提示
lpmo9c的双质粒共表达与培养条件优化
首发时间：2025-02-26
摘要：本研究旨在提升来源于神经孢子neurospora crassa的c4氧化型lpmo（lpmo9c）在毕赤酵母中的异源表达效率。传统单质粒表达体系由于基因拷贝数受限，难以实现高效表达。为克服这一瓶颈，本文采用创新性的双质粒共表达策略，将目的基因nclpmo9c分别构建至ppiczαa和ppic9k质粒中，并整合至毕赤酵母染色体的aox1和his4位点。此策略显著提高了基因拷贝数和表达效率，重组菌酶活性达到0.117u/ml，相较于单质粒体系提升了2.1倍。在此基础上，优化了培养条件，包括初始ph值6.0、接种量3%、甲醇补加量1%及诱导天数4天，最终酶活性进一步提升至0.188u/ml，较未优化前提高了1.6倍。纯化后的lpmo9c分子量显著高于理论值，说明蛋白在毕赤酵母中经历了糖基化修饰，这一修饰提升了蛋白的稳定性和抗降解能力。此外，针对lpmo氧化产物的复杂性，传统的maldi-tof和hpaec方法操作繁琐且耗时，限制了大规模实验的开展。因此，本实验提出并验证了一种基于dns法测定还原糖含量以间接评估lpmo9c氧化水平的新策略。通过建立还原糖浓度与c4氧化产物峰面积的线性关系（r2=0.963），证明该方法具有较高的准确性和可靠性，显著简化了氧化产物的定量分析流程。 综上所述，双质粒共表达策略有效提升了lpmo9c的表达量，优化培养条件进一步增强了酶的产量，且简化的定量方法为lpmo氧化产物的快速分析提供了新途径。上述成果为lpmo类酶在生物质转化领域的工业化应用奠定了坚实基础，展示了毕赤酵母表达系统在基因工程中的广阔潜力。
关键词：
for information in english, please click here
dual plasmid co-expression of lpmo9c and optimisation of culture conditions
abstract：this study aims to enhance the heterologous expression efficiency of c4-oxidative lpmo (lpmo9c) from neurospora crassa in pichia pastoris. traditional single-plasmid expression systems are limited by gene copy number, making it difficult to achieve high-level expression. to overcome this bottleneck, an innovative dual-plasmid co-expression strategy was employed, in which the target gene lpmo9c was separately cdual-plasmid co-expression strategy enhances lpmo productionloned into ppiczαa and ppic9k plasmids and integrated into the aox1 and his4 loci of the pichia pastoris chromosome. this strategy significantly increased the gene copy number and expression efficiency, resulting in a recombinant enzyme activity of 0.117 u/ml, which is 2.1 times higher than that of the single-plasmid system. furthermore, optimization of culture conditions, including an initial ph of 6.0, an inoculum size of 3%, 1% methanol supplementation, and 4 days of induction, led to a further increase in enzyme activity to 0.188 u/ml, 1.6 times higher than before optimization. sds-page analysis of the purified lpmo9c revealed a molecular weight significantly higher than the theoretical value, indicating that the protein underwent glycosylation modification in pichia pastoris, which enhanced its stability and resistance to degradation. additionally, given the complexity of lpmo oxidative products, traditional methods such as maldi-tof and hpaec are time-consuming and labor-intensive, limiting efficient large-scale experiments. therefore, a new strategy was proposed and validated, based on dns assay to indirectly assess the oxidation level of lpmo9c by measuring reducing sugar content. a linear relationship (r2=0.963) was established between reducing sugar concentration and the peak area of c4 oxidative products, demonstrating that this method is accurate, reliable, and significantly simplifies the quantitative analysis of oxidative products. in conclusion, the dual-plasmid co-expression strategy effectively enhanced lpmo9c expression, and optimization of culture conditions further increased enzyme yield. the simplified quantification method provides a new approach for rapid analysis of lpmo oxidative products. these findings lay a solid foundation for the industrial application of lpmo enzymes in biomass conversion and highlight the broad potential of the pichia pastoris expression system in genetic engineering.
keywords：
基金：
论文图表：
引用
导出参考文献
no.****
动态公开评议
共计0人参与
勘误表
lpmo9c的双质粒共表达与培养条件优化
评论
全部评论