scholarly journals Adaptive laboratory evolution triggers pathogen-dependent broad-spectrum antimicrobial potency in Streptomyces

2021 ◽  
Author(s):  
Dharmesh Harwani ◽  
Jyotsna Begani ◽  
Sweta Barupal ◽  
Jyoti Lakhani
Keyword(s):  
Antimicrobial Activity ◽  
Antibiotic Production ◽  
Multidrug Resistant ◽  
Molecular Evidence ◽  
Evolutionary Engineering ◽  
Wild Type ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
E Coli ◽  
Mutant Phenotypes

AbstractIn the present study, adaptive laboratory evolution was used to stimulate antibiotic production in a weak antibiotic-producing Streptomyces strain JB140. The seven different competition experiments utilized three serial passages (three cycles of adaptation-selection of 15 days each) of a weak antibiotic-producing Streptomyces strain (wild-type) against one (biculture) or two (triculture) or three (quadriculture) target pathogens. This resulted in the evolution of a weak antibiotic-producing strain into the seven unique mutant phenotypes that acquired the ability to constitutively exhibit increased antimicrobial activity against bacterial pathogens. The mutant not only effectively inhibited the growth of the tested pathogens but also observed to produce antimicrobial against multidrug-resistant (MDR) E. coli. Intriguingly, the highest antimicrobial activity was registered with the Streptomyces mutants that were adaptively evolved against the three pathogens (quadriculture competition). In contrast to the adaptively evolved mutants, a weak antimicrobial activity was detected in the un-evolved, wild-type Streptomyces. To get molecular evidence of evolution, RAPD profiles of the wild-type Streptomyces and its evolved mutants were compared that revealed significant polymorphism among them. These results demonstrated that competition-based adaptive laboratory evolution method can constitute a platform for evolutionary engineering to select improved phenotypes (mutants) with increased production of antibiotics against targeted pathogens.

scholarly journals Adaptive laboratory evolution triggers pathogen-dependent broad-spectrum antimicrobial potency in Streptomyces

2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Dharmesh Harwani ◽  
Jyotsna Begani ◽  
Sweta Barupal ◽  
Jyoti Lakhani
Keyword(s):  
Antimicrobial Activity ◽  
Antibiotic Production ◽  
Bacterial Pathogens ◽  
Laboratory Model ◽  
Molecular Evidence ◽  
Evolutionary Engineering ◽  
Wild Type ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Mutant Phenotypes

Abstract Background In the present study, adaptive laboratory evolution was used to stimulate antibiotic production in a Streptomyces strain JB140 (wild-type) exhibiting very little antimicrobial activity against bacterial pathogens. The seven different competition experiments utilized three serial passages (3 cycles of adaptation-selection of 15 days each) in which Streptomyces strain (wild-type) was challenged repeatedly to one (bi-culture) or two (tri-culture) or three (quadri-culture) target pathogens. The study demonstrates a simple laboratory model to study the adaptive potential of evolved phenotypes and genotypes in Streptomyces to induce antibiotic production. Results Competition experiments resulted in the evolution of the wild-type Streptomyces strain JB140 into the seven unique mutant phenotypes that acquired the ability to constitutively exhibit increased antimicrobial activity against three bacterial pathogens Salmonella Typhi (NCIM 2051), Staphylococcus aureus (NCIM 2079), and Proteus vulgaris (NCIM 2027). The mutant phenotypes not only effectively inhibited the growth of the tested pathogens but were also observed to exhibit improved antimicrobial responses against one clinical multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC 1021) isolate. In contrast to the adaptively evolved mutants, only a weak antimicrobial activity was detected in the wild-type parental strain. To get molecular evidence of evolution, RAPD profiles of the wild-type Streptomyces and its evolved mutants were compared which revealed significant polymorphism among them. Conclusion The competition-based adaptive laboratory evolution method can constitute a platform for evolutionary engineering to select improved phenotypes (mutants) with increased antibacterial profiles against targeted pathogens.

scholarly journals Adaptive Laboratory Evolution of Cupriavidus necator H16 for Carbon Co-Utilization with Glycerol

2019 ◽  
Vol 20 (22) ◽  
pp. 5737 ◽  
Author(s):  
Miriam González-Villanueva ◽  
Hemanshi Galaiya ◽  
Paul Staniland ◽  
Jessica Staniland ◽  
Ian Savill ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Carbon Sources ◽  
Strain Engineering ◽  
Cupriavidus Necator ◽  
Superior Performance ◽  
Wild Type ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Cupriavidus Necator H16

