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The construction of biocatalytic cascades for the production of chemical precursors is fast becoming one of the most efficient approaches to multi-step synthesis in modern chemistry. However, despite the use of low solvent systems and renewably resourced catalysts in reported examples, many cascades are still dependent on petrochemical starting materials, which as of yet cannot be accessed in a sustainable fashion. Herein, we report the production of the versatile chemical building block cinnamyl alcohol from the primary metabolite and the fermentation product L-phenylalanine. Through the combination of three biocatalyst classes (phenylalanine ammonia lyase, carboxylic acid reductase and alcohol dehydrogenase) the target compound could be obtained in high purity, demonstrable at the 100 mg scale and achieving 53% yield using ambient temperature and pressure in an aqueous solution. This system represents a synthetic strategy in which all components present at time zero are biogenic and thus minimises damage to the environment. Furthermore we extend this biocatalytic cascade by its inclusion in an L-phenylalanine overproducing strain of Escherichia coli. This metabolically engineered strain produces cinnamyl alcohol in mineral media using glycerol and glucose as the carbon sources. This study demonstrates the potential to establish green routes to the synthesis of cinnamyl alcohol from a waste stream such as glycerol derived, for example, from lipase treated biodiesel.

The successful implementation of synthetic biology for chemicals biosynthesis relies on the availability of large libraries of well-characterized enzymatic building blocks. Here we present a scalable pipeline that applies the methodology of synthetic biology itself to bootstrap the creation of such a library. By designing and building a cytochrome P450 enzyme collection and testing it in a custom-made untargeted GC/MS-metabolomics-based approach, we were able to rapidly create and characterize a comprehensive enzyme library for the controlled oxyfunctionalisation of terpene scaffolds with a wide range of activities and selectivities towards several monoterpenes. This novel resource can now be used to access the extensive chemical diversity of terpenoids by pathway engineering and the assembly of biocatalytic cascades to subsequently produce libraries of oxygenated terpenoids and their derivatives for diverse applications, including drug discovery.

Synthetic biology is opening up new opportunities for the sustainable and efficient production of valuable chemicals in engineered microbial factories. Here we review the application of synthetic biology approaches to the engineering of monoterpene/monoterpenoid production, highlighting the discovery of novel catalytic building blocks, their accelerated assembly into functional pathways, general strategies for product diversification, and new methods for the optimization of productivity to economically viable levels. Together, these emerging tools allow the rapid creation of microbial production systems for a wide range of monoterpenes and their derivatives for a diversity of industrial applications.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems are found widespread in bacterial and archaeal genomes and exhibit considerable diversity. However, closer insights into the action of most of the CRISPR modules have remained elusive in particular in Archaea as a result of the lack of suitable in vivo test systems. Here we demonstrate CRISPR/Cas-based immune defence in the hyperthermophilic archaeon Sulfolobus solfataricus. Recombinant variants of the SSV1 virus containing a gene of the conjugative plasmid pNOB8 that represents a target for a corresponding CRISPR spacer in the chromosome were tested in transfection experiments. Almost 100% immunity against the recombinant virus was observed when the chromosomal CRISPR spacer matched perfectly to the protospacer. Different from bacterial systems immunity was still detected, albeit at decreased levels, when mutations distinguished target and spacer. CRISPR/Cas targeting was independent of the transcription of the target gene. Furthermore, a mini-CRISPR locus introduced on the viral DNA with spacers targeting the (non-essential) chromosomal beta-galactosidase gene was unstable in host cells and triggered recombination with the indigenous CRISPR locus. Our experiments demonstrate in vivo activity of CRISPR/Cas in archaea for the first time and suggest that - unlike the recently demonstrated in vitro cleavage of RNA in Pyrococcus- DNA is targeted in this archaeon.

Natural products such as secondary metabolites (e.g., plant terpenoids) are found to be a major source of bioactive compounds. These natural products accumulate as complex mixtures with other related compounds and this chemical complexity adds cost to the downstream recovery and purification of natural products from plant biomass. One aim of synthetic biology and metabolic engineering programmes is to produce such compounds from synthetic gene clusters in heterologous hosts and thereby achieve more targeted and affordable production. Both fungi and bacteria are common hosts for metabolic engineering in industry. Fungal hosts include Penicillium chrysogenum, Saccharomyces cerevisiae, Aspergillus niger and the bacterial hosts Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum. E. coli is often selected as a host given the ease of its genetic manipulation and the long history of using this organism in laboratory‐based bioengineering. The bioengineering of E. coli extends also to feedstock pathways to interface and optimize the production of high value compounds from widely available and inexpensive carbon sources. Genome editing is important in these microbial bioengineering programmes and is needed to isolate stable strains and to optimize production. Herein, this review discusses frequently used methods for genome editing in E. coli in relation to the production of natural compounds and chemicals.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems are found widespread in bacterial and archaeal genomes and exhibit considerable diversity. However, closer insights into the action of most of the CRISPR modules have remained elusive in particular in Archaea as a result of the lack of suitable in vivo test systems. Here we demonstrate CRISPR/Cas-based immune defence in the hyperthermophilic archaeon Sulfolobus solfataricus. Recombinant variants of the SSV1 virus containing a gene of the conjugative plasmid pNOB8 that represents a target for a corresponding CRISPR spacer in the chromosome were tested in transfection experiments. Almost 100% immunity against the recombinant virus was observed when the chromosomal CRISPR spacer matched perfectly to the protospacer. Different from bacterial systems immunity was still detected, albeit at decreased levels, when mutations distinguished target and spacer. CRISPR/Cas targeting was independent of the transcription of the target gene. Furthermore, a mini-CRISPR locus introduced on the viral DNA with spacers targeting the (non-essential) chromosomal beta-galactosidase gene was unstable in host cells and triggered recombination with the indigenous CRISPR locus. Our experiments demonstrate in vivo activity of CRISPR/Cas in archaea for the first time and suggest that - unlike the recently demonstrated in vitro cleavage of RNA in Pyrococcus- DNA is targeted in this archaeon.