Fusion PCR Research Articles

An automated commercial open access assay for detection of Mycoplasma genitalium macrolide resistance.

Azithromycin, a macrolide antibioticum, is the first-line treatment for Mycoplasma genitalium (MG), but resistant MG is an increasing problem. Macrolide resistance-mediated mutations (MRM) has been linked to point mutations in region V of the MG 23S rRNA gene. We have evaluated an open access analyzer (Panther Fusion, Hologic Inc) for detectability of MRM (mutations A2071G and A2072G) and MG wild type (WT) in clinical samples. Also, the agreement of the Panther Fusion assay results with a corresponding established In-house MRM-WT PCR (ABI 7500) was calculated. Left over material from 55 clinical samples positive for MG by the Aptima test (Hologic) based on transcription-mediated amplification (TMA), collected from January to February 2023 in Region Skåne, Sweden, was analyzed. Specific amplification curves were generated for positive controls of MG mutations (A2071G and A2072G) and WT by the Panther Fusion assay. The limit of detection (LOD) was 5.3 copies/mL for WT, 8.1 copies/mL for mutation A2071G, and 81 copies/mL for mutation A2072G. The overall concordance was 91% between the Panther Fusion and the In-house PCR (Kappa 0.621, 95% CI; 0.327-0.914) for detection of WT or MRM in MG-positive clinical samples. The Panther Fusion detected MRM in 20% (11/55) and WT in 62% (34/55) of the samples. The corresponding In-house PCR results were 25% (14/55) and 65% (36/55). In summary, the Panther Fusion assay demonstrated detection of low copy number of MRM and WT of MG. Among clinical samples substantial agreement between the Panther Fusion and In-house PCR results was observed. Integrating MG-analysis (TMA) and MRM-WT assay on the Panther platform could make MRM testing more readily available. However, the Panther Fusion had a lower success rate (82% vs 90%) for macrolide susceptibility testing, hence testing with a complementary method should be considered for samples where neither WT nor MRM MG are detectable.

EP.06F.07 Comparison Between EGFR PCR Alone and NGS with Gene Fusion PCR for Detecting Oncogenic Driver Alteration in Thai NSCLC Patients

EP.06F.07 Comparison Between EGFR PCR Alone and NGS with Gene Fusion PCR for Detecting Oncogenic Driver Alteration in Thai NSCLC Patients

Abundant urinary amino acids activate glutamine synthetase-encoding glnA by two different mechanisms in Escherichia coli.

Growth of uropathogenic Escherichia coli in the bladder induces transcription of glnA which codes for the ammonia-assimilating glutamine synthetase (GS) despite the normally suppressive high ammonia concentration. We previously showed that the major urinary component, urea, induces transcription from the Crp-dependent glnAp1 promoter, but the urea-induced transcript is not translated. Our purpose here was to determine whether the most abundant urinary amino acids, which are known to inhibit GS activity in vitro, also affect glnA transcription in vivo. We found that the abundant amino acids impaired growth, which glutamine and glutamate reversed; this implies inhibition of GS activity. In strains with deletions of crp and glnG that force transcription from the glnAp2 and glnAp1 promoters, respectively, we examined growth and glnA transcription with a glnA-gfp transcriptional fusion and quantitative reverse transcription PCR with primers that can distinguish transcription from the two promoters. The abundant urinary amino acids stimulated transcription from the glnAp2 promoter in the absence of urea but from the glnAp1 promoter in the presence of urea. However, transcription from glnAp1 did not produce a translatable mRNA or GS as assessed by a glnA-gfp translational fusion, enzymatic assay of GS, and Western blot to detect GS antigen in urea-containing media. We discuss these results within the context of the extremely rapid growth of uropathogenic E. coli in urine, the different factors that control the two glnA promoters and possible mechanisms that either overcome or bypass the urea-imposed block of glutamine synthesis during bacterial growth in urine.IMPORTANCEKnowledge of the regulatory mechanisms for genes expressed at the site of infection provides insight into the virulence of pathogenic bacteria. During urinary tract infections-most often caused by Escherichia coli-growth in urine induces the glnA gene which codes for glutamine synthetase. The most abundant urinary amino acids amplified the effect of urea which resulted in hypertranscription from the glnAp1 promoter and, unexpectedly, an untranslated transcript. E. coli must overcome this block in glutamine synthesis during growth in urine, and the mechanism of glutamine acquisition or synthesis may suggest a possible therapy.

