Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (P) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a nonoxygen dependent marker protein, was used as a reporter. Fluorescence and fluorescence emission rates under oxygen-limited conditions were the highest when FbFP was under transcriptional control of P, P and P. The lengths of the E. coli endogenous promoters were shortened by 60%, maintaining their key regulatory elements. This resulted in improved promoter activity in most cases, particularly for P, P and P. Selected promoters were also evaluated using an engineered E. coli strain expressing Vitreoscilla hemoglobin (VHb). The presence of the VHb resulted in a better repression using these promoters under aerobic conditions, and increased the specific growth and fluorescence emission rates under oxygen-limited conditions. These results are useful for the selection of promoters for specific applications and for the design of modified artificial promoters.

Practical applicationIt is known that isogenic microbial population is able to display phenotypic diversification upon environmental changes. In this study, we developed a high throughput methodology based on the use of flow cytometry and the preliminary staining of the cells with Redox Sensor Green (RSG). We demonstrated that RSG is related to the activity of the electron transport chain and www.els-journal.com Page 2 Engineering in Life SciencesThis article is protected by copyright. All rights reserved.2 can be used as an efficient tracker of metabolic activity at the single cell level in different bioreactor configurations. We showed that RSG can be efficiently used to detect carbon limitation and dissolved oxygen limitation in bioreactor. More specifically, our methodology was used in order to assess a potential relationship between bioreactor heterogeneity and microbial population heterogeneity. Unexpectedly, no such correlation was found based on scale-down analysis. AbstractMicrobial heterogeneity in metabolic performances has attracted a lot of attention, considering its potential impact on industrial bioprocesses. However, little is known about the impact of extracellular perturbations (i.e. bioreactor heterogeneity) on cell-to-cell variability in metabolic performances (i.e. microbial population heterogeneity). In this work, we have evaluated the relevance of Redox Sensor Green (RSG) as an exogenous biosensor of metabolic activity at the single cell level. RSG signal is proportional to the activity of the electron transport chain and its signal is strongly affected by metabolic burden, availability of electron final acceptor and side metabolisms (i.e. overflow and mixed acid fermentation). RSG can also be used for the estimation of the impact of scale-down conditions on microbial metabolic robustness. The relationship linking averaged RSG activity and its cell-to-cell variability (noise) has been highlighted but seems unaffected by environmental perturbations.

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BackgroundDissolved oxygen tension (DOT) is hardly constant and homogenously distributed in a bioreactor, which can have a negative impact in the metabolism and product synthesis. However, the effects of DOT on plasmid DNA (pDNA) production and quality have not been thoroughly investigated. In the present study, the effects of aerobic (DOT ≥30% air sat.), microaerobic (constant DOT = 3% air sat.) and oscillatory DOT (from 0 to 100% air sat.) conditions on pDNA production, quality and host performance were characterized.ResultsMicroaerobic conditions had little effect on pDNA production, supercoiled fraction and sequence fidelity. By contrast, oscillatory DOT caused a 22% decrease in pDNA production compared with aerobic cultures. Although in aerobic cultures the pDNA supercoiled fraction was 98%, it decreased to 80% under heterogeneous DOT conditions. The different oxygen availabilities had no effect on the fidelity of the produced pDNA. The estimated metabolic fluxes indicated substantial differences at the level of the pentose phosphate pathway and TCA cycle under different conditions. Cyclic changes in fermentative pathway fluxes, as well as fast shifts in the fluxes through cytochromes, were also estimated. Model-based genetic modifications that can potentially improve the process performance are suggested.ConclusionsDOT heterogeneities strongly affected cell performance, pDNA production and topology. This should be considered when operating or scaling-up a bioreactor with deficient mixing. Constant microaerobic conditions affected the bacterial metabolism but not the amount or quality of pDNA. Therefore, pDNA production in microaerobic cultures may be an alternative for bioreactor operation at higher oxygen transfer rates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0378-x) contains supplementary material, which is available to authorized users.