CN114058522B - 一种高产角鲨烯的酿酒酵母菌、应用及其选育方法 - Google Patents
一种高产角鲨烯的酿酒酵母菌、应用及其选育方法 Download PDFInfo
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Abstract
本发明涉及微生物技术领域,尤其涉及一种高产角鲨烯的酿酒酵母菌、应用及其选育方法。酿酒酵母GS‑A3(Saccharomyces cerevisiae)保藏编号为CCTCCNO M20211191,于2021年9月17日保藏于中国典型培养物保藏中心(CCTCC)。本发明的酿酒酵母GS‑A3在发酵生产角鲨烯中的应用,产量高,周期短,具有产业化潜力。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及一种高产角鲨烯的酿酒酵母菌、应用及其选育方法。
背景技术
角鲨烯(squalene)又名鲨烯,化学名为2,6,10,15,19,23-六甲基-2, 6,10,14,18,22-二十四碳六烯,分子式为C30H50,是由6个异戊二烯连接而成的不饱和三萜类化合物,具有提高超氧化物歧化酶(SOD)活性、增强机体免疫能力、改善性功能、抗衰老、抗疲劳、抗肿瘤等多种生理功能,在化妆品、食品、保健品和医药等领域具有非常广泛的应用。
目前天然的角鲨烯主要是从动植物原料中直接提取。动物来源主要是从深海鲨鱼的肝油中提取,提取率为肝油的50%左右。但随着世界各国对野生鲨鱼的保护越来越严格,而且野生鲨鱼的数量急剧减少,鲨鱼作为角鲨烯来源不可持续;也有从橄榄油、大豆油、山茶油及其他油作物中进行提取分离,但角鲨烯在植物中的含量极低,一般在重量的0.5%左右。动植物生长周期长,原料预处理复杂,成本高和产量不稳定等问题严
重限制了角鲨烯的工业化生产。除此之外,微生物发酵也是角鲨烯的一个来源,微生物发酵法具有条件温和,不受地理和气候影响和容易进行大规模生产等优势,但目前微生物发酵的角鲨烯产量普遍偏低,生产成本较高。
茶粕因含有角鲨烯合成酶抑制剂常用来作为降血脂的良药,发明专利申请201810769428.0就公开了“一种茶粕用作角鲨烯合成酶抑制剂及用于降脂的用途”。茶粕含有角鲨烯合成酶抑制剂,那还能在茶粕中生产发酵的微生物,说明该微生物合成角鲨烯的前体物质充足,即使在角鲨烯合成酶被抑制的情况下,也能合成足够的角鲨烯用于下游代谢产物,确保细胞生长。所以,从茶粕中筛选出能高产角鲨烯的微生物是可能的。
发明内容
有鉴于此,本发明提供了一种高产角鲨烯的酿酒酵母菌、应用及其选育方法。
一株酿酒酵母(Saccharomyces cerevisiae),酿酒酵母GS-A3保藏编号为 CCTCCNO:M20211191,于2021年9月17日保藏于中国典型培养物保藏中心(CCTCC)。
上述酿酒酵母GS-A3在生产角鲨烯中的应用。
上述酿酒酵母GS-A3角鲨烯干重产量为5.36mg/g DCW。
上述酿酒酵母的筛选方法,包括以下步骤:
S1、从茶粕中分离出目标菌株,纯化发酵后,得到菌悬液;
S2、将菌悬液进行诱变处理,得到诱变菌株;
S3、将诱变处理后的诱变菌株培养后,得到预培养菌液,在预培养菌液中加入预设终浓度的特比萘芬作为选择压力,继续培养,至出现正突变;再次,将得到的一系列正突变以10%的接种量转接下一级选择压力的培养基培养,至出现正突变,以此类推,逐级进化筛选,每级选择压力的特比萘芬终浓度增加,直至得到酿酒酵母GS-A3。
进一步地,S2中诱变方法可以为紫外诱变、EMS诱变或ARTP诱变。
进一步地,S3中特比萘芬的预设终浓度为20μg/mL。
进一步地,S3中每级选择压力的特比萘芬终浓度跨度为20μg/mL。
