CN116064266B - 盐胁迫抗性增强的重组酿酒酵母菌及其构建方法与应用 - Google Patents
盐胁迫抗性增强的重组酿酒酵母菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一种盐胁迫抗性增强的重组酿酒酵母菌及其构建方法与应用,属于生物工程技术领域。本发明通过对具有耐盐性的鲁氏酵母菌(Zygosaccharomyces rouxii 3792)克隆脂肪酸合成前体,控制乙酰辅酶A合成的关键基因(ACSS),通过连接到能在酿酒酵母中高拷贝双向表达质粒pY15TEF‑1,转化酿酒酵母(Saccharomyces cerevisiae CEN.PK2‑1C),获得耐盐性能力提升的酿酒酵母基因工程菌。
Description
技术领域
本发明涉及生物工程技术领域,具体而言,涉及一种盐胁迫抗性增强的重组酿酒酵母菌及其构建方法与应用。
背景技术
微生物在发酵和生长过程中容易受到各种环境的干扰,包括外源环境和内源环境。
鲁氏酵母菌(Zygosaccharomyces rouxii)是耐高渗透压的酵母菌,它能在含糖量很高含盐量很高的物料中生长,甚至在饱和食盐条件下仍不能完全抑制它的生长。鲁氏酵母菌是酿制酱油、酱的重要菌种,它能赋予产品乙醇、酯类、糠醛、琥珀酸、呋喃酮等香气成分。因此其被广泛使用于豆瓣、酱油、味噌等传统高盐发酵食品的生产过程中。
酿酒酵母菌(Saccharomyces cerevisiae)是重要的工业模式菌株,广泛应用于酒类食品的生产中。然而,不利的酿造环境因素,例如盐胁迫等胁迫条件,往往使酿酒酵母难以维持高强度的发酵生产。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供一种盐胁迫抗性增强的重组酿酒酵母菌及其构建方法与应用,通过在酿酒酵母菌中过表达乙酰辅酶A合成酶基因(ACSS),可以提高脂肪酸合成能力,进而增强酿酒酵母菌的盐胁迫抗性。
为实现上述目的,本发明构建一种盐胁迫抗性增强的重组酿酒酵母菌。本发明首先将包含有鲁氏酵母菌(Zygosaccharomyces rouxii)的乙酰辅酶A合成酶基因的载体导入酿酒酵母(Saccharomyces cerevisiae)中,发现导入包含乙酰辅酶A合成酶基因的载体的重组菌的脂肪酸合成能力得到提高,进而增强重组菌的盐胁迫耐受能力。
具体地,本发明提供以下技术方案:
第一方面,本发明提供了一种盐胁迫抗性增强的重组酿酒酵母菌,该重组菌中含有外源基因;外源基因包括乙酰辅酶A合成酶基因。
第二方面,本发明提供了上述重组酿酒酵母菌的构建方法,其包括:将乙酰辅酶A合成酶基因连接到表达质粒上,然后将该重组表达质粒导入至酿酒酵母菌中,即获得重组酿酒酵母菌。
第三方面,本发明提供了上述重组酿酒酵母菌在发酵酿造和发酵食品领域中的应用。
第四方面,本发明提供了一种增强酿酒酵母菌盐胁迫抗性的方法,其包括利用上述重组酿酒酵母菌过表达乙酰辅酶A合成酶基因,以控制脂肪酸合成。
本发明具有以下有益效果:
本发明通过克隆表达鲁氏酵母菌ACSS基因,并将其导入酿酒酵母菌中,诱导乙酰辅酶A合成酶基因表达,增强了酿酒酵母脂肪酸的合成能力以及盐胁迫耐受能力。结果表明,在过表达ACSS后,与未过表达菌株相比,脂肪酸总量提高了18.4%,在盐胁迫条件下过表达ACSS菌株的活菌数提高了52.17%。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为实施例1、对比例1的脂肪酸总量结果。
图2为实施例1、对比例1的活菌数统计结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
本发明提供了一种盐胁迫抗性增强的重组酿酒酵母菌,该重组菌中含有外源基因;外源基因包括乙酰辅酶A合成酶基因。
目前,乙酰辅酶A是能源物质代谢的重要中间代谢产物,在体内能源物质代谢中是一个枢纽性的物质。糖、脂肪、蛋白质三大营养物质通过乙酰辅酶A汇聚成一条共同的代谢通路——三羧酸循环和氧化磷酸化,经过这条通路彻底氧化生成二氧化碳和水,释放能量用以ATP的合成。乙酰辅酶A是合成脂肪酸、酮体等能源物质的前体物质,也是合成胆固醇及其衍生物等生理活性物质的前体物质。
本发明通过创造性研究发现,乙酰辅酶A不仅在能源物质代谢中起到关键作用,还发现其能够调节生物体对不利环境的耐受能力。基于此,本发明的发明人将包含有鲁氏酵母菌的乙酰辅酶A合成酶基因的载体导入酿酒酵母中,并诱导乙酰辅酶A的合成,试验结果表明构建的重组菌的脂肪酸合成能力得到提高,进而使更能克服发酵生产中不利的环境因素,特别是盐胁迫环境。
