CN116496917A - 一种高效共利用木糖和葡萄糖的重组酿酒酵母及其应用 - Google Patents
一种高效共利用木糖和葡萄糖的重组酿酒酵母及其应用 Download PDFInfo
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Abstract
本发明涉及一种高效共利用木糖和葡萄糖的重组酿酒酵母及其应用,本发明通过基因工程方法将来自瘤胃壶菌的木糖异构酶XIcl和里氏木霉的木糖转运蛋白XTcl在酿酒酵母中共表达得到高效共利用木糖和葡萄糖的重组酿酒酵母,并通过试验验证了在葡萄糖和木糖共存发酵条件下原始酿酒酵母菌株、木糖异构酶单表达酿酒酵母菌株INVSc‑XI、木糖异构酶和木糖转运蛋白共表达酿酒酵母菌株INVSc‑XI/XT木糖消耗能力,发现在在相同葡萄糖和木糖比例条件下,共表达菌株INVSc‑XI/XT消耗木糖更多,说明本发明的重组酿酒酵母INVSc‑XI/XT可提高木糖和葡萄糖共利用效率,并提高乙醇的产量。
Description
技术领域
本发明涉及木糖利用领域,具体涉及一种高效共利用木糖和葡萄糖的重组酿酒酵母及其应用。
背景技术
木质纤维素完全水解后得到大量的木糖和葡萄糖,已知自然界中利用木糖的微生物有很多,但利用木糖发酵乙醇的有很少,目前不足二百多种,由于存在葡萄糖阻遏现象,树干毕赤酵母(Schefersomyces stipites)、休哈塔假丝酵母(Candida shehatae),优先利用葡萄糖的同时也间接导致了木糖的利用受到抑制。Agbogbo等人通过混合糖发酵发现树干毕赤酵母(S.stipites)也存在葡萄糖阻遏现象,当速效碳源葡萄糖消耗殆尽木糖开始被利用,同时他们还发现葡萄糖用于酵母自身生长所需,而木糖大多用于后期产物合成。表明在发酵体系中混糖不同比例的添加或存在,会影响糖源的利用率从而影响乙醇产量。大多数具有木糖代谢能力的酵母对于高浓度的乙醇、糠醛、乙酸等不具有耐受性,在发酵后期会影响菌株生长甚至衰亡。能利用木糖产乙醇的菌株在自然界中稀少,本身产量又很低,难以用于工业化生产。酿酒酵母被用作酿酒酵母生长繁殖快,基因操作技术成熟,已被广泛用来表达外源蛋白,其作为细胞工厂,可以用于生产许多不同的燃料、化学品、食品配料和药物。相比于其它微生物,酿酒酵母具有较高的乙醇耐受性,因此酿酒酵母适用于利用六碳糖发酵产生物乙醇。然而,酿酒酵母自身不具有五碳糖代谢途径,在保留酿酒酵母自身高效利用葡萄糖的同时开发其对木糖的利用效率,实现混合糖的共利用,目前是一种新的挑战。本实验构建了一株能高效共利用木糖和葡萄糖的酿酒酵母,并评估其在混合糖发酵中的能力。
发明内容
本发明要解决的技术问题是针对以上不足,提供一种高效共利用木糖和葡萄糖的重组酿酒酵母及其应用。
为解决以上技术问题,本发明采用以下技术方案:
一种高效共利用木糖和葡萄糖的重组酿酒酵母,通过基因工程方法将来自瘤胃壶菌的木糖异构酶XIcl和里氏木霉的木糖转运蛋白XTcl通过重组表达载体导入酿酒酵母中,得到高效共利用木糖和葡萄糖的重组酿酒酵母。
进一步地,所述的高效共利用木糖和葡萄糖的重组酿酒酵母在共利用木糖和葡萄糖发酵得到乙醇中的应用。
本发明的有益效果为:
本发明通过基因工程方法将来自瘤胃壶菌的木糖异构酶XIcl和里氏木霉的木糖转运蛋白XTcl在酿酒酵母中共表达,得到高效共利用木糖和葡萄糖的重组酿酒酵母,并通过试验验证了在葡萄糖和木糖共存发酵条件下原始酿酒酵母菌株、木糖异构酶单表达酿酒酵母菌株INVSc-XI、木糖异构酶和木糖转运蛋白共表达酿酒酵母菌株INVSc-XI/XT的木糖消耗能力,发现在相同葡萄糖和木糖比例条件下,共表达菌株INVSc-XI/XT消耗更多木糖,说明本发明的重组酿酒酵母INVSc-XI/XT可提高木糖和葡萄糖共利用效率,并提高乙醇的产量。