Clinically relevant antibiotic resistance genes are linked to a limited set of taxa within gut microbiome worldwide

The acquisition of antimicrobial resistance (AR) genes has rendered important pathogens nearly or fully unresponsive to antibiotics. It has been suggested that pathogens acquire AR traits from the gut microbiota, which collectively serve as a global reservoir for AR genes conferring resistance to all classes of antibiotics. However, only a subset of AR genes confers resistance to clinically relevant antibiotics, and, although these AR gene profiles are well-characterized for common pathogens, less is known about their taxonomic associations and transfer potential within diverse members of the gut microbiota. We examined a collection of 14,850 human metagenomes and 1666 environmental metagenomes from 33 countries, in addition to nearly 600,000 isolate genomes, to gain insight into the global prevalence and taxonomic range of clinically relevant AR genes. We find that several of the most concerning AR genes, such as those encoding the cephalosporinase CTX-M and carbapenemases KPC, IMP, NDM, and VIM, remain taxonomically restricted to Proteobacteria. Even cfiA, the most common carbapenemase gene within the human gut microbiome, remains tightly restricted to Bacteroides, despite being found on a mobilizable plasmid. We confirmed these findings in gut microbiome samples from India, Honduras, Pakistan, and Vietnam, using a high-sensitivity single-cell fusion PCR approach. Focusing on a set of genes encoding carbapenemases and cephalosporinases, thus far restricted to Bacteroides species, we find that few mutations are required for efficacy in a different phylum, raising the question of why these genes have not spread more widely. Overall, these data suggest that globally prevalent, clinically relevant AR genes have not yet established themselves across diverse commensal gut microbiota.

PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines.

Acute myeloid leukemia is a complex heterogeneous disease characterized by the clonal expansion of undifferentiated myeloid precursors. Due to the difficulty in the transfection of blood cells, several hematological models have recently been developed with CRISPR/Cas9, using viral vectors. In this study, we developed an alternative strategy in order to generate CRISPR constructs by fusion PCR, which any lab equipped with basic equipment can implement. Our PCR-generated constructs were easily introduced into hard-to-transfect leukemic cells, and their function was dually validated with the addition of MYBL2 and IDH2 genes into HEK293 cells. We then successfully modified the MYBL2 gene and introduced the R172 mutation into the IDH2 gene within NB4 and HL60 cells that constitutively expressed the Cas9 nuclease. The efficiency of mutation introduction with our methodology was similar to that of ribonucleoprotein strategies, and no off-target events were detected. Overall, our strategy represents a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction.

Virus-Induced Silencing of a Sequence Coding for Loricrin-like Protein in Phytophthora infestans upon Infection of a Recombinant Vector Based on Tobacco Mosaic Virus

Phytophthora infestans is the oomycete responsible for late blight disease of Solanaceae that causes both yield and economic losses. With the aim of reducing plant wilt and high management costs mainly due to wide fungicide applications, alternative eco-sustainable control strategies are needed. RNA interference (RNAi) is a powerful tool for gene function studies that can be accomplished by constitutive transformation or transient expression such as virus-induced gene silencing (VIGS) experiments. VIGS makes use of viruses to deliver sequences homologous to a target gene fragment and trigger RNAi. Indeed, a P. infestans ortholog of plant loricrin-like protein (LLP), named PiLLP, has been silenced using the direct infection of a recombinant vector based on the plant virus tobacco mosaic virus (TMV-PiLLP-1056), aiming to reduce the oomycete sexual reproduction. For this purpose, the gene coding for the green fluorescent protein (GFP) present in the TMV-GFP-1056 vector has been replaced with an antisense construct obtained by fusion PCR of the PiLLP 5′-UTR and 3′-UTR sequences. Here, we show that RNAi can be expressed in the A1 mating type of P. infestans strain 96.9.5.1 by VIGS using the direct infection of TMV-PiLLP-1056. We provide evidence that the recombinant vector can enter, replicate, and persist in mycelia of P. infestans where it induces the partial downregulation of the PiLLP transcript. Compared with the wild-type, the PiLLP-silenced A1 mating type had slower colony growth and a diminished virulence in detached tomato leaflets. This seems to be the first evidence of a constitutive gene downregulation of P. infestans using a recombinant vector based on a plus-sense RNA plant virus.

