trans-2-Decenoic acid is a pivotal α,β-medium-chain unsaturated fatty acid that serves as an essential intermediary in the synthesis of 10-hydroxy-2-decenoic acid and various pharmaceutical compounds. Biosynthesis yield of trans-2-decenoic acid by decanoic acid has significantly improved in recent years; however, the oxidative stress of Escherichia coli at high fatty acid concentrations restricts the conversion rate. Here, we introduced a combination of rational design and metabolic rewiring of the E. coli electron transport chain (ETC) to improve trans-2-decenoic acid production. Overexpressing ubiquinone (UbQ) biosynthesis genes enhanced the expression of ETC complex III: UbQ to reduce reactive oxygen species (ROS) accumulation. Furthermore, applying rotenone to inhibit ETC complex I improved the electron transfer efficiency of complex II. The integration of Vitamin B5 and B2 into the fermentation process increased the activities of fatty acyl-CoA synthetase (MaMACS) and fatty acyl-CoA dehydrogenase (PpfadE). Finally, the constructed E. coli BL21(DE3)(ΔfadBJR/pCDFDuet-1-PpfadE-MaMACS/pRSFDuet-1-sumo-CtydiI-ubiI) strain exhibited a 51.50% decrease in ROS and a 93.33% enhancement in trans-2-decenoic acid yield, reaching 1.45 g/L after 66 h, which is the highest yield reported for flask fermentation. This study reports the feasibility of rewiring the ETC regulation and energy metabolism to improve α,β-UCA biosynthesis efficiency.
Pulse generator circuits based on incoherent feed-forward logic have been developed in bacterial, yeast, and mammalian systems but are typically limited to production of short pulses lasting less than 1 day. To generate longer-lasting pulses, we introduce a feedback-based topology that induces multiday pulsatile gene expression with tunable duration and amplitude in mammalian cells. We constructed the circuit using the PERSIST platform, which consists of entirely post-transcriptional logic, because our experience suggests that this approach may attenuate long-term epigenetic silencing. To enable external regulation of PERSIST regulatory elements, we engineered inducer-stabilized CRISPR endoRNases that respond to FDA-approved drugs, generating small molecule responses with greater than 20-fold change. These inducer-responsive proteins were connected to a two-state cross-repression positive feedback topology to generate the pulse generator circuit architecture. We then optimized circuit design through chromosomal integration of circuit components at varying stoichiometries, resulting in a small library of circuits displaying tunable pulses lasting between two and 6 days in response to a single 24 h input of inducer. We expect that the small molecule-stabilized PERSIST proteins developed will serve as valuable components in the toolbox for post-transcriptional gene circuit development and that tunable post-transcriptional pulse generator circuits in mammalian cells will enable study of endogenous hysteretic gene networks and support advances in cell therapies and organoid engineering.
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