Directed Evolution Of Hyoscyamine 6β-hydroxylase For Efficient Synthesis Of Scopolamine

Scopolamine is one of tropane alkaloid from the plant of Solanaceae,which competes with acetylcholine preventing the transmission of certain nerve impulses of the parasympathetic nervous system,and also could act on central nervous system.Scopolamine has been used as a mydriatic,antispamoidic and anesthetic,which is also used in the treatment of Parkinson’s disease and organic phosphorus pesticide poisoning for its higher pharmacological activity and lower side effects on human body.Scopolamine was biosynthesized by hydroxylation of hyoscyamine and its subsequent epoxidation both catalyzed by hyoscyamine 6β-hydroxylase(H6H)in plant,while scopolamine is normally more rarely than hyoscyamine in plants since the low catalysis efficiency of H6H.In this work,H6H was molecular engineered for the preparation of scopolamine with recombinant E.coli expressing H6H.Firstly,directed evolution was carried out on hyoscyamine 6β-hydroxylase from Anisodus acutangulus(AaH6H)Two "hot spots"(Ser14Pro and Lys97Glu)were found after error prone PCR and site-directed mutagenesis.Six mutants with 0.8-3.4 fold higher hydroxylation activity than wild type(WT)were obtained after site-directed saturation mutagenesis and the combinal mutation.For the best mutant S14P/K97A,its specific hydroxylation activity on hyoscyamine was 75.4 U/g,which was 3.4 higher than the wild type.Secondly,the purification and characterization of WT and S14P/K97A was performed.The enzyme was purified and the molecular weight was determinated of 45 kDa.The optimal pH and temperature for WT and S14P/K97A were pH 8.0 and 35℃.Thermostability was also investigated for WT and S14P/K97A.The half-live of WT were 22.3 h,4.2 h,and 1.7 min at 30℃C,40 ℃,50 ℃ respectively,while for S14P/K97A they were 21.6 h,4.8 h and 1.8 min.Strong inhibiting effects were detected when the enzyme incubated with 10 mM Zn2+,Cu2+,Ni2+ and Co2+.Kinetic study for the in vitro hydroxylation showed that the Km and kcat value of WT were 41.6 μM and 1.0 min-1,while the Km and kcat value of S14P/K97A were 34.8 μM and 3.9 min-1,indicating that the improvement of hydroxylation activity of S14P/K97A was mainly resulted from the increase of kcat.When hyoscyamine was added to the cell culture medium at the final concentration of 100 mg L-1,all of hyoscyamine were converted to scopolamine within 48 h in the medium that cells expressing mutant S14P/K97A,,while only 25%for wild type.When 100 mg L-1 of 6β-hydroxyhyoscyamine was added into the medium,100%of the substrate were converted to scopolamine in 48 h for the mutant,while only 32%for the wild type.Thirdly,biotranformation process of hyoscyamine for the synthesis of scopolamine was optimized.The optimized temperature and IPTG concentration was 16℃ and 0.1 mM for the expression of AaH6H.Further,for the preparation of scopolamine,E.coli harboring mutant Aah6h gene(S14P/K97A)was cultivated in 5 L fermentor and 500 mg L-1 of hyoscyamine were added into the medium.97%of substrate was converted to scopolamine in 34 h and 1.068 mg scopolamine was isolated with the space time yield of 251 mg L-1 d-1,which is much higher than the previously reported ones,showing the great potential of the practical production of scopolamine with recombinant bacteria harboring this modified H6H.