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In recent years, microbioreactor (MBR) systems have evolved towards versatile bioprocess engineering tools. They provide a unique solution to combine higher experimental throughput with extensive bioprocess monitoring and control, which is indispensable to develop economically and ecologically competitive bioproduction processes. MBR systems are based either on down-scaled stirred tank reactors or on advanced shaken microtiter plate cultivation devices. Importantly, MBR systems make use of optical measurements for non-invasive, online monitoring of important process variables like biomass concentration, dissolved oxygen, pH, and fluorescence. The application range of MBR systems can be further increased by integration into liquid handling robots, enabling automatization and, thus standardization, of various handling and operation procedures. Finally, the tight integration of quantitative strain phenotyping with bioprocess development under industrially relevant conditions greatly increases the probability of finding the right combination of producer strain and bioprocess control strategy. This review will discuss the current state of the art in the field of MBR systems and we can readily conclude that their importance for industrial biotechnology will further increase in the near future.

Corynebacterium glutamicum was engineered for the production of L-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum ⌬aceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, L-alanine, and L-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum ⌬aceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and L-alanine towards L-valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum ⌬aceE(pJC4ilvBNCE) produced up to 210 mM L-valine with a volumetric productivity of 10.0 mM h ؊1 (1.17 g l ؊1 h ؊1 ) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose.

In recent years, metabolomics developed to an accepted and valuable tool in life sciences. Substantial improvements of analytical hardware allow metabolomics to run routinely now. Data are successfully used to investigate genotype-phenotype relations of strains and mutants. Metabolomics facilitates metabolic engineering to optimise mircoorganisms for white biotechnology and spreads to the investigation of biotransformations and cell culture. Metabolomics serves not only as a source of qualitative but also quantitative data of intra-cellular metabolites essential for the model-based description of the metabolic network operating under in vivo conditions. To collect reliable metabolome data sets, culture and sampling conditions, as well as the cells' metabolic state, are crucial. Hence, application of biochemical engineering principles and method standardisation efforts become important. Together with the other more established omics technologies, metabolomics will strengthen its claim to contribute to the detailed understanding of the in vivo function of gene products, biochemical and regulatory networks and, even more ambitious, the mathematical description and simulation of the whole cell in the systems biology approach. This knowledge will allow the construction of designer organisms for process application using biotransformation and fermentative approaches making effective use of single enzymes, whole microbial and even higher cells.

BackgroundHigh-throughput methods are widely-used for strain screening effectively resulting in binary information regarding high or low productivity. Nevertheless achieving quantitative and scalable parameters for fast bioprocess development is much more challenging, especially for heterologous protein production. Here, the nature of the foreign protein makes it impossible to predict the, e.g. best expression construct, secretion signal peptide, inductor concentration, induction time, temperature and substrate feed rate in fed-batch operation to name only a few. Therefore, a high number of systematic experiments are necessary to elucidate the best conditions for heterologous expression of each new protein of interest.ResultsTo increase the throughput in bioprocess development, we used a microtiter plate based cultivation system (Biolector) which was fully integrated into a liquid-handling platform enclosed in laminar airflow housing. This automated cultivation platform was used for optimization of the secretory production of a cutinase from Fusarium solani pisi with Corynebacterium glutamicum. The online monitoring of biomass, dissolved oxygen and pH in each of the microtiter plate wells enables to trigger sampling or dosing events with the pipetting robot used for a reliable selection of best performing cutinase producers. In addition to this, further automated methods like media optimization and induction profiling were developed and validated. All biological and bioprocess parameters were exclusively optimized at microtiter plate scale and showed perfect scalable results to 1 L and 20 L stirred tank bioreactor scale.ConclusionsThe optimization of heterologous protein expression in microbial systems currently requires extensive testing of biological and bioprocess engineering parameters. This can be efficiently boosted by using a microtiter plate cultivation setup embedded into a liquid-handling system, providing more throughput by parallelization and automation. Due to improved statistics by replicate cultivations, automated downstream analysis, and scalable process information, this setup has superior performance compared to standard microtiter plate cultivation.

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We recently engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of L: -valine from glucose by inactivation of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes, encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. Based on the first generation of pyruvate-dehydrogenase-complex-deficient C. glutamicum strains, a second generation of high-yield L-valine producers was constructed by successive deletion of the genes encoding pyruvate:quinone oxidoreductase, phosphoglucose isomerase, and pyruvate carboxylase and overexpression of ilvBNCE. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 410 mM (48 g/l) L-valine, showed a maximum yield of 0.75 to 0.86 mol/mol (0.49 to 0.56 g/g) of glucose in the production phase and, in contrast to the first generation strains, excreted neither pyruvate nor any other by-product tested.

Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli to lower the level of NADH and ATP, respectively. We used a global interaction network, comprising of protein interactions, transcriptional regulation, and metabolic networks, to integrate data from transcription profiles, metabolic fluxes, and the metabolite levels. We identified high-scoring networks for the two strains. The results revealed a smaller, but denser network for perturbations of ATP level, compared with that of NADH level. The action of many global transcription factors such as ArcA, Fnr, CRP, and IHF commonly involved both NADH and ATP, whereas others responded to either ATP or NADH. Overexpressing NADH oxidase invokes response in widespread aspects of metabolism involving the redox cofactors (NADH and NADPH), whereas ATPase has a more focused response to restore ATP level by enhancing proton translocation mechanisms and repressing biosynthesis. Interestingly, NADPH played a key role in restoring redox homeostasis through the concerted activity of isocitrate dehydrogenase and UdhA transhydrogenase. We present a reconciled network of regulation that illustrates the overlapping and distinct aspects of metabolism controlled by NADH and ATP. Our study contributes to the general understanding of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli.Microbial metabolic networks function in a coherent fashion to convert the available substrates into biomass and products. Often, intermediates of metabolism are not balanced with respect to their redox and/or energy content. Metabolic cofactors such as NADH and ATP serve in overcoming these constraints. Indeed, these cofactors rank among the most highly connected metabolites in the metabolic networks of most microorganisms (1). A direct consequence of this metabolic structure is that a small change in the concentration of these cofactors is likely to propagate to widespread aspects of metabolism. For example, the synthesis of proteins, lipids, and nucleotides is energetically expensive and would drain cellular ATP and also require NADPH (2). ATP is primarily produced in the electron transport chain, which is fueled by NADH. NADH is produced in the catabolism, relating substrate utilization to biosynthesis and product formation. Therefore, these cofactors hold the potential to serve as targets for altering cellular metabolism.To achieve the desired metabolic changes, it is important to understand the metabolic processes that are specifically controlled by cofactors. NADPH primarily drives anabolic reactions, whereas NADH is the result of catabolism. To fulfill their distinct roles, these two redox couples are generally not in thermodynamic equilibrium. NADPH is primarily produced in the oxidative branch of the pento...