Papers by Ivan-Kresimir Svetec
World Journal of Microbiology and Biotechnology, 2020
High-quality environmentally-friendly bioplastics can be produced by mixing poly-L-lactate with p... more High-quality environmentally-friendly bioplastics can be produced by mixing poly-L-lactate with poly-D-lactate. On an industrial scale, this process simultaneously consumes large amounts of both optically pure lactate stereoisomers. However, because optimal growth conditions of L-lactate producers often differ from those of D-lactate producers, each stereoisomer is produced in a specialised facility, which raises cost and lowers sustainability. To address this challenge, we metabolically engineered Lactobacillus gasseri JCM 1131 T , a bioprocess-friendly and genetically malleable strain of homofermentative lactic acid bacterium, to efficiently produce either pure L-or pure D-lactate under the same bioprocess conditions. Transformation of L. gasseri with plasmids carrying additional genes for L-or D-lactate dehydrogenases failed to affect the ratio of produced stereoisomers, but inactivation of the endogenous genes created strains which yielded 0.96 g of either L-or D-lactate per gram of glucose. In this study, the plasmid pHBintE, routinely used for gene disruption in Bacillus megaterium, was used for the first time to inactivate genes in lactobacilli. Strains with inactivated genes for endogenous lactate dehydrogenases efficiently fermented sugars released by enzymatic hydrolysis of alkali pre-treated wheat straw, an abundant lignocellulose-containing raw material, producing 0.37-0.42 g of lactate per gram of solid part of alkali-treated wheat straw. Thus, the constructed strains are primed to serve as producers of both optically pure L-lactate and D-lactate in the next-generation biorefineries.
International Journal of Molecular Sciences, 2021
A palindrome in DNA consists of two closely spaced or adjacent inverted repeats. Certain palindro... more A palindrome in DNA consists of two closely spaced or adjacent inverted repeats. Certain palindromes have important biological functions as parts of various cis-acting elements and protein binding sites. However, many palindromes are known as fragile sites in the genome, sites prone to chromosome breakage which can lead to various genetic rearrangements or even cell death. The ability of certain palindromes to initiate genetic recombination lies in their ability to form secondary structures in DNA which can cause replication stalling and double-strand breaks. Given their recombinogenic nature, it is not surprising that palindromes in the human genome are involved in genetic rearrangements in cancer cells as well as other known recurrent translocations and deletions associated with certain syndromes in humans. Here, we bring an overview of current understanding and knowledge on molecular mechanisms of palindrome recombinogenicity and discuss possible implications of DNA palindromes i...
Food technology and biotechnology, 2018
Bioethanol production from lignocellulosic hydrolysates requires a producer strain that tolerates... more Bioethanol production from lignocellulosic hydrolysates requires a producer strain that tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous intraspecies hybrid diploids of Saccharomyces cerevisiae by crossing two natural S. cerevisiae isolates, YIIc17_E5 and UWOPS87-2421, a good ethanol producer found in wine and a strain from the flower of the cactus Opuntia megacantha resistant to inhibitors found in lignocellulosic hydrolysates, respectively. Hybrids grew faster than parental strains in the absence and in the presence of acetic and levulinic acids and 2-furaldehyde, inhibitors frequently found in lignocellulosic hydrolysates, and the overexpression of YAP1 gene increased their survival. Furthermore, although originating from the same parental strains, hybrids displayed different fermentative potential in a CO2 production test, suggesting genetic variability that could be used for further selecti...
FEMS Yeast Research, 2017
The fidelity of gene replacement in yeast is influenced by the transformation method as well as t... more The fidelity of gene replacement in yeast is influenced by the transformation method as well as the overall length proportion of the transforming DNA fragment and targeted region in the yeast genome.
FEMS Yeast Research, 2015
Optimized electroporation procedure for transformation of Dekkera/Brettanomyces bruxellensis, a n... more Optimized electroporation procedure for transformation of Dekkera/Brettanomyces bruxellensis, a notorious wine spoilage yeast and a promising bioethanol producer.
DNA Repair, 2014
Gene targeting is extremely efficient in the yeast Saccharomyces cerevisiae. It is performed by t... more Gene targeting is extremely efficient in the yeast Saccharomyces cerevisiae. It is performed by transformation with a linear, non-replicative DNA fragment carrying a selectable marker and containing ends homologous to the particular locus in a genome. However, even in S. cerevisiae, transformation can result in unwanted (aberrant) integration events, the frequency and spectra of which are quite different for ends-out and ends-in transformation assays. It has been observed that gene replacement (ends-out gene targeting) can result in illegitimate integration, integration of the transforming DNA fragment next to the target sequence and duplication of a targeted chromosome. By contrast, plasmid integration (ends-in gene targeting) is often associated with multiple targeted integration events but illegitimate integration is extremely rare and a targeted chromosome duplication has not been reported. Here we systematically investigated the influence of design of the ends-out assay on the success of targeted genetic modification. We have determined transformation efficiency, fidelity of gene targeting and spectra of all aberrant events in several ends-out gene targeting assays designed to insert, delete or replace a particular sequence in the targeted region of the yeast genome. Furthermore, we have demonstrated for the first time that targeted chromosome duplications occur even during ends-in gene targeting. Most importantly, the whole chromosome duplication is POL32 dependent pointing to break-induced replication (BIR) as the underlying mechanism. Moreover, the occurrence of duplication of the targeted chromosome was strikingly increased in the exo1 sgs1 double mutant but not in the respective single mutants demonstrating that the Exo1 and Sgs1 proteins independently suppress whole chromosome duplication during gene targeting.
