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Background Flavonoids are a structurally diverse group of secondary metabolites, predominantly produced by plants, which include a range of compounds with pharmacological importance. Pinocembrin is a key branch point intermediate in the biosynthesis of a wide range of flavonoid subclasses. However, replicating the biosynthesis of these structurally diverse molecules in heterologous microbial cell factories has encountered challenges, in particular the modest pinocembrin titres achieved to date. In this study, we combined genome engineering and enzyme candidate screening to significantly enhance the production of pinocembrin and its derivatives, including chrysin, pinostrobin, pinobanksin, and galangin, in Escherichia coli . Results By implementing a combination of established strain engineering strategies aimed at enhancing the supply of the building blocks phenylalanine and malonyl-CoA, we constructed an E. coli chassis capable of accumulating 353 ± 19 mg/L pinocembrin from glycerol, without the need for precursor supplementation or the fatty acid biosynthesis inhibitor cerulenin. This chassis was subsequently employed for the production of chrysin, pinostrobin, pinobanksin, and galangin. Through an enzyme candidate screening process involving eight type-1 and five type-2 flavone synthases (FNS), we identified Petroselinum crispum FNSI as the top candidate, producing 82 ± 5 mg/L chrysin. Similarly, from a panel of five flavonoid 7- O -methyltransferases (7-OMT), we found pinocembrin 7-OMT from Eucalyptus nitida to yield 153 ± 10 mg/L pinostrobin. To produce pinobanksin, we screened seven enzyme candidates exhibiting flavanone 3-hydroxylase (F3H) or F3H/flavonol synthase (FLS) activity, with the bifunctional F3H/FLS enzyme from Glycine max being the top performer, achieving a pinobanksin titre of 12.6 ± 1.8 mg/L. Lastly, by utilising a combinatorial library of plasmids encoding G. max F3H and Citrus unshiu FLS, we obtained a maximum galangin titre of 18.2 ± 5.3 mg/L. Conclusion Through the integration of microbial chassis engineering and screening of enzyme candidates, we considerably increased the production levels of microbially synthesised pinocembrin, chrysin, pinostrobin, pinobanksin, and galangin. With the introduction of additional chassis modifications geared towards improving cofactor supply and regeneration, as well as alleviating potential toxic effects of intermediates and end products, we anticipate further enhancements in the yields of these pinocembrin derivatives, potentially enabling greater diversification in microbial hosts.

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. Minimized plasmids and their associated design and experimental protocols have been developed to 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.

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.

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 (2S)-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.

The microbial production of fine chemicals provides a promising biosustainable manufacturing solution that has led to the successful production of a growing catalog of natural products and high-value chemicals. However, development at industrial levels has been hindered by the large resource investments required. Here we present an integrated Design–Build-Test–Learn (DBTL) pipeline for the discovery and optimization of biosynthetic pathways, which is designed to be compound agnostic and automated throughout. We initially applied the pipeline for the production of the flavonoid (2 S )-pinocembrin in Escherichia coli , to demonstrate rapid iterative DBTL cycling with automation at every stage. In this case, application of two DBTL cycles successfully established a production pathway improved by 500-fold, with competitive titers up to 88 mg L −1 . The further application of the pipeline to optimize an alkaloids pathway demonstrates how it could facilitate the rapid optimization of microbial strains for production of any chemical compound of interest. Pablo Carbonell et al. present an automated pipeline for the discovery and optimization of biosynthetic pathways for microbial production of fine chemicals. They apply their pipeline to the production of the flavonoid (2S)-pinocembrin in Escherichia coli and show improvement of the pathway by 500-fold.

