Christian Dusny

Single-Cell Analysis in Biotechnology, Systems Biology, and Biocatalysis

Single-cell analysis (SCA) has been increasingly recognized as the key technology for the elucidation of cellular functions, which are not accessible from bulk measurements on the population level. Thus far, SCA has been achieved by miniaturization of established engineering concepts to match the dimensions of a single cell. However, SCA requires procedures beyond the classical approach of upstream processing, fermentation, and downstream processing because the biological system itself defines the technical demands. This review characterizes currently available microfluidics and microreactors for invasive (i.e., chemical) and noninvasive (i.e., biological) SCA. We describe the recent SCA omics approaches as tools for systems biology and discuss the role of SCA in genomics, transcriptomics, proteomics, metabolomics, and fluxomics. Furthermore, we discuss applications of SCA for biocatalysis and metabolic engineering as well as its potential for bioprocess optimization. Finally, we define present and future challenges for SCA and propose strategies to overcome current limitations.

Isolated Microbial Single Cells and Resulting Micropopulations Grow Faster in Controlled Environments

ABSTRACT Singularized cells of Pichia pastoris, Hansenula polymorpha, and Corynebacterium glutamicum displayed specific growth rates under chemically and physically constant conditions that were consistently higher than those obtained in populations. This highlights the importance of single-cell analyses by uncoupling physiology and the extracellular environment, which is now possible using the Envirostat 2.0 concept.

Microfluidic single‐cell analysis links boundary environments and individual microbial phenotypes

Life is based on the cell as the elementary replicative and self-sustaining biological unit. Each single cell constitutes an independent and highly dynamic system with a remarkable individuality in a multitude of physiological traits and responses to environmental fluctuations. However, with traditional population-based cultivation set-ups, it is not possible to decouple inherent stochastic processes and extracellular contributions to phenotypic individuality for two central reasons: the lack of environmental control and the occlusion of single-cell dynamics by the population average. With microfluidic single-cell analysis as a new cell assay format, these issues can now be addressed, enabling cultivation and time-resolved analysis of single cells in precisely manipulable extracellular environments beyond the bulk. In this article, we explore the interplay of cellular physiology and environment at a single-cell level. We review biological basics that govern the functional state of the cell and put them in context with physical fundamentals that shape the extracellular environment. Furthermore, the significance of single-cell growth rates as pivotal descriptors for global cellular physiology is discussed and highlighted by selected studies. These examples illustrate the unique opportunities of microfluidic single-cell cultivation in combination with growth rate analysis, addressing questions of fundamental bio(techno)logical interest.

Taking control over microbial populations: Current approaches for exploiting biological noise in bioprocesses

Phenotypic plasticity of microbial cells has attracted much attention and several research efforts have been dedicated to the description of methods aiming at characterizing phenotypic heterogeneity and its impact on microbial populations. However, different approaches have also been suggested in order to take benefit from noise in a bioprocess perspective, e.g. by increasing the robustness or productivity of a microbial population. This review is dedicated to outline these controlling methods. A common issue, that has still to be addressed, is the experimental identification and the mathematical expression of noise. Indeed, the effective interfacing of microbial physiology with external parameters that can be used for controlling physiology depends on the acquisition of reliable signals. Latest technologies, like single cell microfluidics and advanced flow cytometric approaches, enable linking physiology, noise, heterogeneity in productive microbes with environmental cues and hence allow correctly mapping and predicting biological behavior via mathematical representations. However, like in the field of electronics, signals are perpetually subjected to noise. If appropriately interpreted, this noise can give an additional insight into the behavior of the individual cells within a microbial population of interest. This review focuses on recent progress made at describing, treating and exploiting biological noise in the context of microbial populations used in various bioprocess applications.

