Engineering Saccharomyces Cerevisiae For The Production Of Coumarins From Lignin

As an important renewable resource,lignin is difficult to be utilized due to its intrinsic heterogeneity.To meet the increasing demand for lignin valorization,the biosynthesis of coumarins from lignin was realized in Saccharomyces cerevisiae through design and construction of strains converted lignin monomers.The main work is as follows:1.4-coumarate CoA ligase(4CL)from Petroselinum crispum and feruloyl-CoA6′-hydroxylase(F6′H1)from Arabidopsis thaliana were expressed in yeast,which realized the bioconversion from ferulic acid to scopoletin.The production of scopoletin was 0.36±0.05 mg/L,while 74±23 μg/L of fraxetin was detected.F6′H1 from A.thaliana was first found to hydroxylate scopoletin to produce fraxetin,and the bioconversion of fraxetin from ferulic acid was realized in this study.Besides,the biosynthesis of scopoletin derivatives was explored,and several caffeic acid 3-O-methyltransferase(COMT)were found to methylate fraxetin to produce isofraxidin.2.To improve the production of scopoletin,the effect of fusing 4CL and F6’H1with four linkers in different orientations were explored.The fusion expression with the linker(Gly-Gly-Gly-Gly-Ser)4 between F6′H1 and 4CL increased scopoletin production by 3.3-fold.To increase the stability of the engineered strain,the fusion genes were integrated into HO locus of yeast genome,which led to a titer of 3.42±0.15 mg/L of scopoletin.In addition,six strains contained site-directed mutation of F6′H1 were constructed,but the production of scopoletin was not increased.3.Through the screening of functional genes,the pathway contained4-hydroxyphenylacetate 3-monooxygenase,NADPH-flavin oxidoreductase,COMT,the fusion of F6′H1 and 4CL achieved the bioconversion of scopoletin with a titer of4.98±1.03 mg/L from p-coumaric acid.Furthermore,via the sodium hydroxide pretreatment of corn stover,the lignin hydrolysate was used as substrate to produce scopoletin.The production of scopoletin was 4.79±0.72 mg/L.In this study,the bioconversion from different lignin monomers to coumarins were achieved,which provided a new idea for lignin valorization.