Selections are a powerful tool to obtain microbial strains with superior traits like improved cellulosic sugar utilization. However, the genetic mechanisms behind selections are complex and only partially understood at best. Better understanding of how genetic variation confers growth, fitness effects will allow for improved design of selections. Here the applications and limitations of Elementary Flux Mode (EFM) based modeling of mutant library growth phenotypes will be discussed for E.coli selections on cellulosic sugars.
Trackable Multiplex Recombineering (TRMR) creates a collection of mutants each with either a specific gene overexpressed or knocked-down1. This genetically engineered variance speeds up the discovery and identification of mutants with high growth/fitness thereby indicating mutations/genetic mechanisms that dominate a selection.
Elementary Flux Mode (EFM) Analysis allows for an evaluation of the metabolic pathways of a cell. A recent EFM method2 allows for modeling changes in fluxes in mutants with altered expression of metabolic genes. The method is adapted to assess variability in growth phenotypes in a mutant library. An evaluation of growth (yield) variability is given for different selection conditions (e.g. glucose, xylose growth) and an extension to model mutant fitness in a TRMR library is proposed.
Sugars like glucose and xylose are metabolized via different pathways. Quantifying the variation in metabolic pathway utilization for mutants can help assess its contribution to divergence in selections and could aid in relating it to variation in regulation and (often unanswered) fundamental issues like rate versus yield strategies, the relation between growth and fitness.