Engineering Geobacillus thermoglucosidasius for direct utilisation of holocellulose from wheat straw
Engineering Geobacillus thermoglucosidasius for direct utilisation of holocellulose from wheat straw
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DOI：
10.1186/s13068-019-1540-6
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发表时间：
2019-08-20
影响因子：
6.3
通讯作者：
Zhang, Ying
中科院分区：
文献类型：
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作者：
Bashir, Zeenat;Sheng, Lili;Zhang, Ying
联合生物处理（CBP）是在单一发酵步骤中将木质纤维素转化为所需产物而不添加昂贵的降解酶的过程，代表了化学品和燃料可再生途径的理想解决方案。土芽孢杆菌属的成员能够在升高的温度下生长，并且能够利用来源于木质纤维素的广泛的寡糖。这使得它们非常适合CBP的发展。结果在这项研究中，我们工程化热葡糖苷酸地芽孢杆菌NCIMB 11955利用木质纤维素生物质，在硝酸/氨处理的小麦秸秆的形式，其中没有添加昂贵的水解酶。两种不同的菌株BZ 9和BZ 10通过将来自Thermoanaerobacterium brockii的cglT（β-1，4-葡糖苷酶）基因整合到基因组中并将编码不同纤维素分解酶的基因定位在自主质粒上而产生。菌株BZ 10的质粒携带合成的纤维素体操纵子，其包含来自热纤梭菌的celA（内切葡聚糖酶A）基因和来自褐色嗜热裂菌的cel 6 B（外切葡聚糖酶）;而菌株BZ 9含有编码来自嗜热纤维素菌的celA（多结构域纤维素酶）基因的质粒。所有的基因被成功表达，其编码的产品分泌的功能活性的形式，证明了它们在培养上清液中通过Western印迹和酶法检测。在C. besides CelA酶，这是第一次在异源宿主中实现这种多功能酶的异源生产。两种菌株（BZ 9和BZ 10）在预处理的小麦秸秆上表现出改善的生长，实现更高的最终OD 600并产生更多数量的活细胞。为了证明纤维素乙醇可以由单一生物体直接从木质纤维素生物质中产生，我们在先前工程化的产乙醇G.热葡糖苷酶菌株LS 242。我们观察到重组G.热葡糖苷酶活力分别相当于BZ 9和BZ 10，而G.生长24小时的热葡萄糖苷酶LS 242菌株。结论我们成功地构建了G.本发明的目的是利用热葡糖苷酶来利用真实世界的木质纤维素生物质底物，并证明可以在一个步骤中直接从木质纤维素生物质生产纤维素乙醇。将生物质直接转化为所需产品是CBP的一种新模式，为碳中和和具有成本效益的可持续化学品和燃料生产提供了可能性。
Background A consolidated bioprocessing (CBP), where lignocellulose is converted into the desired product(s) in a single fermentative step without the addition of expensive degradative enzymes, represents the ideal solution of renewable routes to chemicals and fuels. Members of the genus Geobacillus are able to grow at elevated temperatures and are able to utilise a wide range of oligosaccharides derived from lignocellulose. This makes them ideally suited to the development of CBP. Results In this study, we engineered Geobacillus thermoglucosidasius NCIMB 11955 to utilise lignocellulosic biomass, in the form of nitric acid/ammonia treated wheat straw to which expensive hydrolytic enzymes had not been added. Two different strains, BZ9 and BZ10, were generated by integrating the cglT (beta-1,4-glucosidase) gene from Thermoanaerobacter brockii into the genome, and localising genes encoding different cellulolytic enzymes on autonomous plasmids. The plasmid of strain BZ10 carried a synthetic cellulosomal operon comprising the celA (Endoglucanase A) gene from Clostridium thermocellum and cel6B (Exoglucanase) from Thermobifida fusca; whereas, strain BZ9 contained a plasmid encoding the celA (multidomain cellulase) gene from Caldicellulosiruptor bescii. All of the genes were successfully expressed, and their encoded products secreted in a functionally active form, as evidenced by their detection in culture supernatants by Western blotting and enzymatic assay. In the case of the C. bescii CelA enzyme, this is one of the first times that the heterologous production of this multi-functional enzyme has been achieved in a heterologous host. Both strains (BZ9 and BZ10) exhibited improved growth on pre-treated wheat straw, achieving a higher final OD600 and producing greater numbers of viable cells. To demonstrate that cellulosic ethanol can be produced directly from lignocellulosic biomass by a single organism, we established our consortium of hydrolytic enzymes in a previously engineered ethanologenic G. thermoglucosidasius strain, LS242. We observed approximately twofold and 1.6-fold increase in ethanol production in the recombinant G. thermoglucosidasius equivalent to BZ9 and BZ10, respectively, compared to G. thermoglucosidasius LS242 strain at 24 h of growth. Conclusion We engineered G. thermoglucosidasius to utilise a real-world lignocellulosic biomass substrate and demonstrated that cellulosic ethanol can be produced directly from lignocellulosic biomass in one step. Direct conversion of biomass into desired products represents a new paradigm for CBP, offering the potential for carbon neutral, cost-effective production of sustainable chemicals and fuels.