Production Of Limonene And Its Derivative Perillic Acid In Candida Tropicalis Via Metabolic Engineering

Limonene and its derivative perillic acid are a class of monocyclic monoterpenoid compounds with a variety of pharmacological and physiological activities,with great application potential and broad market prospects.These monoterpenoids are widely present in natural plant essential oils,and natural material extraction and chemical synthesis are used in industrial production,while high substrate costs and low conversion rates are the limiting factors for production.Candida tropicalis has a strong omega-oxidation pathway andβ-oxidation pathway,and relatively high intracellular acetyl-Co A levels,which can accumulate higher levels of lipids intracellularly.C.tropicalis is an excellent chassis cell for the efficient production of terpenoids.When systematic metabolic engineering and synthetic biology techniques are used to engineer C.tropicalis,and inexpensive substrates can be used to produce high value-added terpenoids.In this study,the engineered C.tropicalisproducing limonene and its derivative perillaric acid were mainly constructed and optimized.The main results of this study are as follows:（1）An engineered strain of C.tropicalis that can produce limonene was constructed and the effect of truncated limonene synthase on its yield was studied.The LS gene expression cassette was constructed and transformed into the C.tropicalis genome for heterologous expression,and the production of limonene in C.tropicalis was determined,and its yield could reach 0.092 mg?L^-1.The t LS gene that the N-terminal sequence of LS was truncated was expressed in C.tropicalis,and its limonene yield was increased by 2.78-fold that of the initial yield.（2）Effect of subcellular subcellular compartmentalization of limonene pathway for production of limonene in C.tropicalis was studied.The expression cassettes of t LS gene in the mitochondrial and peroxisomes of C.tropicalis were constructed and transformed into the yeast genome to express,both of which can produce limonene.Their yields reached 0.236 mg·L^-1and 0.105 mg?L^-1.It was suggested that t LS hasactivity in mitochondria and peroxisomes,and that its limonene precursor GPP can be transferred from the cytoplasm to mitochondria and peroxisomes.Based on the engineered C.tropicalis overexpressing key enzymes in the MVA pathway,the t LS gene was integrated.The limonene yield was further increased by 31.68-fold in the cytoplasm,which was 12.1-and 8.91-fold higher than in peroxisomes and mitochondrials,respectively.（3）The effect of compartmentalized expression of the key enzyme farnesyl pyrophosphate synthase ERG20 and its mutant ERG20^WW on limonene yield was studied.The overexpression of ERG20 gene significantly reduced the yield of limonene compared with the control strain,which was reduced by 90.51%,23.52%and 2.76%in cytoplasm,peroxisome and mitochondria,respectively.The overexpression of ERG20^WW significantly increased the yield of limonene compared with the control strain,which was increased by 11.33-,5.85-and 0.3-fold in cytoplasm,peroxisome and mitochondria,respectively.By optimizing the fermentation time and the starting sugar concentrations in the fermentation medium,the optimum medium sugar concentration is 60 g?L^-1.The limonene yield reached 141.48 mg?L^-1,which was 1536.8-fold higher than the initial production.（4）Plant-derived CYP71A76 cytochrome P450 was heterologously expressed in C.tropicalis to produce perillic acid,and the yield of perillic acid in the flask reached 7.19 mg?L^-1.In a 5L fermentor,the yield of perillac acid reached 106.69 mg?L^-1,which was increased by13.8-fold that in the flask.