Microbial electrosynthesis with Clostridium ljungdahlii benefits from hydrogen electron mediation and permits a greater variety of products.

Microbial electrosynthesis (MES) is a very promising technology addressing the challenge of carbon dioxide recycling into organic compounds, which might serve as building blocks for the (bio)chemical industry. However, poor process control and understanding of fundamental aspects such as the microbial extracellular electron transfer (EET) currently limit further developments. In the model acetogen Clostridium ljungdahlii, both direct and indirect electron consumption via hydrogen have been proposed. However, without clarification neither targeted development of the microbial catalyst nor process engineering of MES are possible. In this study, cathodic hydrogen is demonstrated to be the dominating electron source for C. ljungdahlii at electroautotrophic MES allowing for superior growth and biosynthesis, compared to previously reported MES using pure cultures. Hydrogen availability distinctly controlled an either planktonic- or biofilm-dominated lifestyle of C. ljungdahlii. The most robust operation yielded higher planktonic cell densities in a hydrogen mediated process, which demonstrated the uncoupling of growth and biofilm formation. This coincided with an increase of metabolic activity, acetate titers, and production rates (up to 6.06 g L-1 at 0.11 g L-1 d-1). For the first time, MES using C. ljungdahlii was also revealed to deliver other products than acetate in significant amounts: here up to 0.39 g L-1 glycine or 0.14 g L-1 ethanolamine. Hence, a deeper comprehension of the electrophysiology of C. ljungdahlii was shown to be key for designing and improving bioprocess strategies in MES research.

Scalable downstream method for the cyclic lipopetide jagaricin.

Cyclic lipopeptides are substances with a high potential to act as antimicrobial agents. Jagaricin, produced by Janthinobacterium agaricidamnosum DSM 9628 and discovered in 2012, is a new member of this class with promising antifungal properties. However, further experiments to investigate future applications and/or conduct chemical derivatization to change properties and toxicity are impossible due to the limited access to jagaricin. Besides a high jagaricin concentration at the end of the fermentation process, a suitable downstream process is essential to generate appropriate amounts with the desired purity. In contrast to other amphiphilic molecules, jagaricin cannot be separated by foam fractionation since it is mainly attached to the surface of the microbial biomass. This technical report presents an overall process chain consisting of 11 individual steps to generate jagaricin in gram scale with a purity of over 95%.

Host nutrition-based approach for biotechnological production of the antifungal cyclic lipopeptide jagaricin.

In today's, society multi-resistant pathogens have become an emerging threat, which makes the search for novel anti-infectives more urgent than ever. A promising class of substances are cyclic lipopeptides like the antifungal jagaricin. Jagaricin is formed by the bacterial mushroom pathogen Janthinobacterium agaricidamnosum. It has shown antifungal activity against human pathogenic fungi like Candida albicans and Aspergillus fumigatus. In addition, jagaricin is nearly non-toxic for plants, which makes it a promising agent for agricultural applications. Cyclic lipopeptides formed by microorganisms originate from their secondary metabolism. This makes it very challenging to determine the inducing factor for product formation, especially for unknown microbial systems like J. agaricidamnosum. In the presented study, a biotechnological process for jagaricin formation was developed, investigating impact factors like the medium, oxygen availability, and phosphate. For this reason, experiments were conducted on microtiter plate, shake flask, and stirred tank bioreactor level. Ultimately, a final maximum jagaricin concentration of 251 mg L-1 (15.5 mgJagaricin∙gCDW-1) could be achieved, which is an increase of approximately 458 % in comparison to previous results in standard glucose medium. This concentration allows the production of significantly higher amounts of jagaricin and enables further experiments to investigate the potential of this substance.

Electrophysiology of the Facultative Autotrophic Bacterium Desulfosporosinus orientis.

