CN111676146B - 一株耐酸酿酒酵母及其应用 - Google Patents

一株耐酸酿酒酵母及其应用 Download PDF

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CN111676146B
CN111676146B CN202010631510.4A CN202010631510A CN111676146B CN 111676146 B CN111676146 B CN 111676146B CN 202010631510 A CN202010631510 A CN 202010631510A CN 111676146 B CN111676146 B CN 111676146B
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saccharomyces cerevisiae
mtpfo
organic acids
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刘龙
陈坚
堵国成
李江华
吕雪芹
孙利
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Jiangnan University
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Abstract

本发明公开了一株耐酸酿酒酵母及其应用，本发明的酿酒酵母(Saccharomyces cerevisiae)MTPfo‑4，于2020年6月10日保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 2020199。在本发明中，利用外源添加苹果酸作为胁迫压力，实验室定向进化筛选得到S.cerevisiae耐酸突变株MTPfo‑4，耐受最低pH为2.44，这也是目前报道的酿酒酵母耐受的最低pH。同时耐受多种有机酸的突变株MTPfo‑4，对外源苹果酸的耐受增加至86.8g/L。进一步对得到的突变株MTPfo‑4进行鉴定，该突变株性状稳定生长良好，同时可以耐受多种有机酸(乳酸、苹果酸、琥珀酸、富马酸、柠檬酸、葡萄糖酸、酒石酸)，另外对无机酸(HCl、H2PO3)也有很强的耐受能力，这在目前S.cerevisiae研究报道中是很难达到的。以期作为耐酸底盘细胞工厂，用于多种短链有机酸的生产。

