Metabolic Engineering Of Saccharomyces Cerevisiae For (S)-linalool Production

Linalool is a typical monoterpene and widely used in food, perfume, medicine andcosmetic industries. In this study, in order to increase the supply of precursor substances andimprove the accumulation of linalool in the fermentation broth, the spatial organization of thekey enzyme molecules of metabolic pathways were transformed, and metabolic flux throughthe isoprenoid biosynthetic pathway were strengthened in Saccharomyces cerevisiae by themeans of molecular biology and metabolic engineering. The main conclusions of this paperwere as follows:1. Heterologous expression of (S)-linalool synthase:(1) Two linalool synthase genesAaLS1and ApLS1derived from Actinidia arguta and Actinidia polygama were expressed inSaccharomyces cerevisiae. The fermentation broth of the transformed strains were detected byGC-MS. The results showed that (S)-linalool production reached59.85g/L afterexpression of the AaLS1gene and (S)-linalool production reached21.32g/L after expressionof the ApLS1gene,(S)-linalool was not detected in the control strain. The accumulation of(S)-Linalool was achieved in Saccharomyces cerevisiae.(2) The expression vectorpY26-AaLS1was introduced into Saccharomyces cerevisiae haploid strains CEN.PK2-1C(MATα), CEN.PK2-1D (MATa) and diploid strain CEN.PK2(MATa/MATα) separately. Bydetection of the fermentation broth, linalool production did not change significantly. Theresults indicated that different mating types may not affect (S)-linalool synthesis.(3) In orderto improve the (S)-linalool yield, the catalytic site at position K197lysine (K) of the farnesyldiphosphate synthase was mutated to glycine (G) and glutamic acid (E) respectively, and theresults showed that the mutations did not have a significant impact on (S)-linalool synthesis.2. The six fusion expression vectors were transformed into the competent cells ofSaccharomyces cerevisiae CEN.PK2respectively.(1) The results showed that the highest(S)-linalool production reached101.55g/L by expression of fusion proteins, the linaloolproduction increased by69.7%compared with expression of (S)-linalool synthase alone.(S)-linalool production was increased by36.67%with a long flexible peptide (GGGGS)3,compared to the short length peptide GGGS,(GGSG)2. The linalool yield increased2.5timesby expression the fusion protein LIS-FPPS compared to FPPS-LIS. The results showed thatthe types of the connecting peptide and composition structure of fusion enzyme are essentialfor the spatial structure of the two enzymes.(2) Ergosterol is a important precursor of sterolsynthsis in the isoprenoid biosynthetic pathway, the changes of the content of ergosterol isable to reflect on the changes of metabolic flux in the isoprenoid pathway. The ergosterolcontent of engineered strains were detected by UV spectrophotometric. The study on changesof the amount of ergosterol synthesis showed that the content of ergosterol was reduced withthe improvement of linalool yield and fusion enzymes could affect the metabolic flux.(3)Ethanol tolerance of the engineering strains showed that ethanol tolerance of the strains didnot change significantly compared with the control strain. It indicated that the fusion proteinsand linalool accumulation might not affect the growth of strains.3. The engineered strain LS02overexpressed the gene of IDI1. Flask fermentation results showed that the (S)-linalool content increased69.6%after regulation the expression of IDI1gene, reached101.46μg/L and the unit cell linalool content increased from8.61μg/g DCWincreased to15.24μg/g DCW. The engineered strains LS03overexpressed the gene of IDI1and tHMG1. The shake flask fermentation results showed that,(S)-linalool yield increased to127.71μg/L and the unit cell linalool content increased by9.4%to16.66μg/g DCW, afterimproved the expression level of HMG-CoA reductase on the basis of regulation of the IDI1gene. By detecting the engineering strain growth curve showed that the cell growth of thestrain expression tHMG1gene increased to a certain extent due to the increased metabolicflux. Generally speaking, linalool accumulation did not have a deadly impact on the growth ofstrains in the process of linalool production. The results showed that the content of ergosterolwas increased from14.26mg/g DCW to17.68mg/g DCW. The regulation of IDI1andtHMG1genes could increase the ergosterol synthesis, and at the same time it might beconducive to strengthening the isoprenoid pathway, increasing the carbon metabolism flux,and ultimately promoting the synthesis of (S)-linalool.