CN110088288B - 用于乙醇生产的改良酵母菌株 - Google Patents
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Abstract
本发明涉及一种重组细胞,优选重组酵母细胞,所述重组细胞包含:a)编码具有甘油‑3‑磷酸脱氢酶活性的酶的基因,其中所述酶对至少NADP+和/或NADPH具有辅因子依赖性;b)编码具有至少NAD+依赖性乙酰化乙醛脱氢酶活性的酶(EC 1.2.1.10)的基因;以及c)在选自GPD1和GPD2的组的至少一个基因中的突变或破坏。所述细胞适用于乙醇生产,在高乙醇产量下具有降低的甘油产量。
Description
技术领域
本发明涉及一种适用于乙醇生产的重组细胞,该细胞用于生产乙醇、丁醇、乳酸、琥珀酸、塑料、有机酸、溶剂、动物饲料补充剂、药物、维生素、氨基酸、酶或化学原料的用途,以及使用所述重组细胞制备发酵产物的方法。
背景技术
通过在功能上替代化石燃料衍生化合物,化学品和运输燃料的微生物生产可以促进向可持续的、低碳的全球经济过渡。燃料乙醇的总工业产量在2015年达到了约1000亿升,预计将进一步增加。酵母Saccharomyces cerevisiae是用于将淀粉衍生和蔗糖衍生的己糖单元转化为乙醇的已确立的微生物细胞工厂,因为它结合了高乙醇产量和生产率与工艺条件下的稳健性。在酵母菌株改良和对基于玉米淀粉和蔗糖的生物乙醇生产的工艺优化方面的努力已经进一步提高了产品产量和生产率。此外,密集的代谢和进化工程研究已经产生了能够有效发酵戊糖木糖和阿拉伯糖的酵母菌株,从而为从木质纤维素水解物进行基于酵母的“第二代”生物乙醇生产铺平了道路。
在工业生物乙醇生产中,碳水化合物原料代表一种最大的成本因素。因此,最大化糖的乙醇产量是关键要求,特别是在第二代工艺中,其乙醇产量和生产率通常仍低于第一代工艺的乙醇产量和生产率。酵母在木质纤维素水解物方面的足够性能还需要对在生物质预处理和水解过程中释放的抑制剂的耐受性。在厌氧条件下,野生型S.cerevisiae菌株需要甘油形成以再氧化在生物合成期间或在与葡萄糖相比被氧化更多的代谢物的生产期间形成的NADH。作为S.cerevisiae中的主要相容性溶质,甘油在渗透抗性方面也起着关键作用。
本发明的一个目的是提供一种新重组细胞,该新重组细胞适用于从碳水化合物厌氧发酵生产乙醇,该新重组细胞与其相应的野生型生物相比具有降低的甘油产量,或者如果该细胞用于乙醇的发酵制备则没有甘油产量。
另一个目的是提供一种用于在厌氧酵母培养物中发酵制备乙醇的新方法,在该方法中没有形成甘油或至少其中形成的甘油少于使用已知的S.cerevisiae菌株的方法中形成的甘油。
可以满足的一个或多个其他目的是根据说明书和/或权利要求显而易见的。
附图说明
图1.葡萄糖的甘油产量的计算。图显示了甘油对照葡萄糖的浓度。菱形:IMX884-I。正方形:IMX884-II。
图2.生物质的乙酸(acetate)消耗率的计算。图显示了乙酸对照生物质的浓度。菱形:IMX884-I。正方形:IMX884-II。
图3.生物质的甘油产量的计算。图显示了甘油对照生物质的浓度。菱形:IMX884-I。正方形:IMX884-II。
图4.葡萄糖的乙酸消耗率的计算。图显示了乙酸对照葡萄糖的浓度。菱形:IMX884-I。正方形:IMX884-II。
图5.合成培养基(20g/L)葡萄糖上的摇瓶培养物的细胞提取物对乙酰-CoA的EutE依赖性还原的比速率。从左到右:S.cerevisiae菌株IMX992(GPD1 GPD2sga1::eutE)、IMX884(GPD1 gpd2::eutE)和IMX776(gpd1::gpsA gpd2::eutE)。数据代表对独立一式两份培养物的测定的平均值±平均偏差。
图6.在甘油和乙酸代谢方面具有不同遗传修饰的S.cerevisiae菌株的厌氧生物反应器分批培养中的生长、葡萄糖消耗和产物形成。培养物在含有180g/L葡萄糖和3g/L乙酸(pH 5)的合成培养基上生长。A,菌株IMX776(gpdl::gpsA gpd2::eutE);B,菌株IMX901(gpdl::gpsA gpd2::eutE ald6△)。符号:·,葡萄糖;■,生物质;□,甘油;○,乙醇;△,乙酸。在IMX776的情况下,在对数生长期结束后立即外部添加乙酸。在IMX901的情况下,在静止期中20小时后外部加入乙酸。
图7.葡萄糖的乙醇产量的计算。图显示了乙醇对照葡萄糖的浓度。菱形:IMX884-I。正方形:IMX884-II。IMX884-I的值大部分与IMX884-II重叠。
图8.葡萄糖的生物质产量的计算。图显示了生物质对照葡萄糖的浓度。菱形:IMX884-I。正方形:IMX884-II。IMX884-I的值与IMX884-II部分重叠。
图9.在甘油和乙酸代谢方面具有不同遗传修饰的S.cerevisiae菌株的厌氧生物反应器分批培养物中的生长、葡萄糖消耗和产物形成。培养物在含有20g/L葡萄糖和3g/L乙酸(pH 5)的合成培养基上生长。A,菌株IME324(GPD1 GPD2);B,菌株IMX992(GPD1GPD2sga1::eutE);C,菌株IMX884(GPD1 gpd2::eutE);D,菌株IMX776(gpd1::gpsAgpd2::eutE);E,菌株IMX901(gpd1::gpsA gpd2::eutE ald6△);F,菌株IMX888(gpd1△gpd2::eutE)。符号:·,葡萄糖;■,生物质;□,甘油;○,乙醇;△,乙酸。小图A-F显示了来自每种菌株的一组两个独立的一式两份培养物的单一代表性培养物。菌株IMX888的数据取自(Papapetridis等人,2016)。
图10.在甘油和乙酸代谢方面具有不同遗传修饰的S.cerevisiae菌株的厌氧生物反应器分批培养物中的生物质和产物产量。培养物在含有20g/L葡萄糖和3g/L乙酸(pH 5)的合成培养基上生长。条形图是指以下经工程化的S.cerevisiae菌株:IME324(GPD1GPD2);IMX992(GPD1 GPD2sga1::eutE);IMX884(GPD1 gpd2::eutE);IMX776(gpd1::gpsAgpd2::eutE);IMX901(gpd1::gpsA gpd2::eutE ald6△);IMX888(gpd1△gpd2::eutE)。A,葡萄糖的生物质产量;B,葡萄糖的乙醇产量(经乙醇蒸发校正的);C,葡萄糖的甘油产量。数据代表对每种菌株的独立一式两份培养物的测量的平均值±平均偏差。菌株IMX888的数据取自(Papapetridis等人,2016)。
图11.S.cerevisiae菌株IMX992(GPD1 GPD2)、IMX884(GPD1gpd2::eutE)和IMX776(gpd1::gpsA gpd2::eutE)在合成培养基(20g/L葡萄糖)上的摇瓶培养物的细胞提取物对二羟丙酮磷酸的NADH依赖性(白色条形)和NADPH依赖性(灰色条形)还原的比速率。数据代表对独立一式两份培养物的测定的平均值±平均偏差。
图12.在甘油和乙酸代谢方面具有不同遗传修饰的S.cerevisiae菌株的厌氧生物反应器分批培养物中的生长、葡萄糖消耗和产物形成。培养物在含有180g/L葡萄糖和3g/L乙酸(pH 5)的合成培养基上生长。A,菌株IMX992(GPD1 GPD2sga1::eutE);B,菌株IMX884(GPD1 gpd2::eutE);C,菌株IMX776(gpdl::gpsA gpd2::eutE);D,菌株IMX901(gpd1::gpsAgpd2::eutE ald6△)。符号:·,葡萄糖;■,生物质;□,甘油;○,乙醇;△,乙酸。小图A-C显示了来自每种菌株的一组两个独立的一式两份培养物的单一代表性培养物。在IMX901的情况下,在对数生长期结束后立即外部添加乙酸。
图13.潜在的细胞质转氢酶循环,由EutE、Acs1/2和Ald6催化而将NADH与NADPH交换。形成的NADPH可用于由GpsA将DHAP还原为甘油。
发明详述
除非另有说明,否则本文不使用数量词修饰时被定义为“至少一个(种)”。
当名词(例如化合物、添加剂等)不使用数量词修饰时,意味着包括复数。因此,当提及例如“基因”之类的特定部分时,这意味着“至少一个/种”该基因,例如“至少一个/种基因”,除非另有说明。如本文所用的术语“或”应理解为“和/或”。
当提及存在几种异构体(例如D对映体和L对映体)的化合物时,该化合物原则上包括该化合物的所有可用于本发明的特定方法的对映异构体、非对映异构体和顺式/反式异构体;特别地当提及诸如化合物时,该化合物包括天然异构体。
术语“发酵”、“发酵性”等在本文中以经典意义使用,即表示工艺在厌氧条件下进行或已经在厌氧条件下进行。厌氧条件在本文中被定义为没有任何氧的条件或酵母细胞(特别是酵母细胞)基本上不消耗氧的条件,并且通常对应于小于5mmol/l.h的氧消耗,特别是小于2.5mmol/l.h或小于1mmol/l.h的氧消耗。更优选地,消耗0mmol/L/h(即,氧消耗为不可检测的)。这通常对应于培养物发酵液中的溶氧浓度小于空气饱和度的5%,特别地溶氧浓度小于空气饱和度的1%或小于空气饱和度的0.2%。
术语“细胞”是指优选作为单一细胞存在的真核生物或原核生物。细胞可选自以下组:真菌、酵母、眼虫类(euglenoids)、古细菌和细菌。
细胞可以特别地选自以下组:由酵母组成的属。
