Rich Organic Nitrogen Impacts Clavulanic Acid Biosynthesis through the Arginine Metabolic Pathway in Streptomyces clavuligerus F613-1.

Clavulanic acid (CA) is the preferred clinical drug for the treatment of infections by ß-lactam antibiotic-resistant bacteria. CA is produced by Streptomyces clavuligerus, and although there have been many reports on the effects of carbon and nitrogen sources on CA production, the mechanisms involved remain unclear. In this study, we found that CA accumulation in S. clavuligerus F613-1 was increased significantly in MH medium, which is rich in organic nitrogen, compared with that in ML medium, which contains half the amount of organic nitrogen present in MH medium. Transcriptome analysis revealed that genes involved in CA biosynthesis, such as ceas1, ceas2, bls1, bls2, cas2, pah2, gcaS, and cad, and arginine biosynthesis, such as argB, argC, argD, argG, argH, argJ, and argR, were upregulated under rich organic nitrogen. Metabolome data revealed notable differences between cultures of F613-1 grown in MH and ML media with regard to levels of key intracellular metabolites, most of which are involved in arginine metabolic pathways, including arginine, glutamine, and glutamic acid. Additionally, supplementation of ML medium with arginine, glutamine, or glutamic acid resulted in increased CA production by S. clavuligerus F613-1. Our results indicate that rich organic nitrogen mainly affects CA biosynthesis by increasing the levels of amino acids associated with the arginine metabolic pathway and activating the expression of the CA biosynthetic gene cluster. These findings provide important insights for improving medium optimization and engineering of S. clavuligerus F613-1 for high-yield production of CA. IMPORTANCE The bacterium Streptomyces clavuligerus is used for the industrial production of the broad-spectrum ß-lactamase inhibitor clavulanic acid (CA). However, much remains unknown about the factors which affect CA yields. We investigated the effects of different levels of organic nitrogen on CA production. Our analyses indicate that higher organic nitrogen levels were associated with increased CA yields and increased levels of arginine biosynthesis. Further analyses supported the relationship between arginine metabolism and CA production and demonstrated that increasing the levels of arginine or associated amino acids could boost CA yields. These findings suggest approaches for improving the production of this clinically important antibiotic.

Stability, compatibility and microbiological activity studies of meropenem-clavulanate potassium.

Meropenem (MEM) and clavulanate potassium have been reported to demonstrate highly effective activity against Mycobacterium tuberculosis. There have been no reports on research into the complex of these chemotherapeutics concerning their mutually dependent stability or microbiological action on other microorganisms. Stability and compatibility studies of MEM/clavulanate potassium were conducted by using an HPLC-DAD method. The antibacterial activity of MEM/clavulanate potassium was tested in vitro against a selection of indicator bacteria strains by determining the MIC as well as analyzing the kinetics of changes in the concentrations of Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes caused by the action of MEM/clavulanate potassium. The stability and compatibility of MEM/clavulanate potassium were examined in aqua pro iniectione, 0.9% NaCl and 5% glucose at room temperature and at 5 °C. The degradation rates of MEM/clavulanate potassium depended on the type of infusion solvent used. Although in aqueous solutions of MEM/clavulanate potassium neither compound showed any mutual impact on the rate of degradation, clavulanate potassium was more labile than MEM. The synergy between these two resulted in a significantly lower value of MIC as compared to the values observed for the individual activity of either compound. The infusion solvent in which compatibility is observed between the components of the mixture MEM/clavulanate potassium is aqua pro iniectione. The complex MEM/clavulanate potassium demonstrates synergic antibacterial activity against P. aeruginosa, S. aureus and L. monocytogenes.

A statistical approach using L(25) orthogonal array method to study fermentative production of clavulanic acid by Streptomyces clavuligerus MTCC 1142.

Clavulanic acid is a naturally occurring antibiotic produced by Streptomyces clavuligerus. The present work reports on clavulanic acid production by Streptomyces clavuligerus MTCC 1142 using one-factor-at-a-time and L(25) orthogonal array. The one-factor-at-a-time method was adopted to investigate the effect of media components (i.e., carbon source, nitrogen source and inoculum concentration) and environmental factors such as pH for clavulanic acid production. Production of clavulanic acid by Streptomyces clavuligerus was investigated using seven different carbon sources (viz. glucose, sucrose, modified starch, rice-bran oil, soybean oil, palm oil, and glycerol) and six different nitrogen sources (viz. peptone, yeast extract, ammonium chloride, ammonium carbonate, sodium nitrate and potassium nitrate). A maximum yield of 140 microg/mL clavulanic acid was obtained in the medium containing soybean oil as a carbon source and yeast extract as nitrogen source. Subsequently, the concentration of soybean flour, soybean oil, dextrin, yeast extract and K2HPO4 were optimized using L25 orthogonal array method. The final optimized medium produced 500 microg/mL clavulanic acid at the end of 96 h as compared to 140 microg/mL before optimization. Synthesis of precursor molecules as a metabolic driving force is of considerable importance in antibiotic synthesis. Attempts to increase the clavulanic acid synthesis by manipulating the anaplerotic flux on C(3) and C(5) precursors by supplementing the medium with arginine, ornithine, proline, valine, leucine, isoleucine, pyruvic acid and alpha-ketoglutarate were successful. Supplementing the optimized medium with 0.1 M arginine and 0.1 M leucine increased the yield of clavulanic acid further to 1100 microg/mL and 1384 microg/mL respectively.