mutant selection
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2022 ◽  
Amir Akhgari ◽  
Bikash Baral ◽  
Arina Koroleva ◽  
Vilja Siitonen ◽  
David P Fewer ◽  

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l-1 in a second round of mutant selection. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g-1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.

2022 ◽  
Kemeng Li ◽  
Jianlu Dai ◽  
Juanjuan Liu ◽  
Tianyi Hao ◽  
Weiqing He

Abstract Background: Carrimycin is a new approved class I antibiotic in China. The novel carrimycin producing strain, Streptomyces spiramyceticus 54IA, was constructed by CRISPR-Cas9 editing system without insertion of antibiotics resistant gene. The problem of low yield limits this strain in large scale fermentation. In this study, the carrimycin production was significantly improved by strain mutagenesis coupled metabolic engineering. Results: The sspD gene is responsible for degradation of triacylglycerol to provide precursors of the polyketide biosynthesis. The extra sspD gene controlled by the promoters of pks and bsm42 genes could moderately enhance carrimycin production. The Bsm42 was identified to play a pathway-specific positive regulator for carrimycin biosynthesis. Due to production of carrimycin significantly enhanced by bsm42 overexpression, the two different length promoters of bsm42 individually ligated with two reporter genes were used to monitor bsm42 expression for screening the higher carrimycin production mutants treated by plasma and ultraviolet. 47% of the 608 selected mutants had higher fermentation titer than the starting strain. The shorter promoter of bsm42 displayed more appropriate for selection of the carrimycin production improved mutants. The F2R-15 mutant had highest titer (1010±30 μg/mL), which was about 9 times higher than that of 54IA strain. Comparative analysis of transcriptome profiles of F2R-15 mutant and 54IA strains found 158 differential expression genes with more than 2 fold-changes. The up-regulated genes were associated with macrolide precursor biosynthesis, macrolide-inactivation, antibiotics transporter, oxidative phosphorylation; while the most down-regulated genes were referring to the primary metabolites synthetic genes and biosynthetic genes of other secondary metabolites. Conclusion: These results suggested that manipulation of the positive regulatory gene bsm42 and traditional mutagenesis coupled with reporter-guided mutant selection method facilitated selection of carrimycin high-yielding mutants.

Aayush Garg ◽  
Milos Ojdanic ◽  
Renzo Degiovanni ◽  
Thierry Titcheu Chekam ◽  
Mike Papadakis ◽  

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1525
Na-Hye Park ◽  
Seung-Jin Lee ◽  
Eon-Bee Lee ◽  
Biruk Tesfaye Birhanu ◽  
Seung-Chun Park

This study aimed to optimize the colistin-based antibacterial therapy to prevent antimicrobial resistance related to biofilm formation in avian pathogenic Escherichia coli (APEC) in chicken. Of all the bacterial isolates (n = 136), 69 were identified as APEC by polymerase chain reaction (PCR). Through a series of antibiotic susceptibility tests, susceptibility to colistin (<2 μg/mL) was confirmed in all isolates. Hence, a mutant selection window (MSW) was determined to obtain colistin-induced resistant bacteria. The minimum inhibitory concentration (MIC) of colistin against the colistin-induced resistant APEC strains ranged from 8 to 16 μg/mL. To identify the inhibitory activity of colistin against the resistant strains, the mutant prevention concentration (MPC) was investigated for 72 h, and the single and multi-dose colistin activities were determined through the time-kill curve against APEC strains. Bacterial regrowth occurred after 12 h at a double MIC50 concentration (1.00 μg/mL), and regrowth was not inhibited even during multiple exposures. However, upon exposure to 8 μg/mL—a concentration that was close to the MPC—the growth of APEC was inhibited, including in the resistant strains. Additionally, colistin-induced resistant strains showed a slower growth compared with the susceptible ones. Colistin-induced resistant APEC strains did not show colistin resistance gene (mcr-1). However, the expression of higher mgrB and phoQ levels was observed in the resistant strains. Furthermore, these strains showed increased formation of biofilm. Hence, the present study indicated that colistin could induce resistance through the increased formation of biofilm in APEC strains by enhancing the expression of phoQ.

