Capturing of themonoterpene olefin limonene produced in Saccharomyces cerevisiae
Capturing of themonoterpene olefin limonene produced in Saccharomyces cerevisiae
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DOI：
10.1002/yea.3038
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发表时间：
2015-01-01
期刊：
影响因子：
2.6
通讯作者：
Beekwilder, Jules
中科院分区：
文献类型：
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作者：
Jongedijk, Esmer;Cankar, Katarina;Beekwilder, Jules
单萜烯烃如柠檬烯是植物化合物，其应用为调味剂和芳香剂、溶剂，并且还可能用于聚合物和燃料化学。我们对面包酵母酿酒酵母进行工程改造，使其表达来自紫苏的（-）-柠檬烯合酶和来自柑橘利蒙的（+）-柠檬烯合酶。这两种蛋白质表达的天然质体靶向信号或在一个截短的形式，其中质体分选信号被删除。用于表达的酵母宿主菌株是AE 9 K197 G，其表达突变型Erg 20酶。这种酶催化香叶基二磷酸的形成，香叶基二磷酸是单萜的前体。测试了几种方法来捕获由酵母产生的柠檬烯。通过戊烷从培养基中提取，或通过添加CaCl 2，然后进行固相微萃取，没有导致可检测到的柠檬烯，表明柠檬烯从培养基中迅速损失。挥发性萜烯（例如柠檬烯）也可能被捕获在发酵期间添加到培养基中的十二烷相中。该方法导致分别使用截短的柑橘和紫苏糖苷酶的菌株中回收0.028mg/l（+）-柠檬烯和0.060mg/l（-）-柠檬烯。在柠檬烯脱氢酶表达菌株的培养期间捕获顶空导致更高的滴度，在0.12mg/l（+）-柠檬烯和0.49mg/l（-）-柠檬烯。这些结果表明，所产生的烯烃的挥发性需要特定的方法来从生物技术生产系统中有效回收这些分子。基因库编号为：KM 015220（紫苏柠檬烯合酶;本研究）; AF 317695（紫苏柠檬烯合酶; Yuba等人，1（柑橘柠檬烯合酶; Lucker等人，).版权所有（c）2014约翰威利父子有限公司
Monoterpene olefins such as limonene are plant compounds with applications as flavouring and fragrance agents, as solvents and potentially also in polymer and fuel chemistry. We engineered baker's yeast Saccharomyces cerevisiae to express a (-)-limonene synthase from Perilla frutescens and a (+)-limonene synthase from Citrus limon. Both proteins were expressed either with their native plastid targeting signal or in a truncated form in which the plastidial sorting signal was removed. The yeast host strain for expression was AE9 K197G, which expresses a mutant Erg20 enzyme. This enzyme catalyses the formation of geranyl diphosphate, which is the precursor for monoterpenes. Several methods were tested to capture limonene produced by the yeast. Extraction from the culture medium by pentane, or by the addition of CaCl2 followed by solid-phase micro-extraction, did not lead to detectable limonene, indicating that limonene is rapidly lost from the culture medium. Volatile terpenes such as limonene may also be trapped in a dodecane phase added to the medium during fermentation. This method resulted in recovery of 0.028mg/l (+)-limonene and 0.060mg/l (-)-limonene in strains using the truncated Citrus and Perilla synthases, respectively. Trapping the headspace during culture of the limonene synthase-expressing strains resulted in higher titres, at 0.12mg/l (+)-limonene and 0.49mg/l (-)-limonene. These results show that the volatile properties of the olefins produced require specific methods for efficient recovery of these molecules from biotechnological production systems. Gene Bank Nos were: KM015220 (Perilla limonene synthase; this study); AF317695 (Perilla limonene synthase; Yuba et al., ); AF514287.1 (Citrus limonene synthase; Lucker et al., ). Copyright (c) 2014 John Wiley & Sons, Ltd.