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Title: Genetic manipulation of metabolic fluxes in Escherichia coli
Author: Okungbowa, Joe
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1991
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The excretion of acetate by E. coli ATCC15224 is a form of metabolite leakage from the central metabolic pathways and a symptom of disproportionate partition of input carbon to metabolites required as precursors for cellular biosynthesis. Preliminary analysis of carbon flux through the central pathways suggested that throughputs of carbon to biosynthetic precursors from the tricarboxylic acid (TCA) cycle may be sub-optimal and limiting for the net synthesis of new cell material and that partition of flux at phosphoeno/pyruvate (PEP) was central to the throughputs of carbon to intermediate metabolites for the synthesis of about 70% of amino acids, 50% of nucleotides and eventually to the output of acetate. Carboxylation of PEP during flux through phosphoeno/pyruvate carboxylase (PEPc) played a primary role in throughput of carbon to the TCA cycle and two biosynthetic precursors-oxaloacetate and 2-oxoglutarate. Using a combination of flux and genetic manipulations, the activity of PEPc and throughput of PEP to oxaloacetate was amplified. Recombinant plasmid pJOE4 (9. 36 kb) which contained the ppc gene encoding PEPc, was isolated from a comprehensive bank of Escherichia coil genomic DNA (in the form of a population of synthetic hybrid plasmids of Sau3A-generated fragments of E. coli chromosomal DNA interposing the unique BamHI recognition sequence of plasmid vector pBR322) by complementing the PEPc lesion in transformants of E. coli PA342. The intrinsic PEPc activity of 24.4 nmol. mg. protein-1, min-1 in E. coli ATCC15224, was overexpressed 75-fold in E. coli ATCC15224-pJOE4 recombinant strain. Frameshift mutation of plasmid pJOE4 by limited deletion of bases at a unique Sacll site on the insert, produced recombinant plasmid pJOE12. No PEPc activity was detected in ultrasonic extracts of recombinant strain-E. coli PA342-pJOE12. Comparison of growth and substrate utilization by E. coli ATCC15224 and ATCC15224-pJOE4 showed that overexpression of PEPc activity diminished fluxes to acetate excretion from 5.12mmol g. dry weight-1, hour-1, to 2.04 mmol. g-1h-1, during aerobic metabolism of glucose. This was equivalent to 60% reduction in acetate excretion. The recombinant strain also excreted 34% less acetate during aerobic growth on pyruvate and 20-25% less acetate on D-gluconate, D-glucuronate respectively. Both strains did not operate fluxes to acetate excretion during fructose and glycerol metabolism. In addition, carbon conversion coefficients by E. coli ATCC15224-pJOE4 were enhanced to a greater or lesser extent, vis-a-vis the parent growing on the various substrates: Y GLUCOSE increased from 90.5g. dry weight, mol-1, to 114. 4p. mol-1, which represented 27% improvement in growth yield, Y FRUCTOSE also increased by 9.4%, y PYRUVATE by 6.7%, Y GLUCURONATE and Y GLUCONATE by 4.7 and 3.0 respectively. These effects were also demonstrated qualitatively in a recombinant strain of E. coli K10 transformed with plasmid pJOE4. Since growth on fructose did not result in acetate excretion, overexpression of PEPc activity probably increased growth yield by diminishing flux around a possible futile cycle between fructose-6-phosphate and fructose-1,6-bisphosphate. The complete abolition of this cycle was also a strong possibility. Overexpression of PEPc activity had no detectable effects on glycerol metabolism where the most important determinants of carbon flux partition were independent of PEP. A significant observation from the analysis of carbon flux in E. coli ATCC15224-pJOE4, was the strong likelihood of a zero flux through pyruvate kinase, as a result of the increased demand for PEP by amplified PEPc activity. This was corroborated by experiments in E. coli HW1387 (pykA, F) which carried lesions for both pyruvate kinases A and F and E. coli HW0760, its parent strain. Y GLUCOSE in E. coli HW0760 was 72g. mol-1, at u= 0.47h-1. Overexpression of PEPc activity in the pyruvate kinase-negative strain brought both growth rate and growth yield to the level obtained in the parent strain. However, these observation were complicated both by slow growth rates and non-excretion of acetate by E. coli HW0760 and E. coli HW1387-pJOE4. It is proposed that the contrasting effects of amplified PEPc activity on acetate excretion and growth yield, was possible through a combination of competitive reduction of flux from PEP to pyruvate via pyruvate kinase on the one hand, and increased oxidation of acetyl-CoA through increased flux of PEP to oxaloacetate, the coreactant/carrier-molecule of acetyl-CoA, on the other. The results demonstrate how a studied manipulation of metabolic fluxes can improve microbial productivity by enhancing conversion of input-carbon to desired outputs and conservation of feed-stock by arresting effluxes to undesirable outputs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available