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.

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.

Ultra-high throughput functional enrichment of large monoamine oxidase (MAO-N) libraries by fluorescence activated cell sorting.

Directed evolution enables the improvement and optimisation of enzymes for particular applications and is a valuable tool for biotechnology and synthetic biology. However, studies are often limited in their scope by the inability to screen very large numbers of variants to identify improved enzymes. One class of enzyme for which a universal, operationally simple ultra-high throughput (>106 variants per day) assay is not available is flavin adenine dinucleotide (FAD) dependent oxidases. The current high throughput assay involves a visual, colourimetric, colony-based screen, however this is not suitable for very large libraries and does not enable quantification of the relative fitness of variants. To address this, we describe an optimised method for the sensitive detection of oxidase activity within single Escherichia coli (E. coli) cells, using the monoamine oxidase from Aspergillus niger, MAO-N, as a model system. In contrast to other methods for the screening of oxidase activity in vivo, this method does not require cell surface expression, emulsion formation or the addition of an extracellular peroxidase. Furthermore, we show that fluorescence activated cell sorting (FACS) of large libraries derived from MAO-N under the assay conditions can enrich the library in functional variants at much higher rates than via the colony-based method. We demonstrate its use for directed evolution by identifying a new mutant of MAO-N with improved activity towards a novel secondary amine substrate. This work demonstrates, for the first time, an ultra-high throughput screening methodology widely applicable for the directed evolution of FAD dependent oxidases in E. coli.

Bioinformatics for the synthetic biology of natural products: integrating across the Design-Build-Test cycle.

Covering: 2000 to 2016Progress in synthetic biology is enabled by powerful bioinformatics tools allowing the integration of the design, build and test stages of the biological engineering cycle. In this review we illustrate how this integration can be achieved, with a particular focus on natural products discovery and production. Bioinformatics tools for the DESIGN and BUILD stages include tools for the selection, synthesis, assembly and optimization of parts (enzymes and regulatory elements), devices (pathways) and systems (chassis). TEST tools include those for screening, identification and quantification of metabolites for rapid prototyping. The main advantages and limitations of these tools as well as their interoperability capabilities are highlighted.

SYNBIOCHEM-a SynBio foundry for the biosynthesis and sustainable production of fine and speciality chemicals.

The Manchester Synthetic Biology Research Centre (SYNBIOCHEM) is a foundry for the biosynthesis and sustainable production of fine and speciality chemicals. The Centre's integrated technology platforms provide a unique capability to facilitate predictable engineering of microbial bio-factories for chemicals production. An overview of these capabilities is described.

GeneGenie: optimized oligomer design for directed evolution.

GeneGenie, a new online tool available at http://www.gene-genie.org, is introduced to support the design and self-assembly of synthetic genes and constructs. GeneGenie allows for the design of oligonucleotide cohorts encoding the gene sequence optimized for expression in any suitable host through an intuitive, easy-to-use web interface. The tool ensures consistent oligomer overlapping melting temperatures, minimizes the likelihood of misannealing, optimizes codon usage for expression in a selected host, allows for specification of forward and reverse cloning sequences (for downstream ligation) and also provides support for mutagenesis or directed evolution studies. Directed evolution studies are enabled through the construction of variant libraries via the optional specification of 'variant codons', containing mixtures of bases, at any position. For example, specifying the variant codon TNT (where N is any nucleotide) will generate an equimolar mixture of the codons TAT, TCT, TGT and TTT at that position, encoding a mixture of the amino acids Tyr, Ser, Cys and Phe. This facility is demonstrated through the use of GeneGenie to develop and synthesize a library of enhanced green fluorescent protein variants.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently.

The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the 'search space' of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (Kd) and catalytic (kcat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving kcat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the 'best' amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole, simultaneously, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape navigation, informed by sequence-activity relationships and coupled to the emerging methods of synthetic biology, offers scope for the development of novel biocatalysts that are both highly active and robust.

High quality de novo genome assembly of the non-conventional yeast Kazachstania bulderi describes a potential low pH production host for biorefineries.

