scholarly journals Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium

2016 ◽  
Vol 82 (10) ◽  
pp. 3032-3041 ◽  
Author(s):  
Anna Lohße ◽  
Isabel Kolinko ◽  
Oliver Raschdorf ◽  
René Uebe ◽  
Sarah Borg ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Large Scale ◽  
Genome Engineering ◽  
Linear Chain ◽  
Gene Clusters ◽  
Magnetotactic Bacteria ◽  
High Yield ◽  
Genomic Amplification ◽  
Content Type ◽  
Powerful Strategy

ABSTRACTMagnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earth's weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties. However, potential applications have been hampered by the difficult cultivation of these fastidious bacteria and their poor yields of magnetosomes. In this study, we found that the size and number of magnetosomes within the cell are controlled by many different Mam and Mms proteins. We present a strategy for the overexpression of magnetosome biosynthesis genes in the alphaproteobacteriumMagnetospirillum gryphiswaldenseby chromosomal multiplication of individual and multiple magnetosome gene clusters via transposition. While stepwise amplification of themms6operon resulted in the formation of increasingly larger crystals (increase of ∼35%), the duplication of all major magnetosome operons (mamGFDC,mamAB,mms6, andmamXY, comprising 29 genes in total) yielded an overproducing strain in which magnetosome numbers were 2.2-fold increased. We demonstrate that the tuned expression of themamandmmsclusters provides a powerful strategy for the control of magnetosome size and number, thereby setting the stage for high-yield production of tailored magnetic nanoparticles by synthetic biology approaches.IMPORTANCEBefore our study, it had remained unknown how the upper sizes and numbers of magnetosomes are genetically regulated, and overproduction of magnetosome biosynthesis had not been achieved, owing to the difficulties of large-scale genome engineering in the recalcitrant magnetotactic bacteria. In this study, we established and systematically explored a strategy for the overexpression of magnetosome biosynthesis genes by genomic amplification of single and multiple magnetosome gene clusters via sequential chromosomal insertion by transposition. Our findings also indicate that the expression levels of magnetosome proteins together limit the upper size and number of magnetosomes within the cell. We demonstrate that tuned overexpression of magnetosome gene clusters provides a powerful strategy for the precise control of magnetosome size and number.

High-yield large scale laser patterning of magnetic nanoparticles

2019 ◽  
Vol 489 ◽  
pp. 165419
Author(s):  
Wenjun Zheng ◽  
Ping Li ◽  
Zhiqiang Wang ◽  
Negar H. Golshan ◽  
Katherine S. Ziemer ◽  
...  
Keyword(s):  
Magnetic Nanoparticles ◽  
Large Scale ◽  
High Yield ◽  
Laser Patterning

Mathematical modeling of the production of magnetic nanoparticles through counter-flow non-premixed combustion for biomedical applications

2021 ◽  
Vol ahead-of-print (ahead-of-print) ◽  
Author(s):  
Shahin Akbari ◽  
Nima Hasanvand ◽  
Sadegh Sadeghi ◽  
Mehdi Bidabadi ◽  
Qingang Xiong
Keyword(s):  
Magnetic Nanoparticles ◽  
Large Scale ◽  
Flame Temperature ◽  
Combustion Process ◽  
Premixed Combustion ◽  
Counter Flow ◽  
Content Type ◽  
Thermophoretic Force ◽  
Mass Fractions ◽  
Carrier Gases

Purpose The widespread usage of magnetic nanoparticles (MNPs) requires their efficient synthesis during combustion process. This study aims to present a mathematical model for the oxidation of MNPs in a counter-flow non-premixed combustion system to produce MNPs, where the key sub-processes during the oxidation reaction are involved. Design/methodology/approach To accurately describe structure of flame and determine distributions of temperature and mass fractions of both reactants and products, equations of energy and mass conservations were solved based on the prevailing assumptions that three regions, i.e. preheating, reaction and oxidizer zones exist. Findings The numerical simulation was first validated against experimental data and characteristics of the combustion process are discussed. Eventually, the influences of crucial parameters such as reactant Lewis numbers, strain rate ratio, particle size, inert gas and thermophoretic force on structure of flame and combustion behavior were examined. The results show that maximum flame temperature can achieve 2,205 K. Replacing nitrogen with argon and helium as carrier gases can increase flame temperature by about 27% and 34%, respectively. Additionally, maximum absolute thermophoretic force was found at approximately 9.6 × 10–8 N. Originality/value To the best of authors’ knowledge, this is the first time to numerically model the preparation of MNPs in a counter-flow non-premixed combustion configuration, which can guide large-scale experimental work in a more effective way.

