Biosynthesis Of D-aldgarose A Novel Two-carbon Branched Sugar

Many biologically active natural products derive their activities from sugar substituents.Changing the structures of these sugars should have a dramatically influence on the biological properties of the parent compounds.As drug resistance becoming more and more serious,and it is much difficult to get new drugs from the nature than before.Structural modification has become an important way to obtain new drugs.Because glycodiversification can improve the compounds' solubility,bioavailability and reduce the side effects,it has been wildly used in modification of antibiotics and antitumor agents.Two key points for biosynthesis glycodiversification are activated sugars and glycosyltransferase.No matter in vivo or in vitro glycodiversification,the biosynthesis pathway of sugars used in must be clear.Although lots of monosaccharides were found in natural compounds,but rarely biosynthesis mechanism have been elucidated.So,investigation on sugar biosynthesis pathway will broaden the sources for biosynthesis glycodiversification.D-aldgarose,a novel 4,6-dideoxysugar with a two-carbon branched-chain and five membered carbornated ring at C-3 position,which was first found in 16-membered antibiotics aldgamycin E in early 1960s,and only been found in aldgamycins type macrolides antibitics till today.Previously,we get a marine derived streptomycete Streptomyces sp.HK-2006-1,which can produce aldgamycins and chalcomycins.It gives us a good chance to investigate the biosynthesis pathway of D-aldgarose.Based on the intergeneric conjugation between Escherichia coli and Streptomyces sp.HK-2006-1,an efficient genetic manipulation system for aldgamycins and chalcomycins produce strain Streptomyces sp.HK-2006-1 has been built.In order to elucidate the biosynthesis pathway of D-aldgarose,we must get the aldgamycins biosynthesis gene cluster in Streptomyces sp.HK-2006-1.Considering the polyketide origins of aldgamycins,we adopted a strategy to locate the biosynthetic gene cluster for aldgamycins by cloning the putative PKS genes using the genome DNA of Streptomyces sp.HK-2006-1 as a template and degenerate primers designed against the highly conserved motif of type I PKS.A band with predicted size of about 750 bp was amplified and was used to disrupte the PKS.The disruption result shows that aldgamycins and chalcomycins are synthesized from one gene cluster.The genome information of Streptomyces sp.HK-2006-1 was obtained by whole genome sequencing,and aldgamycins biosynthesis gene cluster was located by comparing the sequences used for PKS disruption.In the gene cluster,25 ORFs(almK?almB?)were found highly homologous to the reported dihydrochalcomycin biosynthesis gene cluster,which conforms to the fact that Streptomyces sp.HK-2006-1 can produce chalcomycins.And 9 addition ORFs(almC??almD?),which may involoved in the biosynthesis of aldgamyicn,were found downstream of the chalcomyins gene cluster.The boundaries of the aldgamycins biosynthesis gene cluster were determined by in-frame deletion of gene almK,almD? and orf3.The aldgamyicns biosynthesis gene cluster spans 77.73 Kb and encompasse 34 ORFs.Biosynthesis pathway of aldgamycins was predicted by bioinformatics analysis of all genes in the gene cluster.In order to determine the founction of these 9 addition ORFs(almC??almD?),target gene disruption experiments were taken.And the change of secondary metablolites was analysized by HPLC.Gene almD?/almD?,encoding the ?/? subunits of pyruvate dehydrogenase,trigger the biosynthesis of D-aldgarose by form a two-carbon chain to the C-3 of TDP-3-keto-4,6-dideoxyglucose,whereas almC?,encoding an oxidoreductase,initiates the chalcomycins biosynthesis through a 3-keto reduction of TDP-3-keto-4,6-dideoxyglucose.Gene almC?,encoding an O-methyltransferase,methylate the 3-OH of 4,6-dideoxyglucose to finish the D-chalcose pathway.We have demonstrated that aldgamycins and chalcomycins are biosynthesized from a single gene cluster for the first time,and established the entire gene cluster for aldgamycins in which 9 new genes are incorporated with the previously identified chalcomycin gene cluster.The biosynthesis pathway of D-chalcose and D-aldgarose were demonstrated(except the carbonated ring of D-aldgarose).The present study sets the stage for future elucidation of the full biosynthetic pathway for D-aldgarose and D-chalcose,and metabolic engineering for selective production of aldgamycins or chalcomycins.