Searching the Sequence Space for Potent Aptamers Using SELEX in Silico

To isolate functional nucleic acids that bind to defined targets with high affinity and specificity, which are known as aptamers, the systematic evolution of ligands by exponential enrichment (SELEX) methodology has emerged as the preferred approach. Here, we propose a computational approach, SELEX in silico, that allows the sequence space to be more thoroughly explored regarding binding of a certain target. Our approach consists of two steps: (i) secondary structure-based sequence screening, which aims to collect the sequences that can form a desired RNA motif as an enhanced initial library, followed by (ii) sequence enrichment regarding target binding by molecular dynamics simulation-based virtual screening. Our SELEX in silico method provided a practical computational solution to three key problems in aptamer sequence searching: design of nucleic acid libraries, knowledge of sequence enrichment, and identification of potent aptamers. Six potent theophylline-binding aptamers, which were isolated by SELEX in silico from a sequence space containing 4¹³ sequences, were experimentally verified to bind theophylline with high affinity: <i>K</i>_d ranging from 0.16 to 0.52 μM, compared with the dissociation constant of the original aptamer-theophylline, 0.32 μM. These results demonstrate the significant potential of SELEX in silico as a new method for aptamer discovery and optimization

Insights into the Biogenic Amine Metabolic Landscape during Industrial Semidry Chinese Rice Wine Fermentation

Inspired by concerns about food safety, the metabolic landscape of biogenic amines (BAs) was elucidated during industrial semidry Chinese rice wine fermentation. The main fermentation process represented the largest contribution to BA formation, which corresponded to 69.1% (54.3 mg/L). Principal component analysis revealed that total acid and ethanol were strongly correlated with BAs, indicating that BA formation favored acidic and stressful conditions. Other than putrescine (PUT), spermidine (SPD), and spermine (SPM), 5 BAs exhibited strong relationships with the precursor amino acids (<i>R</i>² > 0.85). PUT was mainly decarboxylated from arginine (89.6%) whereas SPD (100%) and SPM (83.1%) were obtained from ornithine. Interestingly, some SPD could convert back to PUT (24.3%). All 8 BAs showed good relationships with lactic acid bacteria (LAB) (<i>R</i>² around 0.75). Moreover, among the five main LAB genera, <i>Lactobacillus</i> had a positive correlation with BA formation

Mixed Starter Culture Regulates Biogenic Amines Formation via Decarboxylation and Transamination during Chinese Rice Wine Fermentation

The utilization of amine-negative starter based on an understanding of nitrogen metabolism is a useful method for controlling biogenic amine (BA) in Chinese rice wine (CRW) fermentation. The contribution of brewing materials to protein degradation was analyzed; wheat Qu protein had no effect, and yeast autolysis generated 10% amino nitrogen. Milling degree of rice was strongly correlated with BAs formation (<i>R</i>² = 0.99). Subsequently, Lactobacillus plantarum and Staphylococcus xylosus were coinoculated as amine-negative starter at an optimized ratio of 1:2. Coinoculation induced a significant reduction in total BAs (43.7%, 44.5 mg L^–1), putrescine (43.0%, 20.4 mg L^–1), tyramine (42.8%, 14.3 mg L^–1), and histamine (42.6%, 3.5 mg L^–1) content. Notably, BAs degradation ability of Staphylococcus xylosus was stronger than the suppression effect of Lactobacillus plantarum, and higher lactic acid bacteria (LAB) amount has a positive correlation with lower BAs content. Overall, mixed strains exerted a synergistic effect in lowering BAs accumulation via decarboxylation and transamination

Protein-Glutaminase Engineering Based on Isothermal Compressibility Perturbation for Enhanced Modification of Soy Protein Isolate

Protein-glutaminase plays a significant role in future food (e.g., plant-based meat) processing as a result of its ability to improve the solubility, foaming, emulsifying, and gel properties of plant-based proteins. However, poor stability, activity, high pressure, and high shear processing environments hinder its application. Therefore, we developed an application-oriented method isothermal compressibility perturbation engineering strategy to improve enzyme performance by simulating the high-pressure environment. The best variant with remarkable improvement in specific activity and half-time, N16M/Q21H/T113E, exhibited a 4.28-fold increase compared to the wild type in specific activity (117.18 units/mg) and a 1.23-fold increase in half-time (472 min), as one of the highest comprehensive performances ever reported. The solubility of the soy protein isolate deaminated by the N16M/Q21H/T113E mutant was 55.74% higher than that deaminated by the wild type, with a tinier particle size and coarser texture. Overall, this strategy has the potential to improve the functional performance of enzymes under complex food processing conditions

