DEAttentionDTA: protein-ligand binding affinity prediction based on dynamic embedding and self-attention.

MOTIVATION: Predicting protein-ligand binding affinity is crucial in new drug discovery and development. However, most existing models rely on acquiring 3D structures of elusive proteins. Combining amino acid sequences with ligand sequences and better highlighting active sites are also significant challenges. RESULTS: We propose an innovative neural network model called DEAttentionDTA, based on dynamic word embeddings and a self-attention mechanism, for predicting protein-ligand binding affinity. DEAttentionDTA takes the 1D sequence information of proteins as input, including the global sequence features of amino acids, local features of the active pocket site, and linear representation information of the ligand molecule in the SMILE format. These three linear sequences are fed into a dynamic word-embedding layer based on a 1D convolutional neural network for embedding encoding and are correlated through a self-attention mechanism. The output affinity prediction values are generated using a linear layer. We compared DEAttentionDTA with various mainstream tools and achieved significantly superior results on the same dataset. We then assessed the performance of this model in the p38 protein family. AVAILABILITY AND IMPLEMENTATION: The resource codes are available at https://github.com/whatamazing1/DEAttentionDTA.

Recent advances of affibody molecules in biomedical applications.

Affibody molecules are 58-amino-acid peptides with a molecular weight of about 6.5 kDa, derived from the Z domain of Staphylococcal Protein A. Since they have been used as substitutes for antibodies in biomedicine, several therapeutic affibody molecules have been developed for clinical use. Additionally, affibody molecules have been designed for a range of different applications. This review focuses on the progress made in the last five years in the field of affibody molecules and their potential uses in medical imaging, especially in oncology and cancer treatment. It covers areas such as molecular imaging, targeted delivery of toxic drugs, and their use in combination with nanoparticles. We also highlight some current biomedical applications where affibody molecules are commonly used as a "guide." Due to their many advantages, affibody molecules offer significant potential for applications in both biochemical and medical fields.

A review on machine learning-powered fluorescent and colorimetric sensor arrays for bacteria identification.

Biosensors have been widely used for bacteria determination with great success. However, the "lock-and-key" methodology used by biosensors to identify bacteria has a significant limitation: it can only detect one species of bacteria. In recent years, optical (fluorescent and colorimetric) sensor arrays are gradually gaining attention from researchers as a new type of biosensor. They can acquire multiple features of a target simultaneously, form a feature pattern, and determine the bacteria species with the help of pattern recognition/machine learning algorithms. Previous reviews in this area have focused on the interaction between the sensor array and bacteria or the materials used to make the sensors. This review, on the other hand, will provide researchers with a better understanding of the field by discussing fluorescent and colorimetric sensor arrays based on the mechanism of optical signal generation. These sensor arrays will be compared based on the identified species. Finally, we will discuss the limitations of these sensor arrays and explore possible solutions.

A Single-Component Blue Light-Induced System Based on EL222 in Yarrowia lipolytica.

Optogenetics has the advantages of a fast response time, reversibility, and high spatial and temporal resolution, which make it desirable in the metabolic engineering of chassis cells. In this study, a light-induced expression system of Yarrowia lipolytica was constructed, which successfully achieved the synthesis and functional verification of Bleomycin resistance protein (BleoR). The core of the blue light-induced system, the light-responsive element (TF), is constructed based on the blue photosensitive protein EL222 and the transcription activator VP16. The results show that the light-induced sensor based on TF, upstream activation sequence (C120)5, and minimal promoter CYC102 can respond to blue light and initiate the expression of GFPMut3 report gene. With four copies of the responsive promoter and reporter gene assembled, they can produce a 128.5-fold higher fluorescent signal than that under dark conditions after 8 h of induction. The effects of light dose and periodicity on this system were investigated, which proved that the system has good spatial and temporal controllability. On this basis, the light-controlled system was used for the synthesis of BleoR to realize the expression and verification of functional protein. These results demonstrated that this system has the potential for the transcriptional regulation of target genes, construction of large-scale synthetic networks, and overproduction of the desired product.

Employing Engineered Enolase Promoter for Efficient Expression of Thermomyces lanuginosus Lipase in Yarrowia lipolytica via a Self-Excisable Vector.

