The shuttle expression vectors pHZGI1 and pHZGI2 were successfully constructed by inserting structural genes of GI containing single mutated site G138P and double mutated site G138P-G247D into E. coli-Streptomyces shuttle vector pHZ-1272, respectively. Then they were transformed into S. lividans TK54 strain by protoplast transformation. SDS-PAGE indicated that two shuttle vectors in TK54 strain expressed obviously specific bands at 42.5 kD after inducted by 2 micrograms/mL thiostrepton. Optical densitometric scan showed that the content of the mutant enzymes GIG138P and GIG138P-G247D were about 19% and 22% of dissoluble proteins, respectively. Western blotting farther proved that GIG138P and GIG138P-G247D were expressed in S. lividans TK54.
Aldose-Ketose Isomerases ; analysis ; genetics ; Blotting, Western ; Genetic Vectors ; Mutation ; Streptomyces ; genetics

L-arabinose isomerase (L-AI) is the key enzyme that isomerizes D-galactose to D-tagatose. In this study, to improve the activity of L-arabinose isomerase on D-galactose and its conversion rate in biotransformation, an L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 was recombinantly expressed and applied in biotransformation. Moreover, its substrate binding pocket was rationally designed to improve the affinity and catalytic activity on D-galactose. We show that the conversion of D-galactose by variant F279I was increased 1.4 times that of the wild-type enzyme. The K_m and k_cat values of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s^-1, respectively, and the catalytic efficiency was increased 8.2 times that of the wild type. When 400 g/L lactose was used as the substrate, the conversion rate of M185A/F279I reached a high level of 22.8%, which shows great application potential for the enzymatic production of tagatose from lactose.
Galactose/metabolism* ; Limosilactobacillus fermentum/genetics* ; Lactose ; Hexoses/metabolism* ; Aldose-Ketose Isomerases/genetics* ; Hydrogen-Ion Concentration

L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of D-galactose and D-tagatose. Given the widespread use of D-tagatose in the food industry, food-grade microorganisms and the derivation of L-AI for the production of D-tagatose is gaining increased attention. In the current study, food-grade strains from different foods that can convert D-galactose to D-tagatose were screened. According to physiological, biochemical, and 16S rDNA gene analyses, the selected strain was found to share 99% identity with Pediococcus pentosaceus, and was named as Pediococcus pentosaceus PC-5. The araA gene encoding L-AI from Pediococcus pentosaceus PC-5 was cloned and overexpressed in E. coli BL21. The yield of D-tagatose using D-galactose as the substrate catalyzed by the crude enzyme in the presence of Mn2+ was found to be 33% at 40 degrees C.
Aldose-Ketose Isomerases ; biosynthesis ; genetics ; Biotransformation ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Galactose ; metabolism ; Genetic Vectors ; genetics ; Hexoses ; metabolism ; Pediococcus ; classification ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics

Two distinct routes (classical mevalonate pathway and a novel mevalonate-independent pathway) are utilized by plants for the biosynthesis of isopentenyl diphosphate, the universal precursor of isoprenoids (Fig. 1). Present researches indicated that taxol was synthesized mainly via non-mevalonate pathway, but not genetic evidence was showed. The second step in non-mevalonate pathway involves an intramolecular rearrangement and subsequent reduction of deoxyxylulose phosphate to yield 2-C-methyl-D-erythritol-4-phosphate, and 1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) with responsibility for this reaction was considered as a key enzyme. As a tool for the isolation of genes in terpenoid biosynthesis in plants, total RNA was prepared from Taxus chinensis suspension cells, a cell type highly specialized for diterpene (taxol). A reverse transcription-PCR strategy based on the design of degenerated oligonucleotides was developed for isolating the gene encoding a gymnosperm homolog of this enzyme from Taxus chinensis. Through sequence analysis by Blast P online, the resulting cDNA showed highly homologous to 1-deoxy-D-xylulose 5-phosphate reductoisomerases, with 95% identification compared with Arabidopsis thaliana (Q9XFS9), 94% with Mentha x piperita (Q9XESO), 80% with Synechococcus elongatus (Q8DK30), 78% with Synechocystis sp. PCC 6803 (Q55663) and Nostoc sp. PCC 7120 (Q8YP49), and 73% with Synechococcus leopoliensis (Q9RKT1). Deduced amino acid sequences were also analyzed by PROSITE, ClustalX (1.81) and Phylio (3.6 alpha), and data present evidence for the existence of this deoxyxyluose phosphate reductoisomerase in Taxus chinensis. This is the first report of the dxr gene cloned from gymnosperm.
Aldose-Ketose Isomerases ; genetics ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Mevalonic Acid ; metabolism ; Multienzyme Complexes ; genetics ; Oxidoreductases ; genetics ; Phylogeny ; RNA ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Taxus ; genetics

Obesity is a great risk factor for type 2 diabetes and certain types of cancer, which become a major burden for public health worldwide. As a classic complex disease, obesity is regarded as the interaction of genetic and environmental factors. However, it is controversial which of these two factors have greater effect on obesity. Several genetic loci have recently been reported to contribute to the development of obesity reported in genome-wide association study (GWAS) these years. GWAS play an important role in complex disease research and explore the potential effect of genetic variance. To further understand the genetic influence on obesity risk, we reviewed and collected articles on Pubmed for genes that reported in recent GWAS. We summarized the publications in GWAS and found 49 candidate genes, which were strongly suggested to relate to obesity risk in human. Despite the findings of this and other similar, contemporary research projects, much of the single nucleotide polymorphism details and underlying mechanism in this field of study remains, to a great extent, unknown. As a result, future studies are needed for obesity risk in human beings.
Aldose-Ketose Isomerases ; genetics ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO ; Brain-Derived Neurotrophic Factor ; genetics ; Genome-Wide Association Study ; trends ; Humans ; Obesity ; genetics ; physiopathology ; Polymorphism, Single Nucleotide ; Proteins ; genetics