术语“酵母”或“酵母细胞”是指一组系统发生上不同的单细胞真菌,其中大多数为子囊菌门(Ascomycota)和担子菌门(Basidiomycota)。芽殖酵母(“真酵母”)被分类为酵母菌目(Saccharomycetales),其中Saccharomyces cerevisiae是最众所周知的物种。
如本文所用的术语“重组(细胞)”或“重组微生物”是指含有这样的核酸的菌株(细胞),该核酸是使用重组DNA技术和/或另一诱变技术进行一个或多个遗传修饰的结果。特别地,重组细胞可包含不存在于对应野生型细胞中的核酸,该核酸已经使用重组DNA技术引入到该菌株(细胞)中(转基因细胞),或者不存在于所述野生型中的核酸是在所述野生型中存在的核酸序列(诸如编码野生型多肽的基因)中——例如使用重组DNA技术或另一种诱变技术(诸如UV辐射)——进行一个或多个突变的结果,或者其中基因的核酸序列已被修饰以将多肽产物(编码其)靶向另一个细胞区室。此外,术语“重组(细胞)”特别地涉及此类菌株(细胞),已经使用重组DNA技术从该菌株(细胞)中去除了DNA序列。
如本文所用的术语“转基因(酵母)细胞”是指含有不天然存在于该菌株(细胞)中并且已经使用重组DNA技术引入该菌株(细胞)中的核酸的菌株(细胞)(即重组细胞)。
本文所用的关于蛋白质或多肽的术语“突变”是指野生型或天然存在的蛋白质或多肽序列中的至少一个氨基酸已经经由对编码这些氨基酸的核酸的诱变而被不同的氨基酸替换、插入,或从该序列中缺失。诱变是本领域中众所周知的方法,并且包括例如借助于PCR或经由寡核苷酸介导的诱变进行的定点诱变,如在Sambrook等人,Molecular Cloning-A Laboratory Manual,第2版,第1-3卷(1989)中所述。如本文所用的关于基因的术语“突变”是指该基因的核酸序列或该基因的调控序列中的至少一个核苷酸已经经由诱变被不同的核苷酸替换,或者已经从该序列中缺失,从而导致功能有定性或定量改变的蛋白质序列的转录或该基因的敲除。
在本发明的上下文中,“改变的基因”具有与突变基因相同的含义。
如本文所用,术语“基因”是指在真核生物中含有核酸聚合酶RNA聚合酶II的模板的核酸序列。将基因转录成mRNA,然后将mRNA翻译为蛋白质。
如本文所用的术语“核酸”包括对单链或双链形式的脱氧核糖核苷酸或核糖核苷酸的聚合物(即多核苷酸)的提及,并且除非另有限制,否则涵盖具有天然核苷酸的基本性质的已知类似物,所述基本性质即它们以类似于天然存在的核苷酸的方式与单链核酸杂交(例如肽核酸)。多核苷酸可以是天然或异源的结构或调控基因的全长序列或子序列。除非另有说明,否则该术语包括对指定序列及其互补序列的提及。因此,具有出于稳定性或其他原因而被修饰的主链的DNA或RNA是“多核苷酸”,如该术语在本文中所用的。此外,仅举两个示例,包含不常见碱基(诸如肌苷)或经修饰的碱基(诸如三苯甲基化碱基)的DNA或RNA是如本文所使用的术语的多核苷酸。应当理解,已经对DNA和RNA进行了多种修饰,该多种修饰用于本领域技术人员已知的许多有用目的。本文采用的术语多核苷酸包括这种化学、酶促或代谢修饰形式的多核苷酸,以及病毒和细胞(尤其包括简单细胞和复杂细胞)特有的DNA和RNA的化学形式。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用,用于指代氨基酸残基的聚合物。该术语用于这样的氨基酸聚合物,在所述氨基酸聚合物中一个或多个氨基酸残基是对应的天然存在的氨基酸的人工化学类似物;以及用于天然存在的氨基酸聚合物。天然存在的氨基酸的这种类似物的基本性质是,当并入蛋白质时,该蛋白质对相同但完全由天然存在的氨基酸组成的蛋白质引发的抗体有特异反应性。术语“多肽”、“肽”和“蛋白质”还包含修饰,所述修饰包括但不限于糖基化、脂质附着、硫酸化、谷氨酸残基的γ-羧化、羟基化和ADP核糖基化。
当参考酶分类(EC)提及酶时,该酶分类是基于由国际生物化学与分子生物学联合会命名委员会(Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology,NC-IUBMB)提供的酶命名法,酶被分类为或可分类为的类别,该命名法可在http://www.chem.qmul.ac.uk/iubmb/enzyme/处找到。还旨在包括没有(尚未)被分类为特定类别但可如此分类的其他合适的酶。
如果在本文中通过引用登录号来提及蛋白质或核酸序列(诸如基因),则除非另有说明,否则该数字特别地用于指代具有可经由www.ncbi.nlm.nih.gov/(在2016年6月14日可得的)找到的序列的蛋白质或核酸序列(基因)。
通过引用遗传密码子,本文中编码多肽的每个核酸序列还描述了核酸的每种可能的沉默变异。术语“保守修饰的变体”适用于氨基酸和核酸序列两者。关于特定的核酸序列,保守修饰的变体是指由于遗传密码子的简并性而编码相同氨基酸序列或保守修饰的氨基酸序列的变体的那些核酸。术语“遗传密码子的简并性”是指大量功能相同的核酸编码任何给定蛋白质的事实。例如,密码子GCA、GCC、GCG和GCU均编码氨基酸丙氨酸。因此,在每个由密码子指定的丙氨酸位置处,可以在不改变编码的多肽的情况下将密码子改变为所述的任何对应密码子。这种核酸变异是“沉默变异”并且代表一种保守修饰的变异。
如本文所用,术语具有特定序列(例如“SEQ ID NO:X”)的多肽的“功能同源物”(或简称“同源物”)是指包含所述特定序列的多肽,前提条件是一个或多个氨基酸被替换、缺失、添加和/或插入,并且该多肽具有(定性地)用于底物转化的相同酶功能。该功能可以通过使用包含重组酵母细胞的测定系统来测试,该重组酵母细胞包含用于在酵母中表达同源物的表达载体,所述表达载体包含可操作地连接到酵母中的功能性启动子的异源核酸序列,并且所述异源核酸序列编码酶促活性为在待测试的酵母细胞中将乙酰辅酶A转化为乙醛的同源多肽,以及评定所述转化是否发生在所述细胞中。候选同源物可以通过使用计算机相似度分析来鉴定。在W02009/013159的实施例2中描述了这种分析的详细示例。技术人员将能够由此推导出如何可以找到合适的候选同源物,并且任选地在密码子(对)优化后,将能够使用如上所述的合适的测定系统来测试此类候选同源物所需的功能。合适的同源物代表与特定多肽具有大于50%,优选60%或更高,特别地至少70%,更特别地至少80%、至少90%、至少95%、至少97%、至少98%或至少99%的氨基酸序列相似性并具有所需的酶功能的多肽。关于核酸序列,术语功能同源物旨在包括由于遗传密码子的简并性而与另一核酸序列不同但编码相同多肽序列的核酸序列。
序列同一性在本文中定义为两个或更多个氨基酸(多肽或蛋白质)序列或两个或更多个核酸(多核苷酸)序列之间的关系,如通过比较序列所确定的。通常,在所比较的序列的全长上比较序列同一性或相似性。在本领域中,“同一性”还意指氨基酸或核酸序列(视情况而定)之间的序列相关性程度,如通过具有此类序列的链之间的匹配所确定的。
氨基酸或核苷酸序列当表现出一定的相似性水平时,被认为是同源的。两个同源的序列表示共同的进化起源。两个同源序列是密切相关还是更远距离相关由“同一性百分比”或“相似性百分比”表示,其分别为高或低。尽管存在争议,但为了表示“同一性百分比”或“相似性百分比”,“同源性水平”或“同源性百分比”经常互换使用。可以使用数学算法来完成序列的比较和对两个序列之间的同一性百分比的确定。本领域技术人员将意识到以下事实:几种不同的计算机程序可用于比对两个序列并确定这两个序列之间的同源性(Kruskal,J.B.(1983)An overview of sequence comparison,在D.Sankoff和J.B.Kruskal(编辑),Time warps,string edits and macromolecules:the theory andpractice of sequence comparison,第1-44页,Addison Wesley中)。两个氨基酸序列之间的同一性百分比可以使用用于比对两个序列的Needleman和Wunsch算法来确定。(Needleman,S.B.和Wunsch,C.D.(1970)J.Mol.Biol.48,443-453)。该算法比对氨基酸序列以及核苷酸序列。Needleman-Wunsch算法已在计算机程序NEEDLE中实现。出于本发明的目的,使用来自EMBOSS程序包的NEEDLE程序(2.8.0版或更高版本,EMBOSS:The EuropeanMolecular Biology Open Software Suite(2000)Rice,P.Longden,I.和Bleasby,A.Trends in Genetics 16,(6),第276-277页,http://emboss.bioinformatics.nl/)。对于蛋白质序列,使用EBLOSUM62来用于替换矩阵。对于核苷酸序列,使用EDNAFULL。可以指定其他矩阵。用于比对氨基酸序列的任选参数是空位开放罚分10和空位延伸罚分0.5。技术人员将理解,当使用不同的算法时,所有这些不同的参数将产生略微不同的结果,但是两个序列的总体同一性百分比不会显著改变。
同源性或同一性是在包括任何空位或延伸的整个比对区域上两个完整序列之间相同匹配的百分比。两个比对序列之间的同源性或同一性计算如下:两个序列在比对中显示出相同氨基酸的对应位置的数目除以包括空位的总比对长度。如本文所定义的同一性可以从NEEDLE获得,并在程序的输出中标记为“同一性(IDENTITY)”。
