Microbial population quality control for enhanced chemical production

Non-genetic, cell-to-cell variation in protein and metabolite concentrations results from naturally-inherent factors, entailing variation in biosynthetic performance. However, the potential effects of heterogeneity on the overall product titer or yield has been rarely determined or addressed, which is particularly important when engineering metabolic pathways to produce useful chemicals. We assess non-genetic variation by direct quantification of bioproduct abundance within subpopulations of an isoclonal culture, and we subsequently develop a generalizable technique to improve bioproduction by continuously selecting for high-performing, non-genetic variants.  We demonstrated a 9-fold variation in free fatty acid (FFA) abundance within an isoclonal FFA-producing culture, and showed that the majority of bioproduct (54%) was generated by a minority of high-performing variants (15% of total). We then devised a technique termed in vivo population quality control (PopQC) to increase the proportion of high-performing variants by coupling product concentration to the expression of a survival gene. We validated PopQC in two biosynthetic pathways and tested two alternative design principles, yielding enhanced bioproduction of both FFA and tyrosine. Employing PopQC in a fed-batch FFA-producing culture resulted in 21.5 g/L titer and 0.5 g/L/hrs productivity. Finally, we confirmed that enhancement of production is achieved through selection of non-genetic variants, and thus we present non-genetic variation as a naturally-inherent phenomenon that can be exploited for industrially-relevant enhancement of biosynthetic performance.