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Biosensors are powerful tools for improving microbial biosynthesis. For example, biosensors can be used to establish feedback control mechanisms to inhibit the growth of low performing cells in a microbial population. In this study, a previously developed biosensor-assisted cell selection system was further engineered to increase the cell selection pressure for improving the bioproduction of 4-hydroxybenzoate (4HB) in E. coli. First, additional sensor protein PobR binding sites were introduced to reduce the binding of PobR controlling the expression of the selection marker gene (antibiotic tetracycline resistance tetA), which stimulated the engineered cells to produce more 4HB to compensate for the tetA expression reduction due to the loss of the PobR binding. Second, the degradation of the PobR sensor protein was promoted using an E. coli ssrA-SspB system to press the cells to make more 4HB. Third, the dosage of antibiotic tetracycline controlling the selection strengths was dynamically increased during the cultivation to align the selection pressure elevation with the cell growth. Our findings show these strategies all effectively improved the 4HB biosynthesis in E. coli and thus demonstrate their potential of wider applications in microbial biosynthesis. • Three strategies were developed to increase the strength of a biosensor-based cell selection mechanism. • Biosynthesis of 4-hydroxybenzoate was used to test the effectiveness of the adopted strategies. • Extra sensor protein binding sites were added to impose higher cell selection pressure. • Accelerating the degradation of the sensor protein also stimulated the cells to produce more product. • The antibiotic dosage was dynamically increased to align the cell selection pressure elevation with the cell growth. [ABSTRACT FROM AUTHOR]

Abstract: Dextromethorphan (DM) is a dextrorotatory morphinan and an over-the-counter non-opioid cough suppressant. We have previously shown that DM protects against LPS-induced dopaminergic neurodegeneration through inhibition of microglia activation. Here, we investigated protective effects of DM against endotoxin shock induced by lipopolysaccharide/d-galactosamine (LPS/GalN) in mice and the mechanism underlying its protective effect. Mice were given multiple injections of DM (12.5mg/kg, s.c.) 30min before and 2, 4h after an injection of LPS/GalN (20μg/700mg/kg). DM administration decreased LPS/GalN-induced mortality and hepatotoxicity, as evidenced by increased survival rate, decreased serum alanine aminotransferase activity and improved pathology. Furthermore, DM was also effective when it was given 30min after LPS/GalN injection. The protection was likely associated with reduced serum and liver tumor necrosis factor alpha (TNF-α) levels. DM also attenuated production of superoxide and intracellular reactive oxygen species in Kupffer cells and neutrophils. Real-time RT-PCR analysis revealed that DM administration suppressed the expression of a variety of inflammation-related genes such as macrophage inflammatory protein-2, CXC chemokine, thrombospondin-1, intercellular adhesion molecular-1 and interleukin-6. DM also decreased the expression of genes related to cell-death pathways, such as the DNA damage protein genes GADD45 and GADD153. In summary, DM is effective in protecting mice against LPS/GalN-induced hepatotoxicity, and the mechanism is likely through a faster TNF-α clearance, and decrease of superoxide production and inflammation and cell-death related components. This study not only extends neuroprotective effect of DM, but also suggests that DM may be a novel compound for the therapeutic intervention for sepsis. [Copyright &y& Elsevier]