Joel R. Cherry

Directed evolution of a fungal peroxidase

The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50°C), and high peroxide concentration (5–10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.

DNA shuffling of subgenomic sequences of subtilisin

DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently created all of the combinations of parental properties, producing a great diversity of property combinations in the progeny enzymes. Thus, molecular breeding, like classical breeding, is a powerful tool for recombining existing diversity to tailor biological systems for multiple functional parameters.

Directed evolution of microbial oxidative enzymes

In the past year, a number of oxidative enzymes have been the target of directed evolution. Catalase reaction specificity has been shifted to peroxidase, the high pH, thermal and oxidative stability of a fungal peroxidase has been dramatically improved, and the substrate specificity of cytochrome P450 has been altered to include substrates that the wild-type enzymes are incapable of oxidizing.

Cloning of the Aspergillus oryzae 5-aminolevulinate synthase gene and its use as a selectable marker.

Abstract The hemA gene encoding 5-aminolevulinate synthase, the first enzyme in heme biosynthesis, was cloned from Aspergillus oryzae and evaluated as a selectable marker for the transformation of filamentous fungi. Deletion of the hemA gene resulted in a lethal phenotype that could be rescued either by the supplementation of culture media with 5-aminolevulinic acid (ALA) or by transformation with the wild-type hemA gene, but not by growth on rich media, nor by the addition of exogenous heme. Transformation of a hemA deletion strain with the hemA gene linked to a lipase expression cassette yielded ALA prototrophs expressing lipase. The hemA gene can therefore be used as a selectable marker for the transformation of A. oryzae.

Community crystal gazing

A selection of individuals from the biotech ecosystem give their views on the challenges facing the sector over the coming years.

Erratum: Community crystal gazing

Nat. Biotechnol. 34, 276–283 (2016); published online 10 March 2016; corrected after print 31 March 2016 In the version of this article initially published, Stanley Crookes name was misspelled as “Crook” in the author list. The error has been corrected in the HTML and PDF versions of the article.