Molecular Dynamics Study of the Structure and Mechanical Properties of Spider Silk Proteins.

Spider silk is renowned for its exceptional toughness, with the strongest dragline silk composed of two proteins, MaSp1 and MaSp2, featuring central repetitive sequences and nonrepetitive terminal domains. Although these sequences to spider silk's strength and toughness, the specific roles of MaSp1 and MaSp2 at the atomic level remain unclear. Using AlphaFold3 models and molecular dynamics (MD) simulations, we constructed models of MaSp1 and MaSp2 and validated their stability.

Ancestral Nitrilase Mining and Semi-Rational Engineering for Enhanced Thermal Stability in Rapeseed Meals-Derived Nitriles Degradation.

Rapeseed meal (RSM), a protein-rich byproduct, holds potential as a high-quality animal feed, but nitrile compounds derived from glucosinolates (GSLs) in RSM pose a toxicity risk. Nitrilases, enzymes that hydrolyze toxic nitriles to carboxylic acids, offer a potential solution for detoxification. However, the low thermal stability of nitrilases restricts their industrial applicability.

Design of a Self-Sufficient Whole-Cell Cascade for the Production of ()-Citronellal from Geraniol.

()-Citronellal is a key chiral precursor of high-value chemicals, such as the best-selling flavor compound (-)-menthol; however, the conventional synthesis suffers from low yield and unsatisfactory enantioselectivity. In this study, we developed a highly atom-efficient hydrogen-borrowing cascade for the synthesis of ()-citronellal from geraniol using alcohol dehydrogenase from K12 (AdhP) and ene-reductase from YJM1341 (OYE2p). The key rate-limiting enzyme, AdhP, was subjected to structure-guided semirational engineering, and the triple mutant AdhP (M3) was obtained that demonstrated a 1.

Identification and analysis of the key genes for Escherichia coli heterologous protein expression by transcriptomic profiling.

Background: Escherichia coli is a frequently used host for heterologous protein expression, but its expression efficiency is hindered by several limitations, such as formation of inclusion bodies and proteolytic degradation.

Methods And Results: In this study, we employed high-density fermentation of heterologous protein production in a 5-L bioreactor, resulting in a yield 2.25 times higher than that of the control group.

Inside-Out Rational Design of Ornithine Cyclodeaminase OCD from by a Multiregion Synergy Strategy for Efficient Synthesis of l-Pipecolic Acid.

Lysine cyclodeaminase (LCD)-mediated synthesis of l-pipecolic acid (l-PA) from l-lysine (l-Lys) is a promising approach. However, only one LCD has been reported, and its inadequate activity limits industrial applications. To address this problem, a substrate analogue-guided enzyme mining strategy was employed.

Hypoxia-Inducible Factor-1α-Activated Protein Switch Based on Allosteric Self-Splicing Reduces Nonspecific Cytotoxicity of Pharmaceutical Drugs.

Protein-based therapeutic agents currently used for targeted tumor therapy exhibit limited penetrability, nonspecific toxicity, and a short circulation half-life. Although targeting cell surface receptors improves cancer selectivity, the receptors are also slightly expressed in normal cells; consequently, the nonspecific toxicity of recombinant protein-based therapeutic agents has not been eliminated. In this study, an allosteric-regulated protein switch was designed that achieved cytoplasmic reorganization of engineered immunotoxins in tumor cells via interactions between allosteric self-splicing elements and cancer markers.

Application of plasmid stabilization systems for heterologous protein expression in Escherichia coli.

Background: Plasmids are the most commonly used vectors for heterologous protein expression in Escherichia coli. However, the plasmid copy number decreases with the segregational instability, which inevitably leads to a decrease in the yield of heterologous protein.

Methods And Results: In this study, plasmid stabilization systems were used to enhance the expression level of heterologous proteins in E.

Rational design of short-chain dehydrogenase DHDR for efficient synthesis of (S)-equol.

(S)-equol, the most influential metabolite of daidzein in vivo, has aroused great attention due to the excellent biological activities. Although existing studies have accomplished the construction of its heterologous synthetic pathway in the context of anaerobicity and inefficiency of natural strains, the low productivity of (S)-equol limits its industrial application. Here, rational design strategies based on decreasing the pocket steric hindrance and fine-tuning the pocket microenvironment to systematically redesign the binding pocket of enzyme were developed and processed to the rate-limiting enzyme dihydrodaidzein reductase in (S)-equol synthesis.

Combining binding pocket mutagenesis and substrate tunnel engineering to improve an (R)-selective transaminase for the efficient synthesis of (R)-3-aminobutanol.

(R)-selective transaminases have the potential to act as efficient biocatalysts for the synthesis of important pharmaceutical intermediates. However, their low catalytic efficiency and unfavorable equilibrium limit their industrial application. Seven (R)-selective transaminases were identified using homologous sequence mining.

Engineering an (R)-selective transaminase for asymmetric synthesis of (R)-3-aminobutanol.

(R)-selective transaminases show promise as catalysts for the asymmetric synthesis of chiral amines, which are building blocks of various small molecule drugs. However, their application is limited by poor substrate acceptance and low catalytic efficiency. Here, a potential (R)-selective transaminase from Fodinicurvata sediminis (FsTA) was identified through a substrate truncating strategy, and used as starting point for enzyme engineering toward catalysis of 4-hydroxy-2-butanone, a substrate that poses challenges in catalysis.

CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii.

