Improvement of FK506 Production in Streptomyces tsukubaensis by Genetic Enhancement of the Supply of Unusual Polyketide Extender Units via Utilization of Two Distinct Site-Specific Recombination Systems

ABSTRACTFK506 is a potent immunosuppressant that has a wide range of clinical applications. Its 23-member macrocyclic scaffold, mainly with a polyketide origin, features two methoxy groups at C-13 and C-15 and one allyl side chain at C-21, due to the region-specific incorporation of two unusual extender units derived from methoxymalonyl-acyl carrier protein (ACP) and allylmalonyl-coenzyme A (CoA), respectively. Whether their intracellular formations can be a bottleneck for FK506 production remains elusive. In this study, we report the improvement of FK506 yield in the producing strainStreptomyces tsukubaensisby the duplication of two sets of pathway-specific genes individually encoding the biosyntheses of these two extender units, thereby providing a promising approach to generate high-FK506-producing strains via genetic manipulation. Taking advantage of the fact thatS. tsukubaensisis amenable to two actinophage (ΦC31 and VWB) integrase-mediated recombination systems, we genetically enhanced the biosyntheses of methoxymalonyl-ACP and allylmalonyl-CoA, as indicated by transcriptional analysis. Together with the optimization of glucose supplementation, the maximal FK506 titer eventually increased by approximately 150% in comparison with that of the original strain. The strategy of engineering the biosynthesis of unusual extender units described here may be applicable to improving the production of other polyketide or nonribosomal peptide natural products that contain pathway-specific building blocks.

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Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

Applied and Environmental Microbiology ◽

10.1128/aem.04190-14 ◽

2015 ◽

Vol 81 (7) ◽

pp. 2481-2488 ◽

Cited By ~ 19

Author(s):

Volker Winstel ◽

Petra Kühner ◽

Bernhard Krismer ◽

Andreas Peschel ◽

Holger Rohde

Keyword(s):

Plasmid Dna ◽

Genetic Manipulation ◽

Current Knowledge ◽

Virulence Determinants ◽

Coagulase Negative Staphylococci ◽

Reliable Technique ◽

Content Type ◽

Research Activities ◽

Wide Range ◽

Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

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A logic model of the implementation of a regional workforce strategy in positive behavioural support

Tizard Learning Disability Review ◽

10.1108/tldr-05-2021-0013 ◽

2021 ◽

Vol ahead-of-print (ahead-of-print) ◽

Author(s):

Steve Noone ◽

Alison Branch ◽

Melissa Sherring

Keyword(s):

Large Scale ◽

Social Care ◽

Building Blocks ◽

Logic Model ◽

Competency Framework ◽

Health Staff ◽

Content Type ◽

Behavioural Support ◽

Health And Social Care ◽

Wide Range

Purpose Positive behavioural support (PBS) as a framework for delivering quality services is recognised in important policy documents (CQC, 2020; NICE, 2018), yet there is an absence in the literature on how this could be implemented on a large scale. The purpose of this paper is to describe a recent implementation of a workforce strategy to develop PBS across social care and health staff and family carers, within the footprint of a large integrated care system. Design/methodology/approach A logic model describes how an initial scoping exercise led to the production of a regional workforce strategy based on the PBS Competence Framework (2015). It shows how the creation of a regional steering group was able to coordinate important developmental stages and integrate multiple agencies into a single strategy to implement teaching and education in PBS. It describes the number of people who received teaching and education in PBS and the regional impact of the project in promoting cultural change within services. Findings This paper demonstrates a proof of concept that it is possible to translate the PBS Competency Framework (2015) into accredited courses. Initial scoping work highlighted the ineffectiveness of traditional training in PBS. Using blended learning and competency-based supervision and assessment, it was possible to create a new way to promote large-scale service developments in PBS supported by the governance of a new organisational structure. This also included family training delivered by family trainers. This builds on the ideas by Denne et al. (2020) that many of the necessary building blocks of implementation already exist within a system. Social implications A co-ordinated teaching and education strategy in PBS may help a wide range of carers to become more effective in supporting the people they care for. Originality/value This is the first attempt to describe the implementation of a framework for PBS within a defined geographical location. It describes the collaboration of health and social care planners and a local university to create a suite of courses built around the PBS coalition competency framework.