Swapan K. Tripathy

Background: Rice (Oryza sativa L.) feeds more than half of the world population, but it is a poor source of zinc (Zn) and iron (Fe). Therefore, there is a need for Zn and Fe- biofortified rice in the food chain. For this, rice breeding needs to be re-oriented to improve the status of grain Zn and Fe content while increasing the yield potential. Identification of micronutrient-rich donors and their association study with agro-economic traits can pave the way for nutritional and food security. Methods: Zinc and iron content of a set of 92 rice genotypes was estimated in the aliquot of seed extract by using an Inductive Coupled Plasma-Optical Emission Spectrophotometer (ICP-OES) to explore donors and to study the degree of association of these micronutrients with agro-economic traits including grain quality traits and seed yield. Result: The top Fe (≥50 ppm) and Zn dense (≥40 ppm) genotypes identified were P44 mutant selection-1, ORCZ 75-3-1, Basudha, Malliphulajhuli, Tikimahsuri and Nikipankhia. P44 mutant Sel.-1 and ORCZ 75-3-1 had good yield potential (44 q ha-1). Grains/panicles and the number of effective bearing tillers/hill maintained an appreciably strong positive association with seed yield, while plant height had an inverse relationship. Grain Fe content positively correlated with panicle length. On the other hand, Zn is positively associated with tillering ability and grains/panicle. Grain Fe and Zn revealed strong a inter se positive association. Interestingly, grain Fe revealed no association, but grain Zn revealed significant positive relationship with seed yield. This envisaged better scope for genetic enhancement of grain Zn content along with substantial increase in grain Fe without any yield penalty.

Uday S. Ganapathy ◽  
Rubén González del Río ◽  
Mónica Cacho-Izquierdo ◽  
Fátima Ortega ◽  
Joël Lelièvre ◽  

Fluoroquinolones – the only clinically used DNA gyrase inhibitors – are effective against tuberculosis (TB) but are in limited clinical use for non-tuberculous mycobacteria (NTM) lung infections due to intrinsic drug resistance. We sought to test alternative DNA gyrase inhibitors for anti-NTM activity. Mycobacterium tuberculosis Gyrase Inhibitors (MGIs), a subclass of Novel Bacterial Topoisomerase Inhibitors (NBTIs), were recently shown to be active against the tubercle bacillus. Here, we show that the MGI EC/11716 not only has potent anti-tubercular activity but is active against M. abscessus and M. avium in vitro . Focusing on M. abscessus , which causes the most difficult to cure NTM disease, we show that EC/11716 is bactericidal, active against drug-tolerant biofilms, and efficacious in a murine model of M. abscessus lung infection. Based on resistant mutant selection experiments, we report a low frequency of resistance to EC/11716 and confirm DNA gyrase as its target. Our findings demonstrate the potential of NBTIs as anti- M. abscessus and possibly broad spectrum anti-mycobacterial agents.

2021 ◽  
Robin Schumann ◽  
Eileen Bischoff ◽  
Severina Klaus ◽  
Sophie Möhring ◽  
Julia Flock ◽  

Decreased susceptibilities of Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of Kelch13, which is the homologue of the redox sensor Keap1 in vertebrates. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of Kelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of Kelch13 resulted in ring-stage survival rates around 40%. While the loss of a potential disulfide bond between residues C580 and C532 had no effect on the artemisinin suceptibility, the thiol group of C473 could not be replaced. We also established a protocol for the production of recombinant Kelch13. In contrast to cysteine-to-serine replacements, common field mutations resulted in misfolded and insoluble protein. In summary, not the redox properties but impaired folding of Kelch13, resulting in a decreased Kelch13 abundance, is the central parameter for mutant selection.

Takahito Toyotome ◽  
Kenji Onishi ◽  
Mio Sato ◽  
Yoko Kusuya ◽  
Daisuke Hagiwara ◽  

