Genome-Driven Discovery of Hygrocins in Streptomyces rapamycinicus.

Ansamycins, represented by the antituberculosis drug rifamycin, are an important family of natural products. To obtain new ansamycins, Streptomyces rapamycinicus IMET 43975 harboring an ansamycin biosynthetic gene cluster was fermented in a 50 L scale, and subsequent purification work led to the isolation of five known and four new analogues, where hygrocin W (2) belongs to benzoquinonoid ansamycins, and the other three hygrocins, hygrocins X-Z (6-8), are new seco-hygrocins. The structures of ansamycins (1-8) were determined by the analysis of spectroscopic (1D/2D NMR and ECD) and MS spectrometric data. The Baeyer-Villiger enzyme which catalyzed the ester formation in the ansa-ring was confirmed through in vivo CRISPR base editing. The discovery of these compounds further enriches the structural diversity of ansamycins.

Analysing Megasynthetase Mutants at High Throughput Using Droplet Microfluidics.

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

Analysis of very low bacterial counts in small sample volumes using angle-resolved light scattering.

Because of its high sensitivity to even small objects and the quick measurement principle, angle-resolved scattering (ARS) measurements exhibit a promising potential as a rapid analysis tool for bacterial cells at small sample sizes and very low numbers of cells. In this study, investigations on scattered light from various bacterial cell samples revealed applicability down to single cell levels, which is a huge benefit compared to conventional methods that depend on time-consuming cellular growth over several hours or even days. With the proposed setup and data analysis method, it is possible to detect scatter differences among cell types, together with the cell concentration.

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.

Monitoring and external control of pH in microfluidic droplets during microbial culturing.

BACKGROUND: Cell-based experimentation in microfluidic droplets is becoming increasingly popular among biotechnologists and microbiologists, since inherent characteristics of droplets allow high throughput at low cost and space investment. The range of applications for droplet assays is expanding from single cell analysis toward complex cell-cell incubation and interaction studies. As a result of cellular metabolism in these setups, relevant physicochemical alterations frequently occur before functional assays are conducted. However, to use droplets as truly miniaturized bioreactors, parameters like pH and oxygen availability should be controlled similar to large-scale fermentation to ensure reliable research. RESULTS: Here, we introduce a comprehensive strategy to monitor and control pH for large droplet populations during long-term incubation. We show the correlation of fluorescence intensity of 6-carboxyfluorescein and pH in single droplets and entire droplet populations. By taking advantage of inter-droplet transport of pH-mediating molecules, the average pH value of several million droplets is simultaneously adjusted in an a priori defined direction. To demonstrate the need of pH control in practice, we compared the fermentation profiles of two E. coli strains, a K12-strain and a B-strain, in unbuffered medium with 5 g/L glucose for standard 1 L bioreactors and 180 pL droplets. In both fermentation formats, the commonly used B-strain E. coli BL21 is able to consume glucose until depletion and prevent a pH drop, while the growth of the K12-strain E. coli MG1655 is soon inhibited by a low pH caused by its own high acetate production. By regulating the pH during fermentation in droplets with our suggested strategy, we were able to prevent the growth arrest of E. coli MG1655 and obtained an equally high biomass yield as with E. coli BL21. CONCLUSION: We demonstrated a comparable success of pH monitoring and regulation for fermentations in 1 L scale and 180 pL scale for two E. coli strains. This strategy has the potential to improve cell-based experiments for various microbial systems in microfluidic droplets and opens the possibility for new functional assay designs.

Biosynthesis of Pseudomonas-Derived Butenolides.

Butenolides are well-known signaling molecules in Gram-positive bacteria. Here, we describe a novel class of butenolides isolated from a Gram-negative Pseudomonas strain, the styrolides. Structure elucidation was aided by the total synthesis of styrolide A. Transposon mutagenesis enabled us to identify the styrolide biosynthetic gene cluster, and by using a homology search, we discovered the related and previously unknown acaterin biosynthetic gene cluster in another Pseudomonas species. Mutagenesis, heterologous expression, and identification of key shunt and intermediate products were crucial to propose a biosynthetic pathway for both Pseudomonas-derived butenolides. Comparative transcriptomics suggests a link between styrolide formation and the regulatory networks of the bacterium.

