谷氨酸棒杆菌是食品安全级菌株,可作为生产高价值产物的优良底盘细胞用于工业生产。它具有良好的分泌系统,是表达重组蛋白的潜在宿主,有极大的研究价值。为优化底盘细胞提高其蛋白表达能力,挖掘并验证谷氨酸棒杆菌中与外源蛋白高表达相关的基因。在谷氨酸棒杆菌重测序的基础上,应用生物信息学对单核苷酸多态性(single-nucleotide polymorphisms, SNP)基因进行挖掘,分析基因保守结构域。通过构建SNP基因过表达和敲除重组菌株,评估其生长情况以及增强型绿色荧光蛋白(enhanced green fluorescence protein, EGFP)和人重组特立帕肽(recombinant human teriparatide, rtPTH)的外源蛋白表达能力。分析并整理了高产量外源蛋白菌株的重测序数据,共有33个SNP突变位点,主要涉及5个基因。其中SNP重组菌株over-2370和ko-973-974的荧光值最高,并且发酵生产rtPTH,均比野生型(wild type,WT)提高了近1倍。该研究成功挖掘出与蛋白表达相关的基因GL002370和GL000974,这些发现将有助于获得优化的底盘细胞,并为深入研究增加蛋白产量的靶基因提供指导。
Food-safe strain Corynebacterium glutamicum can be used as an excellent chassis cell for industrial production of high-value products. It is a potential host for the expression of recombinant proteins due to its good secretion system. In order to optimize the chassis cells and improve their heterologous protein expression ability, genes in C. glutamicum which can highly express heterologous protein were mined and verified. On the basis of whole-genome resequencing of C. glutamicum, bioinformatics was used to mine single-nucleotide polymorphisms(SNP) genes and their conserved domains. The recombinant strains with overexpression or knockout of SNP genes were constructed to evaluate their(SNP) growth and the expression ability of heterologous proteins EGFP and rtPTH. The resequencing data of high heterologous protein production strains were mined. There were 33 SNPs, mapped to five genes. Among them, SNP recombinant strains over-2370 and ko-973-974 had the highest fluorescence intensity, and the production of rtPTH by fermentation was nearly two-folds higher than that of wild type. This study has successfully mined genes involved in the high yield of heterologous protein, GL002370 and GL000974. These findings will help to obtain optimized chassis cells and provide guidance for in-depth research on target genes that can increase protein production.
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