本发明通过在酿酒酵母中共表达木糖异构酶和木糖转运蛋白,实现木糖和葡萄糖的共利用,具有初步成效。提高以混合糖为底物产生物乙醇的效率,对于改善混合糖的工业化利用及生物乙醇的高效生产具有重要的实践意义。
下面结合附图和实施例对本发明进行详细说明。
附图说明
图1为重组载体(pYJSP-xicl)的构建过程;
图2为菌株INVSc和INVSc-XI木糖的消耗折线图;
图3为菌株INVSc、INVSc-XI、INVSc-XI/XT三种菌株在SC培养基中的木糖生长曲线;
图4为菌株INVSc、INVSc-XI、INVSc-XI/XT三种菌株在SC培养基中的木糖消耗曲线;
图5为菌株INVSc、INVSc-XI、INVSc-XI/XT的甘油醛-3-磷酸产量对比图;
图6为重组菌株INVSc-XI/XT不同比例葡萄和木糖的生长曲线;
图7为重组菌株INVSc-XI/XT不同比例葡萄糖和木糖的木糖消耗曲线;
图8为模拟1:5葡萄糖和木糖条件下INVSc、INVSc-XI、INVSc-XI/XT木糖消耗曲线;
图9为模拟1:5葡萄糖和木糖条件下INVSc、INVSc-XI、INVSc-XI/XT乙醇生成曲线。
具体实施方式
以下结合附图对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
一、木糖异构酶重组酿酒酵母S.cerevisiae(INVSc-XI)的构建1.1木糖异构酶表达载体的构建(pYJSP-xicl-Ura-)
构建酿酒酵母的异源表达途径,将木糖异构酶基因构建到368-pgk载体中,引入酵母代谢途径。得到木糖异构酶的表达载体(pYJSP-xicl-Ura-),选择来源于瘤胃壶菌的木糖异构酶基因xicl,进行胞内表达。选择Xba I和Not I做为酶切位点,将目的基因构建到载体上如图1所示。
1.2重组酿酒酵母S.cerevisiae INVSc-XI的转化与筛选
将构建成功的重组质粒(pYJSP-xicl-Ura-)用Hpa I线性化,使其暴露出rDNA。酶切孵育3h,检测酶切条带大小,用乙醇沉淀法回收线性化的质粒,将回收后的线性DNA片段电转入野生型酿酒酵母中,涂至SC筛选平板上,待其长出重组子,挑选长出的菌落进行PCR鉴定得到阳性克隆命名为S.cerevisiae INVSc-XI(INVSc-XI)。
1.3木糖异构酶在酿酒酵母中的表达与检测
挑取验证正确的重组酿酒酵母菌株(INVSc-XI)在YPD液体培养基培养,放置恒温摇床30℃,200rpm。每12h取样,用液氮破菌,使胞内物质流出,将样品跑SDS-PAGE胶,并用半胱氨酸盐酸盐咔唑显色法测定木糖异构酶粗酶活得到在36小时INVSc-XI的最高酶活为36U/ml。
由此,我们已经得到了一个有条带有酶活的木糖异构酶重组酿酒酵母INVSc-XI,为了进一步验证重组菌株对木糖的利用效果,需要将其在以木糖为唯一碳源的培养基中发酵检测木糖的消耗情况,培养条件为30℃,200rpm,每12h取样,每个样品具有三次重复,用HPLC分析得到峰面积值,用标准曲线将其糖消耗量计算出来。以野生酿酒酵母INVSc为对照组,INVSc-XI为实验组,测得结果如图2所示。
