Metabolic Engineering Of Saccharomyces Cerevisiae For De Novo Biosynthesis Of Rubusoside And Rebaudioside M

Steviol glycosides（SGs）are the high-sweetness and low-calorie natural sweeteners,including rubusoside,stevioside（STV）,rebaudioside A（Reb A）,rebaudioside B（Reb B）,rebaudioside D（Reb D）,rebaudioside M（Reb M）,and so on.Reb A is the most commonly used SG in the natural sweetener stevia on the market,while the slight bitterness and astringency taste of Reb A limits its development and application in some products.Reb M has become the new generation of natural sweeteners recently,due to the higher sweetness and better taste.With raised concern about chronic diseases caused by high-sugar and high-calories,such as high blood pressure,diabetes,and obesity,the natural sweeteners market（such as rubusoside and Reb M）is increasing quickly.Traditionally,rubusoside and Reb M are produced by plant extraction,enzymatic conversion,or whole-cell catalysis.However,those methods are limited by the problems of the long plant growth cycle,high extraction cost makes,or low substrates abundance in nature.Comparatively,microbial fermentation producing rubusoside and Reb M from cheap substrates,with the advantages of renewable raw materials,short growth cycle,low cost,green,and sustainability,is an important way to meet market demand in the future.Herein,we engineered the Generally Recognized as Safe strain Saccharomyces cerevisiae CEN.PK2-1C as a chassis for the de novo production of rubusoside and Reb M.The chassis is optimized via many strategies,such as relieving the rate-limiting steps,enhancing yeast tolerance,and re-balancing intracellular metabolic networks.The main research results are as follows:（1）The de novo production chassis cell of rubusoside was constructed.Firstly,the ent-kaurene biosynthesis pathway was constructed by introducing the key gene Gf KS.Then,the GGPP biosynthesis pathway was optimized by overexpressing IDI and t HMG1 and introducing FPS^F112A,which resulted in a 33.7-fold improvement in ent-kaurene titer.Subsequently,the de novo rubusoside production chassis was constructed by introducing the genes in the P450s module（KO,KAH and CRP1）and rubusoside synthesis module（UGT74G1 and UGT85C2）.The rubusoside titer was 4.5 mg/L,and most of them were secreted to the extracellular of the yeast.（2）The rate-limiting steps on the P450s module were eliminated.Firstly,we observed that KO,KAH,and CPR1 were located in the ER by visualization technology.Then,the rate-limiting steps in the P450s module were relieved by endoplasmic reticulum signal peptide truncation,protein fusion expression,and scaffold-free spatial enzyme assembly.After that,steviol titer improved to 40.6 from 5.3 mg/L.Combining the strategy of INO2 overexpression to restore endoplasmic reticulum homeostasis,the yield of the engineered strain reached 67.7 mg/L in shake flasks,and the cell morphology of the engineered strain was irregular and the cell surface was uneven.（3）The efflux pump for rubusoside secretion was mined.Firstly,the 3D protein structure of the plasma membrane efflux pump was obtained based on the Alpha Fold2 database.Then,PDR11 was identified as the main efflux pump for rubusoside secretion in S.cerevisiae through methods including molecular docking,efflux pump inhibition,effector pump knockout,RT-q PCR,and field emission scanning electron microscopy.After strengthening the efflux pump,the production of rubusoside in the shake flask reached 155.6 mg/L,and the cell surface morphology returned to a flat state close to that of the original yeast cell.（4）In silico prediction and experimental operation were applied to re-balance the intracellular metabolic of rubusoside-producing strains.We proved that MSN4 is an important stress response factor to rubusoside for S.cerevisiae by gene knockout and overexpression.Then,based on the genome-scale metabolic model and Opt Knock algorithm,the gene knockout targets in the engineered strains were rapidly predicted,and rubusoside titer increased by about20%after knocking out GAL7.This result suggests that the insufficient supply of UDPG maybe a key factor limiting rubusoside production,as GAL7 is a key competitive enzyme in the UDPG synthesis pathway.After further optimizing the UDPG synthesis pathway,the yield of rubusoside reached 302.1 mg/L in shake flask,and 1368.6 mg/L in 15 L fed-batch bioreactor.（5）The de novo production chassis cell of RebM was constructed and optimized.The Reb M de novo production strain was developed by introducing UGT76G1-MUT and EUGT11 into the rubusoside-producing chassis cell.Then,many engineering strategies,including engineering EUGT11 based on the predicted results of Rosetta Cartesian＿dd G,dynamically regulating UGT76G1-MUT,and overexpressing MSN4,were employed to enhance Reb M production.Finally,Reb M titer reached to 17.6 mg/L form 0.4 mg/L in shake flasks,and 67.0mg/L in 15 L fad-batch bioreactor.