Construction of a doramectin producer mutant from an avermectin-overproducing industrial strain of Streptomyces avermitilis

2009 ◽  
Vol 55 (12) ◽  
pp. 1355-1363 ◽  
Author(s):  
Xuejin Zhao ◽  
Yuanxin Wang ◽  
Shiwei Wang ◽  
Zhi Chen ◽  
Ying Wen ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Fermentation Medium ◽  
Streptomyces Avermitilis ◽  
Industrial Strain ◽  
Agricultural Pesticides ◽  
Double Deletion ◽  
Resistance Markers ◽  
Encoding Gene ◽  
Branched Chain ◽  
Encoding Genes

The avermectin analogue doramectin (CHC-B1), which is produced in mutants that have an altered biosynthesis pathway of avermectin, is one of the most effective agricultural pesticides and antiparasitics. We report here the construction of a bkdF olmA double-deletion mutant lacking one of the branched-chain α-keto acid dehydrogenase encoding genes (bkdF) and the oligomycin PKS encoding gene cluster (olmA) in Streptomyces avermitilis 76-05. We then characterized the production of various antibiotics in cultures of the deletion mutant. In a fermentation medium supplemented with cyclohexanecarboxylic acid, this double mutant produced doramectin and its analogues but no oligomycin. The mutant proved to be genetically stable, without any antibiotic resistance markers inserted into its chromosome, and could potentially become an industrial doramectin-producing strain after further improvement.

scholarly journals Utilizing the ABC Transporter for Growth Factor Production by fleQ Deletion Mutant of Pseudomonas fluorescens

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 679
Author(s):  
Benedict-Uy Fabia ◽  
Joshua Bingwa ◽  
Jiyeon Park ◽  
Nguyen-Mihn Hieu ◽  
Jung-Hoon Ahn
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Pseudomonas Fluorescens ◽  
Abc Transporter ◽  
Transforming Growth Factor Beta ◽  
Deletion Mutant ◽  
Transforming Growth Factor ◽  
Gene Knockout ◽  
Type I ◽  
Double Deletion

Pseudomonas fluorescens, a gram-negative bacterium, has been proven to be a capable protein manufacturing factory (PMF). Utilizing its ATP-binding cassette (ABC) transporter, a type I secretion system, P. fluorescens has successfully produced recombinant proteins. However, besides the target proteins, P. fluorescens also secretes unnecessary background proteins that complicate protein purification and other downstream processes. One of the background proteins produced in large amounts is FliC, a flagellin protein. In this study, the master regulator of flagella gene expression, fleQ, was deleted from P. fluorescens Δtp, a lipase and protease double-deletion mutant, via targeted gene knockout. FleQ directs flagella synthesis, so the new strain, P. fluorescens ΔfleQ, does not produce flagella-related proteins. This not only simplifies purification but also makes P. fluorescens ΔfleQ an eco-friendly expression host because it will not survive outside a controlled environment. Six recombinant growth factors, namely, insulin-like growth factors I and II, beta-nerve growth factor, fibroblast growth factor 1, transforming growth factor beta, and tumor necrosis factor beta, prepared using our supercharging method, were successfully secreted by P. fluorescens ΔfleQ. Our findings demonstrate the potential of P. fluorescens ΔfleQ, combined with our supercharging process, as a PMF.

scholarly journals Functional Genomic Insights into Probiotic Bacillus siamensis Strain B28 from Traditional Korean Fermented Kimchi

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1906
Author(s):  
So-Jeong Heo ◽  
Jong-Hoon Kim ◽  
Mi-Sun Kwak ◽  
Do-Won Jeong ◽  
Moon-Hee Sung
Keyword(s):  
Foodborne Pathogens ◽  
Lipoteichoic Acid ◽  
Essential Amino Acids ◽  
Probiotic Properties ◽  
Oxygen Species ◽  
Branched Chain Fatty Acids ◽  
293 Cells ◽  
Branched Chain ◽  
Scavenging Enzymes ◽  
Encoding Genes

