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The high therapeutic potential of psilocybin, a prodrug of the psychotropic psilocin, holds great promise for the treatment of mental disorders such as therapy-refractory depression, alcohol use disorder and anorexia nervosa. Psilocybin has been designated a 'Breakthrough Therapy' by the US Food and Drug Administration, and therefore a sustainable production process must be established to meet future market demands. Here, we present the development of an in vivo psilocybin production chassis based on repression of l-tryptophan catabolism. We demonstrate the proof of principle in Saccharomyces cerevisiae expressing the psilocybin biosynthetic genes. Deletion of the two aminotransferase genes ARO8/9 and the indoleamine 2,3-dioxygenase gene BNA2 yielded a fivefold increase of psilocybin titre. We transferred this knowledge to the filamentous fungus Aspergillus nidulans and identified functional ARO8/9 orthologs involved in fungal l-tryptophan catabolism by genome mining and cross-complementation. The double deletion mutant of A. nidulans resulted in a 10-fold increased psilocybin production. Process optimization based on respiratory activity measurements led to a final psilocybin titre of 267 mg/L in batch cultures with a space-time-yield of 3.7 mg/L/h. These results demonstrate the suitability of our engineered A. nidulans to serve as a production strain for psilocybin and other tryptamine-derived pharmaceuticals., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

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 ., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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.

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.

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 (ε max ) 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 ε max 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., (© 2022. The Author(s).)

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., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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 (HRMS 2 ) 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., (© 2021 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

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%., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Engineering in Life Sciences published by Wiley‐VCH GmbH.)

Microbial mixed cultures are gaining increasing attention as biotechnological production systems, since they offer a large but untapped potential for future bioprocesses. Effects of secondary metabolite induction and advantages of labor division for the degradation of complex substrates offer new possibilities for process intensification. However, mixed cultures are highly complex, and, consequently, many biotic and abiotic parameters are required to be identified, characterized, and ideally controlled to establish a stable bioprocess. In this review, we discuss the advantages and disadvantages of existing measurement techniques for identifying, characterizing, monitoring, and controlling mixed cultures and highlight promising examples. Moreover, existing challenges and emerging technologies are discussed, which lay the foundation for novel analytical workflows to monitor mixed-culture bioprocesses., Competing Interests: Declaration of Interests There are no interests to declare., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 mg Jagaricin ∙g CDW -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., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Itaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars. Here, we present the stepwise development of a fully consolidated bioprocess (CBP), which is capable of directly converting recalcitrant cellulose into itaconic acid without the need for separate cellulose hydrolysis including the application of commercial cellulases. The process is based on a synthetic microbial consortium of the cellulase producer Trichoderma reesei and the itaconic acid producing yeast Ustilago maydis. A method for process monitoring was developed to estimate cellulose consumption, itaconic acid formation as well as the actual itaconic acid production yield online during co-cultivation., Results: The efficiency of the process was compared to a simultaneous saccharification and fermentation setup (SSF). Because of the additional substrate consumption of T. reesei in the CBP, the itaconic acid yield was significantly lower in the CBP than in the SSF. In order to increase yield and productivity of itaconic acid in the CBP, the population dynamics was manipulated by varying the inoculation delay between T. reesei and U. maydis. Surprisingly, neither inoculation delay nor inoculation density significantly affected the population development or the CBP performance. Instead, the substrate availability was the most important parameter. U. maydis was only able to grow and to produce itaconic acid when the cellulose concentration and thus, the sugar supply rate, was high. Finally, the metabolic processes during fed-batch CBP were analyzed in depth by online respiration measurements. Thereby, substrate availability was again identified as key factor also controlling itaconic acid yield. In summary, an itaconic acid titer of 34 g/L with a total productivity of up to 0.07 g/L/h and a yield of 0.16 g/g could be reached during fed-batch cultivation., Conclusion: This study demonstrates the feasibility of consortium-based CBP for itaconic acid production and also lays the fundamentals for the development and improvement of similar microbial consortia for cellulose-based organic acid production.

