The ability to quickly and reliably assemble DNA constructs is one of the key enabling technologies for synthetic biology. Here we define a new Biopart Assembly Standard for Idempotent Cloning (BASIC), which exploits the principle of orthogonal linker based DNA assembly to define a new physical standard for DNA parts. Further, we demonstrate a new robust method for assembly, based on type IIs restriction enzyme cleavage and ligation of oligonucleotides with single stranded overhangs that determine the assembly order. It allows for efficient, parallel assembly with great accuracy: 4 part assemblies achieved 93% accuracy with single antibiotic selection and 99.7% accuracy with double antibiotic selection, while 7 part assemblies achieved 90% accuracy with double antibiotic selection. The linkers themselves may also be used as composable parts for RBS tuning or the creation of fusion proteins. The standard has one forbidden restriction site and provides for an idempotent, single tier organization, allowing all parts and composite constructs to be maintained in the same format. This makes the BASIC standard conceptually simple at both the design and experimental levels.

Deracemization of racemic chiral tertiary amines has been achieved by combination of an enantioselective amine oxidase, obtained through directed evolution, and ammonia borane in a one-pot process.

Organocatalytic enantioselective desymmetrisation of achiral or meso compounds is a powerful strategy for the construction of enantiomerically enriched complex molecules, often with multiple stereocentres and in high selectivities. Recent years have seen increasing use of organocatalysts in desymmetrisation methodology, in contrast to traditional metal- or enzyme-catalysed reactions, with many impressive advances made in the current decade. This review will provide an overview of the field since 2010, with the aim of highlighting both the practical applications and elegance of enantioselective desymmetrisation to the wider synthetic community.

N-Acetyl amino acid racemases (NAAARs) have demonstrated their potential in the enzymatic synthesis of chiral amino acids, molecules of significant biotechnology interest. In order to identify novel activities and to improve these enzymes by engineering approaches, suitable screening methods are necessary. Previous engineering of the NAAAR from Amycolatopsis Ts-1-60 was achieved by relying on an in vivo selection system that linked the viability of an E. coli l-methionine auxotroph to the activity of the improved enzyme. However, this assay was only suitable for the screening of N-acetyl-d-methionine, therefore limiting the potential to evolve this enzyme toward other natural or non-natural acetylated amino acids. Here, we report the optimization and application of a spectrophotometric microtiter-plate-based assay for NAAAR. The assay is based on the detection of the amino acid reaction product formed by hydrolysis of the N-acylated substrate by an l-amino acid acylase and its subsequent oxidation by an FAD-dependent l-amino acid oxidase (l-AAO). Cofactor recycling of the l-AAO leads to the formation of hydrogen peroxide which is easily monitored using horseradish peroxidase (HRP) and o-dianisidine. This method allowed for the determination of the kinetic parameters of NAAAR and led to the identification of N-acetyl-d-naphthylalanine as a novel NAAAR substrate. This robust method is also suitable for the high-throughput screening of NAAAR mutant gene libraries directly from cell lysates.

Dehydroepiandrosterone (DHEA, 2) is an important endogenous steroid hormone in mammals used in the treatment of a variety of dysfunctions in female and male health, 1 as well as an intermediate in the synthesis of steroidal drugs, such as abiraterone acetate which is used for the treatment of prostate cancer. 2−4 In this manuscript we describe a novel, concise, and cost-efficient route toward DHEA (2) and DHEA acetate (3) from 4-androstene-3,17-dione (4-AD, 1). Crucial to success was the identification of a ketoreductase from Sphingomonas wittichii for the highly regio-and stereoselective reduction of the C3carbonyl group of 5-androstene-3,17-dione (5) to the required 3β-alcohol (2, >99% de). The enzyme displayed excellent robustness and solvent stability under high substrate concentrations (up to 150 g/L).