根据木糖消耗量,可以得出,野生型酿酒酵母不消耗木糖,重组酿酒酵母INVSc-XI72h消耗了1.5g/L的木糖,想要得到工业级的利用还是远远不够,需要一种新的策略来提高木糖的利用率。因此本研究采用木糖转运蛋白来协助木糖异构酶的方式提高木糖的利用。
二、木糖转运蛋白重组酿酒酵母S.cerevisiae INVSc-XT的构建及选择2.1木糖转运蛋白表达载体的构建
木糖由发酵液进入胞内是实现木糖胞内代谢的前提。以实验室保藏的三种不同来源的木糖转运蛋白编码基因为基础,包括xylfgh、gxs1、xtcl,构建重组酿酒酵母表达载体,并分别为命名为pYJSP-xylfgh-Ura-、pYJSP-gxs1-Ura-、pYJSP-xtcl-Leu-。
GXS1来自中间假丝酵母,是一个从分子水平上表征的酵母高亲和性木糖-葡萄糖-H+协运蛋白。Xylfgh来自乙醇嗜热厌氧杆菌,为ATP结合蛋白。XTcl来自里氏木霉,为跨膜糖转运蛋白。在酿酒酵母菌株中表达Gxs1,Xylfgh,XTcl基因。根据SnapGene软件对序列进行分析,选择合适的酶切位点及引物,分别扩增目的基因xylfgh、gxs1、xtcl,与载体pYJSP连接。验证重组表达载体,质粒pYJSP-xtcl-Leu-的酶切时选取合适的酶进行酶切验证,这里根据图谱选择EcoR I双酶切,得到两个片段大小为5158bp和4152bp,结果验证正确。
将构建的质粒pYJSP-xylfgh-Ura-,用Nco I双酶切验证质粒,跑琼脂糖凝胶电泳检测条带,得到两条片段分别为8310bp和2322bp。
2.2重组酿酒酵母S.cerevisiae INVSc-XT的转化及选择
构建成功的质粒pYJSP-xylfgh-Ura-、pYJSP-gxs1-Ura-、pYJSP-xtcl-Leu-电转化酿酒酵母INVSc的感受态细胞,电压选择1.8kV,电压及质粒浓度过高都会导致转化效率低。
将长出的阳性重组子命名为INVSc-XT、INVSc-Gxs1、INVSc-Xylfgh便于后续比较。将这三种重组菌株在YPD中活化培养24h后,转接到以木糖为唯一碳源的Sc培养基中,木糖初始浓度为10g/L。30℃,200rpm条件下发酵培养,每8h取样,取至24h,12000rpm离心收集菌体,液氮研磨破菌,用200μL无菌水溶解,无菌滤头过滤处理样品,将处理好的样品放置进样室跑HPLC测胞内的木糖含量,结果如表1所示:
表1三种转运蛋白在胞内木糖含量
INVSc-XT转运木糖的量为0.09g/L/h,INVSc-Gxs1转运木糖0.048g/L/h,INVSc-Xylfgh表现为不转运木糖,分析原因为在酿酒酵母中Xylfgh不表达,因此没有转运功能,之前虽然有在大肠杆菌中成功表达的案例,可能以酿酒酵母作为表达宿主菌不适用。综上,野生型酿酒酵母缺乏专一高效的木糖转运蛋白,使其不能消耗木糖来获取能力,在各种培养基培养的转基因菌株中,表达XTcl的菌株即INVSc-XT可以转运木糖。细胞内木糖积累分析表明,XTcl是最有效的木糖转运蛋白,具有运输多种糖的能力。选择性表达这种新的转运蛋白的能力应该能够促进共发酵应用(例如,在单一培养中多种菌株的差异生长)和创新使用多种混合原料来驱动一般生长或在复杂的多级工艺设计中驱动子工艺特定的活性。因此选择XTcl转运蛋白基因协助木糖异构酶在酿酒酵母中共表达提高木糖利用率。
2.3共表达重组酿酒酵母S.cerevisiae INVSc-XI/XT的构建
