Random Mutagenesis Library Research Articles

Molecular Engineering L-Aspartate-Alpha-Decarboxylase to Enhance Catalytic Stability and Performance.

L-aspartate-alpha-decarboxylase (ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, which is the decisive step in the biosynthesis of β-alanine. However, the low catalytic stability and efficiency of ADC limit its industrial applications. In this study, a variant of ADC from Bacillus subtilis were used as a starting point for engineering. After constructing a random mutagenesis library by error-prone PCR, followed by high-throughput screening,four substitutions (S7 N, K63 N, A99T, and K113R) were identified. By screening saturation mutagenesis libraries on these positions and computational analysis, two recombined variants N3(S7 N/K63 N/I88 M/A99E/K113R/I126*) and Y1(S7Y/K63 N/I88 M/A99E/K113R/I126*) with improved performance were obtained. Compared to the wild type, the catalytic efficiency and catalytic stability of the best two variants were enhanced up to 95 %(variant N3) and up to 89 %(variant Y1), respectively. In addition, Y1 exhibited 3.37 times improved half-life and 2-fold improved total turnover number. Hydrophilicity analysis and molecular dynamics (MD) simulation revealed that the increased hydrophilicity and steric hindrance of key amino acid residues would affect the catalytic activity and stability. The improved catalytic performance of the variants could be attributed to their enhanced binding capacity to the substrate within the active pocket and the alleviation of mechanism-based inactivation.

Breeding of L-tryptophan high-yield strain by ARTP mutagenesis and high-throughput screening

L-tryptophan is an indispensable essential amino acid with a wide range of applications, which leads to a high demand. Accordingly, the production of L-tryptophan becomes a much-anticipated direction in research and industrial development. While irrational mutagenesis is an effective means to breed industrial strains, how to screen the strains with desirable phenotypes is still a major challenge. In order to improve the efficiency and accuracy of screening L-tryptophan high-yield strains, we used atmospheric and room temperature plasma mutagenesis to construct a random mutant library and then combined it with high-throughput screening in deep-well plates. Using a pseudo-fluorescent protein sensor capable of responding specifically to L-tryptophan, we successfully screened out a strain producing L-tryptophan at a high yield from a random mutagenesis library. The fermentation with the strain in shake flasks produced L-tryptophan at a yield of 1.99 g/L, which was 41.77% higher than that of the starting strain. Finally, the mechanism of high yield of the strain was deciphered by comparative genomics and transcriptomics. The above strategies provide a solid research foundation for further selection and development of high quality L-tryptophan producing strains.

Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries

Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library’s breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

Engineering of a Substrate Affinity Reduced S-Adenosyl-methionine Synthetase as a Novel Biosensor for Growth-Coupling Selection of L-Methionine Overproducers.

Biosensors are powerful tools for monitoring specific metabolites or controlling metabolic flux towards the products in a single cell, which play important roles in microbial cell factory construction. Despite their potential role in metabolic flux monitoring, the development of biosensors for small molecules is still limited. Reported biosensors often exhibit bottlenecks of poor specificity and a narrow dynamic range. Moreover, fine-tuning the substrate binding affinity of a crucial enzyme can decrease its catalytic activity, which ultimately results in the repression of the corresponding essential metabolite biosynthesis and impairs cell growth. However, increasing intracellular substrate concentration can elevate the availability of the essential metabolite and may lead to restore cellular growth. Herein, a new strategy was proposed for constructing whole-cell biosensors based on enzyme encoded by essential gene that offer inherent specificity and universality. Specifically, S-adenosyl-methionine synthetase (MetK) in E. coli was chosen as the crucial enzyme, and a series of MetK variants were identified that were sensitive to L-methionine concentration. This occurrence enabled the engineered cell to sense L-methionine and exhibit L-methionine dose-dependent cell growth. To improve the biosensor's dynamic range, an S-adenosyl-methionine catabolic enzyme was overexpressed to reduce the intracellular availability of S-adenosyl-methionine. The resulting whole-cell biosensor effectively coupled the intracellular concentration of L-methionine with growth and was successfully applied to select strains with enhanced L-methionine biosynthesis from random mutagenesis libraries. Overall, our study presents a universal strategy for designing and constructing growth-coupled biosensors based on crucial enzyme, which can be applied to select strains overproducing high value-added metabolites in cellular metabolism.