Electroautotrophy is a novel and fascinating microbial metabolism, with tremendous potential for CO2 storage and valorization into chemicals and materials made thereof. Research attention has been devoted toward the characterization of acetogenic and methanogenic electroautotrophs. In contrast, here we characterize the electrophysiology of a sulfate-reducing bacterium, Desulfosporosinus orientis, harboring the Wood-Ljungdahl pathway and, thus, capable of fixing CO2 into acetyl-CoA. For most electroautotrophs the mode of electron uptake is still not fully clarified. Our electrochemical experiments at different polarization conditions and Fe0 corrosion tests point to a H2- mediated electron uptake ability of this strain. This observation is in line with the lack of outer membrane and periplasmic multi-heme c-type cytochromes in this bacterium. Maximum planktonic biomass production and a maximum sulfate reduction rate of 2 ± 0.4 mM day-1 were obtained with an applied cathode potential of -900 mV vs. Ag/AgCl, resulting in an electron recovery in sulfate reduction of 37 ± 1.4%. Anaerobic sulfate respiration is more thermodynamically favorable than acetogenesis. Nevertheless, D. orientis strains adapted to sulfate-limiting conditions, could be tuned to electrosynthetic production of up to 8 mM of acetate, which compares well with other electroacetogens. The yield per biomass was very similar to H2/CO2 based acetogenesis. Acetate bioelectrosynthesis was confirmed through stable isotope labeling experiments with Na-H13CO3. Our results highlight a great influence of the CO2 feeding strategy and start-up H2 level in the catholyte on planktonic biomass growth and acetate production. In serum bottles experiments, D. orientis also generated butyrate, which makes D. orientis even more attractive for bioelectrosynthesis application. A further optimization of these physiological pathways is needed to obtain electrosynthetic butyrate production in D. orientis biocathodes. This study expands the diversity of facultative autotrophs able to perform H2-mediated extracellular electron uptake in Bioelectrochemical Systems (BES). We characterized a sulfate-reducing and acetogenic bacterium, D. orientis, able to naturally produce acetate and butyrate from CO2 and H2. For any future bioprocess, the exploitation of planktonic growing electroautotrophs with H2-mediated electron uptake would allow for a better use of the entire liquid volume of the cathodic reactor and, thus, higher productivities and product yields from CO2-rich waste gas streams.

Metabolic engineering of Amycolatopsis japonicum for optimized production of [S,S]-EDDS, a biodegradable chelator.

The actinomycete Amycolatopsis japonicum is the producer of the chelating compound [S,S]-ethylenediamine-disuccinc acid (EDDS). [S,S]-EDDS is an isomer of ethylenediamine-tetraacetic acid (EDTA), an economically important chelating compound that suffers from an extremely poor degradability. Frequent use of the persistent EDTA in various industrial and domestic applications has caused an accumulation of EDTA in soil as well as in aqueous environments. As a consequence, EDTA is the highest concentrated anthropogenic compound present in water reservoirs. The [S,S]-form of EDDS has chelating properties similar to EDTA, however, in contrast to EDTA it is readily biodegradable. In order to compete with the cost-effective chemical synthesis of EDTA, we aimed to optimize the biotechnological production of [S,S]-EDDS in A. japonicum by using metabolic engineering approaches. Firstly, we integrated several copies of the [S,S]-EDDS biosynthetic genes into the chromosome of A. japonicum and replaced the native zinc responsive promoter with the strong synthetic constitutive promoter SP44*. Secondly, we increased the supply of O-phospho-serine, the direct precursor of [S,S]-EDDS. The combination of these approaches together with the optimized fermentation process led to a significant improvement in [S,S]-EDDS up to 9.8 g/L with a production rate of 4.3 mg/h/g DCW.

Synergistic activity of cosecreted natural products from amoebae-associated bacteria.

Investigating microbial interactions from an ecological perspective is a particularly fruitful approach to unveil both new chemistry and bioactivity. Microbial predator-prey interactions in particular rely on natural products as signal or defense molecules. In this context, we identified a grazing-resistant Pseudomonas strain, isolated from the bacterivorous amoeba Dictyostelium discoideum. Genome analysis of this bacterium revealed the presence of two biosynthetic gene clusters that were found adjacent to each other on a contiguous stretch of the bacterial genome. Although one cluster codes for the polyketide synthase producing the known antibiotic mupirocin, the other cluster encodes a nonribosomal peptide synthetase leading to the unreported cyclic lipopeptide jessenipeptin. We describe its complete structure elucidation, as well as its synergistic activity against methicillin-resistant Staphylococcus aureus, when in combination with mupirocin. Both biosynthetic gene clusters are regulated by quorum-sensing systems, with 3-oxo-decanoyl homoserine lactone (3-oxo-C10-AHL) and hexanoyl homoserine lactone (C6-AHL) being the respective signal molecules. This study highlights the regulation, richness, and complex interplay of bacterial natural products that emerge in the context of microbial competition.

