Study On Efficient Bioproduction And Secretion Of Anti-Tumor Ganoderic Acid3-Hydroxy-Lanosta-8,24-Dien-26 Oic Acid (Gahldoa) By Saccharomyces Cerevisiae

Ganoderic acid 3-hydroxy-lanosta-8,24-dien-26 oic acid(GA-HLDOA)is an antitumor triterpenoid derived from the medicinal mushroom Ganoderma lucidum,and is also considered to be the key precursor for biosynthesis of other ganoderic acids with superior antitumor activities.Therefore,efficient biosynthesis of GA-HLDOA is extremly important.In our previous study,overexpression of a Ganoderma cytochrome P450 CYP5150L8 was found to produce 14.5 mg/L of GA-HLDOA in Saccharomyces cerevisiae.In this study,we first used a single tunable system to control the expression of CYP5150L8,and found that appropriate enhancement of CYP5150L8 expression can improve GA-HLDOA production.Using a single tunable system to collectively control the expression of CYP5150L8 and a Ganoderma cytochrome P450 reductase i GLCPR,we found that a moderate increase of i GLCPR expression can improve GA-HLDOA production,but excessive i GLCPR expression may not benefit GA-HLDOA production.Given all this,we finally constructed a dual tunable system to separately control the expression of CYP5150L8 and i GLCPR.As a result,the best engineered strain was able to produce 154.45 mg/L of GA-HLDOA in shake flask under optimal condition,which was 10.7-fold higher than previously reported yield.In the second part of this study,we explored a way for secretion of GAHLDOA.We first introduced the biosynthetic pathway of GA-HLDOA in different S.cerevisiae strains,but found that none of them could secrete GAHLDOA effectively.Interestingly,addition of 2-hydroxypropyl-?-cyclodextrin(HP-?-CD)or 2,6-dimethyl-?-cyclodextrin(DM-?-CD)in the medium enabled a rapid and sufficient secretion of GA-HLDOA in S.cerevisiae.In addition,under the treatments of these cyclodextrins,we did not observe a significant change on cell membrane fluidity.Next,we investigated the effects of cyclodextrin addition on the cellular metabolism using RNA-seq.Overall,the efficient production and secretion of GA-HLDOA platform constructed in this study could not only benefit the widespread application of the antitumor GA-HLDOA,but also provide insights for efficient bioproduction of other triterpenoids.