Why m9 medium

You must have JavaScript enabled in your browser to utilize the functionality of this website. This medium is useful in maintaining positive selection pressure on plasmids coding for substances such as amino acids or vitamins.

It can also be used to maintain bacteria for use with M M9 Broth can be supplemented with specific amino acids or other meolites that allows for specific selection pressures that the researcher may require. This media is supplemented with casamino acids, which provides the bacteria with a rich source of nitrogen that they can use during growth. They also contain potassium and sodium phosphates.

A Available substrate flux from acetyl-CoA to mevalonate may be improved in rpoB -strains, due to previously observed host adaptation upregulating glucose uptake and fermentative metabolism, while downregulating several cellular functions including the tricarboxylic acid TCA cycle LaCroix et al. Simplified sketch of central carbon metabolism shown. B Production of heterologous mevalonate, and C Glucose consumption in wildtype MG and glucose M9 minimal medium-adapted rpoB strains during fermentation.

D Specific uptake and production rate of glucose and mevalonate, respectively, in the glucose to mevalonate conversion phase. Batch growth screens of the medium-adapted strains yielded an improved productivity over the wild type MG starting strain.

In M9 minimal medium with 0. Production-related decreases in the growth rates are not unexpected given the pathway intermediate toxicities of mevalonate biosynthesis Kizer et al. Table 1. In addition to increased mevalonate production rates with the rpoB mutations, the faster initial growth when production is not detected may also contribute to faster formation of product in a bioprocess, though the main gain in production speed appears to arise from the improved specific productivity.

Unlike the pathway-free rpoB mutants LaCroix et al. This indicates that these strains diverted the increased flux to acetyl-CoA to mevalonate. However, the increase in mevalonate production rate did not proportionally follow the increase in glucose uptake rate Figure 1D. Instead, during the mevalonate production phase when glucose was present, the mutants intermittently secreted lactate and succinate as byproducts Table 1 , followed by reuptake upon glucose depletion Supplementary Figures S1 — S3.

This intermittent lactate and succinate accumulation is an indication that there is likely a flux limitation or redox imbalance in the engineered pathway to fully utilize the increased glycolytic flux. Future optimization should target this to take full advantage of the gain in productivity without a trade-off in carbon yield. There are many examples of elimination of both lactate and succinate as byproducts which can be examined King et al.

Increased productivity appeared to be the main outcome of the implemented rpoB EK mutation while end-point titers following 48 h of cultivation did not significantly differ between the different strains.

Process productivity is an important parameter in the design of economic bioprocesses, as it largely determines the bioreactor volume required Ikeda, Thus, an improved productivity can significantly decrease the capital investment necessary for a given process.

As shown in this study, improvements in productivity can result from ALE selecting for improved growth rates. The improved specific productivities in the adapted rpoB mutant strains examined here may be explained by the global adaptation of the metabolism to minimal M9 medium in excess glucose.

The rpoB EK mutation reduces the expression levels of functions involved in environment and stress tolerance, while it increases exponential-phase rates of glucose uptake and acetate production Utrilla et al. Overall, this adaptation likely favors an increased availability of acetyl-CoA for the first specific step of the mevalonate product pathway.

Addition of the pyrE-rph mutation to rpoB EK did not appear to significantly improve productivity further Figure 1D. This non-increase is likely due to the finding that the pyrE-rph -associated fitness gain resulted from restoration of a local biosynthetic defect of MG Jensen, Thus, the effect of this mutation appears to be specific to this biosynthetic pathway and does increase growth rate, but not the glucose uptake rate LaCroix et al.

Improvement of glycolytic flux is generally attractive as a means to increase productivity of a number of metabolic production processes, e. In this work, we demonstrate how a single adaptive point-mutation in the global transcriptional regulator RpoB can elevate the specific glucose uptake rate to increase heterologous mevalonate productivity.

The results of the strategy are supported by rationally optimized productivities of pyruvic acid and mevalonate, respectively, by increased glucose uptake via specific deletions of the genes atpFH ATP synthase F and H subunits and sucA 2-oxoglutarate dehydrogenase Causey et al.

However, high volumetric productivities of, respectively, 6. These volumetric productivities were operating at higher cellular densities which complicates direct comparison. ALE experimentation for strain design has the potential to sample a larger mutation space than rational engineering as it does not require a priori knowledge of what to engineer in a given strain. However, ALE requires a selectable feature. As shown in this brief report, ALE-based substrate and media adaptation is a simple strategy to generate a platform strain with improved productivity of a heterologous metabolite, as resources can be diverted from cellular processes not needed for growth in such a controlled environment to production.

Furthermore, recent alternative strategies employing biosensors to screen for improved glycolytic flux Lehning et al.

However, such approaches are currently limited by the availability of specific biosensors. The results from this study demonstrate the advantage of using key growth-enhancing mutations found in ALE to optimize host strains toward industrial cultivation medium, which results in an improved production rate of a heterologous metabolite.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Barrick, J. Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature , — Causey, T. Engineering Escherichia coli for efficient conversion of glucose to pyruvate.

Conrad, T. RNA polymerase mutants found through adaptive evolution reprogram Escherichia coli for optimal growth in minimal media. Daegelen, P. Dragosits, M. Adaptive laboratory evolution—principles and applications for biotechnology. Cell Fact. Feist, A. Model-driven evaluation of the production potential for growth-coupled products of Escherichia coli. Genee, H. Functional mining of transporters using synthetic selections. Ikeda, M. Amino acid production processes.

Jensen, K. PubMed Abstract Google Scholar. King, Z. Literature mining supports a next-generation modeling approach to predict cellular byproduct secretion. Kizer, L. Application of functional genomics to pathway optimization for increased isoprenoid production.

Koebmann, B. The glycolytic flux in Escherichia coli is controlled by the demand for ATP. LaCroix, R. Use of adaptive laboratory evolution to discover key mutations enabling rapid growth of Escherichia coli K MG on glucose minimal medium. Lehning, C. Assessing glycolytic flux alterations resulting from genetic perturbations in E. Long, C.

Fast growth phenotype of E.