As the design of genetic circuitry for synthetic biology becomes more sophisticated, diverse regulatory bioparts are required. Despite their importance, well-characterized 3-untranslated region (3-UTR) bioparts are limited. Thus, transcript 3-ends require further investigation to understand the underlying regulatory role and applications of the 3-UTR. Here, we revisited the use of Term-Seq in the Escherichia coli strain K-12 MG1655 to enhance our understanding of 3-UTR regulatory functions and to provide a diverse collection of tunable 3-UTR bioparts with a wide termination strength range. Comprehensive analysis of 1,629 transcript 3-end positions revealed multiple 3-termini classes generated through transcription termination and RNA processing. The examination of individual Rho-independent terminators revealed a reduction in downstream gene expression over a wide range, which led to the design of novel synthetic metabolic valves that control metabolic fluxes in branched pathways. These synthetic metabolic valves determine the optimal balance of heterologous pathways for maximum target biochemical productivity. The regulatory strategy using 3-UTR bioparts is advantageous over promoter- or 5-UTR-based transcriptional control as it modulates gene expression at transcription levels without trans-acting element requirements (e.g. transcription factors). Our results provide a foundational platform for 3-UTR engineering in synthetic biology applications.

Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here, we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. These genes characterize the oxidative stress, inflammatory niche, and unfolded protein response as important factors for this modulation. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.