The Method of Screening and Identification of Transcription Factor in Klebsiella.

This protocol describes a method for verifying the specific transcription factor regulating glycerol dehydratase (GDH) expression in Klebsiella. DNA pull-down accompanied with mass spectrometry is used to screen and identify the transcription factor interacting with the promoter region of the key gene in Klebsiella. EMSA method is used to validate the specific binding of the transcription factor to the promoter region in vitro. In addition, the target DNA fragments are constructed by fusion PCR to prepare competent cells from Klebsiella for electrical transformation and further transformed to obtain key gene deletion strains to verify the transcription factor responsible for the target gene expression in Klebsiella.

Construction of crizotinib resistant models with CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutations to explore resistance mechanism and treatment strategy

Targeted therapy is an essential treatment for non-small cell lung cancer (NSCLC) that is always associated with the drug resistance. c-ros oncogene 1 (ROS1) gene point mutation is one of the leading factors causing drug resistance. However, the point mutation cell models of crizotinib are challenging to obtain, causing few reports on the drug resistance mechanism and the treatment strategy. We constructed CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant plasmids by fusion PCR technology and transfected them into A549 cells. Western blot and MTT assay proved that the drug-resistant cell lines were successfully transfected. The transwell assay confirmed that the mutant cells' motor abilities were significantly increased compared with the wild-type group. In addition, focal adhesion kinase (FAK) was significantly increased in mutant cells. Moreover, crizotinib resistance occurred in the mutant cells through the activation of FAK / phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) pathway. Next, crizotinib was combined with defactinib, a FAK inhibitor, to further explore its therapeutic effect. The results showed that the combination could significantly inhibit the proliferation, invasion and migration of mutant cells. In conclusion, we proved that CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant NSCLC cells were resistant to crizotinib through the activation of FAK/PI3K/AKT signaling pathway, and inhibiting FAK/PI3K/AKT signaling pathway activation by defactinib could overcome drug resistance in mutant cells.

Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence.

Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/FRT recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by FRT sites, and delivered the PCR products into R. solanacearum cells through natural transformation. Using this method, we knocked out the epsB and phcA genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several R. solanacearum strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/FRT recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of R. solanacearum pathogenic mechanisms.

Construction of recombinant adenovirus expressing capsid protein of serotype O foot-and-mouth disease virus and analysis of its immunogenicity

In order to construct a recombinant replication deficient human type 5 adenovirus (Ad5) expressing a foot-and-mouth disease virus (FMDV) capsid protein, specific primers for P12A and 3B3C genes of FMDV-OZK93 were synthesized. The P12A and 3B3C genes were then amplified and connected by fusion PCR, and a recombinant shuttle plasmid pDC316-mCMV-EGFP-P12A3B3C expressing the FMDV-OZK93 capsid protein precursor P12A and 3B3C protease were obtained by inserting the P12A3B3C gene into the pDC316-mCMV-EGFP plasmid. The recombinant adenovirus rAdv-P12A3B3C-OZK93 was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system, and the expression was verified by infecting human embryonic kidney cell HEK-293. The humoral and cellular immunity levels of well-expressed and purified recombinant adenovirus immunized mice were evaluated. The results showed that rAdv-P12A3B3C-OZK93 could be stably passaged and the maximum virus titer reached 1×109.1 TCID50/mL. Western blotting and indirect immunofluorescence showed that rAdv-P12A3B3C-OZK93 expressed the FMDV-specific proteins P12A and VP1 in HEK-293 cells. In addition, the PK cell infection experiment confirmed that rAdv-P12A3B3C-OZK93 could infect porcine cells, which is essential for vaccination in pigs. Comparing with the inactivated vaccine group, the recombinant adenovirus could induce higher FMDV-specific IgG antibodies, γ-IFN and IL-10. This indicates that the recombinant adenovirus has good immunity for animal, which is very important for the subsequent development of foot-and-mouth disease vaccine.

Comparative evaluation of Panther Fusion and real-time PCR for detection of Burkholderia pseudomallei in spiked human blood.

