Ajinomoto (Russia)

Cysteine and homocysteine suppressthe growth ofEscherichia coli. It was explained by the inhibition of threonine deaminase (TD) (Harris in J Bacteriol 145(2):1031-1035, 1981). TD inhibition was detected by a decline in its product, 2-ketobutyrate (2-KB). We propose that cysteine or homocysteine may not inhibit TD activity. Instead, 2-KB binds to them forming stable cyclic adducts. This binding possibly leads to isoleucine limitation and growth inhibition.

ABSTRACTBiotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

RNA-based pesticides exert their function by suppressing the expression of an essential gene in the target pest through RNA interference caused by double-stranded RNA (dsRNA). Here, we selected target genes for growth suppression of the solanaceous crop pests ladybird beetle (Henosepilachna vigintioctopunctata) and Colorado potato beetle (Leptinotarsa decemlineata)-the death-associated inhibitor of apoptosis protein 1 gene (diap1), and an orthologous gene of the COPI coatomer protein complex (copI), respectively. We constructed a cost-competitive overproduction system for dsRNA using Corynebacterium glutamicum as a host bacterium. The dsRNA expression unit was equipped with two sets of promoters and terminators derived from coliphage T7, and the convergent expression system was designed to be selectively transcribed by T7 RNA polymerase. This expression system efficiently overproduced both target dsRNAs. On culture in a jar fermentor, the yield of diap1-targeting dsRNA (approximately 360 bp) was > 1 g per liter of culture. Long-chain diap1-targeting dsRNAs (up to around 1 kbp) could be produced without a substantial loss of efficiency. dsRNA accumulated in C. glutamicum significantly suppressed larval growth of H. vigintioctopunctata. The dsRNA expression technology developed here is expected to substantially reduce dsRNA production costs. Our method can be applied for a wide range of industrial uses, including agricultural pest control.Key points• Overexpression of dsRNA was achieved in C. glutamicum using a coliphage T7 system.• The best strain produced > 1 g/L of the target dsRNA species, for use as an insecticide.• The developed system efficiently produced long dsRNA species, up to ~ 1 kbp.

The activity of chorismate synthase, the terminal enzyme of the common aromatic pathway, is absolutely dependent on reduced flavin mononucleotide. The bifunctional chorismate synthase of Saccharomyces cerevisiae (product of the ARO2 gene) can reduce flavin in a reaction that involves NADPH, in contrast to the monofunctional chorismate synthase of Escherichia coli (product of the aro C gene). The latter enzyme does not have the capacity for flavin reduction, and its activity therefore depends on the flavin reductase function of the cell. Chemical synthesis of the structural part of the ARO2 gene that involved the substitution of rare E. coli codons was performed for an in vivo comparison of the two types of chorismate synthase. ARO2 expression was tested in the T7 system, and isogenic E. coli strains TG1Δ aro CPtac-ARO2 and TG1Δ aro CPtac- aro C were obtained. Comparative analysis of proteins from the cell extracts of these strains and in silico assessment of hybrid RBS efficiency showed that the level of AroC protein synthesis in TG1Δ aro CPtac- aro C was higher than the level of ARO2 synthesis in the TG1Δ aro CPtac-ARO2 cells. The introduction of Ptac-ARO2 and Ptac- aro C modifications led to complete recovery of the growth of the aromatic auxotroph TG1Δ aro C on minimal mineral medium supplemented with glucose and restored phenylalanine production in the E. coli strain DV1017Δ aro C, which lacked chorismate synthase activity. The similar positive effects of Ptac- aro C and Ptac-ARO2 on phenylalanine biosynthesis in the DV1017ΔtyrR strain, in which chorismate synthase played a “bottleneck” role, indicated the absence of a limiting effect of reduced flavin on monofunctional chorismate synthase overexpressed in E. coli cells.

The inhibition of mevalonate kinase (MVK) by downstream metabolites is an important mechanism in the regulation of isoprenoid production in a broad range of organisms. The first feedback-resistant MVK was previously discovered in the methanogenic archaeon Methanosarcinamazei. Here, we report the cloning, expression, purification, kinetic characterization and inhibition analysis of MVKs from two other methanogens, Methanosaetaconcilii and Methanocellapaludicola. Similar to the M. mazei MVK, these enzymes were not inhibited by diphosphomevalonate (DPM), dimethylallyl diphosphate (DMAPP), isopentenyldiphosphate (IPP), geranylpyrophosphate (GPP) or farnesylpyrophosphate (FPP). However, they exhibited significantly higher affinity to mevalonate and higher catalytic efficiency than the previously characterized enzyme.

The isocitrate dehydrogenase (MfIDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E. coli as a fused protein. This enzyme is homodimeric,—with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD+ and 63 U mg -1 with NADP+. The MfIDH activity was dependent on divalent cations and Mn2+ enhanced the activity the most effectively. MfIDH exhibited a cofactor-dependent pH-activity profile. The optimum pH values were 8.5 (NAD+) and 6.0 (NADP+).The Km values for NAD+ and NADP+ were 113 μM and 184 μM respectively, while the Km values for DL-isocitrate were 9.0 μM (NAD+), 8.0 μM (NADP+). The MfIDH specificity (kcat/Km) was only 5-times higher for NAD+ than for NADP+. The purified MfIDH displayed maximal activity at 60°C. Heat-inactivation studies showed that the MfIDH was remarkably thermostable, retaining full activity at 50°C and losting ca. 50% of its activity after one hour of incubation at 75°C. The enzyme was insensitive to the presence of intermediate metabolites, with the exception of 2 mM ATP, which caused 50% inhibition of NADP+-linked activity. The indispensability of the N6 amino group of NAD(P)+ in its binding to MfIDH was demonstrated. MfIDH showed high sequence similarity with bacterial NAD(P)+-dependent type I isocitrate dehydrogenases (IDHs) rather than with eukaryotic NAD+-dependent IDHs. The unique double coenzyme specificity of MfIDH potentially resulted from the Lys340, Ile341 and Ala347 residues in the coenzyme-binding site of the enzyme. The discovery of a type I IDH with double coenzyme specificity elucidates the evolution of this subfamily IDHs and may provide fundamental information for engineering enzymes with desired properties.

The review summarizes the main approaches applied during the creation of L-tryptophan producing strains based on Escherichia coli for the industrial production of this amino acid. In addition, some prospects for the further improvement of tryptophan producers to increase their productivity and improve their technological characteristics based on systems metabolic engineering approaches are outlined in the review. These approaches can be used to obtain the producers of other aromatic amino acids and tryptophan precursors or derivatives.