Less sacrifice, more insight: Repeated low-volume sampling of microbioreactor cultivations enables accelerated deep phenotyping of microbial strain libraries.

With modern genetic engineering tools, high number of potentially improved production strains can be created in a short time. This results in a bottleneck in the succeeding step of bioprocess development, which can be handled by accelerating quantitative microbial phenotyping. Miniaturization and automation are key technologies to achieve this goal. In this study, a novel workflow for repeated low-volume sampling of BioLector-based cultivation setup is presented. Six samples of 20 µL each can be taken automatically from shaken 48-well microtiter plates without disturbing cell population growth. The volume is sufficient for quantification of substrate and product concentrations by spectrophotometric-based enzyme assays. From transient concentration data and replicate cultures, valid performance indicators (titers, rates, yields) are determined through process modelling and random error propagation analysis. Practical relevance of the workflow is demonstrated with a set of five genome-reduced C. glutamicum strains that were engineered for Sec-mediated heterologous cutinase secretion. Quantitative phenotyping of this strain library led to the identification of a strain with a 1.6-fold increase in cutinase yield. The prophage-free strain carries combinatorial deletions of three gene clusters (Δ3102-3111, Δ 3263-3301 and Δ 3324-3345) of which the last two likely contain novel target genes to foster rational engineering of heterologous cutinase secretion in C. glutamicum.