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Applied Biochemistry and Microbiology (v.48, #7)
Application of cell technologies for production of plant-derived bioactive substances of plant origin by A. M. Nosov (pp. 609-624).
Bioactive substances (BAS) of plant origin are known to play a very important role in modern medicine. Their use, however, is often limited by availability of plant resources and may jeopardize rare species of medicinal plants. Plant cell cultures can serve as a renewable source of valuable secondary metabolites. To the date, however, only few examples of their commercial use are known. The main reasons for such a situation are the insufficient production of secondary metabolites and high cultivation costs. It is possible to increase the performance of plant cell cultures by one or two orders of magnitude using traditional methods, such as selection of highly productive strains, optimization of the medium composition, elicitation, and addition of precursors of secondary metabolite biosynthesis. The progress in molecular biology methods brought about the advent of new means for increasing of the productivity of cell cultures based on the methods of metabolic engineering. Thus, overexpression of genes encoding the enzymes involved in the synthesis of the target product or, by contrast, repression of these genes significantly influences the cell biosynthetic capacity in vitro. Nevertheless, the attempts of the production of many secondary metabolites in plant cell culture were unsuccessful so far, probably due to the peculiarities of the cell culture as an artificial population of plant somatic cells. The use of plant organ culture or transformed roots (hairy root) could turn to be a considerably more efficient solution for this problem. The production of plant-derived secondary metabolites in yeast or bacteria transformed with plant genes is being studied currently. Although the attempts to use metabolic engineering methods were not particularly successful so far, new insights in biochemistry and physiology of secondary metabolism, particularly in regulation and compartmentation of secondary metabolite synthesis as well as mechanisms of their transport and storage make these approaches promising.
Keywords: Plant cell culture; Secondary metabolism; metabolic engineering
Microbial producers of butanol by O. V. Berezina; N. V. Zakharova; C. V. Yarotsky; V. V. Zverlov (pp. 625-638).
This review is written due to an increased interest in the production of energy carriers and basic substrates of the chemical industry from renewable natural resources. In this review, the microbiological aspects of biobutanol production are reflected and the microbial producers of butanol (both natural, i.e., members of the Clostridium genus, and recombinant), obtained by genetic modification of Clostridia and other microorganisms, are characterised.
Keywords: biofuel; butanol; renewable feedstock; clostridia; recombinant butanol producers; Clostridium acetobutylicum
Modification of E. coli central metabolism to optimize the biotransformation of L-isoleucine into 4-hydroxyisoleucine by enzymatic hydroxylation by A. D. Kivero; A. E. Novikova; S. V. Smirnov (pp. 639-644).
The enzymes of the Entner-Doudoroff pathway (EDP) and/or the oxidative branch of the pentose phosphate pathway (OBPPP) have been inactivated in the E. coli 2Δ (ΔsucAB, ΔaceAK, P L -brnQ) [pEL-IDO] strain, which is used for the biotransformation of L-isoleucine into 4-hydroxyisoleucine (4-HIL) by enzymatic hydroxylation. We established that the inactivation of the EDP in the 2Δ [pEL-IDO] strain does not virtually affect the basic parameters of the process, whereas the blocking of the OBPPP reduces the efficiency of biomass accumulation and L-isoleucine transformation in conversion to glucose. However, the simultaneous inactivation of the enzymes of the EDP and OBPPP in the 2Δ [pEL-IDO] strain allowed us to achieve a considerable improvement of the process, namely, a 20% reduction in glucose consumption and a 35% decrease in biomass yield at a constant L-isoleucine/4-HIL conversion rate at a level of 88%.
Keywords: biotransformation of L-isoleucine; 4-hydroxyisoleucine; hydroxylation; metabolic engineering
Submerged cultivation of Stephania glabra (Roxb.) Miers cells in different systems: Specific features of growth and accumulation of alkaloid stepharine by M. V. Titova; O. V. Reshetnyak; E. A. Osipova; A. I. Osip’yants; N. A. Shumilo; A. V. Oreshnikov; A. M. Nosov (pp. 645-649).
Strains of a Stephania glabra suspension culture grown in flasks and two types of bioreactors (laboratory-scale bubble and pilot-scale stirred reactors) have been compared according to their growth characteristics and accumulation of the alkaloid stepharine. The best characteristics have been recorded for strains 113 and 261. In the case of batch cultivation in flasks, the maximal accumulation of dry biomass by these strains reaches 19–21 g/l; that of the alkaloid stepharine, 0.30–0.35% of dry biomass. The used strains differ in their response to cultivation scale-up from flasks to bioreactors, strain 254 displaying the lowest adaptation to such changes. A bubble reactor is the most beneficial system for submerged cultivation of S. glabra. The absence of detectable stepharine synthesis on the background of a considerable decrease in all growth characteristics of the cultures has been observed when using a pilot stirred bioreactor. The batch cultures of strains 113 and 261 in a bubble bioreactor accumulate 11–16 g/l of dry biomass containing 0.05–0.16% of the alkaloid. It has been shown that strains 113 and 261 retain satisfactory physiological characteristics in a semi-flow regime of a bubble bioreactor. This scale-up scheme can be used for further industrial cultivation.
Keywords: alkaloids; bioreactor; cell suspension culture; Stephania glabra (Roxb.) Miers; stepharine
Protein display on the Yarrowia lipolytica yeast cell surface using the cell wall protein YlPir1 by E. Yu. Yuzbasheva; T. V. Yuzbashev; I. T. Gvilava; S. P. Sineoky (pp. 650-655).
A cell surface display system has been developed in the yeast Yarrowia lipolytica using the cell wall protein YlPir1. The red fluorescent protein TurboFP635 was used as a model protein. The expression plasmid in which the YlPIR1 gene without stop-codon was fused in-frame with the nucleotide sequence encoding TurboFP635 was constructed. The thus obtained recombinant protein complex consists of the YlPir1 the C-terminus of which is fused to the N-terminus of red fluorescent protein. Cell surface display of TurboFP635 was confirmed by fluorescent microscopy. This N-terminal system of protein incorporation in the cell wall could be efficiently applied to the cell surface display of enzymes with active sites located near the C-terminus, for example, lipases.
Keywords: cell wall protein YlPir1; red fluorescent protein; cell surface display; Yarrowia lipolytica