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.

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.

How a 10-epi-Cubebol Synthase Avoids Premature Reaction Quenching to Form a Tricyclic Product at High Purity.

Terpenes are the largest class of natural products and are attractive targets in the fuel, fragrance, pharmaceutical, and flavor industries. Harvesting terpenes from natural sources is environmentally intensive and often gives low yields and purities, requiring further downstream processing. Engineered terpene synthases (TSs) offer a solution to these problems, but the low sequence identity and high promiscuity among TSs are major challenges for targeted engineering. Rational design of TSs requires identification of key structural and chemical motifs that steer product outcomes. Producing the sesquiterpenoid 10-epi-cubebol from farnesyl pyrophosphate (FPP) requires many steps and some of Nature's most difficult chemistry. 10-epi-Cubebol synthase from Sorangium cellulosum (ScCubS) guides a highly reactive carbocationic substrate through this pathway, preventing early quenching and ensuring correct stereochemistry at every stage. The cyclizations carried out by ScCubS potentially represent significant evolutionary expansions in the chemical space accessible by TSs. Here, we present the high-resolution crystal structure of ScCubS in complex with both a trinuclear magnesium cluster and pyrophosphate. Computational modeling, experiment, and bioinformatic analysis identified residues important in steering the reaction chemistry. We show that S206 is crucial in 10-epi-cubebol synthesis by enlisting the nearby F211 to shape the active site contour and prevent the formation of early escape cadalane products. We also show that N327 and F104 control the distribution between several early-stage cations and whether the final product is derived from the germacrane, cadalane, or cubebane hydrocarbon scaffold. Using these insights, we reengineered ScCubS so that its main product was germacradien-4-ol, which derives from the germacrane, rather than the cubebane, scaffold. Our work emphasizes that mechanistic understanding of cation stabilization in TSs can be used to guide catalytic outcomes.

Biotechnological application of Streptomyces for the production of clinical drugs and other bioactive molecules.

Streptomyces is one of the most relevant genera in biotechnology, and its rich secondary metabolism is responsible for the biosynthesis of a plethora of bioactive compounds, including several clinically relevant drugs. The use of Streptomyces species for the manufacture of natural products has been established for more than half a century; however, the tremendous advances observed in recent years in genetic engineering and molecular biology have revolutionised the optimisation of Streptomyces as cell factories and drastically expanded the biotechnological potential of these bacteria. Here, we illustrate the most exciting advances reported in the past few years, with a particular focus on the approaches significantly improving the biotechnological capacity of Streptomyces to produce clinical drugs and other valuable secondary metabolites.

Harnessing intercellular signals to engineer the soil microbiome.

Covering: Focus on 2015 to 2020Plant and soil microbiomes consist of diverse communities of organisms from across kingdoms and can profoundly affect plant growth and health. Natural product-based intercellular signals govern important interactions between microbiome members that ultimately regulate their beneficial or harmful impacts on the plant. Exploiting these evolved signalling circuits to engineer microbiomes towards beneficial interactions with crops is an attractive goal. There are few reports thus far of engineering the intercellular signalling of microbiomes, but this article argues that it represents a tremendous opportunity for advancing the field of microbiome engineering. This could be achieved through the selection of synergistic consortia in combination with genetic engineering of signal pathways to realise an optimised microbiome.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Design and fabrication of recombinant reflectin-based multilayer reflectors: bio-design engineering and photoisomerism induced wavelength modulation.

The remarkable camouflage capabilities of cephalopods have inspired many to develop dynamic optical materials which exploit certain design principles and/or material properties from cephalopod dermal cells. Here, the angle-dependent optical properties of various single-layer reflectin thin-films on Si wafers are characterized within the UV-Vis-NIR regions. Following this, initial efforts to design, fabricate, and optically characterize a bio-inspired reflectin-based multilayer reflector is described, which was found to conserve the optical properties of single layer films but exhibit reduced angle-dependent visible reflectivity. Finally, we report the integration of phytochrome visible light-induced isomerism into reflectin-based films, which was found to subtly modulate reflectin thin-film reflectivity.

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.

Multi-omics Study of Planobispora rosea, Producer of the Thiopeptide Antibiotic GE2270A.

Planobispora rosea is the natural producer of the potent thiopeptide antibiotic GE2270A. Here, we present the results of a metabolomics and transcriptomics analysis of P. rosea during production of GE2270A. The data generated provides useful insights into the biology of this genetically intractable bacterium. We characterize the details of the shutdown of protein biosynthesis and the respiratory chain associated with the end of the exponential growth phase. We also provide the first description of the phosphate regulon in P. rosea. Based on the transcriptomics data, we show that both phosphate and iron are limiting P. rosea growth in our experimental conditions. Additionally, we identified and validated a new biosynthetic gene cluster associated with the production of the siderophores benarthin and dibenarthin in P. rosea. Together, the metabolomics and transcriptomics data are used to inform and refine the very first genome-scale metabolic model for P. rosea, which will be a valuable framework for the interpretation of future studies of the biology of this interesting but poorly characterized species. IMPORTANCE Planobispora rosea is a genetically intractable bacterium used for the production of GE2270A on an industrial scale. GE2270A is a potent thiopeptide antibiotic currently used as a precursor for the synthesis of two compounds under clinical studies for the treatment of Clostridium difficile infection and acne. Here, we present the very first systematic multi-omics investigation of this important bacterium, which provides a much-needed detailed picture of the dynamics of metabolism of P. rosea while producing GE2270A.

