Accumulation in E. coli

P2 Chromium Accumulation in E. coli Cells. The cellular uptake of chromium is presented as milligrams of chromium per dry weight of treated E. coli cells (Table 1). (From Plaper et al., 2002)... 

Based on the available data, a tentative model (Figure 8.6) of the mechanisms responsible for PolyP accumulation in E. coli has been proposed (Ault-Riche et al, 1998 Rao and Kornberg, 1999). 

Although the behavior just described seems relatively simple, transport mechanisms in living cells probably have several more kinetically distinct steps than those assumed for the simple enzyme-substrate reactions underlying the Michaelis-Menten mechanism. For example, as ferric enterobactin is accumulated in E. coli, it has to pass through the outer membrane, the periplasm, and the cytoplasm membrane, and is probably subjected to reduction of the metal in a low-pH compartment or to ligand destruction. 

K. Andry and R. C. Bockrath, Dihydrostreptomycin accumulation in E. coli. Nature, 251... 

Careful empirical selection of the expression platform for carotenogenesis has included selection of the best strains for stability and degree of accumulation and the selection of compatible drug-resistance combinations and low copy number polycistronic plasmids to enhance product accumulation by decrease of metabolic burden." 5 Matthews and Wurtzel discussed culture and induction conditions - that have been explored in most studies. Most efforts to engineer carotenoid biosynthesis in E. coli focused on the genes and enzymes of the pathway and had a modest effect on improved accumulation. For example, substitution and over-expression of a GGPPS that uses IPP directly (discussed in... 

Matthews, P.D. and Wurtzel, E.T., Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase, Appl. Microbiol. Biotechnol. 53, 396, 2000. 

It is worth mentioning that metabolic engineering of E. coli recently provided recombinant strains which synthesized PHAMCL from gluconate. For this, beside phaC2Po or phaClPa> the thioesterase I from E. coli (TesA) [128] or the acyl-ACP thioesterase from Umbellularia californica [129], respectively, were expressed in E. coli. However, the amounts of PHAMCL accumulated in the cells were rather low, and this artificial pathway was not very efficient. 

There are an overwhelming number of studies which successfully demonstrated heterologous expression of PHA synthesis genes in bacteria it will therefore not be possible to mention them all. Establishment of functional active PHA biosynthesis pathways in E. coli requires not only a PHA synthase but also enzymes that allow the conversion of metabolites, which derive from the provided carbon source, into the R isomers of hydroxyacyl-coenzyme A thioesters that are used as a substrate by the respective PHA synthase. Otherwise, no or only marginal amounts of PHAs are accumulated. 

The vast bulk of proteins synthesized naturally by E. coli (i.e. its homologous proteins) are intracellular. Few are exported to the periplasmic space or released as true extracellular proteins. Heterologous proteins expressed in E. coli thus invariably accumulate in the cell cytoplasm. Intracellular protein production complicates downstream processing (relative to extracellular production) as ... 

Figure 8.7 Overview of the manufacture of Betaferon, a recombinant human IFN-(3 produced in E. coli. The product differs from native human IFN-(3 in that it is unglycosylated and cysteine residue 17 had been replaced by a serine residue. E. coli fermentation is achieved using minimal salts/glucose media and product accumulates intracellularly in inclusion body (IB) form. During downstream processing, the lbs are solubilized in butanol, with subseguent removal of this denaturant to facilitate product refolding. After two consecutive gel-filtration steps, excipients are added, the product is filled into glass vials and freeze-dried. It exhibits a shelf life of 18 months when stored at 2-8 °C...

A synthetic DNA cassette was designed that encoded the repeat sequence Lys-25 (Figure 2). This DNA monomer was synthesized, sequenced, and self-ligated to afford a mixture of DNA concatamers. A 3,000 base pair concatameric gene was iolated from this mixture, which encoded a polymer of Lys-25 with a molecular mass of approximately 90 kD. Bacterial expression of the concatameric gene afforded a protein of the expected molecular mass that accumulates to high levels in E. coli (Figure 3). 

A promoter to be used in E. coli should have certain characteristics to render it suitable for high-level production of recombinant protein. First, it must be strong, resulting in the accumulation of protein making up to 10-30% or more of the total cellular protein. Second, it should exhibit a minimal level of basal transcriptional activity. Third, promoters should be capable of induction in a simple and cost-effective manner. 

Functioning in a somewhat different way in E. coli are 6- to 12-residue periplasmic membrane-derived oligosaccharides. These are p-1,2- and P 1,6-linked glucans covalently linked to sn-l-phospho-glycerol, phosphoethanolamine, or succinate (see Fig. 8-28).b e/f They accumulate in the periplasm when cells are placed in a medium of low osmolarity. 

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