Synthetic Biology of Natural Products Engineering: Recent Advances Across the Discover-Design-Build-Test-Learn Cycle.

Advances in genome engineering and associated technologies have reinvigorated natural products research. Here we highlight the latest developments in the field across the discover-design-build-test-learn cycle of bioengineering, from recent progress in computational tools for AI-supported genome mining, enzyme and pathway engineering, and compound identification to novel host systems and new techniques for improving production levels, and place these trends in the context of responsible research and innovation, emphasizing the importance of anticipatory analysis at the early stages of process development.

Bioproduction of methylated phenylpropenes and isoeugenol in .

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 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 production of phenylpropenes in microbial cell factories.

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.

Expanding flavone and flavonol production capabilities in .

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 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 , and a number of gatekeeper (2)-flavanones, from which flavones and flavonols can be derived, have been produced to high titers in using this platform.

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 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.

How a 10--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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 by the Manchester Centre that was achieved in less than 90 days.

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

Cyclic adenosine monophosphate (cAMP) has been known to play an important role in regulating morphological development and antibiotic production in . 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 strains that either produce the secondary metabolite actinorhodin (Act) or lack most secondary metabolite biosynthesis pathways including Act.

Engineering towards production of gatekeeper (2)-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 (2)-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.

Production of the Fragrance Geraniol in Peroxisomes of a Product-Tolerant Baker's Yeast.

Monoterpenoids, such as the plant metabolite geraniol, are of high industrial relevance since they are important fragrance materials for perfumes, cosmetics, and household products. Chemical synthesis or extraction from plant material for industry purposes are complex, environmentally harmful or expensive and depend on seasonal variations. Heterologous microbial production offers a cost-efficient and sustainable alternative but suffers from low metabolic flux of the precursors and toxicity of the monoterpenoid to the cells.

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.

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.

Switchable foldamer ion channels with antibacterial activity.

Synthetic ion channels may have applications in treating channelopathies and as new classes of antibiotics, particularly if ion flow through the channels can be controlled. Here we describe triazole-capped octameric α-aminoisobutyric acid (Aib) foldamers that "switch on" ion channel activity in phospholipid bilayers upon copper(ii) chloride addition; activity is "switched off" upon copper(ii) extraction. X-ray crystallography showed that CuCl complexation gave chloro-bridged foldamer dimers, with hydrogen bonds between dimers producing channels within the crystal structure.