Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications

Lignocellulosic biomass yields after hydrolysis, besides the hexose D-glucose, D-xylose, and L-arabinose as main pentose sugars. In second generation bioethanol production utilizing the yeast Saccharomyces cerevisiae, it is critical that all three sugars are co-consumed to obtain an economically feasible and robust process. Since S. cerevisiae is unable to metabolize pentose sugars, metabolic pathway engineering has been employed to introduce the respective pathways for D-xylose and L-arabinose metabolism. However, S. cerevisiae lacks specific pentose transporters, and these sugars enter the cell with low affinity via glucose transporters of the Hxt family. Therefore, in the presence of D-glucose, utilization of D-xylose and L-arabinose is poor as the Hxt transporters prefer D-glucose. To solve this problem, heterologous expression of pentose transporters has been attempted but often with limited success due to poor expression and stability, and/or low turnover. A more successful approach is the engineering of the endogenous Hxt transporter family and evolutionary selection for D-glucose insensitive growth on pentose sugars. This has led to the identification of a critical and conserved asparagine residue in Hxt transporters that, when mutated, reduces the D-glucose affinity while leaving the D-xylose affinity mostly unaltered. Likewise, mutant Gal2 transporter have been selected supporting specific uptake of L-arabinose. In fermentation experiments, the transporter mutants support efficient uptake and consumption of pentose sugars, and even co-consumption of D-xylose and D-glucose when used at industrial concentrations. Further improvements are obtained by interfering with the post-translational inactivation of Hxt transporters at high or low D-glucose concentrations. Transporter engineering solved major limitations in pentose transport in yeast, now allowing for co-consumption of sugars that is limited only by the rates of primary metabolism. This paves the way for a more economical second-generation biofuels production process

Combined roles of exporters in acetic acid tolerance in Saccharomyces cerevisiae

Acetic acid is a growth inhibitor generated during alcoholic fermentation and pretreatment of lignocellulosic biomass, a major feedstock to produce bioethanol. An understanding of the acetic acid tolerance mechanisms is pivotal for the industrial production of bioethanol. One of the mechanisms for acetic acid tolerance is transporter-mediated secretion where individual transporters have been implicated. Here, we deleted the transporters Aqr1, Tpo2, and Tpo3, in various combinations, to investigate their combined role in acetic acid tolerance. Single transporter deletions did not impact the tolerance at mild acetic acid stress (20 mM), but at severe stress (50 mM) growth was decreased or impaired. Tpo2 plays a crucial role in acetic acid tolerance, while the AQR1 deletion has a least effect on growth and acetate efflux. Deletion of both Tpo2 and Tpo3 enhanced the severe growth defects at 20 mM acetic acid concomitantly with a reduced rate of acetate secretion, while TPO2 and/or TPO3 overexpression in ∆tpo2∆tpo3∆ restored the tolerance. In the deletion strains, the acetate derived from sugar metabolism accumulated intracellularly, while gene transcription analysis suggests that under these conditions, ethanol metabolism is activated while acetic acid production is reduced. The data demonstrate that Tpo2 and Tpo3 together fulfill an important role in acetate efflux and the acetic acid response

Bacterial multi-solute transporters

Bacterial membrane proteins of the SbmA/BacA family are multi-solute transporters that mediate the uptake of structurally diverse hydrophilic molecules, including aminoglycoside antibiotics and antimicrobial peptides. Some family members are full-length ATP-binding cassette (ABC) transporters, whereas other members are truncated homologues that lack the nucleotide-binding domains and thus mediate ATP-independent transport. A recent cryo-EM structure of the ABC transporter Rv1819c from Mycobacterium tuberculosis has shed light on the structural basis for multi-solute transport and has provided insight into the mechanism of transport. Here, we discuss how the protein architecture makes SbmA/BacA family transporters prone to inadvertent import of antibiotics and speculate on the question which physiological processes may benefit from multi-solute transport

TMS-Induced Central Motor Conduction Time at the Non-Infarcted Hemisphere Is Associated with Spontaneous Motor Recovery of the Paretic Upper Limb after Severe Stroke