Komagataella phaffii, a nonconventional yeast, is increasingly attractive to researchers owing to its posttranslational modification ability, strict methanol regulatory mechanism, and lack of Crabtree effect. Although CRISPR-based gene editing systems have been established in K. phaffii, there are still some inadequacies compared to the model organism Saccharomyces cerevisiae.

[Application of live biotherapeutic products and perspective in the treatment of inherited metabolic disease].

Live biotherapeutic products (LBPs) refer to the living bacteria derived from human body intestinal gut or in nature that can be used to treat the human disease. However, the naturally screened living bacteria have some disadvantages, such as deficient therapeutic effect and great divergence, which fall short of the personalized diagnosis and treatment needs. In recent years, with the development of synthetic biology, researchers have designed and constructed several engineered strains that can respond to external complex environmental signals, which speeded up the process of development and application of LBPs.

The identification of a robust leucine dehydrogenase from a directed soil metagenome for efficient synthesis of L-2-aminobutyric acid.

L-2-aminobutyric acid (L-2-ABA) is a chiral precursor for the synthesis of anti-epileptic drug levetiracetam and anti-tuberculosis drug ethambutol. Asymmetric synthesis of L-2-ABA by leucine dehydrogenases has been widely developed. However, the limitations of natural enzymes, such as poor stability, low catalytic efficiency, and inhibition of high-concentration substrates, limit large-scale applications.

Enhancing the Activity of an Alcohol Dehydrogenase by Using "Aromatic Residue Scanning" at Potential Plasticity Sites.

An alcohol dehydrogenase LkADH was successfully engineered to exhibit improved activity and substrate tolerance for the production of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol, an important precursor of ticagrelor. Five potential hotspots were identified for enzyme mutagenesis by using natural residue abundance as an indicator to evaluate their potential plasticity. A semi-rational strategy named "aromatic residue scanning" was applied to randomly mutate these five sites simultaneously by using tyrosine, tryptophan, and phenylalanine as "exploratory residues" to introduce steric hindrance or potential π-π interactions.

Discovery of ER-localized sugar transporters for cellulase production with lac1 being essential.

Background: In the process of cellulose hydrolysis, carbohydrate hydrolysates are transported into cells through membrane transporters, and then affect the expression of cellulase-encoding genes. Sugar transporters play a crucial role in cellulase production in lignocellulolytic fungi, of which relatively few have been functionally validated to date and are all reported to be on cell membrane.

Result: Through transcriptome analysis and qRT-PCR, three putative MFS sugar transporters GST, MFS, and LAC1 were found to display significantly higher mRNA levels in T.

Novel Oxygen-Dependent Degradable Immunotoxin Regulated by the Ubiquitin-Proteasome System Reduces Nonspecific Cytotoxicity.

The use of bacterial toxins as antitumor agents has received considerable attention. Immunotoxins based on antigen recognition of single-chain antibodies have been widely explored for cancer therapy. Despite their impressive killing effect on tumor cells, immunotoxins still display unspecific toxicity with undesired side effects.

Mechanism-Guided Computational Design of ω-Transaminase by Reprograming of High-Energy-Barrier Steps.

ω-Transaminases (ω-TAs) show considerable potential for the synthesis of chiral amines. However, their low catalytic efficiency towards bulky substrates limits their application, and complicated catalytic mechanisms prevent precise enzyme design. Herein, we address this challenge using a mechanism-guided computational enzyme design strategy by reprograming the transition and ground states in key reaction steps.

Roles of PKAc1 and CRE1 in cellulose degradation, conidiation, and yellow pigment synthesis in Trichoderma reesei QM6a.

Purpose: This study aimed to reveal the roles of the protein kinase A catalytic subunit 1 (pkac1) and carbon catabolite repressor cre1 genes in cellulase production by Trichoderma reesei wild-type strain QM6a. Our strategy might be useful to construct a high-yielding cellulase strain for its wide application.

Methods: This paper describes cellulase activity, plate conidiation, and yellow pigment synthesis assays of QM6a with the disruption of pkac1 and cre1.

Induction of cellulase production by Sr in Trichoderma reesei via calcium signaling transduction.

Trichoderma reesei RUT-C30 is a well-known high-yielding cellulase-producing fungal strain that converts lignocellulose into cellulosic sugar for resource regeneration. Calcium is a ubiquitous secondary messenger that regulates growth and cellulase production in T. reesei.

Screening and characterization of a nitrilase with significant nitrile hydratase activity.

Purpose: We screened nitrilases with significant nitrile hydratase activity to exploit their potential in benzylic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on benzylic amide production by nitrilase.

Methods: A sequence-based screening method using previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as "amide-formation hotspots") as molecular probes to identify putative amide-forming nitrilases.

Identification of a novel ene reductase from with potential application in ()-levodione production.

Asymmetric reduction of electronically activated alkenes by ene reductases (ERs) is an attractive approach for the production of enantiopure chiral products. Herein, a novel FMN-binding ene reductase (PaER) from was heterologously expressed in BL21(DE3), and the recombinant enzyme was characterized for its biocatalytic properties. PaER displayed optimal activity at 40 °C and pH 7.

Identification and removal of an inhibitory intermediate to develop an efficient phytosterol bioconversion process using a cyclodextrin-resting cell system.

A classically versatile steroid intermediate, 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD), can be obtained by phytosterol (PS) bioconversion using . In this study, a cyclodextrin-resting cell reaction system with a high concentration of PS (50 g L) was used to produce 9α-OH-AD. However, the inhibitory effect of metabolic intermediates is a key factor limiting production efficiency.