Efficient Conversion of Stevioside to Rebaudioside M in Saccharomyces cerevisiae by a Engineering Hydrolase System and Prolonging the Growth Cycle.

Rebaudioside (Reb) M is an important sweetener with high sweetness, but its low content in Stevia rebaudiana and low catalytic capacity of the glycosyltransferases in heterologous microorganisms limit its production. In order to improve the catalytic efficiency of the conversion of stevioside to Reb M by Saccharomyces cerevisiae, several key issues must be resolved including knocking out endogenous hydrolases, enhancing glycosylation, and extending the enzyme catalytic process. Herein, endogenous glycosyl hydrolase SCW2 was knocked out in S. cerevisiae. The glycosylation process was enhanced by screening glycosyltransferases, and UGT91D2 from S. rebaudiana was identified as the optimum glycosyltransferase. The UDP-glucose supply was enhanced by overexpressing UGP1, and co-expressing UGT91D2 and UGT76G1 achieved efficient conversion of stevioside to Reb M. In order to extend the catalytic process, the silencing information regulator 2 (SIR2) which can prolong the growth cycle of S. cerevisiae was introduced. Finally, combining these modifications produced 12.5 g/L Reb M and the yield reached 77.9% in a 5 L bioreactor with 10.0 g/L stevioside, the highest titer from steviol glycosides to Reb M reported to date. The engineered strain could facilitate the industrial production of Reb M, and the strategies provide references for the production of steviol glycosides.

Genome-wide analysis of ATP-binding cassette (ABC) transporter in Penaeus vannamei and identification of two ABC genes involved in immune defense against Vibrio parahaemolyticus by affecting NF-κB signaling pathway.

The ATP-binding cassette (ABC) transporters have crucial roles in various biological processes such as growth, development and immune defense in eukaryotes. However, the roles of ABC transporters in the immune system of crustaceans remain elusive. In this study, 38 ABC genes were systematically identified and characterized in Penaeus vannamei. Bioinformation analysis revealed that PvABC genes were categorized into ABC A-H eight subfamilies with 17 full-transporters, 11 half transporters and 10 soluble proteins, and multiple immunity-related cis-elements were found in gene promoter regions. Expression analysis showed that most PvABC genes were widely and highly expressed in immune-related tissues and responded to the stimulation of Vibrio parahaemolyticus. To investigate whether PvABC genes mediated innate immunity, PvABCC5, PvABCF1 and PvABCB4 were selected for dsRNA interference experiment. Knockdown of PvABCF1 and PvABCC5 not PvABCB4 increased the cumulative mortality of P. vannamei and bacterial loads in hepatopancreas after infection with V. parahaemolyticus. Further analysis showed that the PvABCF1 and PvABCC5 knockdown decreased expression levels of NF-κB pathway genes and antimicrobial peptides (AMPs). Collectively, these findings indicated that PvABCF1 and PvABCC5 might restrict V. parahaemolyticus challenge by positively regulating NF-κB pathway and then promoting the expression of AMPs, which would contribute to overall understand the function of ABC genes in innate immunity of invertebrates.

A Kind of (t, n) Threshold Quantum Secret Sharing with Identity Authentication.

Quantum secret sharing (QSS) is an important branch of quantum cryptography. Identity authentication is a significant means to achieve information protection, which can effectively confirm the identity information of both communication parties. Due to the importance of information security, more and more communications require identity authentication. We propose a d-level (t,n) threshold QSS scheme in which both sides of the communication use mutually unbiased bases for mutual identity authentication. In the secret recovery phase, the sharing of secrets that only the participant holds will not be disclosed or transmitted. Therefore, external eavesdroppers will not get any information about secrets at this phase. This protocol is more secure, effective, and practical. Security analysis shows that this scheme can effectively resist intercept-resend attacks, entangle-measure attacks, collusion attacks, and forgery attacks.

The mechanism of Megalobrama amblycephala muscle injury repair based on RNA-seq.

Skeletal muscle myogenesis and injury-induced muscle regeneration contribute to muscle formation. Skeletal muscle stem cells, termed satellite cells (SCs), proliferate to repair injured muscle. To identify the molecular mechanism of regeneration after muscle injury as well as the genes related to muscle development in fish, in this study, the immunohistochemistry and the high-throughput RNA sequencing (RNA-seq) analysis were performed after Megalobrama amblycephala muscle was injured by needle stab. The results showed that paired box7-positive (Pax7+) SCs increased, and peaked at 96 to 144 h-post injury (hpi). The 6729 differentially expressed genes (DEGs), including 2125 up-regulated and 4604 down-regulated genes were found. GO terms significantly enriched by DEGs contained intercellular connections, signaling transduction and enzyme activity. KEGG enrichment analysis showed that most of the pathways were related to immunity, metabolism and cells related molecules, including actin skeleton regulation, Epstein Barr virus infection and plaque adhesion. The WGCNA results revealed that actin cytoskeleton and lipid metabolism related genes probably played crucial roles during repair after muscle injury. Collectively, all these results will provide new insights into the molecular mechanisms underlying muscle injury repair of fish.

Transcriptomic analysis of gills in nitrite-tolerant and -sensitive families of Litopenaeus vannamei.

