卷 52, 编号 7 (2016)

The genes frdAB and sdhAB, which encode components of fumarate reductase and succinate dehydrogenase, have been deleted in a recombinant E. coli strain with the inactivated pathways of mixed-acid fermentation and a modified system of glucose transport and phosphorylation upon the heterological expression of the pyruvate carboxylase gene. Under anaerobic conditions, the parental strain efficiently converted glucose to succinic acid without synthesizing notable amounts of fumaric or malic acid. Upon individual deletion of the frdAB genes, the mutant strain fermented glucose to succinic acid less efficiently secreting notable amounts of malic and fumaric acids. Individual deletion of the sdhAB genes in the parental strain did not significantly affect the formation of the main fermentation end-product. The combined inactivation of fumarate reductase and succinate dehydrogenase in the constructed strain enhanced the anaerobic conversion of glucose to fumaric and malic acids with the activation of the glyoxylate bypass and decrease in the contribution of the reductive branch of the TCA cycle to the formation of the target products.

A pNS-cat72 vector was constructed based on the replicative vector pNS2 and reporter gene cat of Tn9 transposone, which encodes chloramphenicol acetyltransferase, in order to clone various gene promoters and to assess their chloramphenicol resistance in Corynebacterium glutamicum strains. The strength of promoters of various genes in the Corynebacterium glutamicum strain GEN1-2 (lys^{CA279T, S317A}), which contains aspartokinase (which is resistant to lysine) and threonine feedback inhibition were studied using this vector. It was found that promoters of the genes eftu, sod, cspB and leuC provide higher level of chloramphenicol resistance than promoters of the genes lysC, pyc, tkt, fbp, which are involved in the control of lysine biosynthesis. It was shown that replacement of the natural promotor ddh by the promotor sod increases the level of transcription almost by ten times, whereas the diaminopimelate dehydrogenase activity is increases by three to four times, which results in a 9% enhancement in lysine production. The investigated set of promoters with different strengths is a necessary tool for the optimization of gene activities and the construction of metabolite-producing strains.

A method was developed for the production and purification of biologically active recombinant human interferon α-2b (rhIFN α-2b) synthesized by expression in Nicotiana benthamiana plants. A gene construct containing a modified hIFN α-2b gene was cloned in two vectors based on tobacco mosaic virus driven by an actin promoter from Arabidopsis thaliana (pA-IFN-A) and cauliflower mosaic virus driven by a 35S promoter (pA-IFN-S). The expression vectors were introduced into the plant cells by agroinfiltration. The maximum rates of synthesis achieved in the case of pA-IFN-A and pA-IFN-S 5 days after agroinfiltration were determined to be 200 and 20 mg per 1 kg of fresh leaves, respectively. The recombinant hIFN α-2b synthesized in the plant showed high antiviral and antitumor activity comparable with that of commercial drug.

Recently published data on the separation and quantification of natural nucleosides and some of their derivatives by thin-layer chromatography on silica gel have been summarized. The use of more than 20 mobile systems for the separation of more than 52 nucleosides and derivatives was discussed; in a few cases, the conditions for their densitometry quantitative analysis after TLC separation were considered. The works performed at GosNIIgenetika on the determination of inosine, thymidine, and acadesine with domestic Sorbfil plates were reviewed in detail.