Engineering of Primary Carbohydrate Metabolism for Increased Production of Actinorhodin in Streptomyces coelicolor

Abstract

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...

Identification and Characterization of Antibiotic-Producing Actinomycetes Isolates

“…2. Similar observation has also been reported with Streptomyces coelicolor, which produces two antibiotics, a blue colored actinorhodin (ACT) and a red colored undecylprodigiosin (Ryu et al, 2006). It has also reported that the actinomycetes often produce two or more antibiotics simultaneously having different spectral peaks (Krassilnikov, 1981).…”

Carbon source regulation of antibiotic production

“…193 For instance, by cloning acetylCoA carboxylase into an expression vector and introducing it into a wild-type strain of S. coelicolor, the conversion of acetyl-CoA into malonyl-CoA was enhanced and channeled to actinorhodin production. 194 In a similar work, deletion of the pfkA2 gene, corresponding to one of the three reported homologs of phosphofructokinase in S. coelicolor, increased actinorhodin and undecylprodigiosin production approximately four times as compared with the wild-type strain. 124 In addition, it is known that G3P and L-arginine are precursors in the biosynthesis of clavulanic acid in S. clavuligerus.…”

GntR family regulator SCO6256 is involved in antibiotic production and conditionally regulates the transcription of myo-inositol catabolic genes in Streptomyces coelicolor A3(2)

“…We do not know whether other targets are related to ACT production, but it is possible that SCO6256 controls ACT biosynthesis via regulating the transcription of these regulatory genes including sigN. As disruption of SCO6256 also increased the transcription of myo-inositol catabolic genes in R2YE medium, the possibility that the enhanced myo-inositol catabolism produced more acetyl-CoA, which is the precursor of ACT biosynthesis (Ryu et al, 2006), could not be excluded.…”