两个比对序列之间的同源性或同一性计算如下:两个序列在比对中显示出相同氨基酸的对应位置的数目除以减去比对中的空位总数后的总比对长度。如本文所定义的同一性可以通过使用NOBRIEF选项从NEEDLE获得,并在程序的输出中标记为“最长同一性”。
本文所公开的核苷酸或氨基酸序列的变体还可以定义为与本文特别公开(例如,在序列表中)的核苷酸或氨基酸序列相比具有一个或若干个替换、插入和/或缺失的核苷酸或氨基酸序列。
任选地,在确定氨基酸相似性程度时,技术人员还可以考虑所谓的“保守性”氨基酸替换,如本领域技术人员所清楚的。保守氨基酸替换是指具有相似侧链的残基的可互换性。例如,一组具有脂肪族侧链的氨基酸是甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸;一组具有脂肪族-羟基侧链的氨基酸是丝氨酸和苏氨酸;一组具有含酰胺侧链的氨基酸是天冬酰胺和谷氨酰胺;一组具有芳族侧链的氨基酸是苯丙氨酸、酪氨酸和色氨酸;一组具有碱性侧链的氨基酸是赖氨酸、精氨酸和组氨酸;并且一组具有含硫侧链的氨基酸是半胱氨酸和甲硫氨酸。在一个实施方式中,保守氨基酸替换组是:缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸-缬氨酸,以及天冬酰胺-谷氨酰胺。本文公开的氨基酸序列的替换变体是这样的变体,其中已去除了所公开序列中的至少一个残基并其位置插入不同残基。优选地,氨基酸变化是保守性的。在一个实施方式中,每种天然存在的氨基酸的保守替换如下:Ala至ser;Arg至lys;Asn至GIn或His;Asp至glu;Cys至Ser或Ala;GIn至Asn;Glu至Asp;Gly至Pro;His至Asn或GIn;Ile至Leu或Val;Leu至Ile或Val;Lys至Arg;GIn或Glu;Met至Leu或Ile;Phe至Met,Leu或Tyr;Ser至Thr;Thr至Ser;Trp至Tyr;Tyr至Trp或Phe;以及Val至Ile或Leu。
本发明的核苷酸序列还可以按照它们分别在中等杂交条件下或优选在严格杂交条件下与本文所公开的特定核苷酸序列的部分杂交的能力来定义。严格杂交条件在本文中被定义为这样的条件,允许具有至少约25个核苷酸,优选约50个核苷酸、75或100个核苷酸,以及最优选约200个或更多个核苷酸的核酸序列在约65℃的温度下在包含约1M盐(优选6×SSC)的溶液或具有相当离子强度的任何其他溶液中进行杂交,并在65℃下以包含约0.1M或更少的盐(优选0.2×SSC)的溶液或具有相当离子强度的任何其他溶液洗涤。优选地,执行杂交过夜,即至少10小时,并且优选地执行洗涤至少一小时,其中洗涤溶液至少更换两次。这些条件将通常允许具有约90%或更高序列同一性的序列的特异性杂交。
中等条件在本文中定义为这样的条件,允许具有至少50个核苷酸,优选约200个或更多个核苷酸的核酸序列在约45℃的温度下在包含约1M盐(优选6×SSC)的溶液或具有相当离子强度的任何其他溶液中杂交,并在室温下以包含约1M盐(优选6×SSC)或具有相当离子强度的任何其他溶液洗涤。
优选地,执行杂交过夜,即至少10小时,并且优选地执行洗涤至少一小时,其中洗涤溶液至少更换两次。这些条件将通常允许具有高达50%序列同一性的序列的特异性杂交。本领域技术人员将能够修改这些杂交条件,以便特异性地鉴定同一性在50%和90%之间变化的序列。
“表达”是指将基因转录成结构RNA(rRNA,tRNA)或信使RNA(mRNA),随后翻译成蛋白质。
如本文所用,关于核酸或蛋白质的“异源”是源自外来物种的核酸或蛋白质,或者如果源自相同物种,则为通过刻意的人为干预而在其组成和/或基因组基因座方面相对于其天然形式发生实质性改变的。例如,与异源结构基因可操作地连接的启动子来自与该结构基因所来源于的物种不同的物种,或者如果来自相同物种,则它们中的一者或两者为相对于它们的原始形式发生实质性改变的。异源蛋白质可以源自外来物种,或者如果来自相同物种,则为通过刻意的人为干预而相对于其原始形式发生实质性改变的。
术语“异源表达”是指异源核酸在宿主细胞中的表达。异源蛋白质在诸如酵母之类的真核宿主细胞系统中的表达是本领域技术人员熟知的。包含编码具有特定活性的酶的基因的核酸序列的多核苷酸可以在这种真核系统中表达。在一些实施方式中,转化/转染的酵母细胞可以用作用于表达酶的表达系统。异源蛋白质在酵母中的表达是众所周知的。Sherman,F.等人,Methods in Yeast Genetics,Cold Spring Harbor Laboratory(1982)是描述了可用于在酵母中表达蛋白质的多种方法的公知著作。两种被广泛利用的酵母是Saccharomyces cerevisiae和Pichia pastoris。用于在Saccharomyces和Pichia中表达的载体、菌株和方案是本领域中已知的,并且可从商业供应商(例如,Invitrogen)获得。合适的载体根据需要通常具有表达控制序列,诸如启动子,包括3-磷酸甘油酸激酶或醇氧化酶,以及复制起点、终止序列等。
如本文所用,“启动子”是指导(结构)基因转录的DNA序列。通常,启动子位于基因的5′区域中,靠近(结构)基因的转录起始位点。启动子序列可以是组成型的、诱导型的或阻抑型的。在一个实施方式中,不需要(外部)诱导物。
如本文所用的术语“载体”包括对常染色体表达载体和对用于整合到染色体中的整合载体的提及。
术语“表达载体”是指线性或环状的DNA分子,该线性或环状的DNA分子包含编码感兴趣的多肽的区段,该区段在提供其转录的附加核酸区段的控制下(即可操作地连接到所述附加核酸区段)。此类附加区段可包括启动子序列和终止子序列,并且可任选地包括一个或多个复制起点、一个或多个选择性标记、增强子、多腺苷酸化信号等。表达载体通常来源于质粒或病毒DNA,或可含有两者的元件。特别地,表达载体包含这样的核酸序列,该核酸序列包含在5′至3′方向上并可操作地连接的以下项:(a)酵母识别的转录和翻译起始区,(b)感兴趣的多肽的编码序列,以及(c)酵母识别的转录和翻译终止区。“质粒”是指自主复制的染色体外DNA,其未整合到微生物的基因组中并且通常自然状态下是环状的。
“整合载体”是指线性或环状的DNA分子,该线性或环状的DNA分子可以整合到微生物的基因组中并提供编码感兴趣的多肽的基因的稳定遗传。整合载体通常包含这样的一个或多个区段,该一个或多个区段包含在提供转录的附加核酸区段的控制下(即可操作地连接到所述附加核酸区段)编码感兴趣的多肽的基因序列。此类附加区段可包括启动子序列和终止子序列,以及通常通过同源重组过程来驱动将感兴趣的基因整合入到靶细胞基因组中的一个或多个区段。通常,整合载体将为可转移到靶细胞中的载体,但该载体具有在该生物体中无功能的复制子。如果在包含感兴趣的基因的区段内包含适当的标记,则可以选择整合该区段。
“宿主细胞”是指含有载体并支持载体的复制和/或表达的细胞。
如本文所用,“转化”是指将外源多核苷酸插入宿主细胞中,而不管用于该插入的方法(例如,直接摄取、转导、f-接合(f-mating)或电穿孔)为何。外源多核苷酸可以保持为非整合载体,例如质粒,或者可以整合到宿主细胞基因组中。
“破坏”是指(或包括)影响对应多肽的翻译或转录和/或影响酶的(比)活性、酶的底物特异性和/或稳定性的所有核酸修饰,诸如核苷酸缺失或替换、基因敲除(等等)。此类修饰可以靶向编码序列或基因的启动子。
在第一方面,本发明提供了一种重组细胞,优选重组酵母细胞,优选适用于生产乙醇,所述细胞包含:
a)编码具有甘油-3-磷酸脱氢酶活性的酶的基因,其中所述酶对至少NADP+和/或对NADPH具有辅因子依赖性;
b)编码具有至少NAD+依赖性乙酰化乙醛脱氢酶活性(EC 1.2.1.10)的酶的基因;以及
c)在选自GPD1和GPD2的组的至少一个基因中的突变或破坏。
如在a)中所定义的编码具有甘油-3-磷酸脱氢酶活性的酶的基因赋予细胞将二羟丙酮磷酸转化为甘油-3-磷酸的能力。S.cerevisiae具有至少两个编码甘油-3-磷酸脱氢酶的基因GPD1和GPD2。GPD1基因是压力诱导的甘油-3-磷酸脱氢酶,其对于在工业发酵条件下(例如在高葡萄糖浓度下,诸如约180g/L)可发生的渗透压力下生长是重要的。GPD1的表达尤其受高渗透压甘油响应途径的调控。因此,在一个实施方式中,GPD2而非GDP1被突变或破坏。然而,发明人已经发现通过使用具有甘油-3-磷酸脱氢酶活性的酶,该酶具有对至少NADP+和/或对NADPH的辅因子依赖性,GPD1和GPD2可以都被突变或破坏,同时细胞能够在高葡萄糖浓度下生长并产生很少的甘油或不产生甘油。因此,在一个实施方式中,GPD1和GPD2两者都被突变或破坏。
在一个实施方式中,具有甘油-3-磷酸脱氢酶活性的酶对NADPH比对NADH具有更低的米氏常数(Km,表示为M)。具有甘油-3-磷酸脱氢酶活性的酶的Km(NADPH)可以是Km(NADH)的至多一半,优选具有甘油-3-磷酸脱氢酶活性的酶的Km(NADPH)与Km(NADH)相比为至多四分之一,更优选至多十分之一,甚至更优选至多1/20、至多1/50,甚至更优选至多1/100、至多1/500,甚至更优选与Km(NADH)相比至多1/000。
在另一个实施方式中,具有甘油-3-磷酸脱氢酶活性的酶对NADPH比对NADH具有更高的最大比活性(Vmax,表达为μmol mg/蛋白质/min)。
具有甘油-3-磷酸脱氢酶活性的酶对NADPH的Vmax可以是对NADH的至少两倍,优选具有甘油-3-磷酸脱氢酶活性的酶的Vmax(NADPH)与其Vmax(NADH)相比为至少四倍,更优选至少10倍、至少50倍、至少100倍,更优选至少500倍,甚至更优选至少1000倍。
