Structural and Functional Analysis of Validoxylamine A 7′-phosphate Synthase ValL Involved in Validamycin A Biosynthesis

Validamycin A (Val-A) is an effective antifungal agent widely used in Asian countries as crop protectant. Validoxylamine A, the core structure and intermediate of Val-A, consists of two C7-cyclitol units connected by a rare C-N bond. In the Val-A biosynthetic gene cluster in Streptomyces hygroscopicus 5008, the ORF valL was initially annotated as a validoxylamine A 7′-phosphate(V7P) synthase, whose encoded 497-aa protein shows high similarity with trehalose 6-phosphate(T6P) synthase. Gene inactivation of valL abolished both validoxylamine A and validamycin A productivity, and complementation with a cloned valL recovered 10% production of the wild-type in the mutant, indicating the involvement of ValL in validoxylamine A biosynthesis. Also we determined the structures of ValL and ValL/trehalose complex. The structural data indicates that ValL adopts the typical fold of GT-B protein family, featuring two Rossmann-fold domains and an active site at domain junction. The residues in the active site are arranged in a manner homologous to that of Escherichia coli (E.coli) T6P synthase OtsA. However, a significant discrepancy is found in the active-site loop region. Also noticeable structural variance is found around the active site entrance in the apo ValL structure while the region takes an ordered configuration upon binding of product analog trehalose. Furthermore, the modeling of V7P in the active site of ValL suggests that ValL might have a similar SNi-like mechanism as OtsA

Microbial Consortia Are Needed to Degrade Soil Pollutants

Soil pollution is one of the most serious environmental problems globally due to the weak self-purification ability, long degradation time, and high cost of cleaning soil pollution. The pollutants in the soil can be transported into the human body through water or dust, causing adverse effects on human health. The latest research has shown that the clean-up of soil pollutants through microbial consortium is a very promising method. This review provides an in-depth discussion on the efficient removal, bio-adsorption, or carbonated precipitation of organic and inorganic pollutants by the microbial consortium, including PAHs, BPS, BPF, crude oil, pyrene, DBP, DOP, TPHP, PHs, butane, DON, TC, Mn, and Cd. In view of the good degradation ability of the consortium compared to single strains, six different synergistic mechanisms and corresponding microorganisms are summarized. The microbial consortium obtains such activities through enhancing synergistic degradation, reducing the accumulation of intermediate products, generating the crude enzyme, and self-regulating, etc. Furthermore, the degradation efficiency of pollutants can be greatly improved by adding chemical materials such as the surfactants Tween 20, Tween 80, and SDS. This review provides insightful information regarding the application of microbial consortia for soil pollutant removal

Recent Advances in Function-based Metagenomic Screening

Metagenomes from uncultured microorganisms are rich resources for novel enzyme genes. The methods used to screen the metagenomic libraries fall into two categories, which are based on sequence or function of the enzymes. The sequence-based approaches rely on the known sequences of the target gene families. In contrast, the function-based approaches do not involve the incorporation of metagenomic sequencing data and, therefore, may lead to the discovery of novel gene sequences with desired functions. In this review, we discuss the function-based screening strategies that have been used in the identification of enzymes from metagenomes. Because of its simplicity, agar plate screening is most commonly used in the identification of novel enzymes with diverse functions. Other screening methods with higher sensitivity are also employed, such as microtiter plate screening. Furthermore, several ultra-high-throughput methods were developed to deal with large metagenomic libraries. Among these are the FACS-based screening, droplet-based screening, and the in vivo reporter-based screening methods. The application of these novel screening strategies has increased the chance for the discovery of novel enzyme genes. Keywords: Metagenomics, Function-based screening, Agar plate screening, Microfluidics, FACS-based screenin

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

Biological Nitrogen Removal Database: A Manually Curated Data Resource

Biological nitrogen removal (BNR) technologies are the most effective approaches for the remediation of environmental nitrogen pollutants from wastewater treatment plants (WWTPs). Presently, research is going on to elucidate the structure and function of BNR microbial communities and optimizing BNR treatment systems to enhance nitrogen removal efficiency. The literature on BNR microbial communities and experimental datasets is not unified across various repositories, while a uniform resource for the collection, annotation, and structuring of these BNR datasets is still unavailable. Herein, we present the Biological Nitrogen Removal Database (BNRdb), an integrated resource containing various manually curated BNR-related data. At present, BNRdb contains 23,308 microbial strains, 46 gene families, 24 enzymes, 18 reactions, 301 BNR treatment datasets, 860 BNR-associated next-generation sequencing datasets, and 6 common BNR bioreactor systems. BNRdb provides a user-friendly interface enabling interactive data browsing. To our knowledge, BNRdb is the first BNR data resource that systematically integrates BNR data from archaeal, bacterial, and fungal communities. We believe that BNRdb will contribute to a better understanding of BNR process and nitrogen bioremediation research

ImDeeplabV3plus with instance selective whitening loss in domain generalization semantic segmentation

Abstract Semantic segmentation is a classical problem in computer vision, which is important in the field of autonomous driving. Although significant progress has been achieved in semantic segmentation, its generalization ability to unknown domains is still challenging. To effectively solve this problem, a semantic segmentation method ImDeeplabV3plus with instance selective whitening loss is proposed in this paper. DeeplabV3plus is selected as the baseline. In order to enhance the representation of the region of interest, the coordinate attention (CA) mechanism is added. To better integrate multiple low‐level features, the adaptively spatial feature fusion (ASFF) is employed to adaptively learn the importance of features at different levels for each location. For preferably coping with the domain changes, an instance selective whitening (ISW) loss is introduced in the early stage of the backbone. The model is trained with the Cityscapes dataset and then applied to the unknown domain RobotCar dataset. Compared with DeeplabV3plus, the authors’ ImDeeplabV3plus model shows 1.29% mIoU improvement. When ISW loss is added, 2.08% improvement in mIoU is achieved compared with ImDeeplabV3plus. Experimental results show that the proposed method is simple and improves the domain generalization ability

Analyses of the Binding between Water Soluble C60 Derivatives and Potential Drug Targets through a Molecular Docking Approach.

Fullerene C60, a unique sphere-shaped molecule consisting of carbon, has been proved to have inhibitory effects on many diseases. However, the applications of C60 in medicine have been severely hindered by its complete insolubility in water and low solubility in almost all organic solvents. In this study, the water-soluble C60 derivatives and the C60 binding protein's structures were collected from the literature. The selected proteins fall into several groups, including acetylcholinesterase, glutamate racemase, inosine monophosphate dehydrogenase, lumazine synthase, human estrogen receptor alpha, dihydrofolate reductase and N-myristoyltransferase. The C60 derivatives were docked into the binding sites in the proteins. The binding affinities of the C60 derivatives were calculated. The bindings between proteins and their known inhibitors or native ligands were also characterized in the same way. The results show that C60 derivatives form good interactions with the binding sites of different protein targets. In many cases, the binding affinities of C60 derivatives are better than those of known inhibitors and native ligands. This study demonstrates the interaction patterns of C60 derivatives and their binding partners, which will have good impact on the fullerene-based drug discovery

Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module.

Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme

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

Docking of C60-16 with acetylcholinesterase: (A) The binding of C60-16 in the active site of acetylcholinesterase. The C60-16 is shown in stick model while the surface of acetylcholinesterase is shown in green, blue and purple (B) The three dimensional analysis of the interactions of C60-16 in the active site of acetylcholinesterase.

<p>The C60-16 makes interactions with Tyr67, Trp81, Gly114, Tyr118, Ser119, Gly120, Tyr127, Trp276, Phe327 and Tyr331.</p