Gene Assembly Research Articles

A Snakemake Toolkit for the Batch Assembly, Annotationand Phylogenetic Analysis of Mitochondrial Genomes and Ribosomal Genes From Genome Skims of Museum Collections.

Low coverage 'genome-skims' are often used to assemble organelle genomes and ribosomal gene sequences for cost-effective phylogenetic and barcoding studies. Natural history collections hold invaluable biological information, yet poor preservation resulting in degraded DNA often hinders polymerase chain reaction-based analyses. However, it is possible to generate libraries and sequence the short fragments typical of degraded DNA to generate genome-skims from museum collections. Here we introduce a snakemake toolkit comprised of three pipelines skim2mito, skim2rrna and gene2phylo, designed to unlock the genomic potential of historical museum specimens using genome skimming. Specifically, skim2mito and skim2rrna perform the batch assembly, annotation and phylogenetic analysis of mitochondrial genomes and nuclear ribosomal genes, respectively, from low-coverage genome skims. The third pipeline gene2phylo takes a set of gene alignments and performs phylogenetic analysis of individual genes, partitioned analysis of concatenated alignments and a phylogenetic analysis based on gene trees. We benchmark our pipelines with simulated data, followed by testing with a novel genome skimming dataset from both recent and historical solariellid gastropod samples. We show that the toolkit can recover mitochondrial and ribosomal genes from poorly preserved museum specimens of the gastropod family Solariellidae, and the phylogenetic analysis is consistent with our current understanding of taxonomic relationships. The generation of bioinformatic pipelines that facilitate processing large quantities of sequence data from the vast repository of specimens held in natural history museum collections will greatly aid species discovery and exploration of biodiversity over time, ultimately aiding conservation efforts in the face of a changing planet.

Genome-wide computational analysis of the dirigent gene family in Solanum lycopersicum

BackgroundDirigent (DIR) genes play a key role in the development of organic products in plants. They confer conformational influence on processes that lack stereoselectivity and regioselectivity through processes that are mostly understood. They are required to produce lignans, which are a unique and widely distributed family of plant secondary metabolites with intriguing pharmacological characteristics and potential role in plant development. DIR genes are implicated in the process of lignification and protect plants from environmental stresses, including biotic and abiotic stresses. Nevertheless, no research has been performed on the DIR gene family in Solanum lycopersicum. This study provides detailed information on the DIR gene family in S. lycopersicum.Methods and resultsThe conserved domain analysis, phylogenetic analysis, evolutionary adaptation, cis-acting elements, proteomic analysis, signal peptide detection, transmembrane potential analysis, sequence identity and similarity analysis, gene assembly, genomic localization, duplication of gene analysis, and evolutionary linkage of 31 potential DIR genes were studied. All these analyses provide a deep understanding of DIR genes in the S. lycopersicum genome that will provide a useful reference for further functional analysis of the DIR genes in S. lycopersicum.ConclusionThis research provides an in-depth and comprehensive explanation of the detailed process and structural characterization of DIR genes in the genome of S. lycopersicum, laying the groundwork for future plant genetic engineering and crop development exploration. This work will provide valuable information for identifying DIR genes in higher plants and support future research on the DIR gene family.

Senataxin and DNA-PKcs Redundantly Promote Non-Homologous End Joining Repair of DNA Double Strand Breaks During V(D)J Recombination.

Non-homologous end joining (NHEJ) is required for repairing DNA double strand breaks (DSBs) generated by the RAG endonuclease during lymphocyte antigen receptor gene assembly by V(D)J recombination. The Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) kinases regulate functionally redundant pathways required for NHEJ. Here we report that loss of the senataxin helicase leads to a significant defect in RAG DSB repair upon inactivation of DNA-PKcs. The NHEJ function of senataxin is redundant with the RECQL5 helicase and the HLTF translocase and is epistatic with ATM. Co-inactivation of ATM, RECQL5 and HLTF results in an NHEJ defect similar to that from the combined deficiency of DNA-PKcs and senataxin or losing senataxin, RECQL5 and HLTF. These data suggest that ATM and DNA-PKcs regulate the functions of senataxin and RECQL5/HLTF, respectively to provide redundant support for NHEJ.