Boosting mediated electron transfer in bioelectrochemical systems with tailored defined microbial cocultures.

Bioelectrochemical systems (BES) hold great promise for sustainable energy generation via a microbial catalyst from organic matter, for example, from wastewater. To improve current generation in BES, understanding the underlying microbiology of the electrode community is essential. Electron mediator producing microorganism like Pseudomonas aeruginosa play an essential role in efficient electricity generation in BES. These microbes enable even nonelectroactive microorganism like Enterobacter aerogenes to contribute to current production. Together they form a synergistic coculture, where both contribute to community welfare. To use microbial co-operation in BES, the physical and chemical environments provided in the natural habitats of the coculture play a crucial role. Here, we show that synergistic effects in defined cocultures of P. aeruginosa and E. aerogenes can be strongly enhanced toward high current production by adapting process parameters, like pH, temperature, oxygen demand, and substrate requirements. Especially, oxygen was identified as a major factor influencing coculture behavior and optimization of its supply could enhance electric current production over 400%. Furthermore, operating the coculture in fed-batch mode enabled us to obtain very high current densities and to harvest electrical energy for 1 month. In this optimized condition, the coulombic efficiency of the process was boosted to 20%, which is outstanding for mediator-based electron transfer. This study lays the foundation for a rationally designed utilization of cocultures in BES for bioenergy generation from specific wastewaters or for bioprocess sensing and for benefiting from their synergistic effects under controlled bioprocess condition.

Strain- and Substrate-Dependent Redox Mediator and Electricity Production by Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa is an important, thriving member of microbial communities of microbial bioelectrochemical systems (BES) through the production of versatile phenazine redox mediators. Pure culture experiments with a model strain revealed synergistic interactions of P. aeruginosa with fermenting microorganisms whereby the synergism was mediated through the shared fermentation product 2,3-butanediol. Our work here shows that the behavior and efficiency of P. aeruginosa in mediated current production is strongly dependent on the strain of P. aeruginosa We compared levels of phenazine production by the previously investigated model strain P. aeruginosa PA14, the alternative model strain P. aeruginosa PAO1, and the BES isolate Pseudomonas sp. strain KRP1 with glucose and the fermentation products 2,3-butanediol and ethanol as carbon substrates. We found significant differences in substrate-dependent phenazine production and resulting anodic current generation for the three strains, with the BES isolate KRP1 being overall the best current producer and showing the highest electrochemical activity with glucose as a substrate (19 µA cm(-2) with ∼150 µg ml(-1) phenazine carboxylic acid as a redox mediator). Surprisingly, P. aeruginosa PAO1 showed very low phenazine production and electrochemical activity under all tested conditions. IMPORTANCE: Microbial fuel cells and other microbial bioelectrochemical systems hold great promise for environmental technologies such as wastewater treatment and bioremediation. While there is much emphasis on the development of materials and devices to realize such systems, the investigation and a deeper understanding of the underlying microbiology and ecology are lagging behind. Physiological investigations focus on microorganisms exhibiting direct electron transfer in pure culture systems. Meanwhile, mediated electron transfer with natural redox compounds produced by, for example, Pseudomonas aeruginosa might enable an entire microbial community to access a solid electrode as an alternative electron acceptor. To better understand the ecological relationships between mediator producers and mediator utilizers, we here present a comparison of the phenazine-dependent electroactivities of three Pseudomonas strains. This work forms the foundation for more complex coculture investigations of mediated electron transfer in microbial fuel cells.

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.

Oxygen allows Shewanella oneidensis MR-1 to overcome mediator washout in a continuously fed bioelectrochemical system.

