Alexander Grünberger

Single-cell microfluidics: opportunity for bioprocess development

Cell-to-cell heterogeneity in microbial biotechnological processes caused by biological (intrinsic) and environmental (extrinsic) fluctuations can have a severe impact on productivity. However, as yet little is known about the complex interplay between environmental reactor dynamics and cellular activity. A few years ago, innovative microfluidic systems were introduced facilitating the spatiotemporal analysis of single cells under well-defined environmental conditions allowing so far unachievable insights into population heterogeneity and bioreactor inhomogeneity. Examples of microfabricated systems include microfluidic cavities harbouring micropopulations of several thousand cells down to femtolitre-size structures entrapping individual bacteria. In well-defined perfusion experiments, central questions in biotechnology regarding, for example, growth, productivity, and heterogeneity on the single-cell level have been addressed for the first time. Microfluidics will take its place as a single-cell analytical technique in biotechnological process and strain characterization.

A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level.

In the continuously growing field of industrial biotechnology the scale-up from lab to industrial scale is still a major hurdle to develop competitive bioprocesses. During scale-up the productivity of single cells might be affected by bioreactor inhomogeneity and population heterogeneity. Currently, these complex interactions are difficult to investigate. In this report, design, fabrication and operation of a disposable picolitre cultivation system is described, in which environmental conditions can be well controlled on a short time scale and bacterial microcolony growth experiments can be observed by time-lapse microscopy. Three exemplary investigations will be discussed emphasizing the applicability and versatility of the device. Growth and analysis of industrially relevant bacteria with single cell resolution (in particular Escherichia coli and Corynebacterium glutamicum) starting from one single mother cell to densely packed cultures is demonstrated. Applying the picolitre bioreactor, 1.5-fold increased growth rates of C. glutamicum wild type cells were observed compared to typical 1 litre lab-scale batch cultivation. Moreover, the device was used to analyse and quantify the morphological changes of an industrially relevant l-lysine producer C. glutamicum after artificially inducing starvation conditions. Instead of a one week lab-scale experiment, only 1 h was sufficient to reveal the same information. Furthermore, time lapse microscopy during 24 h picolitre cultivation of an arginine producing strain containing a genetically encoded fluorescence sensor disclosed time dependent single cell productivity and growth, which was not possible with conventional methods.

Construction of a Prophage-Free Variant of Corynebacterium glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology

ABSTRACT The activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. The genome of the industrial amino acid producer Corynebacterium glutamicum ATCC 13032 contains three prophages (CGP1, CGP2, and CGP3) of so far unknown functionality. Several phage genes are regularly expressed, and the large prophage CGP3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. Here, we present the construction of MB001, a prophage-free variant of C. glutamicum ATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of a C. glutamicum chassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology.

Taking Control over Control: Use of Product Sensing in Single Cells to Remove Flux Control at Key Enzymes in Biosynthesis Pathways

Enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. Here we present an approach to rapidly generate sets of enzymes overriding this control. It is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. The sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened the library in a strain carrying the sensor pSenLys-Spc, which detects l-lysine, l-arginine and l-histidine. Six of the resulting N-acetyl-l-glutamate kinase proteins were further developed and characterized and found to be at least 20-fold less sensitive toward l-arginine inhibition than the wild-type enzyme. Overexpression of the mutein ArgB-K47H-V65A in C. glutamicumΔargR led to the accumulation of 34 mM l-arginine in the culture medium. We also screened mutant libraries of lysC-encoded aspartate kinase and hisG-encoded ATP phosphoribosyltransferase. We isolated 11 LysC muteins, enabling up to 45 mM l-lysine accumulation, and 13 HisG muteins, enabling up to 17 mM l-histidine accumulation. These results demonstrate that in vivo screening of enzyme libraries by using metabolite sensors is extremely well suited to identify high-performance muteins required for overproduction.

Beyond growth rate 0.6: What drives Corynebacterium glutamicum to higher growth rates in defined medium

In a former study we showed that Corynebacterium glutamicum grows much faster in defined CGXII glucose medium when growth was initiated in highly diluted environments [Grünberger et al. (2013b) Biotechnol Bioeng]. Here we studied the batch growth of C. glutamicum in CGXII at a comparable low starting biomass concentration of OD ≈ 0.005 in more detail. During bioreactor cultivations a bi‐phasic growth behavior with changing growth rates was observed. Initially the culture grew with μˆ=0.61±0.02 h−1 before the growth rate dropped to μˆ=0.46±0.02 h−1 . We were able to confirm the elevated growth rate for C. glutamicum in CGXII and showed for the first time a growth rate beyond 0.6 in lab‐scale bioreactor cultivations on defined medium. Advanced growth studies combining well‐designed bioreactor and microfluidic single‐cell cultivations (MSCC) with quantitative transcriptomics, metabolomics and integrative in silico analysis revealed protocatechuic acid as a hidden co‐substrate for accelerated growth within CGXII. The presented approach proves the general applicability of MSCC to investigate and validate the effect of single medium components on microorganism growth during cultivation in liquid media, and therefore might be of interest for any kind of basic growth study. Biotechnol. Bioeng. 2014;111: 359–371.

Beyond growth rate 0.6: Corynebacterium glutamicum cultivated in highly diluted environments

Fast growth of industrial microorganisms, such as Corynebacterium glutamicum, is a direct amplifier for the productivity of any growth coupled or decoupled production process. Recently, it has been shown that C. glutamicum when grown in a novel picoliter bioreactor (PLBR) exhibits a 50% higher growth rate compared to a 1 L batch cultivation [Grünberger et al. (2012) Lab Chip]. We here compare growth of C. glutamicum with glucose as substrate at different scales covering batch cultivations in the liter range down to single cell cultivations in the picoliter range. The maximum growth rate of standard batch cultures as estimated from different biomass quantification methods is

Spatiotemporal microbial single-cell analysis using a high-throughput microfluidics cultivation platform

{\hat {\mu }} = 0.42\pm 0.03\,{\rm h}^{- 1}

Coarse-Grained Modeling of Protein Second Osmotic Virial Coefficients: Sterics and Short-Ranged Attractions

even for microtiter scale cultivations. In contrast, growth in a microfluidic perfusion system enabling analysis of single cells reproducibly reveals a higher growth rate of

Analysis of SOS-induced spontaneous prophage induction in Corynebacterium glutamicum at the single-cell level.

{\hat {\mu }} = 0.62\pm 0.02\,{\rm h}^{- 1}

Polydimethylsiloxane (PDMS) Sub-Micron Traps for Single-Cell Analysis of Bacteria

. When in the same perfusion system cell‐free supernatant from exponentially grown shake flask cultures is used the growth rate of single cells is reduced to