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.
View Article and Find Full Text PDFHuman 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.
View Article and Find Full Text PDFObjectives: 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.
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.
View Article and Find Full Text PDFLacto-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.
View Article and Find Full Text PDFAdv Biochem Eng Biotechnol
February 2016
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.
View Article and Find Full Text PDFThe need for quantitative intracellular metabolome information is central to modern applied biotechnology and systems biology. In most cases, sample preparation and metabolite analysis result in degradation of metabolites and signal suppression due to metabolite instability and matrix effects during LC-MS analysis. Therefore the application of uniformly (U) (13)C-labeled cell extract as an internal standard has gained interest in recent years.
View Article and Find Full Text PDFThe transport of organophosphates across the cytoplasma membrane is mediated by organophosphate:phosphate antiporter proteins. In this work, we present the application of a recombinant phosphoenolpyruvate:phosphate antiporter for isotopic labeling experiments in E. coli strains.
View Article and Find Full Text PDFHuman milk oligosaccharides (HMOs) constitute the third most abundant solid component of human milk. HMOs have been demonstrated to show positive effects on the infant's well-being. Despite numerous studies, more physiological analyses of single compounds are needed to fully elucidate these effects.
View Article and Find Full Text PDFBackground: For the production of L-phenylalanine (L-Phe), two molecules of phosphoenolpyruvate (PEP) and one molecule erythrose-4-phosphate (E4P) are necessary. PEP stems from glycolysis whereas E4P is formed in the pentose phosphate pathway (PPP). Glucose, commonly used for L-Phe production with recombinant E.
View Article and Find Full Text PDFA fed-batch process was studied with lactate and glycerol supply in the growth phase and glycerol supply during L-phenylalanine production with recombinant E. coli K-12. Lactic acid feeding was necessary for growth because the genes encoding the PEP-consuming pyruvate kinase isoenzymes (pykA, pykF) have been deleted.
View Article and Find Full Text PDFFed-batch production of the aromatic amino acid L-phenylalanine was studied with recombinant Escherichia coli strains on a 15 L-scale using glycerol as carbon source. Flux Variability Analysis (FVA) was applied for intracellular flux estimation to obtain an insight into intracellular flux distribution during L-phenylalanine production. Variability analysis revealed great flux uncertainties in the central carbon metabolism, especially concerning malate consumption.
View Article and Find Full Text PDFBackground: The trisaccharide 2'-fucosyllactose (2'-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2'-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2'-FL can be produced enzymatically in vitro and in vivo.
View Article and Find Full Text PDFThe vitamin E family consists of 4 tocopherol and 4 tocotrienol compounds. During recent years, tocotrienols have gained increased interest due to their biological activities that are beyond the vitamin E activity. Here we report the engineering of plasmid-free Escherichia coli strains for an efficient synthesis of 2-methyl-6-geranylgeranyl-benzoquinol (MGGBQ), the central precursor for all four natural tocotrienol compounds.
View Article and Find Full Text PDFBackground: The xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E.
View Article and Find Full Text PDFThe heterologous synthesis of lycopene in non-carotenogenic Escherichia coli required the introduction of the biosynthesis genes crtE, crtB, and crtI. Recombinant E. coli strains, expressing each lycopene biosynthesis gene from Pantoea ananatis using multi-copy plasmid or single-copies after stable chromosomal integration, were cultivated and the formation of lycopene was investigated.
View Article and Find Full Text PDFWe report a method for the integration of expression cassettes into the Escherichia coli chromosome using rare and dispensable sugar degradation gene loci as sites for integration. Clones carrying successfully recombined DNA fragments in the chromosome are easily screened using a solid differential medium containing the respective sugar compound. As an example for the heterologous expression of a complex natural product biosynthesis pathway, we show the stepwise chromosomal integration of the zeaxanthin biosynthesis pathway from Pantoea ananatis into E.
View Article and Find Full Text PDFThe biosynthesis of natural products in a fast growing and easy to manipulate heterologous host system, such as Escherichia coli, is of increasing interest in biotechnology. This procedure allows the investigation of complex natural product biosynthesis and facilitates the engineering of pathways. Here we describe the cloning and the heterologous expression of tocochromanol (vitamin E) biosynthesis genes in E.
View Article and Find Full Text PDFThe enediyne antibiotic calicheamicin (CLM) gamma(1)(I) is a prominent antitumor agent that is targeted to DNA by a novel aryltetrasaccharide comprised of an aromatic unit and four unusual carbohydrates. Herein we report the heterologous expression and the biochemical characterization of the two "internal" glycosyltransferases CalG3 and CalG2 and the structural elucidation of an enediyne glycosyltransferase (CalG3). In conjunction with the previous characterization of the "external" CLM GTs CalG1 and CalG4, this study completes the functional assignment of all four CLM GTs, extends the utility of enediyne GT-catalyzed reaction reversibility, and presents conclusive evidence of a sequential glycosylation pathway in CLM biosynthesis.
View Article and Find Full Text PDFGDP-N-acetyl-d-perosamine is a precursor of the LPS-O-antigen biosynthesis in Escherichia coli O157:H7. Like other GDP-6-deoxyhexoses, GDP-N-acetyl-d-perosamine is supposed to be synthesized via GDP-4-keto-6-deoxy-d-mannose, followed by a transamination- and an acetylation-reaction catalyzed by PerA and PerB. In this study, we have overproduced and purified PerA and PerB from E.
View Article and Find Full Text PDFThe glycosyltransferase AveBI, which is involved in the biosynthesis of the macrolide antihelmintic avermectin (AVM), was characterized in vitro. AveBI was confirmed to catalyze two separate iterative additions of l-oleandrose, and the reversibility of AveBI-catalyzed reaction was also demonstrated. Investigation of sugar nucleotide specificity revealed 10 unique sugar nucleotide substrates which, in combination with five distinct aglycones, led to the production of 50 differentially glycosylated AVM variants.
View Article and Find Full Text PDFGlycosyltransferases (GTs), an essential class of ubiquitous enzymes, are generally perceived as unidirectional catalysts. In contrast, we report that four glycosyltransferases from two distinct natural product biosynthetic pathways-calicheamicin and vancomycin-readily catalyze reversible reactions, allowing sugars and aglycons to be exchanged with ease. As proof of the broader applicability of these new reactions, more than 70 differentially glycosylated calicheamicin and vancomycin variants are reported.
View Article and Find Full Text PDFRebeccamycin and staurosporine represent two broad classes of indolocarbazole glycoside natural products with antitumor properties. Based upon previous sequence annotation and in vivo studies, rebG encodes for the rebeccamycin N-glucosyltransferase, and rebM for the requisite 4'-O-methyltransferase. In the current study, an efficient in vivo biotransformation system for RebG was established in both Streptomyces lividans and Escherichia coli.
View Article and Find Full Text PDF[reaction: see text] The rapid diversification of glycopeptides via glycorandomization reveals that significantly diverse substitutions are tolerated and suggests there may be a synergistic benefit to the construction of mechanistically related natural product core scaffold fusions. This work also further highlights the utility of chemoenzymatic approaches to diversify complex natural product architectures.
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