Yellow polyketide pigment suppresses premature hatching in social amoeba.

Low-molecular-weight natural products from microbes are indispensable in the development of potent drugs. However, their biological roles within an ecological context often remain elusive. Here, we shed light on natural products from eukaryotic microorganisms that have the ability to transition from single cells to multicellular organisms: the social amoebae. These eukaryotes harbor a large number of polyketide biosynthetic genes in their genomes, yet virtually none of the corresponding products can be isolated or characterized. Using complementary molecular biology approaches, including CRISPR-Cas9, we generated polyketide synthase (pks5) inactivation and overproduction strains of the social amoeba Dictyostelium discoideum. Differential, untargeted metabolomics of wild-type versus mutant fruiting bodies allowed us to pinpoint candidate metabolites derived from the amoebal PKS5. Extrachromosomal expression of the respective gene led to the identification of a yellow polyunsaturated fatty acid. Analysis of the temporospatial production pattern of this compound in conjunction with detailed bioactivity studies revealed the polyketide to be a spore germination suppressor.

Ecological Niche-Inspired Genome Mining Leads to the Discovery of Crop-Protecting Nonribosomal Lipopeptides Featuring a Transient Amino Acid Building Block.

Investigating the ecological context of microbial predator-prey interactions enables the identification of microorganisms, which produce multiple secondary metabolites to evade predation or to kill the predator. In addition, genome mining combined with molecular biology methods can be used to identify further biosynthetic gene clusters that yield new antimicrobials to fight the antimicrobial crisis. In contrast, classical screening-based approaches have limitations since they do not aim to unlock the entire biosynthetic potential of a given organism. Here, we describe the genomics-based identification of keanumycins A-C. These nonribosomal peptides enable bacteria of the genus Pseudomonas to evade amoebal predation. While being amoebicidal at a nanomolar level, these compounds also exhibit a strong antimycotic activity in particular against the devastating plant pathogen Botrytis cinerea and they drastically inhibit the infection of Hydrangea macrophylla leaves using only supernatants of Pseudomonas cultures. The structures of the keanumycins were fully elucidated through a combination of nuclear magnetic resonance, tandem mass spectrometry, and degradation experiments revealing an unprecedented terminal imine motif in keanumycin C extending the family of nonribosomal amino acids by a highly reactive building block. In addition, chemical synthesis unveiled the absolute configuration of the unusual dihydroxylated fatty acid of keanumycin A, which has not yet been reported for this lipodepsipeptide class. Finally, a detailed genome-wide microarray analysis of Candida albicans exposed to keanumycin A shed light on the mode-of-action of this potential natural product lead, which will aid the development of new pharmaceutical and agrochemical antifungals.

Structural and Functional Analysis of Bacterial Sulfonosphingolipids and Rosette-Inducing Factor 2 (RIF-2) by Mass Spectrometry-Guided Isolation and Total Synthesis.

We have analyzed the abundance of bacterial sulfonosphingolipids, including rosette-inducing factors (RIFs), in seven bacterial prey strains by using high-resolution tandem mass spectrometry (HRMS2 ) and molecular networking (MN) within the Global Natural Product Social Molecular Networking (GNPS) web platform. Six sulfonosphingolipids resembling RIFs were isolated and their structures were elucidated based on comparative MS and NMR studies. Here, we also report the first total synthesis of two RIF-2 diastereomers and one congener in 15 and eight synthetic steps, respectively. For the total synthesis of RIF-2 congeners, we employed a decarboxylative cross-coupling reaction to synthesize the necessary branched α-hydroxy fatty acids, and the Garner-aldehyde approach to generate the capnine base carrying three stereogenic centers. Bioactivity studies in the choanoflagellate Salpingoeca rosetta revealed that the rosette inducing activity of RIFs is inhibited dose dependently by the co-occurring sulfonosphingolipid sulfobacins D and F and that activity of RIFs is specific for isolates obtained from Algoriphagus.

