Short communication

Regulation of pyruvate dehydrogenase activity in Escherichia coli K12

Pyruvate (pyruvic acid) is a cellular metabolite that is produced at the metabolic junction of tricarboxylic acid and the glycolysis cycle. Pyruvate is employed in a variety of industries, including agricultural, food, pharmaceuticals, and food. Microbial pyruvate synthesis, whether from yeast or bacteria, relies on minimizing pyruvate catabolism while simultaneously limiting the accumulation of several possible by-products. In this review, we cover research into improving pyruvate formation that has focused on both strain development and process development. Strain formation necessitates a strong understanding of carbohydrate metabolism and the several competing enzymes that use pyruvate as a substrate, and it frequently blends traditional isolation and mutation methods with current metabolic engineering techniques. Understanding operational modes and their impact on microbial growth and product generation is essential for process development.

Agricultural biomass remains as one of the commonly found waste on Earth. Although valorisation of these wastes has been studied in detail, the fermentation-based processes still need improvement due to the high cost of hydrolysing enzymes, and the presence of growth inhibitors which constrains the fermentation to produce high-value products. To address these challenges, we developed an integrated process in this study combining abiotic- and bio-catalysis to produce l-tyrosine from corn husk. The first step involved a one-pot hydrolytic hydrogenation tandem reaction without the use of the expensive enzymes, which yielded a mixture of polyols and sugars. Without any purification, these crude hydrolysates can be almost completely utilized by an engineered Escherichia coli strain, which did not exhibit any growth inhibition. The strain produced 0.44 g/L l-tyrosine from 10 g/L crude corn husk hydrolysates, demonstrating the feasibility of converting agricultural biomass into a valuable aromatic amino acid via an integrated process.

The reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO₂ as sole carbon sources. Labeling with ¹³C-formate proved the assimilation of the C1 substrate via the pathway metabolites. Genetic analysis of intermediate isolates revealed a mutational path followed throughout the adaptation process. Mutations were detected affecting the copy number (gene ftfL) or the coding sequence (genes folD and lpd) of genes which specify enzymes implicated in the three steps forming glycine from formate and CO₂, the central metabolite of the synthetic pathway. The mutation R191S present in methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) abolishes the inhibition of cyclohydrolase activity by the substrate formyl-tetrahydrofolate. The mutation R273H in lipoamide dehydrogenase (Lpd) alters substrate affinities as well as kinetics at physiological substrate concentrations likely favoring a reactional shift towards lipoamide reduction. In addition, genetic reconstructions proved the necessity of all three mutations for formate assimilation by the adapted cells. The largely unpredictable nature of these changes demonstrates the usefulness of the evolutionary approach enabling the selection of adaptive mutations crucial for pathway engineering of biotechnological model organisms.

Maintenance of metabolic redox homeostasis is essential to all life and is a key factor in many biotechnological processes. Changes in the redox state of NAD affect metabolic fluxes, mediate regulation and signal transduction, and thus determine growth and productivity. Here we establish an in vivo monitoring system for the dynamics of the cytosolic NADH/NAD⁺ ratio in the basidiomycete Ustilago maydis using the ratiometric fluorescent sensor protein Peredox-mCherry. Metabolic redox dynamics were determined in the cytosol of living cells with high time resolution under biotechnologically relevant conditions, i.e. with high cell density and high aeration. Analytical boundary conditions for reliable analysis were determined, and perturbations in C-, N- or O- availability had marked impact on the cytosolic NADH/NAD⁺ ratio. NAD redox dynamics could be manipulated in lines inducibly expressing a water-forming NADH oxidase as a synthetic reductant sink. The establishment of Peredox-mCherry in U. maydis and the analysis of NAD redox dynamics provides a versatile methodology for the in vivo investigation of cellular metabolism, and contributes fundamental knowledge for rational design and optimization of biocatalysts.

In order to evaluate the mechanical properties of poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)] and its correlation with the LA fraction, P(LA-co-3HB)s with a variety of LA fractions were prepared using recombinant Escherichia coli expressing the LA-polymerizing enzyme and monomer supplying enzymes. The LA-overproducing mutant E. coli JW0885 with a pflA gene disruption was used for the LA-enriched polymer production. The LA fraction was also varied by jar-fermentor based fine-regulation of the anaerobic status of the culture conditions, resulting in LA fractions ranging from 4 to 47 mol%. In contrary to the opaque P(3HB) film, the copolymer films attained semitransparency depending on the LA fraction. Young's modulus values of the P(LA-co-3HB)s (from 148 to 905 MPa) were lower than those of poly(lactic acid) (PLA) (1020 MPa) and P(3HB) (1079 MPa). In addition, the value of elongation at break of the copolymer with 29 mol% LA reached 150%. In conclusion, P(LA-co-3HB)s were found to be a comparatively pliable and flexible material, differing from both of the rigid homopolymers.

Recent metabolic engineering were reviewed for the production of useful metabolites. Although much attention has been paid to metabolic engineering with some practical success, without profound insight into metabolic regulation, the modification of pathways based on the knockout of corresponding genes and/or amplification by plasmids does not necessarily lead to a significant improvement in cell growth and/or the production of specific metabolites. Although much information is available on the genetic regulation, biochemistry, and physiology of cellular metabolism, we are still far from understanding their overall regulation. Metabolic flux distribution is the manifestation of regulation at the enzyme level of the concentrations of intracellular metabolites. Moreover, proteins (including enzymes) are encoded by the corresponding metabolic pathway genes, which are under control of global regulators that respond to the environment. It is strongly desired that different levels of (omics) information be integrated by comparison of the data from different mutants and/or the different cultural environments using a systems biology approach. In particular, the roles of global regulators are quite important for metabolic regulation.