The advent of biotechnology has enabled metabolic engineers to assemble heterologous pathways in cells to produce a variety of products of industrial relevance, often in a sustainable way. However, many pathways face challenges of low product yield. These pathways often suffer from issues that are difficult to optimize, such as low pathway flux and off-target pathway consumption of intermediates. These issues are exacerbated by the need to balance pathway flux with the health of the cell, particularly when a toxic intermediate builds up. Nature faces similar challenges and has evolved vspatial organization strategies to increase metabolic pathway flux and efficiency. Inspired by these strategies, bioengineers have developed clever strategies to mimic spatial organization in nature. This review explores the use of spatial organization strategies, including protein scaffolding and protein encapsulation inside of proteinaceous shells, toward overcoming bottlenecks in metabolic engineering efforts. Expected final online publication date for the Annual Review of Biophysics, Volume 52 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Background: Gene silencing by CpG island hypermethylation often plays a role in colorectal cancer (CRC) progression. Certain regions of the genome, called high confidence differentially-methylated regions (DMRs), are consistently hypermethylated across numerous patient samples. Methods: In this study, we used bioinformatics and bisulfite PCR sequencing of HCT-116 cells to investigate methylation levels at DMRs in the promoters of six genes: DKK3, EN1, MiR34b, SDC2, SPG20, and TLX1. We then investigated whether the anti-cancer drug decitabine, had a demethylating effect at these promoter regions. Results: We found that hypermethylation correlated with lack of transcriptional enhancer binding in these six regions. Importantly, we observed that for all DMRs, decitabine significantly reduced CpG methylation. Decitabine also reduced clonogenic survival, suggesting that there is a correlation between lower CpG island methylation levels and reduced cancerous properties. Conclusions: Our study provided single-nucleotide resolution and revealed hypermethylated CpG sites not shown by previous genome-wide methylation studies. In the future, we plan to perform experiments that demonstrate a causal link between promoter hypermethylation and carcinogenesis and that more accurately model treatments in CRC patients.

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Telomeres are protein‐nucleotide sequences at the ends of chromosomes that serve to prevent the loss of genetic information during cell replication. A telomerase ribonucleoprotein complex includes telomerase reverse transcriptase (TERT) and an RNA template (TER) and is responsible for adding telomeric DNA. Our lab has been studying the biogenesis of TER in Aspergillus nidulans, a molecule that is in part structurally similar to that of Saccharomyces cerevisiae and partly to that of mammalian cells. Research in S. cerevisiae has shown that the telomerase RNA appears to leave the nucleus and is potentially assembled in the cytoplasm. Whether TER migrates to the cytoplasm is not known in A. nidulans, but its ability to form a multi‐nucleate state known as a heterokaryon can be used to determine the localization of TER. We assay for the presence of two genetically distinct nuclei via the heterokaryon test, a molecular tool that has allowed us to deduce whether the RNA products for TER and TERT leave the nucleus. We knocked out the genes for TER and TERT, analyzed the results of the heterokaryon test, and determined if assembly of the active enzyme occurs in the nucleus or the cytoplasm. Our results suggest assembly in the nucleus, a scenario that is similar to humans but not to yeast. These results further suggest the potential of A. nidulans as a useful model organism to study the localization of telomerase components.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Telomeres are protein‐nucleotide sequences at the ends of chromosomes that serve to prevent the loss of genetic information during cell replication. A telomerase ribonucleoprotein complex includes telomerase reverse transcriptase (TERT) and an RNA template (TER) and is responsible for adding telomeric DNA. Our lab has been studying the biogenesis of telomerase in the fungus Aspergillus nidulans. Previous research on TER indicated secondary structural similarities to both Saccharomyces cerevisiae and mammalian cells. However, it was previously unknown whether TER migrates into the cytoplasm during the assembly of telomerase, as in S. cerevisiae, or remains in the nucleus, as in mammalian cells. We now have employed a molecular tool, the unique multi‐nucleate state of A. nidulans termed heterokaryon, to deduce whether the RNA products for TER and TERT leave the nucleus. We knocked out the genes for TER and TERT, analyzed the results of the heterokaryon test, determined whether assembly occurs in the nucleus or cytoplasm, and verified those results with DAPI staining. Our results suggest assembly in the nucleus, a scenario that is similar to humans, but not to yeast. These results further suggest the strength of A. nidulans as a model organism in studying the localization of telomerase components.