Zizhong Tang

Improved thermostability and enzyme activity of a recombinant phyA mutant phytase from Aspergillus niger N25 by directed evolution and site-directed mutagenesis

We previously constructed three recombinant phyA mutant strains (PP-NPm-8, PP-NPep-6A and I44E/T252R-PhyA), showing improved catalytic efficiency or thermostability of Aspergillus niger N25 phytase, by error-prone PCR or site-directed mutagenesis. In this study, directed evolution and site-directed mutagenesis were further applied to improve the modified phytase properties. After one-round error-prone PCR for phytase gene of PP-NPep-6A, a single transformant, T195L/Q368E/F376Y, was obtained with the significant improvements in catalytic efficiency and thermostability. The phytase gene of T195L/Q368E/F376Y, combined with the previous mutant phytase genes of PP-NPep-6A, PP-NPm-8 and I44E/T252R-PhyA, was then sequentially modified by DNA shuffling. Three genetically engineered strains with desirable properties were then obtained, namedQ172R, Q172R/K432R andQ368E/K432R. Among them, Q172R/K432R showed the highest thermostability with the longest half-life and the greatest remaining phytase activity after heat treatment, while Q368E/K432R showed the highest catalytic activity. Five substitutions (Q172R, T195L, Q368E, F376Y, K432R) identified from random mutagenesis were added sequentially to the phytase gene of PP-NPep-6A to investigate how the mutant sites influence the properties of phytase. Characterization and structural analysis demonstrated that these mutations could produce cumulative or synergistic improvements in thermostability or catalytic efficiency of phytase.

Comparative Proteomic Analysis of Differentially Expressed Proteins in Amaranthus hybridus L. Roots Under Cadmium Stress

Amaranthus hybridus L. has great potential for use in phytoremediation of soils contaminated with cadmium (Cd). In this study, we found higher absorption of Cd by the roots of A. hybridus than by its other organs. To understand the mechanism of Cd accumulation in A. hybridus roots, a comparative proteomic approach was used to differentiate the two-dimensional electrophoretic profiles of root proteins in Cd-free and Cd-treated plants. Twenty-eight differentially expressed proteins were successfully identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Of these, 10 were specifically expressed under Cd stress, and another 11 were upregulated and 7 downregulated by >2.5-fold under Cd stress. We observed increased expression of proteins involved in energy metabolism, protein metabolism, stress and defense, and signal transduction. These changes likely enhanced Cd tolerance and enrichment in A. hybridus. The downregulated proteins were mainly involved in the synthesis of microRNAs, cell walls, and other structural components. These observations were further confirmed by quantitative fluorescence PCR. The resulting differences in protein expression patterns suggest that redirection of root cell metabolism might be an important survival mechanism for A. hybridus under Cd stress.

β‐Amyrin synthase from Conyza blinii expressed in Saccharomyces cerevisiae

Conyza blinii H.Lév. is a widely used medicinal herb in southwestern China. The main pharmacological components of C. blinii are a class of oleanane‐type pentacyclic triterpene glycosides known as conyzasaponins, which are thought to be synthesized from β‐amyrin. However, no genes involved in the conyzasaponin pathway have previously been identified. Here, we identify an oxidosqualene cyclase (OSC), a β‐amyrin synthase, which mediates cyclization of 2,3‐oxidosqualene to yield β‐amyrin. Ten OSC sequences were isolated from C. blinii transcript tags. Phylogenetic analysis was used to select the tag Cb18076 as the putative β‐amyrin synthase, named CbβAS. The open reading frame of CbβAS is 2286 bp and encodes 761 amino acids. Its mature protein contains the highly conserved motifs (QXXXGXW/DCTAE) of OSCs and (MWCYCR) of β‐amyrin synthases. Transcription of CbβAS was upregulated 4–24 h after treatment of the seedlings of the C. blinii cultivar with methyl jasmonate. Furthermore, expression of CbβAS in Saccharomyces cerevisiae successfully yielded β‐amyrin. The chemical structures and concentrations of β‐amyrin were confirmed by GC‐MS/MS. The target yeast ultimately produced 4.432 mg·L−1 β‐amyrin. Thus, CbβAS is an OSC involved in conyzasaponin biosynthesis.

