Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871 bp designer eukaryotic chromosome, synIII, which is based on the 316,617 bp native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, tRNAs, transposons and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in “a mater” derivatives resulting from loss of the MATα allele on synIII. The total synthesis of synIII represents the first complete design and synthesis of a eukaryotic chromosome, establishing S. cerevisiae as the basis for designer eukaryotic genome biology.
Carboxylate reductases (CARs) are valuable catalysts for the selective one‐step reduction of carboxylic acids to their corresponding aldehydes. In recent years, numerous new CARs have been made available, studied and applied in the context of biocatalytic syntheses. The preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates represent the two major fields of applications. This review gives a comprehensive overview of the current toolbox of recombinant CARs and their numerous carboxylate substrates. Non‐natural functions of CARs are highlighted and recent insight into the structure‐function relationship of CARs are summarized.
The synthesis of
aldehydes from carboxylic acids has long been
a challenge in chemistry. In contrast to the harsh chemically driven
reduction, enzymes such as carboxylic acid reductases (CARs) are considered
appealing biocatalysts for aldehyde production. Although structures
of single- and didomains of microbial CARs have been reported, to
date no full-length protein structure has been elucidated. In this
study, we aimed to obtain structural and functional information regarding
the reductase (R) domain of a CAR from the fungus Neurospora
crassa (Nc). The NcCAR
R-domain revealed activity for N-acetylcysteamine
thioester (S-(2-acetamidoethyl) benzothioate), which mimics the phosphopantetheinylacyl-intermediate
and can be anticipated as the minimal substrate for thioester reduction
by CARs. The determined crystal structure of the NcCAR R-domain reveals a tunnel that putatively harbors the phosphopantetheinylacyl-intermediate,
which is in good agreement with docking experiments performed with
the minimal substrate. In vitro studies were performed
with this highly purified R-domain and NADPH, demonstrating carbonyl
reduction activity. The R-domain was able to accept not only a simple
aromatic ketone but also benzaldehyde and octanal, which are typically
considered to be the final product of carboxylic acid reduction by
CAR. Also, the full-length NcCAR reduced aldehydes
to primary alcohols. In conclusion, aldehyde overreduction can no
longer be attributed exclusively to the host background.
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