Data_Sheet_1_New Molecular Tools for Regulation and Improvement of A40926 Glycopeptide Antibiotic Production in Nonomuraea gerenzanensis ATCC 39727.PDF

Genome sequencing has revealed that Nonomuraea spp. represent a still largely unexplored source of specialized metabolites. Nonomuraea gerenzanensis ATCC 39727 is the most studied representative species since it produces the glycopeptide antibiotic (GPA) A40926 – the precursor of the clinically relevant antibiotic dalbavancin, approved by the FDA in 2014 for the treatment of acute skin infections caused by multi-drug resistant Gram-positive pathogens. The clinical relevance of dalbavancin has prompted increased attention on A40926 biosynthesis and its regulation. In this paper, we investigated how to enhance the genetic toolkit for members of the Nonomuraea genus, which have proved quite recalcitrant to genetic manipulation. By constructing promoter-probe vectors, we tested the activity of 11 promoters (heterologous and native) using the GusA reporter system in N. gerenzanensis and in Nonomuraea coxensis; this latter species is phylogenetically distant from N. gerenzanesis and also possesses the genetic potential to produce A40926 or a very similar GPA. Finally, the strongest constitutive promoter analyzed in this study, aac(3)IVp, was used to overexpress the cluster-situated regulatory genes controlling A40926 biosynthesis (dbv3 and dbv4 from N. gerenzanensis and nocRI from N. coxensis) in N. gerenzanensis, and the growth and productivity of the best performing strains were assessed at bioreactor scale using an industrial production medium. Overexpression of positive pathway-specific regulatory genes resulted in a significant increase in the level of A40926 production in N. gerenzanensis, providing a new knowledge-based approach to strain improvement for this valuable glycopeptide antibiotic.

基因组测序揭示了诺莫穆拉菌属（Nonomuraea spp.）是尚待充分发掘的具有特殊代谢产物的重要来源。其中，诺莫穆拉菌属的代表性物种诺莫穆拉菌（Nonomuraea gerenzanensis）ATCC 39727因产生糖肽抗生素（GPA）A40926——达巴环素（dalbavancin）的前体，该抗生素于2014年由美国食品药品监督管理局（FDA）批准用于治疗由多重耐药性革兰氏阳性病原体引起的急性皮肤感染，而备受关注。达巴环素的临床相关性促使人们对A40926的生物合成及其调控机制给予更多关注。在本研究中，我们探讨了如何增强诺莫穆拉菌属成员的遗传工具箱，因为这些菌属在遗传操作方面表现出了较强的抗性。通过构建启动子-探针载体，我们利用GusA报告系统在诺莫穆拉菌（N. gerenzanensis）和非诺穆拉菌（N. coxensis）中测试了11个启动子（异源和固有）的活性；后者在系统发育上与N. gerenzanensis相距较远，同时也具有产生A40926或极其相似的GPA的遗传潜力。最终，本研究中分析出的最强组成型启动子aac(3)IVp被用于在诺莫穆拉菌（N. gerenzanensis）中过表达控制A40926生物合成的调控基因簇（来自N. gerenzanensis的dbv3和dbv4，以及来自N. coxensis的nocRI）；在生物反应器规模上，使用工业生产介质评估了表现最佳的菌株的生长和生产力。过表达正调控路径特异性调控基因导致诺莫穆拉菌（N. gerenzanensis）中A40926生产水平显著提高，为这种珍贵的糖肽抗生素的菌株改良提供了一种基于新知识的途径。