Introduction. Melioidosis is an infection that most commonly presents with bacteraemia. Culture-based laboratory methods can result in a significant delay to organism identification. Molecular diagnostic techniques have a high sensitivity and rapid time to diagnosis. A decreased time to diagnosis is likely to improve patient outcomes. Aim. To compare the Panther Fusion automated molecular instrument to an in-house method for the detection of Burkholderia pseudomallei directly from spiked human whole-blood samples. Results. The in-house method detected 11/12 (92 %) samples with a B. pseudomallei concentration of 2.5–4.5×102 c.f.u. ml−1. The Panther was less reliable, detecting only 8/14 (75 %) samples with a similar bacterial concentration. The Panther was able to detect 12/12 (100 %) spiked blood culture-positive samples. Conclusion. The direct detection of B. pseudomallei from patient blood on presentation to a healthcare facility will significantly decrease time to diagnosis. We describe an in-house real-time PCR method with the lowest reported limit of detection to date. Due to lower sensitivity, the Panther Fusion would be best used as a diagnostic method directly from a positive blood culture.

Avermectin B1a production in Streptomyces avermitilis is enhanced by engineering aveC and precursor supply genes.

Avermectins (AVEs) are economically potent anthelmintic agents produced by Streptomyces avermitilis. Among eight AVE components, B1a exhibits the highest insecticidal activity. The purpose of this study was to enhance B1a production, particularly in the high-yielding industrial strain A229, by a combination strategy involving the following steps. (i) aveC gene was engineered to increase B1a:B2a ratio. Three aveC variants (aveC2m, aveC5m, and aveC8m, respectively encoding two, five, and eight amino acid mutations) were synthesized by fusion PCR. B1a:B2a ratio in A229 derivative having kasOp*-controlled aveC8m reached 1.33 (B1a and B2a titers were 8120 and 6124μg/mL). Corresponding values in A229 were 0.99 and 6447 and 6480μg/mL. (ii) β-oxidation pathway genes fadD and fadAB were overexpressed in wild-type (WT) strain and A229 to increase supply of acyl-CoA precursors for AVE production. The resulting strains all showed increased B1a titer. Co-overexpression of pkn5p-driven fadD and fadAB in A229 led to B1a titer of 8537μg/mL. (iii) Genes bicA and ecaA involved in cyanobacterial CO2-concentrating mechanism (CCM) were introduced into WT and A229 to enhance carboxylation velocity of acetyl-CoA and propionyl-CoA carboxylases, leading to increased supply of malonyl- and methylmalonyl-CoA precursors and increased B1a titer. Co-expression of bicA and ecaA in A229 led to B1a titer of 8083μg/mL. (iv) aveC8m, fadD-fadAB, and bicA-ecaA were co-overexpressed in A229, resulting in maximal B1a titer (9613μg/mL; 49.1% increase relative to A229). Our findings demonstrate that the combination strategy we provided here is an efficient approach for improving B1a production in industrial strains.Key points• aveC mutation increased avermectin B1a:B2a ratio and B1a titer.• Higher levels of acyl-CoA precursors contributed to enhanced B1a production.• B1a titer in an industrial strain was increased by 49.1% via a combination strategy.

Genetic Transformation of Candida auris via Homology-Directed Repair Using a Standard Lithium Acetate Protocol.

Reverse genetics is a particularly powerful tool in non-model organisms with known whole-genome sequences enabling the characterization of gene and, thus, protein function via a mutant phenotype. Reverse genetic approaches require genetic manipulation techniques which often need to be specifically developed for non-model organisms; this can be fraught with difficulties. Here, we describe a genetic transformation protocol for the recently emerged human pathogen Candida auris to target the integration of DNA constructs into genomic locations via homology-directed repair using long flanking homologous sequences (>1kb). We detail the generation of DNA constructs for gene deletion with dominant drug resistance markers via fusion PCR, the transformation of these constructs into chemically competent C. auris cells, and the confirmation of correct integration by PCR. This strategy can be adapted to deliver DNA constructs other than deletion cassettes, including promoter exchanges and protein tags.

A Fusion PCR Method for Expressing Genetic Tools in C. elegans.

