Bioproduction of methylated phenylpropenes and isoeugenol in Escherichia coli.

Phenylpropenes are a class of natural products that are synthesised by a vast range of plant species and hold considerable promise in the flavour and fragrance industries. Many in vitro studies have been carried out to elucidate and characterise the enzymes responsible for the production of these volatile compounds. However, there is a scarcity of studies demonstrating the in vivo production of phenylpropenes in microbial cell factories. In this study, we engineered Escherichia coli to produce methylchavicol, methyleugenol and isoeugenol from their respective phenylacrylic acid precursors. We achieved this by extending and modifying a previously optimised heterologous pathway for the biosynthesis of chavicol and eugenol. We explored the potential of six S-adenosyl l-methionine (SAM)-dependent O-methyltransferases to produce methylchavicol and methyleugenol from chavicol and eugenol, respectively. Additionally, we examined two isoeugenol synthases for the production of isoeugenol from coniferyl acetate. The best-performing strains in this study were able to achieve titres of 13 mg L-1 methylchavicol, 59 mg L-1 methyleugenol and 361 mg L-1 isoeugenol after feeding with their appropriate phenylacrylic acid substrates. We were able to further increase the methyleugenol titre to 117 mg L-1 by supplementation with methionine to facilitate SAM recycling. Moreover, we report the biosynthesis of methylchavicol and methyleugenol from l-tyrosine through pathways involving six and eight enzymatic steps, respectively.

Photocobilins integrate B12 and bilin photochemistry for enzyme control.

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B12) can act as a light-sensing chromophore heralded a new field of B12-photobiology. Although microbial genome analysis indicates that photoactive B12-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking. Here we identify and characterise a sub-family of multi-centre photoreceptors, termed photocobilins, that use B12 and biliverdin (BV) to sense light across the visible spectrum. Crystal structures reveal close juxtaposition of the B12 and BV chromophores, an arrangement that facilitates optical coupling. Light-triggered conversion of the B12 affects quaternary structure, in turn leading to light-activation of associated enzyme domains. The apparent widespread nature of photocobilins implies involvement in light regulation of a wider array of biochemical processes, and thus expands the scope for B12 photobiology. Their characterisation provides inspiration for the design of broad-spectrum optogenetic tools and next generation bio-photocatalysts.

Carboxylic acid reductase-dependent biosynthesis of eugenol and related allylphenols.

BACKGROUND: (Hydroxy)cinnamyl alcohols and allylphenols, including coniferyl alcohol and eugenol, are naturally occurring aromatic compounds widely utilised in pharmaceuticals, flavours, and fragrances. Traditionally, the heterologous biosynthesis of (hydroxy)cinnamyl alcohols from (hydroxy)cinnamic acids involved CoA-dependent activation of the substrate. However, a recently explored alternative pathway involving carboxylic acid reductase (CAR) has proven efficient in generating the (hydroxy)cinnamyl aldehyde intermediate without the need for CoA activation. In this study, we investigated the application of the CAR pathway for whole-cell bioconversion of a range of (hydroxy)cinnamic acids into their corresponding (hydroxy)cinnamyl alcohols. Furthermore, we sought to extend the pathway to enable the production of a variety of allylphenols and allylbenzene. RESULTS: By screening the activity of several heterologously expressed enzymes in crude cell lysates, we identified the combination of Segniliparus rugosus CAR (SrCAR) and Medicago sativa cinnamyl alcohol dehydrogenase (MsCAD2) as the most efficient enzymatic cascade for the two-step reduction of ferulic acid to coniferyl alcohol. To optimise the whole-cell bioconversion in Escherichia coli, we implemented a combinatorial approach to balance the gene expression levels of SrCAR and MsCAD2. This optimisation resulted in a coniferyl alcohol yield of almost 100%. Furthermore, we extended the pathway by incorporating coniferyl alcohol acyltransferase and eugenol synthase, which allowed for the production of eugenol with a titre of up to 1.61 mM (264 mg/L) from 3 mM ferulic acid. This improvement in titre surpasses previous achievements in the field employing a CoA-dependent coniferyl alcohol biosynthesis pathway. Our study not only demonstrated the successful utilisation of the CAR pathway for the biosynthesis of diverse (hydroxy)cinnamyl alcohols, such as p-coumaryl alcohol, caffeyl alcohol, cinnamyl alcohol, and sinapyl alcohol, from their corresponding (hydroxy)cinnamic acid precursors but also extended the pathway to produce allylphenols, including chavicol, hydroxychavicol, and methoxyeugenol. Notably, the microbial production of methoxyeugenol from sinapic acid represents a novel achievement. CONCLUSION: The combination of SrCAR and MsCAD2 enzymes offers an efficient enzymatic cascade for the production of a wide array of (hydroxy)cinnamyl alcohols and, ultimately, allylphenols from their respective (hydroxy)cinnamic acids. This expands the range of value-added molecules that can be generated using microbial cell factories and creates new possibilities for applications in industries such as pharmaceuticals, flavours, and fragrances. These findings underscore the versatility of the CAR pathway, emphasising its potential in various biotechnological applications.

