Fig. 2. Activation of mannitol metabolic genes by Cas9-based promoter substitution. (A) Replacement of the native HXT17 promoter by the strong constitutive TDH3 promoter for the HXT17 activation. (B) An alternative promoter substitution strategy for MAN2, which has a paralog. The MAN2 gene was amplified from the upstream region, and another PCR was performed for the coding region with overhanging sequences for in vivo assembly. Next, the CCW12 promoter, MAN2 gene, and the CYC1 terminator were assembled in vivo and integrated at an intergenic region of the genome (Int#1).