Yarrowia lipolytica is progressively being employed as a workhouse for recombinant protein expression. Here, we expanded the molecular toolbox by engineering the enolase promoter (pENO) and developed a new self-excisable vector, and based on this, a combined strategy was employed to enhance the expression of Thermomyces lanuginosus lipase (TLL) in Y. lipolytica. The strength of 11 truncated enolase promoters of different length was first identified using eGFP as a reporter. Seven of the truncated promoters were selected to examine their ability for driving TLL expression. Then, a series of enolase promoters with higher activities were developed by upstream fusing of different copies of UAS1B, and the recombinant strain Po1f/hp16e100-tll harboring the optimal promoter hp16e100 obtained a TLL activity of 447 U/mL. Additionally, a new self-excisable vector was developed based on a Cre/loxP recombination system, which achieved efficient markerless integration in Y. lipolytica. Subsequently, strains harboring one to four copies of the tll gene were constructed using this tool, with the three-copy strain Po1f/3tll showing the highest activity of 579 U/mL. The activity of Po1f/3tll was then increased to 720 U/mL by optimizing the shaking flask fermentation parameters. Moreover, the folding-related proteins Hac1, Pdi, and Kar2 were employed to further enhance TLL expression, and the TLL activity of the optimal recombinant strain Po1f/3tll-hac1-pdi-kar2 reached 1197 U/mL. By using this combined strategy, TLL activity was enhanced by approximately 39.9-fold compared to the initial strain. Thus, the new vector and the combined strategy could be a useful tool to engineer Y. lipolytica for high-level expression of heterologous protein.

A Novel Cre/lox-Based Genetic Tool for Repeated, Targeted and Markerless Gene Integration in Yarrowia lipolytica.

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

Site-Specific Biofunctionalization of Cellulose and Poly(dimethylsiloxane): A Chemoenzymatic Approach for Surface Engineering.

Site-specific, covalent immobilization of protein is of great importance in the design of bioanalytical devices. User-defined covalent coupling of protein onto the surface has been primarily limited to a noncanonical amino acid or cysteine residues. It is desirable to develop a new approach for site-specific biofunctionalization. Herein, we demonstrate a robust and modular chemoenzymatic approach for site-specific, covalent grafting of proteins onto a surface. The synthetic strategy relies on the combination of surface amine functionalization, followed by sortase-mediated coupling. The developed method was validated by site-specific immobilization of two model proteins (glutathione S-transferase and green fluorescent protein) on cellulose and polydimethylsiloxane surfaces via a short recognition motif (LPETG). The covalent coupling of immobilized proteins at the interface was characterized by Fourier Transform Infrared Spectroscopy in attenuated total reflectance mode, X-ray photoelectron spectroscopy, atomic force microscope, and fluorescent microscopy. This enzymatic surface functionalization approach could permit an oriented, homogeneous, and site-specific covalent tethering of LPETG-tag proteins to other materials under mild conditions.

A De Novo Designed Esterase with p-Nitrophenyl Acetate Hydrolysis Activity.

Esterases are a large family of enzymes with wide applications in the industry. However, all esterases originated from natural sources, limiting their use in harsh environments or newly- emerged reactions. In this study, we designed a new esterase to develop a new protocol to satisfy the needs for better biocatalysts. The ideal spatial conformation of the serine catalytic triad and the oxygen anion hole at the substrate-binding site was constructed by quantum mechanical calculation. The catalytic triad and oxygen anion holes were then embedded in the protein scaffold using the new enzyme protocol in Rosetta 3. The design results were subsequently evaluated, and optimized designs were used for expression and purification. The designed esterase had significant lytic activities towards p-nitrophenyl acetate, which was confirmed by point mutations. Thus, this study developed a new protocol to obtain novel enzymes that may be useful in unforgiving environments or novel reactions.

Molecular characterization of the hydroxylase HmtN at 1.3 Å resolution.

Himastatin is a novel antibiotic with antitumor and antibacterial activity. In the himastatin biosynthesis pathway, HmtN is responsible for the hydroxylation of the piperazic acid (Pip) motif. Herein, we present the crystal structures of HmtN (1.3 Å), which is the first structure for a cytochrome P450 involved in the hydroxylation of cyclohexadepsipeptide during himastatin biosynthesis. Structure analysis indicated that almost all the surface of HmtN has negative electrostatic potential, only small patches of positive electrostatic potential can be found. It is worth noting that almost the entire active site of HmtT is negatively charged, while HmtN active site is composed of positive and negative charge. This may be relevant to their specific substrate recognition and different catalytic function. In addition, three channels were observed in HmtN crystal structure; channel 3 may be essential for substrate ingress and egress from the active site to the surface, while channel 1 and channel 2 may be the solvent and water pathway, respectively.

Convolution Neural Network-Based Prediction of Protein Thermostability.

