Efficient utilization of carbon to produce aromatic valencene in Saccharomyces cerevisiae using mannitol as the substrate

Abstract

As a promising brown macroalgal carbohydrate, mannitol has been an alternative renewable feedstock for biofuel and biochemical production, avoiding competition with food production for natural resources. In this study, we describe a cell factory of the evolved Saccharomyces cerevisiae based on adaptive laboratory evolution that efficiently biotransforms mannitol into the high value-added sesquiterpenoid valencene. The evolved S. cerevisiae BN-91A with a series of mutations in the genome exhibited superior performance in mannitol assimilation, with the expression levels of genes involved in mannitol assimilation being dramatically upregulated by 50–480 fold. When grown on mannitol, the BN-91A strain showed a valencene titer of 17.2 mg L⁻¹ and a carbon conversion efficiency of 1.905 mg g⁻¹, which were approximately 3-fold and 7-fold higher than those on glucose, respectively. Further analysis of the valencene synthase coding gene revealed an elevation of its gene dose and transcriptional level under mannitol conditions, which were 3-fold and 5-fold as much as those under glucose conditions, respectively. Moreover, the valencene titer of BN-91A had little to no impact upon salt shock, indicating the competence of BN-91A to produce valencene from macroalgae-derived mannitol. An improved valencene titer of 161.1 mg L⁻¹ was obtained in flasks after applying a combinational engineering strategy including precursor supply enhancement, mannitol uptake facilitation and cofactor regeneration acceleration. Finally, a maximum valencene titer of 5.61 g L⁻¹ 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 application of third-generation renewable biomass marine macroalgae.