Study On Differential Gene Expression And Metabolic Pathway Of Acidiphilium Cryptum DX1-1 Accumulating PHB Under Mixotrophy With Limited Heterotrophy

Poly-β-hydroxybutyrate (PHB) is a kind of polyester that stored in microbial cells. It is a ideal materiel for producing "bio-degradation plastic". Acdiphilium cryptum DX1-1 (CCTCC M208056) was isolated from acidic mine drainage, a site strictly lacking in onganic nutrients. It is a faculty strain capable of accumulation intracellular PHB with ferric iron reduction and sulfur oxidation as well as heterotrophic metabolism.In order to explore the genes related PHB synthesis of it and clarify the molecular mechanism of the effect of culture conditions on PHB accumulation of it, the present work focusing on A. cryptum DX1-1 accumulating PHB are as follows:the growth and accumulation of PHB in heterotrophy, autotrophy and mixotrophy that were provided glucose, sulfur and/or ferric iron, and glucose+sulfur and/or ferric iron as the energy substrates, respectively; cloning, sequencing and homology comparison analysis of related genes of A. cryptum DX1-1 metabolic system; related genes were investigated by RT-qPCR analysis of the differential expressions of genes encoding the key enzymes of PHB, carbon, sulfur, and ferrum metabolisms in heterotrophy, autotrophy and mixotrophy as different energy substrates. And the primary results are as follows:A. cryptum DX1-1 grows well and accumulates great amount of PHB in heterotrophic condition but grows slow and doesn't accumulate PHB in autotrophy, and it can not grow in ferric iron as its only energy source. After adding limited organic carbon source like 0.1% glucose, DX 1-1 also grows better and accumulates more PHB in sulfur and/or ferric iron as the energy substrates than with the some concertration glucose in heterotrophy. The cell amount is up to 9.89×108mL, and the accumulation of PHB is 2.07 g/L in mixtrophic condition as sulfur and ferric iron as the energy substrates. In all conditions, In these culture conditions, the growth and PHB production of the strain decreased in the order in terms of energy substrates:GSF>GS>GF>G>>S, SF>>F (abbreviations see Table 2-2). Referring to genome of A. cryptum JF-5 (JGI, http://genome.orn 1.gov/cgi-bin/JGI_microbial/kegg categories.cgi), twenty-six useful homologous genes as well as the 16S rDNA gene to A. cryptum DX1-1 were successfully cloned, including 6 genes of PHB metabolism,10 gemes of carbon metabolism,7 genes of sulfur metabolism and 3 genes of ferrum metabolism. The sequences of the PCR products of these genes are basically matched to that of strain JF-5.After RT-PCR analysis of the differential expressions under different energy source, the result showed that in the mixotrophic cultures all the key enzyme-encoding genes of PHB, carbon, sulfur and ferrum metabolisms are basically up-regulated, especially PHB polymerase (Arcy₃030), isocitrate dehydrogenase (Acry₀565), Rubiscase (Acry₀565), ribose 5-phosphate isomerase (Acry₁272), ribulose 5-phosphate 3-epimerase (Acry₀827), phosphoadenosine phosphosulfate reductase (Arcy₂800), and sulfite reductase (Arcy₂799) encoding genes were significantly up-graduated.