Teh Min Yan, Ooi Kean Hean, Danny Teo Shun Xiang, Bin Mansoor Mohammad Ehsan, Shaun Lim Wen Zheng, Tan Meng How
School of Chemical and Biomedical Engineering , Nanyang Technological University , 637459 Singapore.
School of Biological Sciences , Nanyang Technological University , 637551 Singapore.
ACS Synth Biol. 2019 Apr 19;8(4):708-723. doi: 10.1021/acssynbio.8b00168. Epub 2019 Mar 22.
The availability of different host chassis will greatly expand the range of applications in synthetic biology. Members of the Acetobacteraceae family of Gram-negative bacteria form an attractive class of nonmodel microorganisms that can be exploited to produce industrial chemicals, food and beverage, and biomaterials. One such biomaterial is bacterial cellulose, which is a strong and ultrapure natural polymer used in tissue engineering scaffolds, wound dressings, electronics, food additives, and other products. However, despite the potential of Acetobacteraceae in biotechnology, there has been considerably little effort to fundamentally reprogram the bacteria for enhanced performance. One limiting factor is the lack of a well-characterized, comprehensive toolkit to control expression of genes in biosynthetic pathways and regulatory networks to optimize production and cell viability. Here, we address this shortcoming by building an expanded genetic toolkit for synthetic biology applications in Acetobacteraceae. We characterized the performance of multiple natural and synthetic promoters, ribosome binding sites, terminators, and degradation tags in three different strains, namely, Gluconacetobacter xylinus ATCC 700178, Gluconacetobacter hansenii ATCC 53582, and Komagataeibacter rhaeticus iGEM. Our quantitative data revealed strain-specific and common design rules for the precise control of gene expression in these industrially relevant bacterial species. We further applied our tools to synthesize a biodegradable cellulose-chitin copolymer, adjust the structure of the cellulose film produced, and implement CRISPR interference for ready down-regulation of gene expression. Collectively, our genetic parts will enable the efficient engineering of Acetobacteraceae bacteria for the biomanufacturing of cellulose-based materials and other commercially valuable products.
不同宿主底盘的可用性将极大地扩展合成生物学的应用范围。革兰氏阴性菌醋杆菌科的成员构成了一类有吸引力的非模式微生物，可用于生产工业化学品、食品和饮料以及生物材料。一种这样的生物材料是细菌纤维素，它是一种用于组织工程支架、伤口敷料、电子产品、食品添加剂和其他产品的坚固且超纯的天然聚合物。然而，尽管醋杆菌科在生物技术方面具有潜力，但在从根本上对细菌进行重新编程以提高性能方面所做的努力却相当少。一个限制因素是缺乏一个经过充分表征的综合工具包，用于控制生物合成途径和调控网络中基因的表达，以优化生产和细胞活力。在这里，我们通过构建一个用于醋杆菌科合成生物学应用的扩展遗传工具包来解决这一缺点。我们在三种不同的菌株中表征了多种天然和合成启动子、核糖体结合位点、终止子和降解标签的性能，这三种菌株分别是木醋杆菌ATCC 700178、汉氏醋杆菌ATCC 53582和莱茵醋杆菌iGEM。我们的定量数据揭示了在这些与工业相关的细菌物种中精确控制基因表达的菌株特异性和通用设计规则。我们进一步应用我们的工具来合成一种可生物降解的纤维素 - 几丁质共聚物，调整所生产的纤维素膜的结构，并实施CRISPR干扰以随时下调基因表达。总的来说，我们的遗传元件将使醋杆菌科细菌能够高效工程化，用于基于纤维素的材料和其他具有商业价值的产品的生物制造。
