Protein engineering for natural product biosynthesis and synthetic biology applications.

As protein engineering grows more salient, many strategies have emerged to alter protein structure and function, with the goal of redesigning and optimizing natural product biosynthesis. Computational tools, including machine learning and molecular dynamics simulations, have enabled the rational mutagenesis of key catalytic residues for enhanced or altered biocatalysis. Semi-rational, directed evolution and microenvironment engineering strategies have optimized catalysis for native substrates and increased enzyme promiscuity beyond the scope of traditional rational approaches.

Synthetic biology, combinatorial biosynthesis, and chemo‑enzymatic synthesis of isoprenoids.

Isoprenoids are a large class of natural products with myriad applications as bioactive and commercial compounds. Their diverse structures are derived from the biosynthetic assembly and tailoring of their scaffolds, ultimately constructed from two C5 hemiterpene building blocks. The modular logic of these platforms can be harnessed to improve titers of valuable isoprenoids in diverse hosts and to produce new-to-nature compounds.

Synthetic biology enabling access to designer polyketides.

The full potential of polyketide discovery has yet to be reached owing to a lack of suitable technologies and knowledge required to advance engineering of polyketide biosynthesis. Recent investigations on the discovery, enhancement, and non-natural use of these biosynthetic gene clusters via computational biology, metabolic engineering, structural biology, and enzymology-guided approaches have facilitated improved access to designer polyketides. Here, we discuss recent successes in gene cluster discovery, host strain engineering, precursor-directed biosynthesis, combinatorial biosynthesis, polyketide tailoring, and high-throughput synthetic biology, as well as challenges and outlooks for rapidly generating useful target polyketides.

Development of a Genetically Encoded Biosensor for Detection of Polyketide Synthase Extender Units in Escherichia coli.

The scaffolds of polyketides are constructed via assembly of extender units based on malonyl-CoA and its derivatives that are substituted at the C2-position with diverse chemical functionality. Subsequently, a transcription-factor-based biosensor for malonyl-CoA has proven to be a powerful tool for detecting malonyl-CoA, facilitating the dynamic regulation of malonyl-CoA biosynthesis and guiding high-throughput engineering of malonyl-CoA-dependent processes. Yet, a biosensor for the detection of malonyl-CoA derivatives has yet to be reported, severely restricting the application of high-throughput synthetic biology approaches to engineering extender unit biosynthesis and limiting the ability to dynamically regulate the biosynthesis of polyketide products that are dependent on such α-carboxyacyl-CoAs.

Specificity of extended O-aryloxycarbonyl hydroxamates as inhibitors of a class C β-lactamase.

Class C β-lactamases have previously been shown to be efficiently inactivated by O-aryloxycarbonyl hydroxamates. O-Phenoxycarbonyl-N-benzyloxycarbonylhydroxylamine (1) and O-phenoxycarbonyl-N-(R)-[(4-amino-4-carboxy-1-butyl)oxycarbonyl]hydroxylamine (2), for example, were found to be effective inactivators. The present paper describes a structure-activity study of these molecules to better define the important structural elements for high inhibitory activity.

Direct analysis of terpenes from biological buffer systems using SESI and IR-MALDESI.

Terpenes are the largest class of natural products with a wide range of applications including use as pharmaceuticals, fragrances, flavorings, and agricultural products. Terpenes are biosynthesized by the condensation of a variable number of isoprene units resulting in linear polyisoprene diphosphate units, which can then be cyclized by terpene synthases into a range of complex structures. While these cyclic structures have immense diversity and potential in different applications, their direct analysis in biological buffer systems requires intensive sample preparation steps such as salt cleanup, extraction with organic solvents, and chromatographic separations.