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Acetaldehyde is an important carbonyl compound commonly detected in wines. A high concentration of acetaldehyde can affect the flavor of wines and result in adverse effects on human health. Alcohol dehydrogenase I (ADH1) in Saccharomyces cerevisiae catalyzes the reduction reaction of acetaldehyde into ethanol in the presence of cofactors, showing the potential to reduce the content of acetaldehyde in wines. In this study, ADH1 was successfully expressed in Pichia pastoris GS115 based on codon optimization. Then, the expression level of ADH1 was enhanced by replacing its promoter with optimized promoters and increasing the copy number of the expression cassette, with ADH1 being purified using nickel column affinity chromatography. The enzymatic activity of purified ADH1 reached 605.44 ± 44.30 U/mg. The results of the effect of ADH1 on the content of acetaldehyde in wine revealed that the acetaldehyde content of wine samples was reduced from 168.05 ± 0.55 to 113.17 ± 6.08 mg/L with the addition of 5 mM NADH and the catalysis of ADH1, and from 135.53 ± 4.08 to 52.89 ± 2.20 mg/L through cofactor regeneration. Our study provides a novel approach to reducing the content of acetaldehyde in wines through enzymatic catalysis. [ABSTRACT FROM AUTHOR]

Acetaldehyde is an important carbonyl compound commonly detected in wines. A high concentration of acetaldehyde can affect the flavor of wines and result in adverse effects on human health. Alcohol dehydrogenase I (ADH1) in Saccharomyces cerevisiae catalyzes the reduction reaction of acetaldehyde into ethanol in the presence of cofactors, showing the potential to reduce the content of acetaldehyde in wines. In this study, ADH1 was successfully expressed in Pichia pastoris GS115 based on codon optimization. Then, the expression level of ADH1 was enhanced by replacing its promoter with optimized promoters and increasing the copy number of the expression cassette, with ADH1 being purified using nickel column affinity chromatography. The enzymatic activity of purified ADH1 reached 605.44 ± 44.30 U/mg. The results of the effect of ADH1 on the content of acetaldehyde in wine revealed that the acetaldehyde content of wine samples was reduced from 168.05 ± 0.55 to 113.17 ± 6.08 mg/L with the addition of 5 mM NADH and the catalysis of ADH1, and from 135.53 ± 4.08 to 52.89 ± 2.20 mg/L through cofactor regeneration. Our study provides a novel approach to reducing the content of acetaldehyde in wines through enzymatic catalysis.

Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from Aspergillus pseudotamarii. pAsPs was for the first time integrated in the genome of yeast strain Komagataella phaffii T07, and then produced in a 5 L bioreactor with an enzyme yield of 150,800 U/mL of culture liquid towards casein. The specific activity of the pAsPs was 7,657,000 U/mg toward casein, 2320 U/mg toward hemoglobin, and 25,344 U/mg toward azocasein per 1 mg of the protein. The enzyme was found to be inhibited by Cu2+. Optimal activity pH was shown in the range of pH 6.5–8.0, and optimal temperature—50–60 °C. The molecular mass of the recombinant protease pAsPs was shown to be 67.5 kDa. Mass-spectrometric analysis confirmed the identity of the amino acid sequence of the obtained pAsPs preparation with the predicted sequence, with 17% coverage and protein score 288. Thus, the novel neutral protease pAsPs is a promising candidate for large-scale use in manufacturing, including the food industry. [ABSTRACT FROM AUTHOR]

Structural rearrangements like copy number variations in the male-specific Y chromosome have been associated with male fertility phenotypes in human and mouse but have been sparsely studied in other mammalian species. Here, we designed digital droplet PCR assays for 7 horse male-specific Y chromosome multicopy genes and SRY and evaluated their absolute copy numbers in 209 normal male horses of 22 breeds, 73 XY horses with disorders of sex development and/or infertility, 5 Przewalski's horses and 2 kulans. This established baseline copy number for these genes in horses. The TSPY gene showed the highest copy number and was the most copy number variable between individuals and breeds. SRY was a single-copy gene in most horses but had 2-3 copies in some indigenous breeds. Since SRY is flanked by 2 copies of RBMY, their copy number variations were interrelated and may lead to SRY-negative XY disorders of sex development. The Przewalski's horse and kulan had 1 copy of SRY and RBMY. TSPY and ETSTY2 showed significant copy number variations between cryptorchid and normal males (P <0.05). No significant copy number variations were observed in subfertile/infertile males. Notably, copy number of TSPY and ETSTY5 differed between successive male generations and between cloned horses, indicating germline and somatic mechanisms for copy number variations. We observed no correlation between male-specific Y chromosome gene copy number variations and male-specific Y chromosome haplotypes. We conclude that the ampliconic male-specific Y chromosome reference assembly has deficiencies and further studies with an improved male-specific Y chromosome assembly are needed to determine selective constraints over horse male-specific Y chromosome gene copy number and their relation to stallion reproduction and male biology. [ABSTRACT FROM AUTHOR]