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The Ndr/LATS Kinase Cbk1 Regulates a Specific Subset of Ace2 Functions and Suppresses the Hypha-to-Yeast Transition in Candida albicans

mBio ◽

10.1128/mbio.01900-20 ◽

2020 ◽

Vol 11 (4) ◽

Author(s):

Rohan S. Wakade ◽

Laura C. Ristow ◽

Mark A. Stamnes ◽

Anuj Kumar ◽

Damian J. Krysan

Keyword(s):

Candida Albicans ◽

Regulatory Network ◽

Transcriptional Analysis ◽

Large Set ◽

Phase Growth ◽

Content Type ◽

Hyphal Morphogenesis ◽

Specific Subset ◽

Wide Range ◽

Yeast Phase

ABSTRACT The regulation of Ace2 and morphogenesis (RAM) pathway is an important regulatory network in the human fungal pathogen Candida albicans. The RAM pathway’s two most well-studied components, the NDR/Lats kinase Cbk1 and its putative substrate, the transcription factor Ace2, have a wide range of phenotypes and functions. It is not clear, however, which of these functions are specifically due to the phosphorylation of Ace2 by Cbk1. To address this question, we first compared the transcriptional profiles of CBK1 and ACE2 deletion mutants. This analysis indicates that, of the large number of genes whose expression is affected by deletion of CBK1 and ACE2, only 5.5% of those genes are concordantly regulated. Our data also suggest that Ace2 directly or indirectly represses a large set of genes during hyphal morphogenesis. Second, we generated strains containing ACE2 alleles with alanine mutations at the Cbk1 phosphorylation sites. Phenotypic and transcriptional analysis of these ace2 mutants indicates that, as in Saccharomyces cerevisiae, Cbk1 regulation is important for daughter cell localization of Ace2 and cell separation during yeast-phase growth. In contrast, Cbk1 phosphorylation of Ace2 plays a minor role in C. albicans yeast-to-hypha transition. We have, however, discovered a new function for the Cbk1-Ace2 axis. Specifically, Cbk1 phosphorylation of Ace2 prevents the hypha-to-yeast transition. To our knowledge, this is one of the first regulators of the C. albicans hypha-to-yeast transition to be described. Finally, we present an integrated model for the role of Cbk1 in the regulation of hyphal morphogenesis in C. albicans. IMPORTANCE The regulation of Ace2 and morphogenesis (RAM) pathway is a key regulatory network that plays a role in many aspects of C. albicans pathobiology. In addition to characterizing the transcriptional effects of this pathway, we discovered that Cbk1 and Ace2, a key RAM pathway regulator-effector pair, mediate a specific set of the overall functions of the RAM pathway. We have also discovered a new function for the Cbk1-Ace2 axis: suppression of the hypha-to-yeast transition. Very few regulators of this transition have been described, and our data indicate that maintenance of hyphal morphogenesis requires suppression of yeast phase growth by Cbk1-regulated Ace2.

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Activation of Antibiotic Production in Bacillus spp. by Cumulative Drug Resistance Mutations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01932-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7799-7804 ◽

Cited By ~ 8

Author(s):

Shigeo Tojo ◽

Yukinori Tanaka ◽

Kozo Ochi

Keyword(s):

Drug Resistance ◽

Antibiotic Production ◽

Transcriptional Analysis ◽

Peptide Antibiotics ◽

Resistance Mutations ◽

Content Type ◽

Drug Resistance Mutations ◽

Wide Range ◽

Bacillus Spp ◽

High Level

ABSTRACTBacillus subtilisstrains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations inB. subtilisstrain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in themthAandrpsLgenes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations inrpoB, which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes ofBacillus amyloliquefaciens, leading to actual production of antibiotic(s).

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Genetic Analysis Reveals the Identity of the Photoreceptor for Phototaxis in Hormogonium Filaments of Nostoc punctiforme

Journal of Bacteriology ◽

10.1128/jb.02374-14 ◽

2014 ◽

Vol 197 (4) ◽

pp. 782-791 ◽

Cited By ~ 35

Author(s):

Elsie L. Campbell ◽

Kari D. Hagen ◽

Rui Chen ◽

Douglas D. Risser ◽

Daniela P. Ferreira ◽

...

