Study Of Saccharomyces Cerevisiae Cell Factory For Efficient Synthesis Of Valencene With Mannitol As Carbon Source

Valencene is a high value-added sesquiterpene compound found in various citrus fruits,which can be used as the preparation of wooden citrus flavor and widely applied in daily fragrance and perfume.Despite the high yield of valencene in Saccharomyces cerevisiae has been achieved in our previous study,it still faces the problem of yield bottleneck.The overflow metabolism of the fermentable substrate glucose causes inefficient flow of carbon to target products other than ethanol,which in turn affects the valencene yield in S.cerevisiae.An effective solution is the combination of the synthesis of valencene with the utilization of mannitol,a non-fermentable carbon source with the property of circumventing the rigid flux of ethanol.Therefore,a mannitol metabolic platform in S.cerevisiae was developed to produce the high valuable sesquiterpene valencene in this study.Based on elementary mode analysis of the metabolic network,the great theoretical potential and network flexibility for the efficient production of valencene from mannitol were revealed.In an attempt to overcome the mannitol utilization bottleneck,two-stage adaptative laboratory evolution(ALE)experiment was carried out to obtain strains with the ability to grow on mannitol.The evolved S.cerevisiae BN-91 A with a series of mutations in genome possessed superior performance on mannitol assimilation,whose expression levels of genes involved in mannitol assimilation were dramatically upregulated by 50-480 folds.It was found that the mutation of global transcriptional repressor TUP1 was responsible for the acquisition of mannitol-assimilating ability in yeast by genome resequencing analysis.The mutation of TUP1 gene resulted in a 20-120-fold upregulation of the expression level of the mannitol gene,which was sufficient for the growth of the strain in mannitol.In addition,the mutation of transcription factor GIS1 and mutations in other coding or non-coding regions further enhanced the mannitol metabolism of the strain,and finally formed the high-efficient mannitol metabolism strain BN-91 A in this study.When grown on mannitol,the BN-91 A strain showed a valencene titer of 17.2 mg/L and a carbon conversion efficiency of 1.905 mg/g,approximately 3-fold and 7-fold higher than those on glucose,respectively.A further analysis of the valencene synthase coding gene revealed an elevation of its gene dose and transcriptional level under mannitol condition,which was 3 folds and 5 folds as much as under glucose condition.Moreover,the valencene titer of BN-91 A got little to no impact upon salt shock of 1M Na Cl,indicating the competence of BN-91 A to product valencene from macroalgae-derived mannitol.An improved valencene titer of 161.1 mg/L was obtained in flask after a combinational engineering strategy including precursor supply enhancement,mannitol uptake facilitation and cofactor regeneration acceleration.Finally,maximum valencene titer of 5.61 g/L was obtained in a fed-batch bioreactor with mannitol feeding.Our findings establish a novel strategy of terpenoid production in S.cerevisiae,which opens a new avenue for applications of third-generation renewable biomass marine macroalgae.