RESUMEN
L-asparaginase is extensively used in the treatment of acute lymphoblastic leukemia and several other lymphoproliferative diseases. In addition to its biomedical application, L-asparaginase is also of prospective use in food industry to reduce the formation of acrylamide, which is classified as probably neurotoxic and carcinogenic to human, and in biosensors for determination of L-asparagine level in medicine and food chemistry. The importance of L-asparaginases in different fields, disadvantages of commercial ferments, and the fact that they are widespread in nature stimuli the search for biobetter L-asparaginases from new producing microorganisms. In this regard, extremofile microorganisms exhibit unique physiological properties such as thermal stability, adaptability to extreme cold conditions, salt and pH tolerance and so provide one of the most valuable sources for novel L-asparaginases. The present review summarizes the recent results on studying the structural, functional, physicochemical and kinetic properties, stability of extremophilic L-asparaginases in comparison with their mesophilic homologues.
Asunto(s)
Asparaginasa/química , Extremófilos/enzimología , AsparaginaRESUMEN
Cephalosporin acid synthetase (CASA) is responsible for specific to synthesis of cephalosporin-acids, its expression in Escherichia coli cells is accompanied by accumulation of unprocessed insoluble precursor. In order to optimize conditions of recombinant CASA production we have studied the effects of several parameters of strain cultivation, including growth media composition, temperature, and inoculation dose. Also plasmids for production of CASA variants with the signal sequence of Erwinia carotovora L-asparaginase (ansCASA) and "leaderless" CASA were created in search of more efficient expression constructs. Removal of the N-terminal secretion signal sequence reduced the production of functionally active CASA more than 10-fold and inhibited strain growth. Insertion of the L-asparaginase signal sequence increased the specific enzyme activity in the resultant recombinant strain. The ansCASA producing strain was used to develop the method of immobilization of the recombinant enzyme on an epoxy-activated macroporous acrylic support. The resultant biocatalyst performed effective synthesis of cefazolin from 3-[(5-methyl-1,3,4-thiadiazol-2-il)-thiomethyl]-7- aminocephalosporanic acid (MMTD-7-ACA) and methyl ester of 1(H)-tetrazolilacetic acid (ÐETzAA), under mild conditions a transformation level of MMTD-7-ACA to cefazolin of 95% is reached.
Asunto(s)
Asparaginasa/metabolismo , Proteínas Bacterianas/metabolismo , Cefazolina/metabolismo , Proteínas Inmovilizadas/metabolismo , Complejos Multienzimáticos/metabolismo , Acrilatos/química , Asparaginasa/genética , Proteínas Bacterianas/genética , Biocatálisis , Clonación Molecular , Medios de Cultivo/química , Escherichia coli/enzimología , Escherichia coli/genética , Expresión Génica , Ingeniería Genética , Proteínas Inmovilizadas/genética , Complejos Multienzimáticos/genética , Pectobacterium carotovorum/química , Pectobacterium carotovorum/enzimología , Plásmidos/química , Plásmidos/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The effects of overexpression of yeast diphosphoinositol polyphosphate phosphohydrolase (DDP1) having endopolyphosphatase activity on inorganic polyphosphate metabolism in Saccharomyces cerevisiae were studied. The endopolyphosphatase activity in the transformed strain significantly increased compared to the parent strain. This activity was observed with polyphosphates of different chain length, being suppressed by 2 mM tripolyphosphate or ATP. The content of acid-soluble and acid-insoluble polyphosphates under DDP1 overexpression decreased by 9 and 28%, respectively. The average chain length of salt-soluble and alkali-soluble fractions did not change in the overexpressing strain, and that of acid-soluble polyphosphate increased under phosphate excess. At the initial stage of polyphosphate recovery after phosphorus starvation, the chain length of the acid-soluble fraction in transformed cells was lower compared to the recipient strain. This observation suggests the complex nature of DDP1 involvement in the regulation of polyphosphate content and chain length in yeasts.
Asunto(s)
Ácido Anhídrido Hidrolasas/genética , Ácido Anhídrido Hidrolasas/metabolismo , Polifosfatos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Expresión Génica , Saccharomyces cerevisiae/enzimologíaRESUMEN
An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of ß-lactam antibiotics of the cephalosporin acids class (cefazolin, cefalotin, cefezole etc.). A comparison of the previously determined genomic sequence of E. coli VKPM B-10182 with a genome of the parent E. coli strain ATCC 9637 was performed. Multiple mutations indicating the long selection history of the strain were detected, including mutations in the genes of RNase and ß-lactamases that could enhance the level of enzyme synthesis and reduce the degree of degradation of the synthesized cephalosporin acids. The CASA gene--a direct homolog of the penicillin G-acylase gene--was identified by bioinformatics methods. The homology of the gene was confirmed by gene cloning and the expression and determination of its enzymatic activity in the reaction of cefazolin synthesis. The CASA gene was isolated and cloned into the original expression vector, resulting in an effective E. coli BL2l(DE3) pMD0107 strain producing CASA.
