The objectives of the current studies were to determine the roles of key enzymes in central carbon metabolism in the context of increased production of antibiotics in Streptomyces coelicolor. Genes for glucose-6-phosphate dehydrogenase and phosphoglucomutase (Pgm) were deleted and those for the acetyl coenzyme A carboxylase (ACCase) were overexpressed. Under the conditions tested, glucose-6-phosphate dehydrogenase encoded by zwf2 plays a more important role than that encoded by zwf1 in determining the carbon flux to actinorhodin (Act), while the function of Pgm encoded by SCO7443 is not clearly understood. The pgm-deleted mutant unexpectedly produced abundant glycogen but was impaired in Act production, the exact reverse of what had been anticipated. Overexpression of the ACCase resulted in more rapid utilization of glucose and sharply increased the efficiency of its conversion to Act. From the current experiments, it is concluded that carbon storage metabolism plays a significant role in precursor supply for Act production and that manipulation of central carbohydrate metabolism can lead to an increased production of Act in S. coelicolor.The wide occurrence of multiply antibiotic-resistant bacterial pathogens of humans has made it urgent to develop new antibiotics. Although over 6,000 different antibiotics have been identified from actinomycetes, these microorganisms are still considered likely to be an important source of further new antibiotics (7). To develop a new antibiotic for clinical application, the compound must be synthesized in sufficient quantities. Random mutation and selection is the tried and tested way to obtain strains producing increased amounts of the targeted compound (11). However, with the advances in genetics, rational or target-directed gene manipulation methods have been developed, and those provide more targeted ways of increasing productivity and discovering novel compounds (12). Such predictive manipulation of central metabolism is likely to be particularly useful at early stages in the testing of new compounds made by genetic manipulation of biosynthetic gene sets in well-characterized surrogate hosts, such as Streptomyces coelicolor or Streptomyces lividans, but the preparation of sufficient quantities of the new compound may be a significant obstacle to further progress.Antibiotics identified from metabolites of microorganisms are classified into several families, such as polyketides, polyethers, macrolides, and ␤-lactams, based on chemical structure similarity and common biosynthetic pathways. The provision of intermediates or precursors from primary/intermediary metabolism is a prerequisite for the biosynthesis of secondary metabolites, and the availability of those molecules is a key factor determining the productivity of antibiotics. These precursors are generally formed through the catabolism of various carbon substrates. S. coelicolor produces blue (actinorhodin [Act])-and red (undecylprodiginines [Red])-pigmented antibiotics, which are synthesized at least in part from the...

In this study, we found that deoxyinosine triphosphate (dITP) could inhibit polymerase chain reaction (PCR) amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from deoxyadenosine triphosphate during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not deoxyuridine triphosphate. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.