Construction Of Phloridzin Yeast Genetic Engineering Strain

Objective:Phloridzin is a 2’-β-D-glucoside of phloretin,which belongs to the dihydrochalcone class of plant flavonoids.Flavonoids are widely distributed,but dihydrochalcone is less common in nature than flavonoids.Root bark glycosides have a variety of important biological activities,including lowering blood sugar,anti-ischemia,lipid-lowering and antioxidant,etc.The high bioavailability makes them promising for development in the pharmaceutical and food industries.The present source of root bark glycosides is mainly isolated from plant extracts,which are often interfered by structurally similar naringenin,flavanones,flavanols and various flavonoid compounds in the extraction process,and the separation and purification process is complicated,which limits its industrialization and application development.Therefore,using synthetic biology methods to synthesize phloridzin glycosides in microorganisms provides an innovative way to obtain phloridzin glycosides with homogeneous quality,easy isolation and purification and stable yield,and accelerates their industrial development and utilization as pharmaceuticals and health products.Methods:In this study,an engineered yeast with high p-coumaric acid production was used as the starting strain to integrate 4CL and CHS genes upstream of phloretin using CRIPSR-Cas9 technology to investigate the effect of different promoters on the yield of phloridzin precursor phloretin.Metabolite analysis and transcriptional analysis were performed on the high phloridzin producing plant,Mucuna pruriens,to screen the key double bond reductase genes of the phloridzin pathway from transcriptome data of different sites.After the full length was obtained using molecular cloning,a yeast expression vector was constructed by seamless splicing technique and transferred into the pre-constructed phloretin chassis strain for functional studies.Finally,the dominant double-bond reductase gene obtained from the screening was integrated into the phloridzin chassis bacteria with glycosyltransferase genes from different sources using CRIPSR-Cas9 technology,and the product was extracted and assayed after fermentation to obtain a phloridzin yeast strain with certain yield.Results:1.In this thesis,using Saccharomyces cerevisiae as the chassis,we selected the 4-coumaroyl-CoA ligase encoding gene At4CL from Arabidopsis thaliana and the chalcone synthase encoding gene HeaCHS from Helianthus annuus as the chassis strain QL11,which was constructed in the laboratory.Subsequently,the CRISPR-Cas9 technology was used to select the constitutive promoter and the naringin chalcone genes.Subsequently,the promoter elements of the engineered yeast were modified by selecting the constitutive promoters PTDH3 and PPGK1,and the inducible promoters PGAL1 and PGAL10,respectively,and an engineered yeast strain LLS2 with excellent performance was successfully screened after shake flask fermentation with the production of 2.33 mg/L of phloretin and 1.13 m/L of naringenin chalcone.2.In this study,the chemical composition of different parts of Lithocarpus litseifolius(Hance)Chun.was examined and the compounds related to the biosynthetic pathway were quantified.The concentration of phloridzin was detected by UPLC-Q-tof-MS in the mature leaves of H.chinensis,with 9.2 mg/g in the young leaves,15.7 mg/g in the old leaves,14.2 mg/g in the stems and 8.0 mg/g in the roots.3.In this study,10 key DBR genes of the upstream pathway consistent with flavonoid accumulation pattern in Mucuna pruriens were screened by transcriptome data analysis.The full length was cloned and yeast expression vectors were constructed for functional studies in a pre-constructed phloretin-engineered yeast strain.The amount of phloretin production was 5.63 mg/L for strain XYX2 and 6.54 mg/L for strain XYX2-Li33822,and the phloretin production was significantly increased to 1.16-fold compared with the non-transfected dehydrogenase strain.The Li33822 gene was identified as a double bond reductase gene capable of mediating the dehydrogenation reaction in yeast.Finally,the MdP2’GT gene from apple and the LiUGT gene from Mucuna pruriens were screened to construct a phloridzin-producing strain,and a new strain,LLS3,with a yield of 14.50 mg/L,was successfully constructed using CRISPR-Cas9 technology.The yeast strain LLS4 was constructed using CRISPR-Cas9 technology to regulate the metabolic homeostasis and to obtain a yeast strain with a certain yield of phloridzin.The amount of phloridzin produced by strain LLS4 was 13.09 mg/L and that of strain LLS5 was 4.19 mg/L.Conclsions:1.First,this chapter used CRISPR-Cas9 gene editing technology to construct a yeast engineered bacterium for the production of phloridzin precursor-phloretin with a yield of 2.33 mg/L by codon optimization and promoter screening and other research strategies,which laid the foundation for the subsequent construction of phloridzin engineered bacteria.2.By detecting the chemical composition in Mucuna pruriens leaf ke,there are differences in phloridzin in different tissue parts,and screening genes highly expressed in leaves from transcriptomes of different parts can be used to obtain efficient catalytic components of the phloretin pathway through functional studies.In this study,we screened and verified the functions of 10 DBR genes in Saccharomyces cerevisiae,identified two functional DBR genes in Mucor leaf ke,and obtained 33822 gene elements that could significantly increase phloretin production by 1.16-fold in chassis bacteria.The 33822 gene element,which significantly increased phloretin production by 1.16-fold in chassis bacteria,was obtained.3.The maximum yield of 13.09 mg/L of phloridzin yeast was obtained by integr ating the double bond reductase gene MdDBR from apple and the glycosyltransferase MdP2’GT from apple and the glycosyltransferase LiUGT from Mucoraceae,respectively.using CRISPR-Cas9 technology on the basis of the chassis bacterium LLS2.