Response of sedimentary microbial community and antibiotic resistance genes to aged Micro(Nano)plastics exposure under high hydrostatic pressure

Several studies reported that the presence of microplastics (MPs)/nanoplastics (NPs) in marine environments can alter microbial community and function. Yet, the impact of aged MPs/NPs on deep sea sedimentary ecosystems under high hydrostatic pressure remains insufficiently explored. Herein, the sedimentary microbial community composition, co-occurrence network, assembly, and transfer of antibiotic resistance genes (ARGs) in response to aged MPs/NPs were investigated. Compared with the control, NPs addition significantly reduced bacterial alpha diversity (p < 0.05), whereas MPs showed no significant impact (p > 0.05). Moreover, networks under NPs exhibited decreased complexity than that under MPs and the control, including edges, average degree, and the number of keystone. The assembly of the microbial community was primarily governed by stochastic processes, and aged MPs/NPs increased the importance of stochastic processes. Moreover, exposure to MPs/NPs for one month decreased the abundance of antibiotic resistance genes (ARGs) (from 94.8 to 36.2 TPM), while exposure for four months increased the abundance (from 40.6 to 88.1 TPM), and the shift of ARGs in sediment was driven by both functional modules and microbial community. This study is crucial for understanding the stress imposed by aged MPs/NPs on sedimentary ecosystems under high hydrostatic pressure.

The Role of Cilia and the Complex Genetics of Congenital Heart Disease.

Congenital heart disease (CHD) can affect up to 1% of live births, and despite abundant evidence of a genetic etiology, the genetic landscape of CHD is still not well understood. A large-scale mouse chemical mutagenesis screen for mutations causing CHD yielded a preponderance of cilia-related genes, pointing to a central role for cilia in CHD pathogenesis. The genes uncovered by the screen included genes that regulate ciliogenesis and cilia-transduced cell signaling as well as many that mediate endocytic trafficking, a cell process critical for both ciliogenesis and cell signaling. The clinical relevance of these findings is supported by whole-exome sequencing analysis of CHD patients that showed enrichment for pathogenic variants in ciliome genes. Surprisingly, among the ciliome CHD genes recovered were many that encoded direct protein-protein interactors. Assembly of the CHD genes into a protein-protein interaction network yielded a tight interactome that suggested this protein-protein interaction may have functional importance and that its disruption could contribute to the pathogenesis of CHD. In light of these and other findings, we propose that an interactome enriched for ciliome genes may provide the genomic context for the complex genetics of CHD and its often-observed incomplete penetrance and variable expressivity.

Development of whole cell biocatalytic system for asymmetric synthesis of esomeprazole with enhancing coenzyme biosynthesis pathway

Esomeprazole is the most popular proton pump inhibitor for treating gastroesophageal reflux disease. Previously, a phenylacetone monooxygenase mutant LnPAMOmu15 (LM15) was obtained by protein engineering for asymmetric synthesis of esomeprazole using pyrmetazole as substrate. To scale up the whole cell asymmetric synthesis of esomeprazole and reduce the cost, in this work, an Escherichia coli whole-cell catalyst harboring LM15 and formate dehydrogenase from Burkholderia stabilis 15516 (BstFDH) were constructed through optimized gene assembly patterns. CRISPR/Cas9 mediated insertion of Ptrc promoter in genome was done to enhance the expression of key genes to increase the cellular NADP supply in the whole cell catalyst, by which the amount of externally added NADP+ for the asymmetric synthesis of esomeprazole decreased to 0.05 mM from 0.3 mM for reducing the cost. After the optimization of reaction conditions in the reactor, the scalable synthesis of esomeprazole was performed using the efficient LM15-BstFDH whole-cell as catalyst, which showed the highest reported space-time yield of 3.28 g/L/h with 50 mM of pyrmetazole loading. Isolation procedure was conducted to obtain esomeprazole sodium of 99.55 % purity and > 99.9 % ee with 90.1 % isolation yield. This work provides the basis for production of enantio-pure esomeprazole via cost-effective whole cell biocatalysis.