Scale-up of an amoeba-based process for the production of the cannabinoid precursor olivetolic acid.

BACKGROUND: The availability of new biological platform organisms to get access to innovative products and processes is fundamental for the progress in biotechnology and bioeconomy. The amoeba Dictyostelium discoideum represents a novel host system that has recently been employed for both the discovery of new natural products and as a cell factory for the production of bioactive compounds such as phytochemicals. However, an essential parameter to evaluate the potential of a new host system is the demonstration of its scalability to allow industrial applicability. Here, we aimed to develop a bioprocess for the production of olivetolic acid, the main precursor of cannabinoids synthesized by a recently engineered D. discoideum strain. RESULTS: In this study, a sophisticated approach is described to scale-up an amoeba-based polyketide production process in stirred tank bioreactors. Due to the shear sensitivity of the cell wall lacking amoebae, the maximum local energy dissipation rate (εmax) was selected as a measure for the hydromechanical stress level among different scales. By performing 1.6-L scale batch fermentations with different stress conditions, we determined a maximum tolerable εmax of 3.9 W/kg for D. discoideum. Further, we used this parameter as scale-up criterion to develop a bioprocess for olivetolic acid production starting from a 7-L stirred tank reactor to the industrially relevant 300-L scale with a product concentration of 4.8 µg/L, a productivity of 0.04 µg/L/h and a yield of 0.56 µg/g glucose. CONCLUSION: We developed a robust and reliable scale-up strategy for amoeba-based bioprocesses and evaluated its applicability for the production of the cannabinoid precursor olivetolic acid. By determining the maximum tolerable hydromechanical stress level for D. discoideum, we were able to scale-up the process from shake flasks to the 300-L stirred tank reactor without any yield reduction from cell shearing. Hence, we showed the scalability and biotechnological exploitation of amoeba-based processes that can provide a reasonable alternative to chemical syntheses or extractions of phytochemicals from plant biomass.

Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin.

Psilocybin, the principal indole alkaloid of Psilocybe mushrooms, is currently undergoing clinical trials as a medication against treatment-resistant depression and major depressive disorder. The psilocybin supply for pharmaceutical purposes is met by synthetic chemistry. We replaced the problematic phosphorylation step during synthesis with the mushroom kinase PsiK. This enzyme was biochemically characterized and used to produce one gram of psilocybin from psilocin within 20 minutes. We also describe a pilot-scale protocol for recombinant PsiK that yielded 150 mg enzyme in active and soluble form. Our work consolidates the simplicity of tryptamine chemistry with the specificity and selectivity of enzymatic catalysis and helps provide access to an important drug at potentially reasonable cost.

Noninvasive tool for optical online monitoring of individual biomass concentrations in a defined coculture.

Cocultures bear great potential in the conversion of complex substrates and process intensification, as well as, in the formation of unique components only available due to inter-species interactions. Dynamic data of coculture composition is necessary for understanding and optimizing coculture systems. However, most standard online determined parameters measure the sum of all species in the reactor system. The kinetic behavior of the individual species remains unknown. Up to now, different offline methods are available to determine the culture composition, as well as the online measurement of fluorescence of genetically modified organisms. To avoid any genetic modification, a noninvasive online monitoring tool based on the scattered light spectrum was developed for microtiter plate cultivations. To demonstrate the potential, a coculture consisting of the bacterium Lactococcus lactis and the yeast Kluyveromyces marxianus was cultivated. Via partial least squares regression of scattered light spectra, the online determination of the individual biomass concentrations without further sampling and analyses is possible. The results were successfully validated by a Coulter counter-analysis, taking advantage of the different cell sizes of both organisms. The findings prove the applicability of the new method to follow in detail the dynamics of a coculture.

Shake flask methodology for assessing the influence of the maximum oxygen transfer capacity on 2,3-butanediol production.