本发明提供的技术方案带来的有益效果是:本发明的酿酒酵母菌,生产角鲨烯中的应用,产量高,周期短,具有产业化潜力。
附图说明
图1为本发明所述酿酒酵母GS-A3的菌落形态特征图;
图2为本发明所述酿酒酵母GS-A3显微镜形态图;
图3为本发明所述酿酒酵母GS-A3发酵过程曲线图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明实施方式作进一步地描述。
下述试验例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的实验材料,如无特殊说明,均为常规生化试剂商店购买所得。以下实验例中的定量实验,均设置三次重复,结果取平均数。
一.菌株的分离、纯化和发酵
1.1菌株的分离
分别从江西上饶,江西宜春和湖南岳阳的8个不同的茶油场采取茶粕,放置在4℃冰箱中备用。
分别取上述8种茶粕各10g加入到装有50mL无菌生理盐水的摇瓶中,以 220r/min在30℃下震荡24h,然后梯度稀释涂布于分离培养基中,于30℃培养箱中静置培养3d,随机选取具有典型酵母菌菌落特征(白色菌落,表面隆起呈半球状,光滑,较湿润,不透明,边缘整齐)的单菌落,划线分离纯化2-3次,斜面保存备用。
分离培养基:马铃薯浸粉3g/L,葡萄糖20g/L,琼脂粉15g/L。
斜面保存培养基:葡萄糖20g/L,蛋白胨20g/L,酵母浸膏10g/L,琼脂 20g/L。
1.2摇瓶发酵
将上述斜面保存的菌种刮一接种环,接种到装有5mL YPD培养基的PA瓶中进行一级培养,220r/min,30℃培养12h后,得到一级种。将一级种接2mL 到含有200mL YPD培养基的摇瓶进行发酵培养5天,得到发酵液,其中,培养条件为220r/min,30℃。离心收集菌体,冷冻干燥至恒重,测其干重;取部分菌体,通过GC-MS测定菌体中角鲨烯的百分含量。
检测流程:取200μL发酵液至2mL圆底离心管中,4000rpm室温下离心5 min,将离心后样品缓慢弃去上清;加入0.2g的0.5mm的玻璃珠至含菌体离心管中,震荡10min;加入1000μL色谱级丙酮至上述离心管中,震荡10min;将上述样品以12000rpm离10min,收集上清,稀释至合适浓度检测。
使用安捷伦7890A气相色谱仪,色谱柱为Agilent HP-5(30mx0.32 mmx0.25 um),柱箱温度:初始温度100℃,保留2min,以10℃/min升至300℃,保留 3min,进样口温度280℃,进样量1uL,柱流量1mL/min,注入分流比1:50,检测器(FID)温度300℃,氮气作为载气,入口压力是12-18psi,模式为恒流模式。
一共摇瓶考察20个菌株,选择一株角鲨烯干重产量高(3.56mg/g DCW)且生物量大(OD600为21.3)的菌株S6作为进化筛选的对象。
二、菌种鉴定
对S6菌种同时用传统形态学观察方法和分子生物学法进行鉴定,步骤如下:
传统形态学观察方法:经过48h固体YPD培养基上生长的菌落特征为乳白色,圆形,菌落球形突出,表面光滑湿润、不透明,其边缘整齐,菌体粘稠易挑起;将菌落进行活体镜检,观察发现,细胞形态为椭圆形,部分菌体有出芽生殖的现象。具体形态见附图1和图2,可初步确定得到此菌株为酿酒酵母。
分子鉴定:以S6菌株基因组为模板,采用真菌rDNA鉴定通用引物ITS1(SEQ ID NO:1)和ITS4(SEQ ID NO:2),Hieff CanaceTM高保真DNA聚合酶进行核糖体DNA区段的PCR扩增,扩增产物由武汉天一华煜基因科技有限公司进行测序,根据测序结果SEQ ID N0:3,利用BLAST软件从Genbank核酸序列数据库中进行同源序列比对,结果显示为酿酒酵母。