Bacillus siamensis strain B28 was previously isolated from traditional Korean fermented kimchi and inhibited expression of the microphthalmia-associated transcription factor and β-catenin in human embryonic kidney 293 cells. Here, we determined the complete genome sequence of strain B28 and compared it with other strains to elucidate its potential probiotic properties. Strain B28 does not contain antibiotic resistance-, hemolysin- or enterotoxin-encoding genes. The genome includes genes related to survival in extreme conditions, adhesion in the gut, and synthesis of the bacteriocin. Considering the potential for enhancement of human health, the strain B28 genome encodes genes related to production of eight essential amino acids, γ-aminobutyric acid, branched-chain fatty acids, γ-glutamyltransferase, and subtilisin. There are genes for the synthesis of uracil, lipoteichoic acid, glutathione, and several reactive oxygen species-scavenging enzymes. Experimentally, strain B28 exhibited sensitivity to eight antibiotics and antibacterial activity against seven foodborne pathogens. B. siamensis B28 is a safe strain with potential for development as a probiotic.

scholarly journals Human Host Range Restriction of the Vaccinia Virus C7/K1 Double Deletion Mutant Is Mediated by an Atypical Mode of Translation Inhibition

2018 ◽  
Vol 92 (23) ◽  
Author(s):  
Gilad Sivan ◽  
Shira G. Glushakow-Smith ◽  
George C. Katsafanas ◽  
Jeffrey L. Americo ◽  
Bernard Moss
Keyword(s):  
Protein Synthesis ◽  
Vaccinia Virus ◽  
Host Range ◽  
Deletion Mutant ◽  
Viral Mrna ◽  
Entry Site ◽  
Range Restriction ◽  
Internal Ribosome Entry ◽  
Host Restriction ◽  
Double Deletion

ABSTRACTReplication of vaccinia virus in human cells depends on the viral C7 or K1 protein. A previous human genome-wide short interfering RNA (siRNA) screen with a C7/K1 double deletion mutant revealed SAMD9 as a principal host range restriction factor along with additional candidates, including WDR6 and FTSJ1. To compare their abilities to restrict replication, the cellular genes were individually inactivated by CRISPR/Cas9 mutagenesis. The C7/K1 deletion mutant exhibited enhanced replication in each knockout (KO) cell line but reached wild-type levels only in SAMD9 KO cells. SAMD9 was not depleted in either WDR6 or FTSJ1 KO cells, suggesting less efficient alternative rescue mechanisms. Using the SAMD9 KO cells as controls, we verified a specific block in host and viral intermediate and late protein synthesis in HeLa cells and demonstrated that the inhibition could be triggered by events preceding viral DNA replication. Inhibition of cap-dependent and -independent protein synthesis occurred primarily at the translational level, as supported by DNA and mRNA transfection experiments. Concurrent with collapse of polyribosomes, viral mRNA was predominantly in 80S and lighter ribonucleoprotein fractions. We confirmed the accumulation of cytoplasmic granules in HeLa cells infected with the C7/K1 deletion mutant and further showed that viral mRNA was sequestered with SAMD9. RNA granules were still detected in G3BP KO U2OS cells, which remained nonpermissive for the C7/K1 deletion mutant. Inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral mRNA, and failure of PKR, RNase L, or G3BP KO cells to restore protein synthesis support an unusual mechanism of host restriction.IMPORTANCEA dynamic relationship exists between viruses and their hosts in which each ostensibly attempts to exploit the other’s vulnerabilities. A window is opened into the established condition, which evolved over millennia, if loss-of-function mutations occur in either the virus or host. Thus, the inability of viral host range mutants to replicate in specific cells can be overcome by identifying and inactivating the opposing cellular gene. Here, we investigated a C7/K1 host range mutant of vaccinia virus in which the cellular gene SAMD9 serves as the principal host restriction factor. Host restriction was triggered early in infection and manifested as a block in translation of viral mRNAs. Features of the block include inhibition of cap-dependent and internal ribosome entry site-mediated translation, sequestration of viral RNA, and inability to overcome the inhibition by inactivation of protein kinase R, ribonuclease L, or G3 binding proteins, suggesting a novel mechanism of host restriction.

scholarly journals Contrasting genetic variation and positive selection followed the divergence of NBS-encoding genes in Asian and European pears

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Manyi Sun ◽  
Mingyue Zhang ◽  
Jugpreet Singh ◽  
Bobo Song ◽  
Zikai Tang ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Disease Resistance ◽  
Positive Selection ◽  
Gene Family ◽  
Selection Pressure ◽  
Black Spot ◽  
Orthologous Gene ◽  
European Pear ◽  
Encoding Gene ◽  
Encoding Genes

