Search

Halohydrin dehalogenases (HHDHs) possess an unnatural activity of introducing functionalities such as N3, CN, NO2 etc., into a molecule through the ring-opening reaction of epoxides. The enantioselectivity of HHDHs is substrate-dependent and not always high enough for synthetic applications. B-group of HHDHs has been neglected in the past, due to observed low enantioselectivity based on performance on a relatively limited number of substrates. Extensive screening of substrates on HheB2 from Mycobacterium sp. GP1 and HheB from Corynebacterium sp. N-1074 was performed. Several highly enantioselective reactions were discovered (E>200), with HheB showing higher enantioselectivity and activity toward larger panel of substrates compared to HheB2. Enzymes HheB and HheB2 are highly homologous ; they differ by only 4 residues. By using site-directed mutagenesis, residues 120 and 125 were found to be responsible for higher enantioselectivity of HheB compared to HheB2. Computational analysis supported experiments and provided evidence that kinetic and thermodynamic parameters of reactions within HheB enzymes are crucial in determining the observed enantioselectivities. Due to remarkable activity and enantioselectivity, B-group HHDHs emerged as a catalyst of choice for the synthesis of bulky tertiary alcohols, as shown in this work.

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 1131T, a bioprocess-friendly and genetically malleable strain of homofermenta- tive 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.

Palindromic sequences in DNA can instigate genetic recombination and genome instability, which can result in devastating conditions such as the Emmanuel syndrome. Palindrome recombinogenicity increases with its size and sequence similarity between palindrome arms, while quasipalindromes with long spacers are less recombinogenic. However, the minimal spacer length, which could reduce or abolish palindrome recombinogenicity in the eukaryotic genome, was never determined. Therefore, we constructed a series of palindromes containing spacers of lengths ranging from 0 (perfect palindrome) to 10 bp and tested their recombinogenicity in yeast Saccharomyces cerevisiae. We found that a 7 bp spacer significantly reduces 126 bp palindrome recombinogenicity, while a 10 bp spacer completely stabilizes palindromes up to 150 bp long. Additionally, we showed that palindrome stimulated recombination rate is not dependent on Mus81 and Yen1 endonucleases. We also compared the recombinogenicity of a perfect 126 bp palindrome and a corresponding quasipalindrome consisting of the same palindrome arms with a stabilising 10 bp spacer in sgs1Δ and rad27Δ backgrounds, since both Sgs1 helicase and Rad27 endonuclease are implicated in preventing hairpin formation at palindromic sequences during replication.

Gene replacement is one of the most essential approaches in construction of the genetically modified yeast strains. However, the fidelity of gene targeting and the effort needed for construction of a particular strain can vary significantly. We investigated the influence of two important factors-the choice of the transformation method and the design of the transforming DNA fragment, which can vary in overall length (including flanking regions and selectable marker) compared to the length of the targeted region in the genome. Gene replacement fidelity was determined in several assays using electroporation and spheroplast transformation, and compared with our previous results obtained by lithium acetate. We have demonstrated clearly that gene targeting fidelity depends on the transformation protocol, being highest for lithium acetate method. In contrast, lower fidelity was observed with electroporation and spheroplast transformation. Additionally, the fidelity also depends on a design of the transformation assay, since a higher overall length ratio of the transforming DNA and targeted region results in higher fidelity. Moreover, the karyotype analysis of the aberrant transformants by qPCR demonstrates that gene targeting can result in diploidisation of haploid strains, most likely via targeted chromosome duplication followed by subsequent duplication of other chromosomes.

In biotechnology, budding yeast Brettanomyces (Dekkera) bruxellensis plays several important roles. On the one hand, it spoils wine by synthesizing “barnyard” and “band-aid” aromas, but it also adds coveted Brett character to some specialty beers and red wines. Furthermore, it can drive bioethanol fermentations and shows potential for second-generation bioethanol production. However, this potential awaits largely untapped, in part because we are still unable to tailor its physiology to the needs of the bioprocess. To tackle this challenge, our group developed the first protocol for effi- cient genetic transformation of B. bruxellensis and we are continuing our work by building much needed genetic toolbox. Here we will discuss challenges and pitfalls encountered during the development of the transformation system, starting from the optimization of transformation method to the complex fate of the transforming DNA which integrates randomly in the genome despite extensive homologies at its ends. We will detail our attempts to evade this obstacle by introducing genes which drive efficient gene targeting in S. cerevisiae. Moreover, we will outline the path for implementing state-of-the-art techniques of genetic manipulation, such as RNA interference and CRISPR/Cas9, as well as roadblocks that could arise on the way