在一些实施方式中,乙酰辅酶A合成酶基因来源于鲁氏酵母菌。
鲁氏酵母菌是耐高渗透压的酵母菌,它能在含糖量很高含盐量很高的物料中生长,甚至在饱和食盐条件下仍不能完全抑制它的生长。鲁氏酵母菌是酿制酱油、酱的重要菌种,它能赋予产品乙醇、酯类、糠醛、琥珀酸、呋喃酮等香气成分。因此其被广泛使用于豆瓣、酱油、味噌等传统高盐发酵食品的生产过程中。
在一些实施方式中,上述鲁氏酵母菌(Z.rouxii)选用的是保藏编号为CGMCCNo.3791的鲁氏酵母菌,其保藏时间为2010年4月29日,保藏地点为中国微生物菌种保藏管理委员会普通微生物中心,地址为北京市朝阳区北辰西路1号院3号。
在一些实施方式中,乙酰辅酶A合成酶基因的NCBI gene ID为ZYGR_0I00290。
在一些实施方式中,乙酰辅酶A合成酶基因的核苷酸序列为SEQ IDNO.1。
在一些实施方式中,重组酿酒酵母菌的出发菌株为酿酒酵母菌CEN.PK2-1C,公开于van Di jken JP et al.,Enzyme Microb.Technol.2000,26:706-714中,基因型为MATaura3-52 leu3-5,112trp1-289his3ΔMAL2-8 c SUC2,由江南大学惠赠。
在一些实施方式中,上述重组酿酒酵母菌的表达质粒为高拷贝双向表达质粒。
在一些实施方式中,上述表达质粒包括pY15TEF-1(Biovector Co.,LTD),其带有亮氨酸LEU标记,由江南大学惠赠。
本发明还提供了上述重组酿酒酵母菌的构建方法,其包括:将乙酰辅酶A合成酶基因连接到表达质粒上,然后将该重组表达质粒导入至酿酒酵母菌中,即获得重组酿酒酵母菌。
具体地,构建方法包括如下步骤:
(1)将两端分别带有Hind III和Xho I两个酶切位点及保护碱基的目的基因ACSS,连接到经过同样酶切的pY15TEF1,转化大肠杆菌DH5a,获得的转化子经菌落PCR验证、提取质粒酶切验证及测序确认为重组质粒pY15TEF1-ACSS。
(2)将构建的重组质粒转化酿酒酵母CEN.PK2-1C,获得盐胁迫抗性提升的重组酿酒酵母。
本发明还提供了上述重组酿酒酵母菌在发酵酿造和发酵食品领域中的应用。
具体地,本发明构建的重组酿酒酵母菌可以应用于发酵食品领域中的豆瓣、酱油、味噌等生产发酵中。
本发明还提供了一种增强酿酒酵母菌盐胁迫抗性的方法,其包括利用上述重组酿酒酵母菌过表达乙酰辅酶A合成酶基因,以控制脂肪酸合成。
在一些实施方式中,过表达为先通过编码乙酰辅酶A合成酶的基因与表达质粒构建含有编码乙酰辅酶A合成酶的基因重组质粒,再将重组质粒导入酿酒酵母菌中,诱导表达乙酰辅酶A合成酶。
在一些实施方式中,酿酒酵母菌为酿酒酵母菌CEN.PK2-1C,其基因型为MATaura3-52 leu3-5,112trp1-289 his3ΔMAL2-8 c SUC2。
在一些实施方式中,乙酰辅酶A合成酶基因来源于鲁氏酵母菌。
在一些实施方式中,鲁氏酵母菌的保藏编号为CGMCC No.3791。
在一些实施方式中,乙酰辅酶A合成酶基因的NCBI gene ID为ZYGR_0I00290。
在一些实施方式中,乙酰辅酶A合成酶基因的核苷酸序列为SEQ ID NO.1。
在一些实施方式中,表达质粒高拷贝双向表达质粒。
在一些实施方式中,表达质粒包括pY15TEF-1。
下面结合具体实施例对本发明进行进一步的阐述。
实施例1
本实施例提供了一种重组酿酒酵母菌及其构建方法。
其中,重组酿酒酵母菌中的外源基因是:核苷酸序列为SEQ ID NO.1的ACSS基因。宿主为:酿酒酵母菌CEN.PK2-1C;载体为pY15TEF-1,均由江南大学惠赠。
构建方法为:
(1)提取鲁氏酵母菌(Z.rouxii CGMCC No.3791)的总RNA,通过PCR获得两端带有Xho I和Hind III两个酶切位点的乙酰辅酶A合成酶基因ACSS。其中克隆ACSS所用的引物为:
F1:CCCTCGAGATGACGGTCAATTATGTATATGCAGG,SEQ IDNO.2;
R1:CCCAAGCTTCTACAATTTTACAGAATCAATCAAACGC,SEQ ID NO.3。
(2)然后连接到经过同样酶切的pY15TEF1质粒,转化大肠杆菌DH5α,获得转化子经菌落PCR验证,提取质粒酶切验证及测序确认为重组质粒pY15TEF1-ACSS。
