Fast and reliable strain characterization ofStreptomyces lividansthrough micro‐scale cultivation

Abstract

Filamentous organisms of the genus Streptomyces play an important role in industrial production processes, due to their extensive secondary metabolism variability, as well as their ability to secrete efficiently large amounts of (heterologous) proteins. While genetic engineering tools are available to rapidly build up large strain libraries, the subsequent strain screening and bioprocess development still constitutes a bottleneck. This is due to the lack of reliable parallelized and accelerated cultivation techniques for morphologically challenging organisms. To address this challenge, we developed an integrated cultivation workflow for Streptomyces lividans based on a parallelized shaken 48-well microtiter-plate (MTP) cultivation device. In a first step, a feasible pre-culture method was identified and validated, revealing high comparability in subsequent main cultivations (coefficient of variation of 1.1% for in-plate replicates and 3.2% between different pre-cultures). When validating the growth performance in 1 mL MTP cultivation against an established 1,000 mL lab-scale cultivation system, highly comparable cultivation patterns were found for online (pH, dissolved oxygen), as well as for offline derived parameters (glucose uptake, cell-dry-weight, and pellet size). Additionally, the two cultivation regimes were compared with respect to transcriptional and protein secretion activity of Streptomyces, showing overall good comparability with minor, but well explainable discrepancies, most probably caused by different energy dissipation (shaking vs. stirring) and adaption effects due to different illumination conditions. Embedded within the presented cultivation workflow, the 1 mL MTP-based parallelized cultivation system seems to be a suitable screening tool for filamentous and industrial relevant organisms like Streptomyces. This can contribute to widen the field of application for these organisms and facilitate screening and early-stage bioprocess development. Biotechnol. Bioeng. 2017;114: 2011-2022. © 2017 Wiley Periodicals, Inc.

Differential transcriptomic analysis reveals hidden light response inStreptomyces lividans

“…This additionally translated into comparable cell dry weight at the investigated sampling points (Table ). Thus, by considering several independent measures no light‐induced differences on the process profile could be observed on the macroscopic level confirming previous results, now delivered with increased statistical significance based on 24 replicate cultivations. Slight differences in cellular morphology could be observed analyzing stationary cultures as illuminated cultures showed a lower projected pellet area (–27%) and a sharper pellet size distribution.…”

“…In this study, S. lividans was cultivated in a parallelized MTP‐based microbioreactor system. While cultures were either run in the dark or exposed to artificial illumination, highly comparable growth performance was observed confirming the results of a previous study . In‐depth metabolic analysis was conducted by transcriptomics.…”

“…Samples for transcriptomic analysis were taken during the late growth phase. Harvesting and RNA isolation was performed as described in Busche, et al rRNA depletion, RNA quality check and cDNA library preparation were performed as described in Koepff, et al For each of the two BL experiments three samples were pooled after RNA isolation and sequenced in paired‐end mode on an Illumina HiSeq 1500 system (San Diego, CA, USA) with 70 bases read length. The raw sequencing read files are available in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under accession number: E‐MTAB‐5623.…”

“…Morphology was investigated using light microscopy and automated image processing for pellet detection and analysis. The detailed procedure is given in Koepff, et al…”

“…To accelerate the upstream part of bioprocess development, a comprehensive standardized screening workflow for S. lividans was introduced recently. The concept is based on the well‐established BioLector® (BL) microbioreactor system and enables 48‐fold parallel cultivation in microtiter plates (MTPs) additionally addressing seed train standardization and cultivation analytics . MTP cultivation could be scaled up successfully to 1 L stirred tank reactors.…”