Enhancing α-farnesene Synthesis Via Peroxisomal Localization Of The Mevalonate Pathway In Yarrowia Lipolytica

Yarrowia lipolytica is an unconventional oleaginous yeast.Due to its sufficient precursor supply,it is suitable for the production of various downstream derivatives of acetyl-CoA.However,when glucose is used as the carbon source,the production of acetyl-CoA is accompanied by the release of carbon dioxide(CO2),causing carbon loss and yield reduction.When fatty acid is used as the substrate,fatty acid undergoes P-oxidation to produce acetyl-CoA,this process does not generate CO2,and has no carbon loss,and has a higher carbon conversion rate.The β-oxidation of fatty acid occurs primarily in the peroxisome.Therefore,in this paper,the mevalonate pathway enzymes were overexpressed and localized in the peroxisome of Y.lipolytica to construct an engineered strain of synthesizing α-farnesene.Fatty acid or lipid was used as a substrate to efficiently synthesize the downstream product of acetyl-CoA,an important terpenoid α-farnesene.First,this study tested an enhanced peroxisomal targeting signal type 1(ePTS1)used in Saccharomyces cerevisiae reported in the literature to efficiently target the protein of interest to the peroxisome in Y.lipolytica.On this basis,using the targeting signal ePTS1,the three genes AtoB,HMGS and HMGR of the mevalonate synthesis pathway were localized to the peroxisome,and the engineered strain MP2 was obtained,enabling Y.lipolytica to efficiently produce mevalonate.By shake flask fermentation,the Y.lipolytica engineered strain MP2 could produce about 2.725 g/L mevalonate with a yield of 0.072 g/g substrate.Although the mevalonate titer of the control strain M1 whose cytoplasm overexpressed the mevalonate synthesis pathway was comparable,the yield was only 0.031 g/g substrate.These results revealed that the peroxisomal localization of the mevalonate synthesis pathway genes enabled Y.lipolytica to produce mevalonate using fatty acids,and the yield of mevalonate was higher than that produced by using glucose in the cytoplasm.The cytosolic mevalonate pathway exists in Y.lipolytica to synthesize terpenoids.α-Farnesene is an acyclic sesquiterpene,a potential biofuel with important application value.On the basis of the high-yield mevalonate strain MP2.the a-farnesene synthase a-FS was overexpressed,so that the engineered strain F1 could synthesize 72 mg/L a-famesene using oleic acid.Seven key enzymes for synthesizing α-farnesene from mevalonate were overexpressed in the cytoplasm,and the titer of α-farnesene was increased.The engineered strain F4,which overexpressed the α-farnesene synthesis pathway genes in the cytoplasm,was fermented with oleic acid for 120 h,and the titer of α-farnesene was 0.365 g/L.At the same time,all the enzymes from mevalonate toα-farnesene synthesis pathway were overexpressed and localized in the peroxisome,the production of α-farnesene was increased,and we get the engineered strain FP3 When the engineered strain FP3 was fermented for 120 h using YPO medium,the titer of α-farnesene reached 0.391 g/L,which was slightly higher than the titer of the strain F4,indicating that the α-farnesene metabolic pathway localized to the peroxisome could avoid metabolic competition in the cytoplasm,which was beneficial to the production of α-farnesene.However,the mevalonate is free to pass through the peroxisome membrane,and so many genes of α-farnesene metabolic pathway may have a negative impact on the localization effect of the enzymes,resulting in theα-farnesene production of the engineered strain FP3 was similar with that of F4.This study also explored the case that the engineered strain FP3 used different kinds of grease to produce α-farnesene.The results showed that the engineered strain FP3 could use different kinds of oils efficiently.Compared with using oleic acid as a carbon source,triolein was more suitable for the production of α-farnesene by the engineered strain FP3.The fed-batch fermentation was carried out in the fermenter,the engineered strain FP3 was fermented for 168 h by using triolein and controlling the pH to 6.0,the titer of α-farnesene was 1.66 g/L,and the yield reached 0.025 g/g substrate.