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Abstract
A biorefinery using sugars derived from lignocellulose is a sustainable route for chemical synthesis. To compete with traditional chemical processes, microbes and bioprocesses must be engineered for improved yield and productivity. Glucose is typically consumed by microbes through glycolytic, pentose phosphate and Entner-Doudroff pathways. Enzymes are responsible for catalyzing the many reactions, and traditionally, overexpression or deletion of genes coding these enzymes has been used to direct carbon to desired pathways. Another approach is to modify key enzymes in metabolism to redirect flow to target pathways. Modulating activity offers a means to fine tune biochemical fluxes to match the microbial needs for growth while optimizing product formation. In this work, two enzymes were studied: Phosphofructokinase 1, which phosphorylates fructose-6-phosphate into fructose-1,6-bisphosphate, and citrate synthase, which converts acetyl-CoA and oxaloacetate into citrate at the start of the tricarboxylic acid cycle.
In a first research study, glycolytic flux was altered by constructing 22 phosphofructokinase (PfkA) variants which each contained single amino acid substitutions at residues associated with mobile loops and adjacent to active site residues. These variants displayed a wide range of growth phenotypes under batch or nitrogen-limited steady-state conditions. Strains containing more severe substitutions showed decreased glucose uptake, decreased acetate formation, decreased intracellular concentrations of glycolytic intermediates, and decreased expression of the pta gene, despite identical growth rates.
In a second study, five citrate synthase (GltA) variants were examined for the formation of an acetyl-CoA-derived product, 3-hydroxybutyrate. GltA variants showed five-fold greater yield of 3-hydroxybutyrate compared to the strain with the wild-type enzyme in a glucose defined medium under batch conditions. A repeated batch process using the GltA[A267T] variant led to 16 g/L 3-hydroxybutyrate with a yield of 0.16 g/g.
In a third study, modifications in both GltA and PfkA were compared for the production of mevalonate, an acetyl-CoA-derived product. Several combined GltA and PfkA variants increased the yield of mevalonate by 50% over the yield obtained by the wild-type strain.
These results demonstrate the benefit of modifying central metabolism at key biochemical junctures to direct glucose into desirable biochemical products.