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Comparative analysis of NADPH-cytochrome P450 reductases from legumes for heterologous production of triterpenoids in transgenic Saccharomyces cerevisiae
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  • Pramesti Istiandari,
  • Pisanee Srisawat,
  • Keita Tamura,
  • Ayaka Chikugo,
  • Hideyuki Suzuki,
  • Shuhei Yasumoto,
  • Hikaru Seki,
  • Ery Fukushima,
  • Toshiya Muranaka
Pramesti Istiandari
Osaka University
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Pisanee Srisawat
Osaka University
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Keita Tamura
Osaka University
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Ayaka Chikugo
Osaka University
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Hideyuki Suzuki
Kazusa DNA Research Institute
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Shuhei Yasumoto
Osaka University
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Hikaru Seki
Osaka University
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Ery Fukushima
Universidad Regional Amaz├│nica IKIAM
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Toshiya Muranaka
Osaka University

Corresponding Author:[email protected]

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Abstract

Triterpenoids are plant specialized metabolites with various pharmacological activities. They are widely distributed in higher plants, such as legumes. Due to its low accumulation in plants, there is a need for improving triterpenoid production. Cytochrome P450 monooxygenases (CYPs) play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations, CYPs require the electrons that are transferred by NADPH-cytochrome P450 reductase (CPR). Plants possess two main CPR classes, class I and class II. CPR classes I and II have been reported to be responsible for primary and specialized (secondary) metabolism, respectively. First, we compared expression levels from three legume species, showing that the expression level of CPR class I was lower and more stable, while that of CPR class II was higher in almost all the samples. By expressing different combinations of CYP families and CPR classes from these three legumes in transgenic yeast, we found that generally CYP716As worked better with CPR-I, while CYP72As worked better with CPR-II. Using engineered yeast strains, CYP88D6 paired with class II GuCPR produced the highest 11-oxo-╬▓-amyrin level. This study provides insight into co-expressing genes from legumes for heterologous production of triterpenoids in yeast.