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An abstract of the article "Construction of a Corynebacterium glutamicum Platform Strain for the Production of High-Value Plant Secondary Metabolites," by J. Marienhagen, N. Kallscheuer, and M. Vogt is presented.

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli . In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.

Glutamate serves as the major cellular amino group donor. In Bacillus subtilis, glutamate is synthesized by the combined action of the glutamine synthetase and the glutamate synthase (GOGAT). The glutamate dehydrogenases are devoted to glutamate degradation in vivo. To keep the cellular glutamate concentration high, the genes and the encoded enzymes involved in glutamate biosynthesis and degradation need to be tightly regulated depending on the available carbon and nitrogen sources. Serendipitously, we found that the inactivation of the ansR and citG genes encoding the repressor of the ansAB genes and the fumarase, respectively, enables the GOGAT-deficient B. subtilis mutant to synthesize glutamate via a non-canonical fumarate-based ammonium assimilation pathway. We also show that the de-repression of the ansAB genes is sufficient to restore aspartate prototrophy of an aspB aspartate transaminase mutant. Moreover, in the presence of arginine, B. subtilis mutants lacking fumarase activity show a growth defect that can be relieved by aspB overexpression, by reducing arginine uptake and by decreasing the metabolic flux through the TCA cycle., (© 2024 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Members of the bacterial phylum Planctomycetota have recently emerged as promising and for the most part untapped sources of novel bioactive compounds. The characterization of more than 100 novel species in the last decade stimulated recent bioprospection studies that start to unveil the chemical repertoire of the phylum. In this study, we performed systematic bioinformatic analyses based on the genomes of all 131 described members of the current phylum focusing on the identification of type III polyketide synthase (PKS) genes. Type III PKSs are versatile enzymes involved in the biosynthesis of a wide array of structurally diverse natural products with potent biological activities. We identified 96 putative type III PKS genes of which 58 are encoded in an operon with genes encoding a putative oxidoreductase and a methyltransferase. Sequence similarities on protein level and the genetic organization of the operon point towards a functional link to the structurally related hierridins recently discovered in picocyanobacteria. The heterologous expression of planctomycetal type III PKS genes from strains belonging to different families in an engineered Corynebacterium glutamicum strain led to the biosynthesis of pentadecyl- and heptadecylresorcinols. Phenotypic assays performed with the heterologous producer strains and a constructed type III PKS gene deletion mutant suggest that the natural function of the identified compounds differs from that confirmed in other bacterial alkylresorcinol producers. KEY POINTS: • Planctomycetal type III polyketide synthases synthesize long-chain alkylresorcinols. • Phylogenetic analyses suggest an ecological link to picocyanobacterial hierridins. • Engineered C. glutamicum is suitable for an expression of planctomycete-derived genes., (© 2024. The Author(s).)

Background: In recent years, the production of inclusion bodies that retain substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) are formed by genetic fusion of an aggregation-inducing tag to a gene of interest via short linker polypeptides. The resulting CatIBs are known for their easy and cost-efficient production, recyclability as well as their improved stability. Recent studies have outlined the cooperative effects of linker and aggregation-inducing tag on CatIB activities. However, no a priori prediction is possible so far to indicate the best combination thereof. Consequently, extensive screening is required to find the best performing CatIB variant., Results: In this work, a semi-automated cloning workflow was implemented and used for fast generation of 63 CatIB variants with glucose dehydrogenase of Bacillus subtilis (BsGDH). Furthermore, the variant BsGDH-PT-CBDCell was used to develop, optimize and validate an automated CatIB screening workflow, enhancing the analysis of many CatIB candidates in parallel. Compared to previous studies with CatIBs, important optimization steps include the exclusion of plate position effects in the BioLector by changing the cultivation temperature. For the overall workflow including strain construction, the manual workload could be reduced from 59 to 7 h for 48 variants (88%). After demonstration of high reproducibility with 1.9% relative standard deviation across 42 biological replicates, the workflow was performed in combination with a Bayesian process model and Thompson sampling. While the process model is crucial to derive key performance indicators of CatIBs, Thompson sampling serves as a strategy to balance exploitation and exploration in screening procedures. Our methodology allowed analysis of 63 BsGDH-CatIB variants within only three batch experiments. Because of the high likelihood of TDoT-PT-BsGDH being the best CatIB performer, it was selected in 50 biological replicates during the three screening rounds, much more than other, low-performing variants., Conclusions: At the current state of knowledge, every new enzyme requires screening for different linker/aggregation-inducing tag combinations. For this purpose, the presented CatIB toolbox facilitates fast and simplified construction and screening procedures. The methodology thus assists in finding the best CatIB producer from large libraries in short time, rendering possible automated Design-Build-Test-Learn cycles to generate structure/function learnings., (© 2024. The Author(s).)