scholarly journals Expanded Natural Product Diversity Revealed by Analysis of Lanthipeptide-Like Gene Clusters in Actinobacteria

2015 ◽  
Vol 81 (13) ◽  
pp. 4339-4350 ◽  
Author(s):  
Qi Zhang ◽  
James R. Doroghazi ◽  
Xiling Zhao ◽  
Mark C. Walker ◽  
Wilfred A. van der Donk
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Gene Cluster ◽  
Posttranslational Modifications ◽  
Large Scale ◽  
Biological Activities ◽  
Gene Clusters ◽  
Chemical Diversity ◽  
Content Type ◽  
Modified Peptides

ABSTRACTLanthionine-containing peptides (lanthipeptides) are a rapidly growing family of polycyclic peptide natural products belonging to the large class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Lanthipeptides are widely distributed in taxonomically distant species, and their currently known biosynthetic systems and biological activities are diverse. Building on the recent natural product gene cluster family (GCF) project, we report here large-scale analysis of lanthipeptide-like biosynthetic gene clusters fromActinobacteria. Our analysis suggests that lanthipeptide biosynthetic pathways, and by extrapolation the natural products themselves, are much more diverse than currently appreciated and contain many different posttranslational modifications. Furthermore, lanthionine synthetases are much more diverse in sequence and domain topology than currently characterized systems, and they are used by the biosynthetic machineries for natural products other than lanthipeptides. The gene cluster families described here significantly expand the chemical diversity and biosynthetic repertoire of lanthionine-related natural products. Biosynthesis of these novel natural products likely involves unusual and unprecedented biochemistries, as illustrated by several examples discussed in this study. In addition, class IV lanthipeptide gene clusters are shown not to be silent, setting the stage to investigate their biological activities.

scholarly journals A Targetron-Recombinase System for Large-Scale Genome Engineering of Clostridia

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Tristan Cerisy ◽  
William Rostain ◽  
Audam Chhun ◽  
Magali Boutard ◽  
Marcel Salanoubat ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Genome Structure ◽  
Anaerobic Bacteria ◽  
Cre Recombinase ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Genomic Deletions ◽  
Cassette Exchange ◽  
Clostridium Phytofermentans

ABSTRACT Clostridia are a group of Gram-positive anaerobic bacteria of medical and industrial importance for which limited genetic methods are available. Here, we demonstrate an approach to make large genomic deletions and insertions in the model Clostridium phytofermentans by combining designed group II introns (targetrons) and Cre recombinase. We apply these methods to delete a 50-gene prophage island by programming targetrons to position markerless lox66 and lox71 sites, which mediate deletion of the intervening 39-kb DNA region using Cre recombinase. Gene expression and growth of the deletion strain showed that the prophage genes contribute to fitness on nonpreferred carbon sources. We also inserted an inducible fluorescent reporter gene into a neutral genomic site by recombination-mediated cassette exchange (RMCE) between genomic and plasmid-based tandem lox sites bearing heterospecific spacers to prevent intracassette recombination. These approaches generally enable facile markerless genome engineering in clostridia to study their genome structure and regulation. IMPORTANCE Clostridia are anaerobic bacteria with important roles in intestinal and soil microbiomes. The inability to experimentally modify the genomes of clostridia has limited their study and application in biotechnology. Here, we developed a targetron-recombinase system to efficiently make large targeted genomic deletions and insertions using the model Clostridium phytofermentans. We applied this approach to reveal the importance of a prophage to host fitness and introduce an inducible reporter by recombination-mediated cassette exchange.

scholarly journals High-Throughput Microfluidic Sorting of Live Magnetotactic Bacteria

2018 ◽  
Vol 84 (17) ◽  
Author(s):  
Andy Tay ◽  
Daniel Pfeiffer ◽  
Kathryn Rowe ◽  
Aaron Tannenbaum ◽  
Felix Popp ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Magnetotactic Bacteria ◽  
Alkaline Treatment ◽  
Genetically Engineered ◽  
Wild Type ◽  
Content Type ◽  
Large Numbers ◽  
Microfluidic Sorting ◽  
Selection Of

