| Myxobacteria are Gram-negative gliding bacteria that are noted for the complicated multicellular social behavior and the excellent ability to produce various bioactive compounds.In the past two decades,about 100 basic structures and 500 structural variants have been discovered in myxobacteria and have been fully characterized chemically,which account for about 3.5%of the presently known secondary metabolites of microbial origin.Nearly 100%of the Sorangium strains produce some kinds of compounds with diverse biological activities.The genetic tools are urgently needed in myxobacteria but to date,transduction and electroporation have been well described only for the model species Myxococcus xanthus.In other myxobacterial taxa and even different Myxococcus species or strains,the genetic performance is usually difficult to achieve or suffers from low efficiency.In Sorangium,the only cellulose-degrader among all of the 17 myxobacterial genera but the producer of almost half of the discovered secondary metabolites from myxobacteria,there are many barriers for genetic performances.The sorangial cells grow slowly,possess multiple antibiotics-resistances,produce abundant extracellular polysaccharides,and tend to aggregate.In Sorangium,conjugation,which was first reported by Jaoua et al.in 1992,is the major genetic tool.In 2002,Pradella et al. adapted the conjugation conditions for S.cellulosum So ce56.However,the conjugation protocol developed for S.cellulosum So ce56 or So ce26 is probably not applicable for other Sorangium strains,due to the discrepant physiological characteristics of Sorangium strains.The Sorangium So0157-2 is a facultative alkaliphile strain,which was isolated from a soil sample collected on ChengHai lake bank.Our previous bioactivity screeninganalysis confirmed that So0157-2 is a promising strain for the production of bioactive secondary metabolites.But using the normal conjugation protocol,there was no visible conjugant on the selection plates.At present,structure modification and analogue synthesis of natural products are the main methods for screening new drugs.The polyketide compounds-epothilones are produced by Sorangium strains,and has generated substantial interests over the last few years in the areas of chemistry,biology,and medicine due to their interesting structure and,more importantly,their activity against numerous cancer cell lines. Polyketides are one important large family of complex natural products.They are synthesized from simple carboxylic acids by sequential reactions catalyzed by the large multienzyme protein complexes called polyketide synthases(PKSs).Modular PKSs constitute a unique class of type I polyketide synthases,exhibiting remarkable diversity in terms of their structures and functions.In resent years,the combinatorial genetic protocols have been applied and confirmed to expand vastly the molecular diversity of these pharmacologically important metabolites,taking advantage of the natural modularity of polyketides.However the most serious problem is that the Sorangium strains have poorly developed the conjugation system and thus lacks flexible genetic tools.The studies of epothilones and other secondary metabolites are lagging.To bypass the serious limitations of Sorangium genetics manipulation, researchers integrated by homologous recombination the entire epothilones gene clusters to heterologous hosts,in which the background genetic methodology is already more highly developed,for the production of value metabolites.But epothilones had great toxicity to heterologous hosts,and the yields were quite low.Sorangium cellulosum So0157-2 is an epothilone producer with good fermentation characteristics.Efficient genetic manipulation methods are needed to establish for epothilone structure modification and regulation controlling in Sorangium cellulosum So0157-2.In this work we discovered two important protocols.One is that we established an efficient conjugation method by the presence of the dual selective antibiotics,which fit for different Sorangium strains even different microorganisms. The other is that we discovered the natural transformation during the development of fruiting bodies.Using the improved conjugation protocol,we achieved in epothilones structure modification in its producing strain.The efficient and flexible conjugation toolbox established in this work meet the major requirements for practical exploitations of combinatorial biosynthesis.The natural transformation was discovered for the first time in myxobacteria,which can afford new ideas and potential means for developing new genetic performance techniques in myxobacteria.Before this work,the selection markers used for Sorangium conjugation protocols was only phlenmycin and hygromycin.However,many Sorangium strains were found to be able to tolerate phleomycin and/or hygromycin,such as So0157-2 and So02007-3.To select a competent marker for Sorangium conjugation,we screened different Sorangium strains on a large scale for their antibiotics-resistances,which revealed that chloramphenicol was one of the few antibiotics to which all of the screened Sorangium strains were sensitive.Thus,a chloramphenicol resistance cassette containing an aphⅡpromoter element and a followed chloramphenicol resistant gene was constructed into the mobilizable plasmid pCVD442.Aider insertion of the fragments of epothilone biosynthetic genes,the resulting plasmids were used for transfer from DH5α(λpir)to S.cellulosum strains with the help of pRK2013.Using the previously described procedures,there was no visible transconjugant from the mating of S.cellulosum So0157-2 cells and the E.coil cells,even adjusting the ratio of the donor and recipient cells,conjugative time,and culture stages. However,when low dosages of the dual selective antibiotics were added into the mating medium,many clones appeared on the selection plates.The mating efficiency for transfer of pCCMT61 containing a 3 kb-insert of epothilone gene fragment was 1-3×10-6.For So0157-2,the genetic manipulation efficiency was improved 100 times, and the background growth was smoothed away.The improved protocol is able to circumvent the barriers for the Sorangium conjugation and is potentially applicable for different Sorangium strains,either sensitive or less sensitive to living E.coli cells.The effect of the dual selective antibiotics was also confirmed in the mating for transfer of the self-transmissible plasmid pRK2013 from E.coli strain DH5αto HB101.The finding is significant for those less-efficient or unachievable conjugation performances, especially in those slowly growing bacteria.By the addition of plasmid pCCK700 to So0003-3,the natural genetic transformation appeared.Our experimental results indicate that competence for the uptake of exogenous DNA materials is able to be transiently developed during the fruiting body formation.We also found that CaCl2 was helpful for the forming of competence stage,and sequence-specific DNA uptake in transformation of Sorangium. Developmental lysis of cells occurs during the morphogenesis and provides a DNA pool,which is suggested to be available for the natural transformation if competence is developed.The discovery of competence of cells in fruiting bodies and natural transformation provides not only a reasonable explanation for the expansion means of myxobacterial genomes,but also new ideas and potential means for developing new genetic performance techniques in myxobacteria.Based on the conjugation method with low dosages of dual selecting antibiotics established by this work,module 7 and module 8 of epothilones synthases gene were used for structure modification.β-ketoacyl-ACP reductase(KR)domain and methyltransferase(MT)domain of module 7 were inactivated by the insertion of chloramphenicol resistance cassette.It was turned out that KR domain in module 7 was inactive and MT domain inactivation in module 7 could lead to production increase of epothiloneA1 and epothiloneA2.Meanwhile the thioesterase(TE)domain was repositioned to module 7,which promote the epothilones specific cleavage in forward,but the products need to be confirmed further.The results were helpful to the combinatorial manipulation of the structures of polyketides,and the resulting products can help to enrich the compounds pool for the selection of drugs.Genome sequencing indicated that 5-10%genome was constituted by PKSs,and it was about twice the capacity for producing polyketides of either Streptomyces coelicolor or Streptomyces avermitilis.Our previous construction of So0157-2 genome library got a cosmid 6 which had high organized similar with epothilones synthases genes.We got 31777 bp sequences by sequencing,in which there was a new polyketide synthase gene,named dx1.There were three modules in dx1,but no acyl-transferase(AT)was found.We did not found NRPS module in dx1.So the dx1 gene was trans-AT module PKS.The inactivation of dx1 make the epothilones product of So0157-2 increased apparently.The exploration of regulatory alteration in So0157-2 can help us not only reconstruct efficient industrial strain,but also find and utilize more secondary metabolites.
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