Michael H. Limberg

Metabolic profile of 1,5-diaminopentane producing Corynebacterium glutamicum under scale-down conditions: Blueprint for robustness to bioreactor inhomogeneities

Performance losses during scale‐up are described since decades, but are still one of the major obstacles for industrial bioprocess development. Consequently, robustness to inhomogeneous cultivation environments is an important quality of industrial production organisms. Especially, Corynebacterium glutamicum was proven to have an outstanding resistance against rapid changes of oxygen and substrate availability as occurring in industrial scale bioreactors. This study focuses on the identification of metabolic key mechanisms for this robustness to get a deeper insight and provide future targets for process orientated strain development. A 1,5‐diaminopentane producing C. glutamicum strain was cultivated in a two compartment scale‐down device to create short‐term environmental changes simulating industrial scale cultivation conditions. Using multi omics based methods, it is shown, that central metabolism is flexibly rearranged under short‐term oxygen depletion and carbon source excess to overcome shortage in NAD+ recycling. In order to balance the redox state, key enzymes for the non‐oxygen dependent fermentative NAD+ regeneration were significantly up‐regulated while parts of non‐essential pathways were down‐regulated. The transfer of the cells back into the well aerated zones with low substrate concentration triggers an additional upregulation of genes for the re‐assimilation of previously formed side products, showing L‐lactate forming and utilizing reactions being active at the same time. Especially L‐lactate as reversible and flexible external buffer for carbon and redox equivalents puts C. glutamicum in a robust position to deal with inhomogeneity in large scale processes. Biotechnol. Bioeng. 2017;114: 560–575.

Plug flow versus stirred tank reactor flow characteristics in two‐compartment scale‐down bioreactor: Setup‐specific influence on the metabolic phenotype and bioprocess performance of Corynebacterium glutamicum

In the last two decades, scale‐down studies based on compartmented reactor setups became the standard procedure to mimic inhomogeneous cultivation conditions. In the academic field and with application to industrial‐scale, two basic scale‐down bioreactor configurations both showing a stirred tank reactor (STR) as main compartment predominate this research field. The connection to a plug flow reactor (PFR) generates oscillatory gradients with a distinct residence time of the culture, while the STR provides a broad residence time distribution leading to more heterogeneous oscillations. The influence of these opposed hydrodynamic profiles for their applicability for scale‐down bioreactor setups as well as their specific influence on the metabolic phenotype of l‐lysine producing Corynebacterium glutamicum DM1800 strain was investigated. Batch cultivations under oscillatory oxygen deprivation and substrate excess were carried out in STR–PFR and STR–STR scale‐down devices. In both setups, the induced inhomogeneity resulted in a reduction of growth rate and increased the l‐lactate and l‐glutamate by‐product formation, while biomass and product yields stayed nearly constant. Apart from differing side‐product levels, very similar results were observed when comparing the metabolic phenotype and bioprocess performance of STR–PFR and STR–STR configuration, although opposed back mixing profiles were present.

A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis

Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector® microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.

Performance loss of Corynebacterium glutamicum cultivations under scale-down conditions using complex media

Substrate and oxygen gradients appear in industrial‐scale fed‐batch processes due to limitations in the achievable power input and concomitantly increased mixing times. In order to mimic these gradients at lab scale, scale‐down reactors are applied. Previous studies in such reactor systems suggest that Corynebacterium glutamicum is robust against oscillatory oxygen and substrate availability in relation to growth and side product accumulation. Usually, defined mineral salt media are applied contrary to the industrial case, in which complex media containing different carbon sources are used. Therefore, this study investigated the cultivation performance using complex medium based on sucrose, molasses, and corn steep liquor in a three‐compartment scale‐down reactor. The reactor consisted of a stirred tank and two plug flow reactor modules. This approach was applied based on assumptions of gradient distributions in bottom‐fed bioreactors. A drastic reduction of growth and volumetric product yield of a cadaverine producing strain was observed while several short chain fatty acids accumulated, among them l‐lactate and acetate. Growth was depleted after several hours of cultivation, while the substrate uptake rate was reduced by 20%. Hence, the main carbon source sucrose accumulated after 10 h of fed‐batch cultivation. Despite growth cessation, neither reduction of cell vitality nor increased cell lysis were observed.