ABSTRACTMagnetic nanoparticles (MNPs) are useful for many biomedical applications, but it is challenging to synthetically produce them in large numbers with uniform properties and surface functionalization. Magnetotactic bacteria (MTB) produce magnetosomes with homogenous sizes, shapes, and magnetic properties. Consequently, there is interest in using MTB as biological factories for MNP production. Nonetheless, MTB can only be grown to low yields, and wild-type strains produce low numbers of MNPs/bacterium. There are also limited technologies to facilitate the selection of MTB with different magnetic contents, such as MTB with compromised and enhanced biomineralization ability. Here, we describe a magnetic microfluidic platform combined with transient cold/alkaline treatment to temporarily reduce the rapid flagellar motion of MTB without compromising their long-term proliferation and biomineralization ability for separating MTB on the basis of their magnetic contents. This strategy enables live MTB to be enriched, which, to the best of our knowledge, has not been achieved with another previously described magnetic microfluidic device that makes use of ferrofluid and heat. Our device also facilitates the high-throughput (25,000 cells/min) separation of wild-typeMagnetospirillum gryphiswaldense(MSR-1) from nonmagnetic ΔmamABMSR-1 mutants with a sensitivity of up to 80% and isolation purity of up to 95%, as confirmed with a gold-standard fluorescent-activated cell sorter (FACS) technique. This offers a 25-fold higher throughput than other previously described magnetic microfluidic platforms (1,000 cells/min). The device can also be used to isolateMagnetospirillum magneticum(AMB-1) mutants with different ranges of magnetosome numbers with efficiencies close to theoretical estimates. We believe this technology will facilitate the magnetic characterization of genetically engineered MTB for a variety of applications, including using MTB for large-scale, controlled MNP production.IMPORTANCEOur magnetic microfluidic technology can greatly facilitate biological applications with magnetotactic bacteria, from selection and screening to analysis. This technology will be of interest to microbiologists, chemists, and bioengineers who are interested in the biomineralization and selection of magnetotactic bacteria (MTB) for applications such as directed evolution and magnetogenetics.

scholarly journals MagCluster: a Tool for Identification, Annotation, and Visualization of Magnetosome Gene Clusters

2022 ◽  
Author(s):  
Runjia Ji ◽  
Wensi Zhang ◽  
Yongxin Pan ◽  
Wei Lin
Keyword(s):  
Large Scale ◽  
Genomic Data ◽  
Gene Clusters ◽  
Magnetotactic Bacteria ◽  
Evolutionary Origin ◽  
Organelle Biogenesis ◽  
Efficient Exploration

Magnetosome gene clusters (MGCs), which are responsible for magnetosome biosynthesis and organization in magnetotactic bacteria (MTB), are the key to deciphering the mechanisms and evolutionary origin of magnetoreception, organelle biogenesis, and intracellular biomineralization in bacteria. Here, we report the development of MagCluster, a Python stand-alone tool for efficient exploration of MGCs from large-scale (meta)genomic data.

scholarly journals Cre-lox-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of Magnetospirillum gryphiswaldense

2010 ◽  
Vol 76 (8) ◽  
pp. 2439-2444 ◽  
Author(s):  
Susanne Ullrich ◽  
Dirk Sch�ler
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Normal Growth ◽  
Magnetotactic Bacteria ◽  
Growth Patterns ◽  
Helper Plasmid ◽  
Magnetospirillum Gryphiswaldense ◽  
Large Deletions ◽  
Unknown Gene ◽  
Resistance Markers

ABSTRACT Magnetosome biomineralization and magnetotaxis in magnetotactic bacteria are controlled by numerous, mostly unknown gene functions that are predominantly encoded by several operons located within the genomic magnetosome island (MAI). Genetic analysis of magnetotactic bacteria has remained difficult and requires the development of novel tools. We established a Cre-lox-based deletion method which allows the excision of large genomic fragments in Magnetospirillum gryphiswaldense. Two conjugative suicide plasmids harboring lox sites that flanked the target region were subsequently inserted into the chromosome by homologous recombination, requiring only one single-crossover event, respectively, and resulting in a double cointegrate. Excision of the targeted chromosomal segment that included the inserted plasmids and their resistance markers was induced by trans expression of Cre recombinase, which leaves behind a scar of only a single loxP site. The Cre helper plasmid was then cured from the deletant strain by relief of antibiotic selection. We have used this method for the deletion of 16.3-kb, 61-kb, and 67.3-kb fragments from the genomic MAI, either in a single round or in subsequent rounds of deletion, covering a region of approximately 87 kb that comprises the mamAB, mms6, and mamGFDC operons. As expected, all mutants were Mag− and some were Mot−; otherwise, they showed normal growth patterns, which indicates that the deleted region is not essential for viability in the laboratory. The method will facilitate future functional analysis of magnetosome genes and also can be utilized for large-scale genome engineering in magnetotactic bacteria.

scholarly journals Identification of a Specific Maleate Hydratase in the Direct Hydrolysis Route of the Gentisate Pathway

