Fermentation Optimization Of Engineered D-limonene-Producing Yarrowia Lipolytica And Construction Of Xylose Metabolic Pathway

D-limonene,a valuable cyclic monoterpene,has wide applications in food,cosmetics and pharmaceutical industries due to its bacteriostat and anticancer physiological function.In the previous work,a metabolically engineered D-limonene-producing Yarrowia lipolytica strain was achieved.In this study,we combined molecular biology engineering and fermentation optimization to further improve the D-limonene production.Furthermore,based on the idea of environmental protection and sustainable development,we attempted to extend the substrate range of Y.lipolytica by introducing xylose metabolic pathway.In the previous study,an engineered Y.lipolytica strain Polf-LN-051 was constructed in our lab,which yielded 6.4 mg/L D-limonene in shake flask.To improve the D-limonene production,we first strengthened the limonene synthetic pathway by expressing an extra copy of NPP synthase gene tNDPSl or limonene synthase gene tLS,resulting in the engineered strain Polf-LN-052 and Polf-LN-053,respectively.The D-limonene production reached 14.88 mg/L in Po1f-LN-053 while overexpressing tNDPS1 did not show positive effect.In order to further improve D-limonene production,some conditions used in shake flask culture of Po1f-LN-053 were optimized.D-limonene production was increased to 58.4 mg/L by using 30 g/L glycerol as main carbon source and supplying 4 g/L citrate as auxiliary carbon source.Finally,the best strain Po1f-LN-062 yielded 165.3 mg/L D-limonene in the fed-batch cultivation with a 1.5 L bioreactor,approximately 26-fold improvement over the titer of the initial strain Po1f-LN-051.Next,we aimed to generate a strain of Y.lipolytica to utilize xylosefor the D-limonene production.Introducing of xylose metabolic genes XYL1,XYL2,yIXKS via integrative plasmid allowed Y.lipolytica to use xylose efficiently.By further integration of limonene synthesis pathway,the engineered strain LYBX03 could produce D-limonene on xylose.However,due to the limitation of gene integration capability of plasmid and auxotroph selection marker,strain LYBX03 could not be further optimized.The newly developed CRISPR-Cas9 system of Y.lipolytica came as a solution.We combined CRISPR-Cas9 knock-in of xylose metabolism genes with D-limonene high-efficient production strategy by introducing D-limonene synthesis pathway plasmid p 1312LLN and the MVA pathway optimization plasmid p 1269HMG1ERG 12.The obtained engineered strain LYBX15 could therefore produce D-limonene up to 9.1 mg/L in shake flask by using xylose as the sole carbon source.