具有甘油-3-磷酸脱氢酶活性的酶的Vmax可以指适当的酶,例如分离形式或纯形式或经纯化形式的酶。技术人员知道如何纯化或分离甘油-3-磷酸脱氢酶。或者,具有甘油-3-磷酸脱氢酶活性的酶的Vmax可以与细胞蛋白质的总量相关,或者与不含细胞的提取物中的蛋白质总量相关。也就是说,可以使用全细胞或无细胞提取物来确定具有甘油-3-磷酸脱氢酶活性的酶的Vmax。
在另一个实施方式中,具有甘油-3-磷酸脱氢酶活性的酶对NADPH比对NADH具有更低的米氏常数和更高的Vmax。
在另一个实施方式中,具有甘油-3-磷酸脱氢酶活性的酶对NADPH比对NADH具有更高的亲和力(Vmax/Km)。具有甘油-3-磷酸脱氢酶活性的酶的Vmax/Km(NADPH)与其Vmax/Km(NADH)相比可为至少两倍,优选具有甘油-3-磷酸脱氢酶活性的酶的Vmax/Km(NADPH)与其Vmax/Km(NADH)相比为至少四倍,更优选至少10倍、至少50倍、至少100倍,更优选至少500倍,甚至更优选至少1000倍。具有甘油-3-磷酸脱氢酶活性的酶的亲和力可以指适当的酶,例如分离形式或纯形式或经纯化形式的酶。技术人员知道如何纯化或分离甘油-3-磷酸脱氢酶。或者,具有甘油-3-磷酸脱氢酶活性的酶的亲和力可以与细胞的蛋白质的总量相关,或者与不含细胞的提取物中的蛋白质总量相关。也就是说,可以使用全细胞或无细胞提取物来确定具有甘油-3-磷酸脱氢酶活性的酶的亲和力。
在一个实施方式中,根据本发明的细胞与其相应的野生型细胞相比,不含或基本上不含或具有降低的NADH依赖性甘油-3-磷酸脱氢酶活性。优选地,细胞与其相应的野生型细胞相比,不含或基本上不含或具有降低的天然(内源)NADH依赖性甘油-3-磷酸脱氢酶活性。为了比较本发明细胞和野生型细胞的活性,优选在相同条件下测量这些活性。
在一个实施方式中,编码具有甘油-3-磷酸脱氢酶活性的酶的基因包含至少一个外源基因,该外源基因可编码具有根据SEQ ID NO:1的氨基酸序列的酶或该酶的具有至少50%,优选至少60%、至少70%,更优选至少80%、至少90%,甚至更优选至少95%的序列同一性的功能同源物。优选的此类基因是gpsA,例如来自Archaeoglobus fulgidus。
根据本发明的细胞可以包含编码具有甘油-3-磷酸脱氢酶活性的酶的改变的内源基因,其中所述改变赋予酶对NADPH增加的亲和力和/或更低的米氏常数和/或更高的最大活性。
编码具有至少NAD+依赖性乙酰化乙醛脱氢酶活性的酶的基因可以编码具有根据SEQ ID NO:2的氨基酸序列的酶或该酶的具有至少50%,优选至少60%、至少70%,更优选至少80%、至少90%,甚至更优选至少95%的序列同一性的功能同源物。优选的此类基因是eutE,例如来自E.coli。
根据本发明的细胞与其相应的野生型细胞相比,可以(基本上)不含或具有降低的NADPH依赖性醛还原酶活性(EC 1.2.1.4)。
根据本发明的细胞的基因组可以包含ALD6或其具有至少50%,优选至少60%、至少70%,更优选至少80%、至少90%,甚至更优选至少95%的序列同一性的功能同源物中的突变。ALD6或功能同源物中的突变可以防止或减少生长中的滞后期(lag phase)。
在一个实施方式中,具有至少NAD+依赖性乙酰化乙醛脱氢酶活性的酶催化乙酰辅酶A可逆转化为乙醛,随后乙醛可逆转化为乙醇,该酶优选包含NAD+依赖性乙酰化乙醛脱氢酶(EC 1.2.1.10)活性和NAD+依赖性醇脱氢酶活性(EC 1.1.1.1)两者。
在一个实施方式中,编码具有至少NAD+依赖性乙酰化乙醛脱氢酶活性的酶的基因编码具有根据SEQ ID NO:3的氨基酸序列的酶或该酶的具有至少50%,优选至少60%、至少70%,更优选至少80%、至少90%,甚至更优选至少95%的序列同一性的功能同源物。优选的此类基因是adhE,例如来自E.coli。
在一个实施方式中,细胞不包含编码具有丙酮酸甲酸裂解酶活性的酶(EC2.3.1.54)的基因。如本文所述,丙酮酸甲酸裂解酶催化至少以下反应(I):
(I)丙酮酸+辅酶A<->甲酸+乙酰辅酶A。
本发明还提供了根据本发明的细胞用于制备乙醇、丁醇、乳酸、琥珀酸、塑料、有机酸、溶剂、动物饲料补充剂、药物、维生素、氨基酸、酶或化学原料,优选乙醇的用途。
本发明还提供了一种用于制备发酵产物的方法,该方法包括从可发酵的碳水化合物制备发酵产物,该可发酵的碳水化合物特别地选自以下项的组:葡萄糖、果糖、蔗糖、麦芽糖、木糖、阿拉伯糖、半乳糖和甘露糖,该制备使用根据本发明的细胞在厌氧条件下进行。所述可发酵碳水化合物优选获自淀粉、纤维素、半纤维素木质纤维素和/或果胶。应理解,可发酵碳水化合物是浆液、悬浮液或液体。
可以将淀粉、木质纤维素和/或果胶与酶组合物接触,其中产生一种或多种糖,并且其中产生的糖被发酵以产生发酵产物,并且其中用根据本发明的细胞进行发酵。
在一个实施方式中,可发酵碳水化合物是生物质水解产物,或包含在生物质水解产物中,该生物质水解产物为诸如玉米秸秆或玉米纤维水解产物。在另一个实施方式中,此类生物质水解产物包含或来源于玉米秸秆和/或玉米纤维。
“水解产物”是指含多糖材料(诸如玉米秸秆、玉米淀粉、玉米纤维或木质纤维素材料),该含多糖材料的多糖通过加入水而解聚以形成单糖和寡糖。水解产物可以通过含多糖材料的酶促水解或酸水解来产生。
生物质水解产物可以是木质纤维素生物质水解产物。本文的木质纤维素包括半纤维素和生物质的半纤维素部分。木质纤维素还包括生物质的木质纤维素级分。合适的木质纤维素材料可在以下列表中找到:果园底料(orchard primings)、树丛(chaparral)、磨坊废弃物(mill waste)、城市木材废弃物、市政废弃物、伐木废弃物、森林疏伐废弃物、短期轮种木本作物、工业废弃物、小麦秸、燕麦秸、水稻秸、大麦秸、黑麦秸、亚麻秸、大豆壳、稻壳、水稻秸、玉米谷蛋白饲料、燕麦壳、甘蔗、玉米秸秆、玉米杆、玉米芯、玉米壳、柳枝稷、芒草、甜高粱、芸苔茎、大豆茎、草原禾草、鸭茅状摩擦禾、狐尾草;甜菜粕、柑橘果实浆、种子壳、纤维素动物粪便、草坪修剪废弃物、棉花、海藻、树木、软木材、硬木材、白杨、松树、灌木丛、草、小麦、小麦秸、甘蔗渣、玉米、玉米壳、玉米棒、玉米粒、来自玉米粒的纤维、来自谷物湿磨或干磨的产物和副产物、城市固体废弃物、废纸、庭院废弃物、草本材料、农业残余物、林业残余物、城市固体废弃物、废纸、纸浆、造纸厂残余物、树枝、灌木、甘蔗、玉米、玉米壳、能源作物、森林、水果、鲜花、谷物、草、草本作物、树叶、树皮、针叶、原木、根、树苗、灌木丛、柳枝稷,树木、蔬菜、水果皮、藤蔓、甜菜粕、小麦麸皮、燕麦壳、硬木材或软木材、由农业加工产生的有机废弃物材料,林业木材废弃物,或它们中的任意两种或更多种的组合。木质纤维素可被认为是潜在的可再生原料,其通常包括多糖纤维素(葡聚糖)和半纤维素(木聚糖、杂木聚糖和木葡聚糖)。另外,一些半纤维素可以作为葡甘露聚糖存在,例如在木材来源的原料中。这些多糖成为可溶性糖(包括单体和多聚体,例如葡萄糖、纤维二糖、木糖、阿拉伯糖、半乳糖、果糖、甘露糖、鼠李糖、核糖、半乳糖醛酸、葡糖醛酸和其他己糖和戊糖)的酶促水解发生于共同作用(acting in concert)的不同酶的作用下。此外,果胶和其他果胶物质如阿拉伯聚糖可占来自非木本植物组织的典型细胞壁干质量的可观的比例(干质量的约四分之一到一半可为果胶)。可以预处理木质纤维素材料。预处理可包括将木质纤维素材料暴露于酸、碱、溶剂、热、过氧化物、臭氧、机械粉碎、碾磨、研磨或快速卸压,或它们中的任意两种或更多种的组合。化学预处理通常与热预处理(例如在150-220℃之间1到30分钟)组合。
本发明方法中的发酵产物可以是乙醇、丁醇、乳酸、琥珀酸、塑料、有机酸、溶剂、动物饲料补充剂、药物、维生素、氨基酸、酶或化学原料中的一种或多种。
在一个实施方式中,相对于可发酵碳水化合物的体积,葡萄糖的浓度为80g/L或更高。也就是说,葡萄糖的初始浓度,即在发酵开始时的浓度,为优选80g/L或更高,优选90g/L或更高、100g/L或更高、110g/L或更高、120g/L或更高、130g/L或更高、140g/L或更高、150g/L或更高、160g/L或更高、170g/L或更高、180g/L或更高。发酵的开始可以是当可发酵的可发酵碳水化合物与本发明的重组细胞接触的时刻。
实施例
方法学实施例
一般分子生物学技术
除非另有说明,否则使用的方法是标准生化技术。合适的通用方法教科书的示例包括Sambrook等人,Molecular Cloning,a Laboratory Manual(1989)和Ausubel等人,Current Protocols in Molecular Biology(1995),John Wiley&Sons,Inc。
菌株繁殖和保持
本实施例中使用的所有S.cerevisiae菌株属于CEN.PK谱系(Entian和2007)(表1)。将S.cerevisiae培养物在含有20g/L葡萄糖的合成培养基(Verduyn等人,1992)中繁殖。将用于质粒克隆的E.coli DH5a培养物在含有100mg/L氨苄青霉素的LB培养基(10g/L细菌培养用胰蛋白胨、5g/L细菌培养用酵母提取物、5g/LNaCl)中繁殖。在向生长培养物中加入无菌甘油(30%v/v)后,将所有菌株保存在-80℃。
表1.S.cerevisiae菌株
表2.质粒
质粒 | 特征 | 来源 |
p426-TEF(空) | 2μ,URA3,TEF1p-CYC1t | Mumberg等,1995 |