2015 ◽  
Vol 81 (17) ◽  
pp. 5753-5760 ◽  
Author(s):  
Kun Liu ◽  
Ying Xu ◽  
Ning-Yi Zhou
Keyword(s):  
Malate Dehydrogenase ◽  
Mutant Strain ◽  
Stop Codon ◽  
Gene Clusters ◽  
High Yield ◽  
Spontaneous Mutant ◽  
Content Type ◽  
Gentisate Pathway ◽  
Pseudomonas Alcaligenes ◽  
Direct Hydrolysis

ABSTRACTIn contrast to the well-characterized and more common maleylpyruvate isomerization route of the gentisate pathway, the direct hydrolysis route occurs rarely and remains unsolved. InPseudomonas alcaligenesNCIMB 9867, two gene clusters,xlnandhbz, were previously proposed to be involved in gentisate catabolism, and HbzF was characterized as a maleylpyruvate hydrolase converting maleylpyruvate to maleate and pyruvate. However, the complete degradation pathway of gentisate through direct hydrolysis has not been characterized. In this study, we obtained from the NCIMB culture collection aPseudomonas alcaligenesspontaneous mutant strain that lacked thexlncluster and designated the mutant strain SponMu. Thehbzcluster in strain SponMu was resequenced, revealing the correct location of the stop codon forhbzIand identifying a new gene,hbzG. HbzIJ was demonstrated to be a maleate hydratase consisting of large and small subunits, stoichiometrically converting maleate to enantiomerically pured-malate. HbzG is a glutathione-dependent maleylpyruvate isomerase, indicating the possible presence of two alternative pathways of maleylpyruvate catabolism. However, thehbzF-disrupted mutant could still grow on gentisate, while disruption ofhbzGprevented this ability, indicating that the direct hydrolysis route was not a complete pathway in strain SponMu. Subsequently, ad-malate dehydrogenase gene was introduced into thehbzG-disrupted mutant, and the engineered strain was able to grow on gentisate via the direct hydrolysis route. This fills a gap in our understanding of the direct hydrolysis route of the gentisate pathway and provides an explanation for the high yield ofd-malate from maleate by thisd-malate dehydrogenase-deficient natural mutant.

scholarly journals Site-Specific Recombination Strategies for Engineering Actinomycete Genomes

2012 ◽  
Vol 78 (6) ◽  
pp. 1804-1812 ◽  
Author(s):  
Simone Herrmann ◽  
Theresa Siegl ◽  
Marta Luzhetska ◽  
Lutz Petzke ◽  
Caroline Jilg ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Cre Recombinase ◽  
Gene Encoding ◽  
Site Specific ◽  
Content Type ◽  
Site Specific Recombination ◽  
Site Specific Integration ◽  
Genomic Regions

ABSTRACTThe feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the generaStreptomyces,Micromonospora, andSaccharothrix. Two different systems based on Cre/loxPand Dre/roxhave been utilized for numerous applications. The activity of the Cre recombinase on the heterospecificloxLEandloxREsites was similar to its activity on wild-typeloxPsites. Moreover, an apramycin resistance marker flanked by theloxLEREsites was eliminated from theStreptomyces coelicolorM145 genome at a surprisingly high frequency (80%) compared to other bacteria. A synthetic gene encoding the Dre recombinase was constructed and successfully expressed in actinomycetes. We developed a marker-free expression method based on the combination of phage integration systems and site-specific recombinases. The Cre recombinase has been used in the deletion of huge genomic regions, including the phenalinolactone, monensin, and lipomycin biosynthetic gene clusters fromStreptomycessp. strain Tü6071,Streptomyces cinnamonensisA519, andStreptomyces aureofaciensTü117, respectively. Finally, we also demonstrated the site-specific integration of plasmid and cosmid DNA into the chromosome of actinomycetes catalyzed by the Cre recombinase. We anticipate that the strategies presented here will be used extensively to study the genetics of actinomycetes.

A Simple Synthesis of the Anticancer Drug Altretamine

2020 ◽  
Vol 17 (8) ◽  
pp. 628-630
Author(s):  
Vu Binh Duong ◽  
Pham Van Hien ◽  
Tran Thai Ngoc ◽  
Phan Dinh Chau ◽  
Tran Khac Vu
Keyword(s):  
Aqueous Solution ◽  
Anticancer Drug ◽  
Potassium Hydroxide ◽  
Large Scale ◽  
Synthesis Method ◽  
Practical Method ◽  
Cyanuric Chloride ◽  
Simple Synthesis ◽  
High Yield ◽  
One Step

A simple and practical method for the synthesis on a large scale of altretamine (1), a wellknown antitumor drug, has been successfully developed. The synthesis method involves the conversion of cyanuric chloride (2) into altretamine (1) by dimethylamination of 2 with an aqueous solution of 40% dimethylamine and potassium hydroxide in 1, -dioxan 4in one step to give altretamine (1) in high yield.

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