The Key Issues Research About Biological Synthesis Of Epothione By Sorangium Cellulosum

Myxobacteria are Gram-negative gliding bacteria, which exhibit complicated multicellular social behavior. They are capable of producing large numbers of bioactive compounds that have unique structures and diverse functions. Myxobacteria are unique among prokaryotes for their social behavior and the morphogenesis of fruiting bodies. Sorangium cellulosum is a species of celluloytic myxobacteria with a very excellent capacity of biological synthesis. The novel bioactive metabolic compounds discovered from Sorangium cellulosum strains occupied up to 48.4% of the total discoveries from myxobacteria,The social Sorangium cellulosum show typical social behaviors, such as cell density–dependent growth patterns and growing close together, Sorangium strains grow extremely slow, dividing once every 16 hours, and, as a result, Their characteristic of complex cooperative growth has been a barrier for separating Sorangium cellulosum , inhibiting the selection of Sorangium cellulosum mutants during the genetic engineering of the epothilone biosynthetic gene cluster, for example.In this work, we used Sorangium cellulosum as a model. A single S.cellulosum cell embedded into alginate gel micro-beads was able to be triggered to form single-cell colonies when the beads were co-incubated with a high concentration of unpacked S.cellulosum. On the basis of the developed method, we purified a S.cellulosum mutant to obtain single colonies using a S.cellulosum strain as the helper. Interestingly, we obtained single colonies with the genetic traits of the mutant. HPLC revealed that the colonies we purified had substantial differences in the production of epothilone. Compared to the new method, the traditional operation is time-consuming and inefficient. We have reason to believe that the method we established will accelerate the rate of progress and improve the efficiency of genetic engineering in S.cellulosum.Epothilones are 16-membered macrolides, produced by Sorangium cellulosum, that act on cancer cells by stabilizing microtubules, the same mechanism of action as Taxol. Biochemical, pharmacological, and clinical studies have shown that epothilones are highly promising agents for cancer treatment. Some epothilones, and their chemically modified derivatives, are in clinical studies. Ixabepilone,one derivative of epothilone, has been approved for clinical use by the U.S. Food and Drug Administration. However, There are several factors,such as low yield and high cost are constraining the cost of epothione production, the resin is an important factor.XAD-16 resin,bought by Rohm and Hass company is the only used in production of epothione,both in pilot experiment and large fermentation.The high price and large demand of XAD-16 resin increase the production costs. In this paper we survey the effect of 40 different domestic resins on epothilones yield of Sorangium cellulosum So0157-2. Four types of resins are proved to improve yield of epothilone while one can significantly promote cell growth. The mix of different advantageous resins has better effect on epothilone yield than single one. Exclusively, the mix of resin D202 and XD-5 with the ratio of 3 to 7 could increase epothilone yield by 8.3 times comparing to single resin XAD-16.we found that the naural production ratio of epothilones A and B sorted into two groups, single epothilone A (we named epoB? strains) or an approximately 2:1 ratio of A to B (we named epoB⁺ strains).we compared the nucleotide and amino acid sequences and the 3D structures of the 10 sequences of AT_ModC2 domains, four amino acid residues in the enzyme active center were altered in parallel to the production ratio shift. Accordingly, the epoB⁺ AT_modC2 gene was site mutated at these four amino acids from Val90, Ala91, Ala95, and Ser196 to Leu90, Val91, Ile95, and Phe196 (the corresponding residues in epoB? strains). These two genes were heterogeneously expressed in Escherichia coli, yielding inclusion body proteins. The capability of mutant AT_modC2 for binding methylmalony-CoA was weakened nearly 80%, contrast with wide ATModC2. MM/M value of two single mutants M95 and M196 were 0.58 and 0.30, which meant the utilizing abilities of methylmalonyl-CoA were only 58% and 30% of the malonyl-CoA. The result indicated that the mutation of 196th site from Ser to Phe was the most effective one. The two newly reported sites 90th and 91th were co-mutated as M90-91, whose MM/M value was 0.35, equal to the single mutant M196. This result proved the idea that these two sites could affect the substrate selectivity of AT_modC2.