Mycofactocin (MFT) is a recently discovered glycosylated redox cofactor, which has been associated with the detoxification of antibiotics in pathogenic mycobacteria, and, therefore, of potential medical interest. The MFT biosynthetic gene cluster is commonly found in mycobacteria, including Mycobacterium tuberculosis , the causative agent of tuberculosis. Since the MFT molecule is highly interesting for basic research and could even serve as a potential drug target, large-scale production of the molecule is highly desired. However, conventional shake flask cultivations failed to produce enough MFT for further biochemical characterization like kinetic studies and structure elucidation, and a more comprehensive study of cultivation parameters is urgently needed. Being a redox cofactor, it can be hypothesized that the oxygen transfer rate (OTR) is a critical parameter for MFT formation. Using the non-pathogenic strain Mycobacterium smegmatis mc 2 155, shake flask experiments with online measurement of the oxygen uptake and the carbon dioxide formation, were conducted under different levels of oxygen supply. Using liquid chromatography and high-resolution mass spectrometry, a 4-8 times increase of MFT production was identified under oxygen-limited conditions, in both complex and mineral medium. Moreover, the level of oxygen supply modulates not only the overall MFT formation but also the length of the glycosidic chain. Finally, all results were scaled up into a 7 L stirred tank reactor to elucidate the kinetics of MFT formation. Ultimately, this study enables the production of high amounts of these redox cofactors, to perform further investigations into the role and importance of MFTs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Peña-Ortiz, Schlembach, Lackner and Regestein.)

Combinatorial biosynthesis has great potential for designing synthetic circuits and amplifying the production of new active compounds. Studies on multienzyme cascades are extremely useful for improving our knowledge on enzymatic catalysis. In particular, the elucidation of enzyme substrate promiscuity can be potentially used for bioretrosynthetic approaches, leading to the design of alternative and more convenient routes to produce relevant molecules. In this perspective, plant-derived polyketides are extremely adaptable to those synthetic biological applications. Here, we present a combination of an in vitro CoA ligase activity assay coupled with a bacterial multigene expression system that leads to precursor-directed biosynthesis of 21 flavonoid derivatives. When the vast knowledge from protein databases is exploited, the herein presented procedure can be easily repeated with additional plant-derived polyketides. Lastly, we report an efficient in vivo expression system that can be further exploited to heterologously express pathways not necessarily related to plant polyketide synthases.

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., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Mycofactocin (MFT) is a redox cofactor belonging to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and is involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis . A preliminary biosynthetic model had been established by bioinformatics and in vitro studies, while the structure of natural MFT and key biosynthetic steps remained elusive. Here, we report the discovery of glycosylated MFT by 13 C-labeling metabolomics and establish a model of its biosynthesis in Mycolicibacterium smegmatis . Extensive structure elucidation including NMR revealed that MFT is decorated with up to nine β-1,4-linked glucose residues including 2- O -methylglucose. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the redox cofactor function of glycosylated MFTs by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results will guide future studies into the biochemical functions and physiological roles of MFT in bacteria., (This journal is © The Royal Society of Chemistry 2020.)

Growth from spores activated a biosynthetic gene cluster in Actinomadura sp. RB29, resulting in the identification of two novel groups of halogenated polyketide natural products, named maduralactomycins and actinospirols. The unique tetracyclic and spirocyclic structures were assigned based on a combination of NMR analysis, chemoinformatic calculations, X-ray crystallography, and 13 C labeling studies. On the basis of HRMS 2 data, genome mining, and gene expression studies, we propose an underlying noncanonical angucycline biosynthesis and extensive post-polyketide synthase (PKS) oxidative modifications.

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., (© 2019 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.)

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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Renewable raw materials in sustainable biorefinery processes pose new challenges to the manufacturing routes of platform chemicals. Beside the investigations of individual unit operations, the research on process chains, leading from plant biomass to the final products like lactic acid, succinic acid, and itaconic acid is increasing. This article presents a complete process chain from wooden biomass to the platform chemical itaconic acid. The process starts with the mechanical pretreatment of beech wood, which subsequently is subjected to chemo-catalytic biomass fractionation (OrganoCat) into three phases, which comprise cellulose pulp, aqueous hydrolyzed hemicellulose, and organic lignin solutions. Lignin is transferred to further chemical valorization. The aqueous phase containing oxalic acid as well as hemi-cellulosic sugars is treated by nanofiltration to recycle the acid catalyst back to the chemo-catalytic pretreatment and to concentrate the sugar hydrolysate. In a parallel step, the cellulose pulp is enzymatically hydrolyzed to yield glucose, which-together with the pentose-rich stream-can be used as a carbon source in the fermentation. The fermentation of the sugar fraction into itaconic acid can either be performed with the established fungi Aspergillus terreus or with Ustilago maydis . Both fermentation concepts were realized and evaluated. For purification, (in situ) filtration, (in situ) extraction, and crystallization were investigated. The presented comprehensive examination and discussion of the itaconate synthesis process-as a case study-demonstrates the impact of realistic process conditions on product yield, choice of whole cell catalyst, chemocatalysts and organic solvent system, operation mode, and, finally, the selection of a downstream concept.

Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli . Globin promoters from Bacillus subtilis , Campylobacter jejuni , Deinococcus radiodurans , Streptomyces coelicolor , Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTR max ) of 7 and 11 mmol L -1  h -1 . Different FbFP fluorescence intensities were observed and the OTR max affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor , the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli .

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., (© 2016 Wiley Periodicals, Inc.)