Anthranilate and its derivatives are important basic chemicals for the synthesis of polyurethanes as well as various dyes and food additives. Today, anthranilate is mainly chemically produced from petroleum-derived xylene, but this shikimate pathway intermediate could be also obtained biotechnologically. In this study, Corynebacterium glutamicum was engineered for the microbial production of anthranilate from a carbon source mixture of glucose and xylose. First, a feedback-resistant 3-deoxy-arabinoheptulosonate-7-phosphate synthase from Escherichia coli, catalysing the first step of the shikimate pathway, was functionally introduced into C. glutamicum to enable anthranilate production. Modulation of the translation efficiency of the genes for the shikimate kinase (aroK) and the anthranilate phosphoribosyltransferase (trpD) improved product formation. Deletion of two genes, one for a putative phosphatase (nagD) and one for a quinate/shikimate dehydrogenase (qsuD), abolished by-product formation of glycerol and quinate. However, the introduction of an engineered anthranilate synthase (TrpEG) unresponsive to feedback inhibition by tryptophan had the most pronounced effect on anthranilate production. Component I of this enzyme (TrpE) was engineered using a biosensor-based in vivo screening strategy for identifying variants with increased feedback resistance in a semi-rational library of TrpE muteins. The final strain accumulated up to 5.9 g/L (43 mM) anthranilate in a defined CGXII medium from a mixture of glucose and xylose in bioreactor cultivations. We believe that the constructed C. glutamicum variants are not only limited to anthranilate production but could also be suitable for the synthesis of other biotechnologically interesting shikimate pathway intermediates or any other aromatic compound derived thereof., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

Background: Phenylpropanoids such as p-coumaric acid represent important precursors for the synthesis of a broad range of plant secondary metabolites including stilbenoids, flavonoids, and lignans, which are of pharmacological interest due to their health-promoting properties. Although extraction from plant material or chemical synthesis is possible, microbial synthesis of p-coumaric acid from glucose has the advantage of being less expensive and more resource efficient. In this study, Corynebacterium glutamicum was engineered for the production of the plant polyphenol precursor p-coumaric acid from glucose., Results: Heterologous expression of the tyrosine ammonia-lyase encoding gene from Flavobacterium johnsoniae enabled the conversion of endogenously provided tyrosine to p-coumaric acid. Product consumption was avoided by abolishing essential reactions of the phenylpropanoid degradation pathway. Accumulation of anthranilate as a major byproduct was eliminated by reducing the activity of anthranilate synthase through targeted mutagenesis to avoid tryptophan auxotrophy. Subsequently, the carbon flux into the shikimate pathway was increased, phenylalanine biosynthesis was reduced, and phosphoenolpyruvate availability was improved to boost p-coumaric acid accumulation. A maximum titer of 661 mg/L p-coumaric acid (4 mM) in defined mineral medium was reached. Finally, the production strain was utilized in co-cultivations with a C. glutamicum strain previously engineered for the conversion of p-coumaric acid into the polyphenol resveratrol. These co-cultivations enabled the synthesis of 31.2 mg/L (0.14 mM) resveratrol from glucose without any p-coumaric acid supplementation., Conclusions: The utilization of a heterologous tyrosine ammonia-lyase in combination with optimization of the shikimate pathway enabled the efficient production of p-coumaric acid with C. glutamicum. Reducing the carbon flux into the phenylalanine and tryptophan branches was the key to success along with the introduction of feedback-resistant enzyme variants., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Microbial synthesis of nutraceutically and pharmaceutically interesting plant polyphenols represents a more environmentally friendly alternative to chemical synthesis or plant extraction. However, most polyphenols are cytotoxic for microorganisms as they are believed to negatively affect cell integrity and transport processes. To increase the production performance of engineered cell factories, strategies have to be developed to mitigate these detrimental effects. Here, we examine the accumulation of the stilbenoid resveratrol in the cell membrane and cell wall during its production using Corynebacterium glutamicum and uncover the membrane rigidifying effect of this stilbenoid experimentally and with molecular dynamics simulations. A screen of free fatty acid supplements identifies palmitelaidic acid and linoleic acid as suitable additives to attenuate resveratrol's cytotoxic effects resulting in a three-fold higher product titer. This cost-effective approach to counteract membrane-damaging effects of product accumulation is transferable to the microbial production of other polyphenols and may represent an engineering target for other membrane-active bioproducts., (© 2023. Springer Nature Limited.)