Journal of Applied Microbiology, 2005
Aims: To investigate the functional role of surface layer proteins (S-layer) in probiotic strain ... more Aims: To investigate the functional role of surface layer proteins (S-layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells. Methods and Results: Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, ca at 45 kDa in L. acidophilus M92. Southern blot with pBK1 plasmid, containing slpA gene, gave a positive signal, suggesting that L. acidophilus M92 has a slpA gene coding for the S-layer proteins. S-layer proteins of this strain are present during all phases of growth. The S-layer proteins appeared when cells treated with 5 mol l)1 LiCl were allowed to grow again. Removal of the S-layer proteins reduced adhesion of L. acidophilus M92 to mouse ileal epithelial cells. Furthermore, the viability of cells without S-layer were reduced in simulated gastric juice at low pH range (2, 2AE5, 3) and simulated pancreatic juice with bile salts (1AE5 and 3 g l)1). S-layer proteins of L. acidophilus M92 were resistant to pepsin and pancreatin, in contrast, the treatment with proteinase K led to a significant proteolysis of the S-layer proteins. Conclusions: These results demonstrated functional role of S-layer; it is responsible for adhesiveness of Lactobacillus acidophilus M92 to mouse ileal epithelial cells and has a protective role for this strain. Significance and Impact of the Study: S-layer proteins have an important role in the establishment of probiotic strain Lactobacillus acidophilus M92 in the gastrointestinal tract.
Current Genetics, 2000
We studied the in¯uence of homology size and polymorphism on the integration of circular plasmids... more We studied the in¯uence of homology size and polymorphism on the integration of circular plasmids into the yeast CYC1 region. The plasmids used also contained the URA3 gene, and the proportion of Ura + transformants resulting from plasmid integration into the CYC1 region was determined by Southern-blot analysis. A size-dependent decrease in integration into the CYC1 region was observed from 858 bp to 363 bp of homology. However, with a homology size of 321, 259 or 107 bp, about 2% of the transformants still contained plasmid molecules integrated in the CYC1 region. A single point mutation in the 858-bp fragment decreased the proportion of integrations to the CYC1 gene, but the presence of additional mutations did not have a cumulative eect. For plasmids isolated in a single-stranded (ss) form, the presence of two or six point mutations did not in¯uence integration. These results were compared with those obtained in other assays designed to study substrate requirements for homologous recombination.
Journal of Fungi
Non-conventional yeasts are increasingly being investigated and used as producers in biotechnolog... more Non-conventional yeasts are increasingly being investigated and used as producers in biotechnological processes which often offer advantages in comparison to traditional and well-established systems. Most biotechnologically interesting non-conventional yeasts belong to the Saccharomycotina subphylum, including those already in use (Pichia pastoris, Yarrowia lypolitica, etc.), as well as those that are promising but as yet insufficiently characterized. Moreover, for many of these yeasts the basic tools of genetic engineering needed for strain construction, including a procedure for efficient genetic transformation, heterologous protein expression and precise genetic modification, are lacking. The first aim of this study was to construct a set of integrative and replicative plasmids which can be used in various yeasts across the Saccharomycotina subphylum. Additionally, we demonstrate here that the electroporation procedure we developed earlier for transformation of B. bruxellensis ca...
Novel approach in developing Saccharomyces cerevisiae hybrid bioethanol producers by mating of natural isolates having desirable traits
Journal of Biotechnology
Brettanomyces bruxellensis : Development of genetic transformation system
Journal of Biotechnology
Construction of novel Saccharomyces cerevisiae hybrid strains resistant to growth and fermentation inhibitors
Journal of Biotechnology
Genome assembly of lactic acid bacteria: The case of Lactobacillus amylovorus DSM 20531T
Journal of Biotechnology, 2016
Construction of Saccharomyces cerevisiae hybrid diploids for bioethanol production
Journal of Biotechnology, 2016
Getting started with Dekkera/Brettanomyces bruxellensis
Journal of Biotechnology, 2016
Genetic side effects accompanying gene targeting in yeast: the influence of short heterologous termini
Yeast, 2007
We investigated the influence of short terminal heterologies on recombination between transformin... more We investigated the influence of short terminal heterologies on recombination between transforming linear DNA fragments and the yeast Saccharomyces cerevisiae genome. The efficiency of plasmid integration to the CYC1 locus (ends-in assay) was decreased more than five-fold when the size of terminal heterology exceeded 28 nucleotides (nt) and a similar inhibitory effect was also observed in the ends-out assay (replacement of the ura3-52 allele by the URA3 gene). Plasmid integration occurred almost exclusively in the target homology and was accompanied by excessive degradation of the heterologous termini. Illegitimate integrations were much more frequent in the ends-out transformation in both the absence (8.9%) and the presence (23.7%) of 45/46 heterologous nucleotides at the ends of the transforming fragment. Interestingly, only about 60% of transformants arose by simple gene replacement, regardless of the presence of heterologous ends, whereas more complex interactions resulted in gene or whole chromosome duplications. Our results warn that different genetic alterations may be introduced in the host strain during ends-out transformation but also indicate possible mechanisms for formation of duplications in the genome.