Uncovering the role of leucine 59 in Renilla luciferase stability and activity with error-prone PCR: Implications for protein engineering

A new variant of Renilla luciferase, named Met C-SRLuc 8, was obtained from a random mutagenesis library and expressed in Escherichia coli BL21 (DE3) plys and purified. The results of the enzyme's binding affinity, kinetic stability, and bioinformatic studies demonstrated that leucine 59, located within the hot-spot foldon in the N-terminal domain of the protein, plays a significant role in the stability and activity of Renilla luciferase. These findings may facilitate the engineering of different variants of this enzyme to achieve thermally stable versions for various biotechnological applications.

Directed evolution of a wax ester synthase for production of fatty acid ethyl esters in Saccharomyces cerevisiae.

Wax ester synthases (WSs) utilize a fatty alcohol and a fatty acyl-coenzyme A (activated fatty acid) to synthesize the corresponding wax ester. There is much interest in developing novel cell factories that can produce shorter esters, e.g., fatty acid ethyl esters (FAEEs), with properties similar to biodiesel in order to use these as transportation fuels. However, ethanol is a poor substrate for WSs, and this may limit the biosynthesis of FAEEs. Here, we implemented a random mutagenesis approach to enhance the catalytic efficiency of a WS from Marinobacter hydrocarbonoclasticus (MhWS2, encoded by the ws2 gene). Our selection system was based on FAEE formation serving as a detoxification mechanism for excessive oleate, where high WS activity was essential for a storage-lipid free yeast to survive. A random mutagenesis library of ws2 was used to transform the storage-lipid free yeast, and mutants could be selected by plating the transformants on oleate containing plates. The variants encoding WS with improved activity were sequenced, and an identified point mutation translated into the residue substitution at position A344 was discovered to substantially increase the selectivity of MhWS2 toward ethanol and other shorter alcohols. Structural modeling indicated that an A344T substitution might affect the alcohol selectivity due to change of both steric effects and polarity changes near the active site. This work not only provides a new WS variant with altered selectivity to shorter alcohols but also presents a new high-throughput selection system to isolate WSs with a desired selectivity. KEY POINTS: • The work provides WS variants with altered substrate preference for shorter alcohols • A novel method was developed for directed evolution of WS of desired selectivity.

Yeast-based evolutionary modeling of androgen receptor mutations and natural selection.

Cancer progression is associated with the evolutionary accumulation of genetic mutations that are biologically significant. Mutations of the androgen receptor (AR) are associated with the development of prostate cancer (PCa) by responding to non-androgenic hormones, and the lack of annotations in their responsiveness to hormone ligands remains a daunting challenge. Here, we have used a yeast reporter system to quickly evaluate the responsiveness of all fifty clinical AR mutations to a variety of steroidal ligands including dihydrotestosterone (DHT), 17β-estradiol (E2), progesterone (PROG), and cyproterone acetate (CPA). Based on an AR-driven reporter that synthesizes histidine, a basic amino acid required for yeast survival and propagation, the yeast reporter system enabling clonal selection was further empowered by combining with a random DNA mutagenesis library to simulate the natural evolution of AR gene under the selective pressures of steroidal ligands. In a time-frame of 1-2 weeks, 19 AR mutants were identified, in which 11 AR mutants were validated for activation by tested steroidal compounds. The high efficiency of our artificial evolution strategy was further evidenced by a sequential selection that enabled the discovery of multipoint AR mutations and evolution directions under the pressure of steroidal ligands. In summary, our designer yeast is a portable reporter module that can be readily adapted to streamline high-throughput AR-compound screening, used as a PCa clinical reference, and combined with additional bioassay systems to further extend its potential.