Background: In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L-histidine producer. Since all known genetic targets contributing to L-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L-histidine biosynthesis., Results: In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol -1 ) in comparison to the starting variant., Conclusions: This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Next-generation bioprocesses of a future bio-based economy will rely on a flexible mix of readily available feedstocks. Renewable energy can be used to generate sustainable CO 2 -derived substrates. Metabolic engineering already enables the functional implementation of different pathways for the assimilation of C1 substrates in various microorganisms. In addition to feedstocks, the benchmark for all future bioprocesses will be sustainability, including the avoidance of CO 2 emissions. Here we review recent advances in the utilization of C1-compounds from different perspectives, considering both strain and bioprocess engineering technologies. In particular, we evaluate methanol as a co-feed for enabling the CO 2 emission-free production of acetyl-CoA-derived compounds. The possible metabolic strategies are analyzed using stoichiometric modeling combined with thermodynamic analysis and prospects for industrial-scale implementation are discussed., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L -1 resveratrol from supplemented p-coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany., (© 2022. The Author(s).)

In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces , are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect but retains its antiviral properties. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level. IMPORTANCE Predation by phages is a major driver of bacterial evolution. As a result, elucidating antiphage strategies is crucial from both fundamental and therapeutic standpoints. While protein-mediated defense mechanisms, like restriction-modification systems or CRISPR/Cas, have been extensively studied, much less is known about the potential antiphage activity of small molecules. Focusing on the model bacteria Escherichia coli and Streptomyces venezuelae, our findings revealed significant antiphage properties of aminoglycosides, a major class of translation-targeting antibiotics produced by Streptomyces . Further, we demonstrate that supernatants from natural aminoglycoside producers protect bacteria from phage propagation, highlighting the physiological relevance of this inhibition. Suppression of phage infection by aminoglycosides did not result from the indirect inhibition of bacterial translation, suggesting a direct interaction between aminoglycosides and phage components. This work highlights the molecular versatility of aminoglycosides, which have evolved to efficiently block protein synthesis in bacterial competitors and provide protection against phages.

The marine bacterium Vibrio natriegens has recently been demonstrated to be a promising new host for molecular biology and next generation bioprocesses. V. natriegens is a Gram-negative, non-pathogenic slight-halophilic bacterium, with a high nutrient versatility and a reported doubling time of under 10 min. However, V. natriegens is not an established model organism yet, and further research is required to promote its transformation into a microbial workhorse. In this work, the potential of V. natriegens as an amino acid producer was investigated. First, the transcription factor-based biosensor LysG, from Corynebacterium glutamicum , was adapted for expression in V. natriegens to facilitate the detection of positively charged amino acids. A set of different biosensor variants were constructed and characterized, using the expression of a fluorescent protein as sensor output. After random mutagenesis, one of the LysG-based sensors was used to screen for amino acid producer strains. Here, fluorescence-activated cell sorting enabled the selective sorting of highly fluorescent cells, i.e. potential producer cells. Using this approach, individual L-lysine, L-arginine and L-histidine producers could be obtained producing up to 1 mM of the effector amino acid, extracellularly. Genome sequencing of the producer strains provided insight into the amino acid production metabolism of V. natriegens . This work demonstrates the successful expression and application of transcription factor-based biosensors in V. natriegens and provides insight into the underlying physiology, forming a solid basis for further development of this promising microbe., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Published by Elsevier B.V. on behalf of International Metabolic Engineering Society.)