将质粒pYJSP-xicl-Ura-和pYJSP-xtcl-Leu-混匀共同转入酿酒酵母中,各取10μL质粒浓度分别为98ng/μL和105ng/μL,电转方法在材料与方法中已叙述。选择Leu-和Ura-双营养缺陷型培养基筛选重组子,培养一段时间,将长出的阳性克隆进行PCR结果的鉴定,分别加入扩增xicl目的基因的引物和xtcl目的基因的引物,进行菌落PCR验证。
将筛选出的阳性克隆接种至SC平板上进行进一步的验证,取无菌水5μL将菌落稀释后点在SC平板上,2-3天后长出菌落,与原始酿酒酵母INVSc对比,重组酿酒酵母INVSc-XI/XT可以在SC筛选平板上生长,原始酿酒酵母INVSc几乎不生长。初步的筛选可以验证INVSc-XI/XT具有消耗木糖作为碳源的功能。
三、评估共表达酿酒酵母S.cerevisiae INVSc-XI/XT糖利用能力3.1木糖为唯一碳源条件下INVSc-XI/XT的木糖利用情况
挑取鉴定正确有功能的菌株INVSc-XI/XT于YPD液体培养基中30℃,200rpm培养24h,转接到以木糖为唯一碳源的SC培养基,调节初始OD600为0.4,相同条件下培养,每12h取样至72h,测生长曲线和糖消耗情况。生长曲线如图3所示,INVSc-XI/XT的最大值为3.6明显高于INVSc-XI和INVSc,其OD600值是INVSc的2.3倍。由生长曲线可以初步推断INVSc-XI/XT可以在木糖唯一碳源上更好的生长。木糖的摄取是最关键的步骤,它决定了酿酒酵母利用木糖作为碳源维持自身生长。检测木糖的消耗量结果如图4所示,分析得INVSc-XI及原始酿酒酵母INVSc很明显没有INVSc-XI/XT所消耗的木糖多。这里测得INVSc-XI菌株72h利用1.9g/L的D-木糖,而INVSc-XI/XT可利用4.4g/L的D-木糖,测其生长曲线OD600值最高为3.6,高于INVSc和INVSc-XI。原始酿酒酵母INVSc不利用木糖,36h后生长曲线呈下降趋势。根据之前的报道分析,在共表达的菌株INVSc-XI/XT中镶嵌在膜上的木糖转运蛋白大多数糖转运蛋白是MFS蛋白,通常具有12个跨膜α-螺旋片段的共同结构基序。木糖转运蛋白上的某个残基可能呈现空间排斥效应,以阻断葡萄糖转运过程,并在D-木糖转运活性中发挥重要作用。D-木糖经镶嵌在细胞膜上的木糖转运蛋白从胞外转运到胞内,木糖在胞内经木糖异构酶异构为木酮糖。
木酮糖进入磷酸戊糖途径(PPP)和糖酵解进一步代谢,木糖异构酶在木糖转运蛋白的协同作用下可以更好的消耗木糖,经过对比,菌株INVSc-XI/XT有效提高了木糖的利用效果。
3.2发酵副产物分析
在以木糖为唯一碳源培养基中,每12h测INVSc、INVSc-XI、INVSc-XI/XT的糖残量,检测出了未知峰的出现,峰面积随时间不断增加。为了分析出未知副产物,利用气相色谱法分析化合物。选用硅烷化衍生化法来处理柱前样品,形成挥发性的衍生物。
分析得副产物为甘油醛-3-磷酸,甘油醛-3-磷酸为糖酵解和糖异生重要的关键体,此外,中间体G-3-P作为前体用于三酰基甘油(TAG)的生物合成甘油磷脂。根据甘油醛-3-磷酸标准曲线,计算得出INVSc、INVSc-XI、INVSc-XI/XT产甘油醛-3-磷酸的量。结果如图5所示,数据揭示了INVSc、INVSc-XI、INVSc-XI/XT在以木糖为唯一碳源的培养基中(木糖浓度为10g/L),分别生成160mg/L、153mg/L、452mg/L的甘油醛-3-磷酸。在酵母细胞体积调节过程中,无机和有机渗透调节物质都会发生细胞内浓度的变化,以平衡细胞外的渗透压力并维持细胞体积的稳态。
