Harnessing biocatalysis to achieve selective functional group interconversion of monomers.

Polymeric materials are ubiquitous to modern life. However, reliance of petroleum for polymeric building blocks is not sustainable. The synthesis of macromolecules from recalcitrant polymer waste feedstocks, such as plastic waste and lignocellulosic biomass, presents an opportunity to bypass the use of petroleum-based feedstocks. However, the deconstruction and transformation of these alternative feedstocks remained limited until recently. Herein, we highlight examples of monomers liberated from the deconstruction of recalcitrant polymers, and more extensively, we showcase the state-of-the-art in biocatalytic technologies that are enabling synthesis of diverse upcycled monomeric starting materials for a wide variety of macromolecules. Overall, this review emphasizes the importance of functional group interconversion as a promising strategy by which biocatalysis can aid the diversification and upcycling of monomers.

Recent developments in enzymatic and microbial biosynthesis of flavor and fragrance molecules.

Flavors and fragrances are an important class of specialty chemicals for which interest in biomanufacturing has risen during recent years. These naturally occurring compounds are often amenable to biosynthesis using purified enzyme catalysts or metabolically engineered microbial cells in fermentation processes. In this review, we provide a brief overview of the categories of molecules that have received the greatest interest, both academically and industrially, by examining scholarly publications as well as patent literature. Overall, we seek to highlight innovations in the key reaction steps and microbial hosts used in flavor and fragrance manufacturing.

T7 Polymerase Expression of Guide RNAs in vivo Allows Exportable CRISPR-Cas9 Editing in Multiple Yeast Hosts.

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.

Yeast Terminator Function Can Be Modulated and Designed on the Basis of Predictions of Nucleosome Occupancy.

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.