Explore the vast range of titles available.

Bacterial degradation of organosulfonates plays an important role in sulfur recycling, and has been extensively studied. However, this process in anaerobic bacteria especially gut bacteria is little known despite of its potential significant impact on human health with the production of toxic H 2 S. Here, we describe the structural and biochemical characterization of an oxygen-sensitive enzyme that catalyzes the radical-mediated C-S bond cleavage of isethionate to form sulfite and acetaldehyde. We demonstrate its involvement in pathways that enables C2 sulfonates to be used as terminal electron acceptors for anaerobic respiration in sulfate- and sulfite-reducing bacteria. Furthermore, it plays a key role in converting bile salt-derived taurine into H 2 S in the disease-associated gut bacterium Bilophila wadsworthia . The enzymes and transporters in these anaerobic pathways expand our understanding of microbial sulfur metabolism, and help deciphering the complex web of microbial pathways involved in the transformation of sulfur compounds in the gut. The C2 sulfonates taurine and isethionate are also present in the anaerobic mammalian gut, where they are converted into toxic H 2 S by sulfate and sulfite-reducing bacteria. Here the authors characterise the O 2 -sensitive enzyme IseG that catalyzes the C-S bond cleavage of isethionate and show that IseG also plays a key role in converting taurine into H 2 S in Bilophila wadsworthia .

Because of extraordinary structural diversity and broad biological activities, natural products have played a significant role in drug discovery. These therapeutically important secondary metabolites are assembled and modified by dedicated biosynthetic pathways in their host living organisms. Traditionally, chemists have attempted to synthesize natural product analogs that are important sources of new drugs. However, the extraordinary structural complexity of natural products sometimes makes it challenging for traditional chemical synthesis, which usually involves multiple steps, harsh conditions, toxic organic solvents, and byproduct wastes. In contrast, combinatorial biosynthesis exploits substrate promiscuity and employs engineered enzymes and pathways to produce novel \"unnatural\" natural products, substantially expanding the structural diversity of natural products with potential pharmaceutical value. Thus, combinatorial biosynthesis provides an environmentally friendly way to produce natural product analogs. Efficient expression of the combinatorial biosynthetic pathway in genetically tractable heterologous hosts can increase the titer of the compound, eventually resulting in less expensive drugs. In this review, we will discuss three major strategies for combinatorial biosynthesis: 1) precursor-directed biosynthesis; 2) enzyme-level modification, which includes swapping of the entire domains, modules and subunits, site-specific mutagenesis, and directed evolution; 3) pathway-level recombination. Recent examples of combinatorial biosynthesis employing these strategies will also be highlighted in this review.

Abstract In vivo biosensors can recognize and respond to specific cellular stimuli. In recent years, biosensors have been increasingly used in metabolic engineering and synthetic biology, because they can be implemented in synthetic circuits to control the expression of reporter genes in response to specific cellular stimuli, such as a certain metabolite or a change in pH. There are many types of natural sensing devices, which can be generally divided into two main categories: protein-based and nucleic acid-based. Both can be obtained either by directly mining from natural genetic components or by engineering the existing genetic components for novel specificity or improved characteristics. A wide range of new technologies have enabled rapid engineering and discovery of new biosensors, which are paving the way for a new era of biotechnological progress. Here, we review recent advances in the design, optimization, and applications of in vivo biosensors in the field of metabolic engineering and synthetic biology.

Editorial Summary Most microbial biosynthetic gene clusters are inactive under laboratory culture conditions. A CRISPR–Cas9 genome-editing approach in Streptomyces species enables the targeted activation of silent gene clusters and production of encoded natural products. Here we report an efficient CRISPR–Cas9 knock-in strategy to activate silent biosynthetic gene clusters (BGCs) in streptomycetes. We applied this one-step strategy to activate multiple BGCs of different classes in five Streptomyces species and triggered the production of unique metabolites, including a novel pentangular type II polyketide in Streptomyces viridochromogenes . This potentially scalable strategy complements existing activation approaches and facilitates discovery efforts to uncover new compounds with interesting bioactivities.

