[Show abstract][Hide abstract] ABSTRACT: Pathway optimization is difficult to achieve owing to complex, nonlinear, and largely unknown interactions of enzymes, regulators, and metabolites. We report a pathway reconstruction using RNA display-derived messenger RNA-enzyme fusion molecules. These chimeras are immobilized by hybridization of their messenger RNA end with homologous capture DNA spotted on a substrate surface. Enzymes thus immobilized retain activity proportional to the amount of capture DNA, allowing modulation of the relative activity of pathway enzymes. Entire pathways can thus be reconstructed and optimized in vitro from genomic information. We provide concept validation with the sequential reactions catalyzed by luciferase and nucleoside diphosphate kinase and further illustrate this method with the optimization of the five-step pathway for trehalose synthesis.
[Show abstract][Hide abstract] ABSTRACT: Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction. This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity. This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum. Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity. In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture. These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway. This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.
[Show abstract][Hide abstract] ABSTRACT: Pyruvate carboxylase was recently sequenced in Corynebacterium glutamicum and shown to play an important role of anaplerosis in the central carbon metabolism and amino acid synthesis of these bacteria.
In this study we investigate the effect of the overexpression of the gene for pyruvate carboxylase (pyc) on the physiology of C. glutamicum ATCC 21253 and ATCC 21799 grown on defined media with two different carbon sources, glucose and lactate. In general, the
physiological effects of pyc overexpression in Corynebacteria depend on the genetic background of the particular strain studied and are determined to a large extent by the interplay between
pyruvate carboxylase and aspartate kinase activities. If the pyruvate carboxylase activity is not properly matched by the
aspartate kinase activity, pyc overexpression results in growth enhancement instead of greater lysine production, despite its central role in anaplerosis
and aspartic acid biosynthesis. Aspartate kinase regulation by lysine and threonine, pyruvate carboxylase inhibition by aspartate
(shown in this study using permeabilized cells), as well as well-established activation of pyruvate carboxylase by lactate
and acetyl coenzyme A are the key factors in determining the effect of pyc overexpression on Corynebacteria physiology.