PDF(Pubmed)
Abstract：
BACKGROUND: S. lividans TK24 is a popular host for the production of small molecules and the secretion of heterologous protein. Within its large genome, twenty-nine non-essential clusters direct the biosynthesis of secondary metabolites. We had previously constructed ten chassis strains, carrying deletions in various combinations of specialized metabolites biosynthetic clusters, such as those of the blue actinorhodin (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red), the coelimycin A (cpk) and the melanin (mel) clusters, as well as the genes hrdD, encoding a non-essential sigma factor, and matAB, a locus affecting mycelial aggregation. Genome reduction was aimed at reducing carbon flow toward specialized metabolite biosynthesis to optimize the production of secreted heterologous protein.
RESULTS: Two of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain.
CONCLUSIONS: The current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.
RESULTS: Two of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain.
CONCLUSIONS: The current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.
摘要：
背景：S.lividansTK24是生产小分子和分泌异源蛋白的流行宿主。在其庞大的基因组中，29个非必需簇指导次级代谢产物的生物合成。我们以前建造了十个底盘菌株，在特殊代谢物生物合成簇的各种组合中携带缺失，例如蓝色的放线运动（行为），钙依赖性抗生素（CDA），十一酸prodigiosin（红色），消除霉素A（cpk）和黑色素（mel）簇，以及hrdD基因，编码非必要的西格玛因子，和MatAB,影响菌丝聚集的位点。基因组减少旨在减少流向专门代谢物生物合成的碳流，以优化分泌的异源蛋白的生产。
结果：这些S.lividansTK24衍生的底盘菌株中的两个显示生物量产量降低约15％，与亲本菌株相比，它们的总天然分泌组质量产量增加了2倍，并且几种分泌蛋白的丰度增加。RNAseq和分泌组的蛋白质组学分析表明，基因组减少导致细胞壁和氧化应激，并伴随着分泌伴侣和secDF的上调，Sec途径组件。有趣的是,分泌的异源蛋白mRFP和mTNFα的量，其中一种菌株，高出12%和70%，分别,比亲本菌株分泌的还要多。
结论：当前的研究描述了一种构建分泌能力增强的底盘菌株的策略，并提出了一个模型，该模型将特化代谢产物生物合成簇的缺失与分泌异源蛋白的生产改善联系起来。
结果：这些S.lividansTK24衍生的底盘菌株中的两个显示生物量产量降低约15％，与亲本菌株相比，它们的总天然分泌组质量产量增加了2倍，并且几种分泌蛋白的丰度增加。RNAseq和分泌组的蛋白质组学分析表明，基因组减少导致细胞壁和氧化应激，并伴随着分泌伴侣和secDF的上调，Sec途径组件。有趣的是,分泌的异源蛋白mRFP和mTNFα的量，其中一种菌株，高出12%和70%，分别,比亲本菌株分泌的还要多。
结论：当前的研究描述了一种构建分泌能力增强的底盘菌株的策略，并提出了一个模型，该模型将特化代谢产物生物合成簇的缺失与分泌异源蛋白的生产改善联系起来。