Most natural proteins exhibit poor thermostability, which limits their industrial application. Computer-aided rational design is an efficient purpose-oriented method that can improve protein thermostability. Numerous machine-learning-based methods have been designed to predict the changes in protein thermostability induced by mutations. However, all of these methods have certain limitations due to existing mutation coding methods that overlook protein sequence features. Here we propose a method to predict protein thermostability using convolutional neural networks based on an in-depth study of thermostability-related protein properties. This method comprises a three-dimensional coding algorithm, including protein mutation information and a strategy to extract neighboring features at protein mutation sites based on multiscale convolution. The accuracies on the S1615 and S388 data sets, which are widely used for protein thermostability predictions, reached 86.4 and 87%, respectively. The Matthews correlation coefficient was nearly double those produced using other methods. Furthermore, a model was constructed to predict the thermostability of Rhizomucor miehei lipase mutants based on the S3661 data set, a single amino acid mutation data set screened from the ProTherm protein thermodynamics database. Compared with the RIF strategy, which consists of three algorithms, i.e., Rosetta ddg monomer, I Mutant 3.0, and FoldX, the accuracy of the proposed method was higher (75.0 vs 66.7%), and the negative sample resolution was simultaneously enhanced. These results indicate that our prediction method more effectively assessed the protein thermostability and distinguished its features, making it a powerful tool to devise mutations that enhance the thermostability of proteins, particularly enzymes.

Inline integration of offset MMF-capillary-MMF structure as a portable and compact fiber-optic surface-enhanced Raman scattering microfluidic chip.

A novel fiber-optic surface-enhanced Raman scattering (SERS) microfluidic chip integrated with an embedded Raman probe is presented and demonstrated. The Raman probe consists of the offset-multimode-fiber (MMF)-capillary-MMF (OMCM) structure and SERS substrate. The probe is embedded in the microfluidic channel to form a compact and portable chip. The chip is employed with a fiber coupler in an all-fiber detection system, which has a good stability compared to the free space focusing by the conventional confocal Raman microscope. The excitation light transmits from both ends of the OMCM probe into the capillary, and the generated Raman scattered signals are collected by two MMFs simultaneously. Experimental results of the Rhodamine 6G (R6G) detection show that the Raman signal intensity increases in a linear pattern at ∼1509 cm-1 with the increase of R6G concentrations. This kind of chip is compact, integrated, and miniaturized for the Raman signal detection. Furthermore, it can be fabricated easily in large quantities at cost, rendering promising applications.

A novel eurythermic and thermostale lipase LipM from Pseudomonas moraviensis M9 and its application in the partial hydrolysis of algal oil.

BACKGROUND: Lipases are regularly used in biotechnology to catalyse the hydrolysis of triglycerides and the synthesis of esters. Microbial lipases in particular have been widely used in a variety of industrial applications. However, the current commercial microbial lipases cannot meet industrial demand due to rapid inactivation under harsh conditions. Therefore, in order to identify more stable enzymes, we isolated novel eurythermic and thermostable lipase(s) from Pseudomonas moraviensis M9. METHODS: Cloning of lipM was based on Touchdown PCR and genome walking, and then recombinant LipM was purified by guanidine hydrochloride and the nickel-nitrilotriacetic acid resins affinity chromatography. Finally, the hydrolysis of algal oil by LipM was analyzed by gas chromatograph-mass spectrometer, thin layer chromatography and gas chromatograph. RESULTS: The lipM gene was first cloned from Pseudomonas moraviensis M9 via Touchdown PCR and genome walking. Sequence analysis reveals that LipM is a member of subfamily I.3 of lipases, and the predicted amino acid sequences of LipM has 82 % identity to lipase LipT from Pseudomonas mandelii JR-1, and 54 % identity to lipase PML from Pseudomonas sp. MIS38 and lipase Lip I.3 from Pseudomonas sp. CR-611. LipM was expressed in Escherichia coli, purified from inclusion bodies, and further biochemically characterized. Purified LipM differed significantly from previously reported subfamily I.3 lipases, and was eurythermic between 10 °C-95 °C. LipM activity was enhanced by Ca(2+), Sr(2+), Mn(2+), and Ba(2+), but sharply inhibited by Cu(2+), Zn(2+), Co(2+), Ni(2+), and EDTA. Compared with other lipases, LipM exhibited medium tolerance to methanol, ethanol, and isopropanol. When applied for hydrolysis of algal oil, LipM could enrich 65.88 % polyunsaturated fatty acids, which include 1.25 % eicosapentaenoic acid, 17.61 % docosapentaenoic acid, and 47.02 % docosahexaenoic acid with derivative glycerides containing 32.46 % diacylglycerols. CONCLUSIONS: A novel eurythermic I.3 subfamily lipase with high tolerance and stability was identified from Pseudomonas moraviensis and biochemically characterized. It will not only improve our understanding of subfamily I.3 lipases, but also provides an ideal biocatalyst for the enrichment of polyunsaturated fatty acids. Pseudomonas moraviensis have been investigated as a potential resource of lipases.

