Ribosome pausing at the AT-rich codons regulates the protein expression of secondary metabolite gene clusters in the Streptomyces tsukubaensis NRRL 18488

Streptomyces tsukubaensis NRRL 18488 is the preferred strain for the production of immunosuppressant agent tacrolimus (FK506). To take full advantage of its genetic potential, systematic understanding of secondary metabolism and related regulatory mechanisms is highly demanded. Here, to this end, we complete its 7.9 Mbp linear genome sequence followed by integrating with multi-omics measurements. With accurate reannotation of FK506 gene cluster, total 2,389 transcription start sites were determined by using primary transcriptome analysis. Integrated analysis of transcriptome and translatome data revealed that secondary metabolic gene clusters, especially FK506 cluster, undergo translational control with decrease in translational efficiency according to the growth. Furthermore, we demonstrated that SD motif has little correlation with ribosome pausing but AT-rich codons delay the translational elongation. Strong ribosome pausing was observed in the rare TTA codon in FK506 cluster. This comprehensive genome-scale analysis provides insight to the translational regulation of secondary metabolism in S. tsukubaensis. Overall design: Transcription abundance and ribosomal profiling analysis of Streptomyces tsukubaensis according to the growth phase

筑波链霉菌（Streptomyces tsukubaensis）NRRL 18488是生产免疫抑制剂他克莫司（tacrolimus, FK506）的首选菌株。为充分挖掘其遗传潜力，系统解析次级代谢途径及其相关调控机制成为迫切需求。为此，我们完成了该菌株7.9 Mbp线性基因组的测序，并结合多组学（multi-omics）检测数据开展整合分析。通过对FK506基因簇进行精准重注释，我们借助原初转录组分析（primary transcriptome analysis）共鉴定出2389个转录起始位点（transcription start sites）。转录组（transcriptome）与翻译组（translatome）的整合分析结果显示，次级代谢基因簇尤其是FK506基因簇存在翻译调控现象：其翻译效率随菌体生长进程逐渐降低。此外，我们证实SD基序（SD motif）与核糖体暂停相关性极低，而AT富集密码子会延缓翻译延伸过程。在FK506基因簇的稀有密码子TTA处，我们观测到显著的核糖体暂停现象。这项全面的基因组规模分析为筑波链霉菌（S. tsukubaensis）次级代谢的翻译调控机制提供了重要见解。实验整体设计：基于菌体生长阶段，对筑波链霉菌开展转录丰度与核糖体谱分析（ribosomal profiling）。