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Natural plant-based flavonoids have drawn significant attention as dietary supplements due to their potential health benefits, including anti-cancer, anti-oxidant and anti-asthmatic activities. Naringenin, pinocembrin, eriodictyol and homoeriodictyol are classified as (2 S)-flavanones, an important sub-group of naturally occurring flavonoids, with wide-reaching applications in human health and nutrition. These four compounds occupy a central position as branch point intermediates towards a broad spectrum of naturally occurring flavonoids. Here, we report the development of Escherichia coli production chassis for each of these key gatekeeper flavonoids. Selection of key enzymes, genetic construct design and the optimization of process conditions resulted in the highest reported titers for naringenin (484 mg/l), improved production of pinocembrin (198 mg/l) and eriodictyol (55 mg/l from caffeic acid), and provided the first example of in vivo production of homoeriodictyol directly from glycerol (17 mg/l). This work provides a springboard for future production of diverse downstream natural and non-natural flavonoid targets. [ABSTRACT FROM AUTHOR]

O-Demethylation by acetogenic or organohalide-respiring bacteria leads to the formation of methyltetrahydrofolate from aromatic methyl ethers. O-Demethylases, which are cobalamin-dependent, three-component enzyme systems, catalyse methyl-group transfers from aromatic methyl ethers to tetrahydrofolate via methylcobalamin intermediates. In this study, crystal structures of the tetrahydrofolate-binding methyltransferase module from a Desulfitobacterium hafniense DCB-2 O-demethylase were determined both in complex with tetrahydrofolate and the product methyltetrahydrofolate. While these structures are similar to previously determined methyltransferase structures, the position of key active-site residues is subtly altered. A strictly conserved Asn is displaced to establish a putative proton-transfer network between the substrate N5 and solvent. It is proposed that this supports the efficient catalysis of methyltetrahydrofolate formation, which is necessary for efficient O-demethylation. [ABSTRACT FROM AUTHOR]

This study describes the identification and the structural and spectroscopic analysis of a cobalamin-binding protein (termed CobDH) implicated in O-demethylation by the organohalide-respiring bacterium Desulfitobacterium hafniense DCB-2. The 1.5 Å resolution crystal structure of CobDH is presented in the cobalamin-bound state and reveals that the protein is composed of an N-terminal helix-bundle domain and a C-terminal Rossmann-fold domain, with the cobalamin coordinated in the base-off/His-on conformation similar to other cobalamin-binding domains that catalyse methyl-transfer reactions. EPR spectroscopy of CobDH confirms cobalamin binding and reveals the presence of a cob(III)alamin superoxide, indicating binding of oxygen to the fully oxidized cofactor. These data provide the first structural insights into the methyltransferase reactions that occur during O-demethylation by D. hafniense. [ABSTRACT FROM AUTHOR]

The transcriptional regulator CprK controls the expression of the reductive dehalogenase CprA in organohalide-respiring bacteria. Desulfitobacterium hafniense CprA catalyses the reductive dechlorination of the terminal electron acceptor o-chlorophenol acetic acid, generating the phenol acetic acid product. It has been shown that CprK has ability to distinguish between the chlorinated CprA substrate and the de-halogenated end product, with an estimated an estimated 104-fold difference in affinity. Using a green fluorescent protein GFPUV-based transcriptional reporter system, we establish that CprK can sense o-chlorophenol acetic acid at the nanomolar level, whereas phenol acetic acid leads to transcriptional activation only when approaching micromolar levels. A structure--activity relationship study, using a range of o-chlorophenol acetic-acid-related compounds and key CprK mutants, combined with pKa calculations on the effector binding site, suggests that the sensitive detection of chlorination is achieved through a combination of direct and indirect readout mechanisms. Both the physical presence of the bulky chloride substituent as well as the accompanying electronic effects lowering the inherent phenol pKa are required for high affinity. Indeed, transcriptional activation by CprK appears strictly dependent on establishing a phenolate--K133 salt bridge interaction, rather than on the presence of a halogen atom per se. As K133 is strictly conserved within the CprK family, our data suggest that physiological function and future applications in biosensing are probably restricted to phenolic compounds. [ABSTRACT FROM AUTHOR]

