Research On The Identification,Application And Rational Engineering Of (S)-Selective Transaminase In The Asymmetric Synthesis Of Chiral Amines

Chiral amines are important pharmaceutical intermediates and are widely used in medicine,agriculture and cosmetics,and approximately 40%of them contain the chiral amine functionality.At present,the synthetic methods of chiral amines are mainly dominated by traditional chemical methods,and the use of enzymatic methods to synthesize chiral amines has become one of the attractive choices due to its mild reaction conditions,high stereoselectivity and simple reaction process.Among these biocatalysts for the synthesis of chiral amines,transaminase is a promising biocatalyst for its stringent stereoselectivity and no requirement for redox cofactors.However,most natural transaminases show prevalent intrinsic problems such as the unacceptance of the most industrial favored amine donor isopropylamine(IPA),low catalytic performance towards high concentration of substrates,unfavorable equilibrium and trace activity for unnatural bulky substrates,which have seriously affected the application process of transaminase industrialization.To overcome these challenges,this paper used different chiral amine precursor ketones as substrates and made the following research from the aspects of transaminase mining,transaminase application and rational modification of transaminase.1)In order to find the transaminases that can tolerate high concentration of ketone substrate,the metagenomic library was specifically excavated in the amidogen-enriched environments established to search for class Ⅲ transaminase,and a robust α-transaminase ATA1012 was identified.It showed signi ficant thermal stability at 30-50℃,with a half-life of up to 160 hours at 50℃,and even longer for 800 hours at 30℃;It could tolerate 2 M amine donor isopropylamine(IPA),and only 2 equivalent of IPA was sufficient to effectively catalyze the target reaction,and the conversion was as high as 100%;It also showed high tolerance of up to 750 mM of 1-Boc-3-piperidone and 1-Boc-3-pyrrolidone.Subsequently,the reaction conditions during the reductive amination process of the enzyme were systematically optimized,including substrate loading,reaction temperature,IPA equivalent and the concentration of the coenzyme 5’-pyridoxal phosphate(PLP).The enzyme can effectively synthesis of 0.75 M of 1-Boc-3piperidone(150 g/L)and 1-Boc-3-pyrrolidone(139 g/L)in 12 h with an ee value>99.9%.At the same time,molecular docking and molecular dynamics were also performed to elucidate the molecular mechanism of the high catalytic efficiency of ATA1012 towards the ketone substrates.The good performance of ATA1012 obviously validates the high efficiency and practicability of the directed metagenomic approach for mining robust biocatalysts,conferring it great potential as a green and highly efficient biocatalyst for the production of pharmaceutically relevant amine-containing molecules,and also providing a reliable template for further protein engineering.2)To further demonstrate the unparalleled power of the directed metagenomic mining strategy and the practicability of the transaminases from the special metagenome,a widely used non-selective commercial herbicide,L-phosphinothricin,was chosen as target chiral amine to be synthesized by asymmetric amination of the corresponding prochiral keto acid 2-oxo-4[(hydroxy)(-methyl)phosphonyls]butyric acid(PPO).The fold-type Ⅰ transaminases(Stransaminase,S-TA)was fully excavated from the metagenome established above,then the recombinant S-TAs were all applied to exploration on the asymmetric synthesis of L-PPT.Finally,two robust S-TA named ATA0611 and ATA1042 were obtained to both have the ability to completely aminate 0.5 M PPO.By introducing the pyruvate decarboxylase(PDC)cascade system to promote the equilibrium to production side and systematically optimize the reaction conditions,the co-expression TA enzyme ATA0611-PDC could completely convert 0.5 M PPO to L-PPT within 30 hours in as low as 2.4 equivalents of L-Ala with ee value of 99.88%.As to another co-expression TA enzyme ATA1042-PDC,the 100%conversion was also observed in 24 hours with only 2 folds of amine donor L-Asp,and the ee value of product L-PPT was>99.9%.Considering the eco-efficiency and superior catalytic properties of ATA1042-PDC,the 0.5 L and 10 L scale-up amination processes mediated by ATA1042-PDC were easily carried out,all providing the 100%conversions in 22 h.Two different green and sustainable synthetic processes of L-PPT by S-TA were established in this section,further indicating the utility of directed metagenomics in the mining of efficient biocatalysts.3)The low catalytic performance toward macromolecular substrates is among one of the major obstacles in the application of ω-transaminase.To conquer this challenge,rational design method was employed to engineer a(S)-selective ω-transaminase BPTA from Paraburkholderia phymatum by reshaping the large binding pocket of the substrate to release the steric hindrance of the small binding pocket toward bulky substrates,and effectively catalyzed the asymmetric amination of target substrate 1-propiophenone.Based on combining molecular docking and dynamic simulation analyses,we identified a non-classical substrate conformation,located in the active site with steric hindrance and undesired interactions,to be responsible for the low catalytic efficiency.By relieving the steric barrier with W82A,we improved the specific activity by 14-fold compared to WT.A π-π stacking interaction was then introduced by M78F and 1284F to strengthen the binding affinity with a large binding pocket to balance the undesired interactions generated by F44.T440Q further enhanced the substrate affinity by providing a more hydrophobic and flexible environment close to the active site entry.Finally,we constructed the quadruple variant M78F/W82A/I284F/T440Q to generate the most productive substrate conformation.The 1-propiophenone catalytic efficiency of the mutant was enhanced by more than 470-fold in kcat/KM,and the conversion increased from 1.3 to 94.4%compared with that of WT,without any stereoselectivity loss(ee>99.9%).Meanwhile,the obtained mutant also showed significant activity improvements towards various aryl alkyl ketones with a small substitution larger than the methyl group ranging between 104～230-fold,demonstrating great potential for the efficient synthesis of aryl alkyl enantiopure amines with steric hindrance in the small binding pocket.The rational design strategy adopted in this section provides a feasible way to engineer such co-transaminases with non-canonical substrate conformations of the bulky substrates.