Co-expression Of CYP716A12 Oxidase And ATR1 Reductase In Saccharomvces Verevisiae AndInvestigation Of Application

As a pentacyclic lupane type of triterpenoid, betulinic acid was reported to display selective cytotoxicity against a number of specific tumor types, a variety of infectious agents such as HIV, malaria and bacteria, and the inflammatory process in general. Recently, considerable in vivo and in vitro evidences have indicated some other important clinical properties, namely, attenuating ethanol-induced liver stellate cell activation, beneficial to hyperglycemia because of suppressing of hepatic glucose production, strengthening immune response, antiobesity through inhibiting pancreatic lipase and protecting against cerebral ischemia-reperfusion injury. Despite its great potential for clinical applications, the insufficient supply of betulinic acid in its natural hosts, birch bark as the major plant source, is a major obstacle in commercializing this compound. Betulin, a precursor of betulinic acid has been successfully converted to betulinic acid by a series of redox reactions in the presence of catalyst, but some issues remain, including low yields, safety, pollutions, and high costs. Microbial biotransformation is another approach for converting betulin to betulinic acid. To the best of our knowledge, two kinds of fungus Armillaria luteo-virens Sacc ZJUQH100-6 and Cunninghamella blakesleeana have been verified to biotransform betulin, still with the problem of low conversion efficiency. Rapid developments in metabolic engineering and synthetic biology provide alternative approaches for the high production of natural products in microbial hosts. However, until now there have been few researches about application of these skills in the biotransformation of betulin to betulinic acid.This study gained P450 oxidase CYP716A12 from Medicago truncatula and P450 reductase ATR1 from Arabidopsis thaliana. We cloned these two genes to bidirection expression vector pESC-ura and then shifted into Saccharomyces cerevisiae W303-1b, named Saccharomyces cerevisiae ZJUQH311 (CCTCC NO:M 2015662). After activation, enrichment culture and induction culture, we tested the product with High Performance Liquid Chromatography Liquid Chromatography (HPLC). It turned out that the productivity of betulinic acid reached 9.67%.This paper studied the way to extract protein and found the protein from the whole protein extraction with no transformation function. We also studied the cell disruption means and found ultrasonication (1.43±0.14 mg microsome protein/50 mL induction culture) gained much more microsome protein than that by grind (1.10±0.16 mg microsome protein/50 mL induction culture) significantly under same conditions (p<0.05). By further optimization of the transformation condition through response surface methodology design, we found the optimum conditions as follows:betulin 40 ?M, NADPH 1.99 mM,9h. Under the optimized conditions, the highest productivity of betulinic acid was 17.79%, improved by 83.97% compared to that before optimization.The successful transformation of microsome protein from Saccharomyces cerevisiae ZJUQH311 laid the theoretical and technical foundation for getting high yield of betulinic acid production using metabolic engineering.