Combining Metabolic Engineering And Protein Engineering For Production Of Amyrin In Yarrowia Lipolytica

Amyrin is a triterpenoid natural product,which has been proven to have various pharmacological activities such as anti-inflammatory,antibacterial,antiviral and antitumor,so the market demand is increasing day by day.The direct extraction of amyrin from plants has the disadvantages of long growth cycle,lack of resources,low content and ecological damage,while the chemical synthesis of amyrin will consume a lot of fossil fuels,causing greenhouse gas emissions and environmental pollution.Due to the above reasons,it is expensive and cannot meet the market demand.In recent years,the rapid development of metabolic engineering and synthetic biology has made it possible to use microbial cell factories to synthesize all kinds of plant natural products such as amyrin heterologously.The use of microbial cell factories to produce amyrin has obvious advantages such as rapidity,simplicity,mild production conditions,environmental friendliness,and green sustainability.Y.lipolytica is an unconventional safety-grade yeast that can grow using a variety of inexpensive carbon sources and produce a variety of high-value metabolites.At the same time,Y.lipolytica contains a high supply of acetyl-Co A in the cells,which can provide abundant precursor substances for the synthesis of terpenoid natural products.Therefore,the yeast has become an excellent microbial chassis for the synthesis of terpenoid natural products such as amyrin.In this study,Y.lipolytica was selected as the chassis organism,and a heterologous high-yielding amyrin producing engineered strain was finally obtained through a combination of strategies such as metabolic engineering,protein engineering and fermentation engineering.The main research contents are as follows:(1)First,the amyrin synthase gene Cr MAS from catharanthus roseus was selected by reading the literature,and then the codon-optimized Cr MAS gene was cloned into the Y.lipolytica expression vector p YLEX1 to construct the recombinant plasmid p YLCr MAS;then linearized.The p YLCr MAS was transferred into the Y.lipolytica chassis strain Po1 g △KU70 to obtain the recombinant yeast strain Po1 g KCr MAS.After fermentation and culture of Po1 g KCr MAS,the fermentation products were qualitatively and quantitatively analyzed by gas chromatography.The results showed that compared with the starting strain Po1 g △KU70,the p YLCr MAS strain could synthesize both α-arosinol and β-arosinol.This result proves that the plant-derived functional genes selected in this study can be expressed and function normally in the chassis of Y.lipolytica.(2)In order to further improve the production of amyrin,the four key functional genes(HMGR,ERG1,ERG9 and ERG20)in the Y.lipolytica mevalonate(MVA)pathway were cloned into p YLEX1 and the corresponding genes were obtained.Then,the expression cassettes were recombined into p YLCr MAS to construct single-gene overexpression recombinant plasmids;finally,the linearized single-gene overexpression recombinant plasmids were respectively transformed into Y.lipolytica chassis strain Po1 g △ KU70 to obtain recombinant plasmids.Yeast strains Polg KERG1,Polg KERG9,Polg KERG20 and Polg KHMGR.These strains were fermented and cultured,and their fermentation products were quantitatively analyzed by gas chromatography.The results showed that the overexpression of the ERG1 gene could significantly increase the production of amyrin,and the corresponding engineering strain Polg KERG1 had a yield of 42.37 mg/L of amyrin,including 26.26mg/L of α-amyrin,16.11 mg/L of β-amyrin.(3)Next,using the method in step(2),the above-mentioned four pathway genes are combined and overexpressed in pairs to obtain 6 engineered strains with different gene combinations.The results showed that the simultaneous overexpression of the HMGR gene and the ERG20 gene led to the highest yield of amyrin,of which the yield of α-amyrin was 36.30 mg/L,and the yield of β-amyrin was 20.10 mg/L.(4)Through the analysis of the production of amyrin and its precursor squalene in the metabolically engineered strains,we speculate that the bottleneck causing the low production of amyrin in the engineered strains may be the enzymatic activity of amyrin synthase itself lower.Therefore,in order to explore the effect of the activity of amyrin synthase on the product yield,we carried out molecular simulation and molecular docking of the enzyme.Through the analysis of residues near the substrate binding site of amyrin synthase,8 sites were selected for amino acid site-directed mutagenesis,namely P240 A,F243A,P251 A,L323A,L324 A,L328A,F331 A,C341A,L323A/L324 A.The above mutation sites were mutated using amino acid sitedirected mutagenesis kit,and the plasmids containing the mutation sites were linearized and transformed into Y.lipolytica chassis strain Po1 g △KU70 to obtain 9recombinant yeast strains.After the strain was fermented and cultivated,its fermentation product was quantitatively analyzed by gas chromatography.The results showed that the L323 A mutation could accelerate the binding and release of the substrate from the binding pocket to the greatest extent,resulting in an increase in the activity of amyrin synthase and an increase in the total production of amyrin.(5)The L323 site of amyrin synthase in the metabolically engineered highyielding strains Po1 g KCHERG9 and Po1 g KCHERG20 was mutated to L323 A.The results showed that after the combination of metabolic engineering and protein engineering strategies,the yields of α-amyrin and β-amyrin in the highest-yielding engineered strain Po1 g KCHERG20A were increased to 69.00 mg/L and 79.58 mg/L,respectively.(6)Finally,the fermentation conditions of the Po1 g KCHERG20A strain were preliminarily optimized,such as temperature,liquid volume and medium,and the optimal fermentation conditions for the engineering strain were determined to be YPD medium,temperature 30 °C,and rotation speed 250r/ min.Under this optimal fermentation condition,the total yield of amyrin increased to 125.42 mg/L,of whichα-amyrin was 102.86 mg/L and β-amyrin was 22.56 mg/L,which were 43 times and15.9 times that of the initial strain,respectively.In conclusion,this study firstly used metabolic engineering to successfully construct a Y.lipolytica cell factory capable of heterologously synthesizing amyrin,and discovered the rate-limiting bottleneck affecting the efficient synthesis of amyrin in this chassis;and then through protein engineering strategies The rational engineering of amyrin synthase was carried out,and an engineered Y.lipolytica strain with a high yield of the target product was finally obtained.The results obtained in this study provided ideas and laid a foundation for the further engineering of Y.lipolytica for efficient production of amyrin in the future.