Antibiotic Overproduction in Streptomyces coelicolor A3(2) Mediated by Phosphofructokinase Deletion

Abstract

Streptomycetes are exploited for production of a wide range of secondary metabolites, and there is much interest in enhancing the level of production of these metabolites. Secondary metabolites are synthesized in dedicated biosynthetic routes, but precursors and co-factors are derived from the primary metabolism. High level production of antibiotics in streptomycetes therefore requires engineering of the primary metabolism. Here we demonstrate this by targeting a key enzyme in glycolysis, phosphofructokinase, leading to improved antibiotic production in Streptomyces coelicolor A3(2). Deletion of pfkA2 (SCO5426), one of three annotated pfkA homologues in S. coelicolor A3(2), resulted in a higher production of the pigmented antibiotics actinorhodin and undecylprodigiosin. The pfkA2 deletion strain had an increased carbon flux through the pentose phosphate pathway, as measured by 13 C metabolic flux analysis, establishing the ATP-dependent PfkA2 as a key player in determining the carbon flux distribution. The increased pentose phosphate pathway flux appeared largely because of accumulation of glucose 6-phosphate and fructose 6-phosphate, as experimentally observed in the mutant strain. Through genome-scale metabolic model simulations, we predicted that decreased phosphofructokinase activity leads to an increase in pentose phosphate pathway flux and in flux to pigmented antibiotics and pyruvate. Integrated analysis of gene expression data using a genome-scale metabolic model further revealed transcriptional changes in genes encoding redox co-factor-dependent enzymes as well as those encoding pentose phosphate pathway enzymes and enzymes involved in storage carbohydrate biosynthesis.

Carbon source regulation of antibiotic production

“…High concentrations of sugar elicit CCR over the use of alternative carbon sources and the synthesis of several secondary metabolites. Glucose repression in S. coelicolor operates at the transcriptional level to repress enzymes involved in the use of glycerol, arabinose, fructose and galactose 122 and this effect seems to be due to either intermediates of carbohydrate catabolism, for example, fructose 1,6-diphosphate and glucose 6-phosphate 123,124 or enzymes of the glucose catabolic pathway, such as glucose kinase. [125][126][127][128] In this regard, it has been reported that mutants of S. coelicolor resistant to the nonutilizable glucose analog, 2-deoxyglucose (DOG), appear to be generally deficient in glucose repression.…”

“…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. G3P is converted into 1,3-diphosphoglicerate (1,3-BPG) by G3P dehydrogenases.…”

Toward Systems Metabolic Engineering of Streptomycetes for Secondary Metabolites Production

“…Examples of metabolic engineering strategies used for the optimization of secondary metabolite production in streptomycete hosts. [83][84][85] Construction of a mutant strain with improved yields C labeling and MFA to construct metabolic network model 84.8% [70][71][72] EFMA on the ascomycin network model for target predictions Strain engineering by overexpression and inactivation of genes Addition of resin HP20 in the growth medium…”

Low Molecular Weight Protein Tyrosine Phosphatases Control Antibiotic Production in Streptomyces coelicolor A3(2)

“…Phosphate limited minimal medium modified from Evan's medium was prepared as described by Borodina et al [27]. The medium contained 30 g/L glucose, 3 mM phosphate and 100 mM ammonia.…”

“…At each time point duplicate samples were taken from plates and OD 450nm , dry cell weight (DCW), pH, glucose consumption and RED and ACT synthesis were monitored. All the analyses were carried out as described by Borodina et al [27].…”