Beyond the bulk: disclosing the life of single microbial cells

Abstract Microbial single cell analysis has led to discoveries that are beyond what can be resolved with population-based studies. It provides a pristine view of the mechanisms that organize cellular physiology, unbiased by population heterogeneity or uncontrollable environmental impacts. A holistic description of cellular functions at the single cell level requires analytical concepts beyond the miniaturization of existing technologies, defined but uncontrolled by the biological system itself. This review provides an overview of the latest advances in single cell technologies and demonstrates their potential. Opportunities and limitations of single cell microbiology are discussed using selected application-related examples.

The MOX promoter in Hansenula polymorpha is ultrasensitive to glucose-mediated carbon catabolite repression.

Redesigning biology towards specific purposes requires a functional understanding of genetic circuits. We present a quantitative in-depth study on the regulation of the methanol-specific MOX promoter system (PMOX) at the single-cell level. We investigated PMOX regulation in the methylotrophic yeast Hansenula (Ogataea) polymorpha with respect to glucose-mediated carbon catabolite repression. This promoter system is particularly delicate as the glucose as carbon and energy source in turn represses MOX promoter activity. Decoupling single cells from population activity revealed a hitherto underrated ultrasensitivity of the MOX promoter to glucose repression. Environmental control with single-cell technologies enabled quantitative insights into the balance between activation and repression of PMOX with respect to extracellular glucose concentrations. While population-based studies suggested full MOX promoter derepression at extracellular glucose concentrations of ∼1 g L(-1), we showed that glucose-mediated catabolite repression already occurs at concentrations as low as 5 × 10(-4) g L(-1) These findings demonstrate the importance of uncoupling single cells from populations for understanding the mechanisms of promoter regulation in a quantitative manner.

An Inert Continuous Microreactor for the Isolation and Analysis of a Single Microbial Cell

Studying biological phenomena of individual cells is enabled by matching the scales of microbes and cultivation devices. We present a versatile, chemically inert microfluidic lab-on-a-chip (LOC) device for biological and chemical analyses of isolated microorganisms. It is based on the Envirostat concept and guarantees constant environmental conditions. A new manufacturing process for direct fusion bonding chips with functional microelectrodes for selective and gentle cell manipulation via negative dielectrophoresis (nDEP) was generated. The resulting LOC system offered a defined surface chemistry and exceptional operational stability, maintaining its structural integrity even after harsh chemical treatment. The microelectrode structures remained fully functional after thermal bonding and were proven to be efficient for single-cell trapping via nDEP. The microfluidic network consisted solely of glass, which led to enhanced chip reusability and minimized interaction of the material with chemical and biological compounds. We validated the LOC for single-cell studies with the amino acid secreting bacterium Corynebacterium glutamicum. Intracellular l-lysine production dynamics of individual bacteria were monitored based on a genetically encoded fluorescent nanosensor. The results demonstrate the applicability of the presented LOC for pioneering chemical and biological studies, where robustness and chemically inert surfaces are crucial parameters for approaching fundamental biological questions at a single-cell level.

Challenging biological limits with microfluidic single cell analysis.