pMEL10 | 2μ,KIURA3,SNR52p-gRNA.CAN1.Y-SUP4t | Mans等.,2015 |
pMEL11 | 2μ,amdS,SNR52p-gRNA.CAN1.Y-SUP4t | Mans等,2015 |
pROS10 | KIURA3-gRNA.CAN1-2mu-gRNA.ADE2 | Mans等,2015 |
pUDI076 | pRS406-TDH3p-eutE-CYC1t | Papapetridis等,2016 |
pUDRi03 | 2μ,KIURA3,SNR52p-gRNA.SGA1.Y-SUP4t | 本发明工作 |
pUDR119 | 2μ,amdS,SNR52p-gRNA.SGA1.Y-SUP4t | van Rossum等,2016 |
pUDR240 | KIURA3-gRNA.GPD1-2mu-gRNA.GPD2 | 本发明工作 |
pUDR264 | 2μ,amdS,SNR52p-gRNA.ALD6.Y-SUP4t | 本发明工作 |
pMK-RQ-gpsA | 递送载体,密码子优化的gpsA ORF | GeneArt,德国 |
构建表达盒和质粒
表2中列出了该实施例中使用的质粒。将表达嵌合gRNA的质粒用于CRISPR/Cas9介导的基因组编辑(Mans等人,2015)。如前所述选择GPD1、GPD2、SGA1和ALD6中的独特Cas9识别序列(Papapetridis等人,2016)。根据制造商的指南,分别用Phusion Hot Start II高保真DNA聚合酶和Dreamtaq聚合酶(Thermo Scientific,Waltham,MA)进行用于构建表达盒的PCR和诊断性PCR。为了构建pUDR240,使用双结合引物5793(表3)和使用pROS10作为模板来对质粒的主链进行PCR扩增。使用引物6965-6966和使用pROS10作为模板来扩增表达靶向GPD1和靶向GPD2的gRNA盒的插入片段。为了构建pUDR103,使用引物5792-5980对pMEL10的质粒骨架进行PCR扩增。使用引物5979-7023和pMEL10作为模板,对靶向SGA1的gRNA表达盒进行PCR扩增。为了构建pUDR264,使用引物5792-5980对pMEL11的质粒骨架进行PCR扩增。使用引物5979-7610和使用pMEL11作为模板,对靶向ALD6的gRNA表达盒进行PCR扩增。在将供应商的方案缩小规模至10μl反应体积后,用Gibson装配克隆试剂盒(New EnglandBiolabs,Ipswich,MA)来装配质粒。在电穿孔转化和用微量制备试剂盒(Sigma-Aldrich,St.Louis,MO)重新分离质粒后,将质粒pUDR240和pUDR264克隆到E.coli DH5a细胞中。通过限制性消化或通过诊断PCR来验证正确的克隆。对于GPD2的单一缺失,用双结合引物5793和用pROS10作为模板来对质粒主链进行PCR扩增。用引物6966和用pROS10作为模板来对表达两个相同的靶向GPD2的gRNA盒的插入片段进行扩增。对于GPD2的单一缺失,将两个质粒片段直接转化到酵母细胞中并在体内装配。
基于高度表达的酵母糖酵解基因的密码子偏好(Wiedemann和Boles,2008),通过GeneArt GmbH(Regensburg,德国)合成Archaeglobus fulgidus gpsA的S.cerevisiae密码子优化版本(SEQ ID NO:4)。用引物7862-7863和用pMK-RQ-gpsA作为模板来对用于用密码子优化的gpsA序列替换GPD1的编码区的整合盒进行PCR扩增。使用pUDI076(Papapetridis等人,2016)作为模板,分别用引物7991-7992或7211-7025来扩增旨在整合到GPD2或SGA1基因座中的E.coli EutE乙酰化乙醛脱氢酶基因(TDH3p-eutE-CYClt)的密码子优化表达盒。整合盒的侧翼为60bp序列,该60bp序列在CRISPR/Cas9介导的所选S.cerevisiae基因组基因座中双链断裂的引入后通过同源重组实现整合。
菌株构建
使用乙酸锂/聚乙二醇法(Gietz和Woods,2002)进行酵母转化。在用质粒pUDR103、pUDR240转化后和进行GPD2的单一缺失后,在含有20g/L葡萄糖的合成培养基琼脂平板(Verduyn等人,1992)上选择转化体。在用质粒pUDR119和pUDR264转化后,如所述进行选择和逆选择(Solis-Escalante等人,2013)。在补充有葡萄糖(20g/L终浓度)和5-氟乳清酸(1g/L终浓度)的YP琼脂平板(10g/L细菌用酵母提取物,20g/L细菌用蛋白胨)上进行对携带URA3的质粒的逆选择。使用诊断菌落PCR来对所选菌落进行基因型分析。
将pUDR119和SGA1侧接的TDH3p-eutE-CYC1t盒共转化到菌株IMX581中,产生菌株IMX992,在所述菌株IMX992中eutE在功能性GPD1和GPD2基因的存在下过表达。
将靶向GPD2的gRNA质粒和侧接GPD2的TDH3p-eutE-CYC1t盒的两个片段共转化到菌株IMX581中,产生菌株IMX884,在所述菌株IMX884中GPD2缺失并且eutE过表达。
将pUDR240、侧接GPD1的gpsA编码序列和侧接GPD2的TDH3p-eutE-CYC1t盒共转化到菌株IMX581中,产生菌株IMX776,在所述菌株IMX776中gpsA从天然GPD1启动子和终止子表达,GPD2缺失并且eutE过表达。
将pUDR264和修复寡核苷酸7608-7609共转化到菌株IMX776中,然后进行pUDR264逆选择,产生菌株IMX901,在所述菌株IMX901中ALD6缺失。
通过用p426-TEF转化IMX581获得空载体参考菌株IME324。
生物反应器分批培养
将厌氧分批培养物在2L生物反应器(Applikon,Schiedam,The Netherlands)中在补充有乙酸(3g/L终浓度)的合成培养基(Verduyn等人,1992)上生长。在乙酸消耗性菌株IMX776和IMX901的高渗透培养物中,当乙酸浓度达到低于1.5g/L的值时,通过加入冰醋酸将乙酸浓度重新设定为3g/L,以防止乙酸限制。在将合成培养基的无机盐组分和乙酸在120℃下高压灭菌20min后,向厌氧生长培养基补充无菌消泡剂C(0.2g/L)(Sigma-Aldrich)、麦角甾醇(10mg/L)、吐温80(420mg/L)和经过滤灭菌的维生素溶液(Verduyn等人;1992)。将葡萄糖溶液在110℃下单独高压灭菌20min,并加入低渗透培养基和高渗透培养基中,终浓度分别为20g/L和180g/L(1M)。用冷冻甘油储备培养物(1mL)接种摇瓶培养物(100mL),并在补充有葡萄糖(20g/L终浓度)的合成培养基上生长。将这些培养物用作相同培养基上的100mL摇瓶预培养物的接种物,将所述培养物在达到对数生长期中期(OD660为4-6)时用于接种厌氧生物反应器培养物,产生为0.15-0.2的初始OD660。通过以0.5L/min的速率连续喷射氮气来维持厌氧条件(<10ppm氧)。使用Norprene管和Viton O形环来最小化到反应器中的氧扩散。在低渗透培养物中,通过加入2M KOH将培养物pH自动控制在5.0。在高渗透培养物中,使用12.5%v/v NH4OH溶液作为滴定剂来防止氮限制。将搅拌速度设定为800rpm,并将温度控制在30℃。通过在冷凝器中将出口气体冷却至4℃来使蒸发最小化。
酶活性测定
通过对在含有20g/L葡萄糖的合成培养基上对数生长的摇瓶培养物(OD660为5-6)进行超声处理(Postma等人,1989)来制备细胞提取物。通过连续分光光度监测340nm处NAD(P)H向NAD(P)+的转化,在30℃下进行酶活性测定。为了确定乙酰化乙醛脱氢酶的活性,将细胞在含有2mMMgCl2和1mM二硫苏糖醇的100mM磷酸钾缓冲液(KPB,pH 7.5)中超声处理。1mL反应混合物含有50mM KPB(pH 7.5)、0.15mM NADH,以及50μL或70μL细胞提取物。通过加入乙酰-CoA至终浓度为0.5mM来开始反应。对于甘油-3-磷酸脱氢酶测定,使用补充有10mMEDTA的20mM Tris-HCl(pH 8.2)缓冲液来收获和储存细胞,并在含有2mM EDTA的20mMTris-HCl(pH 8.2)缓冲液中进行超声处理。1mL反应混合物含有50mM Tris-HCl(pH 6.6)、2mM EDTA、0.15mM NADH或NADPH,以及50μL或70μL细胞提取物。通过加入二羟丙酮磷酸至终浓度为4mM来开始反应。对来自两个独立培养物的样品进行所有测定,酶活性与加入到测定中的细胞提取物的体积成比例。
细胞内甘油测定
从冷冻储备物接种合成培养基(20g/L葡萄糖)上的摇瓶预培养物。达到对数生长期中期后,将细胞用无菌脱矿质水洗涤,并用作在与高渗透生物反应器分批培养相同的培养基上的厌氧摇瓶培养物的接种物。厌氧摇瓶培养物在Bactron厌氧室(SheldonManufacturing,Cornelius,OR)中于30℃生长。收获对数生长期中期培养物并以4000×g离心5min。弃去上清液,将细胞重悬于0.005mol/L H2SO4中并在100℃下温育5分钟。将细胞悬浮液以4000×g离心5分钟,并将上清液用于HPLC分析。
为了计算沉淀体积,使用1.1g/mL的沉淀物平均密度(Bryan等人;PNAS.
