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
and space-time yields of up to 660 g
l
d
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.
•Oxygen and glucose inhomogeneities had no effect on C. glutamicum performance.•pH fluctuations were identified to be one of the most critical scale-up parameters.•Acidic pH levels inhibit the ...metabolic adaption to limiting oxygen supply.•Scale-down simulations are use full to identify most critical scale-up parameters.
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.
Corynebacterium glutamicum is well‐known as an industrial workhorse, most notably for its use in the bulk production of amino acids in the feed and food sector. Previous studies of the effect of ...gradients in scale‐down reactors with complex media disclosed an accumulation of several carboxylic acids and a parallel decrease of growth and product accumulation. This study, therefore, addresses the impact of carboxylic acids, for example, acetate and l‐lactate, on the cultivation of the cadaverine producing strain C. glutamicum DM1945Δact3:Ptuf‐ldcCopt and their potential role in scale up related performance losses. A fluctuating power input in shake flask and stirred tank cultivations with mineral salt was applied to mimic discontinuous oxygen availability.
Results demonstrate, whenever sufficient oxygen was available, C. glutamicum recovered from previously occurring stressful conditions like an oxygen limiting phase. Reassimilation of acids was detected simultaneously. In cultures, which were supplemented with either acetate or l‐lactate, a rapid cometabolization of both acids in presence of glucose was observed, showing conversion rates of 7.8 and 3.8 mmol gcell dry weight−1 hr−1, respectively. Uptake of these acids was accompanied by increased oxygen consumption. Proteins related to oxidative stress response, glycogen synthesis, and the main carbon metabolism were found in altered concentrations under oscillatory cultivation conditions. (Proteomics data are available via ProteomeXchange with identifier PXD012760). Virtually no impact on growth or product formation was observed. We conclude that the reduced growth and product formation in scale‐down cultivations when complex media was used is not caused by the accumulation of carboxylic acids.
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.
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.
: 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.
The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing
(
) 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.
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.
Instead of taking leadership in early 1917 to plan strategy and prepare the country for war, Wilson produced his "peace without victory" speech and continued to pursue mediation. In 1918 and 1919 he ...relied on speeches about grand ideals to sway the Allies and the American public rather than economic pressure, political alliances, or other strategic weapons of influence. Striner's previous scholarship focused on Abraham Lincoln's Civil War leadership, and this book investigates similar questions of wartime leadership and strategic planning.
This paper argues that radical and pacifist peace activists made the Kellogg‐Briand Pact their own for the decade after its creation. While recognizing the treaty's flaws, they co‐opted and adapted ...its meaning, and it influenced many of the goals activists pursued in the 1930s. The pact aided cooperation between peace activists, and both the treaty and Frank B. Kellogg himself played a larger role in the peace movement for a longer period than previously recognized. This argument draws primarily from sources at the Minnesota Historical Society, the WILPF International records at the University of Colorado‐Boulder, the Sophia Smith Collection at Smith College, and the WILPF records in the Swarthmore College Peace Collection.
We have studied the effect of a new, selective lipoxygenase inhibitor, TEI-1338, on alterations in arachidonic acid (20:4) metabolism and breakdown of the blood-aqueous barrier during herpes simplex ...virus (HSV) infection of the rabbit cornea. Synthesis of cyclooxygenase products (prostaglandins) and the lipoxygenase products, hydroxyeicosatet-raenoic acids (HETEs) and leukotriene B4 (LTB4), was measured in vitro by assessing conversion of 1-114C-20:4 in infected and non-infected corneas. TEI-1338 inhibited infection-stimulated lipoxygenase activity in a concentration-dependent manner, without affecting cyclooxygenase activity. Treatment of herpes infection in vivo with TEI-1338 (ocular drops) reduced leakage of protein into aqueous humor, a measure of inflammation-induced deterioration of the blood aqueous barrier. These data indicate that drugs that selectively inhibit lipoxygenase may be useful in the treatment of the inflammatory consequences of herpes keratitis, without exacerbating the infectious process.
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