CN116286939B - 一种提高酿酒酵母核酸产量的方法及应用 - Google Patents
一种提高酿酒酵母核酸产量的方法及应用 Download PDFInfo
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Abstract
本发明涉及一种提高酿酒酵母核酸产量的方法及应用,属于生物工程技术领域。采用外源补充磷酸盐和强化磷酸盐内源转运的策略,通过在超表达磷酸盐转运蛋白PHO84基因基础上添加磷酸盐,进一步提升高核酸酵母的核酸产量,利用该方法培养的酿酒酵母可用于核糖核酸、核苷酸、核苷酸衍生物及酵母抽提物等的制备及生产中,上述产物可被广泛应用于食品、医药、保健品、农业以及畜牧养殖等行业,拥有良好的市场前景。
Description
技术领域
本发明涉及一种提高酿酒酵母核酸产量的方法及应用,属于生物工程技术领域。
背景技术
核糖核酸(简称RNA)不仅在细胞中作为执行重要的生物学功能,对人与动物生长、健康也有极其重要的作用,在医药、保健品、食品和养殖业等诸多领域具有广阔的应用开发前景。由于RNA的生产大部分为微生物发酵所得,酿酒酵母不产生任何毒素,是食品安全级微生物,生长传代速度较快,易培养,并且酿酒酵母本身营养较高,含有丰富的蛋白质、核酸和糖类等营养成分,是公认最理想的RNA来源,提取完核酸的酵母菌体还可作为优质的蛋白质被添加到饲料中,酿酒酵母的核酸含量平均在6%~8%,高于大部分菌株,但仍需要进一步提升才能满足市场需求。因此,选育核酸含量高的酵母菌种就成为了核酸工业生产的关键。
通过菌种选育和改良的方法选育出的高核酸酿酒酵母菌株用于工业化生产,制备出满足市场需求的核酸,还需要优化适于高核酸酿酒酵母菌株的培养条件,比如培养基配方和发酵参数等,以进一步提高高核酸酿酒酵母的核酸产量。倪晓丰等通过单因素以及正交试验获得最优的糖蜜培养基组分为糖蜜、酵母浸粉5%、硫酸铵0.05%、磷酸二氢钾0.05%、硫酸亚铁0.05%、硫酸锌0.10%,在此条件下, 菌株的胞内RNA含量较优化前提高了17.20%(倪晓丰,赵宾,王东旭,彭伟林,陈叶福,肖冬光,郭学武. 硫酸二乙酯化学诱变选育高核糖核酸酿酒酵母及培养基组成优化. 中国酿造. 2018,37(08): 32-36)。俞灿等首先通过单因素优化实验对糖蜜、酵母浸粉、磷酸二氢钾、谷氨酸钠、谷氨酸钠和硫酸亚铁的含量进行了优化,确定了适于影响高核酸酿酒酵母菌株J-5-9的最适发酵培养基为:糖蜜1.8%、酵母浸粉4%、磷酸二氢钾0.01%、谷氨酸钠0.05%、硫酸亚铁0.1%,利用优化发酵培养基和发酵罐中碳、氮和磷源的流加补料工艺,在10 L发酵罐中培养该菌株,RNA含量达到8.11%(俞灿,郑国斌,姚娟,李库,唐冠群,吕江波,王志,陈雄. 富含核糖核酸酿酒酵母的选育及其高密度发酵工艺. 中国酿造. 2016,35(12): 66-71)。李小坤等通过对高核酸酿酒酵母菌株Y17aM3-12进行了培养条件优化,在最佳培养条件下添加磷酸可使Y17aM3的RNA含量提高至119 mg-RNA/g-DCW,对Y17a M3生长没有促进作用亦没有抑制作用(李小坤,王旺,林影,梁书利.常压室温等离子体(ARTP)诱变选育高核酸酿酒酵母. 现代食品科技. 2018,34(12):137-144+238)。
无机磷酸盐(Pi)是构成酿酒酵母核酸、磷脂和许多辅酶的成分;参与碳水化合物代谢主要步骤的磷酸化过程,生成高能磷酸化合物(ATP),贮存和运送能量。可见,对磷的吸收和利用对酿酒酵母的遗传、能量代谢、细胞膜的完整性和细胞内信号传递等生物过程中发挥着重要的作用。磷酸盐在培养基中还具有缓冲作用,可调节培养基的渗透压、pH、氧化还原电位等,是重要的pH缓冲剂。然而,培养酿酒酵母常用的原料(如玉米浆、糖蜜、淀粉水解糖等)中磷含量明显不足,对酿酒酵母菌体生长和核酸产量产生限制。
发明内容
