Constructing Metabolic Engineering Saccharomyces Cerevisiae Strain For Producing Valencene And Nootkatone By Fermentation Using Seaweed Hydrolysate

(+)-Valencene and(+)-nootkatone are high value-added natural products found in plants,which can be used in products such as perfumes,food additives and pesticides.In our previous study,it was found that the non-fermentable carbon source mannitol can avoid the overflow metabolism of the dominant carbon source glucose to ethanol,allowing more energy to flow into the metabolic pathway of terpenoids and maximizing the carbon flux of terpene products.This thesis investigated the effects of different origins and amounts of hydrolytic enzymes on the degree of Seaweed hydrolysis,and explored the biosynthesis of(+)-valencene using Seaweed hydrolysate as a substrate with Saccharomyces cerevisiae as the original strain.Further,the biosynthesis of(+)-nootkatone with Seaweed hydrolysate as the substrate was achieved through enhancement of precursor supply,optimization of key enzymes,adaptive laboratory evolution,and medium optimization.Firstly,this study investigated the effects of different origins and the added volume of hydrolytic enzyme on the hydrolysis effect of Seaweed.It was found that Seaweed from Changdao,Shandong Province released higher mannitol,which was four times higher than that from Xiapu,Fujian Province.And,Seaweed could release20.91 g/L mannitol and 7.31 g/L glucose under the conditions of optimal hydrolytic enzyme amount.Then,the biosynthesis of(+)-valencene was investigated when Seaweed hydrolysate was used as the carbon source.Under the condition of mixed carbon source of mannitol and glucose,the strain was able to maintain normal cell growth and alleviate the Crabtree effect,facilitating the accumulation of(+)-valencene which achieved 102.61 mg/L.When using Seaweed hydrolysate as the substrate,the cells synthesized(+)-valencene up to 108.75 mg/L after optimizing the Seaweed treatment method and fermentation conditions.Based on the above findings,HPO/At CPR and ADH enzymes were overexpressed to construct a biosynthetic pathway of(+)-nootkatone using Seaweed hydrolysate as substrate.Under the condition of mannitol as the only carbon source,the biosynthetic pathway of(+)-nootkatone was successfully constructed by exploring different sources of ADH enzymes to improve the catalytic efficiency of β-nootkatol to(+)-nootkatone and deleting excessive t HMG1 genes to enhance the supply of precursor substances.Further,in order to improve the conversion efficiency of(+)-nootkatone,a recombinant strain of(+)-nootkatone with TUP1 gene deletion was constructed,and the percentage of(+)-nootkatone reached 75% by shake flask fermentation with mannitol as substrate.The yield of(+)-nootkatone was further improved by laboratory adaptive evolution to reach 71.05 mg/L.When Seaweed hydrolysate was used as the carbon source,it was found that the inhibitory factors in the hydrolysate reduced the total terpene content and the percentage of(+)-nootkatone.By optimizing the medium composition,21.25 mg/L of(+)-nootkatone was obtained,which was 2.6 times higher than the yield before optimization.This study provides a green and sustainable technological approach for the production of high value-added terpenoids.And the efficient utilization of Seaweed hydrolysate also provides an excellent example for the application of third-generation renewable biomass resources.