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of high interest in the chemical industry and has wide potential for pharmaceutical applications. We designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of d-xylose for microbial production of PCA. Shake flask cultivation of the engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L -1 ) from d-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-mol PCA per C-mol d-xylose, which corresponds to 38% of the maximum theoretical yield. Under growth-decoupled bioreactor conditions, a comparable PCA titer and a total amount of 16.5 ± 1.1 g PCA could be achieved when d-glucose and d-xylose were combined as orthogonal carbon substrates for biocatalyst provision and product synthesis, respectively. Downstream processing of PCA was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. This resulted in a maximum final purity of 95.4%. The established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Background and Aims: Endoscopic submucosal dissection (ESD) enables the curative resection of early malignant lesions and is associated with reduced recurrence risk. Due to the lack of comprehensive ESD data in the West, the German ESD registry was set up to evaluate relevant outcomes of ESD., Methods: The German ESD registry is a prospective uncontrolled multicenter study. During a 35-month period, 20 centers included 1000 ESDs of neoplastic lesions. The results were evaluated in terms of en bloc, R0, curative resection rates, and recurrence rate after a 3-month and 12-month follow-up. Additionally, participating centers were grouped into low-volume (≤20 ESDs/y), middle-volume (20-50/y), and high-volume centers (>50/y). A multivariate analysis investigating risk factors for noncurative resection was performed., Results: Overall, en bloc, R0, and curative resection rates of 92.4% (95% confidence interval [CI], 0.90-0.94), 78.8% (95% CI, 0.76-0.81), and 72.3% (95% CI, 0.69-0.75) were achieved, respectively. The overall complication rate was 8.3% (95% CI, 0.067-0.102), whereas the recurrence rate after 12 months was 2.1%. High-volume centers had significantly higher en bloc, R0, curative resection rates, and recurrence rates and lower complication rates than middle- or low-volume centers. The lesion size, hybrid ESD, age, stage T1b carcinoma, and treatment outside high-volume centers were identified as risk factors for noncurative ESD., Conclusion: In Germany, ESD achieves excellent en bloc resection rates but only modest curative resection rates. ESD requires a high level of expertise, and results vary significantly depending on the center's yearly case volume., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Lignocellulosic biomass is the most abundant raw material on earth. Its efficient use for novel bio-based materials is essential for an emerging bioeconomy. Possible building blocks for such materials are the key TCA-cycle intermediates α-ketoglutarate and succinate. These organic acids have a wide range of potential applications, particularly in use as monomers for established or novel biopolymers. Recently, Corynebacterium glutamicum was successfully engineered and evolved towards an improved utilization of d-xylose via the Weimberg pathway, yielding the strain WMB2 evo . The Weimberg pathway enables a carbon-efficient C5-to-C5 conversion of d-xylose to α-ketoglutarate and a shortcut route to succinate as co-product in a one-pot process., Methods and Results: C. glutamicum WMB2 evo was grown under dynamic microaerobic conditions on d-xylose, leading to the formation of comparably high amounts of succinate and only small amounts of α-ketoglutarate. Subsequent carbon isotope labeling experiments verified the targeted production route for both products in C. glutamicum WMB2 evo . Fed-batch process development was initiated and the effect of oxygen supply and feeding strategy for a growth-decoupled co-production of α-ketoglutarate and succinate were studied in detail. The finally established fed-batch production process resulted in the formation of 78.4 mmol L -1 (11.45 g L -1 ) α-ketoglutarate and 96.2 mmol L -1 (11.36 g L -1 ) succinate., Conclusion: The developed one-pot process represents a promising approach for the combined supply of bio-based α-ketoglutarate and succinate. Future work will focus on tailor-made down-stream processing of both organic acids from the fermentation broth to enable their application as building blocks in chemical syntheses. Alternatively, direct conversion of one or both acids via whole-cell or cell-free enzymatic approaches can be envisioned; thus, extending the network of value chains starting from cheap and renewable d-xylose., (© 2021 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