3.3INVSc-XI/XT在不同比例葡萄糖和木糖下的木糖利用能力
能够同时利用第二代原料水解物中的己糖和戊糖来高效生产乙醇的工业菌株,对经济转化过程至关重要。为了验证在葡萄糖和木糖共存的条件下菌株INVSc-XI/XT对木糖的利用是否受葡萄糖阻遏(CCR),分析葡萄糖和木糖各自利用的情况,选取了不同比例的葡萄糖和木糖,分别为1:5、1:10、1:100、1:20、1:1、1:2,30℃,200rpm发酵培养,每12h取样测OD600和木糖消耗量结果如图6、7所示。
添加不同浓度的葡萄糖于SC培养基中(木糖的浓度保持10g/L不变),葡萄糖12h利用完,检测上清中木糖含量的变化。数据揭示在添加了葡萄糖与木糖为1:5的情况下木糖的利用更显著,比以木糖为唯一碳源生长的菌株多利用木糖2.2g/L。添加葡萄糖可以使酿酒酵母满足自身生长所需即提高在发酵中的菌体量,菌体量提升可以有效促进木糖的消耗,但是据报道有的细胞含有功能性的SIR2蛋白,对比在2%的葡萄糖培养基中,在0.5%葡萄糖中生长寿命延长约25%。因此推测生物量的增加并不是木糖利用效率提高的主要因素,代谢通路相关的基因变化可能导致木糖的消耗提高。
3.4模拟微氧发酵条件下木糖消耗及产物分析
为了进一步验证INVSc-XI/XT在1:5混合糖比例下木糖的消耗及乙醇产量,将YPD中活化的重组酿酒酵母菌株转接至四联平行发酵罐发酵培养,通过计算机监测并控制发酵温度和转速。每12h取样检测木糖的剩余量及乙醇产量。上述结果已证实1:5的葡萄糖和木糖比可以提高木糖的利用率,因此本实验选择添加2g/L的葡萄糖和10g/L木糖进行培养发酵,检测每12h的糖残量和乙醇产量,葡萄糖在12h可以消耗完全,结果如图8,9所示,在120h内INVSc-XI消耗木糖2.5g/L,INVSc-XI/XT在这里消耗木糖6.5g/L;在96h时,INVSc-XI所得的乙醇产量最高为1.1g/L。INVSc-XI/XT乙醇产量测得最高为2.9g/L。根据监测生长曲线分析可得,发酵可能分为两个时期,前期即36h之前为菌株自身生长时期,通过消耗底物维持生长及传代,后期即36h之后为菌株的代谢时期,这一时期底物的吸收量尤其关键,后期消耗木糖越多乙醇产量越高。从代谢途径解释即当糖酵解通量超过呼吸能力的临界点时,由于丙酮酸通量溢出到乙醇和其他发酵产物,代谢从呼吸切换到发酵。
在木糖和葡萄糖混合培养集中监测这两种糖的利用情况,葡萄糖在最初的12h可以完全利用,木糖前12h里也有所下降,因此分析在转运蛋白的转运作用下,菌株可以减少代谢物阻遏使木糖可以同时被利用,这就增加工业菌株利用木糖和产乙醇的效率。
3.5转录组分析差异基因
为了探究木糖在1:5条件下利用率提高的原因,本研究将1:1比例培养(对照组)的菌株和1:5比例培养(实验组)的菌株取对数生长期,提取总RNA进行转录组分析,经转录组对比分析1052个差异基因发现1:5比例的菌株比1:1比例的菌株基因上调666个基因,通过差异表达基因分析发现,参与TCA循环、乙醛酸循环和有氧呼吸的基因上调。TCA循环是酵母获得能量的主要途径,在这个循环中酵母的糖代谢能力得到提高。其中糖酵解中的乙醇脱氢酶ADH上调,它是乙醛转化为乙醇的关键酶,这也导致后续乙醇产量的提升。与对照相比,糖异生特异性酶磷酸烯醇丙酮酸羧激酶(PCK1)和果糖-1,6-二磷酸酶(FBP1)基因上调。核糖体和MAPK信号通路的差异表达基因数目最多,MAPK是丝裂原活化蛋白激酶,活细胞的一个基本特性是能够感知不断变化的环境条件和各种其他刺激并做出适当反应,酵母经木糖和葡萄糖不同比例的条件刺激,引发MAPK信号通路基因上调,在这种条件下生长的细胞表现出增加的细胞-细胞粘附、增加的细胞-基质粘附和增加的穿透其基质的能力。据报道当葡萄糖变得有限时,Snf1(AMPK)在代谢不同碳源方面具有关键作用。
