One-step assembly and targeted integration of multigene constructs assisted by the I-SceI meganuclease in Saccharomyces cerevisiae

In vivo assembly of overlapping fragments by homologous recombination in Saccharomyces cerevisiae is a powerful method to engineer large DNA constructs. Whereas most in vivo assembly methods reported to date result in circular vectors, stable integrated constructs are often preferred for metabolic engineering as they are required for large-scale industrial application. The present study explores the potential of combining in vivo assembly of large, multigene expression constructs with their targeted chromosomal integration in S. cerevisiae. Combined assembly and targeted integration of a ten-fragment 22-kb construct to a single chromosomal locus was successfully achieved in a single transformation process, but with low efficiency (5% of the analyzed transformants contained the correctly assembled construct). The meganuclease I-SceI was therefore used to introduce a double-strand break at the targeted chromosomal locus, thus to facilitate integration of the assembled construct. I-SceI-assisted integration dramatically increased the efficiency of assembly and integration of the same construct to 95%. This study paves the way for the fast, efficient, and stable integration of large DNA constructs in S. cerevisiae chromosomes.BT/BiotechnologyApplied Science

Simplicity and superrigidity of twin building lattices

Kac-Moody groups over finite fields are finitely generated groups. Most of them can naturally be viewed as irreducible lattices in products of two closed automorphism groups of non-positively curved twinned buildings: those are the most important (but not the only) examples of twin building lattices. We prove that these lattices are simple if the corresponding buildings are irreducible and not of affine type (i.e. they are not Bruhat-Tits buildings). Many of them are finitely presented and enjoy property (T). Our arguments explain geometrically why simplicity fails to hold only for affine Kac-Moody groups. Moreover we prove that a nontrivial continuous homomorphism from a completed Kac-Moody group is always proper. We also show that Kac-Moody lattices fulfill conditions implying strong superrigidity properties for isometric actions on non-positively curved metric spaces. Most results apply to the general class of twin building lattices