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

Irina Borodina,Jeroen Siebring,Jie Zhang,Colin P Smith,Geertje Van Keulen,Lubbert Dijkhuizen,Jens Nielsen

doi:10.1074/jbc.m803105200

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.

Highlights

Streptomyces are well known for their production of biologically active secondary metabolites, with almost two-thirds of all known natural antibiotics being produced by Streptomyces
Polyphosphate kinase gene inactivation in Streptomyces lividans was shown to have an effect on secondary metabolite production as it resulted in accumulation of polyphosphates and activation of actinorhodin production, which is normally silenced in this species (8 –10)
For this reason we studied the effect of pfkA2 deletion on intracellular carbon fluxes, metabolite levels, gene expression, and secondary metabolite production to gain more insight in flux distribution and the link between the fluxes in the primary metabolism and secondary metabolism

Summary

EXPERIMENTAL PROCEDURES

Alignment—The alignment of N-terminal amino acid sequences of S. coelicolor PfkA isoenzymes, including the PfkA N terminus as determined by Alves et al [15], was performed using ClustalX [16]. The samples were boiled in a water bath for 20 min and cooled to room temperature, and absorbance was measured at 625 nm and compared with a glucose standard curve. Lane 1 represents pfkA2 specific PCR product amplified from the S. coelicolor A3(2) reference strain cDNA pool of which any false-positive signals have been removed by restriction digestion. Each 25-␮l phosphate dehydrogenase (G6PDH, EC 1.1.1.49) assays were reaction contained 12.5 ␮l of SYBR Green PCR Master Mix based on the production of NADPH and performed according (Applied Biosystems), 10 ng of cDNA, 10 pmol of each primer, to the protocol of Lessie and Wyk [36] and modified by Butler et and 9.5 ␮l of water. Growth parameters of the S. coelicolor A3(2) reference and ⌬pfkA2 mutant strains as determined in controlled fermentations in defined medium

Carbon balance closurec

Unknown function

NϪM KϪi

Methylmalonate semialdehyde

Findings

DISCUSSION