The time-consuming and laborious characterization of protein or microbial strain designs limits the development of high-performance biocatalysts for biotechnological applications. Here, transcriptional biosensors emerged as valuable tools as they allow for rapid characterization of several thousand variants within a very short time. However, for many molecules of interest, no specific transcriptional regulator determining a biosensor's specificity is available. We present an approach for rapidly engineering biosensor specificities using a semirational transition ligand approach combined with fluorescence-activated cell sorting. In this two-step approach, a biosensor is first evolved toward a more relaxed-ligand specificity before using the resulting variant as the starting point in a second round of directed evolution toward high specificity for several chemically different ligands. By following this strategy, highly specific biosensors for 4-hydroxybenzoic acid, p -coumaric acid, 5-bromoferulic acid, and 6-methyl salicylic acid were developed, starting from a biosensor for the intracellular detection of trans -cinnamic acid.

The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity., (© 2021 The Author(s).)

Synthetic microbial cocultures carry enormous potential for applied biotechnology and are increasingly the subject of fundamental research. So far, most cocultures have been designed and characterized based on bulk cultivations without considering the potentially highly heterogeneous and diverse single-cell behavior. However, an in-depth understanding of cocultures including their interacting single cells is indispensable for the development of novel cultivation approaches and control of cocultures. We present the development, validation, and experimental characterization of an optochemically controllable bacterial coculture on a microcolony level consisting of two Corynebacterium glutamicum strains. Our coculture combines an l-lysine auxotrophic strain together with a l-lysine-producing variant carrying the genetically IPTG-mediated induction of l-lysine production. We implemented two control approaches utilizing IPTG as inducer molecule. First, unmodified IPTG was supplemented to the culture enabling a medium-based control of the production of l-lysine, which serves as the main interacting component. Second, optochemical control was successfully performed by utilizing photocaged IPTG activated by appropriate illumination. Both control strategies were validated studying cellular growth on a microcolony level. The novel microfluidic single-cell cultivation strategies applied in this work can serve as a blueprint to validate cellular control strategies of synthetic mono- and cocultures with single-cell resolution at defined environmental conditions.

Background: The regular arrangement of collecting venules (RAC) and the Kimura-Takemoto classification of atrophic change (KTC) are simple and easy-to-use criteria which have been shown to reliably predict or rule out a Helicobacter pylori infection of the stomach. Although these features have been investigated extensively in Asia, their significance in the West has not been evaluated., Methods: In a series of 200 consecutive gastroscopic examinations (single examiner, single center), the presence or absence of RAC and the KTC grade (open type vs closed type) were recorded prospectively. Helicobacter pylori infection was defined as a positive histology or a positive rapid urease test. Furthermore, multivariate analysis of endoscopic predictors of H. pylori infection based on the Kyoto classification of gastritis was performed., Results: Two hundred patients were examined of which 57 had a H. pylori infection (28%). Both RAC and KTC had excellent negative predictive values of about 90% and sensitivity values of up to 85%. In multivariate analysis, atrophic change and diffuse redness without RAC were significantly associated with H. pylori infection., Conclusion: Regular arrangement of collecting venules and KTC are simple endoscopic features which should be given attention by Western endoscopists and can be easily used to rule out a H. pylori infection of the stomach., (© 2020 The Authors. Digestive Endoscopy published by John Wiley & Sons Australia, Ltd on behalf of Japan Gastroenterological Endoscopy Society.)