Candida albicans utilizes a modified ß-oxidation pathway for the degradation of toxic propionyl-CoA.

Propionyl-CoA arises as a metabolic intermediate from the degradation of propionate, odd-chain fatty acids, and some amino acids. Thus, pathways for catabolism of this intermediate have evolved in all kingdoms of life, preventing the accumulation of toxic propionyl-CoA concentrations. Previous studies have shown that fungi generally use the methyl citrate cycle for propionyl-CoA degradation. Here, we show that this is not the case for the pathogenic fungus Candida albicans despite its ability to use propionate and valerate as carbon sources. Comparative proteome analyses suggested the presence of a modified ß-oxidation pathway with the key intermediate 3-hydroxypropionate. Gene deletion analyses confirmed that the enoyl-CoA hydratase/dehydrogenase Fox2p, the putative 3-hydroxypropionyl-CoA hydrolase Ehd3p, the 3-hydroxypropionate dehydrogenase Hpd1p, and the putative malonate semialdehyde dehydrogenase Ald6p essentially contribute to propionyl-CoA degradation and its conversion to acetyl-CoA. The function of Hpd1p was further supported by the detection of accumulating 3-hydroxypropionate in the hpd1 mutant on propionyl-CoA-generating nutrients. Substrate specificity of Hpd1p was determined from recombinant purified enzyme, which revealed a preference for 3-hydroxypropionate, although serine and 3-hydroxyisobutyrate could also serve as substrates. Finally, virulence studies in a murine sepsis model revealed attenuated virulence of the hpd1 mutant, which indicates generation of propionyl-CoA from host-provided nutrients during infection.

Trace element associated reduction of norleucine and norvaline accumulation during oxygen limitation in a recombinant Escherichia coli fermentation.

BACKGROUND: Norleucine and norvaline belong to a group of non-canonical amino acids which are synthesized as byproducts in the branched chain amino acid metabolism of Escherichia coli. The earlier observed misincorporation of these rare amino acids into recombinant proteins has attracted increasing attention due to the rising use of protein based biopharmaceuticals in clinical application. Experimental data revealed pyruvate overflow inducing conditions, which typically occur in oxygen limited zones of large-scale fermentations as a major reason leading to norvaline and norleucine synthesis during E. coli cultivation. Previous approaches to suppress misincorporation of norleucine and norvaline considered growth media supplementation with the relevant canonical isostructural compounds, but no research was performed on the impact of the overflow metabolism related trace elements molybdenum, nickel and selenium. These elements form essential parts of the formate hydrogen lyase (FHL) metalloprotein complex, which is a key enzyme of anaerobic pyruvate metabolism in E. coli and could therefore represent a crucial connection to the pyruvate accumulation associated biosynthesis of rare amino acids. RESULTS: In this study, the trace element associated response of recombinant antibody producing E. coli to oxygen limitation at high glucose concentration with a special focus on non-canonical amino acids was analysed. During fed-batch cultivation with provoked oxygen limitation and glucose excess norleucine and norvaline were only accumulated in the absence of molybdenum, nickel and selenium. In contrast, the trace element supplemented stress fermentation showed significantly reduced concentrations of these rare amino acids and the major signature fermentation product formate, supporting the correlation between a functional formate hydrogen lyase complex and low unspecific amino acid synthesis under oxygen limitation at high glucose concentration. CONCLUSIONS: The formation of norleucine and norvaline by recombinant E. coli during cultivation with provoked oxygen limitation and glucose excess can be reduced to levels at the detection limit by adding the trace elements molybdenum, selenium and nickel to the fermentation medium. Even under the metabolic burden during induction phase the physiologically available concentrations of non-canonical amino acids remained low. Since our results allow facile process changes that can be easily implemented to avoid the undesirable accumulation of norleucine and norvaline, we consider this study highly interesting for improved process development in E. coli based recombinant drug production and the future development of possible mechanisms to reduce misincorporation events into protein based biopharmaceuticals.

Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach.

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 µm particle C18 reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core-shell particle C18 reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.