Organelle Engineering in Yeast: Enhanced Production of Protopanaxadiol through Manipulation of Peroxisome Proliferation in Saccharomyces cerevisiae

Abstract

Isoprenoids, which are natural compounds with diverse structures, possess several biological activities that are beneficial to humans. A major consideration in isoprenoid production in microbial hosts is that the accumulation of biosynthesized isoprenoid within intracellular membranes may impede balanced cell growth, which may consequently reduce the desired yield of the target isoprenoid. As a strategy to overcome this suggested limitation, we selected peroxisome membranes as depots for the additional storage of biosynthesized isoprenoids to facilitate increased isoprenoid production in Saccharomyces cerevisiae. To maximize the peroxisome membrane storage capacity of S.cerevisiae, the copy number and size of peroxisomes were increased through genetic engineering of the expression of three peroxisome biogenesis-related peroxins (Pex11p, Pex34p, and Atg36p). The genetically enlarged and high copied peroxisomes in S.cerevisiae were stably maintained under a bioreactor fermentation condition. The peroxisome-engineered S.cerevisiae strains were then utilized as host strains for metabolic engineering of heterologous protopanaxadiol pathway. The yields of protopanaxadiol from the engineered peroxisome strains were ca 78% higher than those of the parent strain, which strongly supports the rationale for harnessing the storage capacity of the peroxisome membrane to accommodate the biosynthesized compounds. Consequently, this study presents in-depth knowledge on peroxisome biogenesis engineering in S.cerevisiae and could serve as basic information for improvement in ginsenosides production and as a potential platform to be utilized for other isoprenoids.

Compartmentalization and transporter engineering strategies for terpenoid synthesis

“…Thus, by improving the tolerance of yeast cells to geraniol and compartmentalizing the geraniol-producing enzymes into peroxisomes, the titer of geraniol was found to increase by 80% [ 72 ]. Similar results were also observed in the protopanaxadiol-producing strain [ 73 ]. Furthermore, using peroxisome targeting sequence 1, the non-carotenogenic yeast Pichia pastoris was able to produce 73.9 mg/L lycopene by targeting heterologous carotene-producing enzymes to peroxisomes [ 74 ].…”

“…Furthermore, using peroxisome targeting sequence 1, the non-carotenogenic yeast Pichia pastoris was able to produce 73.9 mg/L lycopene by targeting heterologous carotene-producing enzymes to peroxisomes [ 74 ]. Similar strategies have been used in the biosynthesis of various monoterpenes [ 75 ], sesquiterpenes, including α-humulene [ 76 , 77 ], and triterpenoids, including squalene [ 63 ], β-amyrin [ 78 ], and protopanaxadiol [ 73 ].…”

Recent Advances in Yeast Recombinant Biosynthesis of the Triterpenoid Protopanaxadiol and Glycosylated Derivatives Thereof

“…A possible explanation might be that Ino2p plays a more significant role in phospholipid biosynthesis than Ino4p, and overexpression of INO2 could lead to an imbalance between INO2 expression and its repressor Opi1p, which ultimately leads to activation of the phospholipid biosynthetic pathway of the ER . Alternatively, manipulating the peroxisome proliferation by increasing Pex34p (peroxisome population-regulated protein 34) production while deleting PEX11 (peroxisome population-regulated protein 11) and ATG36 (autophagy-related protein-encoding gene) improved PPD production by 78% (4 mg/L) compared to the original yeast strain . These results indicate that ER size and peroxisome proliferation likely influence PPD production, although the rational for the latter is rather unclear.…”

“…50 Alternatively, manipulating the peroxisome proliferation by increasing Pex34p (peroxisome population-regulated protein 34) production while deleting PEX11 (peroxisome populationregulated protein 11) and ATG36 (autophagy-related proteinencoding gene) improved PPD production by 78% (4 mg/L) compared to the original yeast strain. 51 These results indicate that ER size and peroxisome proliferation likely influence PPD production, although the rational for the latter is rather unclear.…”

Microbial Genetics and Evolution

“…Indeed, Artuso et al [ 14 ] proposed that Aminobacter species could be exploited in bioaugmentation and bioremediation processes, while Gammuto et al [ 13 ] suggested a possible development of new tools based on the use of P. aeruginosa azurin p28 domain for treating cancer diseases. Finally, in another work comprised in this Special Issue, Choi et al [ 19 ] engineered the yeast Saccharomyces cerevisiae by modifying the expression of three peroxisome proliferation-related proteins: the obtained mutant strains had an increased amount of peroxisomes and/or enlarged peroxisomes that provided additional storage space for the accumulation of protopanaxadiol synthesized through the expression of heterologous biosynthetic genes.…”