The successful treatment of Lyme disease (LD) is contingent on accurate diagnosis. However, current laboratory detection assays lack sensitivity in the early stages of the disease. Because delayed diagnosis of LD incurs high healthcare costs and great suffering, new highly sensitive tests are in need. To overcome these challenges, we developed an internally controlled quantitative PCR (Ter-qPCR) that targets the multicopy terminase large subunit (terL) gene encoded by prophages that are only found in LD-causing bacteria. The terL protein helps phages pack their DNA. Strikingly, the detection limit of the Ter-qPCR was analytically estimated to be 22 copies and one bacterial cell in bacteria spiked blood. Furthermore, significant quantitative differences was observed in terms of the amount of terL detected in healthy individuals and patients with either early or late disease. Together, the data suggests that the prophage-targeting PCR has significant power to improve success detection for LD. After rigorous clinical validation, this new test could deliver a step-change in the detection of LD. Prophage encoded markers are prevalent in many other pathogenic bacteria rendering this approach highly applicable to bacterial identification in general.

The successful treatment of Lyme disease (LD) is contingent on accurate diagnosis. However, current laboratory detection assays lack sensitivity in the early stages of the disease. Because delayed diagnosis of LD incurs high healthcare costs and great suffering, new highly sensitive tests are in need. To overcome these challenges, we developed an internally controlled quantitative PCR (Ter-qPCR) that targets the multicopy terminase large subunit (terL) gene encoded by prophages that are only found in LD-causing bacteria. The terL protein helps phages pack their DNA. Strikingly, the detection limit of the Ter-qPCR was analytically estimated to be 22 copies and one bacterial cell in bacteria spiked blood. Furthermore, significant quantitative differences was observed in terms of the amount of terL detected in healthy individuals and patients with either early or late disease. Together, the data suggests that the prophage-targeting PCR has significant power to improve success detection for LD. After rigorous clinical validation, this new test could deliver a step-change in the detection of LD. Prophage encoded markers are prevalent in many other pathogenic bacteria rendering this approach highly applicable to bacterial identification in general. [ABSTRACT FROM AUTHOR]

We present SymPortal (SymPortal.org), a novel analytical framework and platform for genetically resolving the algal symbionts of reef corals using next‐generation sequencing (NGS) data of the ITS2 rDNA. Although the ITS2 marker is widely used to genetically characterize taxa within the family Symbiodiniaceae (formerly the genus Symbiodinium), the multicopy nature of the marker complicates its use. Commonly, the intragenomic diversity resultant from this multicopy nature is collapsed by analytical approaches, thereby focusing on only the most abundant sequences. In contrast, SymPortal employs logic to identify within‐sample informative intragenomic sequences, which we have termed 'defining intragenomic variants' (DIVs), to identify ITS2‐type profiles representative of putative Symbiodiniaceae taxa. By making use of this intragenomic ITS2 diversity, SymPortal is able to resolve genetic delineations using the ITS2 marker at a level that was previously only possible by using additional genetic markers. We demonstrate this by comparing this novel approach to the most commonly used alternative approach for NGS ITS2 data, the 97% similarity clustering to operational taxonomic units (OTUs). The SymPortal platform accepts NGS raw sequencing data as input to provide an easy‐to‐use, standardization‐enforced, and community‐driven framework that integrates with a database to gain resolving power with increased use. We consider that SymPortal, in conjunction with ongoing large‐scale sampling and sequencing efforts, should play an instrumental role in making future sampling efforts more comparable and in maximizing their efficacy in working towards the classification of the global Symbiodiniaceae diversity. [ABSTRACT FROM AUTHOR]

Methylobacterium extorquens AM1 is the model strain for methylotrophic bacteria that metabolize methanol as the sole carbon and energy source. Genetically modified M. extorquens AM1 is used as a methylotrophic cell factory (MeCF) for high value-added chemical production. We tested the Cre-loxP recombination system for its ability to mediate multicopy gene integration of the mvt3 operon (mvt3) in M. extorquens AM1. mvt3 controls the expression of the first three enzymes of the mevalonate synthesis pathway. We assayed for Cre-mediated multigene integration by screening for multicopy mutants via their survival in culture with a high kanamycin concentration (600 μg/mL). We identified mutant strains in which the mevalonate titer was increased by up to 1.9-fold compared with M2 (M. extorquens AM1ΔcelABCΔattTn7::mvt3::loxP) and confirmed mvt3 integration at 2-3 copies per genome. This result demonstrates the feasibility of multicopy integration in M. extorquens AM1 mediated by Cre-loxP recombination and its potential for improving the output of M. extorquens AM1 metabolic pathways, e.g., optimization of terpenoid synthesis. [ABSTRACT FROM AUTHOR]