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, a good ethanol producer found in wine and a strain resistant to inhibitors found in lignocellulosic hydrolysates. Compared to parental strains, hybrids grew faster in the absence, as well as in the presence of acetic and levulinic acid and 2-furaldehyde, inhibitors frequently found in lignocellulosic hydrolysates. It was shown that the overexpression of YAP1 gene, involved in a stress response, further increased the survival. Furthermore, hybrids displayed different fermentative potential in a CO2 production test suggesting genetic variability that could be used for further selection of desirable traits. Therefore, our results suggest that the construction of intraspecies hybrids coupled with use of genetic engineering techniques is a promising approach for an improvement or development of new biotechnologically relevant strains of S. cerevisiae.

Bioethanol production from raw lignocellulosic material requires a producer strain which tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous (hybrid) diploid S. cerevisiae strains by mating two haploid isolates having those desirable traits. In comparison to parental haploids, hybrid diploids were more resistant to several inhibitors frequently found in different lignocellulosic hydrolysates, such as acetic and levulinic acid and 2-furaldehyde. Interestingly, hybrids showed different fermentation ability in a CO2 production test. Our results suggest that construction of intraspecies hybrids coupled with the use of different genetic engineering techniques is a promising approach for the improvement or development of new biotechnologically relevant strains of S. cerevisiae. Additionally, we also show that in two biotechnologically interesting natural isolates percentage of successful gene targeting (gene targeting fidelity) is much lower than in widely used laboratory strains. Furthermore, the most frequent off-targeted event was duplication of a targeted chromosome yielding aneuploids having two copies of targeted chromosome, one containing transformed and the other containing untransformed allele. These results warn us that it could be more difficult to make precise genetic modifications of natural S. cerevisiae strains than expected.

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 exo1Δ sgs1Δ double mutant but not in the respective single mutants demonstrating that the Exo1 and Sgs1 proteins independently suppress whole chromosome duplication during gene targeting.

Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/ PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/microg DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment.

Bioethanol production from raw lignocelullosic material requires a strain of a microorganism which tolerates high ethanol concentrations and presence of growth inhibitors. Therefore we constructed hybrid diploid S.cerevisiae strains by mating two haploids having desirable traits. One haploid was isolated from wine thus tolerating high ethanol concentration and the other has been shown to be resistant to several growth inhibitors. These haploids were mated and resulting homozygous and heterozygous (hybrid) diploids were tested for the ability to produce ethanol and to grow in the presence of several growth inhibitors frequently found in different lignocelullosic hydrolysates (acetic and levulinic acid, 2-furaldehyde). The results showed that some hybrid diploids were more resistant to tested inhibitors than parental haploid strains and homozygous diploids. It is also interesting that constructed strains showed different fermentation ability in CO2 production test, suggesting genetic variability of constructed hybrids. All together, these results suggest that the approach used in this study may help to construct new strains as well as to improve traits of biotechnologically interesting strains.

Yeast Dekkera/Brettanomyces bruxellensis is one of the most common contaminants in wine industry, but also one of the most promising candidates for large-scale bioethanol production. Brettanomyces bruxellensis not only produces and tolerates high ethanol concentrations, but can also ferment cellobiose and adapt to lignocellulose hydrolasate. Furthermore, genome sequences of several B. bruxellensis strains are available, and efforts have been made to develop tools for genetic transformation of this yeast. Previously, we reported a successful transformation using lithium acetate/PEG method and electroporation, however, with very low transformation efficiency (10-20 transformants μg(-1)). Here we describe an optimization of electroporation procedure which resulted in a significant increase of transformation efficiency (2.8 × 10(3) transformants μg(-1)). Several key transformation parameters were optimized including cell growth phase, density of cells in the transformation sample and electroporation settings. We determined that treating the cells with both lithium acetate (100 mM) and dithiothreitol (35 mM) synergistically improves transformation efficiency. Using the described procedure around 500 transformants can be obtained per transformation sample with 180 ng of non- homologous linear transforming fragment. Additionally, several transformants were obtained with less than 1 ng of DNA demonstrating that this procedure is adequate even when very limited amount of DNA is available.