Background: Stroke affects the neuronal networks of the non-infarcted hemisphere. The central motor conduction time (CMCT) induced by transcranial magnetic stimulation (TMS) could be used to determine the conduction time of the corticospinal tract of the non-infarcted hemisphere after a stroke. Objectives: Our primary aim was to demonstrate the existence of prolonged CMCT in the non-infarcted hemisphere, measured within the first 48 h when compared to normative data, and secondly, if the severity of motor impairment of the affected upper limb was significantly associated with prolonged CMCTs in the non-infarcted hemisphere when measured within the first 2 weeks post stroke. Methods: CMCT in the non-infarcted hemisphere was measured in 50 patients within 48 h and at 11 days after a first-ever ischemic stroke. Patients lacking significant spontaneous motor recovery, so-called non-recoverers, were defined as those who started below 18 points on the FM-UE and showed less than 6 points (10%) improvement within 6 months. Results: CMCT in the non-infarcted hemisphere was prolonged in 30/50 (60%) patients within 48 h and still in 24/49 (49%) patients at 11 days. Sustained prolonged CMCT in the non-infarcted hemisphere was significantly more frequent in non-recoverers following FM-UE. Conclusions: The current study suggests that CMCT in the non-infarcted hemisphere is significantly prolonged in 60% of severely affected, is-chemic stroke patients when measured within the first 48 h post stroke. The likelihood of CMCT is significantly higher in non-recoverers when compared to those that show spontaneous motor recovery early post stroke

Efficient, D-glucose insensitive, growth on D-xylose by an evolutionary engineered Saccharomyces cerevisiae strain

Optimizing D-xylose consumption in Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. An evolutionary engineering approach was used to elevate D-xylose consumption in a xylose-fermenting S. cerevisiae strain carrying the D-xylose-specific N367I mutation in the endogenous chimeric Hxt36 hexose transporter. This strain carries a quadruple hexokinase deletion that prevents glucose utilization, and allows for selection of improved growth rates on D-xylose in the presence of high D-glucose concentrations. Evolutionary engineering resulted in D-glucose-insensitive growth and consumption of D-xylose which could be attributed to glucose insensitive D-xylose uptake via a novel chimeric Hxt37 N367I transporter that emerged from a fusion of the HXT36 and HXT7 genes, and a down regulation of a set of Hxt transporters that mediate glucose sensitive xylose transport. RNA sequencing revealed the down-regulation of HXT1 and HXT2 which, together with the deletion of HXT7, resulted in a 21% reduction of the expression of all plasma membrane transporters genes. Morphological analysis showed an increased cell size and corresponding increased cell surface area of the evolved strain, which could be attributed to genome duplication. Mixed strain fermentation of the D-xylose-consuming strain DS71054-evo6 with the D-glucose consuming CEN.PK113-7D strain resulted in decreased residual sugar concentrations and improved ethanol production yields compared to a strain which sequentially consumes D-glucose and D-xylose

Virtual Reality Relaxation for Reducing Perceived Stress of Intensive Care Nurses During the COVID-19 Pandemic

During the COVID-19 pandemic ICU nurses endure high levels of stress. VR relaxation (VRelax, containing 360° immersive environments) provides an easy-to-use and effective means to induce positive affect and reduce perceived stress. We investigated feasibility and immediate effect on perceived stress of VRelax use by ICU nurses during work shifts. ICU nurses working with COVID-19 patients in an academic hospital could use VRelax as a 10-min break during their shift. Primary outcome was the difference between perceived stress immediately before and after VRelax use measured by a single-question VAS-stress scale. Statistically significant difference of the mean VAS-stress before and after use was determined using the paired t student test. A socio-demographic questionnaire, a questionnaire on perceived stress and stress resilience and VRelax user experiences were sent by email. Eighty-six (26%) nurses used VRelax at least once; 77% (N=66) of these filled out the VAS-stress scale before and after use of VRelax. Mean perceived stress lowered with 39.9% after use of VRelax (mean difference=14.0, SD=13.3, p<0.005). Mean score on the perceived stress scale-10 was 11.4 (SD=6.50), mean score on the Connor-Davidson Resilience Scale-10 was 29.0 (SD=5.51). Sixty-two percentage of the ICU nurses thought VRelax was helpful to reduce stress. Main barrier for use was a high workload. It is feasible for nurses to use VRelax in an ICU context. VRelax is an effective intervention to reduce immediate perceived stress and is of added value in stressful situations as during the COVID-19 pandemic, inducing a positive affective state and lowering perceived stress