细胞工厂发酵与特异酶切制备高纯度天然构象甜蛋白thaumatin

High-purity production of native-conformation sweet protein thaumatin via cell factory fermentation and specific enzymatic cleavage

摘要:

索马甜(thaumatin)是一种源于植物丹尼尔奇异果(Thaumatococcus daniellii)的天然甜味蛋白，具有高甜度、低热量、较好的热稳定性与pH稳定性。索马甜可降解为天然氨基酸，是更安全的潜在甜味剂。植物提取该蛋白质面临含量低、作物区域及季节限制等问题，现有微生物细胞工厂通常难以在提高表达量的同时，保证产物的高纯度与天然构象。为解决这一问题，本研究设计并构建了一种融合亲和纯化标签与无痕切除位点的索马甜前体(pThaumatin)蛋白。以毕赤酵母作为表达宿主，通过改造细胞工厂折叠分泌蛋白质的过程与分批补料发酵，提升前体蛋白的折叠与分泌表达效率。经过亲和层析纯化，每升发酵液获得了126 mg高纯度索马甜前体蛋白。随后通过下游无残留蛋白酶切处理去除融合标签，获得与天然序列完全一致的成熟索马甜蛋白(mThaumatin)，转化率接近100%。圆二色谱分析表明，酶切释放所得的索马甜蛋白在二级结构上与天然索马甜高度一致。初步感官评估证实该蛋白甜味显著，功能正常。本研究建立了一条“蛋白质前体设计-细胞工厂分泌表达-无痕酶切后处理”的技术路线，实现了基于微生物发酵的高纯度、天然构象索马甜的制备，为实现甜味蛋白的高纯度表达及天然构象恢复提供了参考。

Abstract:

Thaumatin is a natural sweet-tasting protein derived from the plant Thaumatococcus daniellii, renowned for its high sweetness intensity, low caloric value, and favorable thermal and pH stability. As it can be degraded into natural amino acids, thaumatin represents a potentially safer alternative sweetener. However, plant-based extraction of this protein is constrained by low endogenous content, as well as geographical and seasonal limitations. Meanwhile, existing microbial cell factories often struggle to simultaneously achieve high expression levels and ensure high product purity with natural conformation. To address this bottleneck, this study designed and constructed a thaumatin precursor protein (pThaumatin) incorporating an affinity purification tag and a traceless cleavage site. Using Pichia pastoris as the expression host, the folding and secretory expression efficiency of this precursor protein was enhanced through engineering of the protein folding and secretion pathways in the cell factory, combined with fed-batch fermentation. Following affinity chromatography purification, 126 mg of high-purity thaumatin precursor protein was obtained per liter of fermentation broth. Subsequent downstream processing using residue-free protease cleavage enabled the removal of the fusion tag, yielding mature thaumatin (mThaumatin) with a sequence identical to that of the native protein, achieving a conversion efficiency of nearly 100%. The resulting protein exhibited secondary structure consistent with that of native thaumatin. Circular dichroism spectroscopy revealed that the thaumatin protein released by enzymatic cleavage exhibited high consistency in secondary structure with native thaumatin. Preliminary sensory evaluation confirmed that the protein displayed a pronounced sweet taste, indicating function integrity. This study establishes a technical pipeline encompassing “precursor protein design-cell factory secretory expression-traceless enzymatic post-processing” for the production of high-purity, native-conformation thaumatin via microbial fermentation. This study provides a reference for achieving high-purity expression and the restoration of the native conformation of sweet-tasting proteins.