n -Butanol has several favourable properties as an advanced fuel or a platform chemical. Bio-based production of n -butanol is becoming increasingly important for sustainable chemical industry. Synthesis of n -butanol can be achieved via more than one metabolic pathway. Here we report the metabolic engineering of Saccharomyces cerevisiae to produce n -butanol through a synergistic pathway: the endogenous threonine pathway and the introduced citramalate pathway. Firstly, we characterized and optimized the endogenous threonine pathway; then, a citramalate synthase (CimA) mediated pathway was introduced to construct the synergistic pathway; next, the synergistic pathway was optimized by additional overexpression of relevant genes identified previously; meanwhile, the n -butanol production was also improved by overexpression of keto-acid decarboxylases (KDC) and alcohol dehydrogenase (ADH). After combining these strategies with co-expression of LEU1 (two copies), LEU4 , LEU2 (two copies), LEU5, CimA, NFS1, ADH7 and ARO10 * , we achieved an n -butanol production of 835 mg/L in the final engineered strain, which is almost 7-fold increase compared to the initial strain. Furthermore, the production showed a 3-fold of the highest titer ever reported in yeast. Therefore, the engineered yeast strain represents a promising alternative platform for n -butanol production.

Skatole is a malodorous compound that contributes to the characteristic smell of animal faeces. Although skatole has long been known to originate from bacterial tryptophan fermentation, the enzyme catalysing its formation has so far remained elusive. Here we report the use of comparative genomics for the discovery of indoleacetate decarboxylase, an O 2 -sensitive glycyl radical enzyme catalysing the decarboxylation of indoleacetate to form skatole as the terminal step of tryptophan fermentation in certain anaerobic bacteria. We describe its biochemical characterization and compare it to other glycyl radical decarboxylases. Indoleacetate decarboxylase may serve as a genetic marker for the identification of skatole-producing environmental and human-associated bacteria, with impacts on human health and the livestock industry. Skatole is a bacterial metabolite responsible for boar taint and the objectionable smell of manure. Here, the authors elucidate the final step of skatole biosynthesis, describing the discovery and biochemical characterization of the enzyme catalysing the conversion of indoleacetate into skatole.

Background Streptomyces is renowned for its robust biosynthetic capacity in producing medically relevant natural products. However, the majority of natural products biosynthetic gene clusters (BGCs) either yield low amounts of natural products or remain cryptic under standard laboratory conditions. Various heterologous production hosts have been engineered to address these challenges, and yet the successful activation of BGCs has still been limited. In our search for a valuable addition to the heterologous host panel, we identified the strain Streptomyces sp. A4420, which exhibited rapid initial growth and a high metabolic capacity, prompting further exploration of its potential. Results We engineered a polyketide-focused chassis strain based on Streptomyces sp. A4420 (CH strain) by deleting 9 native polyketide BGCs. The resulting metabolically simplified organism exhibited consistent sporulation and growth, surpassing the performance of most existing Streptomyces based chassis strains in standard liquid growth media. Four distinct polyketide BGCs were chosen and expressed in various heterologous hosts, including the Streptomyces sp. A4420 wild-type and CH strains, alongside Streptomyces coelicolor M1152, Streptomyces lividans TK24, Streptomyces albus J1074, and Streptomyces venezuelae NRRL B-65442. Remarkably, only the Streptomyces sp. A4420 CH strain demonstrated the capability to produce all metabolites under every condition outperforming its parental strain and other tested organisms. To enhance visualization and comparison of the tested strains, we developed a matrix-like analysis involving 15 parameters. This comprehensive analysis unequivocally illustrated the significant potential of the new strain to become a popular heterologous host. Conclusion Our engineered Streptomyces sp. A4420 CH strain exhibits promising attributes for the heterologous expression of natural products with a focus on polyketides, offering an alternative choice in the arsenal of heterologous production strains. As genomics and cloning strategies progress, establishment of a diverse panel of heterologous production hosts will be crucial for expediting the discovery and production of medically relevant natural products derived from Streptomyces .

