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Background: Although efficient l-tryptophan production using engineered Escherichia coli is established from glucose, the use of alternative carbon sources is still very limited. Through the application of glycerol as an alternate, a more sustainable substrate (by-product of biodiesel preparation), the well-studied intracellular glycolytic pathways are rerouted, resulting in the activity of different intracellular control sites and regulations, which are not fully understood in detail. Metabolic analysis was applied to well-known engineered E. coli cells with 10 genetic modifications. Cells were withdrawn from a fed-batch production process with glycerol as a carbon source, followed by metabolic control analysis (MCA). This resulted in the identification of several additional enzymes controlling the carbon flux to l-tryptophan. Results: These controlling enzyme activities were addressed stepwise by the targeted overexpression of 4 additional enzymes (trpC, trpB, serB, aroB). Their efficacy regarding l-tryptophan productivity was evaluated under consistent fed-batch cultivation conditions. Although process comparability was impeded by process variances related to a temporal, unpredictable break-off in l-tryptophan production, process improvements of up to 28% with respect to the l-tryptophan produced were observed using the new producer strains. The intracellular effects of these targeted genetic modifications were revealed by metabolic analysis in combination with MCA and expression analysis. Furthermore, it was discovered that the E. coli cells produced the highly toxic metabolite methylglyoxal (MGO) during the fed-batch process. A closer look at the MGO production and detoxification on the metabolome, fluxome, and transcriptome level of the engineered E. coli indicated that the highly toxic metabolite plays a critical role in the production of aromatic amino acids with glycerol as a carbon source. Conclusions: A detailed process analysis of a new l-tryptophan producer strain revealed that several of the 4 targeted genetic modifications of the E. colil-tryptophan producer strain proved to be effective, and, for others, new engineering approaches could be derived from the results. As a starting point for further strain and process optimization, the up-regulation of MGO detoxifying enzymes and a lowering of the feeding rate during the last third of the cultivation seems reasonable. [ABSTRACT FROM AUTHOR]

Background: In recent years, there has been a growing demand for microbial production of trans-4-hydroxy-L-proline (t4Hyp), which is a value-added amino acid and has been widely used in the fields of medicine, food, and cosmetics. In this study, a multivariate modular metabolic engineering approach was used to remove the bottleneck in the synthesis pathway of t4Hyp. Results: Escherichia coli t4Hyp synthesis was performed using two modules: a α-ketoglutarate (α-KG) synthesis module (K module) and L-proline synthesis with hydroxylation module (H module). First, α-KG attrition was reduced, and then, L-proline consumption was inhibited. Subsequently, to improve the contribution to proline synthesis with hydroxylation, optimization of gene overexpression, promotor, copy number, and the fusion system was performed. Finally, optimization of the H and K modules was performed in combination to balance metabolic flow. Using the final module H1K4 in a shaking flask culture, 8.80 g/L t4Hyp was produced, which was threefold higher than that produced by the W0 strain. Conclusions: These strategies demonstrate that a microbial cell factory can be systematically optimized by modular engineering for efficient production of t4Hyp. [ABSTRACT FROM AUTHOR]