Kazachstania bulderi is a non-conventional yeast species able to grow efficiently on glucose and δ-gluconolactone at low pH. These unique traits make K. bulderi an ideal candidate for use in sustainable biotechnology processes including low pH fermentations and the production of green chemicals including organic acids. To accelerate strain development with this species, detailed information of its genetics is needed. Here, by employing long read sequencing we report a high-quality phased genome assembly for three strains of K. bulderi species, including the type strain. The sequences were assembled into 12 chromosomes with a total length of 14 Mb, and the genome was fully annotated at structural and functional levels, including allelic and structural variants, ribosomal array and mating type locus. This high-quality reference genome provides a resource to advance our fundamental knowledge of biotechnologically relevant non-conventional yeasts and to support the development of genetic tools for manipulating such strains towards their use as production hosts in biotechnological processes.

Telomere-to-telomere genome sequence of the model mould pathogen Aspergillus fumigatus.

The pathogenic fungus Aspergillus fumigatus is a major etiological agent of fungal invasive and chronic diseases affecting tens of millions of individuals worldwide. Draft genome sequences of two clinical isolates (Af293 and A1163) are commonly used as reference genomes for analyses of clinical and environmental strains. However, the reference sequences lack coverage of centromeres, an accurate sequence for ribosomal repeats, and a comprehensive annotation of chromosomal rearrangements such as translocations and inversions. Here, we used PacBio Single Molecule Real-Time (SMRT), Oxford Nanopore and Illumina HiSeq sequencing for de novo genome assembly and polishing of two laboratory reference strains of A. fumigatus, CEA10 (parental isolate of A1163) and its descendant A1160. We generated full length chromosome assemblies and a comprehensive telomere-to-telomere coverage for CEA10 and near complete assembly of A1160 including ribosomal repeats and the sequences of centromeres, which we discovered to be composed of long transposon elements. We envision these high-quality reference genomes will become fundamental resources to study A. fumigatus biology, pathogenicity and virulence, and to discover more effective treatments against diseases caused by this fungus.

GeneORator: An Efficient Method for the Systematic Mutagenesis of Entire Genes.

Directed evolution is a powerful tool for the rapid improvement of a target protein toward a desired fitness criteria, such as activity, specificity, or stability. In order to achieve these desired improvements, it is often beneficial to subject the entirety of the protein to mutagenesis. However, the creation of such libraries by targeted methods (i.e. site-directed mutagenesis) can be a laborious and costly task. Here we outline the GeneORator method, which uses Boolean "OR" logic to introduce specific codon mutations at multiple loci in a single reaction, thereby greatly reducing the experimental workload. The method describes library synthesis using asymmetric PCR, in which mutagenic primers are designed to create OR-type mutations at multiple sites of variation in a two-step protocol. As an example, we show how this can be utilized for controlled and economical mutagenesis of every amino acid codon in a gene.

Baseline proteomics characterisation of the emerging host biomanufacturing organism Halomonas bluephagenesis.

Despite its greener credentials, biomanufacturing remains financially uncompetitive compared with the higher carbon emitting, hydrocarbon-based chemical industry. Replacing traditional chassis such as E. coli with novel robust organisms, are a route to cost reduction for biomanufacturing. Extremophile bacteria such as the halophilic Halomonas bluephagenesis TD01 exemplify this potential by thriving in environments inherently inimical to other organisms, so reducing sterilisation costs. Novel chassis are inevitably less well annotated than established organisms. Rapid characterisation along with community data sharing will facilitate adoption of such organisms for biomanufacturing. The data record comprises a newly sequenced genome for the organism and evidence via LC-MS based proteomics for expression of 1160 proteins (30% of the proteome) including baseline quantification of 1063 proteins (27% of the proteome), and a spectral library enabling re-use for targeted LC-MS proteomics assays. Protein data are annotated with KEGG Orthology, enabling rapid matching of quantitative data to pathways of interest to biomanufacturing.

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.

The Epstein-Barr virus G-protein-coupled receptor contributes to immune evasion by targeting MHC class I molecules for degradation.

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8(+) T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 gamma(1)-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV gamma(2)-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8(+) T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed.

The evolving art of creating genetic diversity: From directed evolution to synthetic biology.

