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酿酒酵母肌醇转运蛋白突变体的构建及其在葡萄糖二酸合成中的应用
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国家重点研发计划(2022YFA0911800);国家自然科学基金(22377039);江苏省杰出青年基金(BK20220089)


The myo-inositol transporter mutants of Saccharomyces cerevisiae: construction and application in glucaric acid production
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    摘要:

    【背景】酿酒酵母(Saccharomyces cerevisiae)自身具有高亲和力的肌醇转运蛋白Itr1,前期构建的酿酒酵母工程菌在合成葡萄糖二酸时可通过Itr1蛋白对外源肌醇进行转运。但肌醇会诱导Itr1蛋白的降解,因此对外源肌醇的低转运效率限制了葡萄糖二酸产量的进一步提高。【目的】研究肌醇转运蛋白Itr1的N端和C端潜在的泛素化位点-赖氨酸残基突变对该蛋白降解的影响,并进一步分析这些突变体对酿酒酵母细胞摄取胞外肌醇的能力和葡萄糖二酸生物合成的影响。【方法】使用融合PCR方法将不同基因元件进行融合,得到用于基因组整合的片段。再通过同源重组在酿酒酵母基因组上整合这些片段,分别构建含Itr1突变体、含Itr1和绿色荧光蛋白(green fluorescent protein, GFP)融合蛋白的工程菌。通过荧光显微镜观察确定融合蛋白的膜定位情况,高效液相色谱(HPLC)方法检测葡萄糖二酸的合成情况,转录组测序和实时荧光定量PCR方法检测胞内基因转录水平的变化。【结果】发现了C端突变对Itr1蛋白降解具有明显的弱化作用,在发酵条件下,N端突变菌株中Itr1蛋白膜定位完整的细胞比例在短期内也有所增加。摇瓶发酵过程中N端突变菌株的葡萄糖二酸产量和菌体生物量较对照菌株都明显提高。进一步发现N端突变菌株中基因ino1inm1被上调,pis1的表达被下调,提高了胞内肌醇的积累并促进了肌醇流向葡萄糖二酸合成的代谢流。在此基础上,进一步进行发酵条件优化,葡萄糖二酸产量达到3.30 g/L,较对照菌株提高了95.3%。【结论】本研究确定了C端突变对肌醇转运蛋白Itr1降解的弱化作用,并发现N端突变可以促进葡萄糖二酸产量的提高,为深入研究肌醇利用及进一步提高葡萄糖二酸产量奠定了理论基础。

    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.

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左方雨,赵运英,邓禹. 酿酒酵母肌醇转运蛋白突变体的构建及其在葡萄糖二酸合成中的应用[J]. 微生物学通报, 2024, 51(11): 4429-4450

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  • 收稿日期:2024-03-11
  • 录用日期:2024-03-29
  • 在线发布日期: 2024-10-31
  • 出版日期: 2024-11-20
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