Fig. 3.

Schematic overview of the isobutanol synthesis pathway, the genetic constructs for identifying respective bottleneck, and the results from the engineered Synechocystis PCC 6803 strains containing different constructs. a Isobutanol synthesis pathway. The heterogenous enzymes are colored in brown: acetolactate synthase (AlsS) from Bacillus subtilis (B. subtilis), acetohydroxy acid isomeroreductase (IlvC), dihydroxy acid dehydratase (IlvD) from E. coli, and α-ketoisovalerate decarboxylase (Kivd) from L. lactis. The endogenous genes are colored in green. The gene-encoding alcohol dehydrogenase used here is the codon-optimized slr1192 (slr1192OP). b Genetic concepts, growth curve, isobutanol titer, SDS-PAGE, and western immunoblot from the engineered strains containing single operon. GOI Gene of interest. Kivd^S286T and slr1192OP-encoded alcohol dehydrogenase were strep-tagged and the other genes were flag-tagged. BCD is the bicistronic design for providing more reliable expression. c Growth curve, isobutanol titer, SDS-PAGE, and Western blot from the engineered strains containing convergent-oriented operons. Kivd^S286T was strep-tagged and the other genes were Flag-tagged. RiboJ is a self-cleaving ribozyme for providing more reliable expression. Size of the proteins are: AlsS: 62 kDa, IlvC: 54 kDa, IlvD: 65 kDa, KivdS^286T: 61 kDa, Sll1981: 60 kDa, Sll1363: 40 kDa, Slr0452: 59 kDa, Slr1192: 36 kDa, Sll0065: 21 kDa, Slr2088: 68 kDa