The ability to engineer biological systems, whether to introduce novel functionality or improved performance, is a cornerstone of biotechnology and synthetic biology. Typically, this requires the generation of genetic diversity to explore variations in phenotype, a process that can be performed at many levels, from single molecule targets (i.e., in directed evolution of enzymes) to whole organisms (e.g., in chassis engineering). Recent advances in DNA synthesis technology and automation have enhanced our ability to create variant libraries with greater control and throughput. This review highlights the latest developments in approaches to create such a hierarchy of diversity from the enzyme level to entire pathways in vitro, with a focus on the creation of combinatorial libraries that are required to navigate a target's vast design space successfully to uncover significant improvements in function.

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.

Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes.

BACKGROUND: Transcription factor-based biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcription factors (aTFs). Therefore, altering the ligand specificity of aTFs towards the detection of new effectors is an important goal. RESULTS: Here, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its ligand specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Ligand recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance. CONCLUSIONS: This is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its ligand specificity to generate a biosensor for aromatic aldehydes.

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.

Experiment and Simulation Reveal How Mutations in Functional Plasticity Regions Guide Plant Monoterpene Synthase Product Outcome.

Monoterpenes (C10 isoprenoids) are a structurally diverse group of natural compounds that are attractive to industry as flavours and fragrances. Monoterpenes are produced from a single linear substrate, geranyl diphosphate, by a group of enzymes called the monoterpene cyclases/synthases (mTC/Ss) that catalyse high-energy cyclisation reactions involving unstable carbocation intermediates. Efforts towards producing monoterpenes via biocatalysis or metabolic engineering often result in the formation of multiple products due to the nature of the highly branched reaction mechanism of mTC/Ss. Rational engineering of mTC/Ss is hampered by the lack of correlation between the active site sequence and cyclisation type. We used available mutagenesis data to show that amino acids involved in product outcome are clustered and spatially conserved within the mTC/S family. Consensus sequences for three such plasticity regions were introduced in different mTC/S with increasingly complex cyclisation cascades, including the model enzyme limonene synthase (LimS). In all three mTC/S studied, mutations in the first two regions mostly give rise to products that result from premature quenching of the linalyl or α-terpinyl cations, suggesting that both plasticity regions are involved in the formation and stabilisation of cations early in the reaction cascade. A LimS variant with mutations in the second region (S454G, C457V, M458I), produced mainly more complex bicyclic products. QM/MM MD simulations reveal that the second cyclisation is not due to compression of the C2-C7 distance in the α-terpinyl cation, but is the result of an increased distance between C8 of the α-terpinyl cation and two putative bases (W324, H579) located on the other side of the active site, preventing early termination by deprotonation. Such insights into the impact of mutations can only be obtained using integrated experimental and computational approaches, and will aid the design of altered mTC/S activities towards clean monoterpenoid products.

GeneORator: An Effective Strategy for Navigating Protein Sequence Space More Efficiently through Boolean OR-Type DNA Libraries.

Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional site-directed methods implicitly utilize the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of genetic variants as the number of mutations increases. We introduce GeneORator, a novel strategy for creating DNA libraries based on the Boolean logical OR operator. Here, a single library is divided into many subsets, each containing different combinations of the desired mutations. Consequently, the effect of adding more mutations on the number of genetic combinations is additive (Boolean OR logic) and not exponential (AND logic). We demonstrate this strategy with large-scale mutagenesis studies, using monoamine oxidase-N ( Aspergillus niger) as the exemplar target. First, we mutated every residue in the secondary structure-containing regions (276 out of a total 495 amino acids) to screen for improvements in kcat. Second, combinatorial OR-type libraries permitted screening of diverse mutation combinations in the enzyme active site to detect activity toward novel substrates. In both examples, OR-type libraries effectively reduced the number of variants searched up to 1010-fold, dramatically reducing the screening effort required to discover variants with improved and/or novel activity. Importantly, this approach enables the screening of a greater diversity of mutation combinations, accessing a larger area of a protein's sequence space. OR-type libraries can be applied to any biological engineering objective requiring DNA mutagenesis, and the approach has wide ranging applications in, for example, enzyme engineering, antibody engineering, and synthetic biology.

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.