Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708, WE, Wageningen, The Netherlands.
Laboratory of Systems and Synthetic Biology, Wageningen University, Stippeneng 4, 6708, WE, Wageningen, The Netherlands.
BMC Biotechnol. 2018 Jun 27;18(1):42. doi: 10.1186/s12896-018-0453-y.
Consolidated bioprocessing (CBP) is a cost-effective approach for the conversion of lignocellulosic biomass to biofuels and biochemicals. The enzymatic conversion of cellulose to glucose requires the synergistic action of three types of enzymes: exoglucanases, endoglucanases and β-glucosidases. The thermophilic, hemicellulolytic Geobacillus thermodenitrificans T12 was shown to harbor desired features for CBP, although it lacks the desired endo and exoglucanases required for the conversion of cellulose. Here, we report the expression of both endoglucanase and exoglucanase encoding genes by G. thermodenitrificans T12, in an initial attempt to express cellulolytic enzymes that complement the enzymatic machinery of this strain.
A metagenome screen was performed on 73 G. thermodenitrificans strains using HMM profiles of all known CAZy families that contain endo and/or exoglucanases. Two putative endoglucanases, GE39 and GE40, belonging to glucoside hydrolase family 5 (GH5) were isolated and expressed in both E. coli and G. thermodenitrificans T12. Structure modeling of GE39 revealed a folding similar to a GH5 exo-1,3-β-glucanase from S. cerevisiae. However, we determined GE39 to be a β-xylosidase having pronounced activity towards p-nitrophenyl-β-D-xylopyranoside. Structure modelling of GE40 revealed its protein architecture to be similar to a GH5 endoglucanase from B. halodurans, and its endoglucanase activity was confirmed by enzymatic activity against 2-hydroxyethylcellulose, carboxymethylcellulose and barley β-glucan. Additionally, we introduced expression constructs into T12 containing Geobacillus sp. 70PC53 endoglucanase gene celA and both endoglucanase genes (M1 and M2) from Geobacillus sp. WSUCF1. Finally, we introduced expression constructs into T12 containing the C. thermocellum exoglucanases celK and celS genes and the endoglucanase celC gene.
We identified a novel G. thermodenitrificans β-xylosidase (GE39) and a novel endoglucanase (GE40) using a metagenome screen based on multiple HMM profiles. We successfully expressed both genes in E. coli and functionally expressed the GE40 endoglucanase in G. thermodenitrificans T12. Additionally, the heterologous production of active CelK, a C. thermocellum derived exoglucanase, and CelA, a Geobacillus derived endoglucanase, was demonstrated with strain T12. The native hemicellulolytic activity and the heterologous cellulolytic activity described in this research provide a good basis for the further development of G. thermodenitrificans T12 as a host for consolidated bioprocessing.
综合生物加工（CBP）是将木质纤维素生物质转化为生物燃料和生物化学品的一种具有成本效益的方法。纤维素转化为葡萄糖需要三种类型的酶的协同作用：外切葡聚糖酶、内切葡聚糖酶和β-葡萄糖苷酶。嗜热、半纤维素分解的 Geobacillus thermodenitrificans T12 被证明具有 CBP 的所需特征，尽管它缺乏纤维素转化所需的内切和外切葡聚糖酶。在这里，我们报告了 G. thermodenitrificans T12 表达内切葡聚糖酶和外切葡聚糖酶编码基因，这是首次尝试表达补充该菌株酶机制的纤维素酶。
使用包含内切和/或外切葡聚糖酶的所有已知 CAZy 家族的 HMM 图谱对 73 株 G. thermodenitrificans 菌株进行了宏基因组筛选。分离并在大肠杆菌和 G. thermodenitrificans T12 中表达了两种属于糖苷水解酶家族 5 (GH5)的假定内切葡聚糖酶 GE39 和 GE40。GE39 的结构建模揭示了与酿酒酵母 GH5 外切-1,3-β-葡聚糖酶相似的折叠。然而，我们确定 GE39 是一种β-木糖苷酶，对 p-硝基苯基-β-D-木吡喃糖苷具有明显的活性。GE40 的结构建模表明其蛋白结构类似于 B. halodurans 的 GH5 内切葡聚糖酶，其内切葡聚糖酶活性通过对 2-羟乙基纤维素、羧甲基纤维素和大麦β-葡聚糖的酶活性得到证实。此外，我们将表达构建体引入含有 Geobacillus sp. 70PC53 内切葡聚糖酶基因 celA 和 Geobacillus sp. WSUCF1 的两种内切葡聚糖酶基因（M1 和 M2）的 T12 中。最后，我们将表达构建体引入含有 C. thermocellum 外切葡聚糖酶 celK 和 celS 基因和内切葡聚糖酶 celC 基因的 T12 中。
我们使用基于多个 HMM 图谱的宏基因组筛选鉴定了一种新型的 G. thermodenitrificans β-木糖苷酶（GE39）和一种新型的内切葡聚糖酶（GE40）。我们成功地在大肠杆菌中表达了这两个基因，并在 G. thermodenitrificans T12 中功能性表达了 GE40 内切葡聚糖酶。此外，还证明了具有活性 CelK（源自 C. thermocellum 的外切葡聚糖酶）和 CelA（源自 Geobacillus 的内切葡聚糖酶）的异源生产，使用菌株 T12。本文所述的天然半纤维素水解活性和异源纤维素水解活性为进一步开发 G. thermodenitrificans T12 作为综合生物加工宿主提供了良好的基础。