522 Random mutagenesis libraries to define interactions between Mycobacterium abscessus and therapeutic phages

522 Random mutagenesis libraries to define interactions between Mycobacterium abscessus and therapeutic phages

Design of a genetically encoded biosensor to establish a high-throughput screening platform for L-cysteine overproduction

Metabolic engineering seeks to rewire the metabolic network of cells for the efficient production of value-added compounds from renewable substrates. However, it remains challenging to evaluate and identify strains with the desired phenotype from the vast rational or random mutagenesis library. One effective approach to resolve this bottleneck is to design an efficient high-throughput screening (HTS) method to rapidly detect and analyze target candidates. L-cysteine is an important sulfur-containing amino acid and has been widely used in agriculture, pharmaceuticals, cosmetics, and food additive industries. However, HTS methods that enable monitoring of L-cysteine levels and screening of the enzyme variants and strains to confer superior L-cysteine biosynthesis remain unavailable, greatly limiting the development of efficient microbial cell factories for L-cysteine production at the industrial scale. Here, we took advantage of the L-cysteine-responsive transcriptional regulator CcdR to develop a genetically encoded biosensor for engineering and screening the L-cysteine overproducer. The in vivo L-cysteine-responsive assays and in vitro electrophoretic mobility shift assay (EMSA) and DNase I footprint analysis indicated that CcdR is a transcriptional activator that specifically interacts with L-cysteine and binds to its regulatory region to induce the expression of target genes. To improve the response performance of the L-cysteine biosensor, multilevel optimization strategies were performed, including regulator engineering by semi-rational design and systematic optimization of the genetic elements by modulating the promoter and RBS combination. As a result, the dynamic range and sensitivity of the biosensor were significantly improved. Using this the excellent L-cysteine biosensor, a HTS platform was established by coupling with fluorescence-activated cell sorting (FACS) and was successfully applied to achieve direct evolution of the key enzyme in the L-cysteine biosynthetic pathway to increase its catalytic performance and to screen the high L-cysteine-producing strains from the random mutagenesis library. These results presented a paradigm of design and optimization of biosensors to dynamically detect metabolite concentrations and provided a promising tool enabling HTS and metabolic regulation to construct L-cysteine hyperproducing strains to satisfy industrial demand.

A high-throughput visual screening method for p-hydroxybenzoate hydroxylase to increase phenolic compounds biosynthesis

BackgroundGallic acid (GA) and pyrogallol are phenolic hydroxyl compounds and have diverse biological activities. Microbial-based biosynthesis, as an ecofriendly method, has been used for GA and pyrogallol production. In GA and pyrogallol biosynthetic pathways, the low hydroxylation activity of p-hydroxybenzoate hydroxylase (PobA) towards 3,4-dihydroxybenzoic acid (3,4-DHBA) limited the high-level biosynthesis of GA and pyrogallol.ResultsThis work reported a high activity PobA mutant (Y385F/T294A/V349A PobA) towards 3,4-DHBA. This mutant was screened out from a PobA random mutagenesis library through a novel naked eye visual screening method. In vitro enzyme assay showed this mutant has a kcat/Km of 0.059 μM−1 s−1 towards 3,4-DHBA, which was 4.92-fold higher than the reported mutant (Y385F/T294A PobA). Molecular docking simulation provided the possible catalytic mechanism explanation of the high activity mutant. Expression of this mutant in E. coli BW25113 (Fʹ) can generate 840 ± 23 mg/L GA from 1000 mg/L 3,4-DHBA. After that, this mutant was assembled into a de novo GA biosynthetic pathway. Subsequently, this pathway was introduced into a 3,4-DHBA-producing strain (E. coli BW25113 (Fʹ)ΔaroE) to achieve 301 ± 15 mg/L GA production from simple carbon sources. Similarly, assembling this mutant into a de novo pyrogallol biosynthetic pathway enabled 129 ± 15 mg/L pyrogallol production.ConclusionsThis work established an efficient screening method and generated a high activity PobA mutant. Assembling this mutant into de novo GA and pyrogallol biosynthetic pathways achieved the production of these two compounds from glucose. Besides, this mutant has great potential for the production of GA or pyrogallol derivatives. The screening method could be used for other GA biosynthesis-related enzymes.

Tuning the catalytic performances of a sucrose isomerase for production of isomaltulose with high concentration.

