Functional Analysis Of Cald Involved In Calcimycin Biosynthesis And The Initial Exploration On Relevant Protein Complex

Calcimycin is a kind of polyther antibiotic containing pyrrole ring,which is widely used to study biological function of divalent cation in cell.Its molecular structure consist of three parts: a pyrrole ring, a sprioketalring and a substituted benzoxazole. Calcimycin is a kind of membranecarrier for transporting divalent cations such as sodium and potassium ionthrough membrane. So Calcimycin is widely used to study divalent cationstransporting through membrane in vitro and in vivo. Besides, Calcimycinhave other biological activities such as anti-fungi, anti-G+bacteria,inducing cell apoptosis and so on. It also is one type of oxidativephosphorylation uncoupler in mammalian cells, inhibiting the activity ofATPase. Calcimycin is of important value for basic research in biology aswell as for applied research. Since discovered, it have been reported inmore than16,000papers.Streptomyces chartreusis NRRL3882can producing Calcimycin.Previous research have revealed the precursor for Calcimycin synthesis, byfeeding with proline, acetic acid, formic acid,3-hydroxy anthranilic acidand methionine labeled with isotope. The complete gene cluster ofCalcimycin were successfully cloned in2011. The Calcimycin biosyntheticpathway was successfully proposed, and parts of genes have beenidentified. To identify the unknown gene, parts of the work were to complementgenes calD into Streptomyces chartreusis NRRL3882respectively, withflag label introduced for subsequent protein purification. HPLC/MS wasused to analyze the fermentation product. Compared with wide-type strainand mutant strain in term of Calcimycin production, aplerosis strain regainthe capability of producing Calcimycin. At the same time, it had beendemonstrated that the flag label would not have an effect on theCalcimycin biosynthesis. Aimed to gain as much protein as possible, thefermentation conditions were optimized in term of fermentation cultureand fermentation period, with Calcimycin production, biomass and celldisruption considerated. As a result, YEME was the optimal culture whichwas used for subsequent large-scale fermentation in30℃for10days.Western blotting analysis was used to comfirm that the target protein withflag label was expressed in Streptomyces chartreusis NRRL3882.Bioinformatic analysis indicated CalD belong to alcohol dehydrogenasefamily, HPLC/MS analysis showed that inactiving of calD caused a greatdecrease in the Calcimycin production in mutant strain. Compared withwide type strain, mutant strain accumulated higher level of3-hydroxylcezomycin and lower level of N-demethylcalcimycin. So it isspeculated that the absense block up most transformation from3-Hydroxylcezomycin to N-demethylcalcimycin, resulting in theaccumulation of3-Hydroxylcezomycin in mutant strain. Base on the abovebioinformative analysis, It was inferred that calD might be responsible forthe oxidative conversion of the hydroxy-cezomycin to keto-cezomycin. Five genes (calA1, calA2, calA3, calA4, calA5) responsible for thepolyketide synthetase biosynthesis were cloned from the gene cluster, toconstruct nine types of recombinant virus: AcNPV-CalA1, AcNPV-CalA,AcNPV-CalA3, AcNPV-CalA4, AcNPV-C5, AcNPV-CalA1His,AcNPV-CalA2StrepIII, AcNPV-CalA3StrepIII, AcNPV-CalA4Flag.Baculovirus expression system was used for protein expression and partsof polyketide synthetase were obtained using affinity chromatography. Itproved that baculovirus expression system could be applied in polyketidesynthetase expression.