Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Smart Mat for Respiratory Activity Detection: Study in a Clinical Setting

We discuss in this paper a study of a smart and unobtrusive mattress in a clinical setting on a population with cardiorespiratory problems. Up to recently, the vast majority of studies with unobtrusive sensors are done with healthy populations. The unobtrusive monitoring of the Respiratory Rate (RR) is essential for proposing better diagnoses. Thus, new industrial and research activity on smart mattresses is targeting respiratory rate in an Internet-of-Things (IoT) context. In our work, we are interested in the performances of a microbend fiber optic sensor (FOS) mattress on 81 subjects admitted in the Cardiac Intensive Care Unit (CICU) by estimating the RR from their ballistocardiograms (BCG). Our study proposes a new RR estimator, based on harmonic plus noise models (HNM) and compares it with known estimators such as MODWT and CLIE. The goal is to examine, using a more representative and bigger dataset, the performances of these methods and of the smart mattress in general. Results of applying these three estimators on the BCG show that MODWT is more accurate with an average mean absolute error (MAE) of 1.97 ± 2.12 BPM. However, the HNM estimator has space for improvements with estimation errors of 2.91 ± 4.07 BPM. The smart mattress works well within a standard RR range of 10–20 breaths-per-minute (BPM) but gets less accurate with a bigger range of estimation. These results highlight the need to test these sensors in much more realistic contexts

Trigger factor interacts with the signal peptide of nascent Tat substrates but does not play a critical role in tat-mediated export.

Twin-arginine translocation (Tat)-mediated protein transport across the bacterial cytoplasmic membrane occurs only after synthesis and folding of the substrate protein that contains a signal peptide with a characteristic twin-arginine motif. This implies that premature contact between the Tat signal peptide and the Tat translocon in the membrane must be prevented. We used site-specific photo-crosslinking to demonstrate that the signal peptide of nascent Tat proteins is in close proximity to the chaperone and peptidyl-prolyl isomerase trigger factor (TF). The contact with TF was strictly dependent on the context of the translating ribosome, started early in biogenesis when the nascent chain left the ribosome near L23, and persisted until the chain reached its full length. Despite this exclusive and prolonged contact, depletion or overexpression of TF had little effect on the kinetics and efficiency of the Tat export process