Fig 1: Adaptation of GoldenBraid2.0 system for integration of constructs in S. cerevisiae genome. a Schematic representation of the GoldenBraid2.0 plasmids adapted for integration of constructs into the genome of S. cerevisiae. b Diagram of the two transcriptional units constructs used in (c-e) (AOH, arms of homology; goi, gene of interest; G418R, kanMX G418 resistance cassette, for (c-d), MTS: Su9, for (e), MTS: MAM33). c Comparison of NifU expression in two transformants after integration in the YPRC?15 and YORW?22 loci of S. cerevisiae strain W303. Anti-NifU western blot of alkaline whole cell extracts from saturated cultures containing glucose as the carbon source were used. Control refers to non-transformed cells. The lower panel shows the corresponding membrane Coomassie stained as a loading control. d Mitochondrial fractionation with lanes corresponding to total cell extract, pelleted cell debris after centrifugation, cytoplasmic and mitochondrial-enriched fractions from cells in (c) (transformant colony one). Anti-NifU WB is shown in the upper panel, anti-HSP60 WB (mitochondrial matrix marker) is the middle panel and anti-tubulin WB (cytoplasmic marker) is the lower panel. e Comparison of NifU expression in two transformants after integration into the YPRC?15 and YORW?22 sites of W303 and CEN.PK S. cerevisiae strains. Saturated cultures containing glucose as the carbon source were used. Control refers to non-transformed cells. The lower panel shows the corresponding membrane Coomassie stained as a loading control