Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae

The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches

Caller experience: A method for evaluating dialog systems and its automatic prediction

In this paper we introduce a subjective metric for evaluating the performance of spoken dialog systems, Caller Experience (CE). CE is a useful metric for tracking the overall performance of a system in deployment, as well as for isolating individual problematic calls in which the system under-performs. The proposed CE metric differs from most performance evaluation metrics proposed in the past in that it is a) a subjective, qualitative rating of the call, and b) provided by expert, external listeners, not the callers themselves. The results of an experiment in which a set of human experts listened to the same calls three times are presented. The fact that these results show a high level of agreement among different listeners, despite the subjective nature of the task, demonstrates the validity of using CE as a standard metric. Finally, an automated rating system using objective measures is shown to perform at the same high level as the humans. This is an important advance, since it provides a way to reduce the human labor costs associated with producing a reliable CE. Index Terms — spoken dialog systems, performance evaluation, inter-rater agreement, classification 1

Functional annotation results.

<p>Proportion of <i>Camptotheca acuminata</i>, <i>Catharanthus roseus</i> and <i>Rauvolfia serpentina</i> transcripts with sequence similarity to the UniRef100 database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052506#pone.0052506-Suzek1" target="_blank">[17]</a>, <i>Arabidopsis thaliana</i> proteome (<a href="http://arabidopsis.org" target="_blank">http://arabidopsis.org</a>), and Pfam domain database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052506#pone.0052506-Punta1" target="_blank">[18]</a>.</p

Monoterpene indole alkaloid pathway.

<p>The key intermediate strictosidine is formed by condensation of tryptamine, which contributes the indole ring, and secologanin, which is produced from the monoterpene geraniol. In various plants, strictosidine is further metabolized to generate over 2,500 monoterpene indole alkaloids. Solid lines indicate single enzymatic steps; dashed lines indicate multiple steps.</p

Hierarchical clustering of expression profiles from sampled tissues from <i>Camptotheca acuminata</i> (A), <i>Catharanthus roseus</i> (B), and <i>Rauvolfia serpentina</i> (C).

<p>Pearson product-moment correlation coefficient of log₂ FPKM (fragments per kb transcript per million mapped reads) expression values among RNA-seq libraries were calculated and clustered using R <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052506#pone.0052506-R1" target="_blank">[39]</a>; negative values were set to zero.</p