Engineered plastic-associated bacteria for biodegradation and bioremediation.

The global plastic waste crisis has triggered the development of novel methods for removal of recalcitrant polymers from the environment. Biotechnological approaches have received particular attention due to their potential for enabling sustainable, low-intensity bioprocesses which could also be interfaced with microbial upcycling pathways to support the emerging circular bioeconomy. However, low biodegradation efficiency of solid plastic materials remains a bottleneck, especially at mesophilic conditions required for one-pot degradation and upcycling.

One-Pot Biosynthesis of Acetone from Waste Poly(hydroxybutyrate).

The plastic waste crisis is catalyzing change across the plastics life cycle. Central to this is increased production and application of bioplastics and biodegradable plastics. In particular, poly(hydroxybutyrate) (PHB) is a biodegradable bioplastic that can be produced from various renewable and waste feedstocks and is a promising alternative to some petrochemical-derived and non-biodegradable plastics.

Overproduction of Native and Click-able Colanic Acid Slime from Engineered .

The fundamental biology and application of bacterial exopolysaccharides is gaining increasing attention. However, current synthetic biology efforts to produce the major component of slime, colanic acid, and functional derivatives thereof have been limited. Herein, we report the overproduction of colanic acid (up to 1.

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 k 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.

Tyramine Derivatives Catalyze the Aldol Dimerization of Butyraldehyde in the Presence of Escherichia coli.

Biogenic amine organocatalysts have transformed the field of synthetic organic chemistry. Yet despite their use in synthesis and to label biomolecules in vitro, amine organocatalysis in vivo has received comparatively little attention - despite the potential of such reactions to be interfaced with living cells and to modify cellular metabolites. Herein we report that biogenic amines derived from L-tyrosine catalyze the self-aldol condensation of butanal to 2-ethylhexenal - a key intermediate in the production of the bulk chemical 2-ethylhexanol - in the presence of living Escherichia coli and outperform many amine organocatalysts currently used in synthetic organic chemistry.

Interfacing non-enzymatic catalysis with living microorganisms.

Interfacing non-enzymatic catalysis with cellular metabolism is emerging as a powerful approach to produce a range of high value small molecules and polymers. In this review, we highlight recent examples from this promising young field. Specifically, we discuss demonstrations of living cells mediating redox processes for biopolymer production, interfacing solar-light driven chemistry with microbial metabolism, and intra- and extracellular non-enzymatic catalysis to generate high value molecules.

Microbial synthesis of vanillin from waste poly(ethylene terephthalate).

Poly(ethylene terephthalate) (PET) is an abundant and extremely useful material, with widespread applications across society. However, there is an urgent need to develop technologies to valorise post-consumer PET waste to tackle plastic pollution and move towards a circular economy. Whilst PET degradation and recycling technologies have been reported, examples focus on repurposing the resultant monomers to produce more PET or other second-generation materials.

The Bipartisan Future of Synthetic Chemistry and Synthetic Biology.

Synthetic biology holds great potential for sustainable chemical synthesis, yet is limited to accessing a relatively small area of chemical space. By interfacing this new technology with the versatility and scope of synthetic chemistry, the best of both worlds can be harnessed to drive a green chemical industry.

S-Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C-Alkylation.

A tandem enzymatic strategy to enhance the scope of C-alkylation of small molecules via the in situ formation of S-adenosyl methionine (SAM) cofactor analogues is described. A solvent-exposed channel present in the SAM-forming enzyme SalL tolerates 5'-chloro-5'-deoxyadenosine (ClDA) analogues modified at the 2-position of the adenine nucleobase. Coupling SalL-catalyzed cofactor production with C-(m)ethyl transfer to coumarin substrates catalyzed by the methyltransferase (MTase) NovO forms C-(m)ethylated coumarins in superior yield and greater substrate scope relative to that obtained using cofactors lacking nucleobase modifications.

Phenylalanine meta-Hydroxylase: A Single Residue Mediates Mechanistic Control of Aromatic Amino Acid Hydroxylation.

The rare nonproteinogenic amino acid, meta-l-tyrosine is biosynthetically intriguing. Whilst the biogenesis of tyrosine from phenylalanine is well characterised, the mechanistic basis for meta-hydroxylation is unknown. Herein, we report the analysis of 3-hydroxylase (Phe3H) from Streptomyces coeruleorubidus.

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.

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.

Fast and Flexible Synthesis of Combinatorial Libraries for Directed Evolution.

Directed evolution (DE) is a powerful tool for optimizing an enzyme's properties toward a particular objective, such as broader substrate scope, greater thermostability, or increased k. A successful DE project requires the generation of genetic diversity and subsequent screening or selection to identify variants with improved fitness. In contrast to random methods (error-prone PCR or DNA shuffling), site-directed mutagenesis enables the rational design of variant libraries and provides control over the nature and frequency of the encoded mutations.

A Tandem Enzymatic sp -C-Methylation Process: Coupling in Situ S-Adenosyl-l-Methionine Formation with Methyl Transfer.

A one-pot, two-step biocatalytic platform for the regiospecfic C-methylation and C-ethylation of aromatic substrates is described. The tandem process utilises SalL (Salinospora tropica) for in situ synthesis of S-adenosyl-l-methionine (SAM), followed by alkylation of aromatic substrates by the C-methyltransferase NovO (Streptomyces spheroides). The application of this methodology is demonstrated for the regiospecific labelling of aromatic substrates by the transfer of methyl, ethyl and isotopically labelled CH CD and CD groups from their corresponding SAM analogues formed in situ.

Structural and Functional Basis of C-Methylation of Coumarin Scaffolds by NovO.

C-methylation of aromatic small molecules by C-methyltransferases (C-MTs) is an important biological transformation that involves C-C bond formation using S-adenosyl-l-methionine (SAM) as the methyl donor. Here, two advances in the mechanistic understanding of C-methylation of the 8-position of coumarin substrates catalyzed by the C-MT NovO from Streptomyces spheroides are described. First, a crystal structure of NovO reveals the Arg116-Asn117 and His120-Arg121 motifs are essential for coumarin substrate binding.

Chemoselective Sequential Click Ligations Directed by Enhanced Reactivity of an Aromatic Ynamine.

Aromatic ynamines or N-alkynylheteroarenes are highly reactive alkyne components in Cu-catalyzed Huisgen [3 + 2] cycloaddition ("click") reactions. This enhanced reactivity enables the chemoselective formation of 1,4-triazoles using the representative aromatic ynamine N-ethynylbenzimidazole in the presence of a competing aliphatic alkyne substrate. The unique chemoselectivity profile of N-ethynylbenzimidazole is further demonstrated by the sequential click ligation of a series of highly functionalized azides using a heterobifunctional diyne, dispelling the need for alkyne protecting groups.

Imidazolium-tagged glycan probes for non-covalent labeling of live cells.

Selective, bioorthogonal and fast labeling of glycoconjugates in living cells is a major challenge for synthetic and cellular biology. Here we report the use imidazolium tagged-mannosamine derivative (ITag-Man) for the non-covalent, rapid and site-specific labeling of sialic acid containing glycoproteins using commercial N-nitrilotriacetate fluorescent reagents in a range of cell lines.