Site-saturation mutagenesis is a powerful tool for protein optimization due to its efficiency and simplicity. A degenerate codon NNN or NNS (K) is often used to encode the 20 standard amino acids, but this will produce redundant codons and cause uneven distribution of amino acids in the constructed library. Here we present a novel small-intelligent strategy to construct mutagenesis libraries that have a minimal gene library size without inherent amino acid biases, stop codons, or rare codons of Escherichia coli by coupling well-designed combinatorial degenerate primers with suitable PCR-based mutagenesis methods. The designed primer mixture contains exactly one codon per amino acid and thus allows the construction of small-intelligent mutagenesis libraries with one gene per protein. In addition, the software tool DC-Analyzer was developed to assist in primer design according to the user-defined randomization scheme for library construction. This small-intelligent strategy was successfully applied to the randomization of halohydrin dehalogenases with one or two randomized sites. With the help of DC-Analyzer, the strategy was proven to be as simple as NNS randomization and could serve as a general tool to efficiently randomize target genes at positions of interest.
bHalohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of R enantiomers of epoxides and -substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low S enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving an S-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (from S enantioselectivity [E S ] ؍ 1.7 to R enantioselectivity [E R ] ؍ 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (E S > 200) and a 5-to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.
SARS-CoV-2
has infected over 128 million people worldwide, and
until a vaccine is developed and widely disseminated, vigilant testing
and contact tracing are the most effective ways to slow the spread
of COVID-19. Typical clinical testing only confirms the presence or
absence of the virus, but rather, a simple and rapid testing procedure
that sequences the entire genome would be impactful and allow for
tracing the spread of the virus and variants, as well as the appearance
of new variants. However, traditional short read sequencing methods
are time consuming and expensive. Herein, we describe a tiled genome
array that we developed for rapid and inexpensive full viral genome
resequencing, and we have applied our SARS-CoV-2-specific genome tiling
array to rapidly and accurately resequence the viral genome from eight
clinical samples. We have resequenced eight samples acquired from
patients in Wyoming that tested positive for SARS-CoV-2. We were ultimately
able to sequence over 95% of the genome of each sample with greater
than 99.9% average accuracy.
Proteins belonging to the NTF2-like superfamily are present in the biosynthetic pathways of numerous polyketide natural products, such as anthracyclins and benzoisochromanequinones. Some have been found to be bona fide polyketide cyclases, but many of them have roles that are currently unknown. Here, the X-ray crystal structures of three NTF2-like proteins of unknown function are reported: those of ActVI-ORFA from Streptomyces coelicolor A3(2) and its homologs Caci_6494, a protein from an uncharacterized biosynthetic cluster in Catenulispora acidiphila, and Aln2 from Streptomyces sp. CM020, a protein in the biosynthetic pathway of alnumycin. The presence of a solvent-accessible cavity and the conservation of the His/Asp dyad that is characteristic of many polyketide cyclases suggest a potential enzymatic role for these enzymes in polyketide biosynthesis.
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