Hemophilia A (HA) is a monogenic disease due to lack of the clotting factor VIII (FVIII). This deficiency may lead to spontaneous joint hemorrhages or life-threatening bleeding but there is no cure for HA until very recently. In this study, we derived induced pluripotent stem cells (iPSCs) from patients with severe HA and used transcription activator-like effector nickases (TALENickases) to target the factor VIII gene ( F8 ) at the multicopy ribosomal DNA (rDNA) locus in HA-iPSCs, aiming to rescue the shortage of FVIII protein. The results revealed that more than one copy of the exogenous F8 could be integrated into the rDNA locus. Importantly, we detected exogenous F8 mRNA and FVIII protein in targeted HA-iPSCs. After they were differentiated into endothelial cells (ECs), the exogenous FVIII protein was still detectable. Thus, it is showed that the multicopy rDNA locus could be utilized as an effective target site in patient-derived iPSCs for gene therapy. This strategy provides a novel iPSCs-based therapeutic option for HA and other monogenic diseases. [ABSTRACT FROM AUTHOR]

To understand the expansion of multicopy microRNA (miRNA) families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miR160 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.Cell Research (2006) 16: 507–518. doi:10.1038/sj.cr.7310062; published online 15 May 2006 [ABSTRACT FROM AUTHOR]

A series of isoforms of α-KT × 14 (short chain potassium channel scorpion toxins) were isolated from the venom of Buthus martensii Karsch by RACE and screening cDNA library methods. These isoforms adding BmKK1 – 3 and BmSKTx1 – 2 together shared high homology (more than 97%) with each other. The result of genomic sequence analysis showed that a length 79bp intron is inserted Ala codes between the first and the second base at the 17th amino acid of signal peptide. The introns of these isoforms also share high homology with those of BmKK2 and BmSKT × 1 reported previously. Sequence analysis of many clones of cDNA and genomic DNA showed that a species population or individual polymorphism of α-KT × 14 genes took place in scorpion Buthus martensii Karsch and accelerated evolution played an important role in the forming process of α-KT × 14 scorpion toxins subfamily. The result of southern hybridization indicated that α-KT × 14 toxin genes existed in scorpion chromosome with multicopies. All findings maybe provided an important evidence for an extensive evolutionary process of the scorpion “pharmacological factory”: at the early course of evolution, the ancestor toxic gene duplicated into a series of multicopy genes integrated at the different chromosome; at the late course of evolution, subsequent functional divergence of duplicate genes was generated by mutations, deletions and insertion. IUBMB Life, 57: 513–521, 2005 [ABSTRACT FROM AUTHOR]

Controlling the copy number of gene expression cassettes is an important strategy to engineer bacterial cells into high-efficiency biocatalysts. Current strategies mostly use plasmid vectors, but multicopy plasmids are often genetically unstable, and their copy numbers cannot be precisely controlled. The integration of expression cassettes into a bacterial chromosome has advantages, but iterative integration is laborious, and it is challenging to obtain a library with varied gene doses for phenotype characterization. Here, we demonstrated that multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) can be achieved by designing a crRNA to target multicopy loci or a crRNA array to target multiple loci in the Escherichia coli genome. Within 5 days without selection pressure, E. coli strains carrying cargos with successively increasing copy numbers (up to 10) were obtained. Recombinant MUCICAT E. coli containing genomic multicopy glucose dehydrogenase expression cassettes showed 2.6-fold increased expression of this important industrial enzyme compared to E. coli harboring the conventional protein-expressing plasmid pET24a. Successful extension of MUCICAT to Tatumella citrea further demonstrated that MUCICAT may be generally applied to many bacterial species.

Sulbactam is a plausible option for treating Acinetobacter infections because of its intrinsic antibacterial activity against the members of the Acinetobacter genus, but the mechanisms of sulbactam resistance have not been fully studied in Acinetobacter baumannii In this study, a total of 2,197 clinical A. baumannii isolates were collected from 27 provinces in China. Eighty-eight isolates with various MICs for sulbactam were selected on the basis of their diverse clonality and underwent multilocus sequence typing (MLST), antimicrobial susceptibility testing, and resistance gene screening. The copy number and relative expression of bla TEM-1D were measured via quantitative PCR and quantitative reverse transcription-PCR, respectively. The genetic structure of multicopy ampC were measured via quantitative PCR and quantitative reverse transcription-PCR, respectively. The genetic structure of multicopy bla TEM-1D was determined using the whole-genome sequencing technology. The cefoperazone-sulbactam resistance rate of the 2,197 isolates was 39.7%. The rate of positivity for bla TEM-1D or IS Aba1-ampC in the sulbactam-nonsusceptible group (64.91% and 78.95%, respectively) was significantly higher than that in the sulbactam-susceptible group (0% and 0%, respectively; P Notably, the genetic structure of the multicopy P < 0.001) varied considerably between the groups expressing ampC with or without upstream IS Aba1 Notably, the genetic structure of the multicopy bla TEM-1D gene in strain ZS3 revealed that bla TEM-1D was embedded within four tandem copies of the cassette IS 26-bla TEM-1D -Tn 3 -IS 26 Therefore, bla TEM-1D and IS Aba1-ampC represent the prevalent mechanism underlying sulbactam resistance in clinical A. baumannii isolates in China. The structure of the four tandem copies of bla TEM-1D first identified may increase sulbactam resistance., (Copyright © 2018 American Society for Microbiology.)