BACKGROUND: Production of 2,3-butanediol from renewable resources is a promising measure to decrease the consumption of fossil resources in the chemical industry. One of the most influential parameters on biotechnological 2,3-butanediol production is the oxygen availability during the cultivation. As 2,3-butanediol is produced under microaerobic process conditions, a well-controlled oxygen supply is the key parameter to control biomass formation and 2,3-butanediol production. As biomass is on the one hand not the final product, but on the other hand the essential biocatalyst, the optimal compromise between biomass formation and 2,3-butanediol production has to be defined. RESULTS: A shake flask methodology is presented to evaluate the effects of oxygen availability on 2,3-butanediol production with Bacillus licheniformis DSM 8785 by variation of the filling volume. A defined two-stage cultivation strategy was developed to investigate the metabolic response to different defined maximum oxygen transfer capacities at equal initial growth conditions. The respiratory quotient was measured online to determine the point of glucose depletion, as 2,3-butanediol is consumed afterwards. Based on this strategy, comparable results to stirred tank reactors were achieved. The highest space-time yield (1.3 g/L/h) and a 2,3-butanediol concentration of 68 g/L combined with low acetoin concentrations and avoided glycerol formation were achieved at a maximum oxygen transfer capacity of 13 mmol/L/h. The highest overall 2,3-butanediol concentration of 78 g/L was observed at a maximum oxygen transfer capacity of 4 mmol/L/h. CONCLUSIONS: The presented shake flask approach reduces the experimental effort and costs providing a fast and reliable methodology to investigate the effects of oxygen availability. This can be applied especially on product and by-product formation under microaerobic conditions. Utilization of the maximum oxygen transfer capacity as measure for the oxygen availability allows for an easy adaption to other bioreactor setups and scales.

Cyclopropanol Warhead in Malleicyprol Confers Virulence of Human- and Animal-Pathogenic Burkholderia Species.

Burkholderia species such as B. mallei and B. pseudomallei are bacterial pathogens causing fatal infections in humans and animals (glanders and melioidosis), yet knowledge on their virulence factors is limited. While pathogenic effects have been linked to a highly conserved gene locus (bur/mal) in the B. mallei group, the metabolite associated to the encoded polyketide synthase, burkholderic acid (syn. malleilactone), could not explain the observed phenotypes. By metabolic profiling and molecular network analyses of the model organism B. thailandensis, the primary products of the cryptic pathway were identified as unusual cyclopropanol-substituted polyketides. First, sulfomalleicyprols were identified as inactive precursors of burkholderic acid. Furthermore, a highly reactive upstream metabolite, malleicyprol, was discovered and obtained in two stabilized forms. Cell-based assays and a nematode infection model showed that the rare natural product confers cytotoxicity and virulence.

Online measurement of viscosity for biological systems in stirred tank bioreactors.

One of the most critical parameters in chemical and biochemical processes is the viscosity of the medium. Its impact on mixing, as well as on mass and energy transfer is substantial. An increase of viscosity with reaction time can be caused by the formation of biopolymers like xanthan or by filamentous growth of microorganisms. In either case the properties of fermentation broth are changing and frequently non-Newtonian behavior are observed, resulting in major challenges for the measurement and control of mixing and mass transfer. This study demonstrates a method for the online determination of the viscosity inside a stirred tank reactor. The presented method is based on online measurement of heat transfer capacity from the bulk medium to the jacket of the reactor. To prove the feasibility of the method, fermentations with the xanthan producing bacterium Xanthomonas campestris pv. campestris B100 as model system were performed. Excellent correlation between offline measured apparent viscosity and online determined heat transfer capacity were found. The developed tool should be applicable to any other process with formation of biopolymers and filamentous growth. Biotechnol. Bioeng. 2017;114: 990-997. © 2016 Wiley Periodicals, Inc.

Efficient evaluation of cellulose digestibility by Trichoderma reesei Rut-C30 cultures in online monitored shake flasks.

