Synthetic co-culture in an interconnected two-compartment bioreactor system: violacein production with recombinant E. coli strains.

The concept of modular synthetic co-cultures holds considerable potential for biomanufacturing, primarily to reduce the metabolic burden of individual strains by sharing tasks among consortium members. However, current consortia often show unilateral relationships solely, without stabilizing feedback control mechanisms, and are grown in a shared cultivation setting. Such 'one pot' approaches hardly install optimum growth and production conditions for the individual partners.

Metabolic control analysis enables rational improvement of E. coli L-tryptophan producers but methylglyoxal formation limits glycerol-based production.

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.

Protein engineering for feedback resistance in 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

The shikimate pathway delivers aromatic amino acids (AAAs) in prokaryotes, fungi, and plants and is highly utilized in the industrial synthesis of bioactive compounds. Carbon flow into this pathway is controlled by the initial enzyme 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS). AAAs produced further downstream, phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), regulate DAHPS by feedback inhibition.

Polyphosphate Kinases Phosphorylate Thiamine Phosphates.

Polyphosphate kinases (PPKs) catalyze the reversible transfer of the γ-phosphate moiety of ATP (or of another nucleoside triphosphate) to a growing chain of polyphosphate (polyP). In this study, we describe that PPKs of various sources are additionally able to phosphorylate thiamine diphosphate (ThP2) to produce thiamine triphosphate (ThP3) and even thiamine tetraphosphate in vitro using polyP as phosphate donor. Furthermore, all tested PPK2s, but not PPK1s, were able to phosphorylate thiamine monophosphate (ThP1) to ThP2 and ThP3 although at low efficiency.

Metabolic control analysis of L-tryptophan producing Escherichia coli applying targeted perturbation with shikimate.

L-tryptophan production from glycerol with Escherichia coli was analysed by perturbation studies and metabolic control analysis. The insertion of a non-natural shikimate transporter into the genome of an Escherichia coli L-tryptophan production strain enabled targeted perturbation within the product pathway with shikimate during parallelised short-term perturbation experiments with cells withdrawn from a 15 L fed-batch production process. Expression of the shikimate/H-symporter gene (shiA) from Corynebacterium glutamicum did not alter process performance within the estimation error.

Opening a Novel Biosynthetic Pathway to Dihydroxyacetone and Glycerol in Mutants through Expression of a Gene Variant () for Fructose 6-Phosphate Aldolase.

Phosphofructokinase (PFK) plays a pivotal role in glycolysis. By deletion of the genes , (encoding the two PFK isoenzymes), and (glucose 6-phosphate dehydrogenase) in K-12, a mutant strain (GL3) with a complete block in glucose catabolism was created. Introduction of plasmid-borne copies of the wild type gene (encoding fructose 6-phosphate aldolase, FSAA) did not allow a bypass by splitting fructose 6-phosphate (F6P) into dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (G3P).

Mechanism of ribosome shutdown by RsfS in Staphylococcus aureus revealed by integrative structural biology approach.

For the sake of energy preservation, bacteria, upon transition to stationary phase, tone down their protein synthesis. This process is favored by the reversible binding of small stress-induced proteins to the ribosome to prevent unnecessary translation. One example is the conserved bacterial ribosome silencing factor (RsfS) that binds to uL14 protein onto the large ribosomal subunit and prevents its association with the small subunit.

Genetic engineering approaches for the fermentative production of phenylglycines.

L-phenylglycine (L-Phg) is a rare non-proteinogenic amino acid, which only occurs in some natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Industrially, more interesting than L-Phg is the enantiomeric D-Phg as it plays an important role in the fine chemical industry, where it is used as a precursor for the production of semisynthetic β-lactam antibiotics. Based on the natural L-Phg operon from Streptomyces pristinaespiralis and the stereo-inverting aminotransferase gene hpgAT from Pseudomonas putida, an artificial D-Phg operon was constructed.

Author Correction: Split intein-mediated selection of cells containing two plasmids using a single antibiotic.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Split intein-mediated selection of cells containing two plasmids using a single antibiotic.

To build or dissect complex pathways in bacteria and mammalian cells, it is often necessary to recur to at least two plasmids, for instance harboring orthogonal inducible promoters. Here we present SiMPl, a method based on rationally designed split enzymes and intein-mediated protein trans-splicing, allowing the selection of cells carrying two plasmids with a single antibiotic. We show that, compared to the traditional method based on two antibiotics, SiMPl increases the production of the antimicrobial non-ribosomal peptide indigoidine and the non-proteinogenic aromatic amino acid para-amino-L-phenylalanine from bacteria.

Metabolic control analysis of L-tryptophan production with Escherichia coli based on data from short-term perturbation experiments.

E. coli strain NT1259 /pF112aroFBL was able to produce 14.3 g L L-tryptophan within 68 h in a fed-batch process from glycerol on a 15 L scale.

2-Ketogluconate Kinase from H16: Purification, Characterization, and Exploration of Its Substrate Specificity.

We have cloned, overexpressed, purified, and characterized a 2-ketogluconate kinase (2-dehydrogluconokinase, EC 2.7.1.

Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites.

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate.

Metabolic Engineering of for -Amino-Phenylethanol and -Amino-Phenylacetic Acid Biosynthesis.