Benzoic acid is one of the most commonly used food preservatives, but currently exclusively produced in petrochemical processes. In this study, a bio-based production pathway using an engineered strain of Pseudomonas taiwanensis is described. In a phenylalanine-overproducing strain, bacterial and plant genes are heterologously expressed to achieve production of benzoate via a β-oxidation pathway. Strategic disruption of the native Pseudomonas benzoate degradation pathway further allows the production of catechol and cis,cis-muconate. Taken together, this work demonstrates new routes for the microbial production of these industrially relevant chemicals from renewable resources., (© 2020 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

L-glutamate (Glu) is the major excitatory transmitter in mammalian brain. Inadequate concentration of Glu in the brain correlates to mood disorder. In industry, Glu is used as a flavour enhancer in food and in foodstuff processing. A high concentration of Glu has several effects on human health such as hypersensitive effects, headache and stomach pain. The presence of Glu in food can be detected by different analytical methods based on chromatography, or capillary electrophoresis or amperometric techniques. We have isolated and characterized a glutamate-binding protein (GluB) from the Gram-positive bacteria Corynebacterium glutamicum. Together with GluC protein, GluD protein and the cytoplasmic protein GluA, GluB permits the transport of Glu in/out of cell. In this study, we have investigated the binding features of GluB as well as the effect of temperature on its structure both in the absence and in the presence of Glu. The results have showed that GluB has a high affinity and selectivity versus Glu (nanomolar range) and the presence of the ligand induces a higher thermal stability of the protein structure., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In recent years, transcriptional biosensors have become valuable tools in metabolic engineering as they allow semiquantitative determination of metabolites in single cells. Although being perfectly suitable tools for high-throughput screenings, application of transcriptional biosensors is often limited by the intrinsic characteristics of the individual sensor components and their interplay. In addition, biosensors often fail to work properly in heterologous host systems due to signal saturation at low intracellular metabolite concentrations, which typically limits their use in high-level producer strains at advanced engineering stages. We here introduce a biosensor design, which allows fine-tuning of important sensor parameters and restores the sensor response in a heterologous expression host. As a key feature of our design, the regulator activity is controlled through the expression level of the respective gene by different (synthetic) constitutive promoters selected for the used expression host. In this context, we constructed biosensors responding to basic amino acids or ring-hydroxylated phenylpropanoids for applications in Corynebacterium glutamicum and Escherichia coli . Detailed characterization of these biosensors in liquid cultures and during single-cell analysis using flow cytometry showed that the presented sensor design enables customization of important biosensor parameters as well as application of these sensors in relevant heterologous hosts., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.

Malonyl-CoA is an important central metabolite serving as the basic building block for the microbial synthesis of many pharmaceutically interesting polyketides, but also fatty acid-derived compounds including biofuels. Especially Saccharomyces cerevisiae, Escherichia coli, and Corynebacterium glutamicum have been engineered towards microbial synthesis of such compounds in recent years. However, developed strains and processes often suffer from insufficient productivity. Usually, tightly regulated intracellular malonyl-CoA availability is regarded as the decisive bottleneck limiting overall product formation. Therefore, metabolic engineering towards improved malonyl-CoA availability is essential to design efficient microbial cell factories for the production of polyketides and fatty acid derivatives. This review article summarizes metabolic engineering strategies to improve intracellular malonyl-CoA formation in industrially relevant microorganisms and its impact on productivity and product range, with a focus on polyketides and other malonyl-CoA-dependent products.Key Points• Malonyl-CoA is the central building block of polyketide synthesis.• Increasing acetyl-CoA supply is pivotal to improve malonyl-CoA availability.• Improved acetyl-CoA carboxylase activity increases availability of malonyl-CoA.• Fatty acid synthesis as an ambivalent target to improve malonyl-CoA supply.

