scholarly journals Morphological Differentiation of Streptomyces clavuligerus Exposed to Diverse Environmental Conditions and Its Relationship with Clavulanic Acid Biosynthesis

Processes ◽  
2020 ◽  
Vol 8 (9) ◽  
pp. 1038
Author(s):  
Jeferyd Yepes-García ◽  
Carlos Caicedo-Montoya ◽  
Laura Pinilla ◽  
León F. Toro ◽  
Rigoberto Ríos-Estepa
Keyword(s):  
Clavulanic Acid ◽  
Defense Mechanism ◽  
Culture Media ◽  
Morphological Differentiation ◽  
Streptomyces Clavuligerus ◽  
Culture Conditions ◽  
Antibiotic Biosynthesis ◽  
Potential Relationship ◽  
Confocal And Electron Microscopy ◽  
Streptomyces Genus

Clavulanic acid (CA) is a potent inhibitor of class A β-lactamase enzymes produced by Streptomyces clavuligerus (S. clavuligerus) as a defense mechanism. Due to its industrial interest, the process optimization is under continuous investigation. This work aimed at identifying the potential relationship that might exist between S. clavuligerus ATCC 27064 morphology and CA biosynthesis. For this, modified culture conditions such as source, size, and age of inoculum, culture media, and geometry of fermentation flasks were tested. We observed that high density spore suspensions (1 × 107 spores/mL) represent the best inoculum source for S. clavuligerus cell suspension culture. Further, we studied the life cycle of S. clavuligerus in liquid medium, using optic, confocal, and electron microscopy; results allowed us to observe a potential relationship that might exist between the accumulation of CA and the morphology of disperse hyphae. Reactor geometries that increase shear stress promote smaller pellets and a quick disintegration of these in dispersed secondary mycelia, which begins the pseudosporulation process, thus easing CA accumulation. These outcomes greatly contribute to improving the understanding of antibiotic biosynthesis in the Streptomyces genus.

scholarly journals Two Proteins with Ornithine Acetyltransferase Activity Show Different Functions in Streptomyces clavuligerus: Oat2 Modulates Clavulanic Acid Biosynthesis in Response to Arginine

2004 ◽  
Vol 186 (19) ◽  
pp. 6501-6507 ◽  
Author(s):  
A. de la Fuente ◽  
J. F. Martín ◽  
A. Rodríguez-García ◽  
P. Liras
Keyword(s):  
Clavulanic Acid ◽  
Streptomyces Clavuligerus ◽  
Culture Conditions ◽  
Transferase Activity ◽  
Reaction Products ◽  
Acid Biosynthesis ◽  
Substrate Specificities ◽  
Acetyltransferase Activity ◽  
Standard Culture

ABSTRACT The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.

scholarly journals Clavulanic Acid Production by Streptomyces clavuligerus: Insights from Systems Biology, Strain Engineering, and Downstream Processing

Antibiotics ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 84
Author(s):  
Víctor A. López-Agudelo ◽  
David Gómez-Ríos ◽  
Howard Ramirez-Malule
Keyword(s):  
Systems Biology ◽  
Clavulanic Acid ◽  
Large Scale ◽  
Enzyme Inhibitor ◽  
Clinical Importance ◽  
Streptomyces Clavuligerus ◽  
Downstream Processing ◽  
Strain Engineering ◽  
Culture Conditions ◽  
Separation And Purification

Clavulanic acid (CA) is an irreversible β-lactamase enzyme inhibitor with a weak antibacterial activity produced by Streptomyces clavuligerus (S. clavuligerus). CA is typically co-formulated with broad-spectrum β‑lactam antibiotics such as amoxicillin, conferring them high potential to treat diseases caused by bacteria that possess β‑lactam resistance. The clinical importance of CA and the complexity of the production process motivate improvements from an interdisciplinary standpoint by integrating metabolic engineering strategies and knowledge on metabolic and regulatory events through systems biology and multi-omics approaches. In the large-scale bioprocessing, optimization of culture conditions, bioreactor design, agitation regime, as well as advances in CA separation and purification are required to improve the cost structure associated to CA production. This review presents the recent insights in CA production by S. clavuligerus, emphasizing on systems biology approaches, strain engineering, and downstream processing.

scholarly journals Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

scholarly journals Characterization of Drug-Resistant Lipid-Dependent Differentially Detectable Mycobacterium tuberculosis

2021 ◽  
Vol 10 (15) ◽  
pp. 3249
Author(s):  
Annelies W. Mesman ◽  
Seung-Hun Baek ◽  
Chuan-Chin Huang ◽  
Young-Mi Kim ◽  
Sang-Nae Cho ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Culture Media ◽  
Multidrug Resistant ◽  
Culture Conditions ◽  
Sputum Smear ◽  
Smear Microscopy ◽  
Solid Culture ◽  
Lowenstein Jensen ◽  
Genetic Mechanisms ◽  
Lipid Resuscitation