其中,SEQ ID NO:1的序列为:TCCGTAGGTGAACCTGCGG,SEQ ID NO: 2的序列为:TCCTCCGCTTATTGATATGC。
三、酿酒酵母的进化与筛选
作为真菌细胞膜的重要组成成分,麦角固醇结构稳定,专性强,对膜结合酶的活性、细胞活力、膜的流动性以及细胞物质运输等起着重要的作用。角鲨烯单加氧酶催化角鲨烯环氧化进入甾醇合成途径,是角鲨烯衍生途径的第一个酶。该酶抑制剂特比萘芬能显著抑制真菌细胞麦角固醇的合成,使麦角固醇合成不足,使真菌的细胞膜受到破坏,同时角鲨烯在细胞中过度蓄积而起杀菌作用。能在含有特比萘芬的培养基中正常生长的,说明该菌合成角鲨烯的途径强劲,即使在角鲨烯单加氧酶被抑制的情况下,也能合成足够的麦角固醇用于下游代谢产物,确保细胞生长且该细胞对高浓度角鲨烯足够耐受,因此长时间不间断地利用高浓度特比萘芬对酵母生产菌株进行应激定向驯化,就有可能获得高产角鲨烯的菌株。
2.1菌株诱变
2.1.1生长曲线的测定
将斜面保存的菌株S6刮一接种环,接种到装有5mL YPD培养基的PA瓶中进行一级培养,220r/min,30℃培养12h左右。将一级种接2mL到含有200mL YPD 培养基的摇瓶进行发酵培养,每隔两个小时,测一下OD600,直到24h结束,得到菌悬液。绘制生长曲线图,得到对数生长期,计算最佳诱变时间。
2.1.2突变菌株库建立
培养菌体至指数生长期,用蒸馏水稀释至约104个细胞/mL,按照常规紫外、 EMS、ARTP诱变方法诱变,获得突变菌株库,进行下一步液体耐受性进化筛选。
具体的,紫外诱变方法:打开紫外灯(15W),预照射30min,使其功率达到稳定。取5mL菌悬液放入直径为90mm的无菌培养皿中,将培养皿平放在距紫外灯30cm(垂直距离)处的磁力搅拌器上,启动磁力搅拌器,一边照射一边搅拌,照射40-45s后将菌液在黑暗中冷冻保存1~2h备用,保存致死率在75%以上。
2.2液体耐受性进化筛选
将诱变处理后的全部菌体细胞平均转接到若干个装有YPD培养基96孔板中,150rpm,30℃预培养8h,然后在预培养菌液中加入20μg/mL终浓度的特比萘芬作为选择压力,继续培养,至出现正突变;再次,将得到的一系列正突变以10%的接种量转接下一级选择压力的培养基培养,至出现正突变,以此类推,逐级进化筛选,每级选择压力的特比萘芬终浓度增加跨度为20μg/mL,直到得到最终的正突变菌株。
2.3固体平板筛选和菌株鉴定
将最终的正突变菌液稀释涂布到最终筛选浓度的特比萘芬浓度YPD固体选择培养基,待菌落长出,挑取较大的菌落20株斜面保存备用。按照1.2所述的摇瓶发酵及含量测定方法对这20个菌株进行细胞中角鲨烯含量测定。
诱变筛选后,经过近三个月的连续定向进化,筛选得到可以耐受200μg/mL 特比萘芬的突变体GS-A3,其角鲨烯干重产量为5.36mg/g DCW;生物量OD600 为22.4。突变体GS-A3于2021年9月17日保藏于中国典型培养物保藏中心,保藏编号为.CCTCC NO:M20211191,分类命名为:Saccharomyces cerevisiae GS-A3,藏地址为:湖北省武汉市武昌区八一路299号武汉大学校内,中国典型培养物保藏中心。
其中,菌株GS-A3的16S rDNA具体序列如下:
四、酿酒酵母GS-A3的发酵