Abstract Background The NBS disease-related gene family coordinates the inherent immune system in plants in response to pathogen infections. Previous studies have identified NBS-encoding genes in Pyrus bretschneideri (‘Dangshansuli’, an Asian pear) and Pyrus communis (‘Bartlett’, a European pear) genomes, but the patterns of genetic variation and selection pressure on these genes during pear domestication have remained unsolved. Results In this study, 338 and 412 NBS-encoding genes were identified from Asian and European pear genomes. This difference between the two pear species was the result of proximal duplications. About 15.79% orthologous gene pairs had Ka/Ks ratio more than one, indicating two pear species undergo strong positive selection after the divergence of Asian and European pear. We identified 21 and 15 NBS-encoding genes under fire blight and black spot disease-related QTL, respectively, suggesting their importance in disease resistance. Domestication caused decreased nucleotide diversity across NBS genes in Asian cultivars (cultivated 6.23E-03; wild 6.47E-03), but opposite trend (cultivated 6.48E-03; wild 5.91E-03) appeared in European pears. Many NBS-encoding coding regions showed Ka/Ks ratio of greater than 1, indicating the role of positive selection in shaping diversity of NBS-encoding genes in pear. Furthermore, we detected 295 and 122 significantly different SNPs between wild and domesticated accessions in Asian and European pear populations. Two NBS genes (Pbr025269.1 and Pbr019876.1) with significantly different SNPs showed >5x upregulation between wild and cultivated pear accessions, and > 2x upregulation in Pyrus calleryana after inoculation with Alternaria alternata. We propose that positively selected and significantly different SNPs of an NBS-encoding gene (Pbr025269.1) regulate gene expression differences in the wild and cultivated groups, which may affect resistance in pear against A. alternata. Conclusion Proximal duplication mainly led to the different number of NBS-encoding genes in P. bretschneideri and P. communis genomes. The patterns of genetic diversity and positive selection pressure differed between Asian and European pear populations, most likely due to their independent domestication events. This analysis helps us understand the evolution, diversity, and selection pressure in the NBS-encoding gene family in Asian and European populations, and provides opportunities to study mechanisms of disease resistance in pear.

scholarly journals Na+/H+Antiport Is Essential for Yersinia pestis Virulence

2013 ◽  
Vol 81 (9) ◽  
pp. 3163-3172 ◽  
Author(s):  
Yusuke Minato ◽  
Amit Ghosh ◽  
Wyatt J. Faulkner ◽  
Erin J. Lind ◽  
Sara Schesser Bartra ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Drug Target ◽  
Deletion Mutant ◽  
Ex Vivo ◽  
Ion Homeostasis ◽  
Content Type ◽  
Double Deletion ◽  
Derivative Strain

ABSTRACTNa+/H+antiporters are ubiquitous membrane proteins that play a central role in the ion homeostasis of cells. In this study, we examined the possible role of Na+/H+antiport inYersinia pestisvirulence and found thatY. pestisstrains lacking the major Na+/H+antiporters, NhaA and NhaB, are completely attenuated in anin vivomodel of plague. TheY. pestisderivative strain lacking thenhaAandnhaBgenes showed markedly decreased survival in blood and blood serumex vivo. Complementation of eithernhaAornhaBintransrestored the survival of theY. pestis nhaA nhaBdouble deletion mutant in blood. ThenhaA nhaBdouble deletion mutant also showed inhibited growth in an artificial serum medium, Opti-MEM, and a rich LB-based medium with Na+levels and pH values similar to those for blood. Taken together, these data strongly suggest that intact Na+/H+antiport is indispensable for the survival ofY. pestisin the bloodstreams of infected animals and thus might be regarded as a promising noncanonical drug target for infections caused byY. pestisand possibly for those caused by other blood-borne bacterial pathogens.

scholarly journals Novel Three-Component Phenazine-1-Carboxylic Acid 1,2-Dioxygenase in Sphingomonas wittichii DP58

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
Qiang Zhao ◽  
Hong-Bo Hu ◽  
Wei Wang ◽  
Xian-Qing Huang ◽  
Xue-Hong Zhang
Keyword(s):  
Carboxylic Acid ◽  
Southern China ◽  
Nitrogen Sources ◽  
Content Type ◽  
Carbon And Nitrogen ◽  
Acid Degradation ◽  
Sphingomonas Wittichii ◽  
Encoding Gene ◽  
Encoding Genes

