Terminators play an important role both in completing the transcription process and impacting mRNA half-life. As such, terminators are an important synthetic component considered in applications such as heterologous gene expression and metabolic engineering. Here, we describe a panel of short (35-70 bp) synthetic terminators that can be used for modulating gene expression in yeast. The best of these synthetic terminator resulted in 3.7-fold more fluorescent protein output and 4.4-fold increase in transcript level compared to that with the commonly used CYC1 terminator. These synthetic terminators offer several advantages over native sequences, including an easily synthesized short length, minimal sequence homology to native sequences, and similar or better performance characteristics than those of commonly used longer terminators. Furthermore, the synthetic terminators are highly functional in both Saccharomyces cerevisiae and an alternative yeast, Yarrowia lipolytica, demonstrating that these synthetic designs are transferrable between diverse yeast species.

Control of gene and protein expression of both endogenous and heterologous genes is a key component of metabolic engineering. While a large amount of work has been published characterizing promoters for this purpose, less effort has been exerted to elucidate the role of terminators in yeast. In this study, we characterize over 30 terminators for use in metabolic engineering applications in Saccharomyces cerevisiae and determine mRNA half-life changes to be the major cause of the varied protein and transcript expression level. We demonstrate that the difference in transcript level can be over 6.5-fold even for high strength promoters. The influence of terminator selection is magnified when coupled with a low-expression promoter, with a maximum difference in protein expression of 11-fold between a high-capacity terminator and the parent plasmid terminator and over 35-fold difference when compared with a no-terminator baseline. This is the first time that terminators have been investigated in the context of multiple promoters spanning orders of magnitude in activity. Finally, we demonstrate the utility of terminator selection for metabolic engineering by using a mutant xylose isomerase gene as a proof-of-concept. Through pairing a high-capacity terminator with a low-expression promoter, we were able to achieve the same phenotypic result as with a promoter considerably higher in strength. Moreover, we can further boost the phenotype of the high-strength promoter by pairing it with a high-capacity terminator. This work highlights how terminator elements can be used to control metabolic pathways in the same way that promoters are traditionally used in yeast. Together, this work demonstrates that terminators will be an important part of heterologous gene expression and metabolic engineering for yeast in the future.

Model-based design of biological parts is a critical goal of synthetic biology, especially for eukaryotes. Here we demonstrate that nucleosome architecture can play a role in defining yeast promoter activity and utilize a computationally-guided approach that can enable both the redesign of endogenous promoter sequences and the de novo design of synthetic promoters. Initially, we use our approach to reprogram native promoters for increased expression and evaluate their performance in various genetic contexts. Increases in expression ranging from 1.5 to nearly 6-fold in a plasmid-based system and up to 16-fold in a genomic context were obtained. Next, we demonstrate that, in a single design cycle, it is possible to create functional, purely synthetic yeast promoters that achieve substantial expression levels (within the top sixth percentile among native yeast promoters). In doing so, this work establishes a unique DNA-level specification of promoter activity and demonstrates predictive design of synthetic parts.

We present a new modeling approach for dividing‐wall columns (DWCs) that is amenable to equation‐oriented flowsheet simulation and optimization. The material, equilibrium, summation, and heat (MESH) equations describing a DWC are highly coupled and nonlinear, making DWC‐based process flowsheets challenging to simulate. Design optimization poses further challenges, typically requiring integer variables to select the number of column stages. To address these difficulties, we represent DWCs as networks of pseudo‐transient (differential‐algebraic) subunit models. We show that these networks have the same steady‐state solution as the original (algebraic) MESH equations, but present significant numerical benefits. We then embed these models in a previously developed pseudo‐transient flowsheet modeling and optimization framework. We further reformulate the models to require only continuous decision variables when selecting the optimal number of stages during design optimization. To illustrate these concepts, we discuss the DWC‐based intensification of the dimethyl ether process. © 2015 American Institute of Chemical Engineers AIChE J, 62: 704–716, 2016

Summary We have developed EumicrobeDBLite—a lightweight comprehensive genome resource and sequence analysis platform for oomycete organisms. EumicrobeDBLite is a successor of the VBI Microbial Database (VMD) that was built using the Genome Unified Schema (GUS). In this version, GUS has been greatly simplified with the removal of many obsolete modules and the redesign of others to incorporate contemporary data. Several dependences, such as perl object layers used for data loading in VMD, have been replaced with independent lightweight scripts. EumicrobeDBLite now runs on a powerful annotation engine developed at our laboratory, called ‘Genome Annotator Lite’. Currently, this database has 26 publicly available genomes and 10 expressed sequence tag (EST) datasets of oomycete organisms. The browser page has dynamic tracks presenting comparative genomics analyses, coding and non‐coding data, tRNA genes, repeats and EST alignments. In addition, we have defined 44 777 core conserved proteins from 12 oomycete organisms which form 2974 clusters. Synteny viewing is enabled by the incorporation of the Genome Synteny Viewer (GSV) tool. The user interface has undergone major changes for ease of browsing. Queryable comparative genomics information, conserved orthologous genes and pathways are among the new key features updated in this database. The browser has been upgraded to enable user upload of GFF files for quick view of genome annotation comparisons. The toolkit page integrates the EMBOSS package and has a gene prediction tool. Annotations for the organisms are updated once every 6 months to ensure quality. The database resource is available at http://www.eumicrobedb.org.