Pathway Evolution Through a Bottlenecking-Debottlenecking Strategy and Machine Learning-Aided Flux Balancing.

The evolution of pathway enzymes enhances the biosynthesis of high-value chemicals, crucial for pharmaceutical, and agrochemical applications. However, unpredictable evolutionary landscapes of pathway genes often hinder successful evolution. Here, the presence of complex epistasis is identifued within the representative naringenin biosynthetic pathway enzymes, hampering straightforward directed evolution. Subsequently, a biofoundry-assisted strategy is developed for pathway bottlenecking and debottlenecking, enabling the parallel evolution of all pathway enzymes along a predictable evolutionary trajectory in six weeks. This study then utilizes a machine learning model, ProEnsemble, to further balance the pathway by optimizing the transcription of individual genes. The broad applicability of this strategy is demonstrated by constructing an Escherichia coli chassis with evolved and balanced pathway genes, resulting in 3.65 g L-1 naringenin. The optimized naringenin chassis also demonstrates enhanced production of other flavonoids. This approach can be readily adapted for any given number of enzymes in the specific metabolic pathway, paving the way for automated chassis construction in contemporary biofoundries.

Combining experimental techniques with molecular dynamics to investigate the impact of different enzymatic hydrolysis of ß-lactoglobulin on the antigenicity reduction.

ß-Lactoglobulin (ß-LG) is one of the major food allergens. Enzymatic hydrolysis is a promising strategy to reduce the antigenicity of ß-LG in industrial production. The relationship between the cleavage sites of ß-LG by protease and its antigenic active sites were explored in this study. Molecular docking and molecular dynamics (MD) were used to analyze the active sites and interaction force of ß-LG and IgG antibody. Whey protein was hydrolyzed by four specific enzymes and the antigenicity of the hydrolysates were determined by ELISA. The results of MD showed that the amino acid residue Gln155 (-4.48 kcal mol-1) played the most important roles in the process of binding. Hydrolysates produced by AY-10, which was the only one with specificity towards cleavage sites next to a Gln, had the lowest antigenicity at the same hydrolysis degree. Antigenicity decrease was related to the energy contribution of the cleavage site in the active sites.

Various machine learning approaches coupled with molecule simulation in the screening of natural compounds with xanthine oxidase inhibitory activity.

Gout is a common inflammatory arthritis associated with various comorbidities, such as cardiovascular disease and metabolic syndrome. Xanthine oxidase inhibitors (XOIs) have emerged as effective substances to control gout. Much attention has been given to the search for natural XOIs. In this study, a molecular database of natural XOIs was created for modeling purposes. Quantitative structure-activity relationship models were developed by combining various machine learning approaches and three descriptor pools. The models revealed several features of XOIs, including hydrophobicity and steric molecular structures. Experimental results showed the xanthine oxidase (XO) inhibitory activity of predicted compounds. Vanillic acid was identified as a promising new XOI candidate, with an IC50 of 0.593 µg mL-1. The functions of hydrogen bonds and hydrophobic interactions in XO activity inhibition were confirmed by molecular docking. This study fills knowledge gaps pertaining to the discovery of natural XOIs and to the interaction mechanisms between XOIs and XO.

Modular engineering of Shiraia bambusicola for hypocrellin production through an efficient CRISPR system.

Shiraia bambusicola exhibits an excellent capability to produce high-value pharmacological drugs, such as hypocrellin. However, less effective molecular tools hamper the processes to discover or exploit these metabolites. To address this issue, the more effective CRISPR/Cas9 system was constructed by optimizing the sgRNA transcription elements and disrupting the endogenous non-homologous end-joining pathway. These tactics prompted the gene-targeting frequency of 100% and simultaneously multiplex genome editing in S. bambusicola. This optimal CRISPR system encouraged us to rewire the entire hypocrellin flux and improve the yield by orchestrating the substrate pool supply, the central hypocrellin pathway, and the antioxidant system. Thus, 8632 mg/L hypocrellin was obtained, resulting in a 12-fold increase than that of the wild-type strain. This engineered S. bambusicola can still endure oxidative stresses from higher target metabolites and sustain an excellent biological activity. This study provides a whole conception to establish the more efficient genome-editing system. Higher conserved transcription elements for sgRNA expressions inspire us to adopt this system for gene modifications of other filamentous fungi. The rational and global biosystems outline will offer guidance to modulate metabolite productivity in other filamentous fungi.

A novel feruloyl esterase with high rosmarinic acid hydrolysis activity from Bacillus pumilus W3.

A novel feruloyl esterase (BpFae12) with rosmarinic acid (RA) hydrolysis activity was isolated from Bacillus pumilus W3 and expressed in Escherichia coli BL21 (DE3). With RA as a substrate, the optimal pH and temperature of BpFae12 were pH 8.0 and 50 °C, respectively. The specific enzyme activity was 12.8 U·mg-1. BpFae12 showed the highest activity and substrate affinity toward RA (Vmax of 13.13 U·mg-1, Km of 0.41 mM). Moreover, it also presented strong hydrolysis performance against chlorogenic acid (190.17 U·mg-1). RA was effectively Hydrolyzed into more bioactive caffeic acid and 3,4-dihydroxyphenyllactic acid by BpFae12, which have potential applications in the food industry.