Back in the 17th century, van Leeuwenhoek was the first to advance into the microbial universe with his simple, but powerful single lens microscope. In a drop of aqueous infusion, he saw for the first time the basic functional and replicating units of life: single cells, in the shape of individual microbes. This observation laid the foundation for modern (micro-) biology. What began with a single cell was followed by centuries of research expanding our knowledge about microbial functionality and cellular processes – with population-based experiments. The central paradigm of microbiological methodology was reductionism, comprising isolation of microorganisms from their natural ecosystems to study them in axenic cultures in artificial environments like shake flasks or bioreactors. This determination of the boundaries of biological systems allowed controlling genetic identity, macroscopic cultivation parameters like availability and type of carbon source, as well as physicochemical parameters. Interactions of microbes with their environment in terms of mass and energy exchanges could be studied by microbial population ecology. Conclusions were and are drawn for idealized hypothetical single cells, de facto serving as wildcards in many areas from population function, to physiology or molecular biology. Yet, cellular individuality in isogenic populations is a fact. To what extent can the knowledge obtained from homogenized results of population experiments be really transferred to individual cells? Strictly speaking, it is not possible to decouple intracellular stochastic processes like molecule location and abundance, and external contributions to cellular individuality due to the lack of environmental control in classical shake flask or bioreactor experiments, including continuous chemostats. Only advances in microstructure technology and its symbiosis with microbiology during the past two decades made it technically and conceptually feasible to tackle these important biological questions. New microfluidic single cell isolation, analysis and cultivation methods, matching the scale of cultivation space and the dimensions of single microorganisms, introduced the possibility to control tiny amounts of liquid volume and manipulate the extracellular environment for defined physicochemical perturbations during the cultivation of single cells. Up to now, this basic concept of microfluidic single cell analysis represents the ultimate increment of the microbiological reductionist paradigm, decoupling environment and dynamics of cellular processes in a controlled single cell microecosystem (Kortmann et al., 2009). We are now witnessing increasing scientific interest and applications of microfluidic single cell analysis. It is becoming a standard technology in many microbiology and biotechnology laboratories, not at least due to the facts that several commercial microcultivation and analysis platforms are already available and established technologies like polydimethylsiloxane (PDMS) molding allow the design and fabrication of custom microfluidic networks, from prototype to final devices in short time periods. Single cells and mixed populations can be spatially and temporally organized in defined habitats. The possibility to control physicochemical cues and interactions between individual microbes in technical or natural ecosystems might well set off a revolution in microbial ecology. Microfluidic single cell assay formats already resulted in staggering insights into the cell as the basic functional biological unit. Examples, to name but a few, cover studies of mutual auxotrophy compensation in mixed, spatially adjacent cultures (Moffitt et al., 2012), ageing in single yeast (Lee et al., 2012) and single bacteria (Wang et al., 2010), interrelation of microbial growth rate and extracellular environment (Dusny et al., 2012), growth and gene expression (Sweedler and Arriaga, 2007) single cell dynamics during nutrient shifts (Boulineau et al., 2013) and effects of spatial confinement, like growth in narrow pores, on reproductive traits (Mannik et al., 2012). Studies like these demonstrate that microfluidic analyses of single cell dynamics, which began as a tender liaison between microengineering and microbiology, develop into basic pillars of systems and synthetic biology. Moreover, microfluidic single cell technologies might soon open to microbiology what was previously reserved to mammalian single cells: simultaneous genome and transcript sequencing (Blainey, 2013) of isolated single cells, even under transient conditions. The technologies are rapidly developing and already enable the mapping of metagenomes in complex microbial consortia. The potential of microfluidic single cell analysis is vast. New microbioreactor concepts and massively improved analytical technologies will allow the formulation and testing of new hypotheses. The main driver for future progress in single cell analysis will not only be the development of new or improved single cell technologies. It will again be the creativity and demand of microbiologists and biotechnologists for specialized (or universal) tools to answer specific biological questions and to understand the functional aspects of a single cell. Conceivable applications for new single cell technologies are manifold. One target is the long discussed cellular designer chassis as a highly specialized microbial factory for understanding functionality but also for engineering specific biotechnological purposes. The rational development of such cell factories assumes that control circuits and complex interconnections and relationships in the intracellular hierarchy, from genome to metabolism and their regulation in response to environmental stimuli, are known. A final systems-level understanding of cellular processes for predicting metabolic activity can only be obtained via controlled single cell experiments comprising all cellular responses to standardized perturbations, unbiased from population activity. Computational modelling of cells as systems and the assignment of catalytic and regulatory function to cellular individuality, and finally the design of custom cells for implementation into specific biotechnological processes will come within reach. We will also be in a position to experimentally address population dynamics and function approaching from the microbial individual. It is also clear that this access to microbial individuality will diversify and differentiate to satisfy future demands of various scientific fields. It might help our understanding of the interplay between the extracellular environment and cellular processes in natural ecosystems, mixed cultures and isogenic populations in technical ecosystems (reactors), as used in environmental applications, medical research and industrial processes. It will also be of utmost importance to adapt basic microbial methods to the analysis of single cell parameters in a classical sense. This applies in particular to a standardized quantification of cellular parameters like rate of biogenesis, maintenance, substrate uptake, as well as product and metabolite efflux. Even most fundamental cellular characteristics, like the specific growth rate of a cell, typically described by biomass or cell volume increase, are inaccessible at the level of a single cell because a unified methodology is lacking. These parameters are the keys for the systematic description of single cells. They will finally identify the individual contribution of single cells to the macroscopic output of microbial populations in processes in natural and technical ecosystems. Reductionism in the area of single cell analysis might even go further. In contrast to top-down approaches, comprising in vivo studies of single cells as whole functional systems, biomimetic bottom-up research based on in vitro assays with liposomes and vesicles as artificial cell-like systems are already emerging and will enrich ‘classical’ life cell approaches (Ullman et al., 2007). Mimicking a biological cell with the cell membrane maintaining a non-equilibrium state of the system with its environment, the analysis of specific features like transmembrane transport or the measurement of enzyme kinetics under in vivo-like reaction conditions will become possible. This will greatly enhance our understanding of processes in a single cell – and in life in general. However, even for this ultimate stage of reductionism, the description of artificial cell-like systems still requires classical (micro-) biological methodology. Microfluidic single cell approaches, in tandem with a holistic understanding of cellular traits at the level of a single cell, will lead to exciting discoveries that deepen our understanding of the single cell as the elementary unit of life.