Obtaining a sucrose isomerase (SIase) with high catalytic performance is of great importance in industrial production of isomaltulose (a reducing sugar). In order to obtain such SIase mutant, a high-throughput screening system in microtiter plate format was developed based on a widely used 2,4-dinitrosalicylic acid (DNS) method for determination of reducing sugar. An SIase from Erwiniasp. Ejp617 (ErSIase) was selected to improve its catalytic efficiency. After screening of ~ 8000 mutants from a random mutagenesis library, Q209 and R456 were identified as beneficial positions. Saturation mutagenesis of the two positions resulted in a double-site mutant ErSIase_Q209S-R456H that showed the highest catalytic efficiency, and its specific activity reached 684 U/mg that is 17.5-fold higher than that of the wild-type ErSIase. By employing the lyophilized Escherichia coli (E. coli) cells harboring ErSIase_Q209S-R456H, a high space-time yield (STY = 3.9kg/(L·d)) was achieved toward 600g/L sucrose. Furthermore, the in silico analysis suggested that the hydrogen bond network was improved and steric hindrance was reduced due to the beneficial substitutions.Key points• A sucrose isomerase mutant with high catalytic efficiency was obtained.• The highest space-time yield was achieved toward high-concentration sucrose.• The optimized H-bond network contributed to the enhanced catalytic efficiency.

Programmable Synthetic Upstream Activating Sequence Library for Fine-Tuning Gene Expression Levels in Saccharomyces cerevisiae.

A wide dynamic range of promoters is necessary for fine-tuning transcription levels. However, weak intensity and narrow dynamic range limit transcriptional regulation via constitutive promoters. The upstream activation sequence (UAS) located upstream of the core promoter is a crucial region that could obviously enhance promoter strength. Herein, we created a random mutagenesis library consisting of 330 different variants based on the UAS of the TDH3 promoter with an ∼37-fold dynamic range by error-prone polymerase chain reaction (PCR) and obtained strong intensity mutant UAS, which was ∼12-fold greater than the wild-type UASTDH3. Analysis of the mutant library revealed 15 strength-enhancing sites and their corresponding bases of the UASTDH3 regions, which provided the impetus for a synthetic library. The resulting 32 768 mutant UAS library was constructed by permutation and combination of the bases of the 15 enhancing sites. To characterize the library, a strength prediction model was built by correlating DNA structural features and UAS strength, which provided a model between UAS sequence and intensity. Following characterization, the UAS library was applied to precisely regulate gene expression in the production of β-carotene, proving that the UAS library would be a useful tool for gene tuning in metabolic engineering. In summary, we designed, constructed, and characterized a UAS library that facilitated precise tuning of transcription levels of target proteins.

Rapid Cloning of Random Mutagenesis Libraries Using PTO-QuickStep.

PTO-QuickStep is a quick and easy molecular cloning technique that allows seamless point integration of a DNA fragment, encoding either a tag or a protein, into any position within a target plasmid. The entire process is conducted in a time-efficient and cost-effective manner, without the need of DNA gel purification and enzymatic restriction and ligation. PTO-QuickStep further innovates protein engineering by providing the possibility of integrating a random mutagenesis step (e.g., error-prone PCR) into the workflow, without compromising the time duration required. Random mutagenesis libraries can be quickly and efficiently cloned into a plasmid of interest, thereby accelerating directed evolution. On top of that, PTO-QuickStep can be utilized for rapid integration of noncoding DNA fragments to modify existing plasmids, making it an excellent tool for synthetic biologists.

Isobutanol tolerance and production of Saccharomyces cerevisiae can be improved by engineering its TATA-binding protein Spt15.

