ABSTRACT
High-throughput screening is a critical part of any industrial strain engineering effort, helping ensure the lowest cost product is produced in the shortest amount of time. Small-scale testing that correlates to manufacturing scale allows rapid strain development with confidence that engineering changes are relevant at-scale. In this review, the current state of high-throughput screening, the technological advances for the next generation strain screening pipeline, and options for implementation are reviewed. New technologies in cell culture (optofluidics) and measurement (acoustic mist ionization mass spectrometry) are highlighted, and special considerations (i.e. cost models, correlation between scales, data quality) are discussed.
Subject(s)
Mass SpectrometryABSTRACT
Antibiotic resistance arising via chromosomal mutations is typically specific to a particular antibiotic or class of antibiotics. We have identified mutations in genes encoding ribosomal components in Mycobacterium smegmatis that confer resistance to several structurally and mechanistically unrelated classes of antibiotics and enhance survival following heat shock and membrane stress. These mutations affect ribosome assembly and cause large-scale transcriptomic and proteomic changes, including the downregulation of the catalase KatG, an activating enzyme required for isoniazid sensitivity, and upregulation of WhiB7, a transcription factor involved in innate antibiotic resistance. Importantly, while these ribosomal mutations have a fitness cost in antibiotic-free medium, in a multidrug environment they promote the evolution of high-level, target-based resistance. Further, suppressor mutations can then be easily acquired to restore wild-type growth. Thus, ribosomal mutations can serve as stepping-stones in an evolutionary path leading to the emergence of high-level, multidrug resistance.
