| Silybin is a flavonolignan compound extracted from the seeds of Silybin,which has important biological activities such as liver protection,anticancer,and antioxidant properties.At present,the production of silybin is mainly extracted from the seed coat of the plant Silybum marianum.However,due to differences in plant growth cycle and planting management level,the content of silybin in different regions and periods of plant Silybum is unstable,and quality control is difficult.Meanwhile,the traditional separation and extraction process of silybin is time-consuming and costly.The biosynthesis of high-value compounds using microorganism is favored due to its free from season,easy control of production quality,and simple separation and purification.However,the microbial biosynthesis of silybin faces problems such as long biosynthetic pathways,insufficient supply of precursors,multiple cofactors,and product inhibition effect on host growth.To achieve efficient microbial synthesis of silybin,the above issues need to be addressed.To explore and remove various limiting factors in the biosynthesis pathway of silybin,the synthesis pathway of silybin was first divided into four modules based on the properties of the intermediates:caffeic acid,ferulic acid,coniferyl alcohol,and silybin.This study comprehensively utilized literature research and transcriptome data to explore new genes that promote phenolic acids and flavonoids,enhance endogenous cofactor synthesis and regeneration,drive the biosynthesis of various phenylpropanoid,and combine multiple pathways to enhance the hydroxylation efficiency of naringenin in Escherichia coli.Finally,an engineering strain for synthesizing silybin was obtained.This study provides a detailed analysis of various limiting factors and their release strategies in the microbial synthesis of silybin,providing a theoretical basis for the microbial heterologous synthesis of complex flavonolignans,which is of great significance for the industrial production of flavonoid lignans.The specific work includes the following:（1）The enhancement of cofactor FAD synthesis and mining transporters from transcriptome data promoted efficient synthesis of caffeic acid.Firstly,the expression level of key genes Fj TAL and Echpa BC for caffeic acid synthesis was optimized and the competitive pathway for L-tyrosine synthesis was knocked out,generating an engineering strain for efficient biosynthesis of caffeic acid.Enhancing the biosynthetic pathway of the cofactor FAD and the expression of previously reported polyphenol transporters promoted the production of caffeic acid.To improve the efficiency of intracellular caffeic acid outward transport and reduce cell growth inhibition,transcriptomic analysis was conducted under caffeic acid and ferulic acid stress to uncover the transporter protein ycj P,which increased the titer of caffeic acid to 775.7mg·L-1.In a 5 L fermenter,the titer of caffeic acid increased to 7922.0 mg·L-1,which is the highest level reported.The construction of a high producing strain of caffeic acid provides excellent chassis cells for the downstream biosynthesis of ferulic acid and coniferyl alcohol.（2）Strengthening the biosynthesis of S-adenosyl-L-methionine and modify the transport pathway promoted the biosynthesis of ferulic acid.To reduce the cytotoxicity of ferulic acid,experiments were conducted on the tolerance of E.coli to ferulic acid.Through transcriptome data mining,it was found that knocking out omp C encoding outer membrane pore proteins promoted ferulic acid synthesis;Subsequently,knocking out ydd G prevented the efflux of L-tyrosine and promote the synthesis of ferulic acid.Secondly,by simulating the molecular docking of caffeic acid O-methyltransferase and caffeic acid,it was found that the 161st,268th,314th,and 317th amino acids of At COMT1 were crucial for the biosynthesis of ferulic acid;By strengthening the synthesis of S-adenosyl-L-methionine,blocking the competitive pathway of L-methionine synthesis,and reducing S-adenosyl-L-methionine consumption,the supply of S-adenosyl-L-methionine was increased,and the titer of ferulic acid was increased to 355.4mg·L-1.The biosynthesis of ferulic acid was optimized in a 5 L fermenter,and the titer of ferulic acid reached 1665.0 mg·L-1.The enhancement of ferulic acid synthesis laid the foundation for the downstream biosynthesis of coniferyl alcohol.（3）Regulating the cofactor pathway and construction of an efflux pump system to promote the synthesis of coniferyl alcohol.Firstly,several biosynthesis routes of coniferyl alcohol were designed and compared,and it was found that the biosynthesis pathway composed of Pc4CL1,Zm CCR,and Zm CAD reached highest efficiency;Secondly,by cofactor regeneration,combined with strategies to block byproduct pathways and knock out competitive pathways,the titer of coniferyl alcohol was increased to 254.6 mg·L-1.Then,the optimized biosynthesis pathway of coniferyl alcohol was transformed into a high-yield strain of ferulic acid.By further optimizing the expression level of Pc4CL1 and linkers engineering,the conversion rate of intermediates was improved,and the titer of coniferyl alcohol reached 630.6 mg·L-1.Finally,heterologous expression of srp B encoding an organic solvent tolerant pump increased the titer of coniferyl alcohol to 742.8 mg·L-1.In a 5 L fermenter,the titer of ferulic acid and coniferyl alcohol reached 195.3 mg·L-1 and 1315.3 mg·L-1,respectively.The efficient biosynthesis of coniferyl alcohol lays the foundation for biosynthesis of silybin by microorganisms.（4）A de novo synthetic strain of silybin was constructed based on multi-channel assembly and modular engineering.Firstly,a biosynthetic pathway for eriodictyol was constructed on the basis of high-yield caffeic acid producing strains;Secondly,the effects of different pathway combinations on the biosynthesis of eriodictyol were compared,and more efficient Echpa BC and tr Th F3’H/CPR were assembled.The titer of eriodictyol reached 33.8 mg·L-1.Then,a biosynthesis pathway of taxifolin was constructed,and the titer of taxifolin reached 58.2 mg·L-1.Basing on transcriptome data from naringenin stress,it was found that overexpression of yhc N enhanced naringenin synthesis.Finally,different synthesis modules of silybin were constructed to achieve biosynthesis of silybin by mono-culture system.In addition,an enzyme catalyzed system for silybin using coniferyl alcohol,taxifolin,and hydrogen peroxide as substrates was constructed and optimized.Under the conditions of 37℃and p H 8,the titer of silybin and isosilybin were 1.8 g·L-1 and 2.2 g·L-1 respectively,with a conversion rate of 49.7%at 1 h. |
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