Production Of Flavonoids By Bio-catalytic System With Multiple Enzymes From Plants

Flavonoids have been extensively studied for their physiological functions in regulating human physiology and treating human diseases.At present,the production methods of flavonoids are mainly through plant extraction and chemical synthesis.However,high-cost of plant extraction and intense of chemical synthesis have limited the large-scale commercial production of flavonoids.Biosynthesis has become the preferred route of flavonoid production because of its low cost,the conditions of reaction are mild.In this paper,the feasibility of producing high value-added flavonoids by biosynthesis was systematically explored,taking representative flavonoids as the research object.Enzymes from different sources of flavonoid synthesis pathways were excavated and screened,and the optimal reaction conditions and characteristics in flavonoid production were studied.Phenylalanine ammonia lyase from Zea mays(ZmPAL2)was applie d to the catalytic system coupled with reaction and separation to efficient production of cinnamic acid.The tyrosine ammonia lyase activity of ZmPAL2 was improved by molecular modification technology.In order to improve the production of p-coumaric acid,an in vitro multi-enzyme combination catalytic system was established and its conditions were optimized to make the cell-free system of naringenin production became possible.In Corynebacterium glutamicum,the promoter of C.glutamicum was mutated by random mutation to obtain different strength of promoters.The metabolic pathway of catechin was successfully constructed in C.glutamicum.The main results of researche are as follows:(1)The heterologous expression and reaction conditions of phenylalanine ammonia lyase(PAL),4-coumaric coenzyme A ligase(4CL),chalcone synthase(CHS)and chalcone isomerase(CHI)from different sources were studied.The phenylalanine ammonia lyase from Zea mays(ZmPAL),4-coumyl-CoA ligase from Oryza Sativa L(Os4CL),chalcone synthase from Mangifera indica(MiCHS)and chalcone isomerase from Nicotiana sylvestris(NsCHI)were selected for the subsequent catalytic experiments.(2)Cinnamic acid was synthesized by crude ZmPAL2 enzyme and a recombinant strain containing ZmPAL2.After optimizing the reaction conditions,the crude ZmPAL2 enzyme can synthesize 5.0 g/L trans-cinnamic acid from 10.0 g/L phenylalanine,and the the recombinant starin containing ZmPAL2 can synthesize 39.6 g/L trans-cinnamic acid from 40.0 g/L phenylalanine in a reaction-isolation coupled system.(3)ZmPAL2 was modified by saturation mutation to obtain F135 H mutant enzyme with higher tyrosinase activity,which was used in the biosynthesis of p-coumaric acid.The enzymatic properties of F135 H mutant enzyme showed that the mutation of F135 H site had no effect on the optimum pH,optimum temperature and pH stability of the enzyme,but slightly improved the temperature stability compared with the wild enzyme.Compared with wild enzymes,mutant enzyme can produce p-coumaric acid by crude enzyme and whole-cell catalysis and the yield is increased by 2.8 times and 4.1 times,respectively.(4)The selected Os4 CL,MiCHS and NsCHI were used to produce naringenin in vitro multi-enzyme catalysis system.The reaction conditions were optimized.Under the optimal reaction conditions,32.0 mg/L of naringenin could be prepared by catalytic reaction within 3 h.(5)Screened the enzymes of catechin biosynthesis pathway from various sources and optimized the catalytic conditions.The catechin biosynthesis pathway was constructed in C.glutamate.Tac promoter of C.glutamicum was mutated to construct promoter libraries with different promoter intensities,which proved that reasonable selection of promoters with different emphasis could improve the yield of flavonoids.In this paper,based on the study of flavonoids synthesized by multi-enzyme catalytic method in vitro,combined with molecular modification technology and the exploration of restrictive factors in metabolic pathway,the synthesis of flavonoids in vitro was realized,which laid a foundation for further development and application of flavonoid natural products synthesis.