Peroxisomal Compartmentalization of the Methylerythritol-4-phosphate Pathway Alleviates Cellular Stress and Enhances Geraniol Production in Saccharomyces cerevisiae

Terpenoids are a diverse class of compounds with significant
application
potential. While prokaryotic bacteria synthesize terpenoids via the
methylerythritol-4-phosphate (MEP) pathway, fungi utilize the mevalonate
(MVA) pathway. The MVA pathway has been widely employed for efficient
terpenoid production in bacteria such as Escherichia
coli, but the MEP pathway performs poorly for biosynthesis
in yeast. In this study, we constructed a compartmentalized MEP pathway
to enhance monoterpenoid production in Saccharomyces
cerevisiae. By introducing a geraniol synthase, we
initially achieved the production of geraniol from glucose. Further
effective incorporation of a cytosolic MEP pathway with nine enzymes
increased geraniol production by 174.5%. However, this also significantly
inhibited cell growth. Overexpression analysis revealed that flavodoxin
and flavodoxin reductase were major contributors to growth inhibition,
which could also be a factor limiting the application of the MEP pathway.
To address these issues, we employed peroxisomal compartmentalization
to isolate the MEP pathway from cytosolic metabolism. This strategy
alleviated growth inhibition and improved geraniol production by 93.18%
compared to that of cytosolic expression. Through additional metabolic
engineering, we optimized peroxisomal geraniol production, achieving
a yield of 30.64 mg/L. Our findings demonstrate the potential of compartmentalized
MEP pathway expression as a viable approach for enhancing terpenoid
biosynthesis in yeast, offering valuable insights for future metabolic
engineering efforts.