E.coli expression

Table 9.1 Evaluation of glycolysis intermediates as substrates for DR5P production by deoxyriboaldolase-expressing E. coli without and with ATP.

Scheme 9.3 DR5P synthesis from glucose and acetaldehyde by baker s yeast and deoxyriboaldolase-expressing E. coli.

The one-pot multi-step enzymatic process described above seemed still impractical on an industrial scale due to the low amount of 2 -deoxyribonucleoside accumula-hon and the low molar yield to added nucleobase (yield to adenine was 33.3%). In addition, it required three kinds of catalyst cells (baker s yeast, deoxyriboaldolase-or phosphopentomutase-expressing E. coli and commercial nucleoside phosphory-lase). It is difficult and complicated to operate the multi-catalysts. If the molar yield of 2 -deoxyribonucleoside to the most expensive material, nucleobase, was improved and the number of catalysts could be reduced, a practical enzymahc process could be developed. 

Prokaryotic expression E. coli Rapid growth with high yields (up to 50% total cell protein) Extensive range of vectors Simple, well-defined growth media Lack of post-translational processing Product may prove toxic to host Product incorrectly folded and inactive High endotoxin content... 

In another study, related to the E. coli K12 proteome, by another group [37], the tryptic digest was first fractionated in 40 fractions by RPLC. Next, Met-containing peptides in these fractions were oxidized by HjOj. These modified proteins were separated from the bulk unmodified proteins by RPLC and fractionated in 8 fractions. These fractions were subsequently analysed by nano-LC-MS-MS on a Q-TOF instrument operated under DDA. After the first RPLC run, an exclusion hst was generated from peptides identified in a MASCOT search of the data, and the sample was then remn. Following this procedure, 754 proteins were identified, which is about 34% of the expressed E. coli K12 proteome. 

Initially, the basis for xylose fermentation in yeasts and fungi was not well understood. Researchers knew that bacteria with xylose isomerase could ferment xylose while fungi with the oxidoreductase uptake system could not, so they sought to express xylose isomerase in S. cerevisiae or other yeasts in order to create an improved xylose fermenter. Ueng et al. [34] cloned the gene for xylose isomerase from E. coli and Chan et al. [35] expressed it in S. pombe. These are the only researchers to have reported success with this approach. Amore et al. [36] expressed the genes from Bacillus and Actinoplanes in S. cerevisiae. Approximately 5 % of the cellular protein consisted of xylose isomerase, but it was not catalytically active. Sarthy et al. [37] expressed E. coli xylose isomerase in S. cerevisiae but found that the protein had only about 10 as much activity as the native protein from E. coli. 

For functional expression, E. coli cultures are grown to late log phase, induced with the addition of isopropyl-y9-D-thioga-... 

EFFICIENT PRODUCTION OF DR5P FROM GLUCOSE AND ACETALDEHYDE VIA FDP BY COUPLING OF ALCOHOLIC FERMENTATION SYSTEM OF BAKER S YEAST AND DEOXYRIBOALDOLASE-EXPRESSING E. COLI... 

Evaluation of Glycolysis Intermediates as the Substrates for 2-Deoxyriboase 5-Phosphate Production by a Deoxyriboaldolase-Expressing E. coli 10B5/pTS8... 

For optimization of DR5P production from the enzymatically prepared FDP and acetaldehyde with deoxyriboaldolase-expressing E. coli 10B5/pTS8, surfactants xylene and polyoxyethylene laurylamine (PL) were added for improvement of the permeation of phosphorylated compounds [10]. Under the preparative reaction... 

Phase-contrast microscopy with a green fluorescent filter (Nikon Eclipse TE300 microscope) Fluorescent-dyed microspheres and GFP-expressing E coli can be viewed under 20X or 40X magnification. 

Transketolase (EC 2.2.1.1), which catalyses condensations between aldehydes and hydroxypyiuvic acid with loss of CO2 (see Vol. 25, Chapter 2, Ref. 9) has been produced in relatively large quantities from an over-expressed E. coli transformant carrying the transketolase gene. This enzynte was fairly non-specific with respect to the aldehyde component, although best results were achieved with a-hydroxyaldehydes. ... 

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