Keyword(s):

Mutational Analysis ◽

Transcriptional Start Site ◽

Gene Clusters ◽

Gliding Motility ◽

Transcriptional Analysis ◽

Nostoc Punctiforme ◽

Recognition Sequence ◽

Content Type ◽

Wide Range ◽

Genes Encoding

In cyanobacterialNostocspecies, substratum-dependent gliding motility is confined to specialized nongrowing filaments called hormogonia, which differentiate from vegetative filaments as part of a conditional life cycle and function as dispersal units. Here we confirm thatNostoc punctiformehormogonia are positively phototactic to white light over a wide range of intensities.N. punctiformecontains two gene clusters (clusters 2 and 2i), each of which encodes modular cyanobacteriochrome–methyl-accepting chemotaxis proteins (MCPs) and other proteins that putatively constitute a basic chemotaxis-like signal transduction complex. Transcriptional analysis established that all genes in clusters 2 and 2i, plus two additional clusters (clusters 1 and 3) with genes encoding MCPs lacking cyanobacteriochrome sensory domains, are upregulated during the differentiation of hormogonia. Mutational analysis determined that only genes in cluster 2i are essential for positive phototaxis inN. punctiformehormogonia; here these genes are designatedptx(forphototaxis) genes. The cluster is unusual in containing complete or partial duplicates of genes encoding proteins homologous to the well-described chemotaxis elements CheY, CheW, MCP, and CheA. The cyanobacteriochrome-MCP gene (ptxD) lacks transmembrane domains and has 7 potential binding sites for bilins. The transcriptional start site of theptxgenes does not resemble a sigma 70 consensus recognition sequence; moreover, it is upstream of two genes encoding gas vesicle proteins (gvpAandgvpC), which also are expressed only in the hormogonium filaments ofN. punctiforme.

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The Genome of the Alga-Associated Marine Flavobacterium Formosa agariphila KMM 3901TReveals a Broad Potential for Degradation of Algal Polysaccharides

Applied and Environmental Microbiology ◽

10.1128/aem.01937-13 ◽

2013 ◽

Vol 79 (21) ◽

pp. 6813-6822 ◽

Cited By ~ 112

Author(s):

Alexander J. Mann ◽

Richard L. Hahnke ◽

Sixing Huang ◽

Johannes Werner ◽

Peng Xing ◽

...

Keyword(s):

Building Blocks ◽

Glycoside Hydrolases ◽

Acid Fermentation ◽

Content Type ◽

Mixed Acid ◽

Marine Habitats ◽

Wide Range ◽

Algal Polysaccharides ◽

Polysaccharide Utilization Loci ◽

Growth Experiments

ABSTRACTIn recent years, representatives of theBacteroideteshave been increasingly recognized as specialists for the degradation of macromolecules.Formosaconstitutes aBacteroidetesgenus within the classFlavobacteria, and the members of this genus have been found in marine habitats with high levels of organic matter, such as in association with algae, invertebrates, and fecal pellets. Here we report on the generation and analysis of the genome of the type strain ofFormosa agariphila(KMM 3901T), an isolate from the green algaAcrosiphonia sonderi.F. agariphilais a facultative anaerobe with the capacity for mixed acid fermentation and denitrification. Its genome harbors 129 proteases and 88 glycoside hydrolases, indicating a pronounced specialization for the degradation of proteins, polysaccharides, and glycoproteins. Sixty-five of the glycoside hydrolases are organized in at least 13 distinct polysaccharide utilization loci, where they are clustered with TonB-dependent receptors, SusD-like proteins, sensors/transcription factors, transporters, and often sulfatases. These loci play a pivotal role in bacteroidetal polysaccharide biodegradation and in the case ofF. agariphilarevealed the capacity to degrade a wide range of algal polysaccharides from green, red, and brown algae and thus a strong specialization of toward an alga-associated lifestyle. This was corroborated by growth experiments, which confirmed usage particularly of those monosaccharides that constitute the building blocks of abundant algal polysaccharides, as well as distinct algal polysaccharides, such as laminarins, xylans, and κ-carrageenans.

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New Insights into Chloramphenicol Biosynthesis in Streptomyces venezuelae ATCC 10712

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04272-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7441-7450 ◽

Cited By ~ 45

Author(s):

Lorena T. Fernández-Martínez ◽

Chiara Borsetto ◽

Juan Pablo Gomez-Escribano ◽

Maureen J. Bibb ◽

Mahmoud M. Al-Bassam ◽

...

Keyword(s):

Gene Cluster ◽

Acyl Carrier Protein ◽

Bioinformatic Analysis ◽

Biosynthetic Gene Cluster ◽

Transcriptional Analysis ◽

Biosynthetic Gene ◽

Phosphopantetheinyl Transferase ◽

Streptomyces Venezuelae ◽

Content Type ◽

New Genes

ABSTRACTComparative genome analysis revealed seven uncharacterized genes,sven0909tosven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916–sven0928) ofStreptomyces venezuelaestrain ATCC 10712 that was absent in a closely relatedStreptomycesstrain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na+/H+antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene,sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest thatsven0929is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway.