With the development of low-carbon economy and renewable resource, fermentation of the pentose sugar xylose to produce ethanol becomes a very hot topic. The recombinant Saccharomyces cerevisiae can be constructed by expressing heterologous xylose isomerase (XI). Because Thermus thermophilus XI (TthXI) does not need cofactor, it has been developed for establishing the utilization pathway of xylose in S. cerevisiae. In this article, we reviewed the progress on xylose isomerase. We first introduced the primary properties, sequence and structure characters of xylose isomerase, and discussed its thermostability. The molecular modification of xylose isomerase, including of substrate specificity and thermostability were discussed in detail. Meanwhile, combined with our own research, we also discussed how to improve the xylose isomerase activity at room temperature. Finally, we suggested perspectives of xylose isomerase.
Aldose-Ketose Isomerases ; chemistry ; genetics ; metabolism ; Catalysis ; Enzyme Stability ; Hot Temperature ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Substrate Specificity

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.
Aldose-Ketose Isomerases ; genetics ; Alpinia ; chemistry ; enzymology ; genetics ; Amino Acid Sequence ; DNA, Complementary ; genetics ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Monoterpenes ; metabolism ; Phylogeny

Our previous research indicated that the Streptomyces pactum Act12 (Act12) had a certain promotional effect on tanshinone accumulation and up-regulated the expression of genes 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in Salvia miltiorrhiza hairy roots. This study focuses on the roles of reactive oxygen species in S. pactum Act12-induced tanshinone production in S. miltiorrhiza hairy roots. The 4% Act12, 4% Act12 + CAT and 4% Act12 + SOD were added to S. miltiorrhiza hairy root and subcultured for 21 days, the dry weight, contents of reactive oxygen species, contents of tanshinones and expression of HMGR and DXR were determined at different harvest-time. The generation of reactive oxygen species (ROS) in S. miltiorrhiza hairy roots was triggered by 4% Act12 treatment. The relative expressions of genes HMGR and DXR in 4% Act12 treatment were 32.4 and 4.8-fold higher than those in the control. And the total tanshinone in the hairy roots was 10.2 times higher than that of the control. The CAT and SOD could significantly inhibit the ROS accumulation and relative expressions of genes HMGR and DXR in 4% Act12 treatment, which induced the total tanshinone content was decreased by 74.6% comparing with the 4% Act12 treatment. ROS mediated Act12-induced tanshinone production. The Act12 may be via the ROS signal channel to activate the tanshinone biosynthesis pathways. Thereby the tanshinon content in hairy roots was increased.
Aldose-Ketose Isomerases ; genetics ; metabolism ; Diterpenes, Abietane ; biosynthesis ; Plant Proteins ; genetics ; metabolism ; Plant Roots ; enzymology ; genetics ; metabolism ; microbiology ; Reactive Oxygen Species ; metabolism ; Salvia miltiorrhiza ; enzymology ; genetics ; metabolism ; microbiology ; Secondary Metabolism ; Streptomyces ; physiology

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.
Acanthamoeba castellanii/*enzymology/genetics/metabolism ; Aldose-Ketose Isomerases/*biosynthesis ; Amebiasis/*pathology ; Benzenesulfonates ; Cell Wall/chemistry/genetics/*metabolism ; Cellulose/biosynthesis ; Down-Regulation ; Encephalitis/parasitology ; Glucosyltransferases/*biosynthesis/genetics ; Keratitis/parasitology ; Microscopy, Electron, Transmission ; RNA Interference ; RNA, Small Interfering

Fosmidomycin (100 micromol x L(-1)) which is the effective inhibitor of DXR, key enzyme in terpenoid MEP pathway, was used to treat with hairy roots of Salvia miltiorrhiza. The treated roots were harvested at 2, 4, 6, 8, 10, 16 and 21 d, mRNA level of SmDXR and tanshinone content in treated and negative control groups were detected. Results found that, after treated with fosmidomycin, color of S. miltiorrhiza hairy roots grew pale gradually comparing with controls; mRNA level of SmDXR in hairy roots varied as a shape of parabolic and the highest value achieved at the sixth day after treatment, then it decreased gradually; Content of four kinds of tanshinones were detected. Among of the four kinds of tanshinones, Tanshinone I content changed relatively little, while content of dihydrotanshinone I, cryptotanshinone and tanshinone II (A) decreased gradually in 21 days. The content of total tanshinones in NC groups was 5, 63 times more than FOS-treated roots in the 21th day. The previous results showed that SmDXR played an important role in the accumulation of tanshinone content in MEP pathway. Once the mRNA level of SmDXR was suppressed, the accumulation of secondary metabolites will be significantly affected.
Aldose-Ketose Isomerases ; genetics ; Diterpenes, Abietane ; metabolism ; Fosfomycin ; analogs & derivatives ; pharmacology ; Gene Expression Regulation, Plant ; drug effects ; Plant Roots ; drug effects ; growth & development ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Salvia miltiorrhiza ; drug effects ; genetics ; growth & development ; metabolism ; Time Factors