Niu Tingting, Cui Yi, Shan Xu, Qin Shuzhen, Zhou Xuejie, Wang Rui, Chang Alan, Ma Nan, Jing Jingjing, He Jianwei
School of Life Sciences, Liaoning University, Shenyang, China.
College of Life and Environmental Sciences, Wenzhou University, Wenzhou, China.
Front Microbiol. 2022 Dec 22;13:1074398. doi: 10.3389/fmicb.2022.1074398. eCollection 2022.
The industrial yeast is widely used as a cell factory to produce proteins, chemicals and advanced biofuels. We have previously constructed strains that overexpress protein disulfide isomerase (PDI), which is a kind of molecular chaperone that can improve the expression of an exogenous protein when they are co-expressed. Chicken cystatin (cC) is a highly thermostable cysteine protease inhibitor and a homologous protein of human cystatin C (HCC). Wild-type cC and the two mutants, I66Q and ΔW (a truncated cC lacking the á-helix 2) represent proteins with different degrees of stability.
Wild-type cC, I66Q and ΔW were each overexpressed in without and with the coexpression of PDI and their extracellular levels were determined and compared. Transcriptomic profiling was performed to compare the changes in the main signaling pathways and cell components (other than endoplasmic reticulum quality control system represented by molecular chaperones) in in response to intracellular folding stress caused by the expression of exogenous proteins with different stabilities. Finally, hub genes hunting was also performed.
The coexpression of PDI was able to increase the extracellular levels of both wild-type cC and the two mutants, indicating that overexpression of PDI could prevent the misfolding of unstable proteins or promote the degradation of the misfolded proteins to some extent. For cells that expressed the I66Q or ΔW mutant, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses of the common DEGs in these cells revealed a significant upregulation of the genes involved in protein processing, but a significant downregulation of the genes enriched in the Ribosome, TCA and Glycolysis/Gluconeogenesis pathways. Hub genes hunting indicated that the most downregulated ribosome protein, C4QXU7 in this case, might be an important target protein that could be manipulated to increase the expression of foreign proteins, especially proteins with a certain degree of instability.
These findings should shed new light on our understanding of the regulatory mechanism in yeast cells that responds to intracellular folding stress, providing valuable information for the development of a convenient platform that could improve the efficiency of heterologous protein expression in
工业酵母被广泛用作细胞工厂来生产蛋白质、化学品和先进生物燃料。我们之前构建了过表达蛋白质二硫键异构酶（PDI）的菌株，PDI是一种分子伴侣，当与外源蛋白共表达时可提高其表达水平。鸡半胱氨酸蛋白酶抑制剂（cC）是一种高度耐热的半胱氨酸蛋白酶抑制剂，与人半胱氨酸蛋白酶C（HCC）是同源蛋白。野生型cC以及两个突变体I66Q和ΔW（一种缺少α-螺旋2的截短型cC）代表了不同稳定性程度的蛋白质。
野生型cC、I66Q和ΔW分别在不共表达和共表达PDI的情况下进行过表达，并测定和比较它们的细胞外水平。进行转录组分析以比较酵母细胞中主要信号通路和细胞成分（分子伴侣所代表的内质网质量控制系统除外）因表达不同稳定性外源蛋白引起的细胞内折叠应激而发生的变化。最后，还进行了枢纽基因筛选。
PDI的共表达能够提高野生型cC和两个突变体的细胞外水平，这表明PDI的过表达能够在一定程度上防止不稳定蛋白的错误折叠或促进错误折叠蛋白的降解。对于表达I66Q或ΔW突变体的酵母细胞，对这些细胞中常见差异表达基因（DEG）进行的基因本体论（GO）和京都基因与基因组百科全书（KEGG）分析显示，参与蛋白质加工的基因显著上调，但核糖体、三羧酸循环（TCA）和糖酵解/糖异生途径中富集的基因显著下调。枢纽基因筛选表明，在这种情况下下调最明显的核糖体蛋白C4QXU7可能是一个重要的靶蛋白，可对其进行调控以提高外源蛋白的表达，尤其是具有一定不稳定性的蛋白。
这些发现将为我们理解酵母细胞中应对细胞内折叠应激的调控机制提供新的思路，为开发一个能够提高酵母中异源蛋白表达效率的便捷平台提供有价值的信息。