Many bioelectrochemical systems (BESs) harness the ability of electrode-active microbes to catalyze reactions between electrodes and chemicals, often to perform useful functions such as wastewater treatment, fuel production, and biosensing. A microbial fuel cell (MFC) is one type of BES, which generates electric power through microbial respiration with an anode as the electron acceptor, and typically with oxygen reduction at the cathode to provide the terminal electron acceptor. Oxygen intrusion into MFCs is typically viewed as detrimental because it competes with anodes for electrons and lowers the coulombic efficiency. However, recent evidence suggests that it does not necessarily lead to lower performances­particularly for the model organism Shewanella oneidensis MR-1. Because flavin-mediated electron transfer is important for Shewanella species, which can produce this electron shuttle endogenuously, we investigated the role of flavins in the performance of pure-culture BESs with S. oneidensis MR-1 with and without oxygen. We found that oxygen increases current production more than twofold under continuously fed conditions, but only modestly increases current production under batch-fed conditions.We hypothesized that oxygen is more beneficial under continuously fed conditions because it allows S. oneidensis to grow and produce flavins at a faster rate, and thus lowers flavin washout. Our conclusions were supported by experiments with a flavin-secretion deficient mutant of S. oneidensis.

Regulated expression of polysaccharide utilization and capsular biosynthesis loci in biofilm and planktonic Bacteroides thetaiotaomicron during growth in chemostats.

Bacteroides thetaiotaomicron is a prominent member of the human distal gut microbiota that specializes in breaking down diet and host-derived polysaccharides. While polysaccharide utilization has been well studied in B. thetaiotaomicron, other aspects of its behavior are less well characterized, including the factors that allow it to maintain itself in the gut. Biofilm formation may be a mechanism for bacterial retention in the gut. Therefore, we used custom GeneChips to compare the transcriptomes of biofilm and planktonic B. thetaiotaomicron during growth in mono-colonized chemostats. We identified 1,154 genes with a fold-change greater than 2, with confidence greater than or equal to 95%. Among the prominent changes observed in biofilm populations were: (i) greater expression of genes in polysaccharide utilization loci that are involved in foraging of O-glycans normally found in the gut mucosa; and (ii) regulated expression of capsular polysaccharide biosynthesis loci. Hierarchical clustering of the data with different datasets, which were obtained during growth under a range of conditions in minimal media and in intestinal tracts of gnotobiotic mice, revealed that within this group of differentially expressed genes, biofilm communities were more similar to the in vivo samples than to planktonic cells and exhibited features of substrate limitation. The current study also validates the use of chemostats as an in vitro "gnotobiotic" model to study gene expression of attached populations of this bacterium. This is important to gut microbiota research, because bacterial attachment and the consequences of disruptions in attachment are difficult to study in vivo.

Microbial catalysis in bioelectrochemical technologies: status quo, challenges and perspectives.

Over the past decade, microbial electrochemical technologies, originally developed from an interesting physiological phenomenon, have evolved from a rush of initiatives for sustainable bioelectricity generation to a multitude of specialized applications in very different areas. Genetic engineering of microbial biocatalysts for target bioelectrochemical applications like biosensing or bioremediation, as well as the discovery of entirely new bioelectrochemical processes such as microbial electrosynthesis of commodity chemicals, open up completely new possibilities. Where stands this technology today? And what are the general and specific challenges it faces not only scientifically but also for transition into commercial applications? This review intends to summarize the recent advances and provides a perspective on future developments.

Exploring phenazine electron transfer interaction with elements of the respiratory pathways of Pseudomonas putida and Pseudomonas aeruginosa.

