Construction Of Yarrowia Lipolytica Strain Producing Naringenin From Glycerol

Naringenin is an important compound in flavonoids,which is catalyzed by related enzymes（methylase,hydroxylase,glycosylase）to derive many bioactive flavonoids.These flavonoids have many applications in food,medicine,cosmetics,and other industries.Currently,naringin and its derivatives are mainly extracted from plants rich in flavonoids,with high extraction costs and environmental friendliness,which greatly weakens the application of naringin and its derivatives.The yield of heterologous biosynthesis of flavonoids is generally low,mostly at the milligram level.Heterologous biosynthesis is a promising production method for developing green and efficient synthesis of naringin and its derivatives.In recent years,studies on Yarrowia lipolytica have shown that it has a strong ability to synthesize flavonoids.This provides a good production platform for the efficient heterologous synthesis of naringenin and its derivatives.In this study,a high yield naringin lipolytic yeast strain was constructed,laying a foundation for the expanded production of naringenin and its derivatives.The main contents of this study are as follows:（1）Enzymes with high catalytic activity are essential for the efficient heterologous synthesis of naringenin.Therefore,phenylalanine（PAL）,tyrosine ammonia lyase（TAL）,and chalcone synthase（CHS）from different species have been screened in Yarrowia lipolytica,and RgPAL,CluTAL,and MdCHS2 with high catalytic activity have been successfully selected.The amount of p-coumaric acid catalysed by RgPAL is more than 4 times that of the previously used EbPAL in the laboratory,and the amount of p-coumaric acid catalysed by CluTAL is nearly 2 times that of the best FjTAL in the previous literature.MdCHS2 is also the best heterologous flavonoid compound currently produced,and can catalyze the production of 165 mg/L of naringin within 96 hours.（2）A biosynthetic pathway of naringenin was constructed using highly active naringenin synthesis related enzymes screened from Yarrowia lipolytica,and the yield of naringenin reached 190 mg/L.（3）The common metabolic targets of shikimic acid were modified,and it was found that the mutants ARO4^K221Land ARO7^G139S,which overexpress aromatic amino acids and are insensitive to feedback,are the most effective for carbon flow directed naringenin synthesis.The accumulation of the intermediate to coumarin acid reached a gram level,while the modification of other common targets of shikimic acid(overexpression of ARO1,ARO2,and ARO3^K225L)has little effect.（4）The bottleneck in the synthesis of coumarin to naringenin was studied.It was found that the catalytic activity of both 4CL and CHS was insufficient.Combined with multiple copies of Eb4CL and MdCHS2,the yield of naringenin gradually increased with the increase in the copy number of Eb4CL and MdCHS2.The yield of naringenin in the four copy strains of Eb4CL and MdCHS2（LW-26）reached 460mg/L.（5）Finally,the fermentation performance of LW-26 was tested in a fermentation tank.In a basic salt medium,using glycerol as the sole carbon source,8.67 g/L naringenin could be produced within 180 hours.