Random Library Research Articles

Mutations of Asn321 and Glu322 Improve Resistance of 4-Hydroxyphenylpyruvate Dioxygenase SpHPPDm to Topramezone.

As a highly efficient 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide, topramezone is an ideal target for herbicide-resistant genetic engineering. In this study, two mutants, K-19 (N321Y) and K-63 (Q166R/E322V), with topramezone resistance increased by 205.3 and 58.5%, respectively, were screened from the random mutation library of SpHPPDm, a topramezone-resistant HPPD mutant that we previously obtained. Sites N321 and E322 were identified as key sites for increased topramezone resistance by single-site mutation analysis. A mutant KB-145 (N321Y/E322K) was further obtained by saturation mutation at sites N321 and E322. The topramezone resistance of KB-145 increased by 955.3% compared to mutant SpHPPDm. In conclusion, this study identifies two new sites that significantly affect the topramezone resistance of SpHPPDm, which provides new insights into the molecular mechanism of herbicide resistance of HPPD, and the acquired mutants have great application potential in the construction of herbicide-resistant crops.

Antimicrobial Peptide Combination Can Hinder Resistance Evolution.

ABSTRACTAntibiotic-resistant microbial pathogens are becoming a major threat to human health. Therefore, there is an urgent need to develop new alternatives to conventional antibiotics. One such promising alternative is antimicrobial peptides (AMPs), which are produced by virtually all organisms and typically inhibit bacteria via membrane disruption. However, previous studies demonstrated that bacteria can rapidly develop AMP resistance. Here, we study whether combination therapy, known to be able to inhibit the evolution of resistance to conventional antibiotics, can also hinder the evolution of AMP resistance. To do so, we evolved the opportunistic pathogen Staphylococcus aureus in the presence of individual AMPs, AMP pairs, and a combinatorial antimicrobial peptide library. Treatment with some AMP pairs indeed hindered the evolution of resistance compared with individual AMPs. In particular, resistance to pairs was delayed when resistance to the individual AMPs came at a cost of impaired bacterial growth and did not confer cross-resistance to other tested AMPs. The lowest level of resistance evolved during treatment with the combinatorial antimicrobial peptide library termed random antimicrobial peptide mixture, which contains more than a million different peptides. A better understanding of how AMP combinations affect the evolution of resistance is a crucial step in order to design “resistant proof” AMP cocktails that will offer a sustainable treatment option for antibiotic-resistant pathogens.IMPORTANCE The main insights gleaned from this study are the following. (i) AMP combination treatment can delay the evolution of resistance in S. aureus. Treatment with some AMP pairs resulted in significantly lower resistance then treatment with either of the individual AMPs. Treatment with a random AMP library resulted in no detectable resistance. (ii) The rate at which resistance to combination arises correlates with the cost of resistance to individual AMPs and their cross-resistance. In particular, combinations to which the least resistance arose involved AMPs with high fitness cost of resistance and low cross-resistance. (iii) No broad-range AMP resistance evolved. Strains that evolved resistance to some AMPs typically remained sensitive to other AMPs, alleviating concerns regarding the evolution of resistance to immune system AMPs in response to AMP treatment.

Isolation of DNA Aptamers for Enteropathogenic Escherichia coli (EPEC) Detection using Bacterial-SELEX Approach

Enteropathogenic Escherichia coli (EPEC) is a Gram-negative pathogenic bacterium that causes diarrheal disease, especially in infants and children. Aptamers are short chain oligonucleotides that have high affinity, specificity, and selectivity to their targets, which have potential to be developed as a method for diagnosing pathogens. In this study, aptamer was isolated through the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method using whole cells bacteria (Bacterial-SELEX) for recognizing pathogenic E. coli EPEC K1.1 which was isolated from children with diarrhea in Indonesia. Ten rounds of bacterial-SELEX procedure were conducted with modification conditions by using Top10, DH5a E. coli cells, Listeria monocytogenes, and Lactobacillus plantarum S34 as counter-selections. The selection process was started with a pool of ssDNA random library consisting of a random base with 40-nucleotides long flanked with fixed primers sequence for aptamer amplification purpose. Short single-stranded DNA amplification was done by symmetric and asymmetric PCR. The highly enriched oligonucleotide pools (pooled 8, 9, and 10) were cloned and the resulting ssDNA aptamers were identified by Sanger DNA sequencing. Finally, twelve aptamers with unique sequences and various secondary structures including G-quadruplex sequence motif within aptamers were obtained as candidates specific aptamer for detection and capturing of EPEC K1.1.