四、结论
利用天然木质纤维素为原料发酵产生物乙醇是一种新的经济策略。自从获得能够发酵木糖的第一个遗传修饰的酿酒酵母菌株以来,已经过去了许多年,这为将丰富的木质纤维素生物质转化为乙醇提供了环境可持续的解决方案。由于木质纤维素生物质水解产物中混合糖难以充分、有效地利用,因此探索高效的木糖转运蛋白功能是最近研究的焦点。大多数涉及酵母中戊糖发酵的工作集中于将异源木糖代谢途径相关基因通过遗传修饰的手段整合进酿酒酵母细胞。而如何将木糖高效转运至酵母细胞内,而不受到己糖的影响是亟待解决的问题。因此将木糖异构酶基因和木糖转运蛋白在酿酒酵母中共表达,在酵母中构建同步转运和代谢木糖的途径,实现木糖和葡萄糖的共利用,改善了混合糖生物发酵产乙醇的效率。主要结论如下:
本发明成功构建了重组菌株INVSc-XI、INVSc-XT、INVSc-Xylfgh、INVSc-GXS1、INVSc-XI/XT,分别验证了其功能。
验证了在以木糖为唯一碳源培养基中INVSc、INVSc-XI、INVSc-XI/XT木糖消耗能力,其中INVSc表现为不利用,测得INVSc-XI菌株72h利用1.9g/L的D-木糖,而INVSc-XI/XT可利用4.4g/L木糖比INVSc-XI提高了2.3倍。
验证了在葡萄糖和木糖共存发酵条件下原始菌株INVSc、木糖异构酶单表达菌株INVSc-XI、木糖异构酶和木糖转运蛋白共表达菌株INVSc-XI/XT木糖消耗能力,发现在1:5葡萄糖和木糖比例条件下,共表达菌株INVSc-XI/XT消耗木糖更多,比不添加葡萄以木糖为唯一碳源的条件下生长的菌株多利用木糖2.2g/L。说明木糖异构酶和木糖转运蛋白共表达菌株INVSc-XI/XT可提高木糖和葡萄糖共利用效率。
转录组分析了1:1和1:5比例的葡萄糖和木糖条件下培养的INVSc-XI/XT,分析了1052个差异基因,糖酵解中的乙醇脱氢酶ADH基因上调及转酮醇酶等基因上调,转录组数据初步揭示了在1:5比例下木糖利用能力提高的原因。
模拟了在1:5葡萄糖和木糖比例条件下发酵并分析INVSc-XI/XT木糖的消耗量及乙醇产量,木糖在108h最高消耗6.5g/L乙醇产量测得最高为2.9g/L。比INVSc-XI多消耗1.8g/L。再次验证了通过木糖异构酶和木糖转运蛋白共表达菌株实现葡萄糖和木糖的共利用并具备产乙醇的能力。
以上所述为本发明最佳实施方式的举例,其中未详细述及的部分均为本领域普通技术人员的公知常识。本发明的保护范围以权利要求的内容为准,任何基于本发明的技术启示而进行的等效变换,也在本发明的保护范围之内。
Claims (3)
1.一种高效共利用木糖和葡萄糖的重组酿酒酵母,其特征在于,通过基因工程方法将来自瘤胃壶菌的木糖异构酶XIcl和里氏木霉的木糖转运蛋白XTcl在酿酒酵母中共表达得到。
2.根据权利要求1所述的高效共利用木糖和葡萄糖的重组酿酒酵母,其特征在于,木糖异构酶XIcl和木糖转运蛋白XTcl通过重组表达载体导入酿酒酵母中,得到高效共利用木糖和葡萄糖的重组酿酒酵母。
3.根据权利要求1所述的高效共利用木糖和葡萄糖的重组酿酒酵母在共利用木糖和葡萄糖发酵得到乙醇中的应用。
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