Wiley & Sons.）。
单萜种类繁多，多天然存在于植物中，所以单萜合成途径研究多以植物为材料。相比于微生物，植物遗传操作较为困难，对其单萜合成相关基因进行定位克隆以及功能分析需耗费较多精力与时间。通常酵母中虽能进行不同单萜之间的生物转化，但不能从头合成单萜，因此目前也还未见有关自然酵母产单萜的报道，已有的用于单萜类生产的工程菌多为转基因株系。
自然状态下，单萜多在植物的腺体、油室等组织中积累，而一些真菌也能产生单萜，如腐生真菌Ceratocystis moniliformis（株系为ATCC12861）在PDB培养基中能产生芳樟醇（Lanza E and Palmer JK. Biosynthesis of monoterpenes by Ceratocystismoniliformis. Phytochemistry, 1977, 16(10): 1555-1560.）。酵母中发现有Kluyveromyces lactis、Torulaspora delbrueckii、Ambrosiozyma monospora、Kloeckera apiculata、Metschnikowia pulcherrima和Candida stellate（Hock R, Benda I andSchreier P. Formation of terpenes by yeasts during alcoholic fermentation.Zeitschrift für Lebensmitteluntersuchung und-Forschung A, 1984, 179(6): 450-452.）等在发酵过程中能积累微量萜烯类物质。已发现的这些野生酵母株系产生的单萜大多含量很低，且仅存在于个别株系中。目前，未见真菌源单萜从头合成能力的研究报道。
与植物不同，大多数酵母缺乏相应的单萜合成酶，所以不具备从头合成单萜的能力（Carrau FM, Medina K, Boido E, Farina L, Gaggero C, Dellacassa E, Versini Gand Henschke PA. De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts. FEMS Microbiol Lett, 2005, 243(1): 107-115.），而部分能积累微量单萜的酵母株系，可能是由甾醇代谢中间产物转化而来（King A and RichardDickinson J. Biotransformation of monoterpene alcohols by Saccharomyces cerevisiae, Torulaspora delbrueckii and Kluyveromyces lactis. Yeast, 2000, 16(6): 499-506.）。利用额外添加单萜的培养基进行培养，发现酿酒酵母具有单萜的生物转化（bioconversion）能力：如（i）香叶醇可转变为香茅醇、芳樟醇、橙花醇和乙酸香叶酯；（ii）橙花醇可转变为香叶醇、芳樟醇和α-松油醇；（iii）芳樟醇可转化α-松油醇以及（iv）香茅醇可转化为乙酸香茅酯等（Gamero A, Manzanares P, Querol A and Belloch C.Monoterpene alcohols release and bioconversion by Saccharomyces species andhybrids. Int J Food Microbiol, 2011, 145(1): 92-97.），不过目前多数单萜生物转化相关的酶基因还未鉴定。
酿酒酵母虽无单萜从头合成能力，但体内GPP短暂积累可被外源（如植物）单萜合成酶作为底物合成单萜。由此以酿酒酵母为生物反应器，通过代谢工程改造，发酵获得单萜类产品得以实现（Oswald M, Fischer M, Dirninger N and Karst F. Monoterpenoidbiosynthesis in Saccharomyces cerevisiae. FEMS Yeast Res, 2007, 7(3): 413-421.）。值得注意的是，由于单萜合成前体GPP供应不足，且单萜本身能抑制麦角固醇合成而具抗菌活性，酿酒酵母普遍对单萜的耐受性较低，从而影响单萜生产潜力（Liu J, ZhangW, Du G, Chen J and Zhou J. Overproduction of geraniol by enhanced precursorsupply in Saccharomyces cerevisiae. J Biotechnol, 2013, 168(4): 446-451.）。目前，食品、化工或医药行业所使用的单萜依然主要由植物提取或化学合成得来，但提取和合成过程中易产生大量有机溶剂或重金属污染，同时也会受到原料供应、农药残留以及分离纯化困难等因素的限制。