Catalytically active inclusion bodies of L-lysine decarboxylase from E. coli for 1,5-diaminopentane production

Sustainable and eco-efficient alternatives for the production of platform chemicals, fuels and chemical building blocks require the development of stable, reusable and recyclable biocatalysts. Here we present a novel concept for the biocatalytic production of 1,5-diaminopentane (DAP, trivial name: cadaverine) using catalytically active inclusion bodies (CatIBs) of the constitutive L-lysine decarboxylase from E. coli (EcLDCc-CatIBs) to process L-lysine-containing culture supernatants from Corynebacterium glutamicum. EcLDCc-CatIBs can easily be produced in E. coli followed by a simple purification protocol yielding up to 43% dry CatIBs per dry cell weight. The stability and recyclability of EcLDCc-CatIBs was demonstrated in (repetitive) batch experiments starting from L-lysine concentrations of 0.1 M and 1 M. EcLDC-CatIBs exhibited great stability under reaction conditions with an estimated half-life of about 54 h. High conversions to DAP of 87–100% were obtained in 30–60 ml batch reactions using approx. 180–300 mg EcLDCc-CatIBs, respectively. This resulted in DAP titres of up to 88.4 g l−1 and space-time yields of up to 660 gDAP l−1 d−1 per gram dry EcLDCc-CatIBs. The new process for DAP production can therefore compete with the currently best fermentative process as described in the literature.

pH fluctuations imperil the robustness of C. glutamicum to short term oxygen limitation

The presence of complex gradients for, e.g., nutrients, oxygen or pH in industrial scale fed batch processes are a major challenge for process performance. To consider such impact of scale-up during laboratory scale process development, scale-down bioreactor simulation, i.e. mimicking inhomogeneous conditions, became the method of choice. However, most scale-down studies simulate combined inhomogeneities of more than one parameter, so that the impact of the individual parameters remains unclear. The presented scale down study addresses this challenge by separating the influence of glucose, pH and oxygen fluctuations in terms of their specific impact in a well-established two compartment scale down device. This was carried out for an 1,5-diaminopentane production process using the industrial production host Corynebacterium glutamicum. Strikingly, oxygen depletion alone showed no effect on the process performance while changes of only one pH unit in acidic as well as alkaline direction reduced the biomass and product formation. Even more pronounced phenotypes up to -13% of μ and -39% of YX/S were observed, when an oscillatory acidic pH shift was combined with dissolved oxygen fluctuations. These losses are accompanied by a missing regulation of fermentative pathways. In conclusion, large-scale C. glutamicum processes seem to be most sensitive to pH variation.

Engineering of Scale-Down Bioreactor Setup: Deciphering Metabolic Phenotype of Corynebacterium glutamicum under Simulated Bioreactor Inhomogeneity

Performance losses during the scale-up from laboratory into production scale and the associated increase of production costs imperil the competitiveness of sustainable biotechnological bulk products. Since the formation of environmental gradients in the largescale was identified as the major obstacle for process scalability, scale-up simulation in the laboratory scale became one of the most important prediction tools. This work hooks up with the so called scale-down strategy by providing a flexible state of the art two compartment scale down device, which is made for easy process parallelization. The setup is composed of two connected and fully controlled stirred tank reactors (STR) implemented in a parallel cultivation platform. In order to validate the STR-STR application for Corynebacterium glutamicum processes, it was compared to an already established scale-down device consisting of a plug flow reactor (PFR) connected to a STR. Apart from differing sideproduct levels, very similar results were observed for the metabolic phenotype and bioprocess performance when sole oxygen perturbations were simulated, although the differing setups provide opposed back mixing profiles. A standardized scale-down workflow for the systematic separation and recombination of critical scale-up parameters was established, including state of the art technology for intracellular metabolite, protein and transcript analysis. Within a comprehensive study of 13 different combinations of oxygen, substrate and pH fluctuations, the work flow was used to investigate the scalability of C. glutamicum based 1,5-diaminopentane production. Thereby C. glutamicum provided an outstanding level of robustness to oxygen and substrate inhomogeneities. Using omics based methods, it was shown, that the flexible rearrangement of central metabolism is key for overcoming shortage in NAD recycling. Furthermore, formed L-lactate served as reversible and flexible external buffer for carbon and redox equivalents. Only acidic and alkaline pH fluctuation of one pH unit compromised C. glutamicum process performance. Especially, acidic pH levels inhibited the regulation of fermentative key pathway and with that the adaption to short term oxygen limitation. This reduced the biomass and product formation in a significant manner. Consequently, pH was identified to be one of the most sensitive parameter for C. glutamicum processes scale-up. Nevertheless, this systematic investigation of intracellular adaption mechanisms provided a comprehensive metabolic characterization of an adaptive and robust microbial strain for large-scale production.