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

Virulence profiling of Shigella flexneri and emergence of serotype 2b as a highly virulent shigellosis causing strain in Pakistan

Bacillary diarrhea caused by Shigella flexneri is mediated by various virulence factors which make it the leading agent of diarrhea in developing countries. Previously, a high prevalence of S. flexneri, associated with diarrhea has been reported in Pakistan but no data is available on their virulence profile. The present study reporting for the first time analysis of various virulence factors among S. flexneri serotypes isolated from clinical (diarrheal stool) and non-clinical (retail raw foods and drinking water) sources. A total of 199 S. flexneri (clinical: 155, raw foods: 22, water: 22) belonging to various serotypes were subjected to virulence genes detection and virulence profiling. The most frequent virulence gene was found to be ipaH (100%), followed by sat (98%), ial (71.3%), set1B (65.8%) and set1A (38.7%). A high level of virulence was detected in serotype 2b as compared to other serotypes as 32.3% of all serotype 2b have the entire set of five virulence genes including ipaH (100%), ial (100%), sat (37.7%), set1A (89.3%), and set1B (100%). Seven different virulence gene profiles (V1 - V7) were detected and the most frequently observed to be V1 (ipaH+, ial+, sat+, set1A+, set1B+) followed by V3 (ipaH+, ial+, sat+, set1B+). The predominant virulence gene pattern in serotype 2b isolated from clinical and non-clinical samples were V1 and V3. Furthermore, about 32% strains belongs to serotype 2b contain the complete set of five virulence genes isolated from patients with high disease severity. In conclusion, the current finding revealed for the first times that serotype 2b was the most virulent strains in both clinical and non-clinical samples in Pakistan. In addition, the virulence of serotype 2b was well correlated with high disease severity