Heterologous Reconstruction Of Baicalein Biosynthetic Pathway In Pichia Pastoris

Scutellaria baicalensis Georgi,a Chinese herbal medicine,is widely used in the treatment of cancer,tumor,respiratory tract infection,hypertension,bleeding and other diseases.There are a variety of flavonoids in S.baicalensis Georgi.The characteristic flavonoids in its root,such as baicalein and baicalin,are the main reasons for its pharmacological activity.Wild S.baicalensis Georgi has been seriously damaged with the increase of market demand.The quality of planted S.baicalensis is unstable.The chemical synthesis method is difficult to meet the needs of pharmaceutical production and use owing to toxic or extreme conditions.The rapid development of synthetic biology makes it possible to synthesize high value-added compounds from various natural sources in heterologous hosts.This project aims to build biosynthetic pathway of baicalein with L-phenylalanine as a precursor,optimize the endogenous biosynthetic pathway of L-phenylalanine and explore methods to relieve the growth inhibition effect of cinnamic acid in P.pastoris.It provids a new strategy for the heterologous synthesis and process research of baicalein and its derived flavonoids in new chassis cells.Firstly,we expressed coenzyme A ligase,chalcone synthase,chalcone isomerase,flavone synthase,flavone-6-hydroxylase from S.baicalensis Georgi and P450 reductase from Arabidopsis thaliana in Pichia pastoris,and realizing the heterologous synthesis of baicalein using cinnamic acid as the substrate.While cinnamic acid is a kind of weak antibacterial acid,which can inhibit the growth of the yeast when it is excessive in the medium.We confirmed that the inhibition effect of the acid radical ion form of cinnamic acid on the growth of the strain was far less than its molecular form through pH control.Furthermore,the inhibitory effect of cinnamic acid on the host was analyzed when methanol,ethanol and glucose were used as carbon sources.The results showed that the growth of P.pastoris was normal under the conditions of ethanol and methanol.When ethanol is used as carbon source,it can also supply malonyl coenzyme A,which is the precursor of the baicalein synthetic pathway,so it is used as preferred carbon source.Secondly,the phenylalanine ammonia lyase from S.baicalensis Georgi could not catalyze the conversion of L-phenylalanine to the intermediate cinnamic acid.Based on its sequence,five other phenylalanine ammonia lyases were screened in NCBI database,and their expression was regulated by constitutive promoter P GAP and ethanol-induced transcriptional signal amplification device(ESAD),with cinnamic acid successfully synthesized.Among them,we obtained 130 mg/L and 230 mg/L cinnamic acid under glucose and ethanol when using PGAP,400 mg/L under ethanol when using ESAD,respectively.And then,phenylalanine ammonia lyase was integrated into the baicalein producing strain previously constructed,and the heterologous synthesis of L-phenylalanine to baicalein was successfully realized.Finally,we expressed the feedback inhibition insensitive mutant of key enzyme in the endogenous L-phenylalanine synthetic pathway which were regulated strictly in natural microorganisms to optimize the expression of this pathway of the strain that can produce cinnamic acid.The optimized strain could synthesize cinnamic acid from basic carbon source and achieve the same yield as the original strain with sufficient L-phenylalanine.In conclusion,we successfully established the biosynthetic pathway of baicalein in P.pastoris,alleviated the inhibition on growth of cinnamic acid by various means and optimizing the biosynthetic pathway of endogenous precursor L-phenylalanine,which laid a foundation for the de novo synthesis of baicalein and other flavonoids in P.pastoris.