Aptamer-based Cell Recognition and Detection

: Cells, regarded as the structural and functional units of organisms, have become one of the most important objects in many research areas. Specific recognition and detection of malignant cells are critical for disease diagnosis, therapy and prognosis. Aptamers are short; single-stranded oligonucleotides screened from a random library by an in vitro technology termed “Systematic Evolution of Ligands by Exponential Enrichment” (SELEX) on the basis of their specific binding to target cargos. With the advantages of small size, easy synthesis, convenient modification, high chemical stability and low immunogenicity, aptamers have attracted broad attention in bioanalysis. Using intact living cells as the selection target, the cell-SELEX technology enables the generation of many aptamers that can specifically recognize molecular signatures of target cells. These aptamers have been extensively utilized in various cell-based research. In this mini-review, we focus on recent advances in aptamer-based recognition and detection of cells, particularly circulating tumor cells (CTCs).

Optimization of Classroom Teaching Strategies for College English Listening and Speaking Based on Random Matrix Theory

Online English teaching systems are more and more widely used in higher education teaching. Based on random matrix theory, this study constructs an optimization model of classroom teaching strategies for college English listening and speaking. By comparing and analyzing the universal properties of the research system and the random system matrix, the model can examine the random properties and special nonrandom properties within the system and solve the quantification problem of college English classroom education. First, based on the existing college English listening and speaking classroom teaching system, with the help of J2EE structure in information technology, the existing college English listening and speaking classroom teaching system is optimized, the customer layer, web layer, business logic layer, and student listening and speaking classroom learning effect management layer are designed, and the system is applied to college English listening and speaking classroom teaching. During the simulation process, a random matrix theoretical dynamics graph library and a subject library were written, so that combining the elements of each vector into a new matrix. Experiments show that the ensemble framework based on ranking learning can not only integrate multiple matrix factorization algorithm models to obtain better recommendation accuracy but also can more fully reflect the eigenvalues of each individual. The experimental results show that when the number of sampling points is 80 and the signal-to-noise ratio is −15 dB, the detection probability is close to 80%, which effectively improves the classroom random detection performance of English teaching and the effect of strategy optimization.

Directed evolution and selection of biostable l-DNA aptamers with a mirror-image DNA polymerase

Mirror-image aptamers made from chirally inverted nucleic acids are nuclease-resistant and exceptionally biostable, opening up opportunities for unique applications. However, the directed evolution and selection of mirror-image aptamers directly from large randomized l-DNA libraries has, to our knowledge, not been demonstrated previously. Here, we developed a ‘mirror-image selection’ scheme for the directed evolution and selection of biostable l-DNA aptamers with a mirror-image DNA polymerase. We performed iterative rounds of enrichment and mirror-image polymerase chain reaction (PCR) amplification of l-DNA sequences that bind native human thrombin, in conjunction with denaturing gradient gel electrophoresis (DGGE) to isolate individual aptamers and l-DNA sequencing-by-synthesis to determine their sequences. Based on the selected l-DNA aptamers, we designed biostable thrombin sensors and inhibitors, which remained functional in physiologically relevant nuclease-rich environments, even in the presence of human serum that rapidly degraded d-DNA aptamers. Mirror-image selection of biostable l-DNA aptamers directly from large randomized l-DNA libraries greatly expands the range of biomolecules that can be targeted, broadening their applications as biostable sensors, therapeutics and basic research tools.

D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses.