Synthetic biology approaches to actinomycete strain improvement.

Their biochemical versatility and biotechnological importance make actinomycete bacteria attractive targets for ambitious genetic engineering using the toolkit of synthetic biology. But their complex biology also poses unique challenges. This mini review discusses some of the recent advances in synthetic biology approaches from an actinomycete perspective and presents examples of their application to the rational improvement of industrially relevant strains.

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.

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.

Investigation of the effects of actinorhodin biosynthetic gene cluster expression and a rpoB point mutation on the metabolome of Streptomyces coelicolor M1146.

The previously reported Streptomyces coelicolor M1146 is commonly used as a host strain for engineering of secondary metabolite production. In this study, absolute quantification of intracellular and extracellular metabolites of M1146 was performed in mid-log phase and stationary phase to observe major metabolites and the changes that occurred during growth. Decreased levels of central carbon metabolites (glycolysis, TCA cycle, and pentose phosphate pathway) and increased levels of amino acids were observed in stationary phase compared to mid-log phase. Furthermore, comparative metabolome analyses of M1146 upon expression of the actinorhodin biosynthetic gene cluster (M1146+ACT), a point mutation on the rpoB gene encoding RNA polymerase beta-subunit (M1152), and both expression of actinorhodin biosynthetic gene cluster and a rpoB point mutation (M1152+ACT) were performed. M1146+ACT showed higher levels of important cofactors, such as ATP, NADPH, and FMN while M1152 led to higher levels of intracellular S-adenosyl-methionine, acyl-CoAs, and extracellular nucleosides compared to M1146. M1152+ACT exhibited the highest levels of actinorhodin with elevated bases, nucleosides, and nucleotides, such as intracellular PRPP (phosphoribosyl phosphate), ATP, along with extracellular inosine, uridine, and guanine compared to the other three strains, which were considered to be combined effects of actinorhodin gene cluster expression and a rpoB point mutation. Metabolites analysis by means of absolute quantification demonstrated changes in precursors of secondary metabolites before and after phosphate depletion in M1146. Comparative metabolome analysis provided further insights into the effects of actinorhodin gene cluster expression along with a rpoB point mutation on the metabolome of S. coelicolor.

Towards the sustainable discovery and development of new antibiotics.

An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.

Host Systems for the Production of Recombinant Spider Silk.

Spider silk is renowned for its impressive mechanical properties. It is one of the strongest known biomaterials, possessing mechanical properties that outmatch both steel and Kevlar. However, the farming of spiders for their silk is unfeasible. Consequently, production of recombinant spider silk proteins (spidroins) in more amenable hosts is an exciting field of research. For large-scale production to be viable, a heterologous silk production system that is both highly efficient and cost effective is essential. Genes encoding recombinant spidroin have been expressed in bacterial, yeast, insect, and mammalian cells, in addition to many other platforms. This review discusses the recent advances in exploiting an increasingly diverse range of host platforms in the heterologous production of recombinant spidroins.

In silico design and automated learning to boost next-generation smart biomanufacturing.

The increasing demand for bio-based compounds produced from waste or sustainable sources is driving biofoundries to deliver a new generation of prototyping biomanufacturing platforms. Integration and automation of the design, build, test and learn (DBTL) steps in centers like SYNBIOCHEM in Manchester and across the globe (Global Biofoundries Alliance) are helping to reduce the delivery time from initial strain screening and prototyping towards industrial production. Notably, a portfolio of producer strains for a suite of material monomers was recently developed, some approaching industrial titers, in a tour de force by the Manchester Centre that was achieved in less than 90 days. New in silico design tools are providing significant contributions to the front end of the DBTL pipelines. At the same time, the far-reaching initiatives of modern biofoundries are generating a large amount of high-dimensional data and knowledge that can be integrated through automated learning to expedite the DBTL cycle. In this Perspective, the new design tools and the role of the learning component as an enabling technology for the next generation of automated biofoundries are discussed. Future biofoundries will operate under completely automated DBTL cycles driven by in silico optimal experimental planning, full biomanufacturing devices connectivity, virtualization platforms and cloud-based design. The automated generation of robotic build worklists and the integration of machine-learning algorithms will collectively allow high levels of adaptability and rapid design changes toward fully automated smart biomanufacturing.

Multi-Omics Analysis of the Effect of cAMP on Actinorhodin Production in Streptomyces coelicolor.

Cyclic adenosine monophosphate (cAMP) has been known to play an important role in regulating morphological development and antibiotic production in Streptomyces coelicolor. However, the functional connection between cAMP levels and antibiotic production and the mechanism by which cAMP regulates antibiotic production remain unclear. In this study, metabolomics- and transcriptomics-based multi-omics analysis was applied to S. coelicolor strains that either produce the secondary metabolite actinorhodin (Act) or lack most secondary metabolite biosynthesis pathways including Act. Comparative multi-omics analysis of the two strains revealed that intracellular and extracellular cAMP abundance was strongly correlated with actinorhodin production. Notably, supplementation of cAMP improved cell growth and antibiotic production. Further multi-omics analysis of cAMP-supplemented S. coelicolor cultures showed an increase of guanine and the expression level of purine metabolism genes. Based on this phenomenon, supplementation with 7-methylguanine, a competitive inhibitor of reactions utilizing guanine, with or without additional cAMP supplementation, was performed. This experiment revealed that the reactions inhibited by 7-methylguanine are mediating the positive effect on growth and antibiotic production, which may occur downstream of cAMP supplementation.