An estimated 15–20% of patients who are treated for pulmonary tuberculosis (TB) are culture-negative at the time of diagnosis. Recent work has focused on the existence of differentially detectable Mycobacterium tuberculosis (Mtb) bacilli that do not grow under routine solid culture conditions without the addition of supplementary stimuli. We identified a cohort of TB patients in Lima, Peru, in whom acid-fast bacilli could be detected by sputum smear microscopy, but from whom Mtb could not be grown in standard solid culture media. When we attempted to re-grow Mtb from the frozen sputum samples of these patients, we found that 10 out of 15 could be grown in a glycerol-poor/lipid-rich medium. These fell into the following two groups: a subset that could be regrown in glycerol after “lipid-resuscitation”, and a group that displayed a heritable glycerol-sensitive phenotype that were unable to grow in the presence of this carbon source. Notably, all of the glycerol-sensitive strains were found to be multidrug resistant. Although whole-genome sequencing of the lipid-resuscitated strains identified 20 unique mutations compared to closely related strains, no single genetic lesion could be associated with this phenotype. In summary, we found that lipid-based media effectively fostered the growth of Mtb from a series of sputum smear-positive samples that were not culturable in glycerol-based Lowenstein–Jensen or 7H9 media, which is consistent with Mtb’s known preference for non-glycolytic sources during infection. Analysis of the recovered strains demonstrated that both genetic and non-genetic mechanisms contribute to the observed differential capturability, and suggested that this phenotype may be associated with drug resistance.

scholarly journals WblAch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpAch

2014 ◽  
Vol 80 (22) ◽  
pp. 6879-6887 ◽  
Author(s):  
Pin Yu ◽  
Shui-Ping Liu ◽  
Qing-Ting Bu ◽  
Zhen-Xing Zhou ◽  
Zhen-Hong Zhu ◽  
...  
Keyword(s):  
Morphological Differentiation ◽  
Binding Activity ◽  
Transcriptional Analysis ◽  
Antibiotic Biosynthesis ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Dna Binding Activity ◽  
In The Wild ◽  
Natamycin Production ◽  
Streptomyces Chattanoogensis

ABSTRACTDetailed mechanisms ofWhiB-like (Wbl) proteins involved in antibiotic biosynthesis and morphological differentiation are poorly understood. Here, we characterize the role of WblAch, aStreptomyces chattanoogensisL10 protein belonging to this superfamily. Based on DNA microarray data and verified by real-time quantitative PCR (qRT-PCR), the expression ofwblAchwas shown to be positively regulated by AdpAch. Gel retardation assays and DNase I footprinting experiments showed that AdpAchhas specific DNA-binding activity for the promoter region ofwblAch. Gene disruption and genetic complementation revealed that WblAchacts in a positive manner to regulate natamycin production. WhenwblAchwas overexpressed in the wild-type strain, the natamycin yield was increased by ∼30%. This provides a strategy to generate improved strains for natamycin production. Moreover, transcriptional analysis showed that the expression levels ofwhigenes (includingwhiA,whiB,whiH, andwhiI) were severely depressed in the ΔwblAchmutant, suggesting that WblAchplays a part in morphological differentiation by influencing the expression of thewhigenes.

Biophysical stimulation for in vitro engineering of functional cardiac tissues

2017 ◽  
Vol 131 (13) ◽  
pp. 1393-1404 ◽  
Author(s):  
Anastasia Korolj ◽  
Erika Yan Wang ◽  
Robert A. Civitarese ◽  
Milica Radisic
Keyword(s):  
Cardiac Tissue ◽  
Culture Media ◽  
Culture Conditions ◽  
Lineage Specification ◽  
Cardiac Tissues ◽  
Cardiac Lineage ◽  
And Function ◽  
Tissue Models

Engineering functional cardiac tissues remains an ongoing significant challenge due to the complexity of the native environment. However, our growing understanding of key parameters of the in vivo cardiac microenvironment and our ability to replicate those parameters in vitro are resulting in the development of increasingly sophisticated models of engineered cardiac tissues (ECT). This review examines some of the most relevant parameters that may be applied in culture leading to higher fidelity cardiac tissue models. These include the biochemical composition of culture media and cardiac lineage specification, co-culture conditions, electrical and mechanical stimulation, and the application of hydrogels, various biomaterials, and scaffolds. The review will also summarize some of the recent functional human tissue models that have been developed for in vivo and in vitro applications. Ultimately, the creation of sophisticated ECT that replicate native structure and function will be instrumental in advancing cell-based therapeutics and in providing advanced models for drug discovery and testing.

scholarly journals BCG Substrains Change Their Outermost Surface as a Function of Growth Media

Vaccines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 40
Author(s):  
Sandra Guallar-Garrido ◽  
Farners Almiñana-Rapún ◽  
Víctor Campo-Pérez ◽  
Eduard Torrents ◽  
Marina Luquin ◽  
...  
Keyword(s):  
Culture Media ◽  
Chemical Properties ◽  
Growth Conditions ◽  
Neutral Red ◽  
Culture Conditions ◽  
Host Cells ◽  
Growth Media ◽  
Phenotypic Characteristics ◽  
Outermost Surface ◽  
Red Dye

Mycobacterium bovis bacillus Calmette-Guérin (BCG) efficacy as an immunotherapy tool can be influenced by the genetic background or immune status of the treated population and by the BCG substrain used. BCG comprises several substrains with genetic differences that elicit diverse phenotypic characteristics. Moreover, modifications of phenotypic characteristics can be influenced by culture conditions. However, several culture media formulations are used worldwide to produce BCG. To elucidate the influence of growth conditions on BCG characteristics, five different substrains were grown on two culture media, and the lipidic profile and physico-chemical properties were evaluated. Our results show that each BCG substrain displays a variety of lipidic profiles on the outermost surface depending on the growth conditions. These modifications lead to a breadth of hydrophobicity patterns and a different ability to reduce neutral red dye within the same BCG substrain, suggesting the influence of BCG growth conditions on the interaction between BCG cells and host cells.

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