BACKGROUND: Pretreated lignocellulosic biomass is considered as a suitable feedstock for the sustainable production of chemicals. However, the recalcitrant nature of cellulose often results in very cost-intensive overall production processes. A promising concept to reduce the costs is consolidated bioprocessing, which integrates in a single step cellulase production, cellulose hydrolysis, and fermentative conversion of produced sugars into a valuable product. This approach, however, requires assessing the digestibility of the applied celluloses and, thus, the released sugar amount during the fermentation. Since the released sugars are completely taken up by Trichoderma reesei Rut-C30 and the sugar consumption is stoichiometrically coupled to oxygen uptake, the respiration activity was measured to evaluate the digestibility of cellulose. RESULTS: The method was successfully tested on commercial cellulosic substrates identifying a correlation between the respiration activity and the crystallinity of the substrate. Pulse experiments with cellulose and cellulases suggested that the respiration activity of T. reesei on cellulose can be divided into two distinct phases, one limited by enzyme activity and one by cellulose-binding-sites. The impact of known (cellobiose, sophorose, urea, tween 80, peptone) and new (miscanthus steepwater) compounds enhancing cellulase production was evaluated. Furthermore, the influence of two different pretreatment methods, the OrganoCat and OrganoSolv process, on the digestibility of beech wood saw dust was tested. CONCLUSIONS: The introduced method allows an online evaluation of cellulose digestibility in complex and non-complex cultivation media. As the measurements are performed under fermentation conditions, it is a valuable tool to test different types of cellulose for consolidated bioprocessing applications. Furthermore, the method can be applied to identify new compounds, which influence cellulase production.

Online monitoring of fermentation processes via non-invasive low-field NMR.

For the development of biotechnological processes in academia as well as in industry new techniques are required which enable online monitoring for process characterization and control. Nuclear magnetic resonance (NMR) spectroscopy is a promising analytical tool, which has already found broad applications in offline process analysis. The use of online monitoring, however, is oftentimes constrained by high complexity of custom-made NMR bioreactors and considerable costs for high-field NMR instruments (>US$200,000). Therefore, low-field (1) H NMR was investigated in this study in a bypass system for real-time observation of fermentation processes. The new technique was validated with two microbial systems. For the yeast Hansenula polymorpha glycerol consumption could accurately be assessed in spite of the presence of high amounts of complex constituents in the medium. During cultivation of the fungal strain Ustilago maydis, which is accompanied by the formation of several by-products, the concentrations of glucose, itaconic acid, and the relative amount of glycolipids could be quantified. While low-field spectra are characterized by reduced spectral resolution compared to high-field NMR, the compact design combined with the high temporal resolution (15 s-8 min) of spectra acquisition allowed online monitoring of the respective processes. Both applications clearly demonstrate that the investigated technique is well suited for reaction monitoring in opaque media while at the same time it is highly robust and chemically specific. It can thus be concluded that low-field NMR spectroscopy has a great potential for non-invasive online monitoring of biotechnological processes at the research and practical industrial scales.

In situ product recovery of single-chain antibodies in a membrane bioreactor.

The demand for biopharmaceuticals, such as monoclonal antibodies, has risen significantly over the last years. To be competitive, continuous production processes that yield consistent product quality and an economic advantage are desirable. In this study, an in situ product recovery process is described, involving use of submerged membranes to recover single-chain antibodies from a continuous fermentation of Hansenula polymorpha yeast cells.Reverse-flow diafiltration (RFD) was applied to prevent cake layer formation. Optimal flux ranges for this process could be identified by a systematic flux step method. The RFD process was optimized, preventing mixing of permeate and unreacted substrate: the space-time yield of antibodies using RFD could be tripled. Increase of the fouling related transmembrane pressure was below 45 Pa min(-1) for all applied dilution rates, indicating that the filtration process was stable. The membrane as well as the feeding mode of RFD did not influence cell viability nor product concentration. A wide range of dilution rates was successfully tested, demonstrating that this process is suitable for industrial applications.

