Fig. 4

Construction of isobutanol and isobutyl acetate ester production strains. The individual genes in the keto-acid degradation and l-valine biosynthetic pathways together were linked together with the other members of the same pathway to create separate modules. For example, in the strain PP110 (a), LlkivD and ScADH7 were linked together by self-cleaving 2A peptide sequence and placed behind the GAP promoter in the integrative expression plasmid, pGAP-Z; Ilv2, Ilv5 and Ilv3 (all from S. cerevisiae) were also linked together by self-cleaving 2A peptide sequence and placed behind the GAP promoter in the integrative expression plasmid, pGAP-Hyg. The individual constructs were sequentially integrated into the yeast genome to create PP110. Similarly, in the isobutanol producer strain PP300 (b), LlkivD and ScADH7 were linked together by self-cleaving 2A peptide sequence and placed behind the GAP promoter in the integrative expression plasmid, pGAP-Z; PpIlv6 and PpIlv2 were linked together by self-cleaving 2A peptide sequence and placed behind the GAP promoter in the integrative expression plasmid, pGAP-Neo; PpIlv5 and PpIlv3, were also linked together by self-cleaving 2A peptide sequence and placed behind the GAP promoter in the integrative expression plasmid, pGAP-Hyg. The individual constructs were sequentially integrated into the yeast genome to create PP300