Expanding flavone and flavonol production capabilities in Escherichia coli.

Flavones and flavonols are important classes of flavonoids with nutraceutical and pharmacological value, and their production by fermentation with recombinant microorganisms promises to be a scalable and economically favorable alternative to extraction from plant sources. Flavones and flavonols have been produced recombinantly in a number of microorganisms, with Saccharomyces cerevisiae typically being a preferred production host for these compounds due to higher yields and titers of precursor compounds, as well as generally improved ability to functionally express cytochrome P450 enzymes without requiring modification to improve their solubility. Recently, a rapid prototyping platform has been developed for high-value compounds in E. coli, and a number of gatekeeper (2S)-flavanones, from which flavones and flavonols can be derived, have been produced to high titers in E. coli using this platform. In this study, we extended these metabolic pathways using the previously reported platform to produce apigenin, chrysin, luteolin and kaempferol from the gatekeeper flavonoids naringenin, pinocembrin and eriodictyol by the expression of either type-I flavone synthases (FNS-I) or type-II flavone synthases (FNS-II) for flavone biosynthesis, and by the expression of flavanone 3-dioxygenases (F3H) and flavonol synthases (FLS) for the production of the flavonol kaempferol. In our best-performing strains, titers of apigenin and kaempferol reached 128 mg L-1 and 151 mg L-1 in 96-DeepWell plates in cultures supplemented with an additional 3 mM tyrosine, though titers for chrysin (6.8 mg L-1) from phenylalanine, and luteolin (5.0 mg L-1) from caffeic acid were considerably lower. In strains with upregulated tyrosine production, apigenin and kaempferol titers reached 80.2 mg L-1 and 42.4 mg L-1 respectively, without the further supplementation of tyrosine beyond the amount present in the rich medium. Notably, the highest apigenin, chrysin and luteolin titers were achieved with FNS-II enzymes, suggesting that cytochrome P450s can show competitive performance compared with non-cytochrome P450 enzymes in prokaryotes for the production of flavones.

Microbial production of the plant flavanone hesperetin from caffeic acid.

OBJECTIVE: Hesperetin is an important O-methylated flavonoid produced by citrus fruits and of potential pharmaceutical relevance. The microbial biosynthesis of hesperetin could be a viable alternative to plant extraction, as plant extracts often yield complex mixtures of different flavonoids making it challenging to isolate pure compounds. In this study, hesperetin was produced from caffeic acid in the microbial host Escherichia coli. We combined a previously optimised pathway for the biosynthesis of the intermediate flavanone eriodictyol with a combinatorial library of plasmids expressing three candidate flavonoid O-methyltransferases. Moreover, we endeavoured to improve the position specificity of CCoAOMT7, a flavonoid O-methyltransferase from Arabidopsis thaliana that has been demonstrated to O-methylate eriodictyol in both the para- and meta-position, thus leading to a mixture of hesperetin and homoeriodictyol. RESULTS: The best performing flavonoid O-methyltransferase in our screen was found to be CCoAOMT7, which could produce up to 14.6 mg/L hesperetin and 3.8 mg/L homoeriodictyol from 3 mM caffeic acid in E. coli 5-alpha. Using a platform for enzyme engineering that scans the mutational space of selected key positions, predicting their structures using homology modelling and inferring their potential catalytic improvement using docking simulations, we were able to identify a CCoAOMT7 mutant with a two-fold higher position specificity for hesperetin. The mutant's catalytic activity, however, was considerably diminished. Our findings suggest that hesperetin can be created from central carbon metabolism in E. coli following the introduction of a caffeic acid biosynthesis pathway.

