Dissertation
Dissertation > Biological Sciences > Microbiology

Comparision of the Metabolic Characteristics of Escherichia Coli Bl21(DE3) and MG1655

Author WangXinRan
Tutor QiQingSheng; HuaQiang
School Shandong University
Course Microbiology
Keywords system biology E.coli MG1655 BL21 riboflavin
CLC Q93
Type Master's thesis
Year 2012
Downloads 207
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In the21st century, with the fast development of biological technology, many achevements have been made in metabolic engineering using microbs as the fermentation hosts. E.coli is often used as host strain in bioindustry, for its advantages such as clear genetic background and low culturing requiremnets. Through intelligent design and modifications of the specific enzymes and pathways, people can finally turn E.coli into a biorefiney cell factory to produce many important materials and medicines. In the mean time of using it as a producing host, more knowlegde about E.coli itself-about the metabolic network and regulation mechanism, will guide us for a better usage of the strain in metabolic engineering.In this paper, methods in system biology were used to compare the metabolism of the molecular coloning host E.coli K-12MG1655and the protein production host B BL21(DE3) under aerobic and anaerobic conditions. We found that under aerobic condition, the growth rate and cell weight of BL21were both lower than MG1655.13C-labeled metabolic analysis showed that BL21had larger flux rate in TCA cycle, while MG1655had more acetate secration. The strong TCA cycle may be the reason for less acetate secration in BL21. From the result of metabolic flux analysis and RT-PCR, we found the glyoxylate shunt in BL21was inactive, the same as in MG1655. The knockout of PtsG, the main glucose transporter in glucose phosphotransferase system resulted in a lower glucose uptake rate, while larger flux partitation in TCA cycle and pentose phosphate pathway in both strains. The reason for flux re-participation was the activation of Crp-cAMP after the inactivation of PtsG. We found the effect of ptsG inactivation was larger in MG1655than to BL21. Maybe some reglution machinesm similar as Crp-cAMP was present in BL21.Except for the differences on growth condition and flux participation, we also found that under aerobic condition, vitamin B2-riboflavin was constitutively secrated in BL21. In this paper, the FAD level was compared in BL21and MG1655, higher level of FAD was detected in BL21, this may be the direct influence of riboflavin accumulation. We know that FAD is an important ligant to many important enzymes in cellular oxidation, the level of FAD may influence the amount and activity of these enzymes, thus influencing the flux of TCA cycle. We conclude that the accumulation of riboflavin may be one of the reasons that BL21has larger TCA flux participation than MG1655. Through the comparision of enzyme activity in BL21and MG1655, we found that the enzyme activity of the dual functional enzyme:riboflavin kmase/FAD synthase (RibF) was lower in BL21than in MG1655. This may be caused by one amino acid substitution in BL21. Thus we conclude that the reason of riboflavin accumulation was the consequence of feedback regluation caused by the inadequate enzyme activity of RibF.Under anaerobic condition, the growth rate and final OD of BL21was low. According to the results of products analysis, we found one of the main products in BL21was lactate, while its secrtion rate in MG1655was very low. After constitutively expressed the pyruvate-formate lysase gene pflB in BL21, we found its growth rate and final OD was remarkably promoted. Under anaerobic conditions, the ATP required for biomass generation was mainly supplied through substrate phosphorylation in fermentation pathways. In central metabolism, the generation of1mole acetate was combined with the generation of1mole ATP. In BL21, when lactate was mainly produced, the generation of acetate was decreased. After the over-expression ofpflB, the generation of acetate in BL21was increased, more ATP was generated, thus improved the growth condition of BL21under anaerobic condition. The knockout of PtsG removed the inhitbation of Crp-cAMP, increased the secration rate of acetate and succinate, but as the the main glucose transporter was absent, the anaerobic growth was still very slow.This research gives us a thorougly understanding of the metabolic network of E.coli, and will help with the further modifications on it.

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