Deficiency mutant *,E .Coli

The same is true of the thiazole acid 40. Although discovered as a growth factor, it is unable to sustain the growth of a thiazole-deficient mutant of E. coli in a liquid medium. It does not decarboxylate in water solution at pH 7. Phosphate 41 (Scheme 17) is also biologically inactive. In any case, if there is only one metabolic route to the thiazole of thiamine, the very structures of 39 and 40 show that they cannot both be intermediates. 

Metabolic pathways containing dioxygenases in wild-type strains are usually related to detoxification processes upon conversion of aromatic xenobiotics to phenols and catechols, which are more readily excreted. Within such pathways, the intermediate chiral cis-diol is rearomatized by a dihydrodiol-dehydrogenase. While this mild route to catechols is also exploited synthetically [221], the chirality is lost. In the context of asymmetric synthesis, such further biotransformations have to be prevented, which was initially realized by using mutant strains deficient in enzymes responsible for the rearomatization. Today, several dioxygenases with complementary substrate profiles are available, as outlined in Table 9.6. Considering the delicate architecture of these enzyme complexes, recombinant whole-cell-mediated biotransformations are the only option for such conversions. E. coli is preferably used as host and fermentation protocols have been optimized [222,223]. 

Fluck, E.R., Poirier, L.A. Ruelius, H.W. (1976) Evaluation of a DNA polymerase-deficient mutant of E. coli for the rapid detection of carcinogens. Chem.-biol. Interact., 15, 219-231... 

Immediately after its identification DNA polymerase I was generally assumed to be the principal chain elongation enzyme. However, an amber mutant of E. coli deficient in DNA polymerase I (gene polA ... 

Fraenkel et al. (17), who isolated mutant strains which had lost the ability to grow on glycerol, succinate, or acetate but grew normally on hexoses or pentoses. These organisms were shown to be deficient in a specific FDPase, which could be distinguished from the nonspecific acid hexosephosphatase present in both mutant or wild-type strains by the fact that the latter was present in the periplasmic space (86) and did not require a divalent metal cation. The properties of the specific FDPase were confirmed with a partially purified preparation (87) the E. coli enzyme was shown to be highly specific for FDP and to be active with very low concentrations of this substance. The requirement for a divalent cation was satisfied by Mg2+, which was far more effective than Mn2+ other divalent cations were either inactive or inhibitory. The partially purified enzyme showed optimum activity at pH 7.8, with very little activity below pH 7 or above pH 9. The enzyme resembled mammalian and Candida FDPases in its sensitivity to low concentrations of AMP it was approximately 50% inhibited at an AMP concentration of 2.5 X 10-° M. 

The investigation of an amino acid biosynthetic pathway in E. coli begins with the accumulation of mutants that are deficient in the capacity to synthesize that amino acid. Mutants of this sort are known as auxotrophs. Each auxotroph bears a mutation in one of the genes that encodes an enzyme required for a step in the biosynthetic pathway of the amino acid. It is possible to determine how many steps are in a particular pathway by the process of complementation analysis (see fig. 11.14). 

Sutton, F., Butler, E.T. Ill Smith, E.T. (1986). Isolation of the structural gene encoding a mutant form of E. coli phos-phoeno/pyruvate carboxylase deficient in regulation by fructose 1,6-bisphosphate. Journal of Biological Chemistry 261, 16078-81. 

Fig. 4.9 Typical generation rate curves of the cell-wall deficient E. coli mutant (right) in comparison with a wild-type E. coli strain (left) in the absence and presence of rifamycin at the concentration indicated.

To test the possibility that the carbamate kinase might be identical with acetokinase in other microorganisms, three deficient E. coli mutants, R185-823, R prototroph, and K -wt, known to have low carbamate kinase activity, were tested for acetyl-P utilization (17). Synthesis of ATP from acetyl-P occurred much faster than from carbamyl-P the ratios of activity for acetyl-P-carbamyl-P ranged from 8 to 20 on the other hand, the enzyme from streptococcus utilizes carbamyl-P twice as fast as acetyl-P this activity ratio. remained unchanged throughout a 40-fold purification of the enzyme (17). 

The important role of polyphosphate kinase in the survival of E. coli under stress and starvation was established by the study of a mutant deficient in the ppkl gene and lacking the most part of PolyP (Rao and Kornberg, 1996 Rao et al., 1998). Mutant cells show no... 

First, polyphosphate kinase is the main enzyme of PolyP synthesis in E. coli and the ppkl-deficient mutants have virtually no PolyP content (Komberg, 1995, 1999 Komberg et al., 1999). Overexpression of ppkl results in a high level of intracellular PolyP (Ohtake et al., 1994 Keasling et al., 1998). Overexpression of both ppk and ppx results in a lower PolyP level and excretion of P from the cells (Keasling et al., 1998). A strain deficient... 

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