Influenza A viruses (IAV) modulate host antiviral responses to promote viral growth and pathogenicity. The non-structural (NS1) protein of influenza A virus has played an indispensable role in the inhibition of host immune responses, especially in limiting interferon (IFN) production. In this study, random site mutations were introduced into the NS1 gene of A/WSN/1933 (WSN, H1N1) via an error prone PCR to construct a random mutant plasmid library. The NS1 random mutant virus library was generated by reverse genetics. To screen out the unidentified NS1 functional mutants, the library viruses were lung-to-lung passaged in mice and individual plaques were picked from the fourth passage in mice lungs. Sanger sequencing revealed that eight different kinds of mutations in the NS1 gene were obtained from the passaged library virus. We found that the NS1 F9Y mutation significantly enhanced viral growth in vitro (MDCK and A549 cells) and in vivo (BALB/c mice) as well as increased virulence in mice. The NS1 D2I mutation attenuated the viral replication and pathogenicity in both in vitro and in vivo models. Further studies demonstrated that the NS1 F9Y mutant virus exhibited systematic and selective inhibition of cytokine responses as well as inhibited the expression of IFN. In addition, the expression levels of innate immunity-related cytokines were significantly up-regulated after the rNS1 D2I virus infected A549 cells. Collectively, our results revealed that the two mutations in the N-terminal of the NS1 protein could alter the viral properties of IAV and provide additional evidence that the NS1 protein is a critical virulence factor. The two characterized NS1 mutations may serve as potential targets for antiviral drugs as well as attenuated vaccine development.

Validation of a random Vibrio parahaemolyticus genomic library by selection of quinolone resistance in a heterologous host.

Vibrio parahaemolyticus is a shellfish-borne pathogen that is a highly prevalent causative agent of inflammatory gastroenteritis in humans. Genomic libraries have proven useful for the identification of novel gene functions in many bacterial species. In this study we prepared a library containing 40 kb fragments of randomly sheared V. parahaemolyticus genomic DNA and introduced this into Escherichia coli HB101 using a commercially available low copy cosmid system. In order to estimate coverage and suitability of the library and potentially identify novel antimicrobial resistance determinants, we screened for the acquisition of resistance to the fluoroquinolone norfloxacin - a phenotype exhibited by V. parahaemolyticus but not the heterologous E. coli host. Upon selection on solid medium containing norfloxacin, 0.52% of the library population was resistant, consistent with the selection of a single resistance locus. End-sequencing identified six distinct insert fragments. All clones displayed fourfold increased norfloxacin MIC compared with E. coli HB101 carrying an empty vector. The common locus contained within resistant clones included qnr, a previously described quinolone resistance gene. These results indicate that the library was unbiased, of sufficient coverage and that heterologous expression was possible. While we hope that this library proves useful for identifying the genetic determinants of complex phenotypes such as those related to virulence, not all norfloxacin resistance genes were detected in our screen. As such, we discuss the benefits and limitations of this approach for identifying the genetic basis of uncharacterized bacterial phenotypes.

In vitro Evolution of Uracil Glycosylase Towards DnaKJ and GroEL Binding Evolves Different Misfolded States

Natural evolution is driven by random mutations that improve fitness. In vitro evolution mimics this process, however, on a short time-scale and is driven by the given bait. Here, we used directed in vitro evolution of a random mutant library of Uracil glycosylase (eUNG) displayed on yeast surface to select for binding to chaperones GroEL, DnaK + DnaJ + ATP (DnaKJ) or E. coli cell extract (CE), using binding to the eUNG inhibitor Ugi as probe for native fold. The CE selected population was further divided to Ugi binders (+U) or non-binders (−U). The aim here was to evaluate the sequence space and physical state of the evolved protein binding the different baits. We found that GroEL, DnaKJ and CE-U select and enrich for mutations causing eUNG to misfold, with the three being enriched in mutations in buried and conserved positions, with a tendency to increase positive charge. Still, each selection had its own trajectory, with GroEL and CE-U selecting mutants highly sensitive to protease cleavage while DnaKJ selected partially structured misfolded species with a tendency to refold, making them less sensitive to proteases. More general, our results show that GroEL has a higher tendency to purge promiscuous misfolded protein mutants from the system, while DnaKJ binds misfolding-prone mutant species that are, upon chaperone release, more likely to natively refold. CE-U shares some of the properties of GroEL- and DnaKJ-selected populations, while harboring also unique properties that can be explained by the presence of additional chaperones in CE, such as Trigger factor, HtpG and ClpB.

