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.

Saccharomyces cerevisiae can serve as a key production platform for biofuels, nutraceuticals, industrial compounds, and therapeutic proteins. Over the recent years, synthetic biology tools and libraries have expanded in yeast to provide newfound control over regulation and synthetic circuits. This review provides an update on the status of the synthetic biology toolbox in yeast for use as a cell factory. Specifically, we discuss the impact of plasmid selection and composition, promoter, terminator, transcription factor, and aptamer selection. In doing so, we highlight documented interactions between these components, current states of development, and applications that demonstrate the utility of these parts with a particular focus on synthetic gene expression control.

Efficient guide RNA expression often limits CRISPR-Cas9 implementation in new hosts. To address this limitation in fungal systems, we demonstrate the utility of a T7 polymerase system to effectively express sgRNAs. Initially, we developed a methodology in Saccharomyces cerevisiae using a modified version of the T7 P266L mutant polymerase with an SV40 nuclear localization signal to allow guide RNA expression immediately downstream of a T7 promoter. To improve targeting efficiency, guide RNA design was found to be tolerant to three mismatches or up to three additional bases appended to the 5' end. The addition of three guanines to a T7-based guide RNA improved guide RNA expression 80-fold and achieved transcriptional output similar to the strong Pol III snr52 promoter. Resulting gene editing and dCas9-guided gene regulation with a T7-based guide RNA was on par with the commonly used snr52 system in S. cerevisiae. Finally, 96% and 60% genome editing efficiencies were achieved in Kluyveromyces lactis and Yarrowia lipolytica respectively with minimal optimization of this system. Thus, T7-based expression of sgRNAs offers an orthogonal method for implementing CRISPR systems in fungal systems.

Improving yeast tolerance to 1-butanol and isobutanol is a step toward enabling high-titer production. To identify previously unknown genetic targets leading to increased tolerance, we establish a tunable RNA interference (RNAi) screening approach. Specifically, we optimized the efficiency and tunability of RNA interference library screening in yeast, ultimately enabling downregulation efficiencies from 0 to 94 %. Using this system, we identified the Hsp70 family as a key regulator of isobutanol tolerance in a single round of screening, with downregulation of these genes conferring up to 64 % increased growth in 12 g/L isobutanol. For 1-butanol, we find through two rounds of iterative screening that the combined downregulation of alcohol dehydrogenase and enolase improves growth up to 3100 % in 10 g/L 1-butanol. Collectively, this work improves the tunability of RNAi in yeast as demonstrated by the discovery of novel effectors for these complex phenotypes.

The design of improved synthetic parts is a major goal of synthetic biology. Mechanistically, nucleosome occupancy in the 3' terminator region of a gene has been found to correlate with transcriptional expression. Here, we seek to establish a predictive relationship between terminator function and predicted nucleosome positioning to design synthetic terminators in the yeast Saccharomyces cerevisiae. In doing so, terminators improved net protein output from these expression cassettes nearly 4-fold over their original sequence with observed increases in termination efficiency to 96%. The resulting terminators were indeed depleted of nucleosomes on the basis of mapping experiments. This approach was successfully applied to synthetic, de novo, and native terminators. The mode of action of these modifications was mainly through increased termination efficiency, rather than half-life increases, perhaps suggesting a role in improved mRNA maturation. Collectively, these results suggest that predicted nucleosome depletion can be used as a heuristic approach for improving terminator function, though the underlying mechanism remains to be shown.

Tuning heterologous gene expression in mammalian production hosts has predominantly relied upon engineering the promoter elements driving the transcription of the transgene. Moreover, most regulatory elements have borrowed genetic sequences from viral elements. Here, we generate a set of 10 rational and 30 synthetic terminators derived from nonviral elements and evaluate them in the HT1080 and HEK293 cell lines to demonstrate that they are comparable in terms of tuning gene expression/protein output to the viral SV40 element and often require less sequence footprint. The mode of action of these terminators is determined to be an increase in mRNA half-life. Furthermore, we demonstrate that constructs comprising completely nonviral regulatory elements (i.e., promoters and terminators) can outperform commonly used, strong viral based elements by nearly 2-fold. Ultimately, this novel set of terminators expanded our genetic toolkit for engineering mammalian host cells.

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In this work, the effect of nitrogen on the double layer (DL) capacitance of nitrogen-doped ordered mesoporous carbon (NOMC) is studied.