Comparison of two methods for designing calorimeters using stirred tank reactors.

Calorimetry is a robust method for online monitoring and controlling bioprocesses in stirred tank reactors. Up to now, reactor calorimeters have not been optimally constructed for pilot scale applications. Thus, the objective of this paper is to compare two different ways for designing reactor calorimeters and validate them. The "heat capacity" method based on the mass flow of the cooling liquid in the jacket was compared with the "heat transfer" method based on the heat transfer coefficient continuously measured in the cultivation of Escherichia coli VH33 in a 50 L stirred tank reactor. It was found that the values of the "heat transfer" method agreed very well with the calculated values from the oxygen consumption. By contrast, the curve of the "heat capacity" method deviated from that of the oxygen consumption calculated with the oxycaloric equivalent. In conclusion, the "heat transfer" method has been proven to have a higher degree of validity than the "heat capacity" method. Thus, it is a better and more robust means to measure heat generation of fermentations in stirred tank bioreactors on a pilot scale.

Non-invasive online detection of microbial lysine formation in stirred tank bioreactors by using calorespirometry.

Non-invasive methods for online monitoring of biotechnological processes without compromising the integrity of the reactor system are very important to generate continuous data. Even though calorimetry has been used in conventional biochemical analysis for decades, it has not yet been specifically applied for online detection of product formation at technical scale. Thus, this article demonstrates a calorespirometric method for online detection of microbial lysine formation in stirred tank bioreactors. The respective heat generation of two bacterial strains, Corynebacterium glutamicum ATCC 13032 (wild-type) and C. glutamicum DM1730 (lysine producer), was compared with the O2 -consumption in order to determine whether lysine was formed. As validation of the proposed calorespirometric method, the online results agreed well with the offline measured data. This study has proven that calorespirometry is a viable non-invasive technique to detect product formation at any time point.

Isolation of sulfonosphingolipids from the rosette-inducing bacterium Zobellia uliginosa and evaluation of their rosette-inducing activity.

The choanoflagellate Salpingoeca rosetta transitions from unicellular to multicellular forms in the presence of bacterial signaling molecules, such as sulfonosphingolipids (RIFs). We set out to characterize the abundance of RIF-like molecules within five different Bacteroidetes strains belonging to different genera. While four strains exhibited similar sulfonosphingolipid profiles with sulfobacin A as the dominant feature, the composition in Z. uliginosa differed distinctively. Targeted isolation yielded four sulfonosphingolipids, including the previously reported flavocristamide A. While none of the sulfonosphingolipids induced rosette formation, a negative impact on choanoflagellate growth and cell density was observed. In contrast, supernatant extracts of Zobellia depleted in sulfonosphingolipid-like features provoked rosette formation in S. rosetta indicating for the presence of yet another morphogenic compound class.

Nonribosomal peptides protect Pseudomonas nunensis 4A2e from amoebal and nematodal predation.

The rhizosphere is a highly competitive environment forcing bacteria to evolve strategies to oppose their enemies. The production of toxic secondary metabolites allows bacteria to counteract predators. In this study, we describe the anti-predator armamentarium of the soil-derived bacterium Pseudomonas nunensis 4A2e. Based on a genome mining approach, we identified several biosynthetic gene clusters coding for nonribosomal peptide synthetases. Generation of gene deletion mutants of the respective clusters shows a loss of defense capabilities. We isolated the novel lipopeptides keanumycin D and nunapeptins B and C, and fully elucidated their structures by a combination of in-depth mass spectrometry experiments, stable isotope labelling, and chemical synthesis. Additionally, investigation of the quorum sensing-dependent biosynthesis allowed us to elucidate parts of the underlying regulation of the biosynthetic machinery. Ecology-inspired bioassays highlight the role of these peptides as a defence strategy against protozoans and led us to find a previously unknown function against the bacterivorous nematode Oscheius myriophilus.

