Dissection Of The Checkpoint Controlling The Apramycin Branched Biosynthetic Pathways

Apramycin is a clinically interesting 2-deoxystreptamine-containing aminoglycoside antibiotic produced by Streptomyces tenebrarius.It exhibits broad-spectrum antibacterial activities,and is often used to treat infections caused by Escherichia coli,Salmonella enteritidis,and mycoplasma,etc.Apramycin has been widely applied in livestock and poultry areas,as well as in routine molecular biology as a selection marker.Structurally,apramycin contains a 4-monosubstitued 2-deoxystreptamine,a 4-NH2-4-deoxy-a-D-Glucose sugar unit,and a highly unique bicyclic octose moiety.Such excellent bioactivity and unusual chemical skeleton make the biosynthesis of apramycin a very attractive project for researchers.However,the assembly logic of apramycin still remains elusive although its biosynethtic gene cluster had been identified ten years ago.This study therefore carried out a series of experiments to solve the above puzzle.Through comprehensive bioinformatic analysis of the apramycin biosynthetic gene cluster,we proposed that aprM/C/S/E/N/D3/D4 may be involved in the production of the intermediate lividamine,aprO/J/K and aprL/D5 may be responsible for the biosynthesis of 4-NH2-4-deoxy-a-D-glucose,aprQ/G/I may participate in the biosynthesis of the unuasual bicyclic octose moiety,and the functions of aprU/F/P in the biosynthesis of apramycin are still unpredictable.Subsequently,we knocked out the C-6' dehydrogenase encoding gene aprQ,and a substantial amount of oxyapramycin,lividamine,and paromamine were found to be accumulated in the mutant strain,suggesting that a homolog of aprQ may exist in the same genome.Genome scanning indeed revealed an aprQ homolog tobQ,which locates in the tobramycin biosynthetic gene cluster.We then constructed a ?aprQ?tobQ double in-frame deletion mutant.HPLC analysis of the metabolites showed that a large amount of lividamine and a few paromamine accumulated in the mutant without the production of apramycin and oxyapramycin,supporting that tobQ can complement aprQ partially.We further investigated the functions of AprQ and TobQ in vitro.The data showed that TobQ could only recognize paromamine,while AprQ could accept both paromamine and lividamine as its substrates with the latter as its physiological substrate.Taken together,these results demonstrate that apramycin and oxyapramycin were produced independently though two parallel biosynthetic pathway,and this is attributed to the broad substrates specificity of AprQ.As for lividamine,we proposed that it may be the product of C3 deoxygenation of paromamine catalyzed by AprD3 and AprD4,because both aprD3 and aprD4 in-frame deletion mutant could accumulate paromamine and oxyapramycin.Bioinformatic analysis coupled with in vitro enzymatic characterization indicated that AprD4 is a novel radical S-adenosyl-L-methionine(SAM)enzyme,which catalyzes the dehydration of paromamine to form 4'-oxo-lividamine,and AprD3 is an NADPH-dependent reductase that catalyzes the reduction of 4'-oxo-lividamine to generate lividamine.Additioanlly,the AprD4/AprD3 enzyme pair does not catalyze the deoxygenation of oxyapramycin to apramycin,supporting that oxyapramycin is not the direct intermediate for generation of apramycin.Instead,oxyapramycin and apramycin are partitioned into two parallel pathways at an early branch point.In summary,a detailed analysis of the biosynthetic gene cluster of apramycin has been carried out in this study,and an important checkpoint that determines the shunt biosynthetic pathway of oxyapramycin has been disclosed to be controlled by AprQ and AprD3/D4.Our results therefore highlight an intriguing parallel pathways in the biosynthesis of apramycin.The insights gleaned from the current investigation may thus facilitate further investigation of the assembly mechanism of the unique octose moiety,and inspire future bioengineering efforts to generate novel sugar-containing natural drugs with improved bioactivities.