C. elegans offer a unique opportunity for understanding computation in neural networks. This is largely due to their relatively compact neural network for which a wiring diagram is available. Recent advances in genetic tools for interrogating neural activity (e.g., optogenetics) make C. elegans particularly compelling as they can be expressed in many different combinations in target individual neurons. Thus, the prospect to decipher principles underlying functionality in neural networks largely depends on the ease by which transgenic animals can be generated. Traditionally, to generate transgenic animals one would inject a plasmid containing the gene of interest under the regulation of the cell- or lineage-specific promoter. This often requires laborious cloning steps of both the gene and the promoter. The Hobert lab has developed a simpler protocol in which linear PCR fragments can be injected to generate transgenic animals. Relying on this PCR fusion-based method, here we provide a detailed protocol that we have optimized for expressing various genetically encoded calcium indicators and optogenetic tools in individual or sets of neurons. We use these simple procedures to generate multiple constructs within a very short time frame (typically 1-2days).

Linking plasmid-based beta-lactamases to their bacterial hosts using single-cell fusion PCR.

The horizonal transfer of plasmid-encoded genes allows bacteria to adapt to constantly shifting environmental pressures, bestowing functional advantages to their bacterial hosts such as antibiotic resistance, metal resistance, virulence factors, and polysaccharide utilization. However, common molecular methods such as short- and long-read sequencing of microbiomes cannot associate extrachromosomal plasmids with the genome of the host bacterium. Alternative methods to link plasmids to host bacteria are either laborious, expensive, or prone to contamination. Here we present the One-step Isolation and Lysis PCR (OIL-PCR) method, which molecularly links plasmid-encoded genes with the bacterial 16S rRNA gene via fusion PCR performed within an emulsion. After validating this method, we apply it to identify the bacterial hosts of three clinically relevant beta-lactamases within the gut microbiomes of neutropenic patients, as they are particularly vulnerable multidrug-resistant infections. We successfully detect the known association of a multi-drug resistant plasmid with Klebsiella pneumoniae, as well as the novel associations of two low-abundance genera, Romboutsia and Agathobacter. Further investigation with OIL-PCR confirmed that our detection of Romboutsia is due to its physical association with Klebsiella as opposed to directly harboring the beta-lactamase genes. Here we put forth a robust, accessible, and high-throughput platform for sensitively surveying the bacterial hosts of mobile genes, as well as detecting physical bacterial associations such as those occurring within biofilms and complex microbial communities.

Molecular Cloning of a New Bi-Functional Fusion Protein of Clostridium perfringens Type A Alpha and Clostridium septicum Alpha Toxin Genes in E. coli

Background: A synthetic construct bi-functional protein fusion includes two protein domains, or proteins bind by a fragment. The synthetic construct is designed to achieve better characterize and new functionality. Therefore, having proper cells is essential for cloning fusion genes. Clostridium perfringens type A produces the alpha-toxin and can cause gas gangrene and gastrointestinal diseases. C. septicum produces the alpha-toxin and can cause non-traumatic and traumatic gas gangrene. Objectives: The current study aimed to investigate molecular cloning of a new bi-functional fusion protein of C. perfringens alpha (cpa) and C. septicum alpha (csa) toxin genes in E. coli TOP10. In silico analysis was used for the chimeric fusion protein structural prediction. Methods: To produce chimeric fusion protein, the alpha-alpha (α-α) fusion gene was designed according to nucleotide sequences of cpa (KY584046.1) and csa (JN793989.2) genes. Tertiary structural prediction and validation of the fusion protein were determined by online software. In the new synthetic construction, α-α fusion protein genes are bind via the linker AEAAAKEAAAKA. The linker was introduced between the two domains by fusion PCR. The synthetic fusion gene was cloned into the pUC57cloning vector and then transferred into the host cell. Results: Analysis of the chimeric protein fusion is showed using the I-TASSER server as C-score equal to -2.68 as well as Rampage software in order to confirm the geometrical model as a natural like protein. Also, 1.0% agarose gel electrophoresis of fusion PCR product and sequencing analysis revealed a DNA fragment length of 2346 bp. Screening gel electrophoresis showed 996 bp length, which the designed linker was contained in it. Gel electrophoresis of extracted and purified recombinant plasmid (pUC57/αα) showed that a pUC57/αα of 5056 bp. The digested recombinant pUC57/αα showed one 2.3 kb (our fusion gene) band and one 2.7 kb (pUC57) band. Conclusions: This study presented a new approach for the fusion of cpa and csa genes based on the fusion PCR strategy. According to the latest information, this is the first time that α-α fusion gene is designed and cloned into a suitable cloning vector.

