Engineering Rhodosporidium Toruloides For Limonene Biosynthesis And Derivatives

Limonene is a valuable monoterpene used in the production of food,pharmaceuticals,cosmetics,biofuels,biological control,commodity chemicals etc.Limonene is obtained from plants(e.g.orange,lemon)and by chemical synthesis.However,those methods are not only low efficient,expensive and always waste natural resources,but also difficult to ensure the production and quality of limonene.Construction of limonene and its derivatives microbial cell factory by metabolic engineering strategies and synthetic biology techniques.Compared with extraction from plants and chemical synthesis,microbial production is provided with the advantages of green,high product purity,easy separation and purification,scalable amplification,and stable supply.In recent years,metabolic engineering and synthetic biology technology have made a lot of progress in the research of microbial synthesis of limonene and its derivatives,including the selection and transformation of chassis microorganisms,the discovery and editing of key enzymes,the microbial synthesis of limonene derivatives,the optimization of fermentation processes,and design,expression,optimization of synthetic pathways.At present,integrating the biosynthetic pathway of monoterpenes and derivatives into the host strain still faces some problems,such as multiple activities of monoterpene synthase,low metabolic flux to target products,and interference of natural homeostasis and cytotoxicity of monoterpenes to host strain.Rhodosporidium toruloides can be used as a chassis microorganism for the biosynthesis of high-value compounds,due to the wide spectrum of substrate utilization and product,better robustness,powerful MVA pathway etc.In this study,the R.toruloides np11 strain was used as the host strain.We characterized the effect of the carotenoid pathway and gene elements(promoter and limonene synthase)on limonene production in yeast.Then,we further improved the limonene synthesis through modular engineering,protein fusion strategy and combinatorial metabolic engineering.This study explored the compartmentalization potential of the peroxisome region of R.toruloides,the N-and C-terminal localization peptides were obtained,and the orthogonal limonene synthesis pathway was targeted to the peroxisome.In addition,we also studied the ability of heterologous biosynthesis of limonene derivatives in R.toruloides.It provides a certain reference to produce high-value compounds in R.toruloides.The results of this paper are as follows:1.The orthogonal limonene heterologous pathway engineered strain for limonene biosynthesis was successfully constructed in R.toruloides np11.The engineered strain produced 6.7 mg/L limonene by introducing neryl diphosphate synthase(NPPS)derived from Solanum habrochaites,limonene synthase(Clt LS1)from Citrus limon and overexpressed truncated endogenous hydroxymethylglutaryl-Co A reductase(HMGR).The highest production of limonene was increased to 37.71 mg/L,when the carotenoid synthesis-deficient strain was used as the host strain,the xylose reductase promoter(p XYL)and the limonene synthase Clt LS1 were used as gene elements.The host strains and gene elements of limonene biosynthesis were preliminarily selected to lay the foundation for subsequent research.2.Combinatorial engineering of R.toruloides for improved limonene production.Limonene production of the engineered strain reached 107.23 mg/L by introducing the key enzymes of the MVA pathway,acetoacetyl-Co A thiolase /HMG-Co A reductase(Ef Mva E)and HMG-Co A synthase(Ef Mva S)from Enterococcus faecalis,and mevalonate kinase(Mm MK)from Methanosarcina mazei.The protein fusion strategy has the effect of reducing the space distance and improving the accessibility between enzyme and substrate,weakening metabolic overflow and enhancing the catalytic efficiency of fusion proteins.In this study,NPPS and Clt LS1 were fused by the protein fusion strategy and the effects of five protein flexible linkers and the forward/reverse fusion protein on limonene production in the engineered strains.It was demonstrated that forward fusion of NPPS and Clt LS1 by GGGS could improve limonene production.The engineered strain produced 139.74 mg/L of limonene,which was a 3.7-fold increase than that of the free enzyme expressed strain.Then these two strategies were combined to further improve the production of limonene in the strain,and the production of limonene reached 393.48 mg/L.Finally,the limonene tolerance experiment of the engineered strains was tested,and a strain that was resistant to limonene was obtained.3.Limonene was synthesized by compartmentalization of peroxisome in R.toruloides.In this study,the red or green fluorescent protein was used as fluorescent marker,and endogenous peroxisome protein Pex11 as reference.The localization effect of peroxisomal targeting peptide type I(PTS1)and type II(PTS2)was verified by the fluorescence co-localization,and the type I(AKL,SKL,AKM and ARL)and II(PTS2-39AA)peroxisomal targeting peptides were obtained.The structure of the peroxisomal membrane protein Pex11 was analyzed,and the sequence of the suspected type II localization peptide in Pex11 was truncated/intercepted.Those sequences were fusion expressions with a red fluorescent protein,and neither truncation nor interception could target the protein to the peroxisome.Finally,the orthogonal pathway of limonene synthesis was targeted to peroxisomes by PTS1(SKL),and the strains produced limonene with titers around 2.0 mg/L.This study provides a scientific reference for the synthesis of high-value compounds by the peroxisomal compartmentalization of R.toruloides.4.We characterized the ability of R.toruloides to synthesize terpenoids by introducing the heterologous biosynthetic pathways of carvone and menthol.Carveol and carvone were synthesized in R.toruloides by introducing geranyl pyrophosphate synthase(GPPS)derived from Abies grandis,(-)-limonene synthase(Mst LS)from Mentha spicata,carveol dehydrogenase(CD)from Mentha × piperita,cytochrome P450 reductase(CPR)from Arabidopsis thaliana and HMGR.Engineered strains were obtained by expressing Mst LS,cytochrome P450(-)-limonene-3-hydroxylase(L3H)and(-)-trans-isopiperitenol dehydrogenase(IPDH),but the target product has not been detected.Combined with the previous experimental results,it may be caused by an insufficient supply of precursors.In conclusion,this study achieved the de novo biosynthesis of limonene and some of its derivatives,and proved the potential of R.toruloides compartmentalization,a potential platform strain for the efficient synthesis of monoterpenoids.