本发明人课题组从刺葡萄皮中分离到了一株能够产单萜的酵母菌株葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24。目前，世界上仅有智利等国报道从葡萄园分离过葡萄酒复膜孢酵母，但未对其进行深入分析，也未见报道该类菌具有产单萜类的能力。
发明内容
本发明所要解决的技术问题是解决目前食品、化工或医药行业中的单萜生产，因植物提取或化学合成带来的有机溶剂或重金属污染的问题，同时解决受到原料供应、农药残留以及分离纯化困难等因素的限制。
由此，本发明提供了一株适合源于开发高产单萜的工程菌株，该菌株为葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24，保藏号为CGMCC No. 13676。
本发明提供所述的葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24在单萜类代谢工程中的应用，包括单萜类的生产和其他发酵产物中增香的应用；例如在酿造葡萄酒中的应用，可与酿酒酵母混合发酵酿造葡萄酒。
所述的应用，其利用所述细菌的发酵液、发酵液提取物进行应用。
本发明所述葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24已于 2017年2月21日保藏于中国微生物菌种保藏管委理员会普通微生物中心（简称CGMCC，地址为：中国北京市朝阳区大屯路），保藏号为CGMCC No.13676，经检测存活。
本发明所述葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24经过在WL营养琼脂培养基上培养48h，其菌落形态与酿酒酵母有所差异，该菌菌落为乳白色，形状不规则，中间微突，边缘放射形毛状，表面不光滑。
本发明葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24从我国湖南省常德市刺葡萄（类型：湘酿1号刺葡萄）果皮上分离、筛选、鉴定得到，是我国首次从葡萄园分离到具有出头合成单萜能力的自然野生酵母，利用本发明的葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24以YPD培养基为原料，能产生很高含量的香茅醇（17.16mg/L）、芳樟醇（3.00mg/L）、α-松油醇（2.03mg/L）、香叶醇（1.36mg/L）等4种单萜（合计23.55mg/L），而对照酿酒酵母RC212只能产生极微量的芳樟醇和α-松油醇，S288C未检测到上述单萜类化合物。由于培养基中仅有葡萄糖作为碳源，因此推断该菌具有从头合成单萜的能力。该能力赋予该菌能够以简单便宜的碳源直接合成单萜类的能力。本发明所述葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24的获得为认识自然界单萜的生物合成提供了独特的材料，并为开发高产单萜的工程菌提供重要的材料与技术支撑。
附图说明
图1 WL培养基上葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24菌落的形态。
图2葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24扫描电镜结果。
图3 三株菌YPD培养液GC-MS检测结果，其中，箭头指示数字表示出峰时间；*表示该位点检测到的质谱特征峰与LA24同位置特征峰一致；▲表示该位点未出峰或质谱特征峰与LA24同位置特征峰不一致（非单萜）。
图4葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24所产单萜类物质质谱鉴定结果。
图5 葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24单萜耐受性结果。
具体实施方式
下述实施例中的方法，如无特别说明，均为常规方法。
