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 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 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 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 Efficient production of d-lactate from methane in a lactate-tolerant strain of Methylomonas sp. DH-1 generated by adaptive laboratory evolution

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Jong Kwan Lee ◽  
Sujin Kim ◽  
Wonsik Kim ◽  
Sungil Kim ◽  
Seungwoo Cha ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Lactic Acid ◽  
Lactate Dehydrogenase ◽  
Value Added ◽  
Wild Type ◽  
Adaptive Laboratory Evolution ◽  
Gene Encoding ◽  
Laboratory Evolution ◽  
Tolerant Strain ◽  
Dehydrogenase Gene

Abstract Background Methane, a main component of natural gas and biogas, has gained much attention as an abundant and low-cost carbon source. Methanotrophs, which can use methane as a sole carbon and energy source, are promising hosts to produce value-added chemicals from methane, but their metabolic engineering is still challenging. In previous attempts to produce lactic acid (LA) from methane, LA production levels were limited in part due to LA toxicity. We solved this problem by generating an LA-tolerant strain, which also contributes to understanding novel LA tolerance mechanisms. Results In this study, we engineered a methanotroph strain Methylomonas sp. DH-1 to produce d-lactic acid (d-LA) from methane. LA toxicity is one of the limiting factors for high-level production of LA. Therefore, we first performed adaptive laboratory evolution of Methylomonas sp. DH-1, generating an LA-tolerant strain JHM80. Genome sequencing of JHM80 revealed the causal gene watR, encoding a LysR-type transcription factor, whose overexpression due to a 2-bp (TT) deletion in the promoter region is partly responsible for the LA tolerance of JHM80. Overexpression of the watR gene in wild-type strain also led to an increase in LA tolerance. When d form-specific lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 was introduced into the genome while deleting the glgA gene encoding glycogen synthase, JHM80 produced about 7.5-fold higher level of d-LA from methane than wild type, suggesting that LA tolerance is a critical limiting factor for LA production in this host. d-LA production was further enhanced by optimization of the medium, resulting in a titer of 1.19 g/L and a yield of 0.245 g/g CH4. Conclusions JHM80, an LA-tolerant strain of Methylomonas sp. DH-1, generated by adaptive laboratory evolution was effective in LA production from methane. Characterization of the mutated genes in JHM80 revealed that overexpression of the watR gene, encoding a LysR-type transcription factor, is responsible for LA tolerance. By introducing a heterologous lactate dehydrogenase gene into the genome of JHM80 strain while deleting the glgA gene, high d-LA production titer and yield were achieved from methane.

scholarly journals Adaptive laboratory evolution of heat tolerance in Streptococcus thermophilus BIOPOP-1 and BIOPOP-2 under gradually increasing heat temperature

2019 ◽  
Author(s):  
Bonggyu Min ◽  
Kkotnim Kim ◽  
Vladimir Li ◽  
Younghoon Kim ◽  
Heebal Kim
Keyword(s):  
Fatty Acid ◽  
Heat Tolerance ◽  
Streptococcus Thermophilus ◽  
Temperature Tolerance ◽  
Fatty Acid Analysis ◽  
Wild Type ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution ◽  
Long Term Storage ◽  
Acid Ratio

Abstract Background Streptococcus thermophilus is one of the lactic acid bacteria (LAB), as such, has been commonly used in the production of fermented dairy foods during past decades. However, LAB including S. thermophilus struggle to survive at temperatures above 60°C, which are applied during manufacturing processes, such as pasteurization that can inhibit cell proliferation and promote cell death. Although there are many studies on S. thermophilus , no research regarding its heat tolerance in over 60°C has been done yet. In this study, a method of adaptive laboratory evolution (ALE) was implemented in an attempt to increase the upper thermal threshold of two S. thermophilus strains of LAB found in South Korea. Results To develop heat tolerant LABs, heat treatment was continuously applied to bacteria by increasing temperature from 60°C until the point of not being detected. The results indicated significant increase in temperature tolerance of ALE strains compared to the wild type (WT) strains. In addition, ALE strains acquired cross protection from various stress conditions like acid, bile salts and salinity. The improved heat tolerance of ALE strains showed higher survival rate during long term storage at sub-zero temperatures compared to the wild type strains. Fatty acid analysis indicated that saturated fatty acid ratio of ALE strains was higher than that of WT cells. Conclusions The results of this study demonstrated that this ALE method can be utilized to improve bacterial tolerance against various environmental stresses. Adaptive evolution under heat stress might be applied in the various industries.

