Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus.

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 can be applied in various yeasts which, together with the constructed plasmids, makes a solid starting point when approaching a transformation of yeasts form the Saccharomycotina subphylum. To provide a proof of principle, we successfully transformed three species from the Schwanniomyces genus (S. polymorphus var. polymorphus, S. polymorphus var. africanus and S. pseudopolymorphus) with high efficiencies (up to 8 × 103 in case of illegitimate integration of non-homologous linear DNA and up to 4.7 × 105 in case of replicative plasmid). For the latter two species this is the first reported genetic transformation. Moreover, we found that a plasmid carrying replication origin from Scheffersomyces stipitis can be used as a replicative plasmid for these three Schwanniomyces species.

SARS-Cov2 S Protein Features Potential Estrogen Binding Site.

RESEARCH BACKGROUND: During the current SARS-CoV2 pandemic, as well as earlier SARS and MERS epidemics, it has been observed that COVID19-positive women on average tend to have milder symptoms and lower fatality rates than men. There is a number of differences between the sexes known to contribute to different immune responses and severity of the disease, one being the effect of estrogen via estrogen receptor signalling. We wondered if estrogen might also affect the SARS-CoV2 more directly, perhaps by binding to the surface glycoprotein (S protein), thus possibly reducing its infectivity. EXPERIMENTAL APPROACH: To assess whether there is a possibility for estrogen binding on the SARS-CoV2 S protein, we used BLAST and HHpred to compare protein sequences of S protein and human estrogen receptor ß, while 3D structures of a potential estrogen binding site and an active site of estrogen receptor ß were visualized and compared using PyMOL. RESULTS AND CONCLUSIONS: By comparing the sequence of SARS-CoV2 S protein with the human estrogen receptor ß, we identified a potential estrogen binding site on S protein and further determined that it also shares notable similarities with the active site of ER ß when observed in 3D structure of their respective proteins. As a control, SARS-CoV2 S protein was compared with the human androgen receptor, and no such similarities were found. The potential estrogen binding site is part of coronavirus S2 superfamily domain, which is involved in host-virus membrane fusion during infection and appears to be conserved throughout the Coronaviridae family. NOVELTY AND SCIENTIFIC CONTRIBUTION: This preliminary communication shows that SARS-CoV2 S protein features a potential estrogen binding site. Hopefully, this will prompt a more comprehensive study on the possibilities of estrogen binding on the S protein and the effect this might confer on the virus infectivity.

Palindromes in DNA-A Risk for Genome Stability and Implications in Cancer.

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 in carcinogenesis. Furthermore, we overview the data on known palindromic sequences in the human genome and efforts to estimate their number and distribution, as well as underlying mechanisms of genetic rearrangements specific palindromic sequences cause.

Metabolically engineered Lactobacillus gasseri JCM 1131 as a novel producer of optically pure L- and D-lactate.

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 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.

Size-dependent antirecombinogenic effect of short spacers on palindrome recombinogenicity.

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.

Novel Approach in the Construction of Bioethanol-Producing Saccharomyces cerevisiae Hybrids§.

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 selection of desirable traits. Therefore, our results suggest that the construction of intraspecies hybrids coupled with the use of genetic engineering techniques is a promising approach for improvement or development of new biotechnologically relevant strains of S. cerevisiae. Moreover, it was found that the success of gene targeting (gene targeting fidelity) in natural S. cerevisiae isolates (YIIc17_E5α and UWOPS87-2421α) was strikingly lower than in laboratory strains and the most frequent off-targeting event was targeted chromosome duplication.