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.

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.