The potential of amoeba-based processes for natural product syntheses.

The identification of novel platform organisms for the production and discovery of small molecules is of high interest for the pharmaceutical industry. In particular, the structural complexity of most natural products with therapeutic potential restricts an industrial production since chemical syntheses often require complex multistep routes. The amoeba Dictyostelium discoideum can be easily cultivated in bioreactors due to its planktonic growth behavior and contains numerous polyketide and terpene synthase genes with only a few compounds being already elucidated. Hence, the amoeba both bears a wealth of hidden natural products and allows for the development of new bioprocesses for existing pharmaceuticals. In this mini review, we present D. discoideum as a novel platform for the production of complex secondary metabolites and discuss its suitability for industrial processes. We also provide initial insights into future bioprocesses, both involving bacterial coculture setups and for the production of plant-based pharmaceuticals.

Engineering the amoeba Dictyostelium discoideum for biosynthesis of a cannabinoid precursor and other polyketides.

Aromatic polyketides are natural polyphenolic compounds with a broad spectrum of pharmacological activities. Production of those metabolites in the model organisms Escherichia coli and Saccharomyces cerevisiae has been limited by the extensive cellular engineering needed for the coordinated biosynthesis of polyketides and their precursors. In contrast, the amoeba Dictyostelium discoideum is a native producer of secondary metabolites and harbors a wide, but largely unexplored, repertoire of genes for the biosynthesis of polyketides and terpenoids. Here we present D. discoideum as an advantageous chassis for the production of aromatic polyketides. By expressing its native and cognate plant polyketide synthase genes in D. discoideum, we demonstrate production of phlorocaprophenone, methyl-olivetol, resveratrol and olivetolic acid (OA), which is the central intermediate in the biosynthesis of cannabinoids. To facilitate OA synthesis, we further engineered an amoeba/plant inter-kingdom hybrid enzyme that produced OA from primary metabolites in two enzymatic steps, providing a shortcut in a synthetic cannabinoid pathway using the D. discoideum host system.

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%.

A novel concept combining experimental and mathematical analysis for the identification of unknown interspecies effects in a mixed culture.

Bacteria in natural habitats only occur in consortia together with various other species. Characterization of bacterial species, however, is normally done by laboratory testing of pure isolates. Any interactions that might appear during growth in mixed-culture are obviously missed by this approach. Existing experimental studies mainly focus on two-species mixed cultures with species specifically chosen for their known growth characteristics, and their anticipated interactions. Various theoretical mathematical studies dealing with mixed cultures and possible interspecies effects exist, but often models cannot be validated due to a lack of experimental data. Here, we present a concept for the identification of interspecies effects in mixed cultures with arbitrary and unknown single-species properties. Model structure and parameters were inferred from single-species experiments for the reproduction of mixed-culture experiments by simulation. A mixed culture consisting of the three-species Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus served as a model system. For species-specific enumeration a quantitative terminal restriction length polymorphism (qT-RFLP) assay was used. Based on models fitted to single-species cultivations, the outcome of mixed-culture experiments was predicted. Deviations of simulation results and experimental findings were then used to design additional single-cell experiments, to modify the corresponding growth kinetics, and to update model parameters. Eventually, the resulting mixed-culture dynamics was predicted and compared again to experimental results. During this iterative cycle, it became evident that the observed coexistence of P. aeruginosa and B. cepacia in mixed-culture chemostat experiments cannot be explained on the basis of glucose as the only substrate. After extension of growth kinetics, that is, for use of amino acids as secondary substrates, mixed-culture simulations represented the experimental findings very well. According to the model structure, as motivated by single-species experiments, the growth of P. aeruginosa and B. cepacia on glucose and amino acids could be assumed to be independent of each other. In contrast, both substrates are taken up simultaneously by S. aureus.