Mef2d regulates mutually exclusive expression of IgZ and IgM isotypes through epigenetic modulation in a zebrafish model

The discovery of IgZ, or its counterpart IgT, represents a novel immunoglobulin isotype (named ζ or τ) found in teleosts, introducing a new member to the existing Ig classes (µ, δ, γ, α, and ε) among vertebrates. The distinctive intrachromosomal organization of ighz and ighm loci implies the necessity of a distinct, mutually exclusive recombination process for the independent generation of IgZ and IgM isotypes. However, the molecular mechanisms governing this process remain elusive. In this study, we unveil the regulatory function of myocyte enhancer factor 2D (Mef2d) in the assembly of ighz genes through epigenetic modulation in a zebrafish model. Mechanistically, Mef2d selectively hinders the recombination of ighm locus in IgZ+ B cells by binding to the 3′Eµ site of the ighm locus and helping establish a repressive modification pattern of H3K4me0/H3K9me2/H3K27me2 in Dµ/Jµ regions through recruiting the co-repressive Sin3/Hdac1 complex with the assistance of cohesin complex and Setdb1/Ezh2 methyltransferases. Consequently, this renders the Dµ/Jµ regions inaccessible to Rag1/2, thus preventing ighm rearrangement. As a pivotal regulator for IgZ isotype production, Mef2d exhibits differential expression in committed IgZ+ B cells, a process regulated by the Il-7/Il-7r-mediated p38 Mapk signaling pathway. These results indicate the existence of a unique isotypic exclusion mechanism underlying recombination between ighz and ighm locus in teleosts. This mechanism highlights an unrecognized strategy for generating diverse isotypes in vertebrates, distinct from the well-established class switch recombination process. This study significantly contributes to our understanding of paradigms, diversifications, and the evolutionary history of vertebrate adaptive immunity.

Transcriptomic analyses provide new insights into immune response of the Procambarus clarkii intestines to Aeromonas hydrophila challenge

In recent years, the death rate of Procambarus clarkii increased sharply due to pathogenic bacterial infections, leading to huge economic losses. To determine the immune response of the intestinal tissues of P. clarkii to bacterial infections, we compared the immune indexes, histopathological damage, and transcriptome data of the control and Aeromonas hydrophila-infected P. clarkii. With the injection of A. hydrophila, it was found that intestinal mucosal folds increased, intestinal villi showed obvious vacuolation, and the tissue structure changed significantly. The results of non-specific immunoenzyme activity showed that the Total protein (TP) content was significantly lower than that of the control group at 24 h, and the Acid phosphatase (ACP) and Alkaline phosphatase (AKP) activities in the treatment group showed a significant increasing trend (p < 0.05). In terms of the transcriptome, an average of 22,927,523 clean reads and 27,866 genes were found in the control and treatment groups through gene assembly and annotation. Among these genes, 567 were identified as differentially expressed genes (DEGs), of which 501 were up-regulated and 66 were down-regulated. Quantitative-polymerase chain reaction (qPCR) analysis of six randomly selected genes was used to verify the RNA-sequencing results. The Gene Ontology enrichment analysis revealed that the DEGs were primarily involved in cell processes, metabolic processes, and biological regulation, while the Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that these DEGs were involved in tyrosine metabolism, glycosphingolipid biosynthesis—ganglionic series, and nitrogen metabolism. We studied the tyrosine metabolic pathway and found that it contributes to the immune defense mechanism of P. clarkii. The results of our study provide important insights into the intestinal immune response of P. clarkii to A. hydrophila infection.

Ginkgolic acid inhibits orthopneumo- and metapneumo- virus infectivity.