An unusual carbon-carbon bond cleavage reaction during phosphinothricin biosynthesis.

Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture. One such compound, phosphinothricin tripeptide, contains the unusual amino acid phosphinothricin attached to two alanine residues. Synthetic phosphinothricin (glufosinate) is a component of two top-selling herbicides (Basta and Liberty), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during phosphinothricin tripeptide biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP). Here we report the in vitro reconstitution of this unprecedented C(sp(3))-C(sp(3)) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-haem iron(ii)-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalysed by the 2-His-1-carboxylate mononuclear non-haem iron family of enzymes.

The two-component GacS-GacA system activates lipA translation by RsmE but not RsmA in Pseudomonas protegens Pf-5.

In Pseudomonas spp., the Gac-Rsm signal transduction system is required for the production of lipases. The current model assumes that the system induces lipase gene transcription mediated through the quorum-sensing (QS) system. However, there are no reports of a QS system based upon N-acyl homoserine lactones or the regulation of lipase gene expression in Pseudomonas protegens. In this study, we investigated the regulatory mechanism acting on lipA expression activated by the Gac-Rsm system in P. protegens Pf-5 through deletion and overexpression of gacA, overexpression of rsmA or rsmE, expression of various lacZ fusions, reverse transcription-PCR analysis, and determination of whole-cell lipase activity. The results demonstrated that the GacS-GacA (GacS/A) system activates lipA expression at both the transcriptional and the translational levels but that the translational level is the key regulatory pathway. Further results showed that the activation of lipA translation by the GacS/A system is mediated through RsmE, which inhibits lipA translation by binding to the ACAAGGAUGU sequence overlapping the Shine-Dalgarno (SD) sequence of lipA mRNA to hinder the access of the 30S ribosomal subunit to the SD sequence. Moreover, the GacS/A system promotes lipA transcription through the mediation of RsmA inhibiting lipA transcription via an unknown pathway. Besides the transcriptional repression, RsmA mainly activates lipA translation by negatively regulating rsmE translation. In summary, in P. protegens Pf-5, the Gac-RsmE system mainly and directly activates lipA translation and the Gac-RsmA system indirectly enhances lipA transcription.

A hydrogel-modified electrochemical biosensor for the rapid detection of ammonia­nitrogen-resistant bacteria.

Ammonia­nitrogen wastewater is one of the main pollutants in the current environment. Rapid detection of microorganisms resistant to ammonia­nitrogen provides a basis for bioremediation of ammonia­nitrogen contaminated sites. This study uses electrochemical analysis for efficiently detecting of ammonia-resistant bacteria, utilizing a commercially available, low-cost screen-printed electrode (SPE) modified with agarose-based hydrogel (gel) or graphene oxide (GO). At the same time, the study employed electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) to monitor bacterial growth, revealing Escherichia coli (E. coli) inhibition upon ammonia­nitrogen addition, while Raoultella terrigena (RN1) and Pseudomonas (RN2) exhibit tolerance. The method provides sensitivity results in <45 min, which is significantly faster than traditional methods. RN1 and RN2 exhibit promising ammonia­nitrogen removal rates, reaching up to 81 % and 92 %, respectively. This study aimed to develop an effective electrochemical method for rapidly detecting the sensitivity of microorganisms to ammonia­nitrogen. The method offers advantages such as high speed, efficiency, and cost-effectiveness, potentially providing valuable microbial resources for mitigating ammonia nitrogen wastewater pollution.

A miniprotein receptor electrochemical biosensor chip based on quantum dots.

Recently protein binders have emerged as a promising substitute for antibodies due to their high specificity and low cost. Herein, we demonstrate an electrochemical biosensor chip through the electronic labelling strategy using lead sulfide (PbS) colloidal quantum dots (CQDs) and the unnatural SARS-CoV-2 spike miniprotein receptor LCB. The unnatural receptor can be utilized as a molecular probe for the construction of CQD-based electrochemical biosensor chips, through which the specific binding of LCB and the spike protein is transduced to sensor electrical signals. The biosensor exhibits a good linear response in the concentration range of 10 pg mL-1 to 1 µg mL-1 (13.94 fM to 1.394 nM) with the limit of detection (LOD) being 3.31 pg mL-1 (4.607 fM for the three-electrode system) and 9.58 fg mL-1 (0.013 fM for the HEMT device). Due to the high sensitivity of the electrochemical biosensor, it was also used to study the binding kinetics between the unnatural receptor LCB and spike protein, which has achieved comparable results as those obtained with commercial equipment. To the best of our knowledge, this is the first example of using a computationally designed miniprotein receptor based on electrochemical methods, and it is the first kinetic assay performed with an electrochemical assay alone. The miniprotein receptor electrochemical biosensor based on QDs is desirable for fabricating high-throughput, large-area, wafer-scale biochips.

