SynBioPub Date : 2024-02-05DOI: 10.3390/synbio2010004
Tsung-Ming Hu, Shih-Hsin Hsu, Hsin-Yao Tsai, M. Cheng
{"title":"Transcriptomic Investigation in CRISPR/Cas9-Mediated GRIK1-, GRIK2-, and GRIK4-Gene-Knockout Human Neuroblastoma Cells","authors":"Tsung-Ming Hu, Shih-Hsin Hsu, Hsin-Yao Tsai, M. Cheng","doi":"10.3390/synbio2010004","DOIUrl":"https://doi.org/10.3390/synbio2010004","url":null,"abstract":"The glutamate ionotropic kainate receptors, encoded by the GRIK gene family, are composed of four subunits and function as ligand-activated ion channels. They play a critical role in regulating synaptic transmission and various synaptic receptors’ processes, as well as in the pathophysiology of schizophrenia. However, their functions and mechanisms of action need to be better understood and are worthy of exploration. To further understand the exact role of the kainate receptors in vitro, we generated kainate-receptor-knockout (KO) isogenic SH-SY5Y cell lines using the CRISPR/Cas9-mediated gene editing method. We conducted RNA sequencing (RNA-seq) to determine the differentially expressed genes (DEGs) in the isogenic edited cells and used rhodamine-phalloidin staining to quantitate filamentous actin (F-actin) in differentiated edited cells. The RNA-seq and the Gene Ontology enrichment analysis revealed that the genetic deletion of the GRIK1, GRIK2, and GRIK4 genes disturbed multiple genes involved in numerous signal pathways, including a converging pathway related to the synaptic membrane. An enrichment analysis of gene–disease associations indicated that DEGs in the edited cell lines were associated with several neuropsychiatric disorders, especially schizophrenia. In the morphology study, fluorescent images show that less F-actin was expressed in differentiated SH-SY5Y cells with GRIK1, GRIK2, or GRIK4 deficiency than wild-type cells. Our data indicate that kainate receptor deficiency might disturb synaptic-membrane-associated genes, and elucidating these genes should shed some light on the pathophysiology of schizophrenia. Furthermore, the transcriptomic profiles for kainate receptor deficiency of SH-SY5Y cells contribute to emerging evidence for the novel mechanisms underlying the effect of kainate receptors and the pathophysiology of schizophrenia. In addition, our data suggest that kainate-receptor-mediated F-actin remodeling may be a candidate mechanism underlying schizophrenia.","PeriodicalId":507619,"journal":{"name":"SynBio","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139865069","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
SynBioPub Date : 2024-01-04DOI: 10.3390/synbio2010003
Mark Hogan, Yuhao Song, Jimmy Muldoon, P. Caffrey
{"title":"Generation of New Glycoanalogues of Polyene Antibiotics by Synthetic Biology—Testing Current Technical Boundaries","authors":"Mark Hogan, Yuhao Song, Jimmy Muldoon, P. Caffrey","doi":"10.3390/synbio2010003","DOIUrl":"https://doi.org/10.3390/synbio2010003","url":null,"abstract":"A number of antifungal drugs are based on polyene macrolides that cause severe side effects. Most of these compounds contain a single aminodeoxysugar, D-mycosamine. Toxicity can be reduced by increasing the extent of glycosylation. The aromatic heptaene 67-121C and two analogues of the degenerate heptaene nystatin have a second sugar attached to the C4′ hydroxyl of mycosamine. Another nystatin analogue has L-digitoxose as a second sugar attached to C35 on the macrolactone ring. The pentaene selvamicin has 4-O-methyl-L-digitoxose at C27, the equivalent position. To assist the production of new antifungals by synthetic biology, we explore further the utility of three classes of polyene glycosyltransferase: extending glycosyltransferases that form disaccharide-containing polyenes, glycosyltransferases that add the L-digitoxose sugars of nystatin A3 and selvamicin, and mycosaminyltransferases that add the primary aminodeoxysugar. In addition, we combine enzymatic hyperglycosylation with a known chemical method for adding sugars to the C3′ amino group of mycosamine. This was used to convert the disaccharide-containing 67-121C heptaene to forms containing branched trisaccharide or tetrasaccharide chains. These analogues are of interest for testing as anti-Leishmania drugs.","PeriodicalId":507619,"journal":{"name":"SynBio","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139386384","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