ABSTRACT Phenazine-1-carboxylic acid, the main component of shenqinmycin, is widely used in southern China for the prevention of rice sheath blight. However, the fate of phenazine-1-carboxylic acid in soil remains uncertain. Sphingomonas wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources for growth. In this study, dioxygenase-encoding genes, pcaA1A2, were found using transcriptome analysis to be highly upregulated upon phenazine-1-carboxylic acid biodegradation. PcaA1 shares 68% amino acid sequence identity with the large oxygenase subunit of anthranilate 1,2-dioxygenase from Rhodococcus maanshanensis DSM 44675. The dioxygenase was coexpressed in Escherichia coli with its adjacent reductase-encoding gene, pcaA3, and ferredoxin-encoding gene, pcaA4, and showed phenazine-1-carboxylic acid consumption. The dioxygenase-, ferredoxin-, and reductase-encoding genes were expressed in Pseudomonas putida KT2440 or E. coli BL21, and the three recombinant proteins were purified. A phenazine-1-carboxylic acid conversion capability occurred in vitro only when all three components were present. However, P. putida KT2440 transformed with pcaA1A2 obtained phenazine-1-carboxylic acid degradation ability, suggesting that phenazine-1-carboxylic acid 1,2-dioxygenase has low specificities for its ferredoxin and reductase. This was verified by replacing PcaA3 with RedA2 in the in vitro enzyme assay. High-performance liquid chromatography–mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) analysis showed that phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation, indicating that PcaA1A2A3A4 constitutes the initial phenazine-1-carboxylic acid 1,2-dioxygenase. This study fills a gap in our understanding of the biodegradation of phenazine-1-carboxylic acid and illustrates a new dioxygenase for decarboxylation. IMPORTANCE Phenazine-1-carboxylic acid is widely used in southern China as a key fungicide to prevent rice sheath blight. However, the degradation characteristics of phenazine-1-carboxylic acid and the environmental consequences of the long-term application are not clear. S. wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources. In this study, a three-component dioxygenase, PcaA1A2A3A4, was determined to be the initial dioxygenase for phenazine-1-carboxylic acid degradation in S. wittichii DP58. Phenazine-1-carboxylic acid was converted to 1,2-dihydroxyphenazine through decarboxylation and hydroxylation. This finding may help us discover the pathway for phenazine-1-carboxylic acid degradation.

scholarly journals Dissemination of imipenem-resistant Acinetobacter baumannii strains carrying the ISAba1–bla OXA-23 genes in a Chinese hospital

2007 ◽  
Vol 56 (8) ◽  
pp. 1076-1080 ◽  
Author(s):  
Hua Zhou ◽  
Bo-Rui Pi ◽  
Qing Yang ◽  
Yun-Song Yu ◽  
Ya-Gang Chen ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Acinetobacter Baumannii ◽  
Environmental Isolates ◽  
Hospital Units ◽  
Wide Range ◽  
Polymyxin E ◽  
Encoding Gene ◽  
Almost All ◽  
Resistance Rates ◽  
Encoding Genes

An outbreak of 95 clinical infections with imipenem-resistant Acinetobacter baumannii in a Chinese hospital was investigated and the carbapenemase-encoding genes and their relationship with ISAba1 of these and a further 16 isolates recovered from the intensive care unit (ICU) environment were analysed. Almost all isolates were resistant to a wide range of antimicrobials; the lowest resistance rates were found for polymyxin E (17.1 %), cefoperazone/sulbactam (30.6 %) and ampicillin/sulbactam (67.6 %). Six pattern types defined by DNA macrorestriction patterns were distinguished among the clinical isolates with dissemination of pattern A (50 isolates) to patients in seven hospital units and pattern B (35 isolates) to eight units; the environmental isolates from ICUs were also of pattern A. All isolates were positive for the bla OXA-66 and bla OXA-23 genes. The OXA-23-encoding gene was located 34 bp downstream of ISAba1. No plasmids were detected and conjugal transfer of resistance was not demonstrated. The bla OXA-23 probe hybridized with 200 and 220 kb ApaI chromosomal fragments for type patterns A and B, respectively.

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