A new Zymomonas mobilis platform strain for the efficient production of chemicals.

Background: Zymomonas mobilis is well known for its outstanding ability to produce ethanol with both high specific productivity and with high yield close to the theoretical maximum. The key enzyme in the ethanol production pathway is the pyruvate decarboxylase (PDC) which is converting pyruvate to acetaldehyde. Since it is widely considered that its gene pdc is essential, metabolic engineering strategies aiming to produce other compounds derived from pyruvate need to find ways to reduce PDC activity.

Construction and comparison of different vehicles for heterologous gene expression in Zymomonas mobilis.

Zymomonas mobilis has the potential to be an optimal chassis for the production of bulk chemicals derived from pyruvate. However, a lack of available standardized and characterized genetic tools hinders both efficient engineering of Z. mobilis and progress in basic research on this organism.

Toward a modeling, optimization, and predictive control framework for fed-batch metabolic cybergenetics.

Biotechnology offers many opportunities for the sustainable manufacturing of valuable products. The toolbox to optimize bioprocesses includes extracellular process elements such as the bioreactor design and mode of operation, medium formulation, culture conditions, feeding rates, and so on. However, these elements are frequently insufficient for achieving optimal process performance or precise product composition.

Integrated biophysical matching of bacterial nanocellulose coronary artery bypass grafts towards bioinspired artery typical functions.

Revascularization via coronary artery bypass grafting (CABG) to treat cardiovascular disease is established as one of the most important lifesaving surgical techniques worldwide. But the shortage in functionally self-adaptive autologous arteries leads to circumstances where the clinical reality must deal with fighting pathologies coming from the mismatching biophysical functionality of more available venous grafts. Synthetic biomaterial-based CABG grafts did not make it to the market yet, what is mostly due to technical hurdles in matching biophysical properties to the complex demands of the CABG niche.

Zymo-Parts: A Golden Gate Modular Cloning Toolbox for Heterologous Gene Expression in .

is a microorganism with extremely high sugar consumption and ethanol production rates and is generally considered to hold great potential for biotechnological applications. However, its genetic engineering is still difficult, hampering the efficient construction of genetically modified strains. In this work, we present Zymo-Parts, a modular toolbox based on Golden-Gate cloning offering a collection of promoters (including native, inducible, and synthetic constitutive promoters of varying strength), an array of terminators and several synthetic ribosomal binding sites and reporter genes.

Enabling anaerobic growth of Escherichia coli on glycerol in defined minimal medium using acetate as redox sink.

Glycerol has become an attractive substrate for bio-based production processes. However, Escherichia coli, an established production organism in the biotech industry, is not able to grow on glycerol under strictly anaerobic conditions in defined minimal medium due to redox imbalance. Despite extensive research efforts aiming to overcome these limitations, anaerobic growth of wild-type E.

Is energy excess the initial trigger of carbon overflow metabolism? Transcriptional network response of carbon-limited Escherichia coli to transient carbon excess.

Background: Escherichia coli adapted to carbon-limiting conditions is generally geared for energy-efficient carbon utilization. This includes also the efficient utilization of glucose, which serves as a source for cellular building blocks as well as energy. Thus, catabolic and anabolic functions are balanced under these conditions to minimize wasteful carbon utilization.

Immobilization of an amino acid racemase for application in crystallization-based chiral resolutions of asparagine monohydrate.

Integration of racemization and a resolution process is an attractive way to overcome yield limitations in the production of pure chiral molecules. Preferential crystallization and other crystallization-based techniques usually produce low enantiomeric excess in solution, which is a constraint for coupling with racemization. We developed an enzymatic fixed bed reactor that can potentially overcome these unfavorable conditions and improve the overall yield of preferential crystallization.

Synthesis of lipid-linked oligosaccharides by a compartmentalized multi-enzyme cascade for the in vitro N-glycosylation of peptides.

A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes.

The Impact of , and Mutations on Growth, Gene Expression and Protein Acetylation in K-12.

Acetate is a characteristic by-product of K-12 growing in batch cultures with glucose, both under aerobic as well as anaerobic conditions. While the reason underlying aerobic acetate production is still under discussion, during anaerobic growth acetate production is important for ATP generation by substrate level phosphorylation. Under both conditions, acetate is produced by a pathway consisting of the enzyme phosphate acetyltransferase (Pta) producing acetyl-phosphate from acetyl-coenzyme A, and of the enzyme acetate kinase (AckA) producing acetate from acetyl-phosphate, a reaction that is coupled to the production of ATP.

Zymomonas mobilis metabolism: Novel tools and targets for its rational engineering.

Zymomonas mobilis is an α-proteobacterium that interests the biofuel industry due to its perfect ethanol fermentation yields. From its first description as a bacterial isolate in fermented alcoholic beverages to date, Z. mobilis has been rigorously studied in directions basic and applied.

Improvement of Acetaldehyde Production in by Engineering of Its Aerobic Metabolism.

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium , this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of , it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration.

All three quinone species play distinct roles in ensuring optimal growth under aerobic and fermentative conditions in E. coli K12.

The electron transport chain of E. coli contains three different quinone species, ubiquinone (UQ), menaquinone (MK) and demethylmenaquinone (DMK). The content and ratio of the different quinone species vary depending on the external conditions.

Temperature-dependent dynamic control of the TCA cycle increases volumetric productivity of itaconic acid production by Escherichia coli.

