The cyanobacterium Synechococcus elongatus PCC 7942 is a potential photosynthetic cell-factory. In this study, two native promoters from S. elongatus PCC 7942 driving the expression of abundant cyanobacterial proteins phycocyanin (P cpcB7942 ) and RuBisCO (P rbc7942 ) were characterized in relation to their sequence features, expression levels, diurnal behavior, and regulation by light and CO 2 , major abiotic factors important for cyanobacterial growth. P cpcB7942 was repressed under high light intensity, but cultivation at higher CO 2 concentration was able to recover promoter activity. On the other hand, P rbc7942 was repressed by elevated CO 2 with a negative regulatory region between 300 and 225 bp. Removal of this region flipped the effect of CO 2 with Rbc225 being activated only at high CO 2 concentration, besides leading to the loss of circadian rhythm. The results from this study on promoter features and regulation will help expand the repertoire of tools for pathway engineering in cyanobacteria.

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the development of new antibacterials. Penicillin V acylases (PVAs) belong to the Ntn hydrolase superfamily, together with AHL acylases. PVAs are exploited widely in the pharmaceutical industry, but their role in the natural physiology of their native microbes is not clearly understood. This report details the characterization of AHL degradation activity by homotetrameric PVAs from two gram-negative plant pathogenic bacteria, Pectobacterium atrosepticum (PaPVA) and Agrobacterium tumefaciens (AtPVA). Both the PVAs exhibited substrate specificity for degrading long-chain AHLs. Exogenous addition of these enzymes into Pseudomonas aeruginosa greatly diminished the production of elastase and pyocyanin and biofilm formation and increased the survival rate in an insect model of acute infection. Subtle structural differences in the PVA active site that regulate specificity for acyl chain length have been characterized, which could reflect the evolution of AHL-degrading acylases in relation to the environment of the bacteria that produce them and also provide strategies for enzyme engineering. The potential for using these enzymes as therapeutic agents in clinical applications and a few ideas about their possible significance in microbial physiology have also been discussed.

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Highlights True fed-batch strategy for high cell density cultivation of E. coli in shake flask. Cybernetic model-based optimization of the feeding recipe. Biomass of 19.9–21.5 g DCW/L, in agreement with the model prediction. Volumetric productivity for tested proteins increased 8–34-fold compared to batch. Scale up of fed-batch recipe to bioreactor resulted in further 2.8-fold increase.