Cupriavidus necator H16 is a non-pathogenic Gram-negative betaproteobacterium that can utilize a broad range of renewable heterotrophic resources to produce chemicals ranging from polyhydroxybutyrate (biopolymer) to alcohols, alkanes, and alkenes. However, C. necator H16 utilizes carbon sources to different efficiency, for example its growth in glycerol is 11.4 times slower than a favorable substrate like gluconate. This work used adaptive laboratory evolution to enhance the glycerol assimilation in C. necator H16 and identified a variant (v6C6) that can co-utilize gluconate and glycerol. The v6C6 variant has a specific growth rate in glycerol 9.5 times faster than the wild-type strain and grows faster in mixed gluconate–glycerol carbon sources compared to gluconate alone. It also accumulated more PHB when cultivated in glycerol medium compared to gluconate medium while the inverse is true for the wild-type strain. Through genome sequencing and expression studies, glycerol kinase was identified as the key enzyme for its improved glycerol utilization. The superior performance of v6C6 in assimilating pure glycerol was extended to crude glycerol (sweetwater) from an industrial fat splitting process. These results highlight the robustness of adaptive laboratory evolution for strain engineering and the versatility and potential of C. necator H16 for industrial waste glycerol valorization.

scholarly journals IN VITRO ANTIMICROBIAL ACTIVITY OF ROOT EXTRACT OF CLITORIA TERNATEA

2017 ◽  
Vol 10 (11) ◽  
pp. 52
Author(s):  
Amita Shobha Rao ◽  
Shobha Kl ◽  
Prathibha Md’almeida ◽  
Kiranmai S Rai
Keyword(s):  
Antimicrobial Activity ◽  
Resistant Strain ◽  
Multidrug Resistant ◽  
Diffusion Method ◽  
Root Extract ◽  
Alcoholic Extract ◽  
Zone Of Inhibition ◽  
Gram Negative ◽  
E Coli ◽  
Multidrug Resistant Strain

  Objective: Infections caused by Gram-negative bacteria are important causes of morbidity and mortality. Extracts of plants and herbs such as Clitorea ternatea are used as diuretic. This work attempts to find out antimicrobial activity of aqueous and alcoholic extract of C. ternatea roots against Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), clinical strains of Klebsiella pneumoniae, and Candida albicans.Methods: The agar well-diffusion method was done using Mueller Hinton agar and Sabouraud’s dextrose agar. The microorganism grown in peptone water was inoculated into culture medium. 4 mm diameter well punched into the agar was filled with 20 μl of aqueous and alcoholic root extracts C. ternatea extracts in various concentrations (100-25 μg/ml). The plates were incubated and antimicrobial activity was evaluated.Results: Aqueous root extract of C. ternatea with the concentration of 100 μg/ml showed zone of inhibition against E. coli (ATCC 25922) 18 mm, P. aeruginosa (ATCC 27853) 14 mm, multidrug resistant strain of K. pneumoniae 15 mm. Alcoholic extract of C. ternatea with the concentration of 100 μg/ml showed zone of inhibition of 35 mm against E. coli (ATCC 25922), P. aeruginosa (ATCC 27853) 22 mm, and multidrug resistant strain of K. pneumoniae 28 mm. C. albicanswas resistant to both extract of C. ternatea root. Conclusions: Alcoholic extract of C. ternatea is a better antibacterial agent against multidrug resistant Klebsiella species and other Gram-negative pathogens. Further, studies are required to identify active substances from the alcoholic extracts of C. ternatea for treating infections.

scholarly journals Antimicrobial Activity of Novel Dendrimeric Peptides Obtained by Phage Display Selection and Rational Modification

2005 ◽  
Vol 49 (7) ◽  
pp. 2665-2672 ◽  
Author(s):  
Alessandro Pini ◽  
Andrea Giuliani ◽  
Chiara Falciani ◽  
Ylenia Runci ◽  
Claudia Ricci ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Multidrug Resistant ◽  
Cytotoxic Effects ◽  
E Coli ◽  
Multiple Antigen ◽  
Antigen Peptide ◽  
Multiple Antigen Peptide ◽  
Residue Substitution ◽  
Peptide Map ◽  
Phage Peptide Library

ABSTRACT A large 10-mer phage peptide library was panned against whole Escherichia coli cells, and an antimicrobial peptide (QEKIRVRLSA) was selected. The peptide was synthesized in monomeric and dendrimeric tetrabranched form (multiple antigen peptide [MAP]), which generally allows a dramatic increase of peptide stability to peptidases and proteases. The antibacterial activity of the dendrimeric peptide against E. coli was much higher than that of the monomeric form. Modification of the original sequence, by residue substitution or sequence shortening, produced three different MAPs, M4 (QAKIRVRLSA), M5 (KIRVRLSA), and M6 (QKKIRVRLSA) with enhanced stability to natural degradation and antimicrobial activity against a large panel of gram-negative bacteria. The MICs of the most potent peptide, M6, were as low as 4 to 8 μg/ml against recent clinical isolates of multidrug-resistant Pseudomonas aeruginosa and members of the Enterobacteriaceae. The same dendrimeric peptides showed high stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells, making them promising candidates for the development of new antibacterial drugs.

