Rustem Saidovich Shakulov

Construction of an l-phenylalanine-producing tyrosine-prototrophic Escherichia coli strain using tyrA ssrA-like tagged alleles

To construct a Phe-producing Tyr+Escherichia coli strain, TyrA (chorismate mutase/prephenate dehydrogenase) activity was varied by engineering a proteolytically unstable protein. The tyrA in the E. coli BW25113 was altered to include ssrA-like tags. The tagged tyrA genes, which ensured different growth rates in M9 medium, were introduced into a Phe-producing strain to replace ΔtyrA. Strains with unstable TyrA-(A)ANDENYALAA proteins had a lower biomass yield and a higher Phe accumulation than strains generating the more stable TyrA-(A)ANDENYALDD. The Tyr/Phe ratio produced by the TyrA-tag strains was 10-fold less than that produced by the TyrAwt strain.

Anaerobic synthesis of succinic acid by recombinant 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 acid 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 through deletion of the genes ackA, pta, poxB, and ldhA (SGM0.1) in E. coli MG1655 strain and by additional introduction of the Bacillus subtilis pyruvate carboxylase (SGM0.1 [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 PL-aceEF-lpdA and SGM0.1 PL-aceEF-lpdA [pPYC] exceeded the corresponding yields of control strains by 2 and 33% in the absence and by 9 and 26% in the presence in media of HCO3− ion. 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 branch of the tricarboxylic acid cycle, is caused by activation of the glyoxylate shunt.

1-Butanol synthesis by Escherichia coli cells through butyryl-CoA formation by heterologous enzymes of clostridia and native enzymes of fatty acid β-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 β-oxidation pathway. It was revealed that when the basic pathways of acetic and lactic acid formation are inactivated due to deletions of 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 dehydrogenase AdhE 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 reversed action of fatty acid β-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.

Comparison of different approaches to activate the glyoxylate bypass in Escherichia coli K-12 for succinate biosynthesis during dual-phase fermentation in minimal glucose media

Two different approaches to activate the glyoxylate bypass in model Escherichia coli K-12 strains for succinate biosynthesis during dual-phase fermentation in minimal glucose media were examined. Inactivation of IclR and FadR, the transcriptional regulators of the aceBAK operon, were insufficient for the involvement of the glyoxylate bypass in anaerobic succinate biosynthesis by strains grown aerobically under glucose-abundant conditions. In contrast, the strains that constitutively expressed the aceEF-lpdA operon coding for the pyruvate dehydrogenase complex could partially synthesise succinate anaerobically via the glyoxylate bypass, even in the presence of intact regulators. The results suggest that the intensive acetyl-CoA formation in the strains constitutively expressing pyruvate dehydrogenase matches the physiological conditions that favour the activation of the glyoxylate bypass.

Metabolic engineering of Escherichia coli for 1,3-butanediol biosynthesis through the inverted fatty acid β-oxidation cycle

The feasibility of 1,3-butanediol biosynthesis through the inverted cycle of fatty acid β-oxidation in Escherichia coli cells was investigated by the rational metabolic engineering approach. CoA-dependent aldehyde dehydrogenase MhpF and alcohol dehydrogenases FucO and YqhD were used as terminal enzymes catalyzing conversion of 3-hydroxybutyryl-CoA to 1,3-butanediol. Constitutive expression of the corresponding genes in E. coli strains, which are deficient in mixed acid fermentation pathways and expressing fàd regulon genes under control of Ptrc-ideal-4 promoter, did not lead to the synthesis of 1,3-butanediol during anaerobic glucose utilization. Additional inactivation of fadE and ydiO genes, encoding acyl-CoA dehydrogenases, also did not cause synthesis of the target product. Constitutive expression of aceEF-lpdA operon genes encoding enzymes of pyruvate dehydrogenase complex led to an increase in anaerobic synthesis of ethanol. Synthesis of 1,3-butanediol was observed with the overexpression of acetyl-CoA C-acetyltransferase AtoB. Constitutive expression of atoB gene in a strain with a basal expression of alcohol/aldehyde dehydrogenase leads to synthesis of 0.3 mM of 1,3-butanediol.

Metabolic engineering of Escherichia coli for the production of succinic acid from glucose

Bio-based succinate production from renewable resources has prospective economic and environmental benefits that caused heightened interest towards the study of succinate-producing microorganisms. The pathways of succinate formation have been well studied, and microorganisms that are capable of biomass convertion into the target substance (bacteria of the genera Actinobacillus, Anaerobiospirillum, and Mannheimia) have been isolated and characterized; however, the realization of economically feasible industrial processes using native producers still remains a challenge. Traditionally, the Escherichia coli bacterium has been used as a workhouse to develop new processes for the biosynthesis of many valuable chemicals due to the extensive knowledge of its metabolism, available genetic tools, and good growth characteristics, combined with low nutrient requirements. This review is focused on modern rational approaches to the construction of recombinant E. coli strains that efficiently produce succinic acid from glucose.

Template-independent synthesis of guanosine tetra- and pentaphosphates on ribosomes.

It is shown that Escherichia coli ribosomes carrying poly(Lys)‐tRNA can form (p)ppGpp in the presence of stringent factor in the absence of the poly(A) template. Template‐independent synthesis of (p)ppGpp is suppressed by tetracycline and partially decreases if deacylated tRNA is omitted.