Based on the recently constructed Escherichia coli itaconic acid production strain ita23, we aimed to improve the productivity by applying a two-stage process strategy with decoupled production of biomass and itaconic acid. We constructed a strain ita32 (MG1655 ΔaceA Δpta ΔpykF ΔpykA pCadCs), which, in contrast to ita23, has an active tricarboxylic acid (TCA) cycle and a fast growth rate of 0.52 hr at 37°C, thus representing an ideal phenotype for the first stage, the growth phase.

Bistability and Nonmonotonic Induction of the lac Operon in the Natural Lactose Uptake System.

The Escherichia coli lac operon is regulated by a positive feedback loop whose potential to generate an all-or-none response in single cells has been a paradigm for bistable gene expression. However, so far bistable lac induction has only been observed using gratuitous inducers, raising the question about the biological relevance of bistable lac induction in the natural setting with lactose as the inducer. In fact, the existing experimental evidence points to a graded rather than an all-or-none response in the natural lactose uptake system.

Model-based metabolic engineering enables high yield itaconic acid production by Escherichia coli.

Itaconic acid is a high potential platform chemical which is currently industrially produced by Aspergillus terreus. Heterologous production of itaconic acid with Escherichia coli could help to overcome limitations of A. terreus regarding slow growth and high sensitivity to oxygen supply.

Racemization of undesired enantiomers: Immobilization of mandelate racemase and application in a fixed bed reactor.

Production of optically pure products can be based on simple unselective synthesis of racemic mixtures combined with a subsequent separation of the enantiomers; however, this approach suffers from a 50% yield limitation which can be overcome by racemization of the undesired enantiomer and recycling. Application of biocatalyst for the racemization steps offers an attractive option for high-yield manufacturing of commercially valuable compounds. Our work focuses on exploiting the potential of racemization with immobilized mandelate racemase.

Enforced ATP futile cycling increases specific productivity and yield of anaerobic lactate production in Escherichia coli.

The manipulation of cofactor pools such as ATP or NAD(P)H has for long been recognized as key targets for metabolic engineering of microorganisms to improve yields and productivities of biotechnological processes. Several works in the past have shown that enforcing ATP futile cycling may enhance the synthesis of certain products under aerobic conditions. However, case studies demonstrating that ATP wasting may also have beneficial effects for anaerobic production processes are scarce.

Basic regulatory principles of Escherichia coli's electron transport chain for varying oxygen conditions.

For adaptation between anaerobic, micro-aerobic and aerobic conditions Escherichia coli's metabolism and in particular its electron transport chain (ETC) is highly regulated. Although it is known that the global transcriptional regulators FNR and ArcA are involved in oxygen response it is unclear how they interplay in the regulation of ETC enzymes under micro-aerobic chemostat conditions. Also, there are diverse results which and how quinones (oxidised/reduced, ubiquinone/other quinones) are controlling the ArcBA two-component system.

Towards a systems level understanding of the oxygen response of Escherichia coli.

Escherichia coli is a facultatively anaerobic bacterium. With glucose if no external electron acceptors are available, ATP is produced by substrate level phosphorylation. The intracellular redox balance is maintained by mixed-acid fermentation, that is, the production and excretion of several organic acids.

A mathematical model of metabolism and regulation provides a systems-level view of how Escherichia coli responds to oxygen.

The efficient redesign of bacteria for biotechnological purposes, such as biofuel production, waste disposal or specific biocatalytic functions, requires a quantitative systems-level understanding of energy supply, carbon, and redox metabolism. The measurement of transcript levels, metabolite concentrations and metabolic fluxes per se gives an incomplete picture. An appreciation of the interdependencies between the different measurement values is essential for systems-level understanding.

Analysis of Escherichia coli mutants with a linear respiratory chain.

The respiratory chain of E. coli is branched to allow the cells' flexibility to deal with changing environmental conditions. It consists of the NADH:ubiquinone oxidoreductases NADH dehydrogenase I and II, as well as of three terminal oxidases.

Kinase activity of ArcB from Escherichia coli is subject to regulation by both ubiquinone and demethylmenaquinone.

Expression of the catabolic network in Escherichia coli is predominantly regulated, via oxygen availability, by the two-component system ArcBA. It has been shown that the kinase activity of ArcB is controlled by the redox state of two critical pairs of cysteines in dimers of the ArcB sensory kinase. Among the cellular components that control the redox state of these cysteines of ArcB are the quinones from the cytoplasmic membrane of the cell, which function in 'respiratory' electron transfer.

A role for EIIA(Ntr) in controlling fluxes in the central metabolism of E. coli K12.

To investigate a possible role of the nitrogen-PTS (PTS(Ntr)) in controlling carbon metabolism, we determined the growth of Escherichia coli LJ110 and of isogenic derivatives, mutated in components of the PTS(Ntr), on different carbon sources. The PTS(Ntr) is a set of proteins homologous to the PEP-dependent phosphotransferase system (C-PTS) that transfers a phosphate group from PEP over EI(Ntr) (encoded by ptsP) and NPr (encoded by ptsO) to EIIA(Ntr) (encoded by ptsN). Strains deleted in ptsN were characterized by a high acetate production coupled to slow growth on glycolytic substrates.

Glucose transport in Escherichia coli mutant strains with defects in sugar transport systems.

In Escherichia coli, several systems are known to transport glucose into the cytoplasm. The main glucose uptake system under batch conditions is the glucose phosphoenolpyruvate:carbohydrate phosphotransferase system (glucose PTS), but the mannose PTS and the galactose and maltose transporters also can translocate glucose. Mutant strains which lack the enzyme IIBC (EIIBC) protein of the glucose PTS have been investigated previously because their lower rate of acetate formation offers advantages in industrial applications.