Low isobutanol tolerance of Saccharomyces cerevisiae limits its application in isobutanol fermentation. Here, we used global transcription machinery engineering to screen mutants with higher isobutanol tolerance and elevated isobutanol titres. TATA-binding protein Spt15 was used as the target of global transcription machinery engineering for improvement of such complex phenotypes. A random mutagenesis library of S. cerevisiae TATA-binding protein Spt15 was constructed and subjected to screening under isobutanol stress. A mutant strain (denoted as spt15-3) with improved isobutanol tolerance was identified. There were three mutations of Spt15 in strain spt15-3, including deletion of A at position -132 nt upstream of initiation codon, insertion of G at position -65 nt upstream of initiation codon and a synonymous mutation at position 315 nt (T → C) downstream of initiation codon. We then metabolically engineered isobutanol synthesis in strains harbouring plasmids YCplac22 containing these Spt15 mutations. Delta integration was used to overexpress ILV3 gene, and 2μ plasmids carrying PGK1p-ILV2 and PGK1p-ARO10 were used to overexpress ILV2 and ARO10 genes. After 24-h micro-aerobic fermentation, Engi-3 produced 0·556 g l-1 isobutanol, which was 404% and 25·3% greater than isobutanol produced by control Engi-1 and engineered Engi-2, respectively. After 28 h, Engi-4 produced 0·459 g l-1 isobutanol, which was 315% and 3·2% greater than isobutanol produced Engi-1 and Engi-2, respectively. RNA-Seq-based transcriptome analysis shows that mutations of Spt15 in strain spt15-3 increased the expression of SPT15. Meanwhile, compared with strain Engi-3, the spt15-3 mutation downregulated the expression of genes involved in the TCA cycle and glyoxylic acid cycle, but increased the expression of genes related to cell stability. This work demonstrates that isobutanol tolerance and production of S. cerevisiae can be improved by engineering its TATA-binding protein Spt15. This study clarified the molecular mechanisms regulating isobutanol production and tolerance in S. cerevisiae.

Characterization of a new heterotrophic nitrification bacterium Pseudomonas sp. strain JQ170 and functional identification of nap gene in nitrite production

Heterotrophic nitrification bacteria play a critical role in nitrogen cycling and pollution removal. However, the underlying nitrification mechanisms are diverse and have rarely been investigated at the genetic level. In this study, the new heterotrophic nitrifier Pseudomonas sp. strain JQ170 was isolated. Strain JQ170 can utilize ammonia (NH4+-N), nitrite (NO2−-N), or nitrate (NO3−-N) as sole nitrogen sources, preferring NH4+-N. A ratio of 96.4% of 1.0 mM NH4+-N was removed in 24 h. The optimum pH, temperature, and carbon source for NH4+-N removal were pH 7.0, 30 °C, and citrate, at a C/N ratio of 9–18, respectively. During the NH4+-N removal process, only NO2−-N but neither hydroxylamine, NO3−-N, nor gaseous nitrogen were detected. A random transposon insertion mutagenesis library of strain JQ170 was constructed. Two NO2−-N-production deficient mutants were screened and transposon insertion sites were located in nap genes (which encode periplasmic NO3−-N reductase Nap). Further gene knockout and complementation of the napA gene confirmed nap as essential for NO2−-N production. The following nitrification processes in strain JQ170 is proposed: NH4+-N to NO3−-N in the cytoplasm; then NO3−-N to NO2−-N in the periplasmic space by Nap; finally, NO2−-N secreted out of cells. Overall, this paper provides new insight towards understanding heterotrophic nitrification at the genetic level.

Establishment of a Biosensor-based High-Throughput Screening Platform for Tryptophan Overproduction.

With the flexibility to fold into complex structures, RNA is well-suited to act as a cellular sensor to recognize environmental fluctuations and respond to changes by regulating the corresponding genes. In this study, we established a high-throughput screening platform to screen tryptophan high-producing strains from a large repertoire of candidate strains. This platform consists of a tryptophan-specific aptamer-based biosensor and fluorescence-activated droplet sorting technology. One mutant strain, with a 165.9% increase in Trp titer compared with the parental strain, was successfully screened from a random mutagenesis library. Sequencing results revealed that a total of 10 single-nucleotide polymorphisms were discovered in the genome of the mutant strain, among which CRP(T29K) was proven to significantly increase Trp production through improving the strain's tolerance of the harsh environment during the stationary phase of the fermentation process. Our results indicate that this strategy has great potential for improving the production of other amino acids in Escherichia coli.

Whole-Cell Biosensors Aid Exploration of Vanillin Transmembrane Transport.

Transcriptional regulatory protein (TRP)-based whole-cell biosensors are widely used nowadays. Here, they were demonstrated to have great potential application in screening cell efflux and influx pumps for small molecules. First, a vanillin whole-cell biosensor was developed by altering the specificity of a TRP, VanR, and strains with improved vanillin productions that were selected from a random genome mutagenesis library by using this biosensor as a high-throughput screening tool. A high intracellular vanillin concentration was found to accumulate due to the inactivation of the AcrA protein, indicating the involvement of this protein in vanillin efflux. Then, the application of this biosensor was extended to explore efflux and influx pumps, combined with directed genome evolution. Elevated intracellular vanillin levels resulting from efflux pump inactivation or influx pump overexpression could be rapidly detected by the whole-cell biosensor, markedly facilitating the identification of genome targets related to small-molecule transmembrane transport, which is of great importance in metabolic engineering.

