SpeedyGenesXL: an Automated, High-Throughput Platform for the Preparation of Bespoke Ultralarge Variant Libraries for Directed Evolution.

Directed evolution of proteins is a highly effective strategy for tailoring biocatalysts to a particular application, and is capable of engineering improvements such as kcat, thermostability and organic solvent tolerance. It is recognized that large and systematic libraries are required to navigate a protein's vast and rugged sequence landscape effectively, yet their preparation is nontrivial and commercial libraries are extremely costly. To address this, we have developed SpeedyGenesXL, an automated, high-throughput platform for the production of wild-type genes, Boolean OR, combinatorial, or combinatorial-OR-type libraries based on the SpeedyGenes methodology. Together this offers a flexible platform for library synthesis, capable of generating many different bespoke, diverse libraries simultaneously.

Prototyping of microbial chassis for the biomanufacturing of high-value chemical targets.

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.

A plasmid toolset for CRISPR-mediated genome editing and CRISPRi gene regulation in Escherichia coli.

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.

Engineering Escherichia coli towards de novo production of gatekeeper (2S)-flavanones: naringenin, pinocembrin, eriodictyol and homoeriodictyol.

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.

Rapid prototyping of microbial production strains for the biomanufacture of potential materials monomers.

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.

Ribosomal Protein L11 Selectively Stabilizes a Tertiary Structure of the GTPase Center rRNA Domain.

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.

An automated pipeline for the screening of diverse monoterpene synthase libraries.

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.

Design and evolution of an enzyme with a non-canonical organocatalytic mechanism.

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1-4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6-10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

SelProm: A Queryable and Predictive Expression Vector Selection Tool for Escherichia coli.

The rapid prototyping and optimization of plasmid-based recombinant gene expression is one of the key steps in the development of bioengineered bacterial systems. Often, multiple genes or gene modules need to be coexpressed, and for this purpose compatible, inducible plasmid systems have been developed. However, inducible expression systems are not favored in industrial processes, due to their prohibitive cost, and consequently the conversion to constitutive expression systems is often desired. Here we present a set of constitutive-expression plasmids for this purpose, which were benchmarked using fluorescent reporter genes. To further facilitate the conversion between inducible and constitutive expression systems, we developed SelProm, a design tool that serves as a parts repository of plasmid expression strength and predicts portability rules between constitutive and inducible plasmids through model comparison and machine learning. The SelProm tool is freely available at http://selprom.synbiochem.co.uk .

Highly multiplexed, fast and accurate nanopore sequencing for verification of synthetic DNA constructs and sequence libraries.

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.

Machine Learning of Designed Translational Control Allows Predictive Pathway Optimization in Escherichia coli.

The field of synthetic biology aims to make the design of biological systems predictable, shrinking the huge design space to practical numbers for testing. When designing microbial cell factories, most optimization efforts have focused on enzyme and strain selection/engineering, pathway regulation, and process development. In silico tools for the predictive design of bacterial ribosome binding sites (RBSs) and RBS libraries now allow translational tuning of biochemical pathways; however, methods for predicting optimal RBS combinations in multigene pathways are desirable. Here we present the implementation of machine learning algorithms to model the RBS sequence-phenotype relationship from representative subsets of large combinatorial RBS libraries allowing the accurate prediction of optimal high-producers. Applied to a recombinant monoterpenoid production pathway in Escherichia coli, our approach was able to boost production titers by over 60% when screening under 3% of a library. To facilitate library screening, a multiwell plate fermentation procedure was developed, allowing increased screening throughput with sufficient resolution to discriminate between high and low producers. High producers from one library did not translate during scale-up, but the reduced screening requirements allowed rapid rescreening at the larger scale. This methodology is potentially compatible with any biochemical pathway and provides a powerful tool toward predictive design of bacterial production chassis.

An automated Design-Build-Test-Learn pipeline for enhanced microbial production of fine chemicals.

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 (2S)-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.

Multifragment DNA Assembly of Biochemical Pathways via Automated Ligase Cycling Reaction.

The microbial production of commodity, fine, and specialty chemicals is a driving force in biotechnology. An essential requirement is to introduce biosynthetic pathways to the target compound(s) into chassis organisms. First suitable enzymes must be selected and characterized, and then genetic pathways must be designed and assembled into suitable expression vectors. The design of these pathways is crucial for balancing the pathway for efficient in vivo activity. This can be achieved through optimization of the pathway regulation by altering transcription and translation rates. The possible permutations of a multigene pathway create a vast design space which is intractable to explore using traditional time-consuming and laborious pathway assembly methods. The advent of multifragment DNA assembly technologies has enabled simultaneous, multiplexed pathway construction allowing an increased capability to sample the design space. Furthermore, the implementation of laboratory automation allows error-reduced, high-throughput (HTP) construction of pathways. In this chapter, we present a workflow that combines automated in silico design of DNA parts followed by pathway assembly using the ligase cycling reaction on robotics platforms, to allow multiplexed assembly of plasmid-borne gene pathways with high efficiency. Details and considerations in designing DNA parts for expression bacterial chassis are discussed followed by laboratory protocols for HTP pathway assembly and screening using robotics platforms. This workflow is employed in the SYNBIOCHEM Synthetic Biology Research Center, providing the capability to assemble over 96 plasmids simultaneously, with over 40% of clones from each assembly harboring the correctly assembled plasmids. This workflow is easy to modify for use in other laboratories and will help to accelerate synthetic biology projects with diverse applications.

