| Erythromycin is an important broad-spectrum macrolide antibiotic.The main industrial production strain of erythromycin is Saccharopolyspora erythraea.Researchers have carried out enormous studies on the genomics,transcriptomics,proteomics and fermentaion process optimization of S.erythraea.However,without the thorough study of intracellular metabolic mechanism of S.erythraea,the integration of genetic manipulation and the fermentation process study would hardly be realized.Thus,the newly constructed genetic engineering strain would need tedious work to optimize its fermentation process,and the clues which were found during the fermentation process optimization were also difficult to be directly and accurately applicable to the genetic manipulation.These problems have blocked the promotion of erythromycin production.In this study,a 13C labeling experiment platform,including the construction of the metabolic model,the optimization of the extraction and determination methods for intracellular metabolites and the estabilishment of the analysis method for isotopic imformation of each metabolites,were constructed.With the help of these platforms,the metabolic characteristics of glucose,propanol and proline in S.erythraea were quantitatively and qualitatively analyzed.The potential genetic modification targets were predicted according to these metabolic imformation.The strain reconstruction targets were validated by the metabolic flux analysis of genetic engineered strains and their physiological traits.Firstly,the metabolomics analysis platform for S.erythraea was established.The extraction methods for intracellular metabolites in S.erythraea were validated,and the relationship between intracellular metabolite pools and the specific erythromycin production rate was studied.Grinding in liquid nitrogen was suitable for acyl-CoAs,phosphorylated sugars,and the boiling ethanol method was the best for organic acids.The changes of intracellular metabolite pools indicated that the activity of TCA and EMP pathway both declined.The specific erythromycin production rate was positively related to methylmalonyl-CoA,propionyl-CoA and many other intracellular metabolites as well.The decrease of propionyl-CoA might be an important cause of the decline of the synthesis of erythromycin.This study makes the metabolomics analysis of Saccharopolyspora erythraea in the industrial erythromycin production process possible.Secondly,the 13C-isotope information analysis platform was improved.According to the principle of low noise and high signal,the suitable GC-MS fragments for amino acids were selected,which guaranteed the validaty of isotopic imformation analysis method for amino acids.For organic acids,the one-to-many method was suitable.The analysis results of the labeling information of the authentic standard of phosphorylated sugars were very close to the theoretical value,which indicated that this method was suitable for phosphorylated sugars analysis.For actyl-CoAs,the one-to-many method was more accurate than the single mass SIM method,but the later was more sensitive.The validaty of the corresponding isotopic imformation analysis method of each metabolite in S.erythraea was assured through the 20%[U-13C]glucose labeling experiment.Thirdly,a 13C labeling experiment platform for S.erythraea was constructed.This platform consists of central carbon metabolic network,carbon transition map,biomass equation and the analysis method for the 13C labeling information of some metabolites.With this platform,the metabolic characteristics of glucose and proline were quatatively analyzed.It was revealed that the ED pathway replaced the EMP pathway to metabolize glucose.The results of 13C metabolic flux analysis showed that the addition of proline in the synthetic medium led to a great change of metabolic flux distribution in S.erythraea and relieved the energy burden of S.erythraea,thus promote the cell growth.Fourthly,the oxidative catabolic metabolic pathway of propanol was revealed with the 13C labeling experiment platform,and the erythromycin titer was increased by 47%via knocking out the relative target gene.Propanol can be catabolized through two pathways(path ? and path ?).The results of the[1-13C]labeling experiment showed that the carbon backbone of propanol can be transferred into TCA cycle via path ?,and path ? is incomplete due to the lack of malonyl-CoA decarboxylase.MutB and sucC,two potential targets for blocking path ?were knocked out separately in the industrial strain S.erythraea E3.The erythromycin titer of S.erythraea E3-AmutB and S.erythraea E3-?sucC were 2.23 times and 1.25 times as the parental strain.Comparing to the original strain,the yield of propanol to erythromycin of S.erythraea E3-?mutB and S.erythraea E3-?sucC were also increased from 0.22 to 0.48 and 0.27(g/g).These results indicated that S.erythraea E3-?mutB was more productive than the other mutant.The results of metabolic flux analysis showed that the reaction of"methylmalonyl-CoA ?succinyl-CoA" was not completely inactivated,and the catabolic rate of propanol leading to TCA cycle was decreaed from 73.1%to 29.8%.The genetically modified strain constructed based on the industrial strain in this study can increase the erythromycin titer and propanol transformation rate,which would benefit the productivity and lower the cost,and promote the competitiveness of erythromycin industry of our country.Finally,a synthetic medium for the production of erythromycin was designed and optimized via a high through-put screening method and the nitrogen and phosphate regulation strategy.The original medium consisted of 38 components according to the need for growth and erythromycin production.The components was reduced in half via a single composition deletion experiment,and the concentration of each component was optimized through the Plackett-Burman design.Then,in a 5 L bioreactor,the growth and erythromycin production behavior was analyzed,a second growth and the selective usage of different groups of amino acids were observed.The number of amino acid component was further reduced via nitrogen regulation.The optimal initial phosphate concentration was obtained via phosphate regulation.The erythromycin titer in the optimized synthetic medium was 1380 mg/L.which was 17 times higher than the synthetic medium previously used in our lab.This synthetic medium can facilitate the profound study of erythromycin metabolism. |
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