Imidazoacridin-6-ones are shown to be potent nanomolar inhibitors of the enzyme NQO2. By use of computational molecular modeling, a reliable QSAR was established, relating inhibitory potency with calculated binding affinity. Further, crystal structures of NQO2 containing two of the imidazoacridin-6-ones have been solved. To generate compounds with reduced off-target (DNA binding) effects, an N-oxide moiety was introduced into the tertiary aminoalkyl side chain of the imidazoacridin-6-ones. This resulted in substantially less toxicity in a panel of eight cancer cell lines, decreased protein binding, and reduced DNA binding and nuclear accumulation. Finally, one of the N-oxides showed potent ability to inhibit the enzymatic function of NQO2 in cells, and therefore, it may be useful as a pharmacological probe to study the properties of the enzyme in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose-ribose bond, and is synthesized from NAD by PAR polymerases. PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose-ribose bonds present in PAR chains; its deficiency leads to cell death. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family. High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease. [ABSTRACT FROM AUTHOR]

The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr)∙S-adenosy1-L-methionine (AdoMet) complex was determined at 2.45-Å resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal "L30-like" putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide Lu-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that TsrrRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme. [ABSTRACT FROM AUTHOR]

Summary: The X-ray crystal structure of a ribosomal L11-rRNA complex with chloroplast-like mutations in both protein and rRNA is presented. The global structure is almost identical to that of the wild-type (bacterial) complex, with only a small movement of the protein α helix away from the surface of the RNA required to accommodate the altered protein residue. In contrast, the specific hydrogen bonding pattern of the mutated residues is substantially different, and now includes a direct interaction between the protein side chain and an RNA base edge and a water-mediated contact. Comparison of the two structures allows the observations of sequence variation and relative affinities of wild-type and mutant complexes to be clearly rationalized, but reinforces the concept that there is no single simple code for protein-RNA recognition. [Copyright &y& Elsevier]

Monoterpenoids are a structurally diverse group of natural products with applications as pharmaceuticals, flavourings, fragrances, pesticides, and biofuels. Recent advances in synthetic biology offer new routes to this chemical diversity through the introduction of heterologous isoprenoid production pathways into engineered microorganisms. Due to the nature of the branched reaction mechanism, monoterpene synthases often produce multiple products when expressed in monoterpenoid production platforms. Rational engineering of terpene synthases is challenging due to a lack of correlation between protein sequence and cyclisation reaction catalysed. Directed evolution offers an attractive alternative protein engineering strategy as limited prior sequence-function knowledge is required. However, directed evolution of terpene synthases is hampered by the lack of a convenient high-throughput screening assay for the detection of multiple volatile terpene products. Here we applied an automated pipeline for the screening of diverse monoterpene synthase libraries, employing robotic liquid handling platforms coupled to GC-MS, and automated data extraction. We used the pipeline to screen pinene synthase variant libraries, with mutations in three areas of plasticity, capable of producing multiple monoterpene products. We successfully identified variants with altered product profiles and demonstrated good agreement between the results of the automated screen and traditional shake-flask cultures. In addition, useful insights into the cyclisation reaction catalysed by pinene synthase were obtained, including the identification of positions with the highest level of plasticity, and the significance of region 2 in carbocation cyclisation. The results obtained will aid the prediction and design of novel terpene synthase activities towards clean monoterpenoid products. [ABSTRACT FROM AUTHOR]

Summary: CRISPR technologies have become standard laboratory tools for genetic manipulations across all kingdoms of life. Despite their origins in bacteria, the development of CRISPR tools for engineering bacteria has been slower than for eukaryotes; nevertheless, their function and application for genome engineering and gene regulation via CRISPR interference (CRISPRi) has been demonstrated in various bacteria, and adoption has become more widespread. Here, we provide simple plasmid‐based systems for genome editing (gene knockouts/knock‐ins, and genome integration of large DNA fragments) and CRISPRi in E. coli using a CRISPR‐Cas12a system. The described genome engineering protocols allow markerless deletion or genome integration in just seven working days with high efficiency (> 80% and 50%, respectively), and the CRISPRi protocols allow robust transcriptional repression of target genes (> 90%) with a single cloning step. The presented minimized plasmids and their associated design and experimental protocols provide efficient and effective CRISPR‐Cas12 genome editing, genome integration and CRISPRi implementation. These simple‐to‐use systems and protocols will allow the easy adoption of CRISPR technology by any laboratory. [ABSTRACT FROM AUTHOR]

