Metabolic Engineering Of Saccharomyces Cerevisiae For Efficient Synthesis Of Limonene

Limonene is a volatile monoterpene compound that is widely used in food,pharmaceutical,fragrance and toiletries.With the rise of synthetic biology,the microbial fermentation of limonene has attracted more and more attention and research.In this study,we first achieved efficient de novo synthesis of limonene in Saccharomyces cerevisiae using a"push-pull-restrain"strategy.By optimizing the precursor supply and cofactor levels,combined with mitochondrial compartment engineering and fermentation optimization,the production efficiency of limonene was significantly improved,and the toxicity of limonene on cell growth was alleviated.The S.cerevisiae cell factory constructed in this study can be used as a chassis for the de novo synthesis of high-value derivatives of limonene,such as menthol.Key findings were as follows:（1）Efficient de novo synthesis of limonene based on the"push-pull-restrain"strategy.De novo synthesis of limonene was achieved by combinatorial optimization of key genes for limonene synthesis and overexpression of related genes in the MVA pathway with a yield of46.96 mg·L^-1.The expression of ERG20 was further dynamically inhibited to limit the competition of squalene synthesis pathway for metabolic flow,and limonene production was increased to 213.08 mg·L^-1.Finally,the copy number of the gene encoding limonene synthase t Lim S was optimized,and the production of limonene reached 640.87 mg·L^-1.（2）Enhancing the supply of precursor acetyl-Co A and cofactor NAPDH.Firstly,the production of limonene was increased to 889.54 mg·L^-1 by knocking out MLS1 and CIT2,which are key genes in the acetyl-coa competitive bypass glyoxylate cycle.Subsequently,the expression of four key genes in the pentose phosphate pathway,GND1,ZWF1,TAL1 and TKL1,and the glutamate dehydrogenase-related genes GDH1 and GDH2 in the cytoplasm were knocked out to achieve the replacement of NADPH,and the yield of limonene was further increased to 1097.43 mg·L^-1.（3）Compartmentalization of limonene synthesis in mitochondria.Firstly,the targeting ability of mitochondrial localization signal peptide MLS and the correct localization of enzymes in limonene synthesis pathway in mitochondria were verified.Subsequently,the production of limonene was gradually increased to 1378.54 mg·L^-1 by reconstruction of limonene synthesis pathway in mitochondria,indicating that reconstruction of limonene synthesis pathway in mitochondria is an effective strategy to promote the production of limonene.（4）Fermentation optimization in shake flask and fermenter.Firstly,the optimal fed-composition,fermentation time and organic phase ratio of biphasic fermentation were determined by shake flask fed-composition optimization.Subsequently,a two-stage fermentation optimization was carried out in a 3-L fermenter to achieve a two-stage conversion of cell growth phase and limonene accumulation phase by controlling the glucose supply.In addition,the connection of the exhaust gas collection device achieved a total limonene yield of2.63 g·L^-1,which is the highest reported level of limonene synthesis by S.cerevisiae.