Optimized quantitative PCR analysis of random DNA aptamer libraries

The quantitative polymerase chain reaction (qPCR) with detection of duplex DNA yield by intercalator fluorescence is a common and essential technique in nucleic acid analysis. We encountered unexpected results when applying standard qPCR methods to the quantitation of random DNA libraries flanked by regions of fixed sequence, a configuration essential for in vitro selection experiments. Here we describe the results of experiments revealing why conventional qPCR methods can fail to allow automated analysis in such cases, and simple solutions to this problem. In particular we show that renaturation of PCR products containing random regions is incomplete in late PCR cycles when extension fails due to reagent depletion. Intercalator fluorescence can then be lost at standard interrogation temperatures. We show that qPCR analysis of random DNA libraries can be achieved simply by adjusting the step at which intercalator fluorescence is monitored so that the yield of annealed constant regions is detected rather than the yield of full duplex DNA products.

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.

CRISPR-Cas Assisted Shotgun Mutagenesis Method for Evolutionary Genome Engineering.

Genome mutagenesis drives the evolution of organisms. Here, we developed a CRISPR-Cas assisted random mutation (CARM) technique for whole-genome mutagenesis. The method leverages an entirely random gRNA library and SpCas9-NG to randomly damage genomes in a controllable shotgunlike manner that then triggers diverse and abundant mutations via low-fidelity repair. As a proof of principle, CARM was applied to evolve the capacity of Saccharomyces cerevisiae BY4741 to produce β-carotene. After seven rounds of iterative evolution over two months, a β-carotene hyperproducing strain, C7-143, was isolated with a 10.5-fold increase in β-carotene production and 857 diverse genomic mutations that comprised indels, duplications, inversions, and chromosomal rearrangements. Transcriptomic analysis revealed that the expression of 2541 genes of strain C7-143 was significantly altered, suggesting that the metabolic landscape of the strain was deeply reconstructed. In addition, CARM was applied to evolve industrially relevant S. cerevisiae CEN.PK2-1C for S-adenosyl-L-methionine production, which was increased 2.28 times after just one round. Thus, CARM can contribute to increasing genetic diversity to identify new phenotypes that could further be investigated by reverse engineering.

Development of highly specific peptibody against Porcine epidemic diarrhea virus

Abstract Porcine epidemic diarrhea virus (PEDV) is an etiological agent of porcine epidemic diarrhea (PED), causing dehydration, vomiting, and acute watery diarrhea. Because it has a high mortality rate in neonatal piglets, PEDV is one of the critical pathogens affecting the swine industry. Since PEDV has shown rapid spreading in herds of swine, early diagnosis of this virus is very important to control the disease. Serological diagnosis test is a simple, rapid method for detecting PEDV infected pigs, and the development of PEDV-specific antibodies is essential for an accurate serological diagnostic test. Because conventional methods of development of monoclonal antibodies are time and hand-consuming work, recombinant antibodies are considered as an alternative strategy for the development of target-specific monoclonal antibodies. Peptibody is one of the promising recombinant antibodies that target-specific peptide is fused with the Fc domain of immunoglobulin G. The peptibody which have a specificity of peptides and immune function of Fc region as advantages have developed for therapeutic and diagnostic purpose. Here, we generated a PEDV-specific peptibody (PEDV-PB) that is fused with PEDV-specific peptide and mouse IgG2b Fc region. PEDV-specific peptide sequences were discovered by random phage display library and PEDV-PB was produced from Chinese hamster ovary (CHO) cells. PEDV-PB showed highly specific binding in several serological tests for PEDV, such as western blotting, immunofluorescence assay, and enzyme-linked immunosorbent assay. Our results suggest that PEDV-PB is very useful for the development of a rapid detection kit for PEDV.

