Recombinant microbe

Murooka, Y, and T. Imanaka, Recombinant Microbes for Industiial and Agricultural Applications, M. Dekker, New York, 1993. 

C. Antiparasitic Drug Discovery Using Recombinant Microbes... 

Further modifications using the same strain of ODC S. cerevisiae reconstituted a bacterial/plant polyamine synthesis pathway in yeast [41], The ODC strain was transformed with plasmids encoding arginine decarboxylase and ag-matine ureohydrolase, which conferred polyamine-independent growth on the recombinant microbe. A similar construction could be used to screen for inhibitors of the homologous enzymes from Apicomplexan protozoa, which synthesize poly amines through this pathway [42]. 

Collections of synthetic compounds are easily screened in recombinant microbes [6,13], Fermentation extracts present more problems, since they may contain nutrients that reduce dependence on the target protein. It proved difficult to screen fermentation extracts in the ODC assay [40], since even trace amounts of polyamines in the extract eliminate dependence on the parasite enzyme. Conversely, the PFK assay was compatible with fermentations. The suitability of fermentation extracts must be empirically determined in each case. 

Risk/Benefit Analysis of Recombinant Microbe-Based Screens... 

Summarized in Table 4, the advantages of an HTS platform based on recombinant microbes are obvious for relatively small screening operations (e.g., parasiticide... 

The unique benefit of recombinant microbes for screening is that target specificity is evaluated early in the process. Compounds that nonspecifically affect enzyme function (by sulfhydryl modification, cation chelation, alkylation, etc.) will not survive the comparative testing phase. The hit rate in our laboratories in these types of screens has been under 0.01% (unpublished observations), which means that additional attention can be focused only on the most promising candidates. In contrast, screens that employ purified recombinant enzymes for HTS often have hit rates >2.5% (unpublished observations). Further characterization of so many candidates requires a considerable effort in both biology and chemistry laboratories. 

The biggest concern over the use of recombinant microbes is that microbial cell walls constitute a permeability barrier for test compounds. Enzyme inhibitors that cannot accumulate in bacterial or yeast cytoplasm will appear as false nega-... 

Undeniably, not all targets are suited for HTS in recombinant microbes. In our own experience, we found that we were unable to complement a PFK strain of S. cerevisiae with the cDNA encoding H. contortus PFK, despite the fact that it was functionally expressed in E. coli (unpublished observations). Conversely, we initially attempted to construct an ODC screen using a strain of E. coli that was unable to synthesize poly amines [40], However, this strain required mutations in multiple genes in order to achieve the polyamine- phenotype, and proved to be too leaky for routine screening [40], The ODC- strain of S. cerevisiae was much better suited for HTS. 

Figure 6. Hydroxylation of alkanes catalyzed by recombinant microbes harboring the alkane monooxygenase (AMO) of P. putida GPol (formerly known as P. oleovorans GPol). A fraction ofthe broad substrate spectrum of AMO is shown [102].

Additional References Murooka, Y. and T. Imanka (ed.). Recombinant Microbes for Industrial and Agricultural Applications, Dekker, NY, 1993. Click, B. R. and J. J. Pasternak, Molecular Biotechnology Principles and Applications of Recombinant DNA, ASM Press, Herndon, VA, 1994. Bajpai, Rakesh K., and Ales Prokop, eds. Recombinant DNA Technology II, Anru of the New York Academy of Sciences, vol. 721,1993. 

M. Ikemi, Industrial chemicals enzymic transformation by recombinant microbes, Bioprocess Technology 1994, 19, 797-813. 

Sano K (1994) Host-vector systems for amino acid-producing coryneform bacteria. In Recombinant microbes for industrial and agricultural applications. Marcel Dekker, p 485... 

Recombinant Microbes for Industrial and Agricultural Applications Muraoka, Y. Imanaka, I. Eds. Marcel Dekker New York, New York, 1993. 

Zollner A, Buchheit D, Meyer MR, Maurer HH, Peters FT, Bureik M (2010) Production of human phase 1 and 2 metabolites by whole-cell biotransformation with recombinant microbes. Bioanalysis 2 1277-1290... 

Vichitsoonthonkul T, Chu YW, Sodhi HS, Saunders G. P-Lactam antibiotics produced by genetically engineered filamentous fungi. Murooka Y, Imanaka T. eds. Recombinant Microbes for Industrial and Agricultural Applications. New York Marcel Dekker, 1994 119-135. 

Virtually all pseudo commodity and commodity chemicals as well as most fine chemicals are synthesized from petroleum feedstocks. It has been estimated that 98% of all chemicals produced in the United States in excess of 2 x 10 kg are synthesized from petroleum and natural gas. By contrast, chemicals isolated as natural products from plants or produced by microbes from carbohydrate feedstocks are typically restricted to ultrafme chemicals and a relatively few fine chemicals. The goal of our research effort is to ascertain how the widest possible spectrum of commodity, pseudo commodity, fine, and ultrafine chemicals can be synthesized from polyol starting materials such as d-glucose, d-xylose, 1-arabinose, and glycerol. These starting materials, in turn, are derived from renewable feedstocks derived from plants such as starch, hemicellulose, cellulose, and oils. A key feature of these conversions is the use of recombinant microbes as synthetic catalysts. 

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