Pseudomonas aeruginosa phenazines contribute to survival under microaerobic and anaerobic conditions by extracellular electron discharge to regulate cellular redox balances. This electron discharge is also attractive to be used for bioelectrochemical applications. However, elements of the respiratory pathways that interact with phenazines are not well understood. Five terminal oxidases are involved in the aerobic electron transport chain (ETC) of Pseudomonas putida and P. aeruginosa. The latter bacterium also includes four reductases that allow for denitrification. Here, we explored if phenazine-1-carboxylic acid interacts with those elements to enhance anodic electron discharge and drive bacterial growth in oxygen-limited conditions. Bioelectrochemical evaluations of terminal oxidase-deficient mutants of both Pseudomonas strains and P. aeruginosa with stimulated denitrification pathways indicated no direct beneficial interaction of phenazines with ETC elements for extracellular electron discharge. However, the single usage of the Cbb3-2 oxidase increased phenazine production, electron discharge, and cell growth. Assays with purified periplasmic cytochromes NirM and NirS indicated that pyocyanin acts as their electron donor. We conclude that phenazines play an important role in electron transfer to, between, and from terminal oxidases under oxygen-limiting conditions and their modulation might enhance EET. However, the phenazine-anode interaction cannot replace oxygen respiration to deliver energy for biomass formation.

Microbial electrosynthesis: opportunities for microbial pure cultures.

Microbial electrosynthesis (MES) is an emerging technology that couples renewable electricity to microbial production processes. Although advances in MES performance have been driven largely by microbial mixed cultures, we see a great limitation in the diversity, and hence value, of products that can be achieved in undefined mixed cultures. By contrast, metabolic control of pure cultures and genetic engineering could greatly expand the scope of MES, and even of broader electrobiotechnology, to include targeted high-value products. To leverage this potential, we advocate for more efforts and activities to develop engineered electroactive microbes for synthesis, and we highlight the need for a standardized electrobioreactor infrastructure that allows the establishment and engineering of electrobioprocesses with these novel biocatalysts.

Enrichment of different taxa of the enigmatic candidate phyla radiation bacteria using a novel picolitre droplet technique.

The candidate phyla radiation (CPR) represents a distinct monophyletic clade and constitutes a major portion of the tree of life. Extensive efforts have focused on deciphering the functional diversity of its members, primarily using sequencing-based techniques. However, cultivation success remains scarce, presenting a significant challenge, particularly in CPR-dominated groundwater microbiomes characterized by low biomass. Here, we employ an advanced high-throughput droplet microfluidics technique to enrich CPR taxa from groundwater. Utilizing a low-volume filtration approach, we successfully harvested a microbiome resembling the original groundwater microbial community. We assessed CPR enrichment in droplet and aqueous bulk cultivation for 30 days using a novel CPR-specific primer to rapidly track the CPR fraction through the cultivation attempts. The combination of soil extract and microbial-derived necromass provided the most supportive conditions for CPR enrichment. Employing these supplemented conditions, droplet cultivation proved superior to bulk cultivation, resulting in up to a 13-fold CPR enrichment compared to a 1- to 2-fold increase in bulk cultivation. Amplicon sequencing revealed 10 significantly enriched CPR orders. The highest enrichment in CPRs was observed for some unknown members of the Parcubacteria order, Cand. Jorgensenbacteria, and unclassified UBA9983. Furthermore, we identified co-enriched putative host taxa, which may guide more targeted CPR isolation approaches in subsequent investigations.

Screening of natural phenazine producers for electroactivity in bioelectrochemical systems.

Mediated extracellular electron transfer (EET) might be a great vehicle to connect microbial bioprocesses with electrochemical control in stirred-tank bioreactors. However, mediated electron transfer to date is not only much less efficient but also much less studied than microbial direct electron transfer to an anode. For example, despite the widespread capacity of pseudomonads to produce phenazine natural products, only Pseudomonas aeruginosa has been studied for its use of phenazines in bioelectrochemical applications. To provide a deeper understanding of the ecological potential for the bioelectrochemical exploitation of phenazines, we here investigated the potential electroactivity of over 100 putative diverse native phenazine producers and the performance within bioelectrochemical systems. Five species from the genera Pseudomonas, Streptomyces, Nocardiopsis, Brevibacterium and Burkholderia were identified as new electroactive bacteria. Electron discharge to the anode and electric current production correlated with the phenazine synthesis of Pseudomonas chlororaphis subsp. aurantiaca. Phenazine-1-carboxylic acid was the dominant molecule with a concentration of 86.1 µg/ml mediating an anodic current of 15.1 µA/cm2 . On the other hand, Nocardiopsis chromatogenes used a wider range of phenazines at low concentrations and likely yet-unknown redox compounds to mediate EET, achieving an anodic current of 9.5 µA/cm2 . Elucidating the energetic and metabolic usage of phenazines in these and other species might contribute to improving electron discharge and respiration. In the long run, this may enhance oxygen-limited bioproduction of value-added compounds based on mediated EET mechanisms.