2010;107:999-1004)。为了将细胞内甘油浓度从g/g干重转化为g/L,使用2.6mL(g/干重)的细胞内体积(Albertyn等人,1994)。
分析方法
如前所述(Guadalupe-Medina等人,2010)进行生物质干重测定、细胞外代谢物的HPLC分析和乙醇蒸发校正。如前所述(Guadalupe-Medina等人,2010)分析培养废气组成,区别为在高渗透条件下用菌株IMX992、IMX884、IMX776和IMX901生长的分批培养物,其中假设每摩尔产生的乙醇形成1mol CO2,根据乙醇产量来计算CO2的产量。在高渗透发酵中进行葡萄糖和乙醇浓度测量之前,用脱矿质水将培养上清液1∶1稀释。根据在对数生长期中期期间取得的最少五个样品计算分批培养中的产物产量和比率(Papapetridis等人,2016)。使用基于在对数生长期中期取得的最少五个样品(对其测量生物质干重和OD660)的校准曲线,基于OD660测量值来计算对应于在对数生长期中期之前(OD660<1)取得的样品的生物质浓度(Papapetridis等人,2016)。计算的示例可以在图1至图4和图7至图8中找到。
表3.用于菌株构建的寡核苷酸引物
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实施例1
在GPD1 GPD2 S.cerevisiae中乙酸还原途径表达的有限影响
为了研究共表达乙酸还原途径与全功能甘油途径的影响,在补充有3g/L乙酸的20g/L葡萄糖上的厌氧、葡萄糖生长生物反应器分批培养物中分析菌株IMX992(GPD1 GPD2sga1::eutE)的生长和产物形成(图9,表4),并与乙酸非还原参考菌株IME324进行比较。在这些条件下,IME324(GPD1 GPD2)显示出2.43mmol/(g生物质)的乙酸消耗(表4)。菌株IMX992(GPD1 GPD2sga1::eutE)显示出3.35mmol/(g生物质)的乙酸消耗,其仅比GPD1 GPD2参考菌株IME324的乙酸消耗高0.92mmol/(g生物质)。与其略微更高的乙酸消耗一致,菌株IMX992的甘油产量相对于菌株IME324仅略微从9.19mmol甘油/(g生物质)降低至8.28mmol甘油/(g生物质)(表4)。显然,在葡萄糖发酵工程化的S.cerevisiae菌株中,基于EutE的乙酸还原不能有效地与全功能天然甘油途径竞争NADH。
实施例2
GPD2缺失通过表达eutE的菌株改善乙酸还原
在菌株IMX884(GPD1 gpd2::eutE)的乙酸补充的厌氧培养物中,eutE表达完全补偿了功能性Gpd2酶的缺乏,无论是在比生长速率方面还是在葡萄糖的生物质产量方面(表4,图9和图10)。与菌株IMX992(GPD1GPD2sga1::eutE)相比,菌株IMX884显示出低4倍的甘油产量(分别为1.92mmol甘油/(g生物质)和8.28mmol甘油/(g生物质)),和相应较高的基于EutE的乙酸消耗(经过针对乙酸非还原参考菌株IME324的乙酸消耗校正,分别为3.34mmol乙酸/(g生物质)和0.92mmol乙酸/(g生物质)),导致葡萄糖的乙醇产量为0.46g/g(图10)。这些结果表明,至少在低渗透培养基中,GPD2的失活使得基于EutE的乙酸还原途径能够有效地与甘油途径竞争氧化还原当量。与乙酸非还原参考菌株IME324中观察到的相比,该工程化策略不仅导致更高的乙酸消耗,而且还导致更高的葡萄糖乙醇产量(表4,图10)。
实施例3
在S.cerevisiae中功能性表达偏好NADPH的G3PDH
如上所述,由A.fulgidus gpsA编码的偏好NADP+的G3PDH的表达可以实现使甘油代谢在酵母渗透抗性和氧化还原平衡中的作用解偶联的策略。为了研究gpsA是否可以在S.cerevisiae中功能性表达,对gpsA的编码序列进行用于在酵母中表达的密码子优化(SEQID NO:4),并整合在菌株IMX581的GPD1基因座处(与eutE在GPD2基因座处的整合一起),从而产生菌株IMX776(gpd1::gpsA gpd2::eutE)。该插入经设计以将gpsA置于GPD1启动子和终止子的控制下,以便能够上调其在高渗透压下的表达(Albertyn等人,1994;Ansell等人,1997)。
在细胞提取物中的酶促活性测定显示,在菌株IMX776中,由gpsA替代天然GPD1基因和GPD2基因导致了对甘油-3-磷酸脱氢酶(G3PDH,图11)的辅因子偏好的切换。gpsA表达菌株显示出对NADPH和NADH的体外活性分别为0.103±0.004μmol/mg蛋白/min和0.006μmol/mg蛋白/min。因此,二羟丙酮磷酸还原的NADPH连接速率和NADH连接速率的比率在菌株IMX776中为参考菌株IMX992中的约500倍高,该参考菌株IMX992表达天然GPD1基因和GPD2基因。
实施例4
在表达gpsA的酵母菌株中乙酸还原增加并且甘油产量降低
在表达gpsA的乙酸还原菌株IMX776(gpd1::gpsA gpd2::eutE)中缺失ALD6,以产生菌株IMX901。在厌氧的、乙酸补充的生物反应器分批培养物中,菌株IMX776(gpd1::gpsAgpd2::eutE)的比生长速率为0.24/h,其比参考菌株IME324(GPD1 GPD2)的比生长速率低约20%。在这些厌氧低渗透培养物中菌株IMX776的生理学,包括生物质形成和乙酸消耗的化学计量在内,与菌株IMX888(gpdlΔ gpd2::eutE)的非常相似(表4,图9和图10)。实际上在菌株IMX776中没有形成细胞外甘油,表明在这些条件下,该菌株中NADPH依赖性甘油生产的体内活性是最小的。与此观点一致,菌株IMX901(gpd1::gpsA gpd2::eutE ald6Δ)在厌氧培养物中的生长和产物形成类似于在这些条件下观察到的菌株IMX776或IMX888的性能。
实施例5
高渗透压下的生长不利地影响gpd2Δ菌株的乙酸还原
为了评定高渗透压对GPD1 gpd2::eutE菌株IMX884中观察到的乙酸还原的影响,将其性能与在1mol/L(180g/L)葡萄糖上生长的厌氧生物反应器分批培养物中的菌株IMX992(GPD1 GPD2sga1::eutE)的性能进行比较。与菌株继续呈对数生长直至葡萄糖被耗尽(图9)的低渗透压培养物不同,高渗透压条件显示出双相生长曲线,其中对数生长期后是第二个更慢的生长期(图12)。
菌株IMX992(GPD1 GPD2sga1::eutE)的初始比生长速率不受将培养基中的葡萄糖浓度增加至1mol/L的影响(表4和表5,图12)。该菌株在高渗透压培养物中的乙酸消耗低于在20g/L葡萄糖上生长期间观察到的(分别为2.67mmol/(g生物质)和3.35mmol/(g生物质))。该观察结果表明,同样在高渗透压条件下,EutE介导的乙酸还原不能有效地与全功能甘油途径竞争NADH。
菌株IMX884(GPD1 gpd2::eutE)显示为在高渗透培养基中的比生长速率比在低葡萄糖浓度上生长的培养物中低10%(表4和表5)。相对于其在低渗透压培养物中的性能,在1mol/L葡萄糖上生长导致细胞外甘油产量增加为3倍(6.34mmol/(g生物质),相对于1.92mmol/(g生物质)),并且乙酸消耗相应减少(2.98mmol/gx,相对于5.77mmol/gx)(表4和表5)。这些变化在很大程度上消除了在低渗透压培养物中观察到的菌株IMX992与IMX884之间甘油产量的四倍差异(表4和表5)。在完全消耗葡萄糖后,乙酸、甘油和乙醇的浓度在两种菌株的高渗透压培养物中达到相似的浓度(图12)。这些结果表明,即使当GPD2缺失时,高渗透压条件也阻碍了基于EutE的乙酸还原途径与甘油途径竞争NADH,这可能是由于渗透压力诱导的GPD1上调。
实施例6
由gpsA取代GPD1和GPD2使氧化还原代谢和渗透调节中甘油形成的作用解偶联
为了测试用偏好NADP+的G3PDH替代酵母NAD+依赖性Gpd同工酶是否可以使渗透调节和氧化还原代谢中甘油形成的作用解偶联,在高渗透压培养中研究菌株IMX776(gpd1::gpsA gpd2::eutE)的生长和产物形成。与菌株IMX992和IMX884不同,菌株IMX776在这些条件下显示出约50h的滞后期(图12),并且其比生长速率比在低渗透压培养物中时低60%(表4和表5)。而在低渗透压条件下,该菌株不产生细胞外甘油,高渗透压分批培养物显示甘油产量为3.29mmol/(g生物质)(表5)。在葡萄糖耗尽后,菌株IMX776在高渗透压培养物中的甘油浓度比对于菌株IMX992(GPD1GPD2sga1::eutE)观察到的低44%(图12)。
菌株IMX776在高葡萄糖培养物中显示出比在低渗透压培养物中低得多的乙酸消耗(表4和表5)。这种差异可能是由通过细胞质NADP+依赖性乙醛脱氢酶Ald6的通量增加,以及细胞质GpsA反应中对NADPH需求的增加导致的。经由将乙醛氧化成乙酸产生NADPH,乙酸随后可以经由乙酰辅酶A合成酶、EutE和NAD+依赖性醇脱氢酶还原成乙醇,将导致NADH再氧化消耗较少的细胞外乙酸(图13)。ALD6的缺失对乙酸还原性gpsA表达S.cerevisiae的厌氧培养物的生理学具有强烈影响。尽管在高渗透压培养物中菌株IMX776(gpd1::gpsA gpd2:eutE)和菌株IMX901(gpd1::gpsA gpd2:eutE ald6△)的比生长速率相似(表5),但完全没有滞后期使后一种菌株的总发酵时间缩短了约35h(图12)。此外,菌株IMX901完全依赖于外源乙酸供应来进行其氧化还原平衡。当在对数生长完成后不提供附加乙酸时,生长和葡萄糖消耗显著减慢(图6)。向菌株IMX776的高渗透压分批培养物中类似地加入乙酸不影响其生长(图6)。
与菌株IMX884和IMX776不同,菌株IMX901在高渗透压培养基上的生物反应器培养物中生长的过程中保持非甘油产生表型,导致与菌株IMX992(GPD1 GPD2sga1::eutE)相比,葡萄糖的乙醇产量提高了13%(表5)。这与在高渗透压培养基上的菌株IMX901的厌氧摇瓶培养物中测量到的细胞内甘油浓度5.3±0.04g/l组合,表明在该菌株中对经由GpsA形成的甘油的完全细胞内保留。当在对数期后立即向菌株IMX901的高渗透压生物反应器培养物中加入附加乙酸时,未检测到细胞外甘油(图12)。然而,当在静止期中20h时加入乙酸时(图6),可检测到低浓度的甘油(<1g/L终浓度)。
表5.在甘油和乙酸代谢方面具有不同遗传修饰的S.cerevisiae菌株的厌氧生物反应器分批培养物中的比生长速率(μ),葡萄糖的生物质、乙醇和甘油产量(Y),以及甘油生产与生物质形成、乙酸消耗与葡萄糖消耗,以及乙酸消耗与生物质形成之间的化学计量关系。培养物在含有180g/L葡萄糖和3g/L乙酸(pH 5)的合成培养基上生长。比生长速率和化学计量从对数生长期中期计算,并表示对独立一式两份培养物的测量值的平均值±平均偏差。
参考文献
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序列表
<110> 帝斯曼知识产权资产管理有限公司
<120> 用于乙醇生产的改良酵母菌株
<130> 32413-WO-PCT
<160> 39
<170> BiSSAP 1.3.6
<210> 1
<211> 335
<212> PRT
<213> Amino acid sequence
<220>
<223> Archaeoglobus fulgidus gpsA
<400> 1
Met Ile Val Ser Ile Leu Gly Ala Gly Ala Met Gly Ser Ala Leu Ser
1 5 10 15
Val Pro Leu Val Asp Asn Gly Asn Glu Val Arg Ile Trp Gly Thr Glu
20 25 30
Phe Asp Thr Glu Ile Leu Lys Ser Ile Ser Ala Gly Arg Glu His Pro
35 40 45
Arg Leu Gly Val Lys Leu Asn Gly Val Glu Ile Phe Trp Pro Glu Gln
50 55 60
Leu Glu Lys Cys Leu Glu Asn Ala Glu Val Val Leu Leu Gly Val Ser
65 70 75 80
Thr Asp Gly Val Leu Pro Val Met Ser Arg Ile Leu Pro Tyr Leu Lys
85 90 95
Asp Gln Tyr Ile Val Leu Ile Ser Lys Gly Leu Ile Asp Phe Asp Asn
100 105 110
Ser Val Leu Thr Val Pro Glu Ala Val Trp Arg Leu Lys His Asp Leu
115 120 125
Arg Glu Arg Thr Val Ala Ile Thr Gly Pro Ala Ile Ala Arg Glu Val
130 135 140
Ala Lys Arg Met Pro Thr Thr Val Val Phe Ser Ser Pro Ser Glu Ser
145 150 155 160
Ser Ala Asn Lys Met Lys Glu Ile Phe Glu Thr Glu Tyr Phe Gly Val
165 170 175
Glu Val Thr Thr Asp Ile Ile Gly Thr Glu Ile Thr Ser Ala Leu Lys
180 185 190
Asn Val Tyr Ser Ile Ala Ile Ala Trp Ile Arg Gly Tyr Glu Ser Arg
195 200 205
Lys Asn Val Glu Met Ser Asn Ala Lys Gly Val Ile Ala Thr Arg Ala
210 215 220
Ile Asn Glu Met Ala Glu Leu Ile Glu Ile Leu Gly Gly Asp Arg Glu
225 230 235 240
Thr Ala Phe Gly Leu Ser Gly Phe Gly Asp Leu Ile Ala Thr Phe Arg
245 250 255
Gly Gly Arg Asn Gly Met Leu Gly Glu Leu Leu Gly Lys Gly Leu Ser
260 265 270
Ile Asp Glu Ala Met Glu Glu Leu Glu Arg Arg Gly Val Gly Val Val
275 280 285
Glu Gly Tyr Lys Thr Ala Glu Lys Ala Tyr Arg Leu Ser Ser Lys Ile
290 295 300
Asn Ala Asp Thr Lys Leu Leu Asp Ser Ile Tyr Arg Val Leu Tyr Glu
305 310 315 320
Gly Leu Lys Val Glu Glu Val Leu Phe Glu Leu Ala Thr Phe Lys
325 330 335
<210> 2
<211> 467
<212> PRT
<213> Amino acid sequence
<220>
<223> E. coli eutE
<400> 2
Met Asn Gln Gln Asp Ile Glu Gln Val Val Lys Ala Val Leu Leu Lys
1 5 10 15
Met Gln Ser Ser Asp Thr Pro Ser Ala Ala Val His Glu Met Gly Val
20 25 30
Phe Ala Ser Leu Asp Asp Ala Val Ala Ala Ala Lys Val Ala Gln Gln
35 40 45
Gly Leu Lys Ser Val Ala Met Arg Gln Leu Ala Ile Ala Ala Ile Arg
50 55 60
Glu Ala Gly Glu Lys His Ala Arg Asp Leu Ala Glu Leu Ala Val Ser
65 70 75 80
