Though highly efficient at fermenting hexose sugars, Saccharomyces cerevisiae has limited ability to ferment five-carbon sugars. As a significant portion of sugars found in cellulosic biomass is the five-carbon sugar xylose, S. cerevisiae must be engineered to metabolize pentose sugars, commonly by the addition of exogenous genes from xylose fermenting fungi. However, these recombinant strains grow poorly on xylose and require further improvement through rational engineering or evolutionary adaptation. To identify unknown genes that contribute to improved xylose fermentation in these recombinant S. cerevisiae, we performed genome-wide synthetic interaction screens to identify deletion mutants that impact xylose utilization of strains expressing the xylose isomerase gene XYLA from Piromyces sp. E2 alone or with an additional copy of the endogenous xylulokinase gene XKS1. We also screened the deletion mutant array to identify mutants whose growth is affected by xylose. Our genetic network reveals that more than 80 nonessential genes from a diverse range of cellular processes impact xylose utilization. Surprisingly, we identified four genes, ALP1, ISC1, RPL20B, and BUD21, that when individually deleted improved xylose utilization of both S. cerevisiae S288C and CEN.PK strains. We further characterized BUD21 deletion mutant cells in batch fermentations and found that they produce ethanol even the absence of exogenous XYLA. We have demonstrated that the ability of laboratory strains of S. cerevisiae to utilize xylose as a sole carbon source is suppressed, which implies that S. cerevisiae may not require the addition of exogenous genes for efficient xylose fermentation.

From the first land plants to the complex gymnosperms and angiosperms of today, environmental conditions have forced plants to develop molecular strategies to surpass natural obstacles to growth and proliferation, and these genetic gains have been transmitted to the following generations. In this long natural process, novel and elaborate mechanisms have evolved to enable plants to cope with environmental limitations. Elements in many signalling cascades enable plants to sense different, multiple and simultaneous ambient cues. A group of versatile master regulators of gene expression control plant responses to stressing conditions. For crop breeding purposes, the task is to determine how to activate these key regulators to enable accurate and optimal reactions to common stresses. In this review, we discuss how plants sense biotic and abiotic stresses, how and which master regulators are implied in the responses to these stresses, their evolution in the life kingdoms, and the domains in these proteins that interact with other factors to lead to a proper and efficient plant response.

In this study, sucrase A (SacA) from Bacillus subtilis was successfully displayed on the outer membrane of Escherichia coli via fusion with the AIDA-I autotransporter from E. coli. The pAIDA-sacA plasmid was constructed by fusing sacA with the ctxB signal sequence and the β-barrel domain of aida gene, enabling surface expression under both aerobic and anaerobic conditions. Functional expression of AIDA–SacA was confirmed by the appearance of reducing sugars in enzymatic assays of sucrose hydrolysis and by acid production on phenol red agar. Structural prediction suggested correct localisation of the catalytic domain on the extracellular surface. Enzymatic characterisation revealed that AIDA-SacA exhibits optimal activity at 40 °C and pH 7. The calculated Km for sucrose was 1.18 mM, while the corresponding Vmax was 2.32 U mL⁻¹. Thermal stability assays showed that the enzyme retained over 80% of its activity after 60 min at 45 °C, indicating notable resistance to moderate temperatures. Metal ion assays indicated that K⁺ enhanced enzymatic activity, while Zn²⁺, Cu²⁺, and Mg²⁺ were inhibitory. SDS-PAGE analysis confirmed the expression of the recombinant fusion protein, with a distinct band at approximately 114 kDa corresponding to the expected size. These results demonstrate the feasibility of employing the AIDA-I system for the surface display of SacA in E. coli, providing a functional platform for future applications in whole-cell biocatalysis.

This study aimed to immobilise sucrase A (SacA) from Bacillus subtilis in E. coli using the AIDA-I system for the whole-cell biocatalysis to transform sucrose to lactate. The pAIDA-sacA plasmid, containing the sacA gene, was fused to the signal peptide of the toxin subunit B from Vibrio cholerae (ctxB) and the autotransporter of the aida gene, encoding a connector peptide and the β-barrel domain of the AIDA-I system. This plasmid was employed to transform E. coli strains W3110, WDHFAK, and WDHFAP, which are unable to naturally use sucrose. These strains were anaerobically cultured in batch fermentations using 10 g L⁻¹ sucrose as the sole carbon source. All strains successfully hydrolysed and fermented sucrose, exhibiting a homolactic profile. Among them, WDHFAP/pAIDA-sacA achieved the highest lactic acid titre of 9.84 ± 0.15 g L⁻¹ and a yield of 0.89 ± 0.02 g g⁻¹. Deletion of the mgsA gene in WDHFAP/pAIDA-sacA confirmed that lactic acid production occurred via the methylglyoxal bypass pathway, as lactic acid titres were reduced by over 80%, while ethanol production increased to 4.27 ± 0.26 g L⁻¹. Adaptive laboratory evolution of WDHFAK/pAIDA-sacA was conducted to improve its capacity and fermentation efficiency under elevated sucrose concentrations. The resultant strain, designated as WDHFAK^EV/pAIDA-sacA, consumed up to 65 g L⁻¹ sucrose, achieving 64.61 ± 1.65 g L⁻¹ lactic acid with a yield of 0.99 ± 0.03 g g⁻¹. These findings underscore AIDA-I-mediated SacA immobilisation as a robust strategy for whole-cell biocatalysis, enabling E. coli strains to efficiently ferment sucrose.