Dedhia, Neilay N. (1995) Metabolic engineering of central carbon metabolism in Escherichia coli : improving the production of biomass and metabolites. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-01252007-075907
The pathway for central carbon metabolism provides precursors for cell biosynthesis and metabolite synthesis along with ATP and NADH. We investigated the metabolic engineering of one of the branches of the central carbon pathways: the pathway of glycogen synthesis and degradation. We were motivated in selecting the glycogen pathway for genetic manipulation by the literature on acetate production in E. coli. The literature indicates that in aerobic cultures the uptake of nutrients occurred faster than the utilization of the precursors, formed from the nutrients, in making biomass and energy. We decided to sequester the excess carbon in glycogen which is a storage polymer. We also devised vectors to degrade the sequestered glycogen. The effects, possible causes of the effects, and potential applications of the sequestering of carbon in the form of glycogen, sometimes combined with engineered degradation of the sequestered glycogen, have been the subject of this thesis.
This manipulation of the glycogen pathway yielded practically useful results. The metabolic engineering was done in an Escherichia coli mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. The sequestering of glycogen was achieved by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under the control of the IPTG-inducible tac promoter. If glycogen overproduction in the ack pta strain grown in complex medium was induced during late log-phase, biomass production increased by 15 - 20% relative to uninduced controls. When glycogen was sequestered and then degraded in E. coli cultures grown in minimal medium, by overamplifying the genes for glycogen synthesis and degradation, then glutamate production was increased almost 3-fold compared to the plasmid-free strain.
When glycogen was sequestered, we observed changes in some of the secreted end-products. We observed that, after overproduction of glycogen, uptake of the previously secreted pyruvate was increased with respect to the control strain, and the CO2 production rate was also increased. These dual observations suggest an increased activity of the gluconeogenic pathways or the TCA cycle. The increase in glutamate, when glycogen sequestering was combined with degradation, also indicate an increase in TCA flux.
Comparison of cAMP levels with and without glycogen overproduction indicate a higher level in cAMP after glycogen is overproduced. There appears to be a tentative link, though not conclusive, between cAMP synthesis and glycogen synthesis pathway. cAMP is a global regulator of central carbon metabolism including many genes of the TCA cycle enzymes. By affecting the TCA flux, cAMP may be one of the causes behind the pleiotropic effects of glycogen overproduction and degradation.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Major Option:||Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||24 October 1994|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||25 Jan 2007|
|Last Modified:||26 Dec 2012 02:29|
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