Glu Thr Gly Met Gly Arg Val Glu Asp Lys Phe Ala Lys Asn Val Ala
85 90 95
Gln Ala Arg Gly Thr Pro Gly Val Glu Cys Leu Ser Pro Gln Val Leu
100 105 110
Thr Gly Asp Asn Gly Leu Thr Leu Ile Glu Asn Ala Pro Trp Gly Val
115 120 125
Val Ala Ser Val Thr Pro Ser Thr Asn Pro Ala Ala Thr Val Ile Asn
130 135 140
Asn Ala Ile Ser Leu Ile Ala Ala Gly Asn Ser Val Ile Phe Ala Pro
145 150 155 160
His Pro Ala Ala Lys Lys Val Ser Gln Arg Ala Ile Thr Leu Leu Asn
165 170 175
Gln Ala Ile Val Ala Ala Gly Gly Pro Glu Asn Leu Leu Val Thr Val
180 185 190
Ala Asn Pro Asp Ile Glu Thr Ala Gln Arg Leu Phe Lys Phe Pro Gly
195 200 205
Ile Gly Leu Leu Val Val Thr Gly Gly Glu Ala Val Val Glu Ala Ala
210 215 220
Arg Lys His Thr Asn Lys Arg Leu Ile Ala Ala Gly Ala Gly Asn Pro
225 230 235 240
Pro Val Val Val Asp Glu Thr Ala Asp Leu Ala Arg Ala Ala Gln Ser
245 250 255
Ile Val Lys Gly Ala Ser Phe Asp Asn Asn Ile Ile Cys Ala Asp Glu
260 265 270
Lys Val Leu Ile Val Val Asp Ser Val Ala Asp Glu Leu Met Arg Leu
275 280 285
Met Glu Gly Gln His Ala Val Lys Leu Thr Ala Glu Gln Ala Gln Gln
290 295 300
Leu Gln Pro Val Leu Leu Lys Asn Ile Asp Glu Arg Gly Lys Gly Thr
305 310 315 320
Val Ser Arg Asp Trp Val Gly Arg Asp Ala Gly Lys Ile Ala Ala Ala
325 330 335
Ile Gly Leu Lys Val Pro Gln Glu Thr Arg Leu Leu Phe Val Glu Thr
340 345 350
Thr Ala Glu His Pro Phe Ala Val Thr Glu Leu Met Met Pro Val Leu
355 360 365
Pro Val Val Arg Val Ala Asn Val Ala Asp Ala Ile Ala Leu Ala Val
370 375 380
Lys Leu Glu Gly Gly Cys His His Thr Ala Ala Met His Ser Arg Asn
385 390 395 400
Ile Glu Asn Met Asn Gln Met Ala Asn Ala Ile Asp Thr Ser Ile Phe
405 410 415
Val Lys Asn Gly Pro Cys Ile Ala Gly Leu Gly Leu Gly Gly Glu Gly
420 425 430
Trp Thr Thr Met Thr Ile Thr Thr Pro Thr Gly Glu Gly Val Thr Ser
435 440 445
Ala Arg Thr Phe Val Arg Leu Arg Arg Cys Val Leu Val Asp Ala Phe
450 455 460
Arg Ile Val
465
<210> 3
<211> 891
<212> PRT
<213> Amino acid sequence
<220>
<223> E. coli adhE
<400> 3
Met Ala Val Thr Asn Val Ala Glu Leu Asn Ala Leu Val Glu Arg Val
1 5 10 15
Lys Lys Ala Gln Arg Glu Tyr Ala Ser Phe Thr Gln Glu Gln Val Asp
20 25 30
Lys Ile Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro
35 40 45
Leu Ala Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val Glu Asp
50 55 60
Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr
65 70 75 80
Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp Asp Thr Phe Gly
85 90 95
Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys Gly Ile Val Pro
100 105 110
Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu
115 120 125
Lys Thr Arg Asn Ala Ile Ile Phe Ser Pro His Pro Arg Ala Lys Asp
130 135 140
Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala Ala Ile Ala Ala
145 150 155 160
Gly Ala Pro Lys Asp Leu Ile Gly Trp Ile Asp Gln Pro Ser Val Glu
165 170 175
Leu Ser Asn Ala Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala
180 185 190
Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro
195 200 205
Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp Glu Thr
210 215 220
Ala Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys Thr Phe
225 230 235 240
Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val Val Val Val Asp
245 250 255
Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Thr His Gly Gly Tyr
260 265 270
Leu Leu Gln Gly Lys Glu Leu Lys Ala Val Gln Asp Val Ile Leu Lys
275 280 285
Asn Gly Ala Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Tyr Lys Ile
290 295 300
Ala Glu Leu Ala Gly Phe Ser Val Pro Glu Asn Thr Lys Ile Leu Ile
305 310 315 320
Gly Glu Val Thr Val Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys
325 330 335
Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu Asp Ala
340 345 350
Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly His Thr
355 360 365
Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala Arg Val Ser Tyr
370 375 380
Phe Gly Gln Lys Met Lys Thr Ala Arg Ile Leu Ile Asn Thr Pro Ala
385 390 395 400
Ser Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser
405 410 415
Leu Thr Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn
420 425 430
Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala
435 440 445
Glu Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg
450 455 460
Gly Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly His Lys
465 470 475 480
Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn Gly Tyr Ala
485 490 495
Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly Val Glu Thr Glu Val
500 505 510
Phe Phe Glu Val Glu Ala Asp Pro Thr Leu Ser Ile Val Arg Lys Gly
515 520 525
Ala Glu Leu Ala Asn Ser Phe Lys Pro Asp Val Ile Ile Ala Leu Gly
530 535 540
Gly Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu
545 550 555 560
His Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile
565 570 575
Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Met
580 585 590
Ile Ala Val Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr Pro Phe
595 600 605
Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu Ala Asp
610 615 620
Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp Ala Asn Leu Val Met
625 630 635 640
Asp Met Pro Lys Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala Val Thr
645 650 655
His Ala Met Glu Ala Tyr Val Ser Val Leu Ala Ser Glu Phe Ser Asp
660 665 670
Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Tyr Leu Pro Ala
675 680 685
Ser Tyr His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg Val His
690 695 700
Ser Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu Gly
705 710 715 720
Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln Phe His Ile Pro
725 730 735
His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val Ile Arg Tyr Asn
740 745 750
Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe Ser Gln Tyr Asp Arg
755 760 765
Pro Gln Ala Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu Gly Leu
770 775 780
Ser Ala Pro Gly Asp Arg Thr Ala Ala Lys Ile Glu Lys Leu Leu Ala
785 790 795 800
Trp Leu Glu Thr Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile Arg
805 810 815
Glu Ala Gly Val Gln Glu Ala Asp Phe Leu Ala Asn Val Asp Lys Leu
820 825 830
Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr
835 840 845
Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr Tyr Gly
850 855 860
Arg Asp Tyr Val Glu Gly Glu Thr Ala Ala Lys Lys Glu Ala Ala Pro
865 870 875 880
Ala Lys Ala Glu Lys Lys Ala Lys Lys Ser Ala
885 890
<210> 4
<211> 1008
<212> DNA
<213> Nucleotide sequence
<220>
<223> Archaeoglobus fulgidus gpsA codon-optimized for S. cerevisiae
<400> 4
atgattgttt ctattttggg tgctggtgct atgggttctg ctttgtctgt tccattggtt 60
gacaacggta acgaagttag aatttggggt accgaattcg acaccgaaat tttgaagtct 120
atttctgctg gtagagaaca cccaagattg ggtgttaagt tgaacggtgt tgaaattttc 180
tggccagaac aattggaaaa gtgtttggaa aacgctgaag ttgttttgtt gggtgtttct 240
accgacggtg ttttgccagt tatgtctaga attttgccat acttgaagga ccaatacatt 300
gttttgattt ctaagggttt gattgacttc gacaactctg ttttgaccgt tccagaagct 360
gtttggagat tgaagcacga cttgagagaa agaaccgttg ctattaccgg tccagctatt 420
gctagagaag ttgctaagag aatgccaacc accgttgttt tctcttctcc atctgaatct 480