(3)将构建好的重组质粒转化酿酒酵母(S.cerevisiae CEN.PK2-1C),提取转化子的质粒,酶切验证,确认为阳性转化子。
对比例1
本对比例构建的重组酿酒酵母为:将空质粒pY15TEF1转化酿酒酵母(S.cerevisiae CEN.PK2-1C),提取转化子的质粒,酶切验证,确认为阳性转化子。
实验例1
本实验例为脂肪酸含量测定,具体步骤如下:
(1)取-80℃保藏于甘油储藏液的实施例1及对比例的菌种:重组酿酒酵母菌S.cerevisiae CEN.PK2-1C pY15TEF1-ACSS和S.cerevisiae CEN.PK2-1C pY15TEF1,以10%(V/V)的接种量接种于种子培养基中,30℃静置培养12h至对数中期。
上述种子培养基的成分包括:6.7g/L无酵母氮源培养基(YNB不含(NH4)2SO4),葡萄糖20g/L,色氨酸Try 0.02g/L,组氨酸His 0.02g/L,尿嘧啶Ura 0.02g/L。
(2)收集菌体进行脂肪酸含量测定,脂肪酸提取和测定的方法公布于Wu et al.,J.Ind.Microbiol.Biotechnol.2012,39:1031-1039中。结果见图1。
从图1中可以发现,在过表达ACSS后,与出发菌株相比,脂肪酸总量提高了18.4%。
实验例2
本实验例为盐胁迫条件下的抗性能力检测,具体步骤如下:
(1)取-80℃保藏于甘油储藏液的实施例1及对比例的菌种:重组酿酒酵母菌S.cerevisiae CEN.PK2-1C pY15TEF1-ACSS和S.cerevisiae CEN.PK2-1C pY15TEF1,以10%(V/V)的接种量接种于种子培养基中,30℃静置培养12h至对数中期,得到种子培养液。
上述种子培养基的成分包括:6.7g/L无酵母氮源培养基(YNB不含(NH4)2SO4),葡萄糖20g/L,色氨酸Try 0.02g/L,组氨酸His 0.02g/L,尿嘧啶Ura 0.02g/L。
(2)以10%(V/V)的接种量将种子培养液接种于盐胁迫培养基中处理4h。
上述盐胁迫培养基的成分包括:6.7g/L无酵母氮源培养基(YNB不含(NH4)2SO4),葡萄糖20g/L,色氨酸Try 0.02g/L,组氨酸His0.02g/L,尿嘧啶Ura 0.02g/L,氯化钠NaCl70.2g/L。
(3)将经盐胁迫处理后的培养液以8000r/min(4℃)离心5min,收集菌体,将菌体重悬于无菌水中,控制初始生物量为0.5OD600/mL,稀释1000倍后,取5μL涂布于固体平板培养基中,30℃条件下静置培养48h,计算菌落数,结果见图2。
上述固体培养基的成分包括:6.7g/L无酵母氮源培养基(YNB不含(NH4)2SO4),葡萄糖20g/L,色氨酸Try 0.02g/L,组氨酸His 0.02g/L,尿嘧啶Ura 0.02g/L,琼脂20g/L。
从图2中可以发现,在过表达ACSS后,与出发菌株相比,ACSS株的活菌数提高了52.17%。由此证明,通过在酿酒酵母菌中过表达控制脂肪酸合成的乙酰辅酶A合成酶基因,可以提高脂肪酸合成能力,进而增强重组菌的盐胁迫耐受能力。
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 (6)
1.一种增强酿酒酵母菌盐胁迫抗性的方法,其特征在于,所述方法包括在酿酒酵母菌中过表达乙酰辅酶A合成酶基因。
2.根据权利要求1所述的增强酿酒酵母菌盐胁迫抗性的方法,其特征在于,所述过表达为先通过编码乙酰辅酶A合成酶的基因与表达质粒构建含有编码乙酰辅酶A合成酶的基因重组质粒,再将重组质粒导入酿酒酵母菌中,诱导表达乙酰辅酶A合成酶。
3.根据权利要求2所述的增强酿酒酵母菌盐胁迫抗性的方法,其特征在于,所述乙酰辅酶A合成酶基因来源于鲁氏酵母菌。
4. 根据权利要求3所述的增强酿酒酵母菌盐胁迫抗性的方法,其特征在于,所述鲁氏酵母菌的保藏编号为CGMCC No.3791。
5. 根据权利要求4所述的增强酿酒酵母菌盐胁迫抗性的方法,其特征在于,所述乙酰辅酶A合成酶基因的NCBI gene ID为ZYGR_0I00290,所述乙酰辅酶A合成酶基因的核苷酸序列为SEQ ID NO.1。
6.根据权利要求5所述的增强酿酒酵母菌盐胁迫抗性的方法,其特征在于,所述酿酒酵母菌为酿酒酵母菌CEN.PK2-1C,其基因型为MATa ura3-52 leu3-5,112 trp1-289 his3ΔMAL2-8 c SUC2。
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