We report the use of cross-linked enzyme aggregates (CLEAs^®) to catalyse the kinetic resolution of tetralol and 1-phenylethanol in a continuous supercritical carbon dioxide (scCO₂) system. We describe the performance of the CLEA from Candida antarctica lipase B (CALB) and compare this to the catalytic activity of Novozym 435 (CALB immobilised on a macroporous acrylic resin). In addition, we report a two-stage reaction with the kinetic resolution of 1-phenylethanol performed in series with the metal (Pd) catalysed hydrogenation of acetophenone. Reactions performed in series have a potential economic advantage as the second, and subsequent reactions do not require additional expenditure of energy for re-pressurization of solvent.

Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence. We performed next generation sequencing using Illumina MiSeq technology on the DSM10061 strain of Clostridium autoethanogenum and observed 243 single nucleotide discrepancies when compared to the published finished sequence (NCBI: GCA_000484505.1), with 59.1 % present in coding regions. These variations were confirmed by Sanger sequencing and subsequent analysis suggested that the discrepancies were sequencing errors in the published genome not true single nucleotide polymorphisms. This was corroborated by the observation that over 90 % occurred within homopolymer regions of greater than 4 nucleotides in length. It was also observed that many genes containing these sequencing errors were annotated in the published closed genome as encoding proteins containing frameshift mutations (18 instances) or were annotated despite the coding frame containing stop codons, which if genuine, would severely hinder the organism’s ability to survive. Furthermore, we have completed a comprehensive manual curation to reduce errors in the annotation that occur through serial use of automated annotation pipelines in related species. As a result, different functions were assigned to gene products or previous functional annotations rejected because of missing evidence in various occasions. We present a revised manually curated full genome sequence for Clostridium autoethanogenum DSM10061, which provides reliable information for genome-scale models that rely heavily on the accuracy of annotation, and represents an important step towards the manipulation and metabolic modelling of this industrially relevant acetogen.

Two iminosugars have been designed and synthesized as potential inhibitors of UDP-Galf transferase, an enzyme involved in Mycobacterium tuberculosis cell wall biosynthesis. The design is based on a proposed model of the transition state for the transferase reaction. One of the two racemic compounds is the first reported inhibitor of the target enzyme from M. smegmatis.