Molecular Engineering Of An NADH-dependent Reductase (ScCR1) From Streptomyces Coelicolor A3 (2)

Statins lipid regulating drug Lipitor can be used to reduce total cholesterol and low density lipoprotein cholesterol levels,which is of great importance in the clinical treatment.Ethyl(S)-4-chloro-3-hydroxybutanoate((S)-CHBE)is a key intermediate for HMG-CoA reductase inhibitors,which is the active ingredient of Lipitor.In the previous work,an NADH-dependent carbonyl reductase ScCR1 has been screened from Streptomyces coelicolor A3(2)by genome mining.Characteristics investigation of ScCR1 indicates that it shows low catalytic activity towards the substrate COBE and its thermostability is also unsatisfactory,limiting its industrial application.The main purpose of this thesis is therefore to improve the catalytic efficiency and thermostability of ScCR1 for practical synthesis of Lipitor intermediate.The research contents are as follows:Firstly,according to the crystal structure of ScCR1 and molecular docking analysis,a semi-rational design strategy targeting the active pocket of ScCR1 was employed to improve its catalytic activity towards COBE.Through semi-rational design strategy and random mutation,variant M3(I158V/P168S)showed much higher catalytic activity towards COBE and its thermostability was also improved slightly.The specific activity of M3 towards COBE was improved from 38.8 U/mg protein to 168 U/mg protein,and its kcat/KM was also increased from 34.9 s-1 mM-1 to 196 s-1 mM-1.Thermostability investigation showed that the T5015 of M3 reached 49.2℃,which was enhanced 1.8℃ compared with the wild type.Secondly,the mutagenesis library was constructed by random mutation across the whole gene sequence using M3 as the parent.As a result,a triple variant M4(A60T/1158V/P168S)that enchanced the thermostability greatly was obtained.Then M4 was used as the new parent for the next evolution step.After a rational design strategy based on FoldX analysis,M7(A60T/I158V/P168S/E149M)was identified with 1.3℃ increase in T5015 as compared with M4.Unfortunately,the activity of M7 towards COBE was reduced to 80%of M4,therefore the variant M4 was chosen for further investigation.In summary,M4,improved the Tm by 7.4℃ over the wild type,and its tolerance against COBE(C5015)was also increased from 34 mM to 162 mM.Meanwhile,the catalytic activity of M4 towards COBE was increased to 260 U/mg protein,which is 6.7-fold higher than that of the wild type(38.8 U/mg protein).Thirdly,the asymmetric reduction of COBE by WT and M4 with 2-propanol as a cosubstrate for NADH regeneration was investigated.In 10 mL water/toluene(1:1,v/v)biphasic system,the catalytic performance of M4 outperforms the wild type either with purified enzyme or freezed-dried cells or lyophilized enzyme powder.Under the reaction conditions of 2.0 g/L lyophilized M4 powders,200 g/L COBE(1.2 M)could be completely converted to(S)-CHBE within 9 h,resulting in a isolated yield of 95%,and>99%ee,and the space time yield was as high as 255 g/(L*d).