[Recombinant Escherichia coli strains deficient in mixed acid fermentation pathways and capable of rapid aerobic growth on glucose with a reduced Crabtree effect].

In this study, we constructed and characterized Escherichia coli strains deficient for mixed acid fermentation pathways, which are capable of rapid aerobic growth on glucose without pronounced bacterial Crabtree effect. The main pathways of production of acetic and lactic acids and ethanol in these strains were inactivated by a deletion of the ackA, pta, poxB, IdhA, and adhEgenes. The phosphoenolpyruvate-dependent phosphotransferase system of glucose transport and phosphorylation was inactivated in the strains by a deletion of the ptsG gene. The possibility of alternative transport and phosphorylation of the carbohydrate substrate was ensured in recombinants by constitutive expression of the galP and glk genes, which encode the low-affinity H+-symporter of D-galactose and glucokinase, respectively. SGMI.0DeltaptsG PtacgalP and SG M1.0DeltaptsG PIglk PtacgalP strains were capable of rapid aerobic growth in a minimal medium containing 2.0 and 10.0 g/l of glucose and secreted only small amounts of acetic acid and trace amounts of pyruvic acid.

[1-butanol synthesis by Escherichia coli cells through butyryl-CoA formation by heterologous enzymes of clostridia and native enzymes of fatty acid beta-oxidation].

Anaerobic biosynthesis of 1-butanol from glucose is investigated in recombinant Escherichia coli strains which form butyryl-CoA using the heterologous enzyme complex of clostridia or as a result of a reversal in the action of native enzymes of the fatty acid beta-oxidation pathway. It was revealed that when the basic pathways of acetic and lactic acid formation are inactivated due to deletions in the ackA, pta, poxB, and ldhA genes, the efficiency of butyryl-CoA biosynthesis and its reduced product, i.e., 1-butanol, by two types of recombinant stains is comparable. The limiting factor for 1-butanol production by the obtained strains is the low substrate specificity of the basic CoA-dependent alcohol/aldehyde AdhE dehydrogenase from E. coli to butyryl-CoA. It was concluded that, in order to construct an efficient 1-butanol producer based on a model strain synthesizing butyryl-CoA as a result of a reversal in fatty acid beta-oxidation enzymes, it is necessary to provide intensive formation of acetyl-CoA and enhanced activity of alternative alcohol and aldehyde dehydrogenases in the cells of a strain.

[Anaerobic synthesis of succinic acid by Escherichia coli strains with activated NAD+ reducing pyruvate dehydrogenase complex].

Effect of constitutive expression of the aceEF-lpdA operon genes coding for the enzymes of NAD+ reducing pyruvate dehydrogenase complex on the anaerobic production of succinic acids from glucose by recombinant Escherichia coli strains was studied. Basic producer strains were obtained by inactivation of the main pathways for synthesis of acetic and lactic acids by deletion of the genes ackA, pta, poxB, and ldhA (SGMO.1) in E. coli strain MG 1655 cells and additional introduction of the Bacillus subtilis pyruvate carboxylase (SG M0.1 [pPYC]). A constitutive expression of the genes aceEF-lpdA in derivatives of the basic strains SGM0.1 PL-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] was provided by replacing the native regulatory region of the operon with the lambda phage PL promoter. Molar yields of succinic acid in anaerobic glucose fermentation by strains SGM0.1 P(L)-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] exceeded the corresponding yields displayed by several control strains (exceeded considerably in the case of the strains with a pyruvate carboxylase activity). It is concluded that an increase in the succinic acid production by strain SGM0.1 PL-aceEF-lpdA [pPYC] as compared with the strains SGM0.1 and SGM0.1 [pPYC], which synthesize this substance in the reductive tricarboxylic acid cycle, is determined by activation of the glyoxylate shunt.