Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.     

CRISPR/Cas9基因编辑技术在致瘤病毒感染研究中的应用

成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),CRISPR/Cas9〕基因编辑技术的发现源于真细菌和古细菌中CRISPR/Cas系统介导的适应性免疫机制研究。该技术利用特异性向导RNA识别靶点基因,引导核酸内切酶Cas9对其切割,并通过同源重组或非同源末端连接完成对目的DNA的编辑。某些病毒感染机体后,可将其基因组整合到宿主细胞基因组中或潜伏于组织中而无法被彻底清除,从而引起持续性感染。本文参考2013年以来CRISPR/Cas9基因组编辑技术的最新相关研究报道,重点综述其在人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)、人乳头瘤病毒(human papillomavirus,HPV )、乙型肝炎病毒(hepatitis B virus, HBV)、 Epstein-Barr病毒(Epstein-Barr virus,EBV)等致瘤病毒感染相关疾病研究中的应用,并概括其作用于这些病毒的有效靶点。     

Structural and functional characterization of Streptococcus pyogenes Cas2 protein under different pH conditions

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute an RNA-guided microbial defense system against invading foreign genetic materials. Cas2 is one of the core Cas proteins found universally in all the subtypes of CRISPR-Cas systems and is required for incorporating new spacers into CRISPR loci. Cas2 homologues from different CRISPR-Cas subtypes were characterized previously as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the crystal structures of Streptococcus pyogenes Cas2 at three different pHs (5.6, 6.5, and 7.5), as well as the results of its nuclease activity assay against double-stranded DNAs at varying pHs (6.0–9.0). Although S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues revealed structural variability and the flexible nature of its putative hinge regions, supporting the hypothesis that conformational switching is important for catalysis. Taken together, our results confirm that Cas2 proteins have pH-dependent nuclease activity against double-stranded DNAs, and provide indirect structural evidence for their conformational changes.     

SSO1450 - A CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are ubiquitous in archaea and eubacteria. It has been suggested that CRISPR and CAS proteins act as an immune system preventing the invasion of foreign genomic elements at the DNA level. The protein SSO1450 from Sulfolobus solfataricus (Sso) P2 belongs to the CAS1 cluster which is one of the core protein clusters most frequently associated with CRISPR sequences. In this study we show that SSO1450 is a high-affinity nucleic acid binding protein. It binds DNA, RNA and DNA-RNA hybrid apparently sequence non-specific in a multi-site binding mode. Furthermore, SSO1450 promotes the hybridization of complementary nucleic acid strands.     

Dual modes of CRISPR-associated transposon homing

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Crystal Structure of the Cmr2–Cmr3 Subcomplex in the CRISPR–Cas RNA Silencing Effector Complex

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci found in prokaryotes are transcribed to produce CRISPR RNAs (crRNAs) that, together with CRISPR-associated (Cas) proteins, target and degrade invading genetic materials. Cmr proteins (Cmr1–6) and crRNA form a sequence-specific RNA silencing effector complex. Here, we report the crystal structures of the Pyrococcus furiosus Cmr2–Cmr3 subcomplex bound with nucleotides (3′-AMP or ATP). The association of Cmr2 and Cmr3 forms an idiosyncratic crevasse, which binds the nucleotides. Cmr3 shares structural similarity with Cas6, which cleaves precursor crRNA for maturation, suggesting the divergent evolution of these proteins. Due to the structural resemblance, the properties of the RNA binding surface observed in Cas6 are well conserved in Cmr3, indicating the RNA binding ability of Cmr3. This surface of Cmr3 constitutes the crevasse observed in the Cmr2–Cmr3 complex. Our findings suggest that the Cmr2–Cmr3 complex uses the crevasse to bind crRNA and/or substrate RNA during the reaction.     

肿瘤坏死因子α突变型斑马鱼在海分枝杆菌感染中的应用研究

肿瘤坏死因子α(tumor necrosis factorα,TNF-α)作为重要的炎性细胞因子,在类风湿关节炎、感染性休克等疾病的发病中起重要作用。为探究TNF-α在宿主抵抗海分枝杆菌感染中的作用,本研究采用成簇的规律间隔的短回文重复序列及其相关蛋白9[clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9]技术,使野生型斑马鱼TNF-α基因发生突变,成功构建并筛选出含有移码突变的TNF-α突变型(TNF-α-/-)斑马鱼。利用原位杂交技术,发现TNF-α-/-斑马鱼中TNF-αmRNA表达水平显著降低。与野生型斑马鱼相比,海分枝杆菌在TNF-α-/-斑马鱼体内扩散和增殖更显著。结果提示,本研究成功构建TNF-α-/-斑马鱼,有助于深入探究TNF-α在斑马鱼抵抗海分枝杆菌感染中的作用及其免疫调节机制。