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The general process of protoplast fusion compared with L-form fusion. Both methods share some steps like the loss of cell wall and cell-cell fusion but differ in other key steps. Fusion in L-forms can lead to multiple possibilities due to polyploidy in daughter cells, whereas reversion to a walled state is essential in protoplast fusion. Differences in chromosome size are for depiction purposes only and not to scale.
Source publication
Filamentous actinobacteria are widely used as microbial cell factories to produce valuable secondary metabolites, including the vast majority of clinically relevant antimicrobial compounds. Secondary metabolites are typically encoded by large biosynthetic gene clusters, which allow for a modular approach to generating diverse compounds through reco...
Contexts in source publication
Context 1
... an alternative was found by stripping away the cell wall altogether. The basic set-up of protoplast fusion involves four consecutive steps: (i) protoplast formation, (ii) protoplast fusion, (iii) recombination between chromosomes within the fused cells, and (iv) reversion of protoplasts to walled cells (Figure 1) [10][11][12]. Protoplasts are obtained by treating walled cells with lysozyme, which degrades the major component of the cell wall, peptidoglycan (PG). ...
Context 2
... alternative method would thus be valuable, especially for generating increased phenotypic diversity and stabilizing fusant strains. Figure 1. The general process of protoplast fusion compared with L-form fusion. ...
Context 3
... of the important consequences of wall deficiency is that the resulting cells are polyploid. Because DNA segregation is likely unregulated during this phase, division of L-forms leads to the uneven distribution of chromosomes among progeny (Figure 1). While some cells inherit a single chromosome, others will inherit multiple chromosomes, with different ratios of the parental chromosomal types. ...
Context 4
... we have observed spontaneous fusion between L-forms (our unpublished results). In contrast to protoplasts, L-form fusion is not followed by regeneration of the cell wall and the subsequent generation of haploid cells (Figure 1). Three potential outcomes are possible after fusion: (i) recombination between chromosomes, (ii) polyploidy where multiple copies of the recombinant genome are present, and (iii) heterokaryosis where both parental genomes are maintained. ...
Similar publications
In an attempt to construct superior Trichoderma harzianum isolates for improving β-glucosidase productivity, protoplast fusion technique was applied. After application of different mutagenic treatments, twenty mutants were chosen to be tested for their resistance or sensitivity against four antifungal agents. Out of them, four isolates were selecte...
Citations
... Different microorganisms require different enzymes (as well as different enzyme amounts and types) when preparing protoplasts. Consequently, different measurements should be made, based on the various microorganisms used [31][32][33]. As one of the traditional methods of protoplast fusion breeding, improving the rate of protoplast regeneration to achieve stable growth of fusion strains under the premise of high mutation lethality is essential [34]. ...
Background
L-lysine is widely used for feed and special diet products. The transformation of fermentation strains plays a decisive role in the development of these industries. Based on the mutation breeding theory and metabolic engineering methods, this study aimed to improve the regeneration rate of high-lethality protoplasts by combining multiple mutagenesis and homologous cell fusion techniques to efficiently concentrate multiple dominant mutations and optimize the L-lysine production strain Escherichia coli QDW.
Results
In order to obtain the best protoplasts, the optimal enzymolysis time was selected as 4 h. The optimal lysozyme concentration was estimated at 0.8 mg/mL, because the protoplast formation rate and regeneration rate reached 90% and 30%, respectively, and their product reached the maximum. In this study, it was necessary that UV mutagenesis be excessive to obtain an expanded mutation library. For high lethality protoplasts, under the premise of minimal influence on its recovery, the optimal time for UV mutagenesis of protoplasts was 7 min, and the optimal time for thermal inactivation of protoplasts at 85 ℃ was 30 min. After homologous fusion, four fusion strains of E. coli were obtained, and their stability was analyzed by flow cytometry. The L-lysine yield of QDW-UH3 increased by 7.2% compared with that of QDW in a fermentation experiment, which promoted the expression of key enzymes in L-lysine synthesis, indicating that the combination of ultraviolet mutagenic breeding and protoplast fusion technology improved the acid-production level of the fusion strain.
Conclusion
This method provides a novel approach for the targeted construction of microbial cell factories.
... On the other hand, cells without a wall are resistant to a whole class of cell wall-targeting antibiotics (penicillins, cephalosporins), transport to the extracellular space is potentially easier, and the cells are stably polyploid. These characteristics can make L-forms a unique model system to study not only cell biology but also questions in the fields of biotechnology, evolution, and the origin of life (34,41,42). First, our use of coiled-coil-directed fusion can be extended to synthetic cells to obtain fusions that increase cellular complexity as well as interspecies fusions. ...
Fusion of cells is an important and common biological process that leads to the mixing of cellular contents and the formation of multinuclear cells. Cell fusion occurs when distinct membranes are brought into proximity of one another and merge to become one. Fusion holds promise for biotechnological innovations, for instance, for the discovery of urgently needed new antibiotics. Here, we used antibiotic-producing bacteria that can proliferate without their cell wall as a model to investigate cell-cell fusion. We found that fusion between genetically distinct cells yields heterokaryons that are viable, contain multiple selection markers, and show increased antimicrobial activity. The rate of fusion induced using physical and chemical methods was dependent on membrane fluidity, which is related to lipid composition as a function of cellular age. Finally, by using an innovative system of synthetic membrane-associated lipopeptides, we achieved targeted fusion between distinctly marked cells to further enhance fusion efficiency. These results provide a molecular handle to understand and control cell-cell fusion, which can be used in the future for the discovery of new drugs.