Recent advances in the peptide-based biosensor designs.

Biosensors have rapidly emerged as a high-sensitivity and convenient detection method. Among various types of biosensors, optical and electrochemical are the most commonly used. Conventionally, antibodies have been employed to ensure specific interaction between the transmission material and analytes. However, there has been increasing recognition of peptides as a promising recognition element for biosensor development in recent years. The use of peptides as recognition elements provides high level of specificity, sensitivity, and stability for the detection process. The combination of peptide designs and optical or electrochemical detection methods has significantly improved biosensor efficacy. These advancements present opportunities for developing biosensors with diverse functions that can be used to lay a strong scientific foundation for the development of personalized medicine and various other fields. This paper reviews the recent advancements in the development and application of peptide-based optical and electrochemical biosensors, as well as their prospects as a sensor type.

Rapid saline-alkali sensitivity testing using hydrogel/gold nanoparticles-modified screen-printed electrodes.

Rapid screening of microorganisms with good saline-alkali tolerance is of great significance for the improvement of saline-alkali land. In this study, a novel electrochemical method was developed for the rapid screening of saline-alkali-tolerant bacteria using a hydrogel/gold nanoparticles-modified screen-printed electrode. Monitoring bacterial growth using electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) yielded a new method to measure saline-alkali sensitivity. The strains were deposited on agarose hydrogel-AuNPs composite-modified electrodes with saline-alkali treatment control at a concentration of 50 mM. The electrochemical-derived growth curve of each bacterial strain was established to monitor the effect of saline-alkaline conditions on bacterial growth. The results showed that E. coli could grow on the hydrogel-AuNPs composite-modified electrodes without saline and alkali, while the growth of E. coli was inhibited after adding saline and alkali to the modified electrodes. In contrast, Paenibacillus lautus (HC_A) and Lysinibacillus fusiformis (HC_B) were able to grow on electrodes containing saline-alkali hydrogel-AuNPs composite modification. This fast growth curves of the strains derived from electrochemical analysis indicate that the possible time for salinity sensitivity results is <45 min. Compared to the traditional bacterial culture method lasting at least 1-2 days, this method has the clear advantages of rapidity, high efficiency, and low cost.

Development of a novel ultrasound- and biocrosslinking-enhanced immobilization strategy with application to food enzymes.

The increasing demand for greener food production makes biocatalysts more desirable than traditional production approaches. One limiting factor for biocatalyst efficiency is the immobilization strategy. In this work, a novel immobilization method was developed with the tyrosine-tag crosslinking mechanism. The immobilization efficiency was further enhanced with ultrasound treatment. Such a strategy was proven to be efficient with food enzyme lipase, d-amino acid oxidase and glucose dehydrogenase when they were immobilized on macroporous resins, amino resins, epoxy resins, and multiwalled carbon nanotubes. For lipase, glucose dehydrogenase and d-amino acid oxidase, the immobilization yield on macroporous resins increased by 20.4%, 21.1% and 24.1%, respectively. In addition, the immobilized enzymes had enhanced reusability, with a high degree of activity (more than 85%) detected after six cycles. Furthermore, the enzyme electrochemical sensors constructed by enzyme crosslinking have higher sensitivity, with peak currents 4-8 times those of sensors with uncrosslinked enzymes. The enzyme immobilization strategy developed in this study paves the way for better application of biocatalysts in the food industry.

Crystal structures of phosphite dehydrogenase provide insights into nicotinamide cofactor regeneration.

The enzyme phosphite dehydrogenase (PTDH) catalyzes the NAD(+)-dependent conversion of phosphite to phosphate and represents the first biological catalyst that has been shown to conduct the enzymatic oxidation of phosphorus. Despite investigation for more than a decade into both the mechanism of its unusual reaction and its utility in cofactor regeneration, there has been a lack of any structural data for PTDH. Here we present the cocrystal structure of an engineered thermostable variant of PTDH bound to NAD(+) (1.7 Å resolution), as well as four other cocrystal structures of thermostable PTDH and its variants with different ligands (all between 1.85 and 2.3 Å resolution). These structures provide a molecular framework for understanding prior mutational analysis and point to additional residues, located in the active site, that may contribute to the enzymatic activity of this highly unusual catalyst.