scholarly journals Improving the Methanol Tolerance of an Escherichia coli Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization

2021 ◽  
Vol 12 ◽  
Author(s):  
R. Kyle Bennett ◽  
Gwendolyn J. Gregory ◽  
Jacqueline E. Gonzalez ◽  
Jie Ren Gerald Har ◽  
Maciek R. Antoniewicz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ribosomal Subunit ◽  
Biological Properties ◽  
Methanol Tolerance ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
E Coli ◽  
Translational Accuracy ◽  
Methanol Utilization ◽  
Carbon Compounds

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.

scholarly journals Activating silent glycolysis bypasses in Escherichia coli

2021 ◽  
Author(s):  
Camillo Iacometti ◽  
Katharina Marx ◽  
Maria Hoenick ◽  
Viktoria Biletskaia ◽  
Helena Schulz-Mirbach ◽  
...  
Keyword(s):  
Triosephosphate Isomerase ◽  
Building Blocks ◽  
Scale Model ◽  
Deletion Strain ◽  
Central Metabolism ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
E Coli ◽  
A Genome ◽  
Serine Biosynthesis

All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli, we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal, the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal immediately operated in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary for implementing a functional methylglyoxal pathway. Furthermore, we engineered the serine shunt which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection we performed an adaptive laboratory evolution study using an enolase deletion strain. The evolved mutants were shown to use the serine shunt. Our study reveals the flexible redesignation of metabolic pathways to create new metabolite links and rewire central metabolism.

scholarly journals Novel Mode Engineering for β-Alanine Production in Escherichia coli with the Guide of Adaptive Laboratory Evolution

2021 ◽  
Vol 9 (3) ◽  
pp. 600
Author(s):  
Jian Xu ◽  
Li Zhou ◽  
Meng Yin ◽  
Zhemin Zhou
Keyword(s):  
Escherichia Coli ◽  
Anaerobic Condition ◽  
Energy Production ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Production Efficiency ◽  
Genetic Mutations ◽  
Cell Factory ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
E Coli

The strategy of anaerobic biosynthesis of β-alanine by Escherichia coli (E. coli) has been reported. However, the low energy production under anaerobic condition limited cell growth and then affected the production efficiency of β-alanine. Here, the adaptive laboratory evolution was carried out to improve energy production of E. coli lacking phosphoenolpyruvate carboxylase under anaerobic condition. Five mutants were isolated and analyzed. Sequence analysis showed that most of the consistent genetic mutations among the mutants were related with pyruvate accumulation, indicating that pyruvate accumulation enabled the growth of the lethal parent. It is possible that the accumulated pyruvate provides sufficient precursors for energy generation and CO2 fixing reaction catalyzed by phosphoenolpyruvate carboxykinase. B0016-100BB (B0016-090BB, recE::FRT, mhpF::FRT, ykgF::FRT, mhpB:: mhpB *, mhpD:: mhpD *, rcsA:: rcsA *) was engineered based on the analysis of the genetic mutations among the mutants for the biosynthesis of β-alanine. Along with the recruitment of glycerol as the sole carbon source, 1.07 g/L β-alanine was generated by B0016-200BB (B0016-100BB, aspA::FRT) harboring pET24a-panD-AspDH, which was used for overexpression of two key enzymes in β-alanine fermentation process. Compared with the starting strain, which can hardly generate β-alanine under anaerobic condition, the production efficiency of β-alanine of the engineered cell factory was significantly improved.

scholarly journals Improving isobutanol productivity through adaptive laboratory evolution in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Aili Zhang ◽  
Yide Su ◽  
Jingzhi Li ◽  
Weiwei Zhang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Metabolic Engineering ◽  
Glucose Concentration ◽  
Evolutionary Engineering ◽  
Whole Genome ◽  
Genome Resequencing ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Whole Genome Resequencing