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Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. Strain MP688

Applied and Environmental Microbiology ◽

10.1128/aem.03740-13 ◽

2013 ◽

Vol 80 (3) ◽

pp. 1082-1090 ◽

Cited By ~ 63

Author(s):

Willem P. Dijkman ◽

Marco W. Fraaije

Keyword(s):

Building Blocks ◽

Degradation Pathway ◽

Enzyme Characterization ◽

Bacterial Degradation ◽

Primary Alcohols ◽

Content Type ◽

Wide Range ◽

Furandicarboxylic Acid ◽

Ph Range

ABSTRACTIn the search for useful and renewable chemical building blocks, 5-hydroxymethylfurfural (HMF) has emerged as a very promising candidate, as it can be prepared from sugars. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a substitute for petroleum-based terephthalate in polymer production. On the basis of a recently identified bacterial degradation pathway for HMF, candidate genes responsible for selective HMF oxidation have been identified. Heterologous expression of a protein fromMethylovorussp. strain MP688 inEscherichia coliand subsequent enzyme characterization showed that the respective gene indeed encodes an efficient HMF oxidase (HMFO). HMFO is a flavin adenine dinucleotide-containing oxidase and belongs to the glucose-methanol-choline-type flavoprotein oxidase family. Intriguingly, the activity of HMFO is not restricted to HMF, as it is active with a wide range of aromatic primary alcohols and aldehydes. The enzyme was shown to be relatively thermostable and active over a broad pH range. This makes HMFO a promising oxidative biocatalyst that can be used for the production of FDCA from HMF, a reaction involving both alcohol and aldehyde oxidations.

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Factors Affecting Molecular Self-Assembly and Its Mechanism

Scientific Research Journal ◽

10.24191/srj.v9i1.5385 ◽

2012 ◽

Vol 9 (1) ◽

pp. 43 ◽

Cited By ~ 1

Author(s):

Hueyling Tan

Keyword(s):

Self Assembly ◽

Building Blocks ◽

Molecular Engineering ◽

Molecular Structures ◽

Polymer Science ◽

New Approach ◽

Factors Affecting ◽

Dna Structures ◽

Self Assembling ◽

Wide Range

Molecular self-assembly is ubiquitous in nature and has emerged as a new approach to produce new materials in chemistry, engineering, nanotechnology, polymer science and materials. Molecular self-assembly has been attracting increasing interest from the scientific community in recent years due to its importance in understanding biology and a variety of diseases at the molecular level. In the last few years, considerable advances have been made in the use ofpeptides as building blocks to produce biological materials for wide range of applications, including fabricating novel supra-molecular structures and scaffolding for tissue repair. The study ofbiological self-assembly systems represents a significant advancement in molecular engineering and is a rapidly growing scientific and engineering field that crosses the boundaries ofexisting disciplines. Many self-assembling systems are rangefrom bi- andtri-block copolymers to DNA structures as well as simple and complex proteins andpeptides. The ultimate goal is to harness molecular self-assembly such that design andcontrol ofbottom-up processes is achieved thereby enabling exploitation of structures developed at the meso- and macro-scopic scale for the purposes oflife and non-life science applications. Such aspirations can be achievedthrough understanding thefundamental principles behind the selforganisation and self-synthesis processes exhibited by biological systems.

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Stereospecific, Palladium-catalyzed C(sp3)–H Alkenylation and Alkynylation of a Proline Derivative Enabled by 8-Aminoquinoline as a Directing Group

10.26434/chemrxiv.12034743 ◽

2020 ◽

Author(s):

Aleksandra Balliu ◽

Aaltje Roelofje Femmigje Strijker ◽

Michael Oschmann ◽

Monireh Pourghasemi Lati ◽

Oscar Verho

Keyword(s):

Carboxylic Acid ◽

Building Blocks ◽

Wide Range ◽

Palladium Catalyzed ◽

Directing Group ◽

High Yields ◽

Vinyl Iodides ◽

Initial Results

In this preprint, we present our initial results concerning a stereospecific Pd-catalyzed protocol for the C3 alkenylation and alkynylation of a proline derivative carrying the well utilized 8‑aminoquinoline directing group. Efficient C–H alkenylation was achieved with a wide range of vinyl iodides bearing different aliphatic, aromatic and heteroaromatic substituents, to furnish the corresponding C3 alkenylated products in good to high yields. In addition, we were able show that this protocol can also be used to install an alkynyl group into the pyrrolidine scaffold, when a TIPS-protected alkynyl bromide was used as the reaction partner. Furthermore, two different methods for the removal of the 8-aminoquinoline auxiliary are reported, which can enable access to both cis- and trans-configured carboxylic acid building blocks from the C–H alkenylation products.

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