PartsGenie: an integrated tool for optimizing and sharing synthetic biology parts.

Motivation: 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. PartsGenie is introduced to facilitate the computational design of such synthetic biology parts, bridging the gap between optimization tools for the design of novel parts, the representation of such parts in community-developed data standards such as Synthetic Biology Open Language, and their sharing in journal-recommended data repositories. Consisting of a drag-and-drop web interface, a number of DNA optimization algorithms, and an interface to the well-used data repository JBEI ICE, PartsGenie facilitates the design, optimization and dissemination of reusable synthetic biology parts through an integrated application. Availability and implementation: PartsGenie is freely available at https://parts.synbiochem.co.uk.

Structure and Biocatalytic Scope of Coclaurine N-Methyltransferase.

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.

Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.

The realization of a synthetic biology approach to microbial (1R,2S,5R)-(-)-menthol (1) production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the Mentha piperita biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida can act as an IPGI, producing (R)-(+)-pulegone ((R)-2) from (+)-cis-isopulegone (3). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of 3 binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream Mentha enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate 1 from 3. This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of 1 in microorganisms.

Zymophore identification enables the discovery of novel phenylalanine ammonia lyase enzymes.

The suite of biological catalysts found in Nature has the potential to contribute immensely to scientific advancements, ranging from industrial biotechnology to innovations in bioenergy and medical intervention. The endeavour to obtain a catalyst of choice is, however, wrought with challenges. Herein we report the design of a structure-based annotation system for the identification of functionally similar enzymes from diverse sequence backgrounds. Focusing on an enzymatic activity with demonstrated synthetic and therapeutic relevance, five new phenylalanine ammonia lyase (PAL) enzymes were discovered and characterised with respect to their potential applications. The variation and novelty of various desirable traits seen in these previously uncharacterised enzymes demonstrates the importance of effective sequence annotation in unlocking the potential diversity that Nature provides in the search for tailored biological tools. This new method has commercial relevance as a strategy for assaying the 'evolvability' of certain enzyme features, thus streamlining and informing protein engineering efforts.

Adenylation Activity of Carboxylic Acid Reductases Enables the Synthesis of Amides.

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.

biochem4j: Integrated and extensible biochemical knowledge through graph databases.

Biologists and biochemists have at their disposal a number of excellent, publicly available data resources such as UniProt, KEGG, and NCBI Taxonomy, which catalogue biological entities. Despite the usefulness of these resources, they remain fundamentally unconnected. While links may appear between entries across these databases, users are typically only able to follow such links by manual browsing or through specialised workflows. Although many of the resources provide web-service interfaces for computational access, performing federated queries across databases remains a non-trivial but essential activity in interdisciplinary systems and synthetic biology programmes. What is needed are integrated repositories to catalogue both biological entities and-crucially-the relationships between them. Such a resource should be extensible, such that newly discovered relationships-for example, those between novel, synthetic enzymes and non-natural products-can be added over time. With the introduction of graph databases, the barrier to the rapid generation, extension and querying of such a resource has been lowered considerably. With a particular focus on metabolic engineering as an illustrative application domain, biochem4j, freely available at http://biochem4j.org, is introduced to provide an integrated, queryable database that warehouses chemical, reaction, enzyme and taxonomic data from a range of reliable resources. The biochem4j framework establishes a starting point for the flexible integration and exploitation of an ever-wider range of biological data sources, from public databases to laboratory-specific experimental datasets, for the benefit of systems biologists, biosystems engineers and the wider community of molecular biologists and biological chemists.

Structures of carboxylic acid reductase reveal domain dynamics underlying catalysis.

Carboxylic acid reductase (CAR) catalyzes the ATP- and NADPH-dependent reduction of carboxylic acids to the corresponding aldehydes. The enzyme is related to the nonribosomal peptide synthetases, consisting of an adenylation domain fused via a peptidyl carrier protein (PCP) to a reductase termination domain. Crystal structures of the CAR adenylation-PCP didomain demonstrate that large-scale domain motions occur between the adenylation and thiolation states. Crystal structures of the PCP-reductase didomain reveal that phosphopantetheine binding alters the orientation of a key Asp, resulting in a productive orientation of the bound nicotinamide. This ensures that further reduction of the aldehyde product does not occur. Combining crystallography with small-angle X-ray scattering (SAXS), we propose that molecular interactions between initiation and termination domains are limited to competing PCP docking sites. This theory is supported by the fact that (R)-pantetheine can support CAR activity for mixtures of the isolated domains. Our model suggests directions for further development of CAR as a biocatalyst.