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Pan, Zhiwei (2007) METABOLIC ENGINEERING OF BACILLUS FOR ENHANCED PRODUCT AND CELLULAR YIELDS. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Microbial cultures usually produce a significant amount of acidic byproducts which can represses cell growth and product synthesis. In addition, the production of acids is a waste of carbon source thereby reduces the product yield and productivity. Metabolic engineering provides a powerful approach to optimize the cellular activities and improve product yields by genetically manipulating specific metabolic pathways. Previous work has identified mutation of Pyruvate Kinase (PYK) as an efficient way to reduce acids production; however, complete abolishment of PYK in Bacillus subtilis resulted in dramatically reduced cell growth rate. In this study, an inducible PYK (iPYK) mutant of B. subtilis was constructed and extensively characterized. The results demonstrated that good cell growth rate and low acetate formation can be attained at an appropriate PYK expression level. In addition, mutation at phosphofructokinase (PFK) on the glycolysis pathway also provides an alternative approach to reduce acetate formation.Two outcomes of the pyk mutant of B. subtilis, high phosphoenolpyruvate (PEP) pool and low acetate concentration, prompted us to investigate the deployment of pyk mutation as an efficient way to improve folic acid and recombinant protein production. The high intracellular PEP and glucose-6-phosphate (G6P) concentration in the pyk mutant led to higher folic acid production by providing abundant synthetic precursors. Additional mutations in the folic acid synthesis pathway, along with the pyk mutation, resulted in 8-fold increase in folic acid production. Recombinant protein was improved two-fold by the pyk mutation due to low acetate formation and longer production time in the pyk mutant. In addition, using glycerol instead of glucose as the carbon source reduced acetate production and improved protein production by 60%.The effect of citrate on acetate production in Bacillus thuringiensis (Bt) was investigated and the continuous culture results showed the effectiveness of citrate on reducing acetate formation. These results indicated pyk may be a potential mutation target to reduce acetate formation in Bt.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairAtaai,
Committee CoChairDomach, Michael
Committee MemberVeser, Götzgveser@pitt.eduGVESER
Committee MemberKoepsel, Richardrrk1@pitt.eduRRK1
Date: 25 September 2007
Date Type: Completion
Defense Date: 29 May 2007
Approval Date: 25 September 2007
Submission Date: 30 May 2007
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Swanson School of Engineering > Chemical Engineering
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: central carbon metabolism; GFP+; metabolic modeling
Other ID:, etd-05302007-093344
Date Deposited: 10 Nov 2011 19:46
Last Modified: 15 Nov 2016 13:44


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