Description

一株耐酸酿酒酵母及其应用
技术领域
本发明涉及一株耐酸酿酒酵母及其应用，属于微生物技术领域。
背景技术
短链有机酸(Short-chain organic acids)作为微生物的天然代谢产物，近年来已广泛用于食品，生物医药，化妆品，洗涤剂，聚合物和纺织品等行业。微生物生产短链有机酸主要包括丙酸，丙酮酸，乳酸，3-羟基丙酸，三羧酸(TCA)循环中间代谢产物α-酮戊二酸，苹果酸，琥珀酸，富马酸和柠檬酸。其中，丙酸的钙盐，钠盐和铵盐具有很强的防腐能力，可作为防腐剂用于动物饲料和人类食品中。衣康酸可制备合成纤维，合成树脂和塑料，其酯衍生物可以用于苯乙烯或聚氯乙烯增塑剂。苹果酸作为天然果汁中重要成份，味道柔和(具有较高的缓冲指数)，具特殊香味，不损害口腔与牙齿，代谢上有利于氨基酸吸收，不积累脂肪，是新一代的食品酸味剂，被生物界和营养界誉为“最理想的食品酸味剂”。柠檬酸是多功能的、无毒的，被FAO/WHO(联合国粮农组织/世界卫生组织)专家委员会认定为安全的食品添加剂，被称为第一食用酸味剂，在饮料工业与酿造酒中，不仅能赋予产品水果风味，而且还有增溶、缓冲、抗氧化等作用，使色素、香气、糖分等成分交融协调，形成调和的口味和香气，同时能增强抗微生物防腐效果。
酿酒酵母(Saccharomyces cerevisiae)，GRAS菌株，可以作为良好的代谢工程平台生产多种有机酸产品，例如丙酮酸、乳酸，苹果酸、琥珀酸、富马酸和衣康酸。在S.cerevisiae中，硫胺素生物合成调控基因中有两个基因tHI2和tHI3，当两个基因被破坏后突变体FMME-002ΔTHI2和FMME-002ΔTHI3都能用于丙酮酸的生产，而且，FMME-002ΔTHI2的丙酮酸盐产量较高，并且当添加0.04m M硫胺素时，丙酮酸的浓度为8.21g/L。为构建富马酸的高产菌株，研究人员通过敲除富马酸酶(Fumarase)，表达来自米曲霉的基因pyc(丙酮酸羧化酶，Pyruvate carboxylase)，最终S.cerevisiae工程菌株的富马酸产量为5.64g/L。在合成衣康酸的改造过程中，敲除酿酒酵母的三个基因ade3，bna2和tes1，高密度发酵，最终滴度为0.168g/L。目前报道的关于S.cerevisiae生产短链有机酸的产量普遍较低，基于市场经济效益考虑很难达到工业化生产水平。
环境pH值是影响细胞代谢和行为的基本信号。因此，对于工程细胞来说，设计和构建底盘细胞响应不同环境pH，拥有越来越复杂的代谢调控功能是至关重要的。在S.cerevisiae中，研究比较系统的是CCW14启动子(stress-responsive yeast promoters，一种应激-反应启动子)。CCW14是一种细胞壁糖蛋白，在柠檬酸(pH 3.5)胁迫下可以被CWI途径激活，提高宿主细胞对酸的耐受能力。研究人员对该启动子进行了进一步压力-诱导合成(stress-inducible synthetic promoters)，得到一系列的合成型启动子，最终筛选得到了耐受能力强的突变体CCW14v5，该启动子的应用与天然启动子TEF1相比，提高了细胞的酸耐受能力，同时在pH 3.0条件下，表达了来源于植物乳杆菌的乳酸脱氢酶基因(ldhL)来生产乳酸，由合成的pH诱导型启动子控制的ldhL菌株(乳酸滴度为2.9–7.9g/L)优于天然启动子TEF1控制的ldhL菌株(乳酸产量为0.72g/L)。
工业生产过程中，基于经济效益的考虑，生产商对有机酸产量要求较高，但在宿主细胞构建过程中，随着有机酸浓度的不断积累形成了低pH环境，对宿主细胞的生长有很大程度的抑制。据报道，丙酸发酵过程中，当浓度达到10g/L时，就已经成了最大的限制因素。另外，丙酮酸，3-羟基丙酸，乳酸，苹果酸，柠檬酸等短链有机酸，在发酵过程中形成的低pH对宿主细胞生长有很强的抑制作用。
目前报道的微生物生产短链有机酸主要是添加中和剂(CaCO3)来调节发酵过程的低pH，生成的产物主要以有机酸盐的形式存在，后续要先经过酸解，才能进一步的分离、纯化。一方面增加了产物分离、纯化的成本和工序，另一方面如果前期添加的中和剂，或者后续酸解所用的酸反应不完全，还会造成资源的浪费和环境的污染。由于细胞本身存在很强的抗逆性，增加了耐酸系统的构建的难度，或者已经开发的耐酸底盘细胞也只能用于一种有机酸的生产，产品谱较窄，无法应用于多种有机酸的生产。
发明内容
为解决上述存在的低pH抑制宿主细胞生长，添加中和剂提高成本、增加后续分离和纯化工序、容易造成环境污染等问题。本发明中为了得到经济有效且发酵过程不添加非中和的宿主细胞(cost-effective non-neutralizing fermentation host strains)，外源添加苹果酸作为筛选压力，对酿酒酵母进行迭代进化，解决酿酒酵母低pH条件下生长及生产短链有机酸的问题，主要发明目的是提高酿酒酵母耐酸能力，构建酿酒酵母耐酸底盘细胞工厂，用于生产多种短链有机酸。
本发明的第一个目的是提供一株耐酸酿酒酵母，命名为酿酒酵母(Saccharomycescerevisiae)MTPfo-4，于2020年6月10日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC M 2020199。
进一步地，所述的酿酒酵母耐受最低pH为2.44。
进一步地，所述的酿酒酵母对外源苹果酸的耐受达到86.6g/L。
本发明的第二个目的是提供所述的酿酒酵母在短链有机酸生产中的应用。
进一步地，所述的应用是采用所述的酿酒酵母作为底盘细胞，构建生产短链有机酸的菌株。
进一步地，所述的短链有机酸为丙酮酸，3-羟基丙酸、乳酸、苹果酸、琥珀酸、富马酸、柠檬酸、葡萄糖酸、酒石酸、糠酸中的一种或多种。
本发明的第三个目的是提供一种包含所述的酿酒酵母的微生物菌剂。
进一步地，所述的微生物菌剂为固体菌剂。
进一步地，所述的微生物菌剂为液体菌剂。
本发明的有益效果：
