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Halogenases: a palette of emerging opportunities for synthetic biology-synthetic chemistry and C-H functionalisation.

ABSTRACT: The enzymatic generation of carbon-halogen bonds is a powerful strategy used by both nature and synthetic chemists to tune the bioactivity, bioavailability and reactivity of compounds, opening up the opportunity for selective C-H functionalisation. Genes encoding halogenase enzymes have recently been shown to transcend all kingdoms of life. These enzymes install halogen atoms into aromatic and less activated aliphatic substrates, achieving selectivities that are often challenging to accomplish using synthetic methodologies. Significant advances in both halogenase discovery and engineering have provided a toolbox of enzymes, enabling the ready use of these catalysts in biotransformations, synthetic biology, and in combination with chemical catalysis to enable late stage C-H functionalisation. With a focus on substrate scope, this review outlines the mechanisms employed by the major classes of halogenases, while in parallel, it highlights key advances in the utilisation of the combination of enzymatic halogenation and chemical catalysis for C-H activation and diversification.

SUBMITTER: Crowe C

PROVIDER: S-EPMC8407142 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Halogenases: a palette of emerging opportunities for synthetic biology-synthetic chemistry and C-H functionalisation.

Crowe Charlotte C Molyneux Samuel S Sharma Sunil V SV Zhang Ying Y Gkotsi Danai S DS Connaris Helen H Goss Rebecca J M RJM

Chemical Society reviews 20210809 17

The enzymatic generation of carbon-halogen bonds is a powerful strategy used by both nature and synthetic chemists to tune the bioactivity, bioavailability and reactivity of compounds, opening up the opportunity for selective C-H functionalisation. Genes encoding halogenase enzymes have recently been shown to transcend all kingdoms of life. These enzymes install halogen atoms into aromatic and less activated aliphatic substrates, achieving selectivities that are often challenging to accomplish u ...[more]

PMID: 34368824

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Project description:BackgroundCoral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Förster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.ResultsHere, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coli BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.ConclusionAvailability of 14 engineered CP genes compared in E. coli, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.

Dataset Information

Halogenases: a palette of emerging opportunities for synthetic biology-synthetic chemistry and C-H functionalisation.

Publications

Halogenases: a palette of emerging opportunities for synthetic biology-synthetic chemistry and C-H functionalisation.

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