Construction and purification of ANK gene deleted recombinant goatpox virus.

Sheeppox virus (SPPV) and goatpox virus (GTPV) are two pathogens of host specificity. Previous studies have hypothesized that ankyrin (ANK) family may play an important role in determining host range of SPPV and GTPV. In order to verify the function of ANK proteins, it is critical to generate and purify the ANK gene deleted GTPV. In this study, the GFP gene as a reporter gene was connected with two homologous arms of ANK gene by fusion PCR. The ANK gene deleted transfer vectors were generated by inserting the PCR products into PET42b, and were transfected into testicular primary cells which were infected by GTPV. The rGTPV were identified as green fluorescence positive and properly purified. The results showed that GFP gene and two homologous arms of ANK gene were connected. The sequence was inserted in PET42b to form ANK deleted transfer vector. ANK deleted rGTPV was generated successfully by transferring vector and GTPV in cells. The ANK deleted rGTPV was purified and identified in this study. The study successfully generated the ANK deleted rGTPV. It overcomes the technical barrier for future studies about the function of ANK genes.

Greatly enhancing the enantioselectivity of PvEH2, a Phaseolus vulgaris epoxide hydrolase, towards racemic 1,2-epoxyhexane via replacing its partial cap-loop

To achieve the kinetic resolution and enantioconvergent hydrolysis of rac-1,2-epoxyhexane, the E value of PvEH2 was enhanced by substituting its partial cap-loop. Based on the experimental results reported previously and computer-aided analysis, the flexible and variable cap-loop, especially its middle segment, was speculated to be related to the catalytic properties of PvEH2. In view of this, four PvEH2's hybrids, Pv2St, Pv2Pv1, Pv2Vr1 and Pv2Vr2, were designed by substituting the middle segment (190EGMGSNLNTSMP201) of a cap-loop in PvEH2 with the corresponding ones in StEH, PvEH1, VrEH1 and VrEH2, respectively. Then, the hybrid-encoding genes, pv2st, pv2pv1, pv2vr1 and pv2vr2, were constructed by fusion PCR, and expressed in E. coli Rosetta(DE3). The expressed hybrid, Pv2St, displayed the highest specific activity of 35.3 U/mg protein towards rac-1,2-epoxyhexane. The corresponding transformant, E. coli/pv2st, exhibited the largest E value of 24.2, which was 11.5-fold that of E. coli/pveh2 expressing PvEH2. The scale-up kinetic resolution of 280 mM rac-1,2-epoxyhexane was carried out using 40 mg dry cells/mL of E. coli/pv2st at 25 °C for 4.5 h, retaining (S)-1,2-epoxyhexane with >99.5% ees and 36.9% yield. Additionally, the chemo-enzymatic enantioconvergent hydrolysis of rac-1,2-epoxyhexane using E. coli/pv2st followed by sulfuric acid produced (R)-hexane-1,2-diol with 73.0% eep and 86.5% yield.

A high efficiency cloning approach of multi-points combinational mutagenesis

The combination of high-quality mutagenesis and effective screening can improve the efficiency of enzyme directed evolution. In this study, a high efficiency cloning construction method of Multi-points Combinational Mutagenesis (MCM) was developed. Efficient multi-point combination mutations were performed in this MCM method by introducing DNA assembly, fusion PCR and hybridization techniques. After optimization, the efficiency of MCM was tested by directed evolution of benzoylformate decarboxylase. The obtained number of Colony Forming Units (CFUs) by electroporation to competent cells E. coli Trelief™ 5α exceeded 10⁶ CFUs/μg DNA. Test results show that 90/100 clones were precisely assembled. The efficiency of simultaneous mutation at 5 sites (L109, L110, H281, Q282 and A460) was up to 88%. Finally, a mutant enzyme (L109Y, L110D, H281G, Q282V and A460M) with a 10-fold increase in kcat/Km was obtained. Therefore, this method can effectively create diverse mutant libraries and promote the rapid development of enzyme directed evolution.