针对目前市场上的酿酒酵母菌株产核酸能力不足、核酸产量不高、生物量有待进一步提升、培养酿酒酵母的原料中磷含量明显不足对酿酒酵母的核酸产量和生物量产生较大限制等的问题,本发明提供一种适于提高酿酒酵母核酸产量的方法,即采用外源补充磷酸盐和强化磷酸盐内源转运的策略,通过在超表达磷酸盐转运蛋白PHO84基因基础上添加磷酸盐,进一步提升高核酸酵母的核酸产量,利用该方法培养的酿酒酵母可用于核糖核酸、核苷酸、核苷酸衍生物及酵母抽提物等的制备及生产中,上述产物可被广泛应用于食品、医药、保健品、农业以及畜牧养殖等行业,拥有良好的市场前景。
本申请的技术方案如下:
一种提高酿酒酵母核酸产量的方法,在酿酒酵母中增加磷酸盐供应可提高酵母的生物量、RNA含量和核酸产量。
优选地,所述的增加磷酸盐供应的方法为:超表达高亲和力磷酸盐转运蛋白PHO84基因和按照磷元素的终浓度0.05%添加磷酸盐。
优选地,所述的高亲和力磷酸盐转运蛋白PHO84基因来源于酿酒酵母,其核苷酸序列如SEQ ID NO.1所示。
本发明的另一目的,保护一种酿酒酵母,所述的酿酒酵母具有高核酸产量,采用上述方法获得。
本发明的另一目的,保护利用上述方法发酵获得的酿酒酵母菌体在核糖核酸、核苷酸、核苷酸衍生物及酵母抽提物的生产中的应用。
本发明的有益效果:
通过超表达酿酒酵母高亲和力磷酸盐膜转运蛋白PHO84基因提高酿酒酵母的RNA产量,将超表达前后的菌株在1 L发酵罐中进行高密度培养,PHO84基因超表达菌株YM84,其RNA含量、生物量和RNA产量分别提高了5.6%、17.4%和10.6%,与在培养基中补充0.05%的磷酸盐产生的效果相当;在过超表达PHO84基因基础上继续补充0.05%的磷酸盐,菌体的RNA含量提高了41.1%,RNA产量提高了46%,在超表达磷酸盐转运蛋白PHO84基因基础上补充磷酸盐来增加细胞磷酸盐供应,产生了意想不到的叠加效果。该方法为提高酿酒酵母RNA产量提高了新思路,同时为以酿酒酵母细胞为原料工业化发酵生产RNA及相关制品提供了数据支持。
附图说明
图1为用于构建重组质粒pJFK过程中扩增到的KanMX基因片段电泳图,M:DNAMarker;1:PCR扩增到的KanMX基因片段。
图2为重组质粒pJFK构建成功的PCR验证电泳图,M:DNA Marker;1-3:以提取的1-3号大肠杆菌转化子提取的质粒为模板,以KanMX-EcoR I-F和KanMX-EcoR I-R为引物,进行PCR扩增的产物。
图3为PCR扩增PHO84基因片段的电泳图,M:DNA Marker;1:PCR扩增到的KanMX基因片段。
图4为pJFK质粒验证电泳图,M:DNA Marker;1:以提取的大肠杆菌转化子质粒为模板,以TEF1p-150-F和SalI-PHO84-R为引物,进行PCR扩增的产物。
图5为转化空质粒pJFK的菌株验证电泳图,M:DNA Marker;1-2:以1-2号转化子中反提的酵母质粒为模板,以TEF1-F和PGK1-t为引物,进行PCR扩增的产物。
图6为转化重组质粒pJFK84的菌株验证电泳图,M:DNA Marker;1-3:以1-3号转化子中反提的酵母质粒为模板,以TEF1p-150-F和SalI-PHO84-R为引物,进行PCR扩增的产物。
图7为超表达PHO84基因和添加0.05%磷酸磷酸二氢钾对高核酸酵母菌株YM832生物量、RNA含量和RNA产量的影响。a:生物量;b:RNA含量;c:RNA产量。
具体实施方式
实施例1:PHO84基因超表达菌株的构建
(1) 构建适于工业菌株的、PHO84基因表达质粒pJFK84