IMPORTANCE Cell-cell fusion is instrumental in introducing different sets of genes in the same environment, which subsequently leads to diversity. There is need for new protocols to fuse cells of different types together for biotechnological applications like drug discovery. We present here wall-deficient cells as a platform for the same. We identify the fluidity of the membrane as an important characteristic for the process of fusion. We demonstrate a cell-specific approach for fusion using synthetically designed peptides yielding cells with modified antibiotic production profiles. Overall, wall-deficient cells can be a chassis for innovative metabolite production by providing an alternative method for cell-cell fusion.
... These disadvantages are clear limitations for the production and the exploitation of original valuable metabolites. The article of Shitut et al. (2020) [28] highlights the potential of new engineered wall-deficient bacteria having the capacity to proliferate without their cell wall. Unlike protoplasts, Lforms are able to stabilize multiple chromosomes over many cycles of division. ...
In recent decades scientific research has demonstrated that the microbial world is infinitely richer and more surprising than we could have imagined. Every day, new molecules produced by microorganisms are discovered, and their incredible diversity has not yet delivered all of its messages. The current challenge of research is to select from the wide variety of characterized microorganisms and compounds, those which could provide rapid answers to crucial questions about human or animal health or more generally relating to society’s demands for medicine, pharmacology, nutrition or everyday well-being.
Keywords: secondary metabolites; microorganisms; biotechnology; screening; production; extraction; bioactive properties; perspectives
Most biosynthetic gene clusters (BGCs) of Actinobacteria are either silent or expressed less than the detectable level. The non-genetic approaches including biological interactions, chemical agents, and physical stresses that can be used to awaken silenced pathways are compared in this paper. These non-genetic induction strategies often need screening approaches, including one strain many compounds (OSMAC), reporter-guided mutant selection, and high throughput elicitor screening (HiTES) have been developed. Different types of genetic manipulations applied in the induction of cryptic BGCs of Actinobacteria can be categorized as genome-wide pleiotropic and targeted approaches like manipulation of global regulatory systems, modulation of regulatory genes, ribosome and engineering of RNA polymerase or phosphopantheteine transferases. Targeted approaches including genome editing by CRISPR, mutation in transcription factors and modification of BGCs promoters, inactivation of the highly expressed biosynthetic pathways, deleting the suppressors or awakening the activators, heterologous expression, or refactoring of gene clusters can be applied for activation of pathways which are predicted to synthesize new bioactive structures in genome mining studies of Acinobacteria. In this review, the challenges and advantages of employing these approaches in induction of Actinobacteria BGCs are discussed. Further, novel natural products needed as drug for pharmaceutical industry or as biofertilizers in agricultural industry can be discovered even from known species of Actinobactera by the innovative approaches of metabolite biosynthesis elicitation.
To effectively combat parasitic weeds, it is essential to develop advanced biocontrol agents capable of thriving in diverse environmental conditions. The objectives of this study were to evaluate the potential of Fusarium oxysporum isolates as biocontrol agents against Egyptian broomrapes in tomato crops and to explore whether protoplast fusion could improve their biocontrol ability. Eleven F. oxysporum isolates significantly reduced the germination of Phelipanche aegyptiaca seeds. Consequently, these isolates were further evaluated for their potential as biological control agents against broomrapes through glasshouse and field experiments. The isolate KGh2-1 of F. oxysporum showed the highest reduction in the number of emerged broomrapes in both trials (p < 0.001). In order to enhance biocontrol activity against broomrapes, protoplast fusion was performed between F. oxysporum and F. equiseti. In comparison to KGh2-1, the fusant strains demonstrated superior ability in inhibiting the growth of broomrapes. The results also showed a clear host specificity of the selected pathogenic isolates and fusants. Protoplast fusion can be recommended as a valuable tool that can efficiently enhance the biocontrol ability of F. oxysporum against broomrapes.
Cell-cell fusion is fundamentally important for tissue repair, virus transmission, and genetic recombination, among other functions. Fusion has been mainly studied in eukaryotic cells and lipid vesicles, while cell-cell fusion in bacteria is less well characterized, due to the cell wall acting as a fusion-limiting barrier. Here we use cell wall-deficient bacteria to investigate the dynamics of cell fusion in bacteria that replicate without their cell wall. Stable, replicating cells containing differently labeled chromosomes were successfully obtained from fusion. We find that the rate of cell-cell fusion depends on the fluidity of cell membranes. Furthermore, we show that not only the efficiency but also the specificity of cell-cell fusion can be controlled via a pair of synthetic membrane-associated lipopeptides. Our results provide a molecular handle to understand and control cell-cell fusion to generate heterokaryotic cells, which was an important step in the evolution of protocells and of increasing importance for the design of synthetic cells.
