Cloning And Identification Of Polyketide Synthase From Huperzia Serrata And Its Application In Combinatorial Synthesis

Huperzia serrata,whose folk name is Qian Ceng Ta,is a member of Lycopodium species.It is a perennial fern and has a long history of clinical application in Chinese folk medicine for treatment of contusions,swellings,alleviating pains and detoxification.H.serrata has become well known since huperizine A was isolated from the plants.HupA has been found to be a potent,reversible and high selective acetylcholinesterase inhibitor(AChEI)and with low toxicity,which made it a promising drug for treatment of symptoms of Alzheimer’s disease(AD).It can also improve learning and relieve the memory deficiencies.Plant polyketide synthases(PKSs)are a class of proteins in plants that produce polyphenols.They catalyze iteratively decarboxylative condensations of a starter coenzyme A(CoA)thioester and C2-units derived from malonyl-CoA to produce linear chain polyketide intermediates.By Claisen,Aldol cyclization,or lactonization,and aromatization,the linear chain polyketide intermediates produce a dazzling array of unnatural small molecules,such as chalcone,stilbene.Differences of starter substrates,numbers of malonyl-CoA condensation and mechanisms of cyclization reactions result in the functional diversity of PKSs,generating a variety of plant secondary metabolites.Owing to the broad range of substrates and the unordinary versatility of catalytic potential,PKSs are ideal tool enzymes to combinatorial synthesis for construction of structurally diverse library which could be used to screen for new drug candidates.HupA mainly comes from wild plant resources.Therefore,plants of the H.serrata are experiencing a rapid decline in China,mostly due to over-harvesting.The organic synthesis involved in many steps and resulted in a very low yield.With the development of biotechnology,the biosynthetic pathway genes have been elucidated that regulate and modify the biosynthesis of intermediates.On the basis of that,it can achieve a directional synthesis or large-scale production of the target product.Reconstruction biosynthesis pathways of taxol,artemisinic acid and indole-type alkaloids in engineered systems have been completed in large-scale production.Therefore,previous feeding experiments were conducted,suggesting that pelletierine is a key intermediate to HupA.Pelletierine coupled with 4PAA/4PAACoA,catalyzed by an unknown enzyme,to form phlegmarine,the general intermediate to all lycopodium alkaloids,which attract the most attention.To some extent,PKSs may involve in the biosynthetic pathway on the similar coupling mechanism of the key reaction.Hence,screening novel plant PKSs from plants and probing their catalytic potentials would be not only advantageous to understand the biosynthesis of plant metabolites but also instructive to enzymatically or biomimetically synthesize structurally diverse and chemically complex unnatural natural products.Many results are list as follows.First of all,using homologous cloning strategy,a new type III PKS(HsPKS3)was successfully cloned from the leaves of H.serrata,excluding the known HsPKS1 and HsPKS2.The function of HsPKS3 is different from HsPKS1 and HsPKS2.HsPKS1 and HsPKS2 catalyze the condensation of one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA to produce chalcone,which may be an important precursor for the biosynthesis of flavonoids in H.serrata.They can also catalyze one molecule of nitrogen-containing bulky N-methylanthraniloyl-CoA and three molecules of malonyl-CoA to produce acridones.However,HsPKS3 catalyzes the head-to-head condensation of two molecules of p-coumaroyl-CoA and one molecule of malonyl-CoA to produce bisdemethoxycurcumin,accompanying with the formation of a C6-C3 scaffold of p-hydroxybenzalacetone.Interestingly,HsPKS3 also accepts nitrogen-containing bulky N-methylanthraniloyl-CoA as a starter to produce quinolone alkaloid.To the best of our knowledge,HsPKS3 is the first type plant PKSs with the multifunctions.Furthermore,on the foundation that the three PKSs have a wide range of substrate,a series of combinatorial systheses were conducted.For one thing,4-coumarate coenzyme A ligase(4CL)and chalcone synthase(CHS),the two pivotal enzymes involved in the biosynthesis of flavonoids were applied to synthesize varied unnatural natural products combinatorially.As a result,8 CoA thioesters were catalyzed by plant-originated 4CL,including a series of phenylpropinoyl analogues and nitrogen-containing five-membered aromatic acid thioesters.Then,25 unnatural natural products including eight flavonoid analogues,16 4-hydroxy-δ-lactones,and one benzalacetone were synthesized by combining HsPKSl and HsPKS2.For another,the curcuminoids-,benzalacetone-,and quinolone-producing HsPKS3 uniquely catalyzes the formation of unnatural 2-substituted quinolones and 1,3-diketones via head-to-head condensation of two completely different substrates.Structurally diverse starters(CoA thioester)including aromatic-CoAs,aliphatic-CoAs,heteroaromatic-CoAs,and bulky anthraniloyl-CoA analogues,and various extenders including 3-oxo-4-phenylbutanoic acid,very long chain 3-oxooctadecanoic acid,and structurally stable β-keto acid thioesters of NAC were used as substrates.As a result,the broad range of substrate of HsPKS3 facilitates synthesizing structurally diverse 2-substituted quinolones and 1,3-diketones.Compounds P07-P11,P13,P14,and P16-P18 were evaluated for their anti-inflammatory activities.The synthesis methodology would be instructive for the construction of a library of structurally diverse small molecules and screening new drug candidates.Lastly,it is the potential functions of HsPKS3 in the biosynthesis of HupA.The similar structure-known protein was chosen as a template,and a successfully predicted structure of HsPKS3 was obtained by homology modelling.Subsequently,we proposed the catalytic mechanism of HsPKS3.Serine 142 was a key residue in catalyzing bisdemethoxycurcumin presumably.Indeed,when serine 142 was substituded with tyrosine,the mutant losts the bisdemethoxycurcumin-forming activity and produces a byproduct.On the other side,4PAA was docked into the receptor(HsPKS3).The compounds’ piperidine ring was closely to the binding site(Cys174),which will instruct to the further catalytic reactions.These are useful to conduct the identification of specific enzymes and the elucidation of the entire pathway of the lycopodium alkaloids.Hence,the attempts on the function of PKSs involved in the coupling mechanism should be made further.