取10uL保存在甘油管的菌株GS-A3划线于含有200μg/mL特比萘芬的YPD 平板上,30℃培养箱培养三天;将平板上长得个较大的点挑3个到含有5mLYPD 培养基的PA瓶中,30℃摇床220rpm培养16h,获得一级种子;将一级种子以1%的转接量转到含有200mL YPD培养基的摇瓶中,30℃摇床220rpm培养12h,获得二级级种子;向5L生物发酵罐中加入2.5L发酵培养基,以10%的转接量接入活化的二级种子液。发酵过程中,温度控制在30℃,pH用浓氨水全程控制在5.0,发酵罐起始转速为200rpm,通气量为1L/min,溶氧100%,随着发酵的进行,OD不断增大,溶氧会不断下降,待降到20%左右,就通过逐步提升转速和通气量控制溶氧在20%左右,直到最大转速和通气;前期通过调整补料一把发酵罐中葡萄糖浓度控制在1g/L以下;发酵30小时后改用补料二,将发酵罐中乙醇浓度维持在1g/L以下,发酵5天。
发酵培养基中包含如下组分:葡萄糖40g/L,酵母提取物5g/L,硫酸铵12g/L,磷酸二氢钾8g/L,七水硫酸镁6.2g/L,硫胺素200mg/L,吡哆素200mg/L,肌醇500mg/L,生物素50mg/L,泛酸钙200mg/L。
补料一组分为:葡萄糖800g/L,酵母提取物50g/L。
补料二组分为:蔗糖800g/L,酵母提取物50g/L。
如图3所示,其中,▲为发酵过程中角鲨烯产量变化曲线,■为发酵过程中酿酒酵母OD600的变化曲线,酿酒酵母GS-A3的最终生物量(0D600)达到247,角鲨烯产量为881mg/L,酵母细胞中角鲨烯含量达到9.41mg/g。
在不冲突的情况下,本文中上述实施例及实施例中的特征可以相互结合。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,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。
序列表
<110> 湖北冠众通科技有限公司
<120> 一种高产角鲨烯的酿酒酵母菌、应用及其选育方法
<160> 1
<210> 1
<211> 522
<212> DNA
<213> 酿酒酵母( Saccharomycescerevisiae)
gaccaatggggcccaaggttaacaacacaacaatttatttattcattaaatttttgtcaa 60
aaacaagaattttggtaactggaaattttaaaatattaaaaactttcaccaacggatttc 120
ttggttttcccatcgatgaagaacccagggaaatgcgatacgtaatgggaattgcagaat 180
tccgggaatcatcgaatctttgaacccccattgccccccttggtattccagggggcatcc 240
ctgtttgagcgtcatttccttttcaaccattttgtttggtagggagggatactttttggg 300
gttaacttgaaattgcgggccttttcattggaggttttttttttccaaagagaggtttct 360
ctgcgtgcttgaggtataatgcaagtacggtcgttttaggttttaccaactgcggctaat 420
cttttttatactgagcgtattggaacgttatcgataagaagagagcgtctaggcgaacaa 480
tgttcttaaagttgacctcaaatcaggtaggagacccgtc at 522
Claims (2)
1.一株酿酒酵母(Saccharomyces cerevisiae),其特征在于,酿酒酵母GS-A3保藏编号为CCTCC NO:M20211191,于2021年9月17日保藏于中国典型培养物保藏中心(CCTCC)。
2.权利要求1所述酿酒酵母GS-A3在生产角鲨烯中的应用。
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