Aromatic amines are an important class of chemicals which are used as building blocks for the synthesis of polymers and pharmaceuticals. In this study we establish a pathway for the biosynthesis of the aromatic amines amino-phenylethanol (PAPE) and amino-phenylacetic acid (4-APA) in . We combined a synthetic -amino-l-phenylalanine pathway with the fungal Ehrlich pathway.

Production of p-amino-L-phenylalanine (L-PAPA) from glycerol by metabolic grafting of Escherichia coli.

Background: The non-proteinogenic aromatic amino acid, p-amino-L-phenylalanine (L-PAPA) is a high-value product with a broad field of applications. In nature, L-PAPA occurs as an intermediate of the chloramphenicol biosynthesis pathway in Streptomyces venezuelae. Here we demonstrate that the model organism Escherichia coli can be transformed with metabolic grafting approaches to result in an improved L-PAPA producing strain.

Fed-batch production of l-tryptophan from glycerol using recombinant Escherichia coli.

l-tryptophan is an essential amino acid of high industrial interest that is routinely produced by microbial processes from glucose as carbon source. Glycerol is an alternative substrate providing a variety of economic and metabolic advantages. Process performance of the recombinant l-tryptophan producer Escherichia coli NT367 was studied in controlled fed-batch processes.

Extended substrate range of thiamine diphosphate-dependent MenD enzyme by coupling of two C-C-bonding reactions.

Carboligations catalyzed by aldolases or thiamine diphosphate (ThDP)-dependent enzymes are well-known in biocatalysis to deliver enantioselective chain elongation reactions. A pyruvate-dependent aldolase (2-oxo-3-deoxy-6-phosphogluconate aldolase [EDA]) introduces a chiral center when reacting with the electrophile, glyoxylic acid, delivering the (S)-enantiomer of (4S)-4-hydroxy-2-oxoglutarate [(S)-HOG]. The ThDP-dependent enzyme MenD (2-succinyl-5-enol-pyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (SEPHCHC synthase)) enables access to highly functionalized substances by forming intermolecular C-C bonds with Michael acceptor compounds by a Stetter-like 1,4- or a benzoin-condensation 1,2-addition of activated succinyl semialdehyde (ThDP adduct formed by decarboxylation of 2-oxoglutarate).

Phosphoenolpyruvate Transporter Enables Targeted Perturbation During Metabolic Analysis of L-Phenylalanine Production With Escherichia coli.

Usually perturbation of the metabolism of cells by addition of substrates is applied for metabolic analysis of production organisms, but perturbation studies are restricted to the endogenous substrates of the cells under study. The goal of this study is to overcome this limitation by making phosphoenolpyruvate (PEP) available for perturbation studies with Escherichia coli producing L-phenylalanine. A production strain overexpressing a PEP-transporter variant (UhpT-D388C) is applied in a standardized fed-batch production-process on a 42 L-scale.

Production of human milk oligosaccharides by enzymatic and whole-cell microbial biotransformations.

Human milk oligosaccharides (HMO) are almost unique constituents of breast milk and are not found in appreciable amounts in cow milk. Due to several positive aspects of HMO for the development, health, and wellbeing of infants, production of HMO would be desirable. As a result, scientists from different disciplines have developed methods for the preparation of single HMO compounds.

Functional expression of a human GDP-L-fucose transporter in Escherichia coli.

Objectives: To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes.

Results: The heterologous expression of the recombinant and codon-adapted human GDP-L-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles.

Novel mode of inhibition by D-tagatose 6-phosphate through a Heyns rearrangement in the active site of transaldolase B variants.

Transaldolase B (TalB) and D-fructose-6-phosphate aldolase A (FSAA) from Escherichia coli are C-C bond-forming enzymes. Using kinetic inhibition studies and mass spectrometry, it is shown that enzyme variants of FSAA and TalB that exhibit D-fructose-6-phosphate aldolase activity are inhibited covalently and irreversibly by D-tagatose 6-phosphate (D-T6P), whereas no inhibition was observed for wild-type transaldolase B from E. coli.

Synthesis of fucosylated lacto-N-tetraose using whole-cell biotransformation.

Fucosylated oligosaccharides present a predominant group of free oligosaccharides found in human milk. Here, a microbial conversion of lactose, D-glucose and L-fucose to fucosylated lacto-N-tetraose by growing Escherichia coli cultures is presented. The recombinant expression of genes encoding for the β1,3-N-acetylglucosaminyltransferase (LgtA) and the β1,3-galactosyltransferase (WbgO) enables the whole-cell biotransformation of lactose to lacto-N-tetraose.

Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose.

Lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc) is one of the most abundant oligosaccharide structures in human milk. We recently described the synthesis of lacto-N-tetraose by a whole-cell biotransformation with recombinant Escherichia coli cells. However, only about 5% of the lactose was converted into lacto-N-tetraose by this approach.

Perturbation Experiments: Approaches for Metabolic Pathway Analysis in Bioreactors.

In the last decades, targeted metabolic engineering of microbial cells has become one of the major tools in bioprocess design and optimization. For successful application, a detailed knowledge is necessary about the relevant metabolic pathways and their regulation inside the cells. Since in vitro experiments cannot display process conditions and behavior properly, process data about the cells' metabolic state have to be collected in vivo.