First Report of Alfalfa Leaf Curl Virus Infecting Alfalfa (Medicago sativa) in China

Alfalfa (Medicago sativa L.) is one of the most important leguminous forage crops globally and has grown in importance in China with ∼4.72 million hectares in 2015. A previous study reported the occurrence of several common viruses (e.g., alfalfa mosaic virus [AMV], white clover mosaic virus, bean yellow mosaic virus, and cowpea mosaic virus) in alfalfa fields in China, in Anning District and Gaolan County of Lanzhou, and Jingtai County of Baiyin City, Gansu Province (Zhou et al. 2016). In April 2017 a survey was conducted to identify viruses infecting alfalfa in a temperate region: Yuanyang County, Henan Province (N35°01′, E113°43′). From one field, 117 alfalfa leaf samples with symptoms of macular mosaic (n = 26), mottle mosaic (21), etiolation (1), shrinkage (27), mosaic shrinkage (25), and dwarfism (17) were collected. Total RNA was extracted from a composite sample comprising six leaves exhibiting each above-mentioned symptom using an EASYspin Plant Micro RNA Rapid Extraction Kit (Aidlab Biotechnologies, Beijing, China) following the manufacturer’s guidelines. The RNA was then subjected to high-throughput sequencing of small RNA to detect any viruses using the Illumina Hiseq4000 platform at Biomarker Technologies (Beijing, China). Over 20 million clean reads between 18 and 35 nt were obtained. Using Velvet 1.0 software (Zerbino and Birney 2008), 6,942 contigs from 33 to 416 nt were assembled. Although the majority of the contigs were mapped to the host genome, 58, 13, and 10 contigs were mapped to AMV (Alfamovirus, Bromoviridae, reference sequences [RSs] AMV-RNA1 NC_001495, AMV-RNA2 NC_002024, and AMV-RNA3 NC_002025), alfalfa dwarf virus (ADV, Rhabdovirus, Rhabdoviridae, RS KP205452), and alfalfa leaf curl virus (ALCV, Capulavirus, Geminiviridae, RS KX574859), respectively. The 10 projected ALCV contigs totaled 1,113 nt, accounting for 40.47% of the complete ALCV RS of 2,750 nt. The nucleotide sequence identity between these contigs and the ALCV RS ranged from 96 to 100%. Total DNA was then extracted from the composite sample using a New Plant Genomic DNA Rapid Extraction Kit (Aidlab Biotechnologies) following the manufacturer’s guidelines. The presence of ALCV in the pooled sample was confirmed by PCR using an ALCV-specific primer pair, ALCV-1F 5′-TGGAATATTGTGCTGCTTGG-3′ and ALCV-1R 5′-ATTTTGGGACTTGTGCTCCA-3′ (Roumagnac et al. 2015), and subsequent cloning and Sanger sequencing. The 785-nt-long amplicon, deposited in GenBank as MK422439, exhibited a sequence identity of 96% to the ALCV RS. To further confirm the result, the full genome of ALCV in the pooled sample was amplified by fusion PCR using improved primer pair ALCV-F 5′-CCCTGGCCTGCTAAAGTGGCCCAATTCAACATGG-3′ and ALCV-R 5′-CCAGGGGGCCTTATTCCTCTGGGACCG-3′ adapted from Bernardo et al. (2016), cloned, and sequenced. The complete 2,750-bp sequence was deposited in GenBank as MK422438. BLASTn analysis indicated the sequence shared 90 and 98% identity with previously reported ALCV isolates from France (KT214370) (Roumagnac et al. 2015) and Argentina (MG792039) (Bejerman et al. 2018), respectively, thus confirming the ALCV identity of the virus. To reveal whether ALCV was associated with the symptoms, all samples were subjected to PCR with the primer pair ALCV-1F/ALCV-1R. ALCV was detected in 38 of 117 samples: in 8 of 26 samples with macular mosaic, 5 of 21 with mottle mosaic, 11 of 27 with shrinkage, 8 of 25 with mosaic shrinkage, and 6 of 17 with dwarfism, but not in the single plant showing etiolation. Interestingly, AMV was detected in the pooled sample as well as all 117 individual samples by RT-PCR, whereas ADV failed to be detected by RT-PCR in any of the samples. These results demonstrated that ALCV was present in alfalfa in China, and it often coinfected alfalfa plants with AMV. This is the first report of ALCV infecting alfalfa in China. The distribution of ALCV within alfalfa-growing regions, the identity of potential virus reservoirs, and the economic impact have yet to be determined. The results help expand understanding of this virus and help in selection of virus-free propagation material for resistant breeding of alfalfa and production of high-quality forage.