Maximizing protein production by keeping cells at optimal secretory stress levels using real-time control approaches.

Optimizing the production of recombinant proteins is a problem of major industrial and pharmaceutical importance. Secretion of the protein by the host cell considerably simplifies downstream purification processes. However, for many proteins, this is also the limiting production step. Current solutions involve extensive engineering of the chassis cell to facilitate protein trafficking and limit protein degradation triggered by excessive secretion-associated stress. Here, we propose instead a regulation-based strategy in which induction is dynamically adjusted to an optimal strength based on the current stress level of the cells. Using a small collection of hard-to-secrete proteins, a bioreactor-based platform with automated cytometry measurements, and a systematic assay to quantify secreted protein levels, we demonstrate that the secretion sweet spot is indicated by the appearance of a subpopulation of cells that accumulate high amounts of proteins, decrease growth, and face significant stress, that is, experience a secretion burnout. In these cells, adaptations capabilities are overwhelmed by a too strong production. Using these notions, we show for a single-chain antibody variable fragment that secretion levels can be improved by 70% by dynamically keeping the cell population at optimal stress levels using real-time closed-loop control.

Phosphorylation of mRNA-Binding Proteins Puf1 and Puf2 by TORC2-Activated Protein Kinase Ypk1 Alleviates Their Repressive Effects.

Members of the Puf family of RNA-binding proteins typically associate via their Pumilio homology domain with specific short motifs in the 3'-UTR of an mRNA and thereby influence the stability, localization and/or efficiency of translation of the bound transcript. In our prior unbiased proteome-wide screen for targets of the TORC2-stimulated protein kinase Ypk1, we identified the paralogs Puf1/Jsn1 and Puf2 as high-confidence substrates. Earlier work by others had demonstrated that Puf1 and Puf2 exhibit a marked preference for interaction with mRNAs encoding plasma membrane-associated proteins, consistent with our previous studies documenting that a primary physiological role of TORC2-Ypk1 signaling is maintenance of plasma membrane homeostasis. Here, we show, first, that both Puf1 and Puf2 are authentic Ypk1 substrates both in vitro and in vivo. Fluorescently tagged Puf1 localizes constitutively in cortical puncta closely apposed to the plasma membrane, whereas Puf2 does so in the absence of its Ypk1 phosphorylation, but is dispersed in the cytosol when phosphorylated. We further demonstrate that Ypk1-mediated phosphorylation of Puf1 and Puf2 upregulates production of the protein products of the transcripts to which they bind, with a concomitant increase in the level of the cognate mRNAs. Thus, Ypk1 phosphorylation relieves Puf1- and Puf2-mediated post-transcriptional repression mainly by counteracting their negative effect on transcript stability. Using a heterologous protein-RNA tethering and fluorescent protein reporter assay, the consequence of Ypk1 phosphorylation in vivo was recapitulated for full-length Puf1 and even for N-terminal fragments (residues 1-340 and 143-295) corresponding to the region upstream of its dimerization domain (an RNA-recognition motif fold) encompassing its two Ypk1 phosphorylation sites (both also conserved in Puf2). This latter result suggests that alleviation of Puf1-imposed transcript destabilization does not obligatorily require dissociation of Ypk1-phosphorylated Puf1 from a transcript. Our findings add new insight about how the TORC2-Ypk1 signaling axis regulates the content of plasma membrane-associated proteins to promote maintenance of the integrity of the cell envelope.

Colistin Treatment Affects Lipid Composition of Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii (A. baumannii) causes severe and often fatal healthcare-associated infections due partly to antibiotic resistance. There are no studies on A. baumannii lipidomics of susceptible and resistant strains grown at lethal and sublethal concentrations. Therefore, we analyzed the impact of colistin resistance on glycerolipids' content by using untargeted lipidomics on clinical isolate. Nine lipid sub-classes were annotated, including phosphatidylcholine, rarely detected in the bacterial membrane among 130 different lipid species. The other lipid sub-classes detected are phosphatidylethanolamine (PE), phosphatidylglycerol (PG), lysophosphatidylethanolamine, hemibismonoacylglycerophosphate, cardiolipin, monolysocardiolipin, diacylglycerol, and triacylglycerol. Under lethal and sublethal concentrations of colistin, significant reduction of PE was observed on the resistant and susceptible strain, respectively. Palmitic acid percentage was higher at colistin at low concentration but only for the susceptible strain. When looking at individual lipid species, the most abundant PE and PG species (PE 34:1 and PG 34:1) are significantly upregulated when the susceptible and the resistant strains are cultivated with colistin. This is, to date, the most exhaustive lipidomics data compilation of A. baumannii cultivated in the presence of colistin. This work is highlighting the plasma membrane plasticity used by this gram-negative bacterium to survive colistin treatment.