Identification of the genetic elements involved in biofilm formation by Salmonella enterica serovar Tennessee using mini-Tn10 mutagenesis and DNA sequencing

Biofilms are known to contribute to bacterial tolerance to desiccation and survival in low moisture foods. However, the molecular mechanisms underlying biofilm formation have not been fully elucidated. This study identified some of the genes that are implicated in biofilm formation by Salmonella enterica serovar Tennessee, the “peanut butter outbreak” strain. Mini-Tn10 mutagenesis was used in the study to generate random transposon insertion libraries. The ability of selected mutants in forming biofilms was compared with their wildtype parent using the crystal violet binding assay. Mutants forming significantly less (P ≤ 0.05) biofilm compared to their wildtype parent were selected for whole-genome sequencing. Mini-Tn10 insertion sites on mutant genomes were identified by comparing the acquired sequencing data with those in the Genbank using the BLAST search. In total, 56 mutants were obtained, and five were selected for further analysis according to the result of the biofilm assay. Sequencing analysis revealed that the mini-Tn10 interrupted the S. enterica genes that encode bacterial cell membrane lipoprotein, DNA topoisomerase III, attachment and invasion locus protein, bacteriocin immunity protein, and cell division protein. The information generated from the research should be useful in the control fo S. enterica in low-moisture foods and their production environments.

Development of RNA G-quadruplex (rG4)-targeting L-RNA aptamers by rG4-SELEX.

RNA G-quadruplex (rG4)-SELEX is a method that generates L-RNA aptamers to target an rG4 structure of interest, which can be applied to inhibit G-quadruplex-mediated interactions that have important roles in gene regulation and function. Here we present a Protocol Extension substantially modifying an existing SELEX protocol to describe in detail the procedures involved in performing rG4-SELEX to identify rG4-specific binders that can effectively suppress rG4-peptide and rG4-protein associations. This Protocol Extension improves the speed of aptamer discovery and identification, offering a suite of techniques to characterize the aptamer secondary structure and monitor binding affinity and specificity, and demonstrating the utility of the L-RNA aptamer. The previous protocol mainly describes the identification of RNA aptamers against proteins of interest, whereas in this Protocol Extension we present the development of an unnatural RNA aptamer against an RNA structure of interest, with the potential to be applicable to other nucleic acid motifs or biomolecules. rG4-SELEX starts with a random D-RNA library incubated with the L-rG4 target of interest, followed by binding, washing and elution of the library. Enriched D-aptamer candidates are sequenced and structurally characterized. Then, the L-aptamer is synthesized and used for different applications. rG4-SELEX can be carried out by an experienced molecular biologist with a basic understanding of nucleic acids. The development of rG4-targeting L-RNA aptamers expands the current rG4 toolkit to explore innovative rG4-related applications, and opens new doors to discovering novel rG4 biology in the near future. The duration of each selection cycle as outlined in the protocol is ~2 d.

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.

Biosensor-driven, model-based optimization of the orthogonally expressed naringenin biosynthesis pathway

BackgroundThe rapidly expanding synthetic biology toolbox allows engineers to develop smarter strategies to tackle the optimization of complex biosynthetic pathways. In such a strategy, multi-gene pathways are subdivided in several modules which are each dynamically controlled to fine-tune their expression in response to a changing cellular environment. To fine-tune separate modules without interference between modules or from the host regulatory machinery, a sigma factor (σ) toolbox was developed in previous work for tunable orthogonal gene expression. Here, this toolbox is implemented in E. coli to orthogonally express and fine-tune a pathway for the heterologous biosynthesis of the industrially relevant plant metabolite, naringenin. To optimize the production of this pathway, a practical workflow is still imperative to balance all steps of the pathway. This is tackled here by the biosensor-driven screening, subsequent genotyping of combinatorially engineered libraries and finally the training of three different computer models to predict the optimal pathway configuration.ResultsThe efficiency and knowledge gained through this workflow is demonstrated here by improving the naringenin production titer by 32% with respect to a random pathway library screen. Our best strain was cultured in a batch bioreactor experiment and was able to produce 286 mg/L naringenin from glycerol in approximately 26 h. This is the highest reported naringenin production titer in E. coli without the supplementation of pathway precursors to the medium or any precursor pathway engineering. In addition, valuable pathway configuration preferences were identified in the statistical learning process, such as specific enzyme variant preferences and significant correlations between promoter strength at specific steps in the pathway and titer.ConclusionsAn efficient strategy, powered by orthogonal expression, was applied to successfully optimize a biosynthetic pathway for microbial production of flavonoids in E. coli up to high, competitive levels. Within this strategy, statistical learning techniques were combined with combinatorial pathway optimization techniques and an in vivo high-throughput screening method to efficiently determine the optimal operon configuration of the pathway. This “pathway architecture designer” workflow can be applied for the fast and efficient development of new microbial cell factories for different types of molecules of interest while also providing additional insights into the underlying pathway characteristics.