Synthetic biology is typified by developing novel genetic constructs from the assembly of reusable synthetic DNA parts, which contain one or more features such as promoters, ribosome binding sites, coding sequences and terminators. While repositories of such parts exist to promote their reuse, there is still a need to design novel parts from scratch. PartsGenie, freely available at http://parts.synbiochem.co.uk, is introduced to facilitate the computational design of such synthetic biology parts. PartsGenie has been designed to bridge the gap between optimisation tools for the design of novel parts, the representation of such parts in community-developed data standards such as Synthetic Biology Open Language (SBOL), and their sharing in journal-recommended data repositories. Consisting of a drag-and-drop web interface, a number of DNA optimisation algorithms, and an interface to the well-used data repository JBEI ICE, PartsGenie facilitates the design, optimisation and dissemination of reusable synthetic biology parts through a single, integrated application. PartsGenie can therefore be used as a single, stand-alone tool, or integrated into larger synthetic biology pipelines that are being developed in the SYNBIOCHEM centre and elsewhere.

The primary aim of the whole-body vibration (WBV) investigation was to determine if g-force is a valid measurement of vibration exercise intensity (VEI). For this purpose, twelve healthy lean adults (male, 7; female 5; age, 29.4 ± 6.4 years; height 171.4 ± 4.9 cm; weight, 67.4 ± 10.4 kg; BMI, 22.9 ± 2.6 kg/m2) voluntarily participated for the study. To examine the relationship between g-force and VEI, a 3-way (4-muscle group x 4-frequency x 2-amplitude) repeated measures design was employed, which analyzed electromyography (EMG) muscle activity in the vastus lateralis (V.L.), vastus inedialis (V.M.), gastrocnemius lateralis (G.L.) and gastrocnemius medialis (G.M.) muscles during WBV exercise. Participants, performed a standard unloaded isometric high-squat position (knee angle 30 degrees, hip angle 30 degrees, feet [middle toe to middle toe] 30cm apart) while at eight different levels (k = 8) of g-force. The levels of g-force were derived from four different preset frequency settings (30, 35, 40, and 50 Hz) measured at both the low and high amplitude settings. Each participant underwent four trials at each of the amplitudes. In each trial a participant experienced all four preset frequencies and a control condition. The VEI was operationally defined as the WBV-induced percent increase in EMGrms compared with the control condition (no vibration). Control data was expressed as a percent of maximum voluntary contraction (MVC), thus allowing for inter-subject comparisons. The results of the study clearly indicated that the highest g-forces were not associated with the highest VEI responses. The highest VEI responses were found at the lower frequencies (30 and 35 Hz) with exception of the G.M. (40 Hz). Furthermore the higher amplitude produced an approximately 35% higher VEI response than the low amplitude. Based on the findings it was concluded that g-force is not a valid measurement of VET. Additionally, the study revealed that both a large degree of inter-subject variability in VET responses exists and that WBV only produces a small amount of muscle activity when expressed relative to MVC data. Thus, it was concluded that WBV should only be used as a supplement to traditional exercise prescription, not as a replacement.

The leaf essential oils of Agonis fragrans, isolated by steam distillation and solvent extraction, were analyzed by GC and GC/MS. The major components identified in five of the six samples investigated were 1,8-cineole (28%-34% and 12%-26%, respectively) and α-pinene (14%-28 % and 12%-18% respectively). The remaining sample was almost devoid of 1, 8-cineole (1% in oil and 0% in extract) with higher concentrations of α-pinene (22% and 39%), linalool (25% and 18%) and (+)-(1S, 5R)-myrtenol (20% and 12% respectively). The compositional variation within the species indicated that a breeding project could identify and use the best composition for commercial development.

A research project was undertaken to utilize Ultrasonic Consolidation (UC) and Direct-write (DW) to fabricate an advanced miniature synthetic aperture radar (SAR) antenna. This research necessitated fundamental research relating to the processes of UC and DW. The design of an enclosure for the SAR antenna was desired that could minimize the losses due to misalignment and other issues while decreasing the mass compared to current systems. In order to develop such a design there were numerous tests that needed to be performed for both DW and UC. The results from the testing as well as the general design of the SAR antenna formulate a base for design guidelines to be built upon in the future. The successful fabrication of a prototype SAR enclosure demonstrates the usefulness of UC and DW as well as the validity of the design parameters determined through testing.