Isothermal Compressibility Perturbation as a Protein Design Principle for T1 Lipase Stability–Activity Trade-Off Counteracting

Given the widely existing stability–activity trade-off in enzyme evolution, it is still a goal to obtain enzymes embracing both high activity and stability. Herein, we employed an isothermal compressibility (βT) perturbation engineering (ICPE) strategy to comprehensively understand the stability–activity seesaw-like mechanism. The stability and activity of mutants derived from ICPE uncovered a high Pearson correlation (r = 0.93) in a prototypical enzyme T1 lipase. The best variant A186L/L188M/A190Y exhibited a high Tm value up to 78.70 °C, catalytic activity of 474.04 U/mg, and a 73.33% increase in dimethyl sulfoxide resistance compared to the wild type, one of the highest comprehensive performances reported to date. The elastic activation mechanism mediated by conformational change with a ΔβT range of −6.81 × 10–6 to −1.90 × 10–6 bar–1 may account for the balancing of stability and activity to achieve better performing enzymes. The ICPE strategy deepens our understanding of stability–activity trade-off and boosts its applications in enzyme engineering

Computer-Aided Reconstruction and Application of <i>Bacillus halodurans</i> S7 Xylanase with Heat and Alkali Resistance

β-1,4-Endoxylanase is the most critical hydrolase for xylan degradation during lignocellulosic biomass utilization. However, its poor stability and activity in hot and alkaline environments hinder its widespread application. In this study, BhS7Xyl from Bacillus halodurans S7 was improved using a computer-aided design through isothermal compressibility (βT) perturbation engineering and by combining three thermostability prediction algorithms (ICPE-TPA). The best variant with remarkable improvement in specific activity, heat resistance (70 °C), and alkaline resistance (both pH 9.0 and 70 °C), R69F/E137M/E145L, exhibited a 4.9-fold increase by wild-type in specific activity (1368.6 U/mg), a 39.4-fold increase in temperature half-life (458.1 min), and a 57.6-fold increase in pH half-life (383.1 min). Furthermore, R69F/E137M/E145L was applied to the hydrolysis of agricultural waste (corncob and hardwood pulp) to efficiently obtain a higher yield of high-value xylooligosaccharides. Overall, the ICPE-TPA strategy has the potential to improve the functional performance of enzymes under extreme conditions for the high-value utilization of lignocellulosic biomass

Exploring the Mutational Robustness of Nucleic Acids by Searching Genotype Neighborhoods in Sequence Space

To assess the mutational robustness of nucleic acids, many genome- and protein-level studies have been performed, where nucleic acids are treated as genetic information carriers and transferrers. However, the molecular mechanisms through which mutations alter the structural, dynamic, and functional properties of nucleic acids are poorly understood. Here we performed a SELEX in silico study to investigate the fitness distribution of the l-Arm-binding aptamer genotype neighborhoods. Two novel functional genotype neighborhoods were isolated and experimentally verified to have comparable fitness as the wild-type. The experimental aptamer fitness landscape suggests the mutational robustness is strongly influenced by the local base environment and ligand-binding mode, whereas bases distant from the binding pocket provide potential evolutionary pathways to approach the global fitness maximum. Our work provides an example of successful application of SELEX in silico to optimize an aptamer and demonstrates the strong sensitivity of mutational robustness to the site of genetic variation

Directed Evolution of the UDP-Glycosyltransferase UGT_BL1 for Highly Regioselective and Efficient Biosynthesis of Natural Phenolic Glycosides

The O-glycosylation of polyphenols for the synthesis of glycosides has garnered substantial attention in food research applications. However, the practical utility of UDP-glycosyltransferases (UGTs) is significantly hindered by their low catalytic efficiency and suboptimal regioselectivity. The concurrent optimization of the regioselectivity and activity during the glycosylation of polyphenols presents a formidable challenge. Here, we addressed the long-standing activity–regioselectivity tradeoff in glycosyltransferase UGTBL1 through systematic enzyme engineering. The optimal combination of mutants, N61S/I62M/D63W/A208R/P218W/R282W (SMWRW1W2), yielded a 6.1-fold improvement in relative activity and a 17.3-fold increase in the ratio of gastrodin to para-hydroxybenzyl alcohol-4′-O-β-glucoside (with 89.5% regioselectivity for gastrodin) compared to those of the wild-type enzyme and ultimately allowed gram-scale production of gastrodin (1,066.2 mg/L) using whole-cell biocatalysis. In addition, variant SMWRW1W2 exhibited a preference for producing phenolic glycosides from several substrates. This study lays the foundation for the engineering of additional UGTs and the practical applications of UGTs in regioselective retrofitting