Structural and Functional Biochemistry, Laboratory of Proteomics and Metabolic Engineering of Prokaryotes, Department of Life Sciences and Systems Biology, University of Torino, Torino, Italy.
Department of Applied Science and Technology, Politecnico of Turin, Torino, Italy.
Biotechnol J. 2021 Aug;16(8):e2100064. doi: 10.1002/biot.202100064. Epub 2021 Jun 7.
Engineering microbial strains combining efficient lignocellulose metabolization and high-value chemical production is a cutting-edge strategy towards cost-sustainable 2 generation biorefining. Here, protein components of the Clostridium cellulovorans cellulosome were introduced in Lactococcus lactis IL1403, one of the most efficient lactic acid producers but unable to directly ferment cellulose. Cellulosomes are protein complexes with high cellulose depolymerization activity whose synergistic action is supported by scaffolding protein(s) (i.e., scaffoldins). Scaffoldins are involved in bringing enzymes close to each other and often anchor the cellulosome to the cell surface. In this study, three synthetic scaffoldins were engineered by using domains derived from the main scaffoldin CbpA and the Endoglucanase E (EngE) of the C. cellulovorans cellulosome. Special focus was on CbpA X2 and EngE S-layer homology (SLH) domains possibly involved in cell-surface anchoring. The recombinant scaffoldins were successfully introduced in and secreted by L. lactis. Among them, only that carrying the three EngE SLH modules was able to bind to the L. lactis surface although these domains lack the conserved TRAE motif thought to mediate binding with secondary cell wall polysaccharides. The synthetic scaffoldins engineered in this study could serve for assembly of secreted or surface-displayed designer cellulosomes in L. lactis.
将高效木质纤维素代谢和高价值化学品生产相结合的工程微生物菌株是成本可持续的第二代生物炼制的前沿策略。在这里，将纤维素分解菌 Clostridium cellulovorans 细胞表面展示纤维素酶复合物的蛋白组件引入到乳酸乳球菌 IL1403 中，IL1403 是生产乳酸效率最高的菌株之一，但不能直接发酵纤维素。细胞表面展示纤维素酶复合物是具有高纤维素解聚活性的蛋白复合物，其协同作用由支架蛋白（即支架蛋白）支持。支架蛋白参与使酶彼此靠近，并经常将细胞表面展示纤维素酶复合物锚定在细胞表面。在这项研究中，使用源自主要支架蛋白 CbpA 和 C. cellulovorans 细胞表面展示纤维素酶复合物的内切葡聚糖酶 E（EngE）的结构域，设计了三种合成支架蛋白。特别关注可能参与细胞表面锚定的 CbpA X2 和 EngE S-层同源（SLH）结构域。重组支架蛋白成功地在乳酸乳球菌中引入并分泌。其中，只有携带三个 EngE SLH 模块的支架蛋白能够与乳酸乳球菌表面结合，尽管这些结构域缺乏被认为介导与次生细胞壁多糖结合的保守 TRAE 基序。在这项研究中设计的合成支架蛋白可用于在乳酸乳球菌中组装分泌或表面展示的设计细胞表面展示纤维素酶复合物。