Azole resistance of Aspergillus fumigatus is a global problem. The major resistant mechanism is a cytochrome P 450 14-α sterol demethylase Cyp51A alteration such as mutation(s) in the gene and the acquisition of a tandem repeat in the promoter. Although other azole tolerances and resistant mechanisms such as hmg1 (a 3-hydroxy-3-methylglutaryl-coenzyme-A reductase gene) mutation are known, few reports have described studies elucidating non-Cyp51A resistance mechanisms. This study explored genes contributing to azole tolerance in A. fumigatus by in vitro mutant selection with tebuconazole, an azole fungicide. After three-round selection, we obtained four isolates with low susceptibility to tebuconazole. These isolates also showed low susceptibility to itraconazole and voriconazole. Comparison of the genome sequences of the obtained isolates and the parental strain revealed a non-synonymous mutation in MfsD for a major facilitator superfamily protein (Afu1g11820, R337L mutation) in all isolates. Furthermore, non-synonymous mutations in AgcA for a mitochondrial inner membrane aspartate/glutamate transporter (Afu7g05220, E535Stop mutation), UbcD for a ubiquitin-conjugating enzyme E2 (Afu3g06030, T98K mutation), AbcJ for an ABC transporter (Afu3g12220, G297E mutation), and RttA for a putative protein r esponsible for t ebuconazole t olerance (Afu7g04740, A83T mutation), were found in at least one isolate. Disruption of the agcA gene led to decreased susceptibility to azoles. Reconstruction of the A83T point mutation in RttA led to decreased susceptibility to azoles. Reversion of T98K mutation to wild type in UbcD led to decreased susceptibility to azoles. These results suggest that these mutations contribute to lowered susceptibility to medical azoles and agricultural azole fungicides.

2021 ◽  
Vol 11 (1) ◽  
Nayara Helisandra Fedrigo ◽  
Danielle Rosani Shinohara ◽  
Josmar Mazucheli ◽  
Sheila Alexandra Belini Nishiyama ◽  
Floristher Elaine Carrara-Marroni ◽  

AbstractThe emergence of polymyxin resistance in Gram-negative bacteria infections has motivated the use of combination therapy. This study determined the mutant selection window (MSW) of polymyxin B alone and in combination with meropenem and fosfomycin against A. baumannii strains belonging to clonal lineages I and III. To evaluate the inhibition of in vitro drug resistance, we investigate the MSW-derived pharmacodynamic indices associated with resistance to polymyxin B administrated regimens as monotherapy and combination therapy, such as the percentage of each dosage interval that free plasma concentration was within the MSW (%TMSW) and the percentage of each dosage interval that free plasma concentration exceeded the mutant prevention concentration (%T>MPC). The MSW of polymyxin B varied between 1 and 16 µg/mL for polymyxin B-susceptible strains. The triple combination of polymyxin B with meropenem and fosfomycin inhibited the polymyxin B-resistant subpopulation in meropenem-resistant isolates and polymyxin B plus meropenem as a double combination sufficiently inhibited meropenem-intermediate, and susceptible strains. T>MPC 90% was reached for polymyxin B in these combinations, while %TMSW was 0 against all strains. TMSW for meropenem and fosfomycin were also reduced. Effective antimicrobial combinations significantly reduced MSW. The MSW-derived pharmacodynamic indices can be used for the selection of effective combination regimen to combat the polymyxin B-resistant strain.

2021 ◽  
Vol 39 (1) ◽  
pp. 20
Maria Fatima Palupi ◽  
Eli Nugraha ◽  
Meutia Hayati ◽  
Neneng Atikah

Mutant prevention concentration (MPC) is an in vitro test used to determine the lowest drug concentration needed to inhibit the growth of a single-step-mutant bacterial subpopulation. The purpose of this study was to determine the MPC value of ciprofloxacin against pathogenic Escherichia coli to obtained the range of mutant selection windows (MSW) of ciprofloxacin. Ciprofloxacin is a quinolone group that is included in the Highest Priority Critically Important Antimicrobials for Human Medicine but is also used for the treatment of bacterial infections in production animals. Twenty-four of pathogenic E. coli isolates sensitive to ciprofloxacin were tested to obtain MPC values and minimum inhibitory concentration (MIC) values. Test the MPC and MIC values to get the MSW range is done by the method of agar dilution. Mueller-Hinton agar containing standard ciprofloxacin was inoculated with 1010 cfu E. coli for the MPC test and 104 for the MIC test. Based on the MPC test results, the MPC value of ciprofloxacin was 4-64 μg / mL (22.96 ± 19.07 μg / mL) and there was one isolate which had an MPC> 256 μg / mL. These results give a wide range of MSW with a lower limit of the MIC value of 0.25 - 2 µg / mL (0.55 ± 0.37 µg / mL) to the upper limit of the MPC value of 4-64 µg / mL (22.96 ± 19.07 μg / mL). Based on the results of this MPC assessment it can be concluded that the dose of ciprofloxacin in production animals has a wide range of MSW that is allow for single-step mutants.

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