Production of a functional cell wall-anchored minicellulosome by recombinant Clostridium acetobutylicum ATCC 824.
Production of a functional cell wall-anchored minicellulosome by recombinant Clostridium acetobutylicum ATCC 824.
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DOI：
10.1186/s13068-016-0526-x
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发表时间：
2016
影响因子：
6.3
通讯作者：
Minton NP
中科院分区：
文献类型：
--
作者：
Willson BJ;Kovács K;Wilding-Steele T;Markus R;Winzer K;Minton NP
使用化石燃料已经站不住脚了。它们不仅是一种有限的资源，而且它们的使用正在通过污染和全球变暖破坏环境。需要替代的、环境友好的、可再生的化学品和燃料来源。到目前为止，重点一直放在使用木质纤维素作为微生物发酵的原料上。然而，它对解构的抗拒使经济进程的发展极具挑战性。一种解决方案是产生一种适合于联合生物处理(CBP)的生物体，即一种既能水解木质纤维素又能发酵释放的糖的生物体，这是合成生物学的一个重要目标。我们的目标是使用合成生物学通过引入纤维素体(一种结合到称为支架蛋白的支架蛋白上的纤维素分解酶的复合体)来将产溶剂菌C.Acetobuylicum发展为CBP有机体。在以前的工作中，我们能够展示通过重组的乙酰丁酸杆菌菌株在体内生产来自高温细胞杆菌的微小纤维素体，并旨在在这一成功的基础上进行开发，解决以前策略的潜在问题。利用等位基因偶联交换(ACE)技术，将纤维素体酶Cel9G、Cel48F和Xyn10A的基因与来源于纤维素酶CipC的小咖啡因一起整合到乙酰丁酸菌基因组中。通过平板生长、分批发酵和糖释放实验，对重组纤维素体锚定在细胞表面的可能性进行了评价，并对重组菌株的降解性能进行了测定。我们已经能够通过重组乙酰丁酸杆菌菌株合成并在体内组装一个四组分的微纤维素体。此外，我们已经能够通过使用天然的索酸酶系统将一个微小的纤维素体锚定在乙酰丁酸杆菌的细胞壁上。重组菌株在木聚糖上的生长表型有所改善，从几种底物(包括未经处理的粉状麦草)中释放的还原糖增加。这构成了一个重要的里程碑，朝着开发一种真正适合CBP的纤维素分解菌株。本文的在线版本(doi：10.1186/s13068-0160526-x)包含补充材料，授权用户可以使用。
The use of fossil fuels is no longer tenable. Not only are they a finite resource, their use is damaging the environment through pollution and global warming. Alternative, environmentally friendly, renewable sources of chemicals and fuels are required. To date, the focus has been on using lignocellulose as a feedstock for microbial fermentation. However, its recalcitrance to deconstruction is making the development of economic processes extremely challenging. One solution is the generation of an organism suitable for use in consolidated bioprocessing (CBP), i.e. one able to both hydrolyse lignocellulose and ferment the released sugars, and this represents an important goal for synthetic biology. We aim to use synthetic biology to develop the solventogenic bacterium C. acetobutylicum as a CBP organism through the introduction of a cellulosome, a complex of cellulolytic enzymes bound to a scaffold protein called a scaffoldin. In previous work, we were able to demonstrate the in vivo production of a C. thermocellum-derived minicellulosome by recombinant strains of C. acetobutylicum, and aim to develop on this success, addressing potential issues with the previous strategy. The genes for the cellulosomal enzymes Cel9G, Cel48F, and Xyn10A from C. cellulolyticum were integrated into the C. acetobutylicum genome using Allele-Coupled Exchange (ACE) technology, along with a miniscaffoldin derived from C. cellulolyticum CipC. The possibility of anchoring the recombinant cellulosome to the cell surface using the native sortase system was assessed, and the cellulolytic properties of the recombinant strains were assayed via plate growth, batch fermentation and sugar release assays. We have been able to demonstrate the synthesis and in vivo assembly of a four-component minicellulosome by recombinant C. acetobutylicum strains. Furthermore, we have been able to anchor a minicellulosome to the C. acetobutylicum cell wall by the use of the native sortase system. The recombinant strains display an improved growth phenotype on xylan and an increase in released reducing sugar from several substrates including untreated powdered wheat straw. This constitutes an important milestone towards the development of a truly cellulolytic strain suitable for CBP. The online version of this article (doi:10.1186/s13068-016-0526-x) contains supplementary material, which is available to authorized users.