Biodegradation of Feather Waste Keratin by the Keratin-Degrading Strain Bacillus subtilis 8

Bacillus subtilis 8 is highly efficient at degrading feather keratin. We observed integrated feather degradation over the course of 48 h in basic culture medium while studying the entire process with scanning electron microscopy. Large amounts of ammonia, sulfite, and L-cysteic acid were detected in the fermented liquid. In addition, four enzymes (gamma-glutamyltranspeptidase, peptidase T, serine protease, and cystathionine gamma-synthase) were identified that play an important role in this degradation pathway, all of which were verified with molecular cloning and prokaryotic expression. To the best of our knowledge, this report is the first to demonstrate that cystathionine gamma-synthase secreted by B. subtilis 8 is involved in the decomposition of feather keratin. This study provides new data characterizing the molecular mechanism of feather degradation by bacteria, as well as potential guidance for future industrial utilization of waste keratin.

The Potential Role of Auxin and Abscisic Acid Balance and FtARF2 in the Final Size Determination of Tartary Buckwheat Fruit

Tartary buckwheat is a type of cultivated medicinal and edible crop with good economic and nutritional value. Knowledge of the final fruit size of buckwheat is critical to its yield increase. In this study, the fruit development of two species of Tartary buckwheat in the Polygonaceae was analyzed. During fruit development, the size/weight, the contents of auxin (AUX)/abscisic acid (ABA), the number of cells, and the changes of embryo were measured and observed; and the two fruit materials were compared to determine the related mechanisms that affected fruit size and the potential factors that regulated the final fruit size. The early events during embryogenesis greatly influenced the final fruit size, and the difference in fruit growth was primarily due to the difference in the number of cells, implicating the effect of cell division rate. Based on our observations and recent reports, the balance of AUX and ABA might be the key factor that regulated the cell division rate. They induced the response of auxin response factor 2 (FtARF2) and downstream small auxin upstream RNA (FtSAURs) through hormone signaling pathway to regulate the fruit size of Tartary buckwheat. Further, through the induction of fruit expansion by exogenous auxin, FtARF2b was significantly downregulated. The FtARF2b is a potential target for molecular breeding or gene editing.

Research on homology modeling, molecular docking of the cellulase and highly expression of the key enzyme (Bgl) in Pichia pastoris

Cellulose is the most abundant and renewable biological resource on earth. As nonrenewable resources are becoming scarce, cellulose is expected to become a major raw material for food, energy, fuel and other products. 1,4-β-glucosidase (Bgl), as a kind of cellulose, can be degraded cellulose into industrial available glucose. In this study, we constructed mutants of Bgl with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. On the basis of the high-activity mutations were got (N347S and G235 M) by using site-directed mutagenesis and screening methods and introduced in the Pichia pastoris expression system, the enzymatic properties of mutant enzymes were analysed. Assays of the activity of the purified Bgl revealed that the two mutants exhibited increased activity. The pPICZαA-G235 M and pPICZαA-N347S mutants exhibited a >33.4% and 44.8% increase in specific activity respectively, with similar pH, temperature and metal ion requirements, compared to wild-type Bgl. These findings would be good foundation for improving production properties of Bgl in the future.

Insights into the correlation between Physiological changes in and seed development of tartary buckwheat ( Fagopyrum tataricum Gaertn . )

BackgroundTartary buckwheat (Fagopyrum tataricum Gaertn.) is a widely cultivated medicinal and edible crop with excellent economic and nutritional value. The development of tartary buckwheat seeds is a very complex process involving many expression-dependent physiological changes and regulation of a large number of genes and phytohormones. In recent years, the gene regulatory network governing the physiological changes occurring during seed development have received little attention.ResultsHere, we characterized the seed development of tartary buckwheat using light and electron microscopy and measured phytohormone and nutrient accumulation by using high performance liquid chromatography (HPLC) and by profiling the expression of key genes using RNA sequencing with the support of the tartary buckwheat genome. We first divided the development of tartary buckwheat seed into five stages that include complex changes in development, morphology, physiology and phytohormone levels. At the same time, the contents of phytohormones (gibberellin, indole-3-acetic acid, abscisic acid, and zeatin) and nutrients (rutin, starch, total proteins and soluble sugars) at five stages were determined, and their accumulation patterns in the development of tartary buckwheat seeds were analyzed. Second, gene expression patterns of tartary buckwheat samples were compared during three seed developmental stages (13, 19, and 25 days postanthesis, DPA), and 9 765 differentially expressed genes (DEGs) were identified. We analyzed the overlapping DEGs in different sample combinations and measured 665 DEGs in the three samples. Furthermore, expression patterns of DEGs related to phytohormones, flavonoids, starch, and storage proteins were analyzed. Third, we noted the correlation between the trait (physiological changes, nutrient changes) and metabolites during seed development, and discussed the key genes that might be involved in the synthesis and degradation of each of them.ConclusionWe provided abundant genomic resources for tartary buckwheat and Polygonaceae communities and revealed novel molecular insights into the correlations between the physiological changes and seed development of tartary buckwheat.