Efficient Synthesis Of Farnesene By Yarrowia Lipolytica

Yarrowia lipolytica,as model of unconventional yeast,is widely used in basic research and industrial production.It is widely considered as an ideal host for the production of lipid biofuels.The advantages of using Y.lipolytica as a cell factory to produce various products are as follows:it can use various types of substrates;survive in a wide range of pH and osmotic stress environments;and it has relatively matured genetic operation tools and high metabolic flux of acetyl-CoA.Terpenoids are a large and diverse family with important medical and industrial value.At present,many studies have used Y.lipolytica to produce terpenoids,but the titer of most products is still at a low level.Famesene is a sesquiterpene of the terpenoid family.It has great market application potential in agriculture,industry,bioenergy and other fields.Microbial production of famesene is an attractive way for its sustaibale production.Although the synthesis of farnesene has been realized in Y.lipolytica,its production is far lower than that in E.coli and S.cerevisiae.In the first part of the study,we took α-farnesene as representative example to explore the potential of Y.lipolytica to synthesize terpenoids.We divided the afarnesene synthesis pathway into upstream mevalonate synthesis module and downstream a-famesene synthesis module.In order to strengthen the mevalonate synthesis module,we introduced the coding genes of AtoB from Escherichia coli and NADH dependent HMGR from Bordetella petrii to Y.lipolytica,and constructed the genes expression library through NHEJ mediated gene integration into the genome,and successfully screened strain AHH12 with high mevalonate titer from the library.The mevalonate titer of AHH12 reached 1.96 g/L at 96 h in shake flask fermentation.Taking AHH12 strain as the platform,the a-famesene synthesis module was optimized,including protein fusion,overexpression multi copy integration and key genes in metabolic pathway,and the recombinant strain F5 was obtained.Combining with the optimization of fermentation conditions,F5 accumulated 25.55 g/L afarnesene in the final fed-batch fermentation,which was the highest terpenoid titer in Y.lipolytica reported at that time,showing that Y.lipolytica had great terpenoids synthesis potential.This study also shows that NHEJ mediated genome integration and genes overexpression library screening strategy are effective method to obtain strains with high genes expression and production.Y.lipolytica can grow vigorously on the oil substrate.Comparing with the glucose substrate,oil substrate has higher theoretical conversion of farnesene,which are 0.723 g/g oleic acid and 0.252 g/g glucose respectively.Based on these advantages,F5 strain was further optimized to efficiently synthesise a-farnesene from cheap oleic and alleviate the problem of large accumulation of by-products.We found that comparing using glucose as substrate,the α-farnesene titer of F5 was slightly higher when using lipids substrate,and identified that the insufficient expression of ERG 12 is the key factor that limiting the further increase of α-farnesene titer.The recombinant strain F10 was obtained by increasing the copy number of key genes and overexpressing VHb to optimize the oxygen utilization capacity.Combining with the optimization of fermentation conditions,the α-farnesene titer and yield of F10 were 10.2 g/L and 0.1 g/g oleic acid,respectively,higher than 7.4 g/L and 0.038 g/g glucose in the same fermentation period of F5.At the same time,the accumulation of main by-products was also significantly reduced.Our results show that Y.lipolytica has the ability to efficiently synthesize terpenoids from lipids.In the third part of the study,we engineered Y.lipolytica to synthesize βfarnesene efficiently from lipids through combining metabolic engineering with organelle engineering and protein engineering.Firstly,we screened β-farnesene synthase of Artemisia annua(AacFS)with higher activity in Y.lipolytica from different plant sources.The catalytic efficiency of AacFS was further improved through protein engineering,and the β-farnesene titer of obtained mutant AanFSK197T/F180H was 2.8-fold higher than that of wild type.Subsequently,through localizing mevalonate synthetic pathway into peroxisome and cytoplasm,the strain CP7 with high accumulation of mevalonate from oleic acid was obtained.We enhanced the metabolic intensity of CP7 strain from mevalonate to β-farnesene synthesis,including the effective fusion of AanFSK197T/F180H and ERG20,overexpression of all genes of metabolic pathway,multi copy integration of key genes,and the β-farnesene titer of recombinant strain reached 1.56 g/L.Strengthening[3-oxidation process increased β-farnesene titer to 2.52 g/L.through regulating fatty acid anabolism,the β-famesene titer of the recombinant strain Q26 reached 3.34 g/L,which was 221.7-fold higher than that of the initial strain,and meanwhile,the yield was 0.17 g/g oleic acid,intracellular lipids accumulation also significantly decreased.Interestingly,the β-farnesene titer and yield of Q26 was 2.16 g/L and 0.031 g/g glucose respectively when using glucose as substrate,the titer and yield were 3.64 g/L and 0.16 g/g WCO when using WCO as substrate at 96 h of shake flask fermentation.The results highlight the advantages of synthesizing β-farnesene from lipids by the engineering strain,and can efficiently convert WCO into farnesene.Combining with the optimization of fermentation conditions,the β-farnesene titer of Q26 strain reached 35.2 g/L in fedbatch fermentation,which is the highest β-farnesene production reported in Y.lipolytica.The β-farnesene titer also reached 31.9 g/L from WCO substrate.Our study proved that the production of farnesene from waste lipids by Y.lipolytica is a potential research,and provides a reference for further industrial application.We also proved that the combination of organelle engineering,metabolic engineering and protein engineering strategy is an efficient method to significantly improve the production of target products in Y.lipolytica.In general,in this study,Y.lipolytica strain was engineered for farnesene production,which proved that Y.lipolytica has great terpenoids synthesis potential and can synthesize farnesene from cheap oil with higher conversion rate;metabolic engineering combined with organelle engineering and protein engineering strategy can effectively improve terpenoids production;the final farnesene titer further shortens the distance between theoretical research and industrial production of microbial synthesis of farnesene.