E. coli mutants

A mutant E. coli strain LS5218 (fadR atoC) was employed for the synthesis of P(3HB-co-3HV) copolymer since this mutant strain constitutively expresses the enzymes involved in the transport and utilization of short chain fatty acids [58, 59]. P(3HB-co-3HV) could be synthesized by a recombinant E. coli strain LS5218 harboring the R. eutropha PHA biosynthesis genes when propionic acid or valeric acid was added as a cosubstrate [58,60]. The P(3HB-co-3HV) copolymer consisting of up to 40 mol% of 3HV could be produced. An alternative method that allowed synthesis of P(3HB-co-3HV) using propionic acid or valeric... 

Strains of the genera of Serratia, Bacillus, Pseudomonas, and a mutant E. Coli [378,379] were isolated and deposited in the Japanese Agency, namely, Pseudomonas sp. KUKK-1 PERM BP-4708, Pseudomonas sp. KUKK-2 PERM BP-4709, Pseudomonas sp. KUKK-3 PERM BP-4710, Bacillus sp. KUKK-4 PERM BP-4712, Bacillus sp. KUKK-5 PERM BP-4713, E. coli KUKK-6FERM BP-4714, Serratia sp. KUKK-7 PERM BP-4715, and Pseudomonas sp. KUKK-8 BP-4716. 

Fig. 1. Construction of plasmid pl04613C2ReABstb/ which is used to transform/ad mutant E. coli strains for production of SCL-MCL PHA copolymers.

The inherent substrate specificities of Pseudomonas sp. 61-3 PHA synthases (PhaClPs and PhaC2Ps) are rather low toward 3HB monomer (17,18), and the fad mutant E. coli strain used in our study cannot generate enough 3HB monomers from the (3-oxidation pathway (21,26). Therefore, we introduced the R. eutropha phaABRe genes to generate enough more 3HB... 

Fig. 2. Metabolic pathways for PHA biosyntheis in fad mutant E. coli strains used in this study. Enoyl-CoA hydratase, epimerase, and 3-ketoacyl-CoA or ACP reductase have been suggested to supply PHA precursors from inhibited b-oxidation pathway. The crosses indicate inactivation of corresponding enzymes. The question mark represents uncharacterized enzyme. Enzymes involved in the metabolic pathways shown have been described previously FabG (21,32), YfcX (24,33), MaoC (34), PhaA (36), and PhaB (36).

A major breakthrough that has facilitated the identification of active mutant DNA polymerases has been the use of E. coli recA718polA12, a bacteria that encodes a temperature-sensitive mutant DNA polymerase I. At elevated temperatures the mutant E. coli fails to form colonies unless complemented by a DNA polymerase that can effectively substitute for DNA polymerase I [34], The E. coli recA71S polA12 strain was first utilized in identification of active mutants of rat DNA Pol-/ and then in analyzing mutations in HIV reverse transcriptase [35], In both situations, complementation required very active mutant enzymes and it was only feasible to screen libraries that contained thousands of mutant genes. 

Construction of a mutant E.coli strain capable of producing hydrogen in the presence of nitrate... 

Elimination of a branched pathway - construction of a LDH-A mutant E.coli... 

The ability of the LamB synthetic signal sequences to inhibit protein translocation in vitro correlates with their activity in vivo (L. Chen and P. C. Tai, personal communication). The wild-type and mutant E. coli LamB signal sequences described above (Section III,H) were added to the cell-free translation/translocation system of Chen et al. (1985). The wild-type peptide blocks translocation of OmpA and alkaline phosphatase 50% inhibition is reached at a peptide concentration of 1-2 fiM. 

Methionine is overproduced by auxotrophic and analogue-resistant mutants. E. coli K-12 mutant resistant to methionine analogues (norleucine, ethionine, and methyl methionine) produced 2mg/ml of methionine. A multianalogue-resistant mutant of Corynebacterium lilium resistant to ethionine, norleucine, and methionine sulfoxide yielded 2.3 g/L methionine in a 5L fermenter.Methionine production from different analogue-resistant yeasts are also reported in the late 1970s and 1980s. ° Mondal et al. have reported methionine production from methionine-analogue-resistant mutants of Brevibacterium heali. 

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