Abstract Background: Isobutanol is an ideal second-generation biofuels due to its lower hygroscopicity, higher energy density and higher-octane value. However, isobutanol is toxic to production organisms. To improve isobutanol productivity, adaptive laboratory evolution method was carried out to improve the tolerance of Saccharomyces cerevisiae toward higher isobutanol and higher glucose concentration.Results: We evolved the laboratory strain of S. cerevisiae W303-1A by using EMS (ethyl methanesulfonate) mutagenesis followed by adaptive laboratory evolution. The evolved strain EMS39 with significant increase in growth rate and viability in media with higher isobutanol and higher glucose concentration was obtained. Then, metabolic engineering of the evolved strain EMS39 as a platform for isobutanol production were carried out. Delta integration method was used to over-express ILV3 gene and 2μ plasmids carrying ILV2, ILV5 and ARO10 were used to over-express ILV2, ILV5 and ARO10 genes in the evolved strain EMS39 and wild type W303-1A. And the resulting strains was designated as strain EMS39V2δV3V5A10 and strain W303-1AV2δV3V5A10, respectively. Our results shown that isobutanol titers of the evolved strain EMS39 increased by 30% compared to the control strain. And isobutanol productivity of strain EMS39V2δV3V5A10 increased by 32.4% compared to strain W303-1AV2δV3V5A10. Whole genome resequencing and analysis of site-directed mutagenesis of the evolved strain EMS39 have identified important mutations. In addition, RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from EMS39.Conclusions: With the aim of increase productivity of isobutanol in S. cerevisiae, improving tolerance toward higher isobutanol and higher glucose concentration via EMS mutagenesis followed by adaptive evolutionary engineering was conducted. An evolved strain EMS39 with significant increase in growth rate and viability had been obtained. And metabolic engineering of the evolved strain as a platform for isobutanol production was carried out. Furthermore, analysis of whole genome resequencing and transcriptome sequencing were also carried out.

scholarly journals Antimicrobial Activity Evaluation of Tebipenem (SPR859), an Orally Available Carbapenem, against a Global Set of Enterobacteriaceae Isolates, Including a Challenge Set of Organisms

2019 ◽  
Vol 63 (6) ◽  
Author(s):  
S. J. Ryan Arends ◽  
Paul R. Rhomberg ◽  
Nicole Cotroneo ◽  
Aileen Rubio ◽  
Robert K. Flamm ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Broth Microdilution ◽  
Wild Type ◽  
Content Type ◽  
E Coli

ABSTRACT The antimicrobial activity of tebipenem and other carbapenem agents were tested in vitro against a set of recent clinical isolates responsible for urinary tract infection (UTI), as well as against a challenge set. Isolates were tested by reference broth microdilution and included Escherichia coli (101 isolates), Klebsiella pneumoniae (208 isolates), and Proteus mirabilis (103 isolates) species. Within each species tested, tebipenem showed equivalent MIC50/90 values to those of meropenem (E. coli MIC50/90, ≤0.015/0.03 mg/liter; K. pneumoniae MIC50/90, 0.03/0.06 mg/liter; and P. mirabilis MIC50/90, 0.06/0.12 mg/liter) and consistently displayed MIC90 values 8-fold lower than imipenem. Tebipenem and meropenem (MIC50, 0.03 mg/liter) showed equivalent MIC50 results against wild-type, AmpC-, and/or extended-spectrum β-lactamase (ESBL)-producing isolates. Tebipenem also displayed MIC50/90 values 4- to 8-fold lower than imipenem against the challenge set. All carbapenem agents were less active (MIC50, ≥8 mg/liter) against isolates carrying carbapenemase genes. These data confirm the in vitro activity of the orally available agent tebipenem against prevalent UTI Enterobacteriaceae species, including those producing ESBLs and/or plasmid AmpC enzymes.

scholarly journals Evolutionary engineering of E. coli MG1655 for tolerance against isoprenol

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Heiko Babel ◽  
Jens O. Krömer
Keyword(s):  
Target Genes ◽  
Inhibitory Concentration ◽  
Biofuel Production ◽  
Evolutionary Engineering ◽  
Adaptive Laboratory Evolution ◽  
Biotechnological Production ◽  
Knock Out ◽  
E Coli ◽  
Major Target ◽  
As Toxicity

Abstract Background Isoprenol is the basis for industrial flavor and vitamin synthesis and also a promising biofuel. Biotechnological production of isoprenol with E. coli is currently limited by the high toxicity of the final product. Adaptive laboratory evolution (ALE) is a promising method to address complex biological problems such as toxicity. Results Here we applied this method successfully to evolve E. coli towards higher tolerance against isoprenol, increasing growth at the half-maximal inhibitory concentration by 47%. Whole-genome re-sequencing of strains isolated from three replicate evolutions at seven time-points identified four major target genes for isoprenol tolerance: fabF, marC, yghB, and rob. We could show that knock-out of marC and expression of mutated Rob H(48) → frameshift increased tolerance against isoprenol and butanol. RNA-sequencing showed that the deletion identified upstream of yghB correlated with a strong overexpression of the gene. The knock-out of yghB demonstrated that it was essential for isoprenol tolerance. The mutated Rob protein and yghB deletion also lead to increased vanillin tolerance. Conclusion Through ALE, novel targets for strain optimization in isoprenol production and also the production of other fuels, such as butanol, could be obtained. Their effectiveness could be shown through re-engineering. This paves the way for further optimization of E. coli for biofuel production.

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