Abstract: Organohalide respiration is used by a limited set of anaerobic bacteria to derive energy from the reduction of halogenated organic compounds. The enzymes that catalyze the reductive dehalogenation reaction, the reductive dehalogenases, represent a novel and distinct class of cobalamin and Fe-S cluster dependent enzymes. Until now, biochemical studies on reductive dehalogenases have been hampered by the lack of a reliable protein source. Here we present an efficient and robust heterologous production system for the reductive dehalogenase PceA from Dehalobacter restrictus. Large quantities of Strep-tagged PceA fused to a cold-shock induced trigger factor could be obtained from Escherichia coli. The recombinant enzyme was conveniently purified in milligram quantities under anaerobic conditions by StrepTactin affinity chromatography, and the trigger factor could be removed through limited proteolysis. Characterization of the purified PceA by UV–Vis and electron paramagnetic resonance (EPR) spectroscopy reveal that the recombinant protein binds methylcobalamin in the base-on form after proteolytic cleavage of the trigger factor, and that 4Fe-4S clusters can be chemically reconstituted under anoxic conditions. This study demonstrates a novel PceA production platform that allows further study of this new enzyme class. [Copyright &y& Elsevier]

Metabolic engineering technologies have been employed with increasing success over the last three decades for the engineering and optimization of industrial host strains to competitively produce high-value chemical targets. To this end, continued reductions in the time taken from concept, to development, to scale-up are essential. Design-Build-Test-Learn pipelines that are able to rapidly deliver diverse chemical targets through iterative optimization of microbial production strains have been established. Biofoundries are employing in silico tools for the design of genetic parts, alongside combinatorial design of experiments approaches to optimize selection from within the potential design space of biological circuits based on multi-criteria objectives. These genetic constructs can then be built and tested through automated laboratory workflows, with performance data analysed in the learn phase to inform further design. Successful examples of rapid prototyping processes for microbially produced compounds reveal the potential role of biofoundries in leading the sustainable production of next-generation bio-based chemicals. [ABSTRACT FROM AUTHOR]

Bio-based production of industrial chemicals using synthetic biology can provide alternative green routes from renewable resources, allowing for cleaner production processes. To efficiently produce chemicals on-demand through microbial strain engineering, biomanufacturing foundries have developed automated pipelines that are largely compound agnostic in their time to delivery. Here we benchmark the capabilities of a biomanufacturing pipeline to enable rapid prototyping of microbial cell factories for the production of chemically diverse industrially relevant material building blocks. Over 85 days the pipeline was able to produce 17 potential material monomers and key intermediates by combining 160 genetic parts into 115 unique biosynthetic pathways. To explore the scale-up potential of our prototype production strains, we optimized the enantioselective production of mandelic acid and hydroxymandelic acid, achieving gram-scale production in fed-batch fermenters. The high success rate in the rapid design and prototyping of microbially-produced material building blocks reveals the potential role of biofoundries in leading the transition to sustainable materials production. Image 1 • An automated biomanufacturing pipeline is benchmarked for the production of material monomers. • Over 85 days, 160 genes were screened for activity, assembled into 115 unique pathways and tested for in vivo production. • E. coli production strains were successfully constructed to produce 17 target compounds at competitive titers. • Scale-up potential is demonstrated through enantioselective production of mandelic acid targets at gram-scale in bioreactors. • The high success rate demonstrates the capabilities of biofoundries to rapidly prototype microbial production strains. [ABSTRACT FROM AUTHOR]

