Bacterial Cellulose Synthase Modification and Export

细菌纤维素合酶修饰及出口

• 批准号: RGPIN-2020-06637
• 负责人: Weadge, Joel
• 金额: $ 2.33万
• 依托单位: Wilfrid Laurier University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741348
• 关键词: Bacterial; Cellulose; Synthase; Modification; Export

Many bacteria form highly resistant biofilms composed of extracellular polysaccharides, like cellulose, that promote the adherence to various biotic and abiotic surfaces. The biofilm matrix also affords bacteria protection from immune system components, antimicrobial agents, detergents and harsh environmental factors. Bacterial cellulose is also gaining industrial significance, as it has unique physical/chemical properties that provide significant advantages when compared to algal- or plant-derived cellulose (eg., nano-scale, pliable and durable). Even though the biological and industrial implications of bacterial cellulose are expanding, the synthesis, modification and export of this polymer by bacteria is still being elucidated. The bcsABZC genes comprise the core bacterial cellulose synthase genes, however, important neighboring loci have also been noted for proper polymer production, modification and release. For example, bcsEFG and wssFGHI have been implicated in the installation of phosphoethanolamine (pEtN) or acetyl (Ac) groups onto newly synthesized cellulose chains, respectively. Despite the growing number of bacteria identified with these genes for distinct cellulose synthases, biochemical verification of the precise activities of the inherent enzymes that produce these uniquely modified polymers is lacking. Adding to this ambiguity, the associated cellulases that are predicted to release cellulose chains from the cell into the biofilm matrix are from diverse glycosyl hydrolase (GH) families. BcsZ enzymes in Gram negatives belong to GH-8 and BglX is a GH-3, but the CcsZ Gram positive homologs in Clostridia are GH-5 enzymes. The biological reason for this deviation and how these enzymes specifically recognize their respectively decorated cellulose polymers is unknown. This research proposal aims to address these deficiencies by a gene-to-molecule approach. We will phenotypically analyse environmental conditions that lead to cellulose biofilm formation and link this to expression of specific genes for our panel of organisms containing novel synthase complexes. The distinct cellulose-active enzymes from these synthases (Ac-/pEtN-transferases and GH enzymes) will be biochemically characterized in parallel to uncover molecular determinants responsible for polymer recognition, modification and turnover. These combined results will solve the ambiguity regarding fundamental requirements/synergies of enzymes in the distinct synthases of Gram negatives and positives and relate this to the importance of specific polymers to bacterial colonization and persistence. Exploitation of our toolkit (strains, enzymes, substrates and assays) will also be used to develop biocatalytic alternatives for the installment of desirable functionalities and physicochemical properties to cellulose polymers for various bioapplications. Thus, this combined research has immense potential for basic discoveries, as well as avenues that enhance further applied research.

许多细菌形成由细胞外多糖（如纤维素）组成的高抗性生物膜，其促进对各种生物和非生物表面的粘附。生物膜基质还可以保护细菌免受免疫系统成分、抗菌剂、清洁剂和恶劣环境因素的影响。细菌纤维素也获得了工业意义，因为它具有独特的物理/化学性质，与藻类或植物来源的纤维素（例如，纳米级、柔韧和耐用）。尽管细菌纤维素的生物和工业意义正在扩大，但细菌对这种聚合物的合成、修饰和输出仍在阐明中。bcsABZC基因包含核心细菌纤维素合酶基因，然而，重要的邻近基因座也被注意到用于适当的聚合物产生、修饰和释放。例如，bcsEFG和wssFGHI分别涉及磷酸乙醇胺（pEtN）或乙酰基（Ac）基团到新合成的纤维素链上的安装。尽管越来越多的细菌与这些不同的纤维素酶基因的鉴定，产生这些独特的改性聚合物的固有酶的精确活动的生化验证是缺乏的。除了这种模糊性之外，预测将纤维素链从细胞释放到生物膜基质中的相关纤维素酶来自不同的糖基水解酶（GH）家族。革兰氏阴性菌中的BcsZ酶属于GH-8，BglX是GH-3，但梭菌中的CcsZ革兰氏阳性同源物是GH-5酶。这种偏差的生物学原因以及这些酶如何特异性地识别它们各自修饰的纤维素聚合物是未知的。这项研究计划旨在通过基因到分子的方法来解决这些缺陷。我们将表型分析导致纤维素生物膜形成的环境条件，并将其与含有新型合酶复合物的生物体的特定基因的表达联系起来。这些酶（Ac-/pEtN-转移酶和GH酶）的不同的纤维素活性酶将进行生化表征，以揭示负责聚合物识别，修饰和周转的分子决定因素。这些组合结果将解决关于革兰氏阴性和阳性的不同酶中酶的基本要求/协同作用的模糊性，并将其与特定聚合物对细菌定殖和持久性的重要性联系起来。我们的工具包（菌株，酶，底物和测定）的开发也将用于开发生物催化替代品，用于各种生物应用的纤维素聚合物的理想功能和物理化学性质的安装。因此，这种综合研究具有巨大的基础发现潜力，以及加强进一步应用研究的途径。