Asunto(s)
Cefalosporinas/metabolismo , Escherichia coli/enzimología , Ligasas/genética , Clonación Molecular , Escherichia coli/genética , Genoma Bacteriano , Ligasas/aislamiento & purificación , Ligasas/metabolismo , Penicilina Amidasa/genética , Penicilina Amidasa/metabolismo , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
The role of exopolyphosphatase PPX1 in polyphosphate metabolism in yeasts has been studied in strains of Saccharomyces cerevisiae with inactivated PPX1 and PPN1 genes transformed by the expression vector carrying the yeast PPX1 gene. Exopolyphosphatase activity in transformant strains increased 90- and 40-fold compared to the ΔPPX1 and ΔPPN1 strains, respectively. The purified recombinant exopolyphosphatase PPX1 was similar to the PPX1 of wild strains in its substrate specificity and requirement for divalent metal cations. It was more active with tripolyphosphate and low molecular mass polyphosphates than with high molecular mass polyphosphates and required Mg2+ for its activity. The high level of recombinant PPX1 expression caused no decrease in polyphosphate content in the cells of the transformant. This fact suggests the restricted role of PPX1 in polyphosphate metabolism in yeasts.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Polifosfatos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Ácido Anhídrido Hidrolasas/genética , Expresión Génica , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Transcript levels of several key genes responsible for cephalosporin C (CPC) biosynthesis and transport have been determined using qPCR analysis of Acremonium chrysogenum strains differing more than 100-fold in the levels of CPC production. The expression of genes involved in the final steps of CPC production was significantly increased in the high-producing RNCM F-4081D strain compared to the wild-type ATCC 11550 strain. Different dynamics in the course of cultivation was observed for the genes known to be involved in the transport of CPC intermediates between subcellular compartments. Overall, comparative expression analysis showed balanced and fine-tuned expression of the genes responsible for CPC biosynthesis and transport in the genetically selected A. chrysogenum RNCM F-4081D strain, reflecting its capacity to overcome known CPC biosynthesis "bottlenecks" and produce CPC of high yield and purity.
Asunto(s)
Acremonium/genética , Acremonium/metabolismo , Vías Biosintéticas/genética , Cefalosporinas/metabolismo , Perfilación de la Expresión Génica , Acremonium/crecimiento & desarrollo , Transporte Biológico/genética , Mutación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The role of exopolyphosphatase PPN1 in polyphosphate metabolism in fungi has been studied in strains of Saccharomyces cerevisiae transformed by the yeast PPN1 gene and its ortholog of the fungus Acremonium chrysogenum producing cephalosporin C. The PPN1 genes were expressed under a strong constitutive promoter of the gene of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae in the vector pMB1. The yeast strain with inactivated PPN1 gene was transformed by the above vectors containing the PPN1 genes of S. cerevisiae and A. chrysogenum. Exopolyphosphatase activity in the transformant with the yeast PPN1 increased 28- and 11-fold compared to the mutant and parent PPN1 strains. The amount of polyphosphate in this transformant decreased threefold. Neither the increase in exopolyphosphatase activity nor the decrease in polyphosphate content was observed in the transformant with the orthologous PPN1 gene of A. chrysogenum, suggesting the absence of the active form of PPN1 in this transformant.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Proteínas Fúngicas/metabolismo , Polifosfatos/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácido Anhídrido Hidrolasas/genética , Acremonium/genética , Proteínas Fúngicas/genética , Expresión Génica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Vectors for the expression of the CefT transporter of the MFS family in Acremonium chrysogenum--a producer of beta-lactam antibiotic cephalosporin C--and in Saccharomyces cerevisiae as a fusion with the cyan fluorescent protein (CFP) have been created. The subcellular localization of the CefT-CFP hybrid protein in yeast cells has been investigated. It was shown that the CefT-CFP hybrid protein is capable of complementation of the qdr3, tpo 1, and tpo3 genes encoding for orthologous MFS transporters of Saccharomycetes, making the corresponding strains resistant to spermidine, ethidium bromide, and hygromycin B. High-yield strain VKM F-4081D of A. chrysogenum, expressing the cefT-cfp fusion, was obtained by an agrobacteria conjugated transfer. It was also shown that the constitutive expression of cefT in A. chrysogenum VKM F-4081D led to a change in the biosynthetic profiles of cephalosporin C and its precursors. This resulted in a 25-35% decrease in the finite product accumulated in the cultural liquid with a simultaneous increase in the concentration of its intermediators.
Asunto(s)
Acremonium/metabolismo , Antibacterianos/metabolismo , Proteínas Portadoras/metabolismo , Cefalosporinas/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Saccharomyces cerevisiae/metabolismo , Acremonium/genética , Transporte Biológico , Proteínas Portadoras/genética , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Vectores Genéticos/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Saccharomyces cerevisiae/genéticaRESUMEN
Using pulse electrophoresis in controlled homogenous electric field we conducted molecular karyotyping of highly-productive and laboratory strains of Acremonium chrysogenum generating antibiotic cephalosporin C (cefC). Differences in size of several chromosomes of highly active strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the highly active strain was not associated with alteration of localization and copy number ofcephalosporin C biosynthesis and transport genes. A cluster of "early" cefC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the "late genes", on chromosome II (2.3 Mb). Both clusters are presented as a single copy perA. chrysogenum genome in the wild-type and in CB26/8 producer strains. Based on comparative analysis of laboratory and industrial cefC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.