下述实施例中，WL营养琼脂培养基配方如下：
酵母提取物，4g/L；蛋白胨，5g/L；葡萄糖，50g/L；KH2PO4，0.055g/L；KCl，0.425g/L；CaCl2，0.125g/L；MgSO4，0.125g/L；FeCl3，0.0025g/L；MnSO4，0.0025g/L；溴甲酚绿，0.022g/L；琼脂，20g/L；pH = 6. 5。
YPD培养基配方如下：
葡萄糖，20g/L；蛋白胨，20g/L；酵母抽提物，10g/L；自然pH值，121℃，灭菌15min。固体培养基加入20g/L琼脂，微波炉加热完全融化后灭菌。
实施例 1、葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24的分离、纯化及其鉴定
本发明人课题组前期分别从中国湖南的两个刺葡萄主产地中方县和澧县，共采集四个样本：中方县的湘珍珠，米葡萄，紫秋1号及澧县的湘酿1号，并从这四个不同刺葡萄品种/类型的果皮表面和自然发酵的四个不同时期（果实破碎后、发酵启动时期、发酵旺盛时期及发酵结束时期）分离到酵母菌405株，通过WL培养基的形态分类和D1/D2区域26S序列比对，将405株酵母菌分为13个不同的种，其中分离自发酵各个时期最多的非酿酒酵母种主要是H. uvarum和H. opuntiae，其次为酿酒酵母，其他非酿酒酵母株系较少且分布离散。Interdelta分析将酿酒酵母分为5个类型。
在酵母菌分离鉴定过程中，我们发现一株酵母在培养皿中具有明显的玫瑰和类似植物香精油的香味，后经GC-MS检测发现该菌能产生香茅醇等4种单萜类。该菌编号为LA24，分离自澧县湘酿1号果皮表面，其菌落形态与酿酒酵母有所差异。该菌菌落为乳白色，形状不规则，中间微突，边缘放射形毛状，表面不光滑（图1）。通过D1/D2区域26S序列比对发现该菌与Saccharomycopsis vini遗传关系最近。Saccharomycopsis vini与酿酒酵母同为Saccharomycetales 酵母目。Saccharomycopsis属共十多个种。目前，对该属酵母研究还很少，只有巴西、智利等国Saccharomycopsis vini从葡萄园中分离的报道，但未见有深入研究，也未有见到该类菌具有产单萜能力的报道。
该菌株经过形态学观察及26S序列分析后对所有鉴定结果综合分析后，鉴定 LA24为葡萄酒复膜孢酵母（Saccharomycopsis vini）的新株。具体鉴定结果如下：
形态学观察方法和结果：经过3天培养，LA24在YPD固体培养基上菌落形态与酿酒酵母有所差异。该菌菌落为乳白色，形状不规则，中间微突，边缘放射形毛状，表面不光滑（图1）；扫描电镜检测结果显示酵母菌体呈椭球形，0.7-0.9um×1.4-2.8um，单个排列，形成假菌丝（图2）。显微观察发现LA24与酿酒酵母一样，其细胞可以以出芽方式生长。
酵母的26s rDNA D1/D2 区序列具有显著的种间差异性，可作为鉴别酵母菌种的分类依据，酵母的26s rDNA D1/D2 区序列分析鉴定方法和结果如下：
（1）提取酵母菌基因组DNA；
（2）LA24菌株26s rDNA D1/D2 区序列扩增：
扩增正反向引物序列如下（Kurtzman and Robnett 1998）：
NL1（5’-GCATATCAATAAGCGGAGGAAAAG-3’）
NL4（5’-GGTCCGTGTTTCAAGA-3’）
扩增体系（50ul体系）：1×PCR buffer；1.5mol/L MgCl2；10mmol/L dNTP；引物NL1和NL4各为0.2mmol/L；Taq酶0.3U；模板DNA （10ng-1ug/uL）；加灭菌双蒸水定容至50ul。
扩增反应条件为：95℃ 5min（预变性）；94℃ 1min（变性），52℃1min（退火），72℃80s（延伸），循环36次；72℃ 8min（补充延伸）。
26s rDNA D1/D2 区序列分析：将PCR扩增产物进行测序，测序引物同PCR引物NL1或NL4 ；根据测序结果，利用BLAST软件从GenBank核酸序列数据库中进行同源序列搜索，比较供试菌株与已知酵母菌株相应序列的相似程度。
根据目前酵母菌分子系统学研究领域所流行的观点，即具有相同或相似26s rDNAD1/D2 区（序列同源性> 99% ）的菌株属于同一物种。根据序列分析发现测试菌株LA24与基因库参考菌株葡萄酒复膜孢酵母Saccharomycopsis vini KY109520.1 26s rDNA D1/D2区的序列同源性最高为99%，因此可以确认鉴定结果，即LA24为葡萄酒复膜孢酵母Saccharomycopsis vini。
