M. V. Dumina

Polyphosphates and exopolyphosphatase activities in the yeast Saccharomyces cerevisiae under overexpression of homologous and heterologous PPN1 genes.

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.

Complete mitochondrial genome of the cephalosporin-producing fungus Acremonium chrysogenum.

Abstract Complete nucleotide sequence of the 27,266 bp mitochondrial genome of the сephalosporin C producing fungus Acremonium chrysogenum have been determined using whole genome shotgun sequencing approach. The circular mapping molecule encodes a usual set of mitochondrial proteins and RNA genes, including large and small ribosomal RNAs, 19 proteins and 26 tRNA genes and contains 2 introns. All structural genes are located on one strand and are apparently transcribed in one direction. Comparative analysis of this and previously sequenced Pezizomycotina mtDNAs revealed more extensive similarity between A. chrysogenum genome and those of Fusarium clade and specific synthenic patterns characteristic of Hypocrealean mitogenomes. Phylogenetic analysis based on catenated mitochondrial protein sequences confirmed current taxonomic position of A. chrysogenum within Hyprocreales and related taxa.

PPX1 gene overexpression has no influence on polyphosphates in Saccharomyces cerevisiae.

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.

Flor yeast strains from culture collection: Genetic diversity and physiological and biochemical properties

Sixteen flor yeast strains from the Magarach Collection of the Microorganisms for Winemaking (Yalta, Crimea), which are used for production of sherry, were analyzed for morphophysiological, cultural, and biochemical properties. Long-term storage did not affect their viability or the preservation of major properties, such as their flor- and aldehyde-forming abilities, and the ability to produce wines with typical sherry properties. Significant variation in the strains was observed mainly in the aldehyde-forming and flor-forming abilities and flor properties. Interdelta typing was shown to be the most informative technique to study the genetic diversity of flor yeast strains. Certain correlations between genetic polymorphisms and the enological properties of the strains were observed. The presence of a 24-bp long deletion in the ITS1 spacer of the ribosomal gene cluster, a typical feature of Spanish flor yeast strains, is correlated with a high level of production of aldehydes and acetales, efficient flor formation, and the ability to produce high quality sherry. The presence of a specific deletion in the promoter of the FLO11 gene appeared to be less informative, since the aldehyde and acetal production and flor formation abilities of such strains were variable. The studies of intraspecies genetic polymorphism by various molecular markers have revealed a high degree of phylogenetic closeness of some yeast flor strains from different geographic regions.

Polyphosphates and Polyphosphatase Activity in the Yeast Saccharomyces cerevisiae during Overexpression of the DDP1 Gene.

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.

Cephalosporin-acid synthetase of Escherichia coli strain VKPM B-10182: Genomic context, gene identification, producer strain production

An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of β-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 β-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 BL21(DE3) pMD0107 strain producing CASA.

Role of acetyl-CoA Synthetase and LovE Regulator Protein of Polyketide Biosynthesis in Lovastatin Production by Wild-Type and Overproducing Aspergillus terreus Strains

The expression of two key genes of lovastatin (LOV) biosynthesis has been studied in two Aspergillus terreus strains characterized by a more than hundredfold difference in the LOV yield. As compared to the wild-type strain ATCC 20542, in the overproducing strain 43-16 significantly enhanced expression level of LOV biosynthetic genes (by 5–50 times), transcription factor lovE (by 3–20 times), and the acs gene, which encodes acetyl-CoA synthetase (by two times), was detected. To improve the efficiency of LOV biosynthesis, recombinant A. terreus strains constitutively expressing the ACS1 gene from Saccharomyce scerevisiae or the lovE gene from A. terreus have been designed by metabolic engineering methods. According to the obtained results, the expression of ACS1 in strain 43-16 results in the suppression of lovC and lovD production and a more than 25% reduction of LOV production. In the case of low-active ATCC 20542/ACS1 recombinants, the expression level of lov genes remains almost unchanged, while the expression of the endogenous asc1 gene is enhanced and the LOV yield increases by 117%. Constitutive overexpression of the lovE gene in the ATCC 20452 strain results in the increase of mRNA levels of biosynthetic lov-genes and is accompanied by a significant (6–10 times) increase in the LOV yield.

Draft Genome Sequence of Escherichia coli Strain VKPM B-10182, Producing the Enzyme for Synthesis of Cephalosporin Acids

ABSTRACT Escherichia coli strain VKPM B-10182, obtained by chemical mutagenesis from E. coli strain ATCC 9637, produces cephalosporin acid synthetase employed in the synthesis of β-lactam antibiotics, such as cefazolin. The draft genome sequence of strain VKPM B-10182 revealed 32 indels and 1,780 point mutations that might account for the improvement in antibiotic synthesis that we observed.