Simultaneously down-regulation of multiplex branch pathways using CRISPRi and fermentation optimization for enhancing...

The production of β-amyrin in Saccharomyces cerevisiae is still low due to the inability of effectively regulating the endogenous metabolic pathway for competitive synthesis of β-amyrin precursors. In this study, we focused on two branches of β-amyrin synthetics pathway that consume β-amyrin precursors (2,3-oxidosqualene and cytosolic acetyl-CoA) and regulated related genes (ADH1, ADH4, ADH5, ADH6, CIT2, MLS2 and ERG7). We developed a CRISPRi method by constructing a multi-gRNA plasmid to down-regulate the seven genes simultaneously, which is reported for the first time in S. cerevisiae. The average transcription inhibition efficiency of the seven genes reached as high as 75.5%. Furthermore, by optimizing the fermentation condition (including pH, inoculum size, initial glucose concentration and feed of glucose or ethanol) and increasing extracellular transportation via supplying methyl-β-cyclodextrin, β-amyrin concentration of engineered strain SGibSdCg increased by 44.3% compared with the parent strain SGib, achieving 156.7 mg/L which was the highest concentration of β-amyrin reported in yeast. The one-step down-regulation of multiple genes using CRISPRi showed high efficiency and promising future in improving the yields of natural products.