Identification Of Limonene Tolerance Related Genes And Construction Of Limonene Synthesis Strains In Yarrowia Lipolytica

Yarrowia lipolytica is an important unconventional oleaginous yeast.Due to its high flux of tricarboxylic acid cycle,and the ability to utilize a variety of cheap carbon sources,as well as high tolerance to a variety of organic compounds,high salt concentrations and pH values,it is an excellent host for natural products synthesis.In addition,Y.lipolytica has mevalonate pathway,which can provide precursors for the synthesis of terpenoids and has potential for the production of terpenoids.Limonene is an important cyclic monoterpene,which is widely used in medicine and industry due to its antioxidant,antiviral and aroma properties.At present,microorganisms such as bacteria and yeasts have been successfully used in limonene biosynthesis,but due to the severe cytotoxicity of limonene to the cells,the titer of microbial synthesis is usually low.In order to analyze the key mechanism of limonene tolerance and to construct strains that efficiently produce limonene,a genome-scale random insertion mutagenesis library was constructed in Y.lipolytica to screen limonene tolerant strains and locate the key genes,and the mechanism of limonene tolerance was explored through reverse metabolic engineering.On this basis,the limonene synthase was introduced and the genes in mevalonate pathway was overexpressed to construct limonene production strain of Y.lipolytica.To identify the key genes for improving limonene tolerance,the genome-scale random insertion mutagenesis library was constructed in Y.lipolytica.Y.lipolytica tends to integrate DNA fragments into the genome through non-homologous end joining(NHEJ)repair which can achieve random insertion.Using this feature,we constructed the random insertion mutagenesis library.We first fused the LEU2 expression cassette with a strong promoter UT8 in tandem,and transformed the LEU2-UT8 integration fragment into Y.lipolytica strains for random insertion.The resulting insertion library could not only achieve gene deletion or inhibition,but also activate gene expression when the insertion fragment are located in promoter regions.These features is conducive to screen tolerance strains.Through NHEJ-mediated random integration,we constructed a mutagenesis library with a capacity of approximately 2×105 transformants.The library was cultivated in the presence of 2000 mg/L and 3000 mg/L limonene and the tolerance strains were obtained on plates supplemented with 2000 mg/L and 3000 mg/L limonene.To identify insertion sites and target genes,the colonies from the plates containing 2000 mg/L and 3000 mg/L limonene were randomly picked,and the insertion sites were determined.We found that most of insertions in the mutation strains are located at the same site,which is 735 bp upstream of the YALI1_D28825g gene.This gene is similar to the NIP1 gene of Saccharomyces cerevisiae and encoding a subunit of eIF3,the eukaryotic translation initiation factor.We detected the expression level of NIP1 gene in the mutation strains,and found that the expression level of NIP1 gene in the mutation strains was increased compared with the control strain AHH regardless of whether the strain was under limonene stress.In order to explore the mechanism of limonene tolerance,we used reverse metabolic engineering to overexpress the NIP1 gene in AHH strain using promoters of different strengths to test the effect on limonene tolerance.When NIP1 was overexpressed with the strong promoter UT8,the growth lag phase of the strains was visibly shortened and limonene tolerance was significantly improved in the medium supplemented with 1500 mg/L limonene.Therefore,the increased expression level of NIP1 gene may be an important reason for the limonene tolerance of Y.lipolytica.In order to obtain limonene-producing strains,we selected limonene tolerance strain LT-2g and mevalonate-producing strain AHH as starting strains to construct limonene biosynthesis pathway.First,we introduced the limonene synthase derived from Perilla frutescens(PfLS)to achieve the accumulation of limonene in Y.lipolytica(0.52 mg/L and 2.65 mg/L).In order to increase the flux of GPP,the direct precursor of limonene synthesis,a double-point mutation was introduced in the endogenous ERG20 to obtain mERG20(ERG20F88W-N119W).The fermentation results showed that the overexpression of mERG20 increased the production of limonene to 5.22 mg/L and 26.92 mg/L,respectively,which was about 10 times that of LS only.We further overexpressed other enzymes of the mevalonate pathway in Y.lipolytica to improve limonene titer.Through the combined expression of IDI,ERG12,GPPS,ERG8,ERG19 and mERG20,the final limonene titer obtained by the engineering strain was 38.88 mg/L by shake-flask fermentation.In this study,the key genes affecting limonene tolerance were screened by random insertion of mutagenesis library construction,and limonene synthesis strains were preliminarily constructed,which laid a solid foundation for revealing the mechanism of limonene tolerance in Y.lipolytica and achieving the efficient synthesis of limonene.