Background: The phenylbutanoid 4-(4-hydroxyphenyl)butan-2-one, commonly known as raspberry ketone, is responsible for the typical scent and flavor of ripe raspberries. Chemical production of nature-identical raspberry ketone is well established as this compound is frequently used to flavor food, beverages and perfumes. However, high demand for natural raspberry ketone, but low natural abundance in raspberries, render raspberry ketone one of the most expensive natural flavoring components., Results: In this study, Corynebacterium glutamicum was engineered for the microbial synthesis of the character impact compound raspberry ketone from supplemented p-coumaric acid. In this context, the NADPH-dependent curcumin/dihydrocurcumin reductase CurA from Escherichia coli was employed to catalyze the final step of raspberry ketone synthesis as it provides a hitherto unknown benzalacetone reductase activity. In combination with a 4-coumarate: CoA ligase from parsley (Petroselinum crispum) and a monofunctional benzalacetone synthase from Chinese rhubarb (Rheum palmatum), CurA constitutes the synthetic pathway for raspberry ketone synthesis in C. glutamicum. The resulting strain accumulated up to 99.8 mg/L (0.61 mM) raspberry ketone. In addition, supplementation of other phenylpropanoids allowed for the synthesis of two other naturally-occurring and flavoring phenylbutanoids, zingerone (70 mg/L, 0.36 mM) and benzylacetone (10.5 mg/L, 0.07 mM)., Conclusion: The aromatic product portfolio of C. glutamicum was extended towards the synthesis of the flavoring phenylbutanoids raspberry ketone, zingerone and benzylacetone. Key to success was the identification of CurA from E. coli having a benzalacetone reductase activity. We believe, that the constructed C. glutamicum strain represents a versatile platform for the production of natural flavoring phenylbutanoids at larger scale.

Aim:  To test the usability and user experience of a newly self-developed eLearning tool to teach PET/CT and CT to undergraduate medical students., Methods:  The eLearning tool permits to display PET and CT DICOM images web-based. It contains a healthy subject with anatomical annotations and a clinical case study. The usability and user experience of the eLearning tool was evaluated in undergraduate medical students of the medical faculty of the RWTH Aachen. We applied a survey based on different already existing and validated questionnaires such as System Usability Scale (SUS) and User Experience Questionnaire (UEQ-S) as well as specific questions regarding the eLearning tool., Results:  38 volunteers (9 males) participated in our study. Applying the SUS resulted in a mean of 82.24, and a median of 83.75. This positive evaluation is supported by the results of the UEQ-S that were 2.2 with regard to the pragmatic quality, 2.3 with regard to hedonic quality and 2.3 for overall quality indicating a very positive evaluation. In the free-text answers, students emphasised easy and intuitive use of the eLearning tool that was additionally described as interesting and exciting. The students also positively mentioned the case study and the possibility of practice-based learning. Negative aspects were mainly problems with synchronisation of the PET and CT images., Conclusion:  The positive evaluation is encouraging and form a foundation for further development of the eLearning module. It may be the basis for the implementation of a sustainable blended learning concept in the nuclear medicine curriculum., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Aim:  Molecular imaging and therapy as applied in nuclear medicine represent an important contribution to modern medicine. The university curricula of medical schools permit to convey knowledge of nuclear medicine. In this study, the status quo of nuclear medicine teaching at German medical schools shall be surveyed for the first time., Methods:  In cooperation with the chairs and/or lecturers, the topics organizational conditions, scope of teaching, teaching methods, examinations, evaluations, composition of teaching staff, interest of the students in the field of nuclear medicine, as well as suggestions for improvement of teaching were recorded., Results:  All 36 public German medical schools participated in the survey. In these medical schools a median of 12 hours of nuclear medicine is being taught. 52 % of the teaching consists of teacher-centered learning. 18 medical schools teach additional elective courses in nuclear medicine. Nine 9 persons (median) per department are involved in the teaching. The examinations, in all but one school, follow a multiple choice format. The students' interest in nuclear medicine is perceived by the educators to be predominately medium to high., Conclusions:  Potential improvement might be achieved with regard to the quantity of teaching lessons, innovative methods of teaching, design of the exams, the establishment of a nationwide e-learning platform, as well as measures to increase the motivation of teachers., Competing Interests: Die Autoren geben an, dass kein Interessenkonflikt besteht., (© Georg Thieme Verlag KG Stuttgart · New York.)