Journal of Medical Microbiology, 2011
In recent years, an evident rise in the frequency of candidaemia caused by non-albicans Candida s... more In recent years, an evident rise in the frequency of candidaemia caused by non-albicans Candida species has been reported. In this paper we present three cases of clinically manifested candidaemia caused by Candida utilis in neonatal patients hospitalized in the same neonatal intensive care unit within a 6 month period. To the authors' knowledge, only two cases of C. utilis candidaemia have been reported in the literature to date, but neither of these involved newborns. Clinical resolution and elimination of C. utilis from the blood were achieved using liposomal amphotericin B or caspofungin in all patients. Abbreviations: CRP, C-reactive protein; CVC, central venous catheter; NICU, neonatal intensive care unit; RAPD, random amplification of polymorphic DNA.
Synbiotic effect of Lactobacillus helveticus M92 and prebiotics on the intestinal microflora and immune system of mice
Journal of Dairy Research, 2009
The synbiotic effect of the oral treatment of Swiss albino mice with milk-based diets supplemente... more The synbiotic effect of the oral treatment of Swiss albino mice with milk-based diets supplemented with Lactobacillus helveticus M92 and various kinds of prebiotics was investigated. Survival, competition, adhesion and colonization, as well as, immunomodulating capability of Lb. helveticus M92, in synbiotic combination, in the gastrointestinal tract (GIT) of mice, were monitored. After the mice were fed with synbiotics, the lactic acid bacteria (LAB) counts in faeces were increased and reduction of enterobacteria and sulphite-reducing clostridia was observed. Similar results were obtained in homogenates of small and large intestine of mice on the 1st and 14th day, after feeding with synbiotics. After the mice were orally given viable Lb. helveticus M92 cells, alone or in combination with prebiotic, the concentration of faecal SIgA and total serum IgA antibodies from all immunized mice were higher compared with the control. The specific humoral immune response was not evoked after or...
Palindrome content of the yeast Saccharomyces cerevisiae genome
Current Genetics, 2005
Palindromic sequences are important DNA motifs involved in the regulation of different cellular p... more Palindromic sequences are important DNA motifs involved in the regulation of different cellular processes, but are also a potential source of genetic instability. In order to initiate a systematic study of palindromes at the whole genome level, we developed a computer program that can identify, locate and count palindromes in a given sequence in a strictly defined way. All palindromes, defined as identical inverted repeats without spacer DNA, can be analyzed and sorted according to their size, frequency, GC content or alphabetically. This program was then used to prepare a catalog of all palindromes present in the chromosomal DNA of the yeast Saccharomyces cerevisiae. For each palindrome size, the observed palindrome counts were significantly different from those in the randomly generated equivalents of the yeast genome. However, while the short palindromes (2-12 bp) were under-represented, the palindromes longer than 12 bp were over-represented, AT-rich and preferentially located in the intergenic regions. The 44-bp palindrome found between the genes CDC53 and LYS21 on chromosome IV was the longest palindrome identified and contained only two C-G base pairs. Avoidance of coding regions was also observed for palindromes of 4-12 bp, but was less pronounced. Dinucleotide analysis indicated a strong bias against palindromic dinucleotides that could explain the observed short palindrome avoidance. We discuss some possible mechanisms that may influence the evolutionary dynamics of palindromic sequences in the yeast genome.
DNA repair, 2009
Palindromic and quasi-palindromic sequences are important DNA motifs found in various cis-acting ... more Palindromic and quasi-palindromic sequences are important DNA motifs found in various cis-acting genetic elements, but are also known to provoke different types of genetic alterations. The instability of such motifs is clearly size-related and depends on their potential to adopt secondary structures known as hairpins and cruciforms. Here we studied the influence of palindrome size on recombination between two directly repeated copies of the yeast CYC1 gene leading to the loss of the intervening sequence ("pop-out" recombination). We show that palindromes inserted either within one copy or between the two copies of the CYC1 gene become recombinogenic only when they attain a certain critical size and we estimate this critical size to be about 70 bp. With the longest palindrome used in this study (150 bp) we observed a more than 20-fold increase in the pop-out recombination. In the sae2/com1 mutant the palindromestimulated recombination was completely abolished. Suppression of palindrome recombinogenicity may be crucial for the maintenance of genetic stability in organisms containing a significant number of large palindromes in their genomes, like humans.
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Papers by Ivan-Kresimir Svetec