Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
Biotechnology Training Program, Northwestern University, Evanston, IL 60208, USA.
Nucleic Acids Res. 2020 May 21;48(9):5169-5182. doi: 10.1093/nar/gkaa167.
One primary objective of synthetic biology is to improve the sustainability of chemical manufacturing. Naturally occurring biological systems can utilize a variety of carbon sources, including waste streams that pose challenges to traditional chemical processing, such as lignin biomass, providing opportunity for remediation and valorization of these materials. Success, however, depends on identifying micro-organisms that are both metabolically versatile and engineerable. Identifying organisms with this combination of traits has been a historic hindrance. Here, we leverage the facile genetics of the metabolically versatile bacterium Acinetobacter baylyi ADP1 to create easy and rapid molecular cloning workflows, including a Cas9-based single-step marker-less and scar-less genomic integration method. In addition, we create a promoter library, ribosomal binding site (RBS) variants and test an unprecedented number of rationally integrated bacterial chromosomal protein expression sites and variants. At last, we demonstrate the utility of these tools by examining ADP1's catabolic repression regulation, creating a strain with improved potential for lignin bioprocessing. Taken together, this work highlights ADP1 as an ideal host for a variety of sustainability and synthetic biology applications.
合成生物学的一个主要目标是提高化学制造的可持续性。天然存在的生物系统可以利用多种碳源，包括对传统化学处理构成挑战的废物流，如木质素生物质，为这些材料的修复和增值提供了机会。然而，成功取决于识别具有代谢多功能性和可工程性的微生物。具有这种组合特征的生物的识别一直是一个历史性的障碍。在这里，我们利用代谢多功能细菌不动杆菌 ADP1 易于遗传的特性，创建了简单快速的分子克隆工作流程，包括基于 Cas9 的单步无标记和无痕基因组整合方法。此外，我们创建了一个启动子文库、核糖体结合位点 (RBS) 变体，并测试了前所未有的数量的合理整合的细菌染色体蛋白表达位点和变体。最后，我们通过检查 ADP1 的分解代谢抑制调节来展示这些工具的实用性，创建了一个具有改善木质素生物加工潜力的菌株。总之，这项工作突出了 ADP1 作为各种可持续性和合成生物学应用的理想宿主。
