Cell-Free Production of Pentacyclic Triterpenoid Compound Betulinic Acid from Betulin by the Engineered Saccharomyces cerevisiae

Abstract

Betulinic acid is a product of plant secondary metabolism which has shown various bioactivities. Several CYP716A subfamily genes were recently characterized encoding multifunctional oxidases capable of C-28 oxidation. CYP716A12 was identified as betulin C-28 oxidase, capable of modifying betulin. This study aimed to induce the transformation of betulin to betulinic acid by co-expressing enzymes CYP716A12 from Medicago truncatula and ATR1 from Arabidopsis thaliana in Saccharomyces cerevisiae. The microsome protein extracted from the transgenic yeast successfully catalyzed the transformation of betulin to betulinic acid. We also characterized the optimization of cell fragmentation, protein extraction method, and the conversion conditions. Response surface methodology was implemented, and the optimal yield of betulinic acid reached 18.70%. After optimization, the yield and the conversion rate of betulin were increased by 83.97% and 136.39%, respectively. These results may present insights and strategies for the sustainable production of betulinic acid in multifarious transgenic microbes.

Synthesis, Characterization of Liposomes Modified with Biosurfactant MEL-A Loading Betulinic Acid and Its Anticancer Effect in HepG2 Cell

“…The supernatant was centrifuged at 12,000 rpm for 20 min, and the liposome precipitate was combined with methanol and chloroform (1:1) to destroy the structure. The content of betulin in the liposomes was determined referring to the method of Wu et al [33] as detection wavelength 210 nm, Reverse C18 Column (250 mm × 4.6 mm id, 4 μm; Waters), mobile phase acetonitrile: water = 91:9, flow rate 1.0 mL/min under 30 °C. Encapsulation efficiency of the liposomes was calculated by the following formula:EE (%) = (encapsulated betulinic acid)/(encapsulated betulinic acid+unencapsulated betulinic acid) × 100%…”

Betulin and betulinic acid: triterpenoids derivatives with a powerful biological potential

“…In terms of BA biosynthesis, two major steps are to be distinguished-lupeol synthesis by 2,3-oxidosqualene cyclisation and, subsequently, oxidation in the C 28 position by the enzymes of cytochrome P450 (CYP) (Wu et al 2017). Several genes encoding the enzymes critical for these reactions have been characterised.…”

Stimulatory Effects of Oleci Acid and Fungal Elicitor on Betulinic Acid Production by Submerged Cultivation of Medicinal Mushroom Inonotus obliquus

“…For example, fatty acids of Coix lacryma-jobi oil increased polysaccharide production by directly affecting the synthesis level of phosphoglucose isomerase and α-phosphoglucomutase at different stage [ 10 ]. BA is synthesized mainly via the mevalonate pathway where acetyl-CoA is converted through a series of chemical reactions to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), to mevalonate (MVA), to isopentenyl-pyrophosphate (IPP), to farnesyl diphosphate (FPP), to squalene, and finally to betulinic acid [ 7 , 12 ]. Some enzymes are involved in the synthesis of betulinic acid including HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), farnesyl pyrophosphate synthase (FPS) and squalene synthase (SQS) [ 47 ].…”

“…Betulin is the precursor material of BA. In recent times, several reports have demonstrated that BA could be prepared through biotransformation process [ 11 , 12 , 13 ]. Meanwhile, betulin was used stimulatory agent to enhance mycelial biomass and production of exo-polysaccharides from I. obliquus [ 8 ].…”