Functional Identification Of Type Ⅲ Polyketide Synthase DrPKSl And DrPKS2in Deinococcus Radiodurans

Polyketides were a kind of secondary metabolite with different structures and biological activities.They showed all kinds of pharmacological properties, including antibacterium, fungistasis, anti-parasite,antioxidation and anticancer etc. They were synthesized by polyketide synthase （PKS） via a series ofcondensation of acyl-CoA, cyclization and decoration. Deinococcus radiodurans was famous for itsoutstanding ability of abiotic stress resistance such as ionizing radiation resistance; UV radiationresistance; oxidation resistance and desiccation resistance. In D. radiodurans R1, there were two typeⅢ PKSs, which we named DrPKS1（DR₂091, drpks1） and DrPKS2（DR_A0326, drpks2）. Thefunction of DrPKS1and DrPKS2was still unclear.In this research, we analyzed these two genes and proteins encoded by them via bioinformationmethod, constructed single mutant strains and double mutant strain of drpks1and drpks2, as well asprotein expression E. coli strains. By a series of HPLC analysis of secondary metabolite andphysiological and biochemical experiments associate with abiotic stress resistance, we identified thefunction of these two type Ⅲ PKSs, as followed the results.drpks1gene encoded360aa,38.1kDa protein with1083bp, drpks2gene encoded438aa,46.6kDaprotein with1317bp. Compared with these bacterial type Ⅲ PKSs of which the function was alreadycharacterized, RePKS in Rhizobium etli shared the highest sequence identity in40.06%with DrPKS1and BpsA in Bacillus subtilis shared the second highest in39.30%. DrPKS2shared the highest but only28.34%sequence identity with AgqA in Actinoplanes missouriensis. Both DrPKS1and DrPKS2contained the classic αβαβα fold structure and extremely conservative Cys, His and Asn catalyticallyactive sites.The result of fluorescence quantitive real-time PCR in different growth phases indicated theexpression of drpks1happened in late stabilization phase and drpks2was always in trace expression.The HPLC analysis of secondary metabolites from mutant strains and wild strain showed that therewere two remain peaks in the sample of the double mutant strain while the wild strain doesn’t. Theabsorption spectrum parameters of these secondary metabolites were UV （methanol）: λmax=200,220,290nm.The growth curve of mutant strains and wild strain show that the biomass of drpks2mutant typestrain was higher than other three strains since early logarithmic phase and the colour of drpks1culturemedia was lighter than the other three strains in48-60h incubation. In other stress resistant experimentsuch as H2O2,15%ethanol,3M NaCl,3M sorbitol and UV, there were no distinctions between thesestrains.In order to do in vitro experiment, we constructed the protein expression E. coli strains for DrPKS1,DrPKS2and DGoPKS which is the homological protein of DrPKS1in D. gobiensis. The HPLC analysisof secondary metabolite of protein expression strains showed these were a series of product remainpeaks in DrPKS1and DGoPKS. These products were assumed in similar structure with product in D.radiodurans double mutant type strain because of the similar absorption spectrum parameters UV （methanol）: λmax=205,225and290nm.A series of abiotic stress resistance experiments were done to test these protein expression E. colistrains, with empty vector as the control. After1mM H2O2stress for30min, the survival rate of threeprotein expression strains was2-3folds lower than control, DrPKS2expression strain was1fold lowerthan DrPKS1and DGoPKS strains. After2M NaCl stress for2h, the resistance of three proteinexpression strains was2fold lower than control, and no different between them. These results indicatedthat DrPKS1may associate with abiotic stress resistance.This research basically identified the function of type Ⅲ PKS in D. radiodurans, successfullyconstruct the protein expression E. coli strains, and lay a good basis for further protein purification andin vitro experiment. The exact structure of substrate and product and the pathway of these type Ⅲ PKSin D. radiodurans was still unclear, and further study of the crystal structure of enzyme, the reactionbetween active sites and product structure, and the mechanism of condensation, cyclization anddecoration were all needed.