Let's talk about flux or the importance of (intracellular) reaction rates

The physiology of cells, determined by all extracellular production and consumption rates, intracellular reaction rates and the growth rate, is of fundamental interest in such different fields of the life sciences as microbiology, biotechnology and medical applications. In this Crystal Ball, I emphasize the importance of metabolic reaction rates, that is fluxes, for our understanding of metabolic network operation and envisage a time in which fluxes can be changed at will. Indeed, the ultimate goal of many fields of the life sciences is to understand cell physiology and hence the flux distribution, which characterizes the metabolic state of a cell. Based on this information, it will be possible to design strategies, for example, to avoid certain phenotypes (applications in infection biology and cancer research) or to redirect fluxes (applications in industrial biotechnology to increase rate, yield and titre of a product of interest). I argue that we are now in the lucky position that we can measure extraand intracellular fluxes, that we have well-characterized genetic tools for changing enzyme activities, and that we have fermentation capabilities to maintain optimal environmental conditions for a microorganism. Rationally changing a flux, however, requires a solid understanding of how the interplay of all mentioned aspects (and more) result in the observed flux distributions. Before describing some of these developments, we have to agree on the importance of the goal to precisely manipulate cellular fluxes. In many research projects, fluxes are in fact ignored, despite the ultimate goal implicitly being to understand metabolic network operation. For example, complex interactions between signalling pathways is often studied while ignoring cell physiology, although many cellular processes are growth rate dependent. And in industrial biotechnology, often the titre of the product of interest is communicated as this value quantifies overall performance of the biocatalytic reaction, implicitly even the product production rate (i.e. the flux towards the product) assuming a defined experimental time. Of course, when the fermentation is reported in the context of the entire process, titre is central; as it considers physical parameters including product solubility, the yield of the product on a given substrate and product inhibition of the biocatalyst besides other aspects. However, this is not the only critical value relevant to the process. In Metabolic Engineering, we propose to execute rational strain engineering, with the performance of the (microbial) cell in focus. Still, often end-point concentrations (e.g. g L ) and not specific rates normalized to the cell dry weight used (e.g. gproduct per gCDW per hour) are reported. When manipulating enzyme activities (e.g. using a stronger promoter or a more active enzyme variant), one implicitly aims for a change in flux at a given network node; the subsequent outcome of this flux change being a change in product concentration. A focus on end-point assays potentially misses interesting clones that show a significant improvement in production rate, hence achieve the maximal titre long before the end-point. However, there currently is no facile method to observe cellular fluxes on a broad scale. A potential answer discussed in the literature to the challenge of measuring fluxes in vivo is the application of in vivo metabolite sensors that respond to metabolite concentrations using fluorescent reporter systems. Cells carrying these sensors can be cultivated under constant fluorescence measurements conditions. A wide variety of fluorescent reporters is available, so metabolite sensors that report intracellular concentrations via fluorescence can be used for an ever increasing number of metabolites. It seems likely that we will see ever more advanced sensors with, for example, increased linear dynamic range for the analyte-to-sensor signal, lower interference from host components and lower interference of host metabolism. However, it has to be clear that these sensors measure first of all concentrations and not fluxes! In combination with a dedicated host that has a defined flux Received 15 October, 2016; accepted 16 October, 2016. *For correspondence. E-mail lars.blank@rwth-aachen.de; Tel. +49 (0)241 80-26600; Fax +49 (0)241 80-622180. Microbial Biotechnology (2017) 10(1), 28–30 doi:10.1111/1751-7915.12455