为了在酿酒酵母工业菌株中表达PHO84基因,我们首先构建了具有G418抗性基因的表达质粒pJFK,具体构建过程:以质粒pUG6为模板,利用引物KanMX-EcoRI-F(5’-GGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGGACATGGAGGCCCAGAATAC-3’)和KanMX-EcoRI-R(5’-ACAAAGTATGCATTGTGGTACCGAGCTCGAATTTTCTTAGACCAGTATAGCGACCAGCATTC -3’)进行PCR扩增,得到1300 bp左右的G418抗性基因KanMX的基因片段(图1),PCR扩增条件为95℃预变性3min,95℃变性45 s,52℃退火15 s,72℃延伸1.5 min,30个循环,72℃终延伸5 min。然后利用EcoRI单酶切酵母附加体质粒pJFE3,然后利用Gibson法将KanMX基因片段与酶切后的pJFE3连接,连接液转化大肠杆菌DH5α,利用含有100 mg/L氨苄青霉素的LB培养基筛选转化子,挑选3个转化子,接种至含有100 mg/L氨苄青霉素的LB中,经37℃培养12 h后,提取质粒,以质粒为模板,利用引物KanMX-EcoRI-F和KanMX-EcoRI-R为模板,经PCR验证,这3个转化子可以扩增出1300 bp左右的条带(图2),说明KanMX已成功连接到pJFE3上,获得重组质粒pJFK。
然后,将酿酒酵母的PHO84基因连接到pJFK的TEF1启动子和PGK1终止子之间,获得重组质粒pJFK84,具体构建过程:以酿酒酵母YM83的基因组DNA为模板,利用引物BamHI-PHO84-F(5’- CGCGGATCCATGAGTTCCGTCAATAAAGATAC -3’)和SalI-PHO84-R(5’- TTCCGCGGCCGCTATGGCCGACGTCGACTTATGCTTCATGTTGAAGTTGAG -3’) 进行PCR扩增,得到1700 bp的PHO84基因片段(图3),PCR扩增条件为95℃预变性3 min,95℃变性45 s,52℃退火15 s,72℃延伸1.5 min,30个循环,72℃终延伸5 min。利用限制性内切酶BamHI和SalI分别双酶切PHO84基因片段和pJFK,然后进行连接,连接液转化大肠杆菌DH5α,利用含有100 mg/L氨苄青霉素的LB培养基筛选转化子,挑选3个转化子,接种至含有100 mg/L氨苄青霉素的LB中,经37℃培养12 h后,提取质粒,以质粒为模板,利用引物BamHI-PHO84-F和PGK1-t(5’-CAGGAAACAGCTATGAC-3’)进行PCR扩增,经PCR验证,这3个转化子均可以扩增出2000 bp左右的条带(图4),说明PHO84已成功定向连接到pJFK上的TEF1启动子和PGK1终止子之间,获得重组质粒pJFK84。
(2) 表达质粒转化高核酸酿酒酵母菌株YM83
利用PEG-LiAc介导的酿酒酵母转化法将空质粒pJFK和重组质粒pJFK84分别转化前期选育的高核酸酵母菌株YM83(保藏编号为CGMCC No. 25730),利用含有400 mg/L G418的YPD平板筛选转化子,分别挑选pJFK和pJFK84的转化子进行培养,并从中反提质粒。然后分别以反提的酵母质粒为模板,利用引物TEF1-F(5’-ACCCAAGCACAGCATACTA-3’)和PGK1-t进行PCR扩增验证pJFK(PCR扩增条件为95℃预变性3 min,95℃变性45 s,52℃退火15 s,72℃延伸1 min,30个循环,72℃终延伸5 min),经验证,均扩增到了500 bp左右的条带(图5),符合预期大小,说明空质粒pJFK成功转化到YM83中,获得的重组菌株命名为YM832。以反提到的酵母质粒为模板,利用引物BamHI-PHO84-F和PGK1-t进行PCR扩增验证pJFK84(PCR扩增条件为95℃预变性3 min,95℃变性45 s,52℃退火15 s,72℃延伸1.5 min,30个循环,72℃终延伸5 min),经验证PCR扩增得到2000 bp左右的条带(图6),符合预期大小,说明表达质粒pJFK84成功转化到YM83中,获得的重组菌株命名为YM84。
实施例2:PHO84超表达提高了酿酒酵母的生物量、RNA含量及核酸产量
