Abstract:[Background] Saccharomyces cerevisiae has the high-affinity myo-inositol transporter Itr1, and we have constructed a S. cerevisiae strain capable of transporting exogenous myo-inositol via Itr1 during the synthesis of glucaric acid. However, the Itr1 degradation induced by myo-inositol hinders the efficient transport of exogenous myo-inositol, limiting the potential for increasing the glucaric acid production. [Objective] To study the effects of mutating lysine residues at potential ubiquitination sites located at the N-terminus and C-terminus of Itr1 on the degradation of Itr1 and further investigate the effects of these mutations on extracellular myo-inositol uptake and glucaric acid biosynthesis in budding yeast cells. [Methods] Different genetic elements were fused by fusion PCR, and the obtained fragments were then integrated on the S. cerevisiae genome by homologous recombination to construct engineered stains containing Itr1 mutants and Itr1 and GFP (green fluorescent protein, GFP) fusion protein, respectively. The membrane localization of the fusion protein was visualized by fluorescence microscopy. The production of glucaric acid was quantified by HPLC, RNA sequencing and qRT-PCR were employed to analyze changes in the intracellular gene transcription levels. [Results] The C-terminal mutation weakened Itr1 degradation. The proportion of cells with complete Itr1 membrane localization in the N-terminal mutant strain increased in a short term of fermentation. The glucaric acid production and biomass of the N-terminal mutant strain during shake flask fermentation significantly increased compared with those of the control strain. Furthermore, the N-terminal mutant strain presented up-regulated expression of ino1 and inm1 and down-regulated expression of pis1, which increased the accumulation of intracellular myo-inositol and facilitated the flow of myo-inositol to glucaric acid synthesis. On this basis, the fermentation conditions were optimized, after which the glucaric acid titer reached 3.30 g/L, 95.3% higher than that of the control strain. [Conclusion] This study confirmed the weakening effect of the C-terminal mutation on the degradation of Itr1 and discovered that the N-terminal mutation increased glucaric acid production. The findings lay a theoretical foundation for further improving myo-inositol utilization and glucaric acid production.