Study on the inhibitory effect of selective estrogen receptor modulators on Streptococcus mutans.

To screen small-molecule antibacterial drugs and investigate the antibacterial effect and mechanism of selective estrogen receptor modulators (SERMs) against Streptococcus mutans (S.mutans). The minimum inhibitory concentration of 426 Food and Drug Administration (FDA)-approved small-molecule drugs against S. mutans was determined using the microdilution method, and the target of SERMs acting on S. mutans was explored by employing a random transposon mutant library. Among the 426 FDA-approved SERMs, toremiphene, tamoxifen, clomiphene, and raloxifene exhibited excellent antibacterial effects against S.mutans. Results of mutant library screening showed that the two mutant strains were resistant to clomiphene. The gene sequence of the resistant strains showed that the transposon insertion sites were located in the genes of smu_546 and smu_874. SERMs, such as toremifene, tamoxifen, clomiphene, and raloxifene, exerted obvious antibacterial effects on S. mutans, and their targets may be proteins expressed by smu_546 and smu_874 gene.

Improved Productivity of Astaxanthin from Photosensitive Haematococcus pluvialis Using Phototaxis Technology.

Haematococcus pluvialis is a microalgae actively studied for the production of natural astaxanthin, which is a powerful antioxidant for human application. However, it is economically disadvantageous for commercialization owing to the low productivity of astaxanthin. This study reports an effective screening strategy using the negative phototaxis of the H. pluvialis to attain the mutants having high astaxanthin production. A polydimethylsiloxane (PDMS)-based microfluidic device irradiated with a specific light was developed to efficiently figure out the phototactic response of H. pluvialis. The partial photosynthesis deficient (PP) mutant (negative control) showed a 0.78-fold decreased cellular response to blue light compared to the wild type, demonstrating the positive relationship between the photosynthetic efficiency and the phototaxis. Based on this relationship, the Haematococcus mutants showing photosensitivity to blue light were selected from the 10,000 random mutant libraries. The M1 strain attained from the phototaxis-based screening showed 1.17-fold improved growth rate and 1.26-fold increases in astaxanthin production (55.12 ± 4.12 mg g−1) in the 100 L photo-bioreactor compared to the wild type. This study provides an effective selection tool for industrial application of the H. pluvialis with improved astaxanthin productivity.

A Novel SELEX Based on Immobilizing Libraries Enables Screening of Saxitoxin Aptamers for BLI Aptasensor Applications.

Saxitoxin (STX) is one of the potent marine biotoxins that has high rate of lethality. However, there are no effective treatments at present, and the existing detection methods need to be further explored because of ethical problems or technical limitations. In this work, oligonucleotide aptamers toward STX were screened based on immobilizing libraries on Immobilized Metal-Chelate (IMC), such as Ni-NTA Sepharose, and the IMC-SELEX was conducted by the G-quadruplex library and the random library, respectively. Aptamer 45e (from the G-quadruplex library) and aptamer 75a were obtained after optimization, and aptamer 45e turned out to have a higher affinity toward STX. Furthermore, it was found that the hydrogen bonding and the van der Waals forces (VDW) played major roles in the high efficiency and specificity between STX and 45e by means of molecular docking and dynamics simulation. Based on this, aptamer 45e-1 with the Kd value of 19 nM was obtained by further optimization, which was then used to construct a simple, label-free and real-time optical BLI aptasensor for the detection of STX. This aptasensor showed good reproducibility and stability. In summary, with the advantages of screening aptamers of high efficiency and specificity toward the targets, the proposed IMC-SELEX provides a promising screening strategy for discovering aptamers, which could be used as the potential molecular recognition elements in the fields of biomedicine, food safety and environmental monitoring.