The human respiratory syncytial virus (hRSV) and the human metapneumovirus (hMPV) are important human respiratory pathogens from the Pneumoviridae family. Both are responsible for severe respiratory tract infections in infants, young children, elderly individuals, adults with chronic medical conditions, and immunocompromised patients. Despite their large impact on human health, vaccines for hRSV were only recently introduced, and only limited treatment options exist. Here we show that Ginkgolic acid (GA), a natural compound from the extract of Ginkgo biloba, with known antiviral properties for several viruses, efficiently inhibits these viruses' infectivity and spread in cultures in a dose-dependent manner. We demonstrate that the drug specifically affects the entry step during the early stages on the viruses' life cycle with no effect on post-entry and late stage events, including viral gene transcription, genome replication, assembly and particles release. We provide evidence that GA acts as an efficient antiviral for members of the Pneumoviridae family and has the potential to be used to treat acute infections.

Enzymatic Assembly of Small Synthetic Genes with Repetitive Elements.

Gene synthesis efficiency has greatly improved in recent years but is limited when it comes to repetitive sequences, which results in synthesis failure or delays by DNA synthesis vendors. This represents a major obstacle for the development of synthetic biology since repetitive elements are increasingly being used in the design of genetic circuits and design of biomolecular nanostructures. Here, we describe a method for the assembly of small synthetic genes with repetitive elements: First, a gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden-Gate-compatible overhangs. Then, synthons are made by oligo extension and finally assembled into a synthetic gene by Golden Gate Assembly. We demonstrate the method by constructing eight challenging genes with repetitive elements, e.g., multiple repeats of RNA aptamers and RNA origami scaffolds with multiple identical aptamers. The genes range in size from 133 to 456 base pairs and are assembled with fidelities of up to 87.5%. The method was developed to facilitate our own specific research but may be of general use for constructing challenging and repetitive genes and, thus, a valuable addition to the molecular cloning toolbox.

Screening of E. coli O157:H7 AI-2 QS inhibitors and their inhibitory effect on biofilm formation in combination with disinfectants

Food processing environments contaminated with E. coli O157:H7 and forming biofilms will be a continuous source of contamination, leading to foodborne illness and food spoilage. Now, the main countermeasure is chemical disinfectants, but the use of disinfectants may cause the development of drug-resistant bacteria and other negative effects. Therefore, there is an urgent need to find novel antimicrobial agents. Quorum-sensing inhibitors (QSI) are a promising strategy to control biofilm formation. This study aimed to screen the optimal QSI of E. coli O157:H7 from plant-derived small molecules by biofilm and QS inhibitory activities and to validate their effectiveness in combination with disinfectants. In addition, the inhibition mechanism of gallic acid on biofilm formation was further explored. Gallic acid was screened as the QSI of E. coli O157:H7, and its AI-2 inhibitory activity against LuxS/AI-2 QS system reached 95.54% and 98.89% after treatment with 1/2 MIC gallic acid for 4 h and 6 h, respectively. It inhibited biofilm formation by affecting flagellar assembly, chemotaxis, and expression of adhesion factor-related genes, which interfered with the transition of bacteria from reversible to irreversible adhesion. In beef broth simulation system, the inhibition rate was greater than 90.00% in all gallic acid and benzalkonium chloride combination groups. Therefore, gallic acid can be a potential antimicrobial synergist in the food industry, combined with disinfectants to control E. coli O157:H7 biofilms while avoiding the development of resistance.

A novel computational pipeline for var gene expression augments the discovery of changes in the Plasmodium falciparum transcriptome during transition from in vivo to short-term in vitro culture

The pathogenesis of severe Plasmodium falciparum malaria involves cytoadhesive microvascular sequestration of infected erythrocytes, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 variants are encoded by the highly polymorphic family of var genes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches for var gene profiling and classification of predicted binding phenotypes in clinical P. falciparum isolates (Wichers et al., 2021), which represented a major technical advance. Building on this, we report here a novel method for var gene assembly and multidimensional quantification from RNA-sequencing that outperforms the earlier approach of Wichers et al., 2021, on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can interrogate the var transcriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role of var genes in malaria pathogenesis. We applied this new method to investigate changes in var gene expression through early transition of parasite isolates to in vitro culture, using paired sets of ex vivo samples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Modest but unpredictable var gene switching and convergence towards var2csa were observed in culture, along with differential expression of 19% of the core transcriptome between paired ex vivo and generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences about in vivo phenotype and behaviour.