The Study of Bacillus Subtils Antimicrobial Activity on Some of the Pathological Isolates

2019 ◽  
Vol 9 (02) ◽  
Author(s):  
Hussein A Kadhum ◽  
Thualfakar H Hasan2
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Bacterial Growth ◽  
Broad Spectrum ◽  
Inhibitory Activity ◽  
Bacterial Pathogens ◽  
Inhibitory Ability ◽  
Selection Of

The study involved the selection of two isolates from Bacillus subtilis to investigate their inhibitory activity against some bacterial pathogens. B sub-bacteria were found to have a broad spectrum against test bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. They were about 23-30 mm and less against Klebsiella sp. The sensitivity of some antibodies was tested on the test samples. The results showed that the inhibitory ability of bacterial growth in the test samples using B. subtilis extract was more effective than the antibiotics used.

scholarly journals Engineering improved ethylene production: Leveraging systems Biology and adaptive laboratory evolution

2021 ◽  
Author(s):  
Sophie Vaud ◽  
Nicole Pearcy ◽  
Marko Hanževački ◽  
Alexander M.W. Van Hagen ◽  
Salah Abdelrazig ◽  
...  
Keyword(s):  
Systems Biology ◽  
Ethylene Production ◽  
Adaptive Laboratory Evolution ◽  
Laboratory Evolution

scholarly journals The Mutant Phenotype Associated With P-Element Alleles of the vestigial Locus in Drosophila melanogaster May Be Caused by a Readthrough Transcript Initiated at the P-Element Promoter

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1665-1672 ◽  
Author(s):  
Ross B Hodgetts ◽  
Sandra L O'Keefe
Keyword(s):  
Rna Secondary Structure ◽  
Insertion Site ◽  
Large Body ◽  
P Element ◽  
Wild Type ◽  
Subtle Difference ◽  
Pronounced Difference ◽  
Internal Repeat ◽  
Mutant Phenotypes

Abstract We report here the isolation of a new P-element-induced allele of the vestigial locus vg2a33, the molecular characterization of which allows us to propose a unifying explanation of the phenotypes of the large number of vestigial P-element alleles that now exists. The first P-element allele of vestigial to be isolated was vg21, which results in a very weak mutant wing phenotype that is suppressed in the P cytotype. By destabilizing vg2a33 in a dysgenic cross, we isolated the vg2a33 allele, which exhibits a moderate mutant wing phenotype and is not suppressed by the P cytotype. The new allele is characterized by a 46-bp deletion that removes the 3′-proximal copy of the 11-bp internal repeat from the P element of vg21. To understand how this subtle difference between the two alleles leads to a rather pronounced difference in their phenotypes, we mapped both the vg and P-element transcription units present in wild type and mutants. Using both 5′-RACE and S1 protection, we found that P-element transcription is initiated 19 bp farther upstream than previously thought. Using primer extension, the start of vg transcription was determined to lie 435 bp upstream of the longest cDNA recovered to date and upstream of the P-element insertion site. Our discovery that the P element is situated within the first vg exon has prompted a reassessment of the large body of genetic data on a series of alleles derived from vg21. Our current hypothesis to explain the degree of variation in the mutant phenotypes and their response to the P repressor invokes a critical RNA secondary structure in the vg transcript, the formation of which is hindered by a readthrough transcript initiated at the P-element promoter.

scholarly journals Analysis of maxillopedia Expression Pattern and Larval Cuticular Phenotype in Wild-Type and Mutant Tribolium

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 721-731 ◽  
Author(s):  
Teresa D Shippy ◽  
Jianhua Guo ◽  
Susan J Brown ◽  
Richard W Beeman ◽  
Robin E Denell
Keyword(s):  
Rna Interference ◽  
Expression Pattern ◽  
Tribolium Castaneum ◽  
Expression Patterns ◽  
Ectopic Expression ◽  
Homeotic Gene ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Similar Expression ◽  
Mutant Phenotypes

Abstract The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

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