Abstract
BackgroundUnderstanding the process of amino acid fermentation as a comprehensive system is a challenging task. Previously, we developed a literature-based dynamic simulation model, which included transcriptional regulation, transcription, translation, and enzymatic reactions related to glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and the anaplerotic pathway of Escherichia coli. During simulation, cell growth was defined such as to reproduce the experimental cell growth profile of fed-batch cultivation in jar fermenters. However, to confirm the biological appropriateness of our model, sensitivity analysis and experimental validation were required.ResultsWe constructed an l-glutamic acid fermentation simulation model by removing sucAB, a gene encoding α-ketoglutarate dehydrogenase. We then performed systematic sensitivity analysis for l-glutamic acid production; the results of this process corresponded with previous experimental data regarding l-glutamic acid fermentation. Furthermore, it allowed us to predicted the possibility that accumulation of 3-phosphoglycerate in the cell would regulate the carbon flux into the TCA cycle and lead to an increase in the yield of l-glutamic acid via fermentation. We validated this hypothesis through a fermentation experiment involving a model l-glutamic acid-production strain, E. coli MG1655 ΔsucA in which the phosphoglycerate kinase gene had been amplified to cause accumulation of 3-phosphoglycerate. The observed increase in l-glutamic acid production verified the biologically meaningful predictive power of our dynamic metabolic simulation model.ConclusionsIn this study, dynamic simulation using a literature-based model was shown to be useful for elucidating the precise mechanisms involved in fermentation processes inside the cell. Further exhaustive sensitivity analysis will facilitate identification of novel factors involved in the metabolic regulation of amino acid fermentation.
Highlights
Understanding the process of amino acid fermentation as a comprehensive system is a challenging task
Sensitivity analysis for L-glutamic acid production Using a dynamic simulation model, we calculated the difference in L-glutamic acid production yields in response to variation in gene copy number
L-glutamic acid production would be decreased. We suggest that this is why moderate enhancement of pdhR expression could have the same effect on pyruvate concentration as pykF amplification
Summary
Understanding the process of amino acid fermentation as a comprehensive system is a challenging task. Due to the progress in -omics studies and bioinformatics, including computer simulation, we can integrate knowledge from different levels such as that pertaining to gene expression, protein expression, and metabolite concentrations. This information is useful for designing a strain for producing a particular substance [2]. Mathematical modeling of the tryptophan operon in Escherichia coli clearly showed that TrpR and TrpL were responsible for high and low intracellular tryptophan concentrations, respectively [3] This knowledge formed the theoretical basis for engineering a tryptophan-production strain [4]
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