Background: Although the biocatalytic conversion of cellulosic biomass could replace fossil oil for the production of various compounds, it is often not economically viable due to the high costs of cellulolytic enzymes. One possibility to reduce costs is consolidated bioprocessing (CBP), integrating cellulase production, hydrolysis of cellulose, and the fermentation of the released sugars to the desired product into one process step. To establish such a process, the most suitable cellulase-producing organism has to be identified. Thereby, it is crucial to evaluate the candidates under target process conditions. In this work, the chosen model process was the conversion of cellulose to the platform chemical itaconic acid by a mixed culture of a cellulolytic fungus with Aspergillus terreus as itaconic acid producer. Various cellulase producers were analyzed by the introduced freeze assay that measures the initial carbon release rate, quantifying initial cellulase activity under target process conditions. Promising candidates were then characterized online by monitoring their respiration activity metabolizing cellulose to assess the growth and enzyme production dynamics., Results: The screening of five different cellulase producers with the freeze assay identified Trichoderma   reesei and Penicillium   verruculosum as most promising. The measurement of the respiration activity revealed a retarded induction of cellulase production for P.   verruculosum but a similar cellulase production rate afterwards, compared to T.   reesei . The freeze assay measurement depicted that P.   verruculosum reaches the highest initial carbon release rate among all investigated cellulase producers. After a modification of the cultivation procedure, these results were confirmed by the respiration activity measurement. To compare both methods, a correlation between the measured respiration activity and the initial carbon release rate of the freeze assay was introduced. The analysis revealed that the different initial enzyme/cellulose ratios as well as a discrepancy in cellulose digestibility are the main differences between the two approaches., Conclusions: With two complementary methods to quantify cellulase activity and the dynamics of cellulase production for CBP applications, T.   reesei and P.   verruculosum were identified as compatible candidates for the chosen model process. The presented methods can easily be adapted to screen for suitable cellulose degrading organisms for various other applications.

Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (P vgb ) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a nonoxygen dependent marker protein, was used as a reporter. Fluorescence and fluorescence emission rates under oxygen-limited conditions were the highest when FbFP was under transcriptional control of P adhE , P pfl and P vgb . The lengths of the E. coli endogenous promoters were shortened by 60%, maintaining their key regulatory elements. This resulted in improved promoter activity in most cases, particularly for P adhE , P pfl and P narK . Selected promoters were also evaluated using an engineered E. coli strain expressing Vitreoscilla hemoglobin (VHb). The presence of the VHb resulted in a better repression using these promoters under aerobic conditions, and increased the specific growth and fluorescence emission rates under oxygen-limited conditions. These results are useful for the selection of promoters for specific applications and for the design of modified artificial promoters.

Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their application in heterologous protein production. However, plasmids can be lost during cell growth, if no selection pressure like, e.g., antibiotics is used, hampering the production of the desired protein and endangering the economic success of a biotechnological production process. Thus, in this study the Continuously Operated Shaken BIOreactor System (COSBIOS) is applied as a tool for fast parallel testing of strain stability and operation conditions and to evaluate measures to counter such plasmid loss. In specific, by applying various ampicillin concentrations, the lowest effective ampicillin dosage is investigated to secure plasmid stability while lowering adverse ecological effects. A significant difference was found in the growth rates of plasmid-bearing and plasmid-free cells. The undesired plasmid-free cells grew 30% faster than the desired plasmid-bearing cells. During the testing of plasmid stability without antibiotics, the population fraction of plasmid-bearing cells rapidly decreased in continuous culture to zero within the first 48 h. An initial single dosage of ampicillin did not prevent plasmid loss. By contrast, a continuous application of a low dosage of 10 µg/mL ampicillin in the feed medium maintained plasmid stability in the culture. Consequently, the COSBIOS is an apt reactor system for measuring plasmid stability and evaluating methods to enhance this stability. Hence, decreased production of heterologous protein can be prevented. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1418-1425, 2016., (© 2016 American Institute of Chemical Engineers.)

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.

The butanol yield of the classic fermentative acetone-butanol-ethanol (ABE) process has been enhanced in the past decades through the development of better strains and advanced process design. Nevertheless, by-product formation and the incomplete conversion of intermediates still decrease the butanol yield. This study demonstrates the potential of increasing the butanol yield from glycerol though the addition of small amounts of butyric acid. The impact of butyric acid was investigated in a 7L stirred tank reactor. The results of this study show the positive impact of butyric acid on butanol yield under pH controlled conditions and the metabolic stages were monitored via online measurement of carbon dioxide formation, pH value and redox potential. Butyric acid could significantly increase the butanol yield at low pH values if sufficient quantities of primary carbon source (glycerol) were present., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (© 2015 Wiley Periodicals, Inc.)