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Hot Red Asian Habitat Bacteria Trehalose Synthase Gene Cloning, Expression And Trehalose Metabolism Pathway

Posted on:2010-05-23Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y M ZhuFull Text:PDF
GTID:1110360302957672Subject:Microbiology
Abstract/Summary:PDF Full Text Request
Trehalose is widely used in food processing industry, agriculture, pharmaceutical industry and many other fields, because of its ability to protect the biological molecules. In industrial production of trehalose, trehalose synthase (TreS) has been getting so much concern. That was because TreS can convert maltose into trehalose in one step reaction, and the cheaper raw material can be derived from starch hydrolysates. Screening of trehalose synthase gene and building a high-expression strain become key factors in decreasing the cost of trehalose industrial production.At the early study of our laboratory, a thermophilic strain CBS-01 producing trehalose synthase was isolated from geothermal water. The preliminary analysis of the TreS showed that the optimal reaction temperature is 50℃, and the optimum reaction pH is 6.5. The enzyme can maintain more than 80% of its activity below 50℃and at pH range of 5.0-9.0. In view of the trehalose synthase with strong stability, it is necessary to clone the TreS gene and build engineering strains producing TreS for trehalose industrial production. On the other hand, it is necessary to study the structure, reaction mechanism and thermoadapatation mechanism of the enzyme, so that modified TreS with stronger thermostability and a favorable conversion rate could be obtained by molecular biological technology.First of all, this strain producing trehalose synthase was identified. It was rod, Gram-negative, aerobic, and could produce dark red pigment. The optimum temperature and pH of its growth are 55℃~60℃and 7.5~8.5, respectively. At physiological and biochemical experiments, the strain CBS-01 showed the same results as the standard strains of Meiothermus ruber ATCC 35948. The G+C content of its genome was 62.1%, and the DNA-DNA hybridization rate with M. ruber ATCC 35948 was 63.9%. The sequence of 16s rDNA gene of CBS-01 showed the greatest identity to M. ruber in Genbank. Based on the above experimental results, the strain producing TreS was identified as Meiothermus ruber.Secondly, the trehalose synthase gene was cloned. According to the conserved amino acid sequences of trehalose synthase reported, degenerate primers were designed for PCR. A fragment of 762 bp was obtained. The deduced amino acid sequence of the DNA product had high identity to other trehalose synthase, indicating that a partial putative TreS gene was isolated from M. ruber. On this basis, A 6200 bp DNA sequence containing three open reading frames was isolated, by using linker mediated PCR, TAIL-PCR, and PCR-based method for screening genomic DNA library. The length of the three ORFs were 1350, 690, and 2889 bp, encoding 449, 229, and 962 amino acids, respectively. Sequence alignment analysis revealed that the deduced amino acid sequences of these ORFs were potential trehalose 6-phosphate synthase (TPS), trehalose 6-phosphate phosphatase (TPP) and trehalose synthase (TreS), respectively. Hence, there were two trehalose synthetic pathways in M. ruber CBS-01 and these three genes are likely to exist in the same operon-like structure. The potential TreS from M.ruber showed the highest homology to those of Thermus genus, and the molecular weight was much larger than those from mesophilic bacteria, indicating the TreS was likely to have better thermal stability. At the same time, it was found that the potential trehalose synthase belonged to theα-amylase family. These results indicated a putative TreS gene was isolated.Third, an engineering strain producing TreS was built, and protein expression conditions were optimized. The potential trehalose synthase gene was transformed into E. coli and induced to express. The activity of crude enzyme solution was detected by using high-performance liquid chromatography (HPLC). The result revealed that the recombinant protein showed the activity of trehalose synthase, and could catalyze the conversion reaction of maltose into trehalose with glucose as the byproduct. This novel gene has been assigned in GenBank, the accession No. is EU443098. In order to increase the yield of trehalose synthase in E. coli, the 5' terminal nucleotides of the TreS were optimized by using PCR-based site-directed mutagenesis technology. At the same time, conditions of protein expression were also optimized, and the optimum conditions were below: using pET-21a as expression vector and E. coli Rosetta-gami (DE3) as host bacterium, the induction was carried out at 20℃, low speed, and overnight. The results suggested the formation of inclusion bodies was decreased, and the yield of soluble and active protein was increased. The TreS activity was 0.60 U/ml broth by fermentation of E. coli engineering strain, and it was about 8-fold than that of M. ruber.Fourth, the recombinant TreS was purified and its enzymatic properties were studied in detail. The TreS was purified by using NTA-Ni column affinity chromatography. The kinetic values of the enzyme were determined, and glucose was found a competitive inhibitor of the reaction. The activity of the recombinant TreS was 80 U/mg, the optimum reaction temperature was 50℃, the optimum pH was 6.0-7.0. It was found that the enzyme could maintain 90% of its activity at a wide temperature (4-60℃) and pH range (pH 4.0-8.5). Because of its stability, the TreS proved very suitable for industrial production of trehalose. Meanwhile, it was found that low reaction temperature could decrease the formation of glucose, and increased the yield of trehalose.Fifth, the vector for expressing the truncated TreS without C-terminal domain was constructed. It was found that the TreSΔC protein did not have the TreS activity. According to the Native-PAGE results, TreS was a tetramer, but TreSΔC was a dimer. The results suggested that the C-terminal domain of TreS played an important role in the formation of the tetramer, which had the TreS activity. However, TreSΔC without C-terminal domain could not form a tetramer, so its activity lost.Sixth, the trehalose biosynthetic pathway of M. ruber was studied. The potential TPS and TPP gene were transformed into E. coli, and the recombinant proteins were purified by using NTA-Ni column. The results of thin layer chromatography (TLC) revealed that the two proteins showed the activity of TPS and TPP, respectively. The novel genes have been assigned in GenBank, the accession No. were FJ360766 and FJ360767. At the same time, it was found that M. ruber could accumulate 6-phosphate trehalose as the compatible solute in response to water stress imposed by salt. The results suggested that the TPP expression of M. ruber was defective, so the 6-trehalose phosphate producted by TPS could not be converted into trehalose.
Keywords/Search Tags:trehalose, Meiothermus ruber, trehalose synthase (TreS), trehalose 6-phosphate synthase (TPS), trehalose 6-phosphate phosphatase (TPP), cloning and expression, trehalose synthetic pathway
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