Study On Breeding And Metabolic Pathway Of Acidiphilium Cryptum DX1-1 Accumulating PHB

PHB is a kind of degradable microorganism material, besides the similar characteristics with chemosynthetic macromolecules, it also shows some other characteristics that are not obtained by ordinary chemosynthetic macromolecules, such as, good optical property, low oxygen permibility, anti-ultraviolet radiation, bio-degradability, bio-compatibility, piezoelectricity, and anti-cruor, etc.. It has thus a promising prospect in its application and has attracted more and more attention.A new bacterial strain, designated as strain DX1-1, was isolated from an acid mine drainage of Dexing Copper Mine, Jiangxi Province, China. Basing on morphology, biochemical and physiological characterization, strain DX1-1 was identified as facultative autotrophic bacterium. It grew at temperature ranging from 20℃to 35℃and at initial pH values ranging from 2.0 to 5.0. It was affiliated to the genus Acidiphilium according to the phylogenetic tree derived from 16S rRNA sequence alignment. The capacity of producing polymer granules of strain DX1-1 was found by Transmission Electron Microscopy (TEM). The polymer granules was extracted and detected by ultraviolet-visible (UV) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. The results show that it has the same character with poly-β-hydroxybutyrate (PHB). It suggests that strain DX1-1 might have the potential of producing biodegradable materials (PHB).Four methods were employed to extract PHB from strain DX1-1. There is advantage and disadvantage for each method. Chloroform-sodium hypochlorite method is the best in extracting PHB form Acidiphilium cryptum DX1-1. The extraction rate reaches 73%, the purification rate is 97% and molecular weight is 326 kD. The PHB extracted by this method was then analyzed by UV spectroscopy, FT-IR spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The results show that PHB from strain DX1-1 have the same biochemical structure and character with PHB standard. Mass spectrometer (MS) analysis reveals that the long chain of PHB is destroyed when treated by chloroform-sodium hypochlorite. The differential scanning calorimetry (DSC) of PHB shows PHB from Acidiphilium cryptum DX1-1 has low degree of crystallinity which makes the PHB have a wider range of applications.The culture condition of strain DX1-1 for producing PHB was optimized. Glucose and KNO3 were found to be the best carbon and nitrogen substrates for the production of PHB. The L16 (43) orthogonal experiment improved the yield to 19.56g/L when 40g/L glucose as carbon source,15g/L KNO3 as nitrogen source and pH 3.0 were adopted. Then the cells growth curve and PHB production curve were measured under the optimum culture condition. It was found that the process of PHB accumulating was a growth-associated process. The strain DX1-1 was irradiated respectively by UV and Co60 to improve PHB production. The results indicated that the effect of UV better than using Co60. One strain of the UV mutagenized called UV60-3 has the highest PHB production yield, showing final PHB concentration of 28.56 g/L. Further research about the best culture condition of the mutant was done. The optimum nitrogen source was 30g/L (NH4)2SO4, the final PHB concentration reaches to 30.57g/L.The time, yield and the expression of the PHB accumulation-related genes of Acidiphilium cryptum DX1-1 were investigated under four different initial C/N ratios 1.2,2.4,7.5, and 24. The results of time and yield of PHB accumulation show that the initial C/N ratio 2.4 was optimum for strain DX1-1 to accumulate PHB, both higher and lower initial C/N ratios did not favor that process. Total proteins and intracellular proteins were extracted from strain DX1-1 cultivated under different initial C/N ratios. The protein plots in 2-DE maps were abundant, dispersed and clear, suggesting that the 2-DE technique developed in this study could absolutely meet the requirement of further proteomics study. Eleven proteins pots in total proteins and 9 proteins pots in intracellular proteins which resolved differentially expression were chosen. The 2-DE and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF/TOF MS) were used to identify the proteins. No proteins which catalyze PHB metabolsim were found by this method. But the results show that PHB metabolism is related to metabolism of proteins, fatty acids, and nucleic acids and factor of transcription besides carbon metabolism. The mRNAs of the 19 genes encoding those proteins were detected by Real-Time PCR. The results show that the changes of expression for the 19 genes were consistent with the changes in their mRNAs.Based on the genome of Acidiphilium cryptum JF-5, thirteen PHB accumulation related genes in strain JF-5 were chosen and successfully cloned from strain DX1-1. The differential expressions of the 13 functional genes, in different C/N ratios as cited above, were then studied by Real-time PCR. The results show that all the 13 genes were most upregulated when the initial C/N ratio was 2.4, and among which the gene Acry₃030 encoding poly-β-hydroxybutyrate polymerase and Acry₀626 encoding acetyl-CoA synthetase were much more upregulated than the other genes, which prove that they play the most important role for PHB accumulation and acetate is the main initial substance for PHB accumulation for strain DX1-1. Potential regulatory motifs analysis shows that the genes related to PHB accumulation are regulated by different promoters and that the motif has weak similarity to the model promoters, suggesting that PHB-metabolism in Acidiphilium cryptum may be mediated by a different mechanism.Acidiphilium cryptum DX1-1 can grow slowly with sulfur as energy source. However, growth cycle shortened and cell density increased with glucose added. It was found that PHB reached 13.51g/l with 0.1% glucose added when sulfur as energy source. The expression of genes related to PHB metabolism, carbon fixation and sulfur metabolism in different culture media were detected by Real-time PCR. The results show that glucose could induce the expression of genes related to sulfur metabolism and carbon fixation. When sulfur was used as the only energy sources, the expression of the genes related to PHB metabolism and carbon fixation down-regulared while the genes related to sulfur metabolism up-regulated, which was according with the growth state of keeping the cells alive.