Engineering Yarrowia Lipolytica For High Production Of Fatty Alcohols And Triterpenes

The coordination between heterologous synthesis and other relative metabolism is critical to heterologous microbial production.Here,with the aim of fatty alcohol and triterpene overproduction in Yarrowia lipolytica,this study has focused on developing an efficient strategy to break through the following key bottleneck.How to coordinate indirect pathways to further improve lipid and terpene synthesis after the optimization of their major metabolism?For improving fatty alcohol synthesis,we developed a novel strategy to coordinate fatty alcohol synthesis with glycolysis which achieved a?pull-and-push‘effect to improve fatty alcohol production.Transcript profiling indicated that genes in carbohydrate metabolism were up-regulated significantly in response to higher fatty alcohol production.Based on it,11 glycolysis promoters were screened to express fatty acyl-Co A reductase(FAR)to corelate the fatty alcohol production with the up-regulated carbohydrate metabolism,and the fatty alcohol production reached to 557 mg/L when FAR was expressed by the promoter of P_FBAin.RNA-seq and q RT-PCR analysis demonstrated that a?pull-and-push‘effect caused by the coordination system dynamically enhanced the product synthesis flux from top to bottom,which was also testified and intensified by doubled glucose concentration.After manipulating structural and regulatory genes of lipid metabolism,the final strain JLFL-46 achieved up to 5.75 g/L fatty alcohol production from modified YPD medium(containing 91 g/L glucose)in shake flasks,which represented the highest titer reported to date.For improving triterpene synthesis,the lipid metabolism was disturbed and we found that increased proportion of unsaturated fatty acids was contributed to the extraction pull of triterpene synthesis during two-phase fermentation and indirect increased the precursor supply of mevalonate(MVA)pathway,hereby improved the triterpene production.Compared overexpression of key genes in MVA pathway(HMG1,ERG1,ERG9),combining disturbance of lipid metabolism(overexpression of OLE1 with deletion of PAH1 and DGK1)increased lupeol production more significantly(from 28.99 mg/L to 137.5 mg/L,374%higher increase).By combining these two strategies,the final strain JLLU-46 achieved up to 411.7 mg/L lupeol from modified YPD medium(containing 45.5 g/L glucose and 6 g/L pyruvate)in shake flasks,which was 32.2-fold higher than the initial titer.Metabolic and transcriptional analysis indicated that the disturbance of lipid metabolism increased proportion of unsaturated fatty acids,which promoted the extraction pull of terpene synthesis during two-phase fermentation and activated sterol synthesis to enhanced the synthesis flux from MVA pathway.Moreover,this strategy was also applied to improve other triterpene and sesquiterpene production,which achieved 1.76 time of?-amyrin production,8.83 time of?-amyrin production,5.86 time of total sesquiterpenes(longifolene,longipinene and longicyclene)production,respectively.In summary,this work explored the applications of coordination with indirect pathways on fatty alcohol and triterpene biosynthesis,which also offered a feasible and effective reference for manipulation of fatty acid-derived chemicals and terpene chemicals production.