Biosynthesis of cannabigerol and cannabigerolic acid: the gateways to further cannabinoid production.

Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. The native cannabinoid biosynthetic pathway of Cannabis sativa generates cannabigerolic acid (CBGA), the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analog of this compound, cannabigerol (CBG), represents an alternate gateway into the cannabinoid space as a substrate either to non-canonical cannabinoid synthase homologs or to synthetic chemical reactions. Herein, we describe the identification and repurposing of aromatic prenyltransferase (AtaPT), which when coupled with native enzymes of C. sativa can form an Escherichia coli production system for CBGA in cell lysates and CBG in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics toward CBGA production for subsequent use in a proof-of-concept lysate system. For the first time, we show a synthetic biology platform for CBG biosynthesis in E. coli cells by employing AtaPT under an optimized microbial system. Our results have therefore set the foundation for sustainable production of well-researched and rarer cannabinoids in an E. coli chassis. Graphical Abstract.

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.

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.

Structure of the Cannabis sativa olivetol-producing enzyme reveals cyclization plasticity in type III polyketide synthases.

In the native pathway to therapeutic cannabinoid biosynthesis in Cannabis sativa, the three-step production of a key intermediate, olivetolic acid, is catalysed by the enzymes tetraketide synthase (TKS; linear tetraketide intermediate production in two stages) and olivetolic acid cyclase (OAC; final C2 â†’ C7 aldol condensation). In the absence of OAC, a nonenzymatic C2 â†’ C7 decarboxylative aldol condensation of the tetraketide intermediate occurs forming olivetol. TKS is a type III polyketide synthase, and the question arises why it is unable to form olivetolic acid directly, but instead forms this unwanted side product. We determined the TKS, CoA complex structure, and performed structurally guided mutagenesis studies to identify potential residues responsible for cyclization pathway discrimination in type III polyketide synthases. Prior studies suggested an 'aldol switch' is necessary to allow linear tetraketide intermediate release prior to cyclization, thereby enabling subsequent olivetolic acid production by OAC. However, our studies do not support the presence of a universal or predictable 'aldol switch' consensus sequence. Instead, we propose the mode of ordered active site water activation between type III polyketide synthases catalysing different cyclization mechanisms is subtle and homologue-specific. Our work indicates that subtle structural variations between homologous enzymes can have a major mechanistic impact on the catalytic outcome. This highlights the importance of embedding high-resolution structural analysis of multiple enzyme homologues with classical site-directed mutagenesis studies when investigating highly similar enzymes with different mechanistic pathway outcomes. ENZYMES: TKS, EC 2.3.1.206; OAC, EC 4.4.1.26; chalcone synthase, EC 2.3.1.74; stilbene synthase, EC 2.3.1.95; 2-PS, EC 2.3.1.-. ACCESSION NUMBERS: The atomic coordinates and structure factors for the crystal structure of TKS have been deposited in the Protein Data Bank with accession number 6GW3.

Enzymatic Carboxylation of 2-Furoic Acid Yields 2,5-Furandicarboxylic Acid (FDCA).

