Gene Cloning, Expression And Enzyme Properties Of Trehalose-6-Phosphate Synthase 1 From Metarhizium Anisopliae

Metarhizium anisopliae, an entomopathogenic fungus, is used as commercial biological control agent of locust. As an entomopathogenic microorganism, there are many advantages in the use of the fungi such as non-residue, non-resistance and non-resurgence except for the active penetration into the host cuticle and the sustaining control for locusts. At present, there are more than 10 strains of Metarhizium anisopliae registered for commercialization. But large scale use of the fungal bio-control agents is limited partially due to the adaptability to the circumstance and the failure of conidia retaining pathogenicity during long term storage.Trehalose, an important component in fungal spores, is a disaccharide which protects against several environmental stresses, such as heat, desiccation, salt, cold, freeze thawing, radical, hypoxia and organic solvent by its protection for protein and bimolecular lamellar lipid membrane. Moreover, there is a strong correlation between intracellular trehalose accumulation and prolonged storage time of conidia from filamentous fungi. So it can be speculated that increasing trehalose content in conidia, prolonging conidial storage time and enhancing its environmental stability may be feasible through changing trehalose metabolism pathway by the application of genetic engineering.The main pathway for trehalose biosynthesis is the condensation reaction of UDP-glucose (UDPG) and glucose-6-phosphate (G-6-P) to give trehalose-6-phosphate (T-6-P) catalyzed by T-6-P synthase (TPS: EC 2.4.1.15), which is the key enzyme for biosynthesis of the disaccharide. Subsequently T-6-P is dephosphorylated to trehalose by a specific T-6-P phosphatase (TPP: EC 3.1.3.12) (2, 6). In Escherichia coli, trehalose is synthesized by the two separate enzymes, encoded by the genes otsA and otsB, respectively. This is different from Saccharomyces cerevisiae, in which trehalose is synthesized by a multisubunit complex (designated as TPS complex). The TPS complex is composed at least by four subunits TPS1 with TPS activity; TPS2 with TPP activity, and regulatory subunits of TPS3/TSL1. TPS1/TPS plays a key role in the biosynthesis of trehalose. Based on the protection from trehalose, many researches showed that the overexpression of TPS1 gene would enhance the resistence of the host to the abiotic stresses while the disruption of the gene decreased the resistence. In addition, TPS1 has showed abundant biological funtions, such as its effects on conidiogenesis, spore germination, plant embryo development and pathogenesis of fungi. Moreover, TPS1 also involved in the control of the influx of sugar into glycosis and the sugar induced signalling. However, It should be noted that the function and its mechanism of TPS1 gene and enzyme are far from illumination by now.TPS1 inspired great insterest of researchers by its key role in the resistence on abiotic stresses and abundant biological functions. For M. anisopliae, TPS1 raised a new idea to solve the problems in the shelf time and the stress resistence to the enviroment. Unfortunately, no report is available on the TPS1 gene and the corresponding enzyme properties in the fungi so far, despite their potential importance for the stress resistance of the spore. Thus, the fungi become the focus of our research. We tried to clone the TPS1 gene in the fungi, analyze its sequence properties, investigated its copy number in the fungi`s genome and study its expression at different development stages. Futhermore, the TPS1 was heterogenously expressed in yeast Pichia pastoris expression system. The recombinant protein was purified and the properties of the purified protein was analyzed. The results attained from the studies above would not only provide more knowledge to the functions of TPS1, but also provide an essential work for the development of more storable, more stable, and more business perspective fungi pesticide by genetic engineering. The main results are as follows:1) The TPS1 gene was cloned successfully from M. anisopliae CQMa102 by PCR, RT-PCR and RACE. The cDNA and its deduced protein sequences were deposited in GenBank (accession number AY954915). The gene existed one copy in the fungi`s genome, and its DNA sequence contained two introns. The 1837-bp cDNA sequence contained an 1554-bp single open reading frame encoded a protein of 517 amino acids, an 87-bp 5'untranslated region and an 195-bp 3'untranslated region. Analysis of the amino acid sequences by computer using the NCBI database and BLAST revealed that the TPS1 belonged to glycosyltransferase_GTB_type superfamily. The three-dimensional structure of the protein was constructed by homology modeling method, and the typical structure of the proteins in GTB_type superfamily was found also in the TPS1. As GTB proteins, TPS1 of M. anisopliae also had distinct N- and C- terminal domains each containing a typical Rossmann fold. There was a cleft to separate the two domains.2) By the investigation on the TPS1 gene expression at different development stages of M. anisopliae CQMa102, it could be found that no expression could be detected in spores, a small amount of expression could be detected after incubated 3h (the spore germination) in 1/4SDA liquid and then increased continously until 24h (the early exponential growth), the expression decreased at 48h (the later exponential growth) and could hardly be detected at 64h (the stationary growth).3) M. anisopliae CQMa102 TPS1 was expressed heterogenously in Pichia pastoris KM71, and the recombinant protein was purified. The purified protein was≈58 kDa with a (His)6 tag at C-terminal. The TPS1catalytic function of the recombinant protein approved the accuracy of our work in the gene cloning. It should be noted that the expression system will provide sufficient amounts of recombinant TPS1 for future structural characterization of the protein.4) The properties of recombinant TPS1 were examined. The optimal temperature was 40°C and the protein reserved nice activity in 35-50°C(more than 60% activity). The optimal pH was about 6.5 and the the protein reserved nice activity in pH5-7.5. The high optimal temperature and the broadly active temperature and pH range should be adaptable to the key role of TPS1 in the resistence to abiotic stresses. The pH range reserved nice activity contain the pH of the fungi`s host, locust in which the pH of its hemolymph is 6.0, so the active pH range of the recombinant protein should be adaptable to the essential catalytic function at the early stage of the infection on locusts. Phosphate was confirmed its inhibition to the activity of the recombinant protein, the activity decreased rapidly as the concentrations of phosphate increased. The Km value of recombinant TPS1 for UDP-glucose and glucose-6-phosphate was 9.6 mM and 3.9 mM, respectively, and the enzyme was highly specific to glucose-6-phosphate for glucosyl acceptor,...