Promoters play a central role in controlling gene regulation; however, a small set of promoters is used for most genetic construct design in the yeast Saccharomyces cerevisiae. Generating and utilizing models that accurately predict protein expression from promoter sequences would enable rapid generation of useful promoters and facilitate synthetic biology efforts in this model organism. We measure the gene expression activity of over 675,000 sequences in a constitutive promoter library and over 327,000 sequences in an inducible promoter library. Training an ensemble of convolutional neural networks jointly on the two data sets enables very high (R 2 > 0.79) predictive accuracies on multiple sequence-activity prediction tasks. We describe model-guided design strategies that yield large, sequence-diverse sets of promoters exhibiting activities higher than those represented in training data and similar to current best-in-class sequences. Our results show the value of model-guided design as an approach for generating useful DNA parts.

Covering: 2006 to 2018 Phytochemicals are important sources for the discovery and development of agricultural and pharmaceutical compounds, such as pesticides and medicines. However, these compounds are typically present in low abundance in nature, and the biosynthetic pathways for most phytochemicals are not fully elucidated. Heterologous production of phytochemicals in plant, bacterial, and yeast hosts has been pursued as a potential approach to address sourcing issues associated with many valuable phytochemicals, and more recently has been utilized as a tool to aid in the elucidation of plant biosynthetic pathways. Due to the structural complexity of certain phytochemicals and the associated biosynthetic pathways, reconstitution of plant pathways in heterologous hosts can encounter numerous challenges. Synthetic biology approaches have been developed to address these challenges in areas such as precise control over heterologous gene expression, improving functional expression of heterologous enzymes, and modifying central metabolism to increase the supply of precursor compounds into the pathway. These strategies have been applied to advance plant pathway reconstitution and phytochemical production in a wide variety of heterologous hosts. Here, we review synthetic biology strategies that have been recently applied to advance complex phytochemical production in heterologous hosts.

AbstractPromoters play a central role in controlling gene regulation; however, a small set of promoters is used for most genetic construct design in the yeast Saccharomyces cerevisiae. Generating and utilizing models that accurately predict protein expression from promoter sequences would enable rapid generation of novel useful promoters and facilitate synthetic biology efforts in this model organism. We measured the gene expression activity of over 675,000 unique sequences in a constitutive promoter library, and over 327,000 sequences in an inducible promoter library. Training an ensemble of convolutional neural networks jointly on the two datasets enabled very high (R2 > 0.79) predictive accuracies on multiple sequence-activity prediction tasks. We developed model-guided design strategies which yielded large, sequence-diverse sets of novel promoters exhibiting activities similar to current best-in-class sequences. In addition to providing large sets of new promoters, our results show the value of model-guided design as an approach for generating useful DNA parts.

Cyanogenic glycosides are defense compounds found in a wide range of plant species, including many crops. We demonstrate that the cyanogenic glucoside dhurrin, naturally found in sorghum, can be produced at high titers in Saccharomyces cerevisiae , constituting the first report of cyanogenic glycoside production in a microbe. Genetic modifications to increase the supply of the dhurrin precursor tyrosine enabled dhurrin production in excess of 80 mg/L. The dhurrin-producing yeast strain was used as a chassis to investigate previously uncharacterized enzymes identified close to the biosynthetic gene cluster containing the dhurrin pathway enzymes. This work shows the potential of heterologous expression in yeast to facilitate investigations of plant cyanogenic glycoside pathways.