表1酵母LA24和RC212的26s rDNA D1/D2区域序列分析
RC212*：表示商业化活性干酵母Saccharomyces cerevisiae RC212, 购自法国Lalemand公司。
采用两种分类鉴定方法对LA24进行鉴定，其鉴定结果完全符合葡萄酒复膜孢酵母菌株的特点，故可将LA24鉴定为葡萄酒复膜孢酵母Saccharomycopsis vini，已于 2017年2月21日将该菌保藏于中国微生物菌种保藏管委理员会普通微生物中心（简称CGMCC，地址为：中国北京市朝阳区大屯路），保藏号为CGMCC No.13676，经检测存活。
实施例2、葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24的产单萜能力检测
将葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24与商业酿酒酵母RC212和酿酒酵母S288C进行平行培养，采用YPD培养基培养至稳定期，通过固相萃取后，利用GC-MS检测细胞外的代谢产物，并采用标准曲线法进行了定量（以mg/培养基体积计）（张明霞.葡萄酒香气变化规律研究—着重于关键酿造工艺对葡萄酒香气的影响.中国农业大学博士学位论文.2007）。结果发现LA24能产生很高含量的香茅醇（17.16mg/L）、芳樟醇（3.00mg/L）、α-松油醇（2.03mg/L）、香叶醇（1.36mg/L），而RC212能产生极微量的芳樟醇和α-松油醇，S288C未检测到上述单萜类化合物（表2、图3、图4）。我们对课题组分离的5个类型酿酒酵母共选择10株进行了培养和GC-MS分析，也发现3株能产生痕量的芳樟醇和α-松油醇，但均不产生香茅醇和香叶醇。目前，葡萄酒复膜孢酵母仅在智利等葡萄园有见分离的报道，更未见该菌具有产单萜类能力的报道。本发明是首次葡萄酒复膜孢酵母株系具有产单萜类的能力。
表2 酵母培养液GC-MS检测结果
注：a，表示峰面积平均值用于参考标准曲线进行含量的判断；n.d，未检出。
基于LA24合成单萜的能力，课题组进行了其他方面的验证。通过YPD培养基分别添加葡萄糖、蔗糖发现LA24均能正常生长，且均能产生4种单萜；将LA24种子菌液按1：100接种至YPD液体培养基。每隔12h取前一培养物按1：100接种至新YPD液体培养基，共继代22次，发现LA24依然有产单萜能力。
针对LA24单萜耐受性：将菌液（OD600=1）中分别添加终浓度0mg/L（对照）、100mg/L、200mg/L、400mg/L、800mg/L的芳樟醇处理4h后，梯度稀释，取1ul点YPD固体平板生长2d，发现稀释100倍前800mg/L芳樟醇处理LA24均能生长（图5A），而S288C稀释100倍前400mg/L芳樟醇处理生长受抑制。另外，将种子菌液置于添加了芳樟醇终浓度分别为0~800mg/L（同上）的YPD固体平板中，梯度稀释，培养2d（点样1ul），发现大于800mg/L芳樟醇中LA24不再生长，随浓度增加芳樟醇对LA24生长有抑制，但400mg/L中依然能够生长（图5B），而S288C在≥200mg/L不再生长。这表明LA24具有极高的单萜类耐受性，可作为理想的单萜类生产工程菌。
Claims (6)
1.一种葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24，保藏号为CGMCC No.13676。
2.如权利要求1所述的葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24在单萜类代谢工程中的应用，所述单萜类为香茅醇、芳樟醇、α-松油醇或香叶醇。
3.如权利要求2所述的应用，其特征在于：包括单萜类的生产和葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24发酵产物中增香的应用。
4.如权利要求3所述的应用，其特征在于：在酿造葡萄酒中的应用。
5.如权利要求4所述的应用，其特征在于：与酿酒酵母混合发酵酿造葡萄酒。
6.如权利要求3所述的应用，其特征在于：其利用所述葡萄酒复膜孢酵母（Saccharomycopsis vini）LA24的发酵液、发酵液提取物进行应用。
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