Nitrite stress is a major environmental factor that limits aquatic animal growth, reproduction and survival. Even so, some shrimps still can withstand somewhat high concentrations of nitrite environment. However, few studies have been conducted about the tolerance molecular mechanism of Litopenaeus vannamei in the high concentration nitrite. To identify the genes and pathways involved in the regulation of nitrite tolerance, we performed comparative transcriptomic analysis in the L. vannamei nitrite-tolerant (NT) and nitrite-sensitive (NS) families, and untreated shrimps were used as the control group. After 24 h of nitrite exposure (NaNO2, 112.5 mg/L), a total of 1521 and 868 differentially expressed genes (DEGs) were obtained from NT compared with NS and control group, respectively. Functional enrichment analysis revealed that most of these DEGs were involved in immune defense, energy metabolism processes and endoplasmic reticulum (ER) stress. During nitrite stress, energy metabolism in NT was significantly enhanced by activating the related genes expression of oxidative phosphorylation (OXPHOS) pathway and tricarboxylic acid (TCA) cycle. Meanwhile, some DEGs involved in innate immunity- related genes and pathways, and ER stress responses also were highly expressed in NT. Therefore, we speculate that accelerated energy metabolism, higher expression of immunity and ER related genes might be the important adaptive strategies for NT in relative to NS under nitrite stress. These results will provide new insights on the potential tolerant molecular mechanisms and the breeding of new varieties of nitrite tolerant L. vannamei.

Full-length transcriptome of Misgurnus anguillicaudatus provides insights into evolution of genus Misgurnus.

Reconstruction and annotation of transcripts, particularly for a species without reference genome, plays a critical role in gene discovery, investigation of genomic signatures, and genome annotation in the pre-genomic era. This study generated 33,330 full-length transcripts of diploid M. anguillicaudatus using PacBio SMRT Sequencing. A total of 6,918 gene families were identified with two or more isoforms, and 26,683 complete ORFs with an average length of 1,497 bp were detected. Totally, 1,208 high-confidence lncRNAs were identified, and most of these appeared to be precursor transcripts of miRNAs or snoRNAs. Phylogenetic tree of the Misgurnus species was inferred based on the 1,905 single copy orthologous genes. The tetraploid and diploid M. anguillicaudatus grouped into a clade, and M. bipartitus showed a closer relationship with the M. anguillicaudatus. The overall evolutionary rates of tetraploid M. anguillicaudatus were significantly higher than those of other Misgurnus species. Meanwhile, 28 positively selected genes were identified in M. anguillicaudatus clade. These positively selected genes may play critical roles in the adaptation to various habitat environments for M. anguillicaudatus. This study could facilitate further exploration of the genomic signatures of M. anguillicaudatus and provide potential insights into unveiling the evolutionary history of tetraploid loach.

Genome-wide analysis of poplar SAUR gene family and expression profiles under cold, polyethylene glycol and indole-3-acetic acid treatments.

Small auxin-up RNA (SAUR) proteins play an important role in the regulation of plant growth and development. Here, we identified 105 SAUR genes and comprehensively analyzed them in Populus trichocarpa. Based on the phylogenetic relationships, the PtSAURs were classified into ten subfamilies. Of the 105 PtSAURs, 100 were randomly distributed along the nineteen chromosomes, while the remaining genes were located along unassigned scafoolds. These genes mainly evolved through segmental duplications. In total, 94 PtSAURs contained no introns, and each group had a similar conserved motif structure. A promoter analysis revealed various cis-elements related to growth, development and stress responses, and a synteny analysis established orthologous relationships among SAURs in Arabidopsis, rice, grape and poplar. The qRT-PCR and tissue expression analyses indicated that PtSAURs show different expression levels in various tissues in response to different treatments. PtSAUR53 was located on the nuclear and plasma membrane by conducting subcellular localization analysis. This study provides a comprehensive overview of poplar SAUR proteins and a foundation for further investigations for functional analysis of SAURs in poplar growth and development. At the same time, it will be valuable to further study the poplar SAUR genes to reveal their biological effects.

Genome-wide identification, classification, and expression of phytocyanins in Populus trichocarpa.

MAIN CONCLUSION: 74 phytocyanin genes were identified in the Populus trichocarpa genome. Phylogenetic analysis grouped the PC proteins into four subfamilies (UCs, PLCs, SCs, and ENODLs). Closely related PC proteins share similar motifs, implying similar functions. Expression profiles of PtPC genes were analyzed in response to drought and salt-stress. Phytocyanins (PCs) are blue copper proteins associated with electron carrier activity that have a large influence on plant growth and resistance. The majority of PCs are chimeric arabinogalactan proteins (AGPs). In this work, we identified 74 PC genes in Populus trichocarpa and analyzed them comprehensively. Based on the ligands composition of copper-binding sites, glycosylation state, the domain structure and spectral characteristics of PC genes, PCs were divided into four subfamilies [uclacyanins (UCs), plantacyanins (PLCs), stellacyanins (SCs) and early nodulin-like proteins (ENODLs)], and phylogenetic relationship analysis classified them into seven groups. All PtPCs are randomly distributed on 17 of the 19 poplar chromosomes, and they appear to have undergone expansion via segmental duplication. Eight PtPCs do not contain introns, and each group has a similar conserved motif structure. Promoter analysis revealed cis-elements related to growth, development and stress responses, and established orthology relationships of PCs between Arabidopsis and poplar by synteny analysis. Expression profile analysis and qRT-PCR analysis showed that PtPCs were expressed widely in various tissues. Quantitative real-time RT-PCR analysis of PC genes expression in response to salt and drought stress revealed their stress-responses profiles. This work provides a theoretical basis for a further study of stress resistance mechanisms and the function of PC genes in poplar growth and development.