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Item type:Publication, Access status: without files , Alterations in the transcription factors GntR1 and RamA enhance the growth and central metabolism of Corynebacterium glutamicum(2018-07); ; ; ; Evolution, i.e. the change in heritable characteristics of biological populations over successive generations, has created the diversity of life that exists today. In this study we have harnessed evolution to create faster growing mutants of Corynebacterium glutamicum, i.e. to debottleneck growth rate of this highly important industrial workhorse. After approximately 1500 generations of Adaptive Laboratory Evolution (ALE) in defined minimal medium with glucose, we obtained faster growing mutants with specific growth rate as high as 0.64 h−1 as compared with 0.45 h−1 for the wild type, and this 42% improvement is the highest reported for C. glutamicum to date. By genome resequencing and inverse metabolic engineering, we were able to pinpoint two mutations contributing to most of the growth improvement, and these resided in the transcriptional regulators GntR1 (gntR1-E70K) and RamA (ramA-A52V). We confirmed that the two mutations lead to alteration rather than elimination of function, and their introduction in the wild-type background resulted in a specific growth rate of 0.62 h−1. The glycolytic and pentose phosphate pathway fluxes had both increased significantly, and a transcriptomic analyses supported this to be associated with increased capacity. Interestingly, the observed fast growth phenotype was not restricted to glucose but was also observed on fructose, sucrose and xylose, however, the effect of the mutations could only be seen in minimal medium, and not rich BHI medium, where growth was already fast. We also found that the mutations could improve the performance of resting cells, under oxygen-deprived conditions, where an increase in sugar consumption rate of around 30% could be achieved. In conclusion, we have demonstrated that it is feasible to reprogram C. glutamicum into growing faster and thus enhance its industrial potential.Publicationtype: Journal Article138 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Discovery of feed-forward regulation in L-tryptophan biosynthesis and its use in metabolic engineering of E. coli for efficient tryptophan bioproduction(2018-05); ; ; The L-tryptophan (Trp) biosynthesis pathway is highly regulated at multiple levels. The three types of regulations identified so far, namely repression, attenuation, and feedback inhibition have greatly impacted our understanding and engineering of cellular metabolism. In this study, feed-forward regulation is discovered as a novel regulation of this pathway and explored for engineering Escherichia coli for more efficient Trp biosynthesis. Specifically, indole glycerol phosphate synthase (IGPS) of the multifunctional enzyme TrpC from E. coli is found to be feed-forward inhibited by anthranilate noncompetitively. Surprisingly, IGPS of TrpC from both Saccharomyces cerevisiae and Aspergillus niger was found to be feed-forward activated, for which the glutamine aminotransferase domain is essential. The anthranilate binding site of IGPS from E. coli is identified and mutated, resulting in more tolerant variants for improved Trp biosynthesis. Furthermore, expressing the anthranilate-activated TrpC from A. niger in a previously engineered Trp producing E. coli strain S028 made the strain more robust in growth and more efficient in Trp production in bioreactor. It not only increased the Trp concentration from 19 to 29 g/L within 42 h, but also improved the maximum Trp yield from 0.15 to 0.18 g/g in simple fed-batch fermentations, setting a new level to rationally designed Trp producing strains. The findings are of fundamental interest for understanding and re-designing dynamics and control of metabolic pathways in general and provide a novel target and solution to engineering of E. coli for efficient Trp production particularly.Publicationtype: Journal Article162 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Efficient production of the Nylon 12 monomer ω-aminododecanoic acid methyl ester from renewable dodecanoic acid methyl ester with engineered Escherichia coli(2016-07-01); ; ; ; The expansion of microbial substrate and product scopes will be an important brick promoting future bioeconomy. In this study, an orthogonal pathway running in parallel to native metabolism and converting renewable dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to 12-aminododecanoic acid methyl ester (ADAME), a building block for the high-performance polymer Nylon 12, was engineered in Escherichia coli and optimized regarding substrate uptake, substrate requirements, host strain choice, flux, and product yield. Efficient DAME uptake was achieved by means of the hydrophobic outer membrane porin AlkL increasing maximum oxygenation and transamination activities 8.3 and 7.6-fold, respectively. An optimized coupling to the pyruvate node via a heterologous alanine dehydrogenase enabled efficient intracellular L-alanine supply, a prerequisite for self-sufficient whole-cell transaminase catalysis. Finally, the introduction of a respiratory chain-linked alcohol dehydrogenase enabled an increase in pathway flux, the minimization of undesired overoxidation to the respective carboxylic acid, and thus the efficient formation of ADAME as main product. The completely synthetic orthogonal pathway presented in this study sets the stage for Nylon 12 production from renewables. Its effective operation achieved via fine tuning the connectivity to native cell functionalities emphasizes the potential of this concept to expand microbial substrate and product scopes.Publicationtype: Journal Article132