Application And Analysis Of Carbon Source Metabolic Fluxes Through Central Pathways In Streptomyces Rimosus Using¹³C Labeling Technique

Industrially, Streptomyces rimosus is an oxytetracycline (OTC) producing bacterium. Oxytetracycline, one of the tetracycline antibiotics, is widely used in the fields of agriculture and fisheries. Recently, the improvement of the oxytetracycline production at industrial scale has been one of important research issues. In this thesis, the13C labeling technique is adopted to identify the metabolic flux distributions of glucose through central metabolic pathways in Streptomyces rimosus cells, then construct a genome-wide metabolic network model. The results can help identify related key gene targets for oxytetracycline production improvement. The results are showed as follows:1. Development of an inorganic nitrogen synthetic medium suitable for13C metabolic flux analysis (13C-MFA).Twelve kinds of organic nitrogen sources and nine kinds of inorganic nitrogen sources was optimized for Streptomyces rimosus strain M4018growth and oxytetracycline synthesis. S4synthetic medium which contains KNO3as the main nitrogen source was obtained, then the response surface methodology was taken to optimize other compositions of S4. The cell dry weight of S.rimosus M4018grown on the optimized S4medium reached6.32g/L, and oxytetracycline yield of145.6mg/L was achieved, which was very close to the OTC level using organic nitrogen as main nitrogrn source. Importantly, the optimized S4synthetic medium excluding organic nitrogen source was suitable for13C metabolic flux analysis(13C-MFA) and prevented it from the interference of organic N source as an alternative carbon source during OTC fermentation.2. Reconstruction of the Streptomyces rimosus central metabolic network model.Based on the known Streptomyces rimosus M4018genomic information and biomass composition tested in this thesis, the central carbon metabolic network model of S.rimosus M4018was reconstructed, and verified and corrected by the13C-labeled experimental results. The built model contained two kinds of anaplerosis pathways besides glycolysis, pentose phosphate pathway and TCA cycle as well. Furthermore, lack of ED pathway was identified in S.rimosus cells by100%of1-13C labeled glucose analysis, which laid the foundation for13C-MFA application in metabolic investigation on S.rimosus.3. Establishment of the13C metabolic flux analysis method of S.rimosus. Based the feed experiments with100%1-13C labeled glucose and20%U-13C labeled glucose, and the13C metabolic flux analysis was performed for S.rimosus growth. The effect of the primary metabolic pathway gene zwfl (encoding glucose-6-phoosphate dehydrogenase, G6PDH) and mini PKS gene oxyABC in OTC synthesis pathway on the central carbon metabolic flux distribution of S. rimosus M4018was quantatively investigated, respectively. The results showed that when the zwfl gene was knocked-out or the oxyABC gene was duplicated, the oxytetracycline production by M4018was markedly enhanced. As measured by13C labeling experiments analysis, it also showed a decreased flux through the PPP pathway, and an increased flux in the EMP pathway and TCA cycle in both mutants. It indicates that the NADPH formation by PPP was exceeded to the requirement of NADPH for oxytetracycline biosynthesis, while the EMP pathway can provide more the desired precursor acetyl-CoA of oxytetracycline biosynthesis to improve OTC synthesis.