High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass.
High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass.
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DOI：
10.1186/s13068-016-0569-z
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发表时间：
2016
影响因子：
6.3
通讯作者：
Scotti N
中科院分区：
文献类型：
--
作者：
Castiglia D;Sannino L;Marcolongo L;Ionata E;Tamburino R;De Stradis A;Cobucci-Ponzano B;Moracci M;La Cara F;Scotti N
利用植物生物质生产生物燃料是一个复杂的多步骤过程，具有重要的经济负担。已经寻求了几种生物技术方法来降低生物燃料的生产成本。本研究的目的是探索从嗜热和超嗜热细菌和古生菌中分别产生三个编码(Hemi)纤维素酶的基因在烟草质体体中的产生，并测试它们在生产第二代生物燃料的重要工业预处理生物质原料(A.donax)的生物转化中的应用。所选择的内切葡聚糖酶、内切β-1，4-木聚糖酶和β-葡萄糖苷酶在烟草质体体中表达，蛋白质产量占总可溶性蛋白(TSP)的2%~75%以上。内切葡聚糖酶(高达2%TSP)的积累使植物表型发生变化，其严重程度与酶产量直接相关。最严重的幼苗致死表型是由于内切葡聚糖酶蛋白与类囊体结合而导致的叶绿体发育受阻。内切β-1，4-木聚糖酶和β-葡萄糖苷酶的产量很高，不会对植物生长产生不利影响，热处理(分别为105.4和255.4单位/毫克)可使这两种酶的产量增加4倍。这两种来自叶绿体的生物催化剂都保留了天然或重组表达的酶的主要特征，但有有趣的区别。叶绿体来源的木聚糖酶和β-葡萄糖苷酶的嗜热性分别高于重组大肠杆菌和天然重组菌。在50摄氏度和60摄氏度下进行的生物转化实验表明，叶绿体衍生的酶能够水解经工业处理的巨型芦苇生物质。特别是，在60℃下用叶绿体来源的木聚糖酶取代商业酶，可以提高木糖的回收率和水解率；而用木聚糖酶和β-葡萄糖苷酶替代商业鸡尾酒产生的葡萄糖水平与商业鸡尾酒相似，木糖产率在整个24-72小时范围内总是更高。本研究中所描述的极高的嗜热酶和超嗜热酶的产量、稳定性和生物转化效率表明，叶绿体转化是一种真正具有成本效益的纤维素酶生产平台。本文的在线版本(doi：10.1186/s130680160569-z)包含补充材料，授权用户可以使用。
Biofuels production from plant biomasses is a complex multi-step process with important economic burdens. Several biotechnological approaches have been pursued to reduce biofuels production costs. The aim of the present study was to explore the production in tobacco plastome of three genes encoding (hemi)cellulolytic enzymes from thermophilic and hyperthermophilic bacterium and Archaea, respectively, and test their application in the bioconversion of an important industrially pretreated biomass feedstock (A. donax) for production of second-generation biofuels. The selected enzymes, endoglucanase, endo-β-1,4-xylanase and β-glucosidase, were expressed in tobacco plastome with a protein yield range from 2 % to more than 75 % of total soluble proteins (TSP). The accumulation of endoglucanase (up to 2 % TSP) gave altered plant phenotypes whose severity was directly linked to the enzyme yield. The most severe seedling-lethal phenotype was due to the impairment of plastid development associated to the binding of endoglucanase protein to thylakoids. Endo-β-1,4-xylanase and β-glucosidase, produced at very high level without detrimental effects on plant development, were enriched (fourfold) by heat treatment (105.4 and 255.4 U/mg, respectively). Both plastid-derived biocatalysts retained the main features of the native or recombinantly expressed enzymes with interesting differences. Plastid-derived xylanase and β-glucosidase resulted more thermophilic than the E. coli recombinant and native counterpart, respectively. Bioconversion experiments, carried out at 50 and 60 °C, demonstrated that plastid-derived enzymes were able to hydrolyse an industrially pretreated giant reed biomass. In particular, the replacement of commercial enzyme with plastid-derived xylanase, at 60 °C, produced an increase of both xylose recovery and hydrolysis rate; whereas the replacement of both xylanase and β-glucosidase produced glucose levels similar to those observed with the commercial cocktails, and xylose yields always higher in the whole 24–72 h range. The very high production level of thermophilic and hyperthermophilic enzymes, their stability and bioconversion efficiencies described in this study demonstrate that plastid transformation represents a real cost-effective production platform for cellulolytic enzymes. The online version of this article (doi:10.1186/s13068-016-0569-z) contains supplementary material, which is available to authorized users.