Vol 54, No 3 (2018)

This review examines the main features of natural phytoalexines of flavonoid and stilbenoid natures, which are secondary metabolism products in numerous plants widely used as biologically active substances in the medicine, pharmacology, and agricultural plants protection. We considered the role of flavonoids and stilbenes in phytoimmune and antistress responses in plants, bactericide antifungul, and antiviral effects towards microorganisms, and the wide medical application for a number of mammalian pathologies. The main achievements in the metabolic engineering of flavonoids in microbial biotechnologies are discussed.

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.

Medical bioremediation is a unique strategy of targeting pathogenic compounds with an exogenous enzyme of microbial origin. The objective of this study was to isolate and screen the microorganisms from diverse environmental samples for their ability to catabolize 7-ketocholesterol. Isolation of bacterial strains was performed and molecular identification was carried out by amplification and sequencing of 16S rDNA for 4 the best degrader isolates. Degradation was confirmed on the basis of UV spectrophotometric and HPLC analysis. Four bacterial isolates, showing high catabolic activity towards 7-ketocholesterol were isolated: Alcanivorax jadensis IP4 (accession number KP309836; sea water sediment), Streptomyces auratus IP2 (accession number KP309837; soil), Serratia marcescens IP3 (accession number KP309838; soil) and Thermobifida fusca IP1 (accession number KM677184; manure piles). All the isolates were capable of utilizing 7-ketocholesterol as the sole organic substrate, resulting in its mineralisation. The most rapid degradation was observed with A. jadensis IP4 followed by T. fusca IP1. The degradation was followed and analyzed by HPLC. A. jadensis IP4 removed 7-ketocholesterol below detection levels within 8 days.

The screening of three strains of lactic acid bacteria identified as Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus helveticus showed significant antagonistic activity against Klebsiella pneumoniae strains characterized by multiple antibiotic resistance. Lactobacilli cocultivated with the Klebsiella strains inhibited their growth 20 to 86% on the first and second days, respectively. Exoproteome analysis of L. rhamnosus cocultivated with K. pneumoniae revealed the induction of peptidoglycan hydrolases, including extracellular lytic transglycosylases, family II (MltA), and endopeptidases capable of disrupting the peptidoglycan bacterial cell wall.

A comparative study of soybean and rapeseed protein hydrolysis by protosubtilin, an original Russian enzyme preparation widely used in animal feed production, has been performed. SDS-PAG electrophoresis, HPLC, and mass spectrometry have been employed to analyze the obtained products. The soybean protein isolate used for hydrolysate production was obtained from a commercial supplier, and rapeseed proteins were prepared from the meal by alkali extraction. Low molecular weight impurities were removed by ultrafiltration. The degree of protein hydrolysis has been shown to depend on the substrate-to-enzyme preparation ratio, hydrolysis time, and protein concentration. Rapeseed protein hydrolysis by protosubtilin at an enzyme/protein ratio of 1: 20 and hydrolysis time of 20 h resulted in complete cleavage of the proteins present in the raw material and the accumulation of oligopeptides (molecular weight less than 14 kDa) and free amino acids, which accounted for 53 and 8% of the initial protein weight, respectively. In contrast to rapeseed proteins, soybean proteins showed considerable gelling at the initial stages of hydrolysis, and the formation of insoluble hydrolysis-resistant fragments was observed. The soluble part of the hydrolysate contained short oligopeptides and free amino acids, which accounted for 13% of the initial protein weight only.

Comparative analysis of the influence of arachidonic acid and its derivatives, 3-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic and 20-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acids, on the formation of sexual and asexual reproductive structures in Neurospora crassa demonstrated that the presence and location of the hydroxyl group in unsaturated fatty acid determine the biological effect of the compound. In the presence of 5 μM of arachidonic acid, 20-HETE, and 3-HETE, vegetative spore formation in the light decreased by 45, 31, and 40%, respectively, indicating a similarity in the mechanisms of their action on the light-dependent asexual reproduction of the fungus. However, the effects of these compounds on the sexual process of N. crassa were radically different. Gas chromatography–mass spectrometry (GC–MS) revealed a fourfold increase in the monounsaturated oleic acid content in N. crassa cells after the addition of arachidonic acid. In the same time, the effect was not observed after the addition of 20-HETE. On the other hand, the increase in the linoleic acid content in the fungal mycelia was higher with 20-HETE.

Peroxisomal malate dehydrogenase (EC 1.1.1.37) with a specific activity of 533 U/mg (144-fold purification) and a yield of 5% was obtained in a homogeneous state by a purification scheme including sucrose gradient centrifugation from maize mesophyll. The Michaelis constants for the forward and reverse reactions were determined to be 11.6 mM and 256 μM, and the pH optimum was 9.5 and 9.0, respectively. Analysis of the molecular weight of the native enzyme and its subunits showed that the peroxisomal malate dehydrogenase was a homodimer. It was established that the isolated and purified isoform of the enzyme had a higher affinity for malate and NAD⁺ in comparison with the mitochondrial and cytoplasmic isoforms.

The effect of N-phenyl-2-naphthylamine (a negative allelopathic compound present in root exudates of pea (Pisum sativum L.)) on the plankton culture titer and biofilm density, as well as on the activity of adenylate cyclase signal system components and virulence factors (endo- and exocellulases and pectinases) in the causative agent of potato Clavibacter michiganensis ssp. sepedonicus ring rot (strain 6889) has been studied. N-phenyl-2-naphthylamine at a concentration of 9 μM was shown to have no effect on the growth of these bacteria, in both plankton culture and biofilms, or on the activity of the above parameters. An inhibitory effect was exhibited by N-phenyl-2-naphthylamine at a concentration of 45 μM. There was a sharp change in the concentration of intra- and extracellular cAMP levels, which was due to modulation of soluble phosphodiesterase and adenylate cyclase activities: the activation of adenylate cyclase was higher than that of phosphodiesterase in the plankton culture, and significant inhibition of adenylate cyclase and activation of phosphodiesterase were observed in biofilms. Furthermore, N-phenyl-2-naphthylamine had no effect on the activity of transmembrane adenylate cyclase in either bacteria phenotype. Under these conditions, the pectinases and cellulases activities did not change in the plankton culture, but they were significantly stimulated in both biofilms and isolated enzyme forms.