RNAs are well-suited to act as cellular sensors that detect and respond to metabolite changes in the environment, due to their ability to fold into complex structures. Here, we introduce a genome-wide strategy called PARCEL that experimentally identifies RNA aptamers in vitro, in a high-throughput manner. By applying PARCEL to a collection of prokaryotic and eukaryotic organisms, we have revealed 58 new RNA aptamers to three key metabolites, greatly expanding the list of natural RNA aptamers. The newly identified RNA aptamers exhibit significant sequence conservation, are highly structured and show an unexpected prevalence in coding regions. We identified a prokaryotic precursor tmRNA that binds vitamin B2 (FMN) to facilitate its maturation, as well as eukaryotic mRNAs that bind and respond to FMN, suggesting FMN as the second RNA-binding ligand to affect eukaryotic expression. PARCEL results show that RNA-based sensing and gene regulation is more widespread than previously appreciated in different organisms. Riboswitches recognize and respond to specific metabolites by altering gene expression. Here, the authors developed a high-throughput method (PARCEL) to experimentally identify RNA aptamers across transcriptomes.

The phenolic metabolite p‐cresol is a byproduct of tyrosine fermentation by certain strictly anaerobic bacteria, including the human gut pathogen Clostridium difficile, with toxic effects on the host and intestinal microbiota. The only enzyme in this biochemical pathway characterized to date is the glycyl radical enzyme p‐hydroxyphenylacetate (HPA) decarboxylase (HPAD), which catalyzes the terminal step. Here we report the identification and characterization of enzymes for anaerobic degradation of tyrosine to HPA in the model p‐cresol‐producing bacterium Clostridium scatologenes. In this pathway, tyrosine is first converted to p‐hydroxyphenylpyruvate (HPP), followed by net oxidation to HPA by a trio of enzymes HPP: ferredoxin oxidoreductase (HpdDEFG), phosphate HPA‐transferase (HpdJ) and HPA kinase (HpdK). Each step is thermodynamically reversible, and the pathway is coupled to net generation of ATP and reduced ferredoxin. A bioinformatics search reveals that gene clusters containing a similar trio of enzymes are widespread in anaerobic Firmicutes bacteria, suggesting analogous pathways for the degradation of other amino acids. These findings clarify the oxidative pathways by which anaerobic gut bacteria convert aromatic amino acids into a major class of aromatic metabolites including HPA and p‐cresol, deepening our understanding of microbiota–host metabolic interactions. In anaerobic bacteria, tyrosine serves as an electron donor in the oxidative Stickland fermentation branch, generating p‐hydroxyphenylacetate, ATP and reduced ferredoxin for decarboxylation into p‐cresol.

Halogenation of pyrrole requires strong electrophilic reagents and often leads to undesired polyhalogenated products. Biocatalytic halogenation is a highly attractive approach given its chemoselectivity and benign reaction conditions. While there are several reports of enzymatic phenol and indole halogenation in organic synthesis, corresponding reports on enzymatic pyrrole halogenation have been lacking. Here we describe the in vitro functional and structural characterization of PrnC, a flavin-dependent halogenase that can act on free-standing pyrroles. Computational modeling and site mutagenesis studies identified three key residues in the catalytic pocket. A moderate resolution map using single-particle cryogenic electron microscopy reveals PrnC to be a dimer. This native PrnC can halogenate a library of structurally diverse pyrrolic heterocycles in a site-selective manner and be applied in the chemoenzymatic synthesis of a chlorinated analog of the agrochemical fungicide Fludioxonil. Pyrroles are an important scaffold in medicinal chemistry with various bioactivities; however, the selective chemical halogenation of pyrroles remains challenging. Here, the authors develop an enzymatic site-selective chlorination of pyrrolic heterocycles by a flavin-dependent halogenase PrnC and apply it to the chemoenzymatic synthesis of a chlorinated analogue of the fungicide Fludioxonil.