将转化空质粒pJFK的菌株YM832和转化重组质粒pJFK84的菌株YM84分别接种至含有400 mg/L G418的YPD液体中,振荡培养12~24 h,活化2次后,将菌悬液接种至1 LMultifors平行发酵罐中(装液总量为600 mL),调整起始OD600为1.5,接种前在485 mL YP培养基中添加15 mL葡萄糖(即1.5 g),使葡萄糖终浓度为3 g/L,接种2 h后根据流加速率方程(F=0.242e0.298t),持续流加100 mL葡萄糖(10.5 g)至16 h,使酿酒酵母的比生长速率(μ)维持在为0.29左右,将发酵罐发酵参数设定为30 ℃,pH维持在 5.5左右,通气量为1 vvm,搅拌转速200~1000 rpm,溶氧为30%,取样并测定菌株在16 h的菌体生物量、RNA含量和RNA产量,结果如图7所示,与出发菌株YM832相比,超表达PHO84基因的YM84菌株的菌体生物量提高了17.4%(图7a),RNA含量提高了5.6%(图7b),RNA产量为1404 mg/L,提高了10.6%(图7c)。
实施例3:添加磷酸盐提高了酿酒酵母的生物量、RNA含量及核酸产量
将菌株YM832和YM84分别接种至含有400 mg/L G418的YPD液体中,振荡培养12~24h,活化2次后,再次转接菌液到新鲜的含有400 mg/L G418的YPD中,调整起始浓度OD600至0.2左右,30℃培养震荡培养至对数中期(OD600为1.0左右),经无菌水清洗两次重悬后,将其接种至1 L Multifors平行发酵罐中(装液总量为600 mL),调整起始OD600为1.5,接种前在485 mL含有0.05%(按照磷元素的终浓度计算)KH2PO4的YP培养基中添加15 mL葡萄糖(即1.5g),使葡萄糖终浓度为3 g/L,在接种2 h后根据流加速率方程(F=0.242e0.298t),持续流加100 mL葡萄糖(10.5 g)至16 h,使酿酒酵母的比生长速率(μ)维持在为0.29左右,将发酵罐发酵参数设定为30 ℃,pH维持在5.5左右,通气量为1 vvm,搅拌转速200~1000 rpm,溶氧为30%,取样并测定测定菌株在16 h的生物量、RNA含量和RNA产量,结果如图7所示,与不添加磷酸盐的条件相比,YM832在添加磷酸盐后,生物量提高了5.6%(图7a),RNA含量提高了8.0%(图7b),RNA产量提升至1439 mg/L,提高了13.3%(图7c);与超表达PHO84基因的YM84菌株相比,添加500 mg/L的磷酸盐后,菌体的生物量变化不大(图7a),RNA含量提高了41.1%(图7b),RNA产量提升至2050 mg/L,提高了46%(图7c);与不添加磷酸盐的对照菌株YM832相比,菌株YM84在添加500 mg/L的磷酸盐后,菌体的生物量提高了51.6%(图7a),RNA含量提高了13%(图7b),RNA产量提高了61.41%(图7c)。
实验结果表明,前期选育的高核酸酵母菌株YM832处于磷饥饿状态,添加磷酸盐或超表达磷酸盐转运蛋白PHO84增加磷的供应,均能提升菌体的生物量、RNA含量和RNA产量,我们的前期研究结果显示YM832菌株在高于500 mg/L磷酸盐的YPD条件下生长受到抑制,说明高浓度磷酸盐对酿酒酵母细胞的生长产生抑制作用,同时使用两种策略(即在超表达磷酸盐转运蛋白PHO84基因基础上补充磷酸盐)增加磷酸盐供应,菌体的生物量虽与YM84相当,但RNA含量和RNA产量有了较显著的提高,可产生意想不到的叠加效果。
Claims (1)
1.一种提高酿酒酵母核酸产量的方法,其特征在于,在酿酒酵母中增加磷酸盐供应可提高酵母的生物量、RNA含量和核酸产量;
所述的增加磷酸盐供应的方法为:超表达高亲和力磷酸盐转运蛋白PHO84基因和按照磷元素的终浓度0.05%添加磷酸盐;
所述的高亲和力磷酸盐转运蛋白PHO84基因来源于酿酒酵母,其核苷酸序列如SEQ IDNO.1所示。
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