| Penicillin is an important broad-spectrum ?-lactam antibiotic,mainly produced by the fermentation of the filamentous fungus Penicillum chrysogenum.According to statistics,the global antibiotic drug market reached 43.81 billion US dollars in 2016,of which penicillin drugs accounted for 47%of the antibiotic market and the demand for penicillin has increased year by year;At the same time,penicillin intermediate 6-APA is a synthetic raw material for the synthesis of other semi-synthetic antibiotics such as amoxicillin and ampicillin.However,the scale-up of the fermentation process of penicillin is often faced with the problem of reduced production capacity,which has become an important bottleneck for further exploring the "potential" of Penicillium chrysogenum.In this thesis,we have carried out the following research using the tool of quantitative metabolomics:In this paper,we investigated the effect of altered trehalose biosynthesis on both cell growth and penicillin production using the mutant strains of P.chrysogenum ?tpsl and P.chrysogenum ?tps2,which were previously constructed in the laboratory.Experimental results showed that,in the culture of glucose-limited chemostat with a dilution rate of 0.05 h-1,the biomass yield of trehalose pathway-deficient strains decreased by 2.7%and 13.5%,respectively,and the oxygen consumption per unit substrate of engineering strains decreased by 14.6%and 10.1%,respectively,and the metabolic efficiency is slightly improved;however,the penicillin productivity was dropped by 50%in the trehalose mutant strains.Comparison with a high-yielding P.chrysogenum strain revealed that the original and mutant strains had a lower glucose uptake capacity but higher intracellular levels of free amino acids.Flux estimates through the central carbon metabolism showed distinctive difference in the upper part of the glycolysis and in the pentose phosphate pathway but comparable flux through the TCA cycle.Combining,the striking phenotypic effects observed in the trehalose mutants of P.chrysogenum indicated that trehalose metabolism plays an important role in metabolic regulation and is central to maintaining higher penicillin productivity under glucose-limited cultures.Meanwhile,formate dehydrogenase has been confirmed in Penicillium chrysogenum for formate oxidation for an extra supply of ATP,and co-assimilation of glucose and formate has the potential to increase penicillin production and biomass yield.In this study,the steady-state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose-limited chemostat cultures at the dilution rates(D)of both 0.03 h-1 and 0.05 h-1 were determined to evaluate quantitative impact on the physiology of a high-yielding P.chrysogenum strain.It was observed that an equimolar addition of formate was conducive to the increase in both biomass yield and penicillin production at D=0.03 h-1,while this is not the case at D=0.05 h-1.In addition,a higher cytosolic redox status(NADH/NAD+),a higher intracellular glucose level and a lower penicillin productivity were only observed upon formate addition at D=0.05 h-1,which were virtually absent at D=0.03 h-1.As a conclusion,the results demonstrated that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose-limited chemostat cultivations of P.chrysogenum is not only dependent on the formate:glucose ratio as published before,but also on the specific growth rate.The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial-scale scenario. |
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