tctgctaaca agatgaagga aattttcgaa accgaatact tcggtgttga agttaccacc 540
gacattattg gtaccgaaat tacctctgct ttgaagaacg tttactctat tgctattgct 600
tggattagag gttacgaatc tagaaagaac gttgaaatgt ctaacgctaa gggtgttatt 660
gctaccagag ctattaacga aatggctgaa ttgattgaaa ttttgggtgg tgacagagaa 720
accgctttcg gtttgtctgg tttcggtgac ttgattgcta ccttcagagg tggtagaaac 780
ggtatgttgg gtgaattgtt gggtaagggt ttgtctattg acgaagctat ggaagaattg 840
gaaagaagag gtgttggtgt tgttgaaggt tacaagaccg ctgaaaaggc ttacagattg 900
tcttctaaga ttaacgctga caccaagttg ttggactcta tttacagagt tttgtacgaa 960
ggtttgaagg ttgaagaagt tttgttcgaa ttggctacct tcaagtaa 1008
<210> 5
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 5
ccaaatgcga catgagtcac 20
<210> 6
<211> 18
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 6
acggacctat tgccattg 18
<210> 7
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 7
ttgttcaatg gatgcggttc 20
<210> 8
<211> 22
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 8
tggtcgacag atacaatcct gg 22
<210> 9
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 9
atcccgggtg gaaactaaac 20
<210> 10
<211> 18
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 10
aggcacaagc ctgttctc 18
<210> 11
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 11
tcctcggtag atcaggtcag 20
<210> 12
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 12
acggtgagct ccgtattatc 20
<210> 13
<211> 33
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 13
gttttagagc tagaaatagc aagttaaaat aag 33
<210> 14
<211> 24
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 14
gatcatttat ctttcactgc ggag 24
<210> 15
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 15
tattgacgcc gggcaagagc 20
<210> 16
<211> 17
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 16
cgaccgagtt gctcttg 17
<210> 17
<211> 104
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 17
gtgcgcatgt ttcggcgttc gaaacttctc cgcagtgaaa gataaatgat cgggcaagga 60
cgtcgaccat agttttagag ctagaaatag caagttaaaa taag 104
<210> 18
<211> 104
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 18
gtgcgcatgt ttcggcgttc gaaacttctc cgcagtgaaa gataaatgat cccaagaatt 60
cccattattc ggttttagag ctagaaatag caagttaaaa taag 104
<210> 19
<211> 120
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 19
gttgataacg gactagcctt attttaactt gctatttcta gctctaaaac gaagaattcc 60
agtggtcaat gatcatttat ctttcactgc ggagaagttt cgaacgccga aacatgcgca 120
<210> 20
<211> 120
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 20
gttgataacg gactagcctt attttaactt gctatttcta gctctaaaac aattcagagc 60
tgttagccat gatcatttat ctttcactgc ggagaagttt cgaacgccga aacatgcgca 120
<210> 21
<211> 120
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 21
tagaagaaaa aacatcaaga aacatcttta acatacacaa acacatacta tcagaataca 60
tgtaccaacc tgcatttctt tccgtcatat acacaaaata ctttcatata aacttacttg 120
<210> 22
<211> 120
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 22
caagtaagtt tatatgaaag tattttgtgt atatgacgga aagaaatgca ggttggtaca 60
tgtattctga tagtatgtgt ttgtgtatgt taaagatgtt tcttgatgtt ttttcttcta 120
<210> 23
<211> 79
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 23
tatatttgat gtaaatatct aggaaataca cttgtgtata cttctcgctt ttcttttatt 60
gcaaattaaa ccttcgagc 79
<210> 24
<211> 91
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 24
gcatagaaca ttatccgcgg aaacgggtat taggggtgag ggtgaataag gaaagtcagg 60
gaaatcgggc aagctggagc tcagtttatc a 91
<210> 25
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 25
tggtattggc agtttcgtag 20
<210> 26
<211> 22
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 26
gttatgagaa atgacataat gc 22
<210> 27
<211> 80
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 27
gtattttggt agattcaatt ctctttccct ttccttttcc ttcgctcccc ttccttatca 60
cacaggaaac agctatgacc 80
<210> 28
<211> 80
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 28
ataactgtag taatgttact agtagtagtt gtagaacttg tgtataatga taaattggtt 60
gccgcaaatt aaagccttcg 80
<210> 29
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 29
cgaacaagtt gtcaaggctg 20
<210> 30
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 30
gcatcgacca aaacacaacg 20
<210> 31
<211> 90
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 31
gcatagaaca ttatccgcgg aaacgggtat taggggtgag ggtgaataag gaaagtcagg 60
gaaatcgggc gtacaatgcc tggcatgttc 90
<210> 32
<211> 80
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 32
tatatttgat gtaaatatct aggaaataca cttgtgtata cttctcgctt ttcttttatt 60
ctcgtccagt tgagtctagc 80
<210> 33
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 33
acaattcaga gctgttagcc 20
<210> 34
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 34
acactgctga accagtcaag 20
<210> 35
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 35
caccaccgaa tggaactctg 20
<210> 36
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 36
ggtgctggtg ctatgggttc 20
<210> 37
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 37
ggtttcggtg acttgattgc 20
<210> 38
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 38
gagtccaaca acttggtgtc 20
<210> 39
<211> 20
<212> DNA
<213> Nucleotide sequence
<220>
<223> Primer
<400> 39
accggtaata gcaacggttc 20
Claims (18)
1.一种重组酵母细胞,其包含:
a)编码具有甘油-3-磷酸脱氢酶活性的酶的基因,其中所述酶对至少NADP+和/或对NADPH具有辅因子依赖性;
b)编码具有至少NAD+依赖性乙酰化乙醛脱氢酶活性(EC 1.2.1.10)的酶的基因;以及
c)在选自GPD1和GPD2的组的至少一个基因中的突变或破坏。
2.根据权利要求1所述的细胞,其中所述具有甘油-3-磷酸脱氢酶活性的酶对NADPH比对NADH具有更高的亲和力和/或更低的米氏常数和/或更高的最大活性。
3.根据权利要求1或2所述的细胞,其中所述编码具有甘油-3-磷酸脱氢酶活性的酶的基因包含至少一个外源基因。
4.根据权利要求3所述的细胞,其中所述基因编码具有根据SEQ IDNO:1的氨基酸序列的酶或所述酶的具有至少50%序列同一性的功能同源物。
5.根据权利要求1或2所述的细胞,其中所述编码具有至少NAD+依赖性乙酰化乙醛脱氢酶活性的酶的基因编码具有根据SEQ ID NO:2的氨基酸序列的酶或所述酶的具有至少50%序列同一性的功能同源物。
6.根据权利要求1或2所述的细胞,与其相应的野生型细胞相比,所述细胞不含NADPH依赖性醛还原酶活性(EC 1.2.1.4)或具有降低的NADPH依赖性醛还原酶活性(EC 1.2.1.4)。
7.根据权利要求1或2中任一项所述的细胞,其中所述细胞的基因组包含ALD6或其具有至少50%序列同一性的功能同源物中的突变。
8.根据权利要求1或2所述的细胞,其中所述具有至少NAD+依赖性乙酰化乙醛脱氢酶活性的酶催化乙酰辅酶A向乙醛的可逆转化和随后乙醛向乙醇的可逆转化。
9.根据权利要求8所述的细胞,其中所述酶包括NAD+依赖性乙酰化乙醛脱氢酶(EC1.2.1.10)活性和NAD+依赖性醇脱氢酶活性(EC 1.1.1.1)两者。
10.根据权利要求1或2所述的细胞,其中所述编码具有至少NAD+依赖性乙酰化乙醛脱氢酶活性的酶的基因编码具有根据SEQ ID NO:3的氨基酸序列的酶或所述酶的具有至少50%序列同一性的功能同源物。
11.根据权利要求1或2所述的细胞,所述细胞不包含编码具有丙酮酸甲酸裂解酶活性的酶的基因。
12.根据权利要求1至11中任一项所述的细胞用于制备塑料、有机酸、溶剂、动物饲料补充剂、药物、酶或化学原料的用途。
13.根据权利要求1至11中任一项所述的细胞用于制备乙醇、丁醇、乳酸、琥珀酸、维生素或氨基酸的用途。
14.一种用于制备发酵产物的方法,所述方法包括从可发酵的碳水化合物制备发酵产物,所述制备使用根据权利要求1至11中任一项所述的细胞在厌氧条件下进行。
15.根据权利要求14所述的方法,其中所述可发酵的碳水化合物获自淀粉、木质纤维素和/或果胶。
16.根据权利要求14或15所述的方法,其中所述可发酵的碳水化合物选自以下项的组:葡萄糖、果糖、蔗糖、麦芽糖、木糖、阿拉伯糖、半乳糖、纤维二糖和甘露糖。
17.根据权利要求16所述的方法,其中葡萄糖的浓度为80g/L或更高。
18.根据权利要求14或15所述的方法,其中所述发酵产物是乙醇。
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PCT/EP2017/083242 WO2018114758A1 (en) | 2016-12-20 | 2017-12-18 | Improved yeast strains for ethanol production |
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DK3559246T3 (da) | 2022-09-05 |
CA3045739A1 (en) | 2018-06-28 |
PL3559246T3 (pl) | 2022-09-05 |
ES2924108T3 (es) | 2022-10-04 |
US10913928B2 (en) | 2021-02-09 |
EP3559246B1 (en) | 2022-06-15 |
EP3559246A1 (en) | 2019-10-30 |
US20200095536A1 (en) | 2020-03-26 |
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BR112019011093A2 (pt) | 2019-10-22 |
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