protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has a wide range of pharmacological activities and high medicinal values. It is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme
to increase the low metabolic flux through PPDS and increase the productivity of protopanaxadiol production, enzyme PPDS is modified through transmembrane domain truncation and construction of self-sufficient PPDS-ATR1 fusion proteins. The fusion enzymes exhibit approximately 4.5fold increase in catalytic activity and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. The engineered yeast carrying fusion protein effectively converts 96.8% of dammarenediol-II into protopanaxadiol, bioreactor in fed-batch fermentation. Construction of four fusion proteins named as PPDS-linker1-46tATR1, PPDSlinker2-46tATR1, PPDS-linker3-46tATR1, and PPDS-nolinker-46tATR1, respectively. In addition, 31tPPDS heme domain and 46tATR1 reductase domain are linked by polypeptide GSTSSGSG. Method optimization, overview. Evaluation of the oxidative stress in yeast cells induced by co-expression of PPDS and ATR1
gene PqD12H, expression of PqD12H in Saccharmoyces cerevisiae strain WAT21 from plasmid pAUR123-PqD12H, RT-PCR enzyme expression analysis, recombinant coexpression with dammarenediol synthase DS leads to increased accumulation of ginsenosides, i.e. protopanaxatriol protopanaxadiol. compared to control, both PqDS and PqD12H have higher accumulation of mRNA in the transgenic hairy roots and the increase ratio decreased slightly over culture time. Yeast with single recombinant vector pAUR123-PqD12H is unable to express protopanaxadiol without exogenous dammarenediol-II
recombinant expression of wild-type and mutant fusion proteins in Saccharomyces cerevisiae strain W303-1a. coexpression of wild-type enzyme with dammarenediol-II synthase from Panax ginseng and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). The low metabolic flux through PPDS results in a low productivity of protopanaxadiol, but is increased with the fusion enzymes
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auxin treatment, particularly 2,4-dichlorophenoxyacetic acid, strongly enhances the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes and thus the production of dammarenediol-II and protopanaxadiol (PPD)