A. Yu. Skorokhodova

Tuning the Expression Level of a Gene Located on a Bacterial Chromosome

A new method of constructing a set of bacterial cell clones varying in the strength of a promoter upstream of the gene of interest was developed with the use of Escherichia coli MG1655 and lacZ as a reporter. The gist of it lies in constructing a set of DNA fragments with tac-like promoters by means of PCR with the consensus promoter Ptac and primers ensuring randomization of the four central nucleotides in the −35 region. DNA fragments containing the tac-like promoters and a selective marker (CmR) were used to replace lacI and the regulatory region of the lactose operon in E. coli MG1655. Direct LacZ activity assays with independent integrant clones revealed 14 new promoters (out of 44 = 256 possible variants), whose strength varied by two orders of magnitude: LacZ activity in the corresponding strains gradually varied from 102 Miller units with the weakest promoter to 104 Miller units with consensus Ptac Sequencing of the modified promoters showed that randomization of three positions in the −35 region is sufficient for generating a representative promoter library, which reduces the number of possible variants from 256 to 64. The method of constructing a set of clones varying in expression of the gene or operon of interest is promising for modern metabolic engineering.

A new method for the construction of translationally coupled operons in a bacterial chromosome

A new method for the construction of translationally coupled operons in a bacterial chromosome was developed on the basis of the recombineering approach. The method includes the in vitro construction of an artificial operon with an efficiently translated proximal cistron, its insertion into the Escherichia coli chromosome, the modification of the operon via Red-driven insertion of a special “Junction” with an excisable selective marker into the intercistronic region of the initial operon, and the excision of the marker. The Junction structure was designed and tested. The Junction consists of three components. The first component is the E. coli rplC-rplD intercistronic region and serves for placing the TAA codon of the proximal gene in the SD sequence (TAAGGAG) of rplD. The second component is the CmR gene flanked by λattL/R sites in such a fashion that the residual λattB site after λInt/Xis-driven excision of the marker does not contain termination codons in frame with ATG of rplD. The third component is the E. coli trpE-trpD intercistronic region which is added so that TGA of trpE acts a termination codon of the new open reading frame (ORF), while the overlapping (TGATG) ATG of trpD is in the position of the initiation codon of the distal gene of the original operon. The general design of the Junction provides the conversion of the original two-cistron operon into a three-cistron operon with translationally coupled genes, where the coupling of the artificial ORF (rplD’-λattB-’trpE) with the proximal gene is due to the rplC-rplD intercistronic region and its coupling with the distal gene is due to trpE-trpD. The strategy was experimentally implemented to construct an artificial operon Ptac-aroG4-serA5, where the expression the distal serA5 gene was optimized owing to translational coupling in a three-cistron operon.

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.

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 in 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, ldhA, and adhE genes. 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. The resulting SGM1.0ΔptsG PtacgalP and SGM1.0ΔptsG PLglk 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.

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.

Effect of extra- and intracellular sources of CO2 on anaerobic utilization of glucose by Escherichia coli strains deficient in carboxylation-independent fermentation pathways

The effect of extra- and intracellular CO2 sources on anaerobic glucose utilization by Escherichia coli strains deficient in the main pathways of mixed acid fermentation and possessing a modified system of glucose transport and phosphorylation was studied. Intracellular CO2 generation in the strains was ensured resulting from the oxidative decarboxylation of pyruvic acid by pyruvate dehydrogenase. Endogenous CO2 formation by pyruvate dehydrogenase stimulated anaerobic glucose consumption by the strains due to the involvement in the fermentation process of condensation reactions between oxaloacetic acid and acetyl-CoA. The availability of an external CO2 source (dissolved in medium sodium bicarbonate) promoted utilization of carbohydrate substrate by favoring the predominant participation in the fermentation of reactions directly dependent on phosphoenolpyruvate carboxylation. The positive effect of the availability of exogenous СО2 was sharply decreased in recombinant strains with the impaired functionality of the reductive branch of the tricarboxylic acid cycle. As a result, intracellular СО2 generation coupled to acetyl-CoA formation promoted anaerobic glucose utilization by cells of the corresponding mutants more markedly than the presence in the medium of dissolved sodium bicarbonate.

Study on aerobic biosynthesis of 4-hydroxybutyric acid by Escherichia coli cells upon heterologous expression of the 2-ketoglutarate decarboxylase gene

The Mycobacterium tuberculosis Rv1248c (kgd) gene has been expressed in the recombinant Escherichia coli strain with the inactivated pathways of mixed-acid fermentation and anaerobic generation of acetyl-CoA, and also with modified system of glucose transport and phosphorylation, and altered regulation of ydfG gene encoding NADPH-dependent dehydrogenase of hydroxy carboxylic acids. It was found that with the intensive 2-ketoglutarate formation during aerobic glucose utilization, 4-hydroxybutyrate synthesis could be resulted not only from the direct conversion of 2-ketoglutarate to succinate semialdehyde by the heterologous enzymatic activity, but also from the involvement of respective tricarboxylic acid cycle intermediate in a cascade of native biochemical reactions. Induced expression of the 2-ketoglutarate decarboxylase gene in the recombinant strain provided an efficient conversion of 2-ketoglutarate to succinate semialdehyde derivatives, while the concentration of synthesized 4-hydroxybutyric acid reached 0.3 mM and has apparently been limited by the activity of the enzyme responsible for the terminal stage of precursor reduction.

Construction of a Synthetic Bypass for Improvement of Aerobic Synthesis of Succinic Acid through the Oxidative Branch of the Tricarboxylic Acid Cycle by Recombinant Escherichia coli Strains

The effect of the introduction of a synthetic bypass, providing 2-ketoglutarate to succinate conversion via the intermediate succinate semialdehyde formation, on aerobic biosynthesis of succinic acid from glucose through the oxidative branch of the tricarboxylic acid cycle in recombinant Escherichia coli strains has been studied. The strain lacking the key pathways of acetic, lactic acid and ethanol formation from pyruvate and acetyl-CoA and possessing modified system of glucose transport and phosphorylation was used as a chassis for the construction of the target recombinants. The operation of the glyoxylate shunt in the strains was precluded resulting from the deletion of the aceA, aceB, and glcB genes encoding isocitrate lyase and malate synthases A and G. The constitutive activity of isocitrate dehydrogenase was ensured due to deletion of isocitrate dehydrogenase kinase/phosphatase gene, aceK. Upon further inactivation of succinate dehydrogenase, the corresponding strain synthesized succinic acid from glucose with a molar yield of 24.9%. Activation of the synthetic bypass by the induced expression of Mycobacterium tuberculosis 2-ketoglutarate decarboxylase gene notably increased the yield of succinic acid. Functional activity of the synthetic bypass in the strain with the inactivated glyoxylate shunt and opened tricarboxylic acid cycle led to 2.7-fold increase in succinate yield from glucose. As the result, the substrate to the target product conversion reached 67.2%. The respective approach could be useful for the construction of the efficient microbial succinic acid producers.