在本发明中，利用外源添加短链有机酸苹果酸作为胁迫压力，实验室定向进化筛选得到S.cerevisiae耐酸突变株MTPfo-4，耐受最低pH为2.44，这也是目前报道的酿酒酵母耐受的最低pH。同时耐受多种有机酸的突变株MTPfo-4，对外源苹果酸的耐受增加至86.6g/L。进一步对得到的突变株MTPfo-4进行鉴定，该突变株性状稳定生长良好，同时可以耐受多种有机酸(乳酸、苹果酸、琥珀酸、富马酸、柠檬酸、葡萄糖酸、酒石酸)，另外对无机酸(HCl、H2PO3)也有很强的耐受能力，这在目前已知的S.cerevisiae研究报道中是很难达到的。以期作为耐酸底盘细胞工厂，用于多种短链有机酸的生产。
生物材料保藏：
酿酒酵母(Saccharomyces cerevisiae)MTPfo-4，于2020年6月10日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC M 2020199。
附图说明
图1为耐酸酿酒酵母的进化与筛选；
图2为酿酒酵母及其突变株耐受苹果酸生长曲线；
图3为突变株MTPfo-4对其它酸(pH 2.44)的耐受分析；
图4为突变株MTPfo-4对其它酸(pH 3.0)的耐受分析。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：耐酸酿酒酵母细胞的进化与筛选
以酿酒酵母(Saccharomyces cerevisiae CEN.PK2-1C)为出发菌株，外源添加不同浓度的苹果酸，定向进化筛选得到耐受更低pH的酿酒酵母突变株。本发明中，通过添加不同浓度的苹果酸，控制pH逐级降低(从pH 6.0开始，外源苹果酸的添加量为4.6g/L)，每级pH中细胞进化到能够生长后，平级培养进行富集(目标是该pH下细胞在48h的OD600达到稳定，前后两次没有明显增加；目的是增加突变细胞的数量，使该pH下细胞生长更加稳定)后再进行下一级pH的进化。其中，初始pH 6.0至pH 4.0每级pH跨度为0.2，即6.0、5.8、5.6···4.2、4.0；pH 4.0至pH 3.5每级pH跨度为0.1，即4.0、3.9、3.8···3.6、3.5；pH 3.5至pH3.0，每级跨度为0.05，即3.5、3.45、3.4···3.05、3.0；pH 3.0至pH 2.8，每级跨度为0.02，即3.0、2.98、2.96···2.82、2.80；pH低于2.8时，每级pH跨度为0.01，即2.8、2.79、2.78···2.45、2.44。在初始pH 6.0至pH 4.0时细胞生长较快，可能可以直接从pH 4.0开始进化，但为了得到更加稳定的耐酸菌株，本发明建议从pH 6.0开始采用迭代进化法(Iterative evolution)每一级pH得到的进化菌株将作为下一次传代的出发菌株，直到进化出耐受更低pH的突变体。
如图1所示，经过近四个月的连续定向进化，筛选得到可以耐受86.6g/L苹果酸(pH2.44)的突变体MTPfo-4。每阶段的pH条件下培养48h后，培养基的pH维持稳定，没有出现升高，反而有降低的趋势。例，突变体MTPfo-4在pH 2.44条件下培养48h后，pH稳定在2.42-2.44。
在pH 2.44条件下，培养48h后OD600达到18.5，结果显示该突变株可以稳定生长，这也是目前报道的S.cerevisiae能耐受的最低pH。
筛选得到的酿酒酵母突变体MTPfo-4已于2020年6月10日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC M2020199。
实施例2：耐酸酿酒酵母细胞性状分析
为了验证得到的S.cerevisiae突变株MTPfo-4在低pH条件下的稳定性，将MTPfo-4与出发菌株CEN.PK2-1C在不同外源苹果酸添加量的情况下进行生长比对。外源苹果酸的添加浓度(g/L)分别为0、30、40、50、60、70、80、90、100，每隔12h取样，至72h，测定菌株的生长曲线，测定结果如图2所示，与出发菌株CEN.PK2-1C相比，突变株MTPfo-4在外源添加苹果酸的条件下生长有显著提高，对外源添加苹果酸有很强的耐受能力。
实施例3：耐酸酿酒酵母细胞耐受其它酸的能力分析
为了分析突变株MTPfo-4对其它酸的耐受能力，本发明中通过外源添加10种酸，包括2种无机酸HCl、H3PO4，8种有机酸乳酸、苹果酸、富马酸、琥珀酸、酒石酸、糠酸、葡萄糖酸、柠檬酸，进行细胞耐酸能力的分析，控制初始pH 2.44，每12h取样，至72h，通过测定OD600来分析细胞耐酸性能。结果如图3所示，与出发菌株CEN.PK2-1C相比，MTPfo-4对其中6种酸HCl、H3PO4、乳酸、苹果酸、柠檬酸、葡萄糖酸有较强的耐受性，但是，在该pH条件下，所有10种酸对CEN.PK2-1C的生长有强烈的抑制作用。由于pH 2.44条件下，富马酸、琥珀酸、糠酸、酒石酸对突变株MTPfo-4也有抑制作用，所以，在本发明中，将以上4种酸初始pH调至3.0，每12h取样，至72h，通过测定OD600再一次分析细胞的耐酸能力。结果如图4所示，与出发菌株CEN.PK2-1C相比，MTPfo-4对上述4种酸有较强的耐受性，同样在该pH条件下，所有4种酸对CEN.PK2-1C的生长有强烈的抑制作用。结合上述所有结果表明突变株MTPfo-4
除了对苹果酸耐受以外，对其它酸也有很强的耐受能力，有作为耐酸底盘细胞生产多种有机酸的潜力。并进一步开发用于多种短链有机酸的生产。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (6)

1.一株耐酸酿酒酵母，其特征在于，命名为酿酒酵母（Saccharomycescerevisiae）MTPfo-4，于2020年6月10日保藏于中国典型培养物保藏中心，保藏地址为中国武汉武汉大学，保藏编号为CCTCC M 2020199。
2.权利要求1所述的酿酒酵母在短链有机酸生产中的应用。
3.根据权利要求2所述的应用，其特征在于，所述的短链有机酸为丙酮酸，3-羟基丙酸、乳酸、苹果酸、琥珀酸、富马酸、柠檬酸、葡萄糖酸、酒石酸、糠酸中的一种或多种。
4.一种包含权利要求1所述的酿酒酵母的微生物菌剂。
5.根据权利要求4所述的微生物菌剂，其特征在于，所述的微生物菌剂为固体菌剂。
6.根据权利要求4所述的微生物菌剂，其特征在于，所述的微生物菌剂为液体菌剂。
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