l-Arabinose triggers its own uptake via induction of the arabinose-specific Gal2p transporter in an industrial Saccharomyces cerevisiae strain

Bioethanol production processes with Saccharomyces cerevisiae using lignocellulosic biomass as feedstock are challenged by the simultaneous utilization of pentose and hexose sugars from biomass hydrolysates. The pentose uptake into the cell represents a crucial role for the efficiency of the process. The focus of the here presented study was to understand the uptake and conversion of the pentose l-arabinose in S. cerevisiae and reveal its regulation by d-glucose and d-galactose. Gal2p—the most prominent transporter enabling l-arabinose uptake in S. cerevisiae wild-type strains—has an affinity for the transport of l-arabinose, d-glucose, and d-galactose. d-Galactose was reported for being mandatory for inducing GAL2 expression. GAL2 expression is also known to be regulated by d-glucose-mediated carbon catabolite repression, as well as catabolite inactivation. The results of the present study demonstrate that l-arabinose can be used as sole carbon and energy source by the recombinant industrial strain S. cerevisiae DS61180. RT-qPCR and RNA-Seq experiments confirmed that l-arabinose can trigger its own uptake via the induction of GAL2 expression. Expression levels of GAL2 during growth on l-arabinose reached up to 21% of those obtained with d-galactose as sole carbon and energy source. l-Arabinose-induced GAL2 expression was also subject to catabolite repression by d-glucose. Kinetic investigations of substrate uptake, biomass, and product formation during growth on a mixture of d-glucose/l-arabinose revealed impairment of growth and ethanol production from l-arabinose upon d-glucose depletion. The presence of d-glucose is thus preventing the fermentation of l-arabinose in S. cerevisiae DS61180. Comparative transcriptome studies including the wild-type and a precursor strain delivered hints for an increased demand in ATP production and cofactor regeneration during growth of S. cerevisiae DS61180 on l-arabinose. Our results thus emphasize that cofactor and energy metabolism demand attention if the combined conversion of hexose and pentose sugars is intended, for example in biorefineries using lignocellulosics.

Neue Einblicke in die Welt isolierter Mikroben

Single cell analysis is one of the key technologies for the investigation of cellular functionality. The physical laws at the microscale can be exploited with microfluidic bioreactors for decoupling a cell from the population bias and for cultivating and analyzing it in controllable artificial micro - environments. This gives direct access to the cellular physiology of the minimal unit of life, the single cell.