The biological production of FDCA is of considerable value as a potential replacement for petrochemical-derived monomers such as terephthalate, used in polyethylene terephthalate (PET) plastics. HmfF belongs to an uncharacterized branch of the prenylated flavin (prFMN) dependent UbiD family of reversible (de)carboxylases and is proposed to convert 2,5-furandicarboxylic acid (FDCA) to furoic acid in vivo. We present a detailed characterization of HmfF and demonstrate that HmfF can catalyze furoic acid carboxylation at elevated CO2 levels in vitro. We report the crystal structure of a thermophilic HmfF from Pelotomaculum thermopropionicum, revealing that the active site located above the prFMN cofactor contains a furoic acid/FDCA binding site composed of residues H296-R304-R331 specific to the HmfF branch of UbiD enzymes. Variants of the latter are compromised in activity, while H296N alters the substrate preference to pyrrole compounds. Solution studies and crystal structure determination of an engineered dimeric form of the enzyme revealed an unexpected key role for a UbiD family wide conserved Leu residue in activity. The structural insights into substrate and cofactor binding provide a template for further exploitation of HmfF in the production of FDCA plastic precursors and improve our understanding of catalysis by members of the UbiD enzyme family.

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.

Mobilising ion mobility mass spectrometry for metabolomics.

Chromatography-based mass spectrometry approaches (xC-MS) are commonly used in untargeted metabolomics, providing retention time, m/z values and metabolite-specific fragments, all of which are used to identify and validate an unknown analyte. Ion mobility-mass spectrometry (IM-MS) is emerging as an enhancement to classic xC-MS strategies, by offering additional ion separation as well as collision cross section (CCS) determination. In order to apply such an approach to a metabolomics workflow, verified data from metabolite standards is necessary. In this work we present experimental DTCCSN2 values for a range of metabolites in positive and negative ionisation modes using drift tube-ion mobility-mass spectrometry (DT-IM-MS) with nitrogen as the buffer gas. The value of DTCCSN2 measurements for application in metabolite identification relies on a robust technique that acquires measurements of high reproducibility. We report that the CCS values found for 86% of metabolites measured in replicate have a relative standard deviation lower than 0.2%. Examples of metabolites with near identical mass are demonstrated to be separated by ion mobility with over 4% difference in DTCCSN2 values. We conclude that the integration of ion mobility into current LC-MS workflows can aid in small molecule identification for both targeted and untargeted metabolite screening.

Real-Time Screening of Biocatalysts in Live Bacterial Colonies.

Screening of bacterial colonies to identify new biocatalytic activities is a widely adopted tool in biotechnology, but is constrained by the requirements for colorimetric or tag-based detection methods. Herein we report a label-free screening platform for biotransformations in live colonies using desorption electrospray ionization coupled with ion mobility mass spectrometry imaging (DiBT-IMMS). The screening method is demonstrated for both ammonia lyases and P450 monooxygenases expressed within live bacterial colonies and is shown to enable multiplexing of enzyme variants and substrate libraries simultaneously.

A Comprehensive and Effective Mass Spectrometry-Based Screening Strategy for Discovery and Identification of New Brassinosteroids from Rice Tissues.

The exploration and identification of new brassinosteroid (BR) compounds is critical to improve the biosynthetic research of BRs and expand the chemodiversity of active BRs. However, traditional methods are labor-intensive, time-consuming, and less sensitive. Here, we present a facile screening strategy for discovering and identifying novel BRs from plant tissues based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). A total of 14 potential BRs were discovered from only 1 g of rice tissues and structurally elucidated by following a MS-based clue, acquired through multiple reaction monitoring (MRM) data-dependent enhanced product ion (EPI) scan, high resolution MS, and MS survey-dependent MS/MS. One of the 14 candidates was identified as 6-deoxo-28-homotyphasterol, a brand new BR compound that is reported for the first time in the BRs biosynthesis pathway. Detailed comparison with reference standards and quantitative level analysis in rice BR mutants confirmed the availability of the other candidates. This effective, yet simple method provides an efficient way to find more and more chemically new BR biosynthetic intermediates in plants, which is significant for complementing the biosynthesis and metabolism network of BRs. This strategy may also be used to discover unknown compounds of other plant hormone species as well as their key metabolites.

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.

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.

Rapid and sensitive monitoring of biocatalytic reactions using ion mobility mass spectrometry.

The combination of stable isotope labelling with direct infusion ion mobility mass spectrometry (IM-MS) enabled qualitative and quantitative monitoring of biocatalytic reactions with reduced analysis times, enhanced sensitivity and µL-level assay volumes. The new approach was demonstrated by applying to both lipase and monooxygenase enzymes, including multi-substrate screening.