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.

Insights into Streptomyces coelicolor A3(2) growth and pigment formation with high-throughput online monitoring.

Streptomyces species are intensively studied for their ability to produce a variety of natural products. However, conditions influencing and leading to product formation are often not completely recognized. Therefore, in this study, high-throughput online monitoring is presented as a powerful tool to gain in-depth understanding of the cultivation of the model organism Streptomyces coelicolor A3(2). Through online measurements of oxygen transfer rate and autofluorescence, valuable information about availability of nutrients and product formation patterns of the pigments actinorhodin and undecylprodigiosin can be obtained and explained. Therefore, it is possible to determine the onset of pigmentation and to study in detail the influencing factors thereof. One factor identified in this study is the filling volume of the cultivation vessel. Slight variations led to varying pigmentation levels. By combining optical and metabolic online monitoring techniques, the correlation of the filling volume with pigmentation could be explained as a result of different growth trajectories caused by varying specific power inputs and their influence on the pellet formation of the filamentous system. Finally, experiments with the addition of supernatant from unpigmented and pigmented cultures could highlight the applicability of the presented approach to study quorum sensing and cell-cell interaction.

The oxygen dilemma: The challenge of the anode reaction for microbial electrosynthesis from CO2.

Microbial electrosynthesis (MES) from CO2 provides chemicals and fuels by driving the metabolism of microorganisms with electrons from cathodes in bioelectrochemical systems. These microorganisms are usually strictly anaerobic. At the same time, the anode reaction of bioelectrochemical systems is almost exclusively water splitting through the oxygen evolution reaction (OER). This creates a dilemma for MES development and engineering. Oxygen penetration to the cathode has to be excluded to avoid toxicity and efficiency losses while assuring low resistance. We show that this dilemma derives a strong need to identify novel reactor designs when using the OER as an anode reaction or to fully replace OER with alternative oxidation reactions.

Advantages of optical fibers for facile and enhanced detection in droplet microfluidics.

Droplet microfluidics offers a unique opportunity for ultrahigh-throughput experimentation with minimal sample consumption and thus has obtained increasing attention, particularly for biological applications. Detection and measurements of analytes or biomarkers in tiny droplets are essential for proper analysis of biological and chemical assays like single-cell studies, cytometry, nucleic acid detection, protein quantification, environmental monitoring, drug discovery, and point-of-care diagnostics. Current detection setups widely use microscopes as a central device and other free-space optical components. However, microscopic setups are bulky, complicated, not flexible, and expensive. Furthermore, they require precise optical alignments, specialized optical and technical knowledge, and cumbersome maintenance. The establishment of efficient, simple, and cheap detection methods is one of the bottlenecks for adopting microfluidic strategies for diverse bioanalytical applications and widespread laboratory use. Together with great advances in optofluidic components, the integration of optical fibers as a light guiding medium into microfluidic chips has recently revolutionized analytical possibilities. Optical fibers embedded in a microfluidic platform provide a simpler, more flexible, lower-cost, and sensitive setup for the detection of several parameters from biological and chemical samples and enable widespread, hands-on application much beyond thriving point-of-care developments. In this review, we examine recent developments in droplet microfluidic systems using optical fiber as a light guiding medium, primarily focusing on different optical detection methods such as fluorescence, absorbance, light scattering, and Raman scattering and the potential applications in biochemistry and biotechnology that are and will be arising from this.