The GTPase Center (GAC) RNA domain in bacterial 23S rRNA is directly bound by ribosomal protein L11, and this complex is essential to ribosome function. Previous cocrystal structures of the 58-nucleotide GAC RNA bound to L11 revealed the intricate tertiary fold of the RNA domain, with one monovalent and several divalent ions located in specific sites within the structure. Here, we report a new crystal structure of the free GAC that is essentially identical to the L11-bound structure, which retains many common sites of divalent ion occupation. This new structure demonstrates that RNA alone folds into its tertiary structure with bound divalent ions. In solution, we find that this tertiary structure is not static, but rather is best described as an ensemble of states. While L11 protein cannot bind to the GAC until the RNA has adopted its tertiary structure, new experimental data show that L11 binds to Mg2+-dependent folded states, which we suggest lie along the folding pathway of the RNA. We propose that L11 stabilizes a specific GAC RNA tertiary state, corresponding to the crystal structure, and that this structure reflects the functionally critical conformation of the rRNA domain in the fully assembled ribosome. Image 1 • GAC RNA structures in the crystal and cocrystals are the same. • L11 stabilizes the functional GAC tertiary fold. • L11 binds RNA during Mg2+-dependent folding trajectory. • L11 prevents misfolding of tertiary structure. [ABSTRACT FROM AUTHOR]

Synthetic biology utilizes the Design–Build–Test–Learn pipeline for the engineering of biological systems. Typically, this requires the construction of specifically designed, large and complex DNA assemblies. The availability of cheap DNA synthesis and automation enables high-throughput assembly approaches, which generates a heavy demand for DNA sequencing to verify correctly assembled constructs. Next-generation sequencing is ideally positioned to perform this task, however with expensive hardware costs and bespoke data analysis requirements few laboratories utilize this technology in-house. Here a workflow for highly multiplexed sequencing is presented, capable of fast and accurate sequence verification of DNA assemblies using nanopore technology. A novel sample barcoding system using polymerase chain reaction is introduced, and sequencing data are analyzed through a bespoke analysis algorithm. Crucially, this algorithm overcomes the problem of high-error rate nanopore data (which typically prevents identification of single nucleotide variants) through statistical analysis of strand bias, permitting accurate sequence analysis with single-base resolution. As an example, 576 constructs (6 × 96 well plates) were processed in a single workflow in 72 h (from Escherichia coli colonies to analyzed data). Given our procedure's low hardware costs and highly multiplexed capability, this provides cost-effective access to powerful DNA sequencing for any laboratory, with applications beyond synthetic biology including directed evolution, single nucleotide polymorphism analysis and gene synthesis. [ABSTRACT FROM AUTHOR]

Abstract: Benzylisoquinoline alkaloids (BIAs) are a structurally diverse family of plant secondary metabolites, which have been exploited to develop analgesics, antibiotics, antitumor agents, and other therapeutic agents. Biosynthesis of BIAs proceeds via a common pathway from tyrosine to (S)‐reticulene at which point the pathway diverges. Coclaurine N‐methyltransferase (CNMT) is a key enzyme in the pathway to (S)‐reticulene, installing the N‐methyl substituent that is essential for the bioactivity of many BIAs. In this paper, we describe the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture. The specificity of CNMT was also explored with a range of natural and synthetic substrates as well as co‐factor analogues. Knowledge from this study could be used to generate improved CNMT variants required to produce BIAs or synthetic derivatives. [ABSTRACT FROM AUTHOR]

Abstract: Benzylisoquinoline alkaloids (BIAs) are a structurally diverse family of plant secondary metabolites, which have been exploited to develop analgesics, antibiotics, antitumor agents, and other therapeutic agents. Biosynthesis of BIAs proceeds via a common pathway from tyrosine to (S)‐reticulene at which point the pathway diverges. Coclaurine N‐methyltransferase (CNMT) is a key enzyme in the pathway to (S)‐reticulene, installing the N‐methyl substituent that is essential for the bioactivity of many BIAs. In this paper, we describe the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture. The specificity of CNMT was also explored with a range of natural and synthetic substrates as well as co‐factor analogues. Knowledge from this study could be used to generate improved CNMT variants required to produce BIAs or synthetic derivatives. [ABSTRACT FROM AUTHOR]

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles. [ABSTRACT FROM AUTHOR]

Carboxylic acid reductases (CARs) catalyze the reduction of a broad range of carboxylic acids to aldehydes using the cofactors adenosine triphosphate and nicotinamide adenine dinucleotide phosphate, and have become attractive biocatalysts for organic synthesis. Mechanistic understanding of CARs was used to expand reaction scope, generating biocatalysts for amide bond formation from carboxylic acid and amine. CARs demonstrated amidation activity for various acids and amines. Optimization of reaction conditions, with respect to pH and temperature, allowed for the synthesis of the anticonvulsant ilepcimide with up to 96 % conversion. Mechanistic studies using site-directed mutagenesis suggest that, following initial enzymatic adenylation of substrates, amidation of the carboxylic acid proceeds by direct reaction of the acyl adenylate with amine nucleophiles. [ABSTRACT FROM AUTHOR]

