Engineering Polyketide/Steroidal Scaffold Through Acyl-CoA Synthase And P450 Hydroxylase

Polyketides and steroids cover tens of thousands of natural products and more than 500 natural/semi-synthetic/synthetic drugs,which are important sources of new drug discovery.However,due to the complex structure,inert carbon atom reaction and lack of efficient carbon skeleton editing methods,the structure modification of their carbon skeletons is extremely challenging.The integration of non-natural extension units and biological hydroxylation are important strategy for engineering polyketide and steroid scaffold,but they are constrained by the bottlenecks of insufficient diversity of acyl-CoA and low catalytic efficiency and poor selectivity of hydroxylase enzymes.This dissertation focuses on the development of novel acyl-CoA synthases and steroidal C14-hydroxylase,and on this basis,establishes a method for editing polyketone backbones based on extension units and 14-hydroxysteroid skeleton synthesis based on C-H oxidation.Acyl-CoAs are requisite precursors in primary and secondary metabolic pathway,particularly,in polyketide biosynthesis,acyl-CoAs are responsible for starter units and extender units biosynthesis,thus,its structural diversity and efficient synthesis are crucial for polyketides structure engineering.The reported acyl-CoA synthetase（ACS）has a narrow substrate spectrum and is regulated by acylation of a key lysine residue（Lys-A10）in Motif A10,which limits the efficient bioavailability of acyl-CoAs in organisms.In this work,we identified a novel ACS（UkaQ）from the UK-2A biosynthetic pathway,which naturaly lacks Lys-A10 and is not regulated by endo-acylation,and exhibits unprecedented substrate tolerance.Enzyme engineering significantly improved its stability,substrate scope and catalytic activity.In total,38carboxylic acid substrates were effectively catalyzed by the best mutant UkaQ^FAVto form corresponding thioesters.By utilizing the enzymatic cascade reaction of UkaQ^FAV-CCRs(Ant E^V350G)and UkaQ^FAV-ACCase（Arm13-ACCase）,20 carboxylic acids were efficiently converted to the corresponding malonyl-CoAs-type products.At last,the UkaQ^FAV-CCRs cascade was introduced into an artificially biosynthetic pathway of deacylated antimycin,and 6 novel halobenzyl-containing antimycin analogs were successfully synthesized by precursor feeding.C14-functionalized steroids belong to a unique class of steroids with important biological activities.However,the lack of efficient methods to access C14-functionalized steroids impede related steroidal drug discovery.Herein we report a modular chemoenzymatic approach to access diversified C14-functionalized steroids.We first identified a novel C14α-hydroxylase（CYP14A）from Cochliobolus lunatus with high catalytic efficiency and substrate promiscuity.Protein engineering of CYP14A generated two best variants I111L-M115K and I11L-V124W that greatly improved the C14-hydroxy regioselectivity.Based on this efficient biocatalytic method,a range of C14α-OH steroids with C17 side chain were prepared in good yields which served as a versatile handle to install diversified functional groups（e.g.epoxide,β-OH,F,Cl,N₃,Seph,alkyl,NCO,cylcopropane）at C14 position through hydrofunctionalization.Furthermore,the synthetic utility of this powerful chemoenzymatic methodology was demonstrated by performing a 7-step semisynthesis of periplogenin and the diversity-oriented synthesis of（+）-digitoxigenin and its three diastereomers in a concise manner.This study identified a novel acyl-CoA synthase and steroid 14-hydroxylase.Based on the above findings,an efficient biosynthesis method of editing polyketide carbon skeleton based on extended unit diversity and steroid C14-functionalization was established.These provide a proven method for carbon skeleton editing and modification of polyketides and steroids.