A novel computational pipeline for var gene expression augments the discovery of changes in the Plasmodium falciparum transcriptome during transition from in vivo to short-term in vitro culture.

The pathogenesis of severe Plasmodium falciparum malaria involves cytoadhesive microvascular sequestration of infected erythrocytes, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 variants are encoded by the highly polymorphic family of var genes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches for var gene profiling and classification of predicted binding phenotypes in clinical P. falciparum isolates (Wichers et al., 2021), which represented a major technical advance. Building on this, we report here a novel method for var gene assembly and multidimensional quantification from RNA-sequencing that outperforms the earlier approach of Wichers et al., 2021, on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can interrogate the var transcriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role of var genes in malaria pathogenesis. We applied this new method to investigate changes in var gene expression through early transition of parasite isolates to in vitro culture, using paired sets of ex vivo samples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Modest but unpredictable var gene switching and convergence towards var2csa were observed in culture, along with differential expression of 19% of the core transcriptome between paired ex vivo and generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences about in vivo phenotype and behaviour.

Metagenomic probing toward an atlas of the taxonomic and metabolic foundations of the global ocean genome

The global ocean genome (the pool of genes in marine organisms and the functional information they encode) is a major, untapped resource for science and society with a growing range of biotechnology applications in sectors such as biomedicine, energy, and food. Shotgun sequencing and metagenomics can now be used to catalog the diversity of ocean microbial life and to explore its functional potential, but has been limited by sample coverage, access to suitable sequencing platforms, and computational capacity. Here we provide a novel synthesis of the global ocean genome based on analysis of 2,102 sampled ocean metagenomes, with gene assembly and annotation via the KAUST Metagenome Analysis Platform (KMAP) Global Ocean Gene Catalog 1.0 containing 308.6 million gene clusters. Taxonomically, we report the distribution of marine genes across the tree of life and different ocean basins and depth zone biomes. Functionally, we map its relationship to protein families and biogeochemical processes, including the major microbial metabolic pathways that process three elements that play fundamental roles in biogeochemical cycles and are relevant to climate change. These data extend our understanding of the complex, dynamic nature of the ocean microbiome and its metabolic capabilities. Further research is of critical global importance both to unlock the potential of the ocean genome and to understand and predict the effects of human-induced changes, including pollution and climate change. Further hypothesis-driven research should target under-sampled deep sea and benthic microbial communities using enhanced metagenomic methods, to better understand marine ecosystem functioning. Investment in the necessary computational capacity is essential, as are suitable intellectual property frameworks.

Genome-wide identification, classification and expression analysis of MYB gene family in coconut (Cocos nucifera L.).

MYB transcription factors regulate the growth, development, and secondary metabolism of plant species. To investigate the origin of color variations in coconut pericarp, we identified and analyzed the MYB gene family present in coconut. According to the sequence of MYB genes in Arabidopsis thaliana, homologous MYB gene sequences were found in the whole genome database of coconut, the conserved sequence motifs within MYB proteins were analyzed by Motif Elicitation (MEME) tool, and the sequences without conservative structure were eliminated. Additionally, we employed RNA-seq technology to generate gene expression signatures of the R2R3-MYB genes across distinctive coconut parts exhibiting diverse colors. To validate these profiles, we conducted quantitative PCR (qPCR). Through comprehensive genome-wide screening, we successfully identified a collection of 179 MYB genes in coconut. Subsequent phylogenetic analysis categorized these 179 coconut MYB genes into 4-subfamilies: 124 R2R3-MYB, 4 3R-MYB types, 4 4R-MYB type, and 47 unknown types. Furthermore, these genes were further divided into 34 subgroups, with 28 of these subgroups successfully classified into known subfamilies found in Arabidopsis thaliana. By mapping the CnMYB genes onto the 16 chromosomes of the coconut genome, we unveiled a collinearity association between them. Moreover, a preservation of gene structure and motif distribution was observed across the CnMYB genes. Our research encompassed a thorough investigation of the R2R3-MYB genes present in the coconut genome, including the chromosomal localization, gene assembly, conserved regions, phylogenetic associations, and promoter cis-acting elements of the studied genes. Our findings revealed a collection of 12 R2R3-MYB candidate genes, namely CnMYB8, CnMYB15, CnMYB27, CnMYB28, CnMYB61, CnMYB63, CnMYB68, CnMYB94, CnMYB101, CnMYB150, CnMYB153, and CnMYB164. These genes showed differential expressions in diverse tissues and developmental stages of four coconut species, such as CnMYB68, CnMYB101, and CnMYB28 exhibited high expression in majority of tissues and coconut species, while CnMYB94 and CnMYB164 showed lower expression. These findings shed light on the crucial functional divergence of CnMYB genes across various coconut tissues, suggesting these genes as promising candidate genes for facilitating color development in this important crop.