A practical and efficient biocatalytic synthesis of aromatic d-amino acids has been developed, based on the reductive amination of the corresponding α-keto acids via a recombinant whole cell system composed of an engineered dehydrogenase and cofactor recycling apparatus. The reaction was shown to give excellent enantioselectivity (≥98%) and good yields at the preparative scale across a broad range of substrates. Additionally, the structure of the variant enzyme was solved to allow rationalisation of the observed reaction rates. The engineered whole cell catalyst was also used to mediate the production of d-phenylalanine derivatives from racemic mixtures and cheaper l-amino acids by combining it with an enantiocomplementary deaminase. [ABSTRACT FROM AUTHOR]

Queuosine (Q) is a hypermodified RNA base that replaces guanine in the wobble positions of 5'-GUN-3' tRNA molecules. Q is exclusively made by bacteria, and the corresponding queuine base is a micronutrient salvaged by eukaryotic species. The final step in Q biosynthesis is the reduction of the epoxide precursor, epoxyqueuosine, to yield the Q cyclopentene ring. The epoxyqueuosine reductase responsible, QueG, shares distant homology with the cobalamin-dependent reductive dehalogenase (RdhA), however the role played by cobalamin in QueG catalysis has remained elusive.Wereport the solution and structural characterization of Streptococcus thermophilus QueG, revealing the enzyme harbors a redox chain consisting of two [4Fe-4S] clusters and a cob(II)alamin in the base-off form, similar to RdhAs. In contrast to the shared redox chain architecture, the QueG active site shares little homology with RdhA, with the notable exception of a conserved Tyr that is proposed to function as a proton donor during reductive dehalogenation. Docking of an epoxyqueuosine substrate suggests the QueG active site places the substrate cyclopentane moiety in close proximity of the cobalt. Both the Tyr and a conserved Asp are implicated as proton donors to the epoxide leaving group. This suggests that, in contrast to the unusual carbon-halogen bond chemistry catalyzed by RdhAs, QueG acts via Co-C bond formation. Our study establishes the common features of Class III cobalamindependent enzymes, and reveals an unexpected diversity in the reductive chemistry catalyzed by these enzymes. [ABSTRACT FROM AUTHOR]

Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis. [ABSTRACT FROM AUTHOR]

Ligand-dependent control of gene expression is essential for gene functional analysis, target validation, protein production, and metabolic engineering. However, the expression tools currently available are difficult to transfer between species and exhibit limited mechanistic diversity. Here we demonstrate how the modular architecture of purine riboswitches can be exploited to develop orthogonal and chimeric switches that are transferable across diverse bacterial species, modulating either transcription or translation, to provide tunable activation or repression of target gene expression, in response to synthetic non-natural effector molecules. Our novel riboswitch—ligand pairings are shown to regulate physiologically important genes required for bacterial motility in Escherichia coli and cell morphology in Bacillus subtilis. These findings are relevant for future gene function studies and antimicrobial target validation, while providing new modular and orthogonal regulatory components for deployment in synthetic biology regimes. [ABSTRACT FROM AUTHOR]

The ability to independently control the expression of multiple genes by addition of distinct small-molecule modulators has many applications from synthetic biology, functional genomics, pharmaceutical target validation, through to gene therapy. Riboswitches are relatively simple, small-molecule-dependent, protein-free, mRNA genetic switches that are attractive targets for reengineering in this context. Using a combination of chemical genetics and genetic selection, we have developed riboswitches that are selective for synthetic "nonnatural" small molecules and no longer respond to the natural intracellular ligands. The orthogonal selectivity of the riboswitches is also demonstrated in vitro using isothermal titration calorimetry and x-ray crystallography. The riboswitches allow highly responsive, dose-dependents orthogonally selective, and dynamic control of gene expression in vivo. It is possible that this approach may be further developed to reengineer other natural riboswitches for application as small-molecule responsive genetic switches in both prokaryotes and eukaryotes. [ABSTRACT FROM AUTHOR]