Identification of genetic determinants of hemolytic activity of Riemerella anatipestifer using random transposon mutagenesis

Riemerella anatipestifer causes epizootic infectious disease in poultry resulting in serious economic losses especially to the duck industry. In our previous study, R. anatipestifer was found to lyse duck erythrocytes in vitro. In the present study, a random Tn4351 mutagenesis library of hemolytic R. anatipestifer strain SX containing 4000 mutants was constructed to investigate the genetic basis of hemolytic activity. Thirty mutants with reduced hemolytic activity and one with increased hemolytic activity were screened and insertions in 24 genes were identified. Of these genes, four were predicted to encode outer membrane proteins, one encoded a cytoplasmic membrane protein, 11 encoded cytoplasmic proteins, and eight encoded proteins with unknown locations. Based on current annotations of the R. anatipestifer genomes, of the 24 genes, 7 (29.17%) were involved in iron utilization. The hemolytic activities of the complemented strains M2 (pRES-Riean_0790) and M18 (pRES-Riean_0653) were restored, indicating that both Riean_0653 and Riean_0790 are involved in the hemolytic activity of strain SX. However, the recombinant proteins rRiean_0317, rRiean_0790, rRiean_0653, rRiean_1027, rRiean_1143, and rRiean_1561 had no hemolytic activity, suggesting that none were hemolysins.

Droplet-Based Microfluidic High-Throughput Screening of Enzyme Mutant Libraries Secreted by Yarrowia lipolytica.

Yarrowia lipolytica has emerged as an attractive solution for screening enzyme activities thanks to the numerous tools available for heterologous protein production and its strong secretory ability. Nowadays, activity screening for improved enzymes mostly relies on the evaluation of independent clones in microtiter plates. However, even with highly robotized screening facilities, the relatively low throughput and high cost of the technology do not enable the screening of large diversities, which significantly reduce the probability of isolating improved variants. Droplet-based microfluidics is an emerging technology that allows the high-throughput and individual picoliter droplets manipulation and sorting based on enzymatic substrate fluorescence. This technology is an attractive alternative to microtiter plate screenings with higher throughputs and drastic reduction of working volume and cost.Here, we present a droplet-based microfluidic platform for the screening of libraries expressed in the yeast Y. lipolytica, from the generation of a random mutagenesis library of a heterologous enzyme and its expression in Y. lipolytica to the droplet-based microfluidic procedures composed of cell encapsulation and growth and activity screening or sorting of improved clones.

Elucidating the Role of Virulence Traits in the Survival of Pathogenic E. coli PI-7 Following Disinfection.

Reuse and discharge of treated wastewater can result in dissemination of microorganisms into the environment. Deployment of disinfection strategies is typically proposed as a last stage remediation effort to further inactivate viable microorganisms. In this study, we hypothesize that virulence traits, including biofilm formation, motility, siderophore, and curli production along with the capability to internalize into mammalian cells play a role in survival against disinfectants. Pathogenic E. coli PI-7 strain was used as a model bacterium that was exposed to diverse disinfection strategies such as chlorination, UV and solar irradiation. To this end, we used a random transposon mutagenesis library screening approach to generate 14 mutants that exhibited varying levels of virulence traits. In these 14 isolated mutants, we observed that an increase in virulence traits such as biofilm formation, motility, curli production, and internalization capability, increased the inactivation half-lives of mutants compared to wild-type E. coli PI-7. In addition, oxidative stress response and EPS production contributed to lengthening the lag phase duration (defined as the time required for exposure to disinfectant prior to decay). However, traits related to siderophore production did not help with survival against the tested disinfection strategies. Taken together, the findings suggested that selected virulence traits facilitate survival of pathogenic E. coli PI-7, which in turn could account for the selective enrichment of pathogens over the non-pathogenic ones after wastewater treatment. Further, the study also reflected on the effectiveness of UV as a more viable disinfection strategy for inactivation of pathogens.