Locus folding mechanisms determine modes of antigen receptor gene assembly.

The dynamic folding of genomes regulates numerous biological processes, including antigen receptor (AgR) gene assembly. We show that, unlike other AgR loci, homotypic chromatin interactions and bidirectional chromosome looping both contribute to structuring Tcrb for efficient long-range V(D)J recombination. Inactivation of the CTCF binding element (CBE) or promoter at the most 5'Vβ segment (Trbv1) impaired loop extrusion originating locally and extending to DβJβ CBEs at the opposite end of Tcrb. Promoter or CBE mutation nearly eliminated Trbv1 contacts and decreased RAG endonuclease-mediated Trbv1 recombination. Importantly, Trbv1 rearrangement can proceed independent of substrate orientation, ruling out scanning by DβJβ-bound RAG as the sole mechanism of Vβ recombination, distinguishing it from Igh. Our data indicate that CBE-dependent generation of loops cooperates with promoter-mediated activation of chromatin to juxtapose Vβ and DβJβ segments for recombination through diffusion-based synapsis. Thus, the mechanisms that fold a genomic region can influence molecular processes occurring in that space, which may include recombination, repair, and transcriptional programming.

Rapid Targeted Assembly of the Proteome Reveals Evolutionary Variation of GC Content in Avian Lice.

Nucleotide base composition plays an influential role in the molecular mechanisms involved in gene function, phenotype, and amino acid composition. GC content (proportion of guanine and cytosine in DNA sequences) shows a high level of variation within and among species. Many studies measure GC content in a small number of genes, which may not be representative of genome-wide GC variation. One challenge when assembling extensive genomic data sets for these studies is the significant amount of resources (monetary and computational) associated with data processing, and many bioinformatic tools have not been optimized for resource efficiency. Using a high-performance computing (HPC) cluster, we manipulated resources provided to the targeted gene assembly program, automated target restricted assembly method (aTRAM), to determine an optimum way to run the program to maximize resource use. Using our optimum assembly approach, we assembled and measured GC content of all of the protein-coding genes of a diverse group of parasitic feather lice. Of the 499 426 genes assembled across 57 species, feather lice were GC-poor (mean GC = 42.96%) with a significant amount of variation within and between species (GC range = 19.57%-73.33%). We found a significant correlation between GC content and standard deviation per taxon for overall GC and GC3, which could indicate selection for G and C nucleotides in some species. Phylogenetic signal of GC content was detected in both GC and GC3. This research provides a large-scale investigation of GC content in parasitic lice laying the foundation for understanding the basis of variation in base composition across species.

Microbial Cell Factories in the Bioeconomy Era: From Discovery to Creation.

