Selection auxotrophic complementation

Smiley, J. A., andBenkovic, S.J. (1994). Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxotrophic Escherichia coli antibodies for orotate decarboxylation. Proc, Natl. Acad. Sci. USA, 91(18), 8319-8323. 

There exists evidence indicating that despite the action of natural selection to increase enzyme selectivity by various means, ranging from shaping the active site itself to regulation of enzyme expression and activity, numerous cross-reactions and breaches of specificity occur, not just in vitro, but in particular within living cells. Such cross-reactivities are often unraveled by the analysis of auxotrophic knockout strains that lack a crucial enzyme. These deficiencies are often complemented by other enzymes, or even other enzyme pathways,... 

Over the past 30 years, a wide range of selection markers have become available for P pastoris. Recessive markers, which require a mutation in the host genome that is complemented by the marker, mostly derive from amino acid or nucleotide synthesis pathways, as traditionally applied for other yeasts or bacteria as well (Table 19.3). The disadvantage of these auxotrophic markers is the possibility... 

Puck and his colleagues have selected from mutagenized Chinese hamster cells in culture, many glycine auxotrophs, all of which fall into one of four complementation classes [105]. Each class is apparently due to a different specific enzyme defect in the pathway of glycine synthesis [105]. Although no regulatory mutants were reportedly found, the techniques are now available [106,107] to begin such a search. 

The electric field pulse treatment of a suspension of mixed protoplasts of auxotrophic yeast strains in the presence of PEG and Ca -ions strongly enhances the fusion of protoplasts as detected by selection of prototrophic colonies on solid minimal medium. In our experiments the number of prototrophic colonies formed from protoplast suspensions to which an electric field pulse of proper strength had been applied was much higher than from unaffected suspensions (see Table 2). These colonies are the result of complementation of auxotrophic deficiency of the parental strains. The latter are not able to grow and to form colonies on unsupplemented minimal medium. The optimal field strength in the case of intraspecific fusion is about 3 to 5 kV/cm for Sm.lipolytica (Table 2). 

Substrate-Dependent Selection of Acyl-ACP Desaturases by Complementation of an E. coli Unsaturated Fatty Acid Auxotroph... 

The long-/medium-chain acyl-ACP pools of E. coli are enriched in 14 0- and 16 0-ACP but contain little or no 18 0-ACP (6). Consistent with this, we have found that 14 0- and 16 0-ACP desaturases e.g., A -14 0-ACP desaturase, A -16 0-ACP desaturase, and the milkweed acyl-ACP desaturase) complement the E. coli unsaturated fatty acid auxotroph when grown at 30 C, but 18 0-ACP desaturases e.g., A -18 0-ACP desaturase) do not complement these cells at this temperature (Fig. 1). As an extension of this observation, we are now in the process of introducing random and site-specific mutations into the A -18 0-ACP desaturase and then selecting for mutant enzymes with increased specificity for 14 0- and 16 0-ACP by complementation of the E. coli unsaturated fatty acid auxotroph. 

While crude extracts of wild type E. coli readily incorporate radio labelled C2-units from malonyl-CoA into long chain fatty acids (Figure 3 left), extracts from fatty acid requiring mutants do not do so. This was the basis for the first FAS complementation tests in E. coli [25] iti which equal amounts of crude extracts from two such auxotrophic mutants were mixed and assayed. Positive results indicated that a FAS moiety defective in one mutant strain was functional and present in the other mutant strain, and vice versa. In cerulenin based complementation assays, the antibiotic replaces one of the mutant strains by selectively inactivating one or more of the condensing enzymes. Low concentrations eliminate... 

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