SynBioPub Date : 2024-01-04DOI: 10.3390/synbio2010002
Saad Alrashdi, Federica Casolari, K. Kyeremeh, Hai Deng
{"title":"Chemo-Enzymatic Synthesis of Bioactive Carbazole Derivatives","authors":"Saad Alrashdi, Federica Casolari, K. Kyeremeh, Hai Deng","doi":"10.3390/synbio2010002","DOIUrl":"https://doi.org/10.3390/synbio2010002","url":null,"abstract":"Carbazoles are key scaffolds of either antimicrobial/antiviral alkaloid natural products or therapeutics. As such, access to structurally diverse indole-containing carbazoles has attracted considerable attention. In this report, a pilot study is described using biotransformation to provide carbazoles that contain various acyl substituents. The biotransformation system contains the thiamine-diphosphate (ThDP)-dependent enzyme NzsH, the FabH-like 3-ketoacyl-ACP synthase NzsJ, and the aromatase/cyclase NzsI, encoded in the biosynthetic gene cluster (nzs) of the bacterial carbazole alkaloid natural product named neocarazostatin A. The utilization of a range of acyl-SNACs (synthetic acyl-thioester analogues of the native substrate) together with indole-3-pyruvate and pyruvate in the designed biotransformation system allows production of carbazole derivatives. Our results demonstrate that this three-enzyme system displays a considerable substrate profile toward acyl donors for production of carbazoles with different acyl substituents. Finally, two more enzymes were included in the biotransformation system: the tryptophan synthase stand-alone β-subunit variant, PfTrpB, generated from directed evolution in the literature, and a commercially available L-amino acid oxidase (LAAO). The addition of these two enzymes allows the transformation to start with indole building blocks to provide carbazoles with modifications in the indole ring system.","PeriodicalId":507619,"journal":{"name":"SynBio","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-01-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139386285","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
SynBioPub Date : 2023-11-16DOI: 10.3390/synbio1030015
Sergii Krysenko
{"title":"Impact of Nitrogen-Containing Compounds on Secondary Metabolism in Streptomyces spp.—A Source of Metabolic Engineering Strategies","authors":"Sergii Krysenko","doi":"10.3390/synbio1030015","DOIUrl":"https://doi.org/10.3390/synbio1030015","url":null,"abstract":"Actinobacteria from the genus Streptomyces feature complex primary and secondary metabolism, developmental cycle, and ability to produce a variety of natural products. These soil bacteria are major producers of antibiotics and other bioactive compounds and have been extensively investigated due to the medical and industrial relevance of Streptomyces-derived secondary metabolites. However, the genetic toolbox for Streptomyces engineering as well as yield optimization strategies for the production of relevant metabolites are limited. On the one hand, the genetic potential of these organisms has not been fully utilized due to many “silent” or poorly expressed biosynthetic gene clusters, whose activation depends on environmental stimuli and nutrient availability. On the other hand, these GC-rich Gram-positive bacteria are difficult to manipulate, and traditional genetic manipulation strategies are time-consuming and have low efficiency. Recent studies of Streptomyces metabolism and genomes provided new insights into possibilities to overcome these challenges. In this review, advances and approaches for Streptomyces manipulations and secondary metabolite production optimization are discussed. Special focus is given to understanding the interplay between primary and secondary metabolism in Streptomyces and the supply of nitrogen-containing compounds into secondary metabolism. Existing strategies to manipulate cellular metabolism in Streptomyces are reviewed.","PeriodicalId":507619,"journal":{"name":"SynBio","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2023-11-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139267948","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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