Microbial cell factories (MCFs) are extensively used to produce a wide array of bioproducts, such as bioenergy, biochemical, food, nutrients, and pharmaceuticals, and have been regarded as the "chips" of biomanufacturing that will fuel the emerging bioeconomy era. Biotechnology advances have led to the screening, investigation, and engineering of an increasing number of microorganisms as diverse MCFs, which are the workhorses of biomanufacturing and help develop the bioeconomy. This review briefly summarizes the progress and strategies in the development of robust and efficient MCFs for sustainable and economic biomanufacturing. First, a comprehensive understanding of microbial chassis cells, including accurate genome sequences and corresponding annotations; metabolic and regulatory networks governing substances, energy, physiology, and information; and their similarity and uniqueness compared with those of other microorganisms, is needed. Moreover, the development and application of effective and efficient tools is crucial for engineering both model and nonmodel microbial chassis cells into efficient MCFs, including the identification and characterization of biological parts, as well as the design, synthesis, assembly, editing, and regulation of genes, circuits, and pathways. This review also highlights the necessity of integrating automation and artificial intelligence (AI) with biotechnology to facilitate the development of future customized artificial synthetic MCFs to expedite the industrialization process of biomanufacturing and the bioeconomy.

Chromosome-level genome assembly of the glass catfish ( Kryptopterus vitreolus) reveals molecular clues to its transparent phenotype.

Glass catfish ( Kryptopterus vitreolus) are notable in the aquarium trade for their highly transparent body pattern. This transparency is due to the loss of most reflective iridophores and light-absorbing melanophores in the main body, although certain black and silver pigments remain in the face and head. To date, however, the molecular mechanisms underlying this transparent phenotype remain largely unknown. To explore the genetic basis of this transparency, we constructed a chromosome-level haplotypic genome assembly for the glass catfish, encompassing 32 chromosomes and 23 344 protein-coding genes, using PacBio and Hi-C sequencing technologies and standard assembly and annotation pipelines. Analysis revealed a premature stop codon in the putative albinism-related tyrp1b gene, encoding tyrosinase-related protein 1, rendering it a nonfunctional pseudogene. Notably, a synteny comparison with over 30 other fish species identified the loss of the endothelin-3 ( edn3b) gene in the glass catfish genome. To investigate the role of edn3b, we generated edn3b -/- mutant zebrafish, which exhibited a remarkable reduction in black pigments in body surface stripes compared to wild-type zebrafish. These findings indicate that edn3b loss contributes to the transparent phenotype of the glass catfish. Our high-quality chromosome-scale genome assembly and identification of key genes provide important molecular insights into the transparent phenotype of glass catfish. These findings not only enhance our understanding of the molecular mechanisms underlying transparency in glass catfish, but also offer a valuable genetic resource for further research on pigmentation in various animal species.

Optimizing the Metal Bioreduction Process in Recombinant Shewanella azerbaijanica Bacteria: A Novel Approach via mtrC Gene Cloning and Nitrate-Reducing Pathway Destruction.

Environmental pollution is growing every day in terms of the increase in population, industrialization, and urbanization. Shewanella azerbaijanica is introduced as a highly potent bacterium in metal bioremediation. The mtrC gene was selected as a cloning target to improve electron flux chains in the EET (extracellular electron transfer) pathway. Using the SDM (site-directed mutagenesis) technique, the unique gene assembly featured the mtrC gene sandwiched between two napD/B genes to disrupt the nitrate reduction pathway, which serves as the primary metal reduction competitor. Shew-mtrC gene construction was transferred to expression plasmid pET28a (+) in the expression host bacteria (E. coli BL21 and S. azerbaijanica), in pUC57, cloning plasmid, which was transferred to the cloning host bacteria E. coli Top10 and S. azerbaijanica. All cloning procedures (i.e., synthesis, insertion, transformation, cloning, and protein expression) were verified and confirmed by precise tests. ATR-FTIR analysis, CD, western blotting, affinity chromatography, SDS-PAGE, and other techniques were used to confirm the expression and structure of the MtrC protein. The genome sequence and primers were designed according to the submitted Shewanella oneidensisMR-1 genome, the most similar bacteria to this native species. The performance of recombinant S. azerbaijanica bacterium in metal bioremediation, as sustainable strategy, has to be verified by more research.