The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3'→5' exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement inediting accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete therkDandhomEgenes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters.

The current diminishing returns in finding useful antibiotics and the occurrence of drug-resistant bacteria call for the need to find new antibiotics. Moreover, the whole genome sequencing revealed that the biosynthetic potential of Streptomyces, which has produced the highest numbers of approved and clinical-trial drugs, has been greatly underestimated. Considering the known gene editing tool kits were arduous and inefficient, novel and efficient gene editing system are desirable. Here, we developed an engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) combined with the counter selection system CodA(sm), the D314A mutant of cytosine deaminase, to rapidly and effectively edit Streptomyces genomes. In-frame deletion of the actinorhodin polyketide chain length factor gene actI-ORF2 was created in Streptomyces coelicolor M145 as an illustration. This CRISPR/Cas9-CodA(sm) combined system strikingly increased the frequency of unmarked mutants and shortened the time required to generate them. We foresee the system becoming a routine laboratory technique for genome editing to exploit the great biosynthetic potential of Streptomyces and perhaps for other medically and economically important actinomycetes.

Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-L-methionine(SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.

天然微生物药物的生物合成存在产量低、组分复杂、周期长、严谨调控等特征，同时与药物生物合成相关的催化基因、调节基因、抗性基因和外排基因等成簇排列，初具模块化特征。在充分挖掘、解析和优化多种生物合成元件、模块、系统及高效底盘的基础上，合成生物学整合工程学理念，采用先设计、后实验的策略，修补和强化微生物药物合成机器，高效对接优良底盘，实现微生物药物合成的高产、高效、降耗和减排，最终推动微生物药物产业升级。

English abstract is not available.

DNA克隆和组装技术是重要的分子生物学工具。近年来，随着合成生物学的飞速发展，对大片段DNA元件的快速有效组装就显得尤为关键。同时，各种DNA克隆和组装技术也竞相发展起来。通过对基于非典型酶切连接、PCR、同源重组、单链退火拼接等原理发展起来的各种DNA克隆和组装技术进行综述，为合成生物学的进一步发展提供有效的操作工具。

DNA cloning and assembly techniques are essential tools for molecular biology research. With the recent advances in synthetic biology, efficient and fast assembly of large DNAincluding anumber of genes is becoming more and more important. Meanwhile, a variety of DNA assembly methods are also developed very quickly. In this paper, various DNA assembly methods based on a typical enzyme digestion and ligation, PCR, homologous recombination, single strand annealing and splicing are summarized for providing effective technique tools for the further development of synthetic biology.

庆大霉素是临床上重要的氨基糖苷类抗生素。近年来，伴随现代生物技术水平的发展，人们对该抗生素有了更深入的了解。本文综述了近年来国内外关于庆大霉素的作用机制和耐药机制、生物合成途径和结构改造，及其新活性等方面研究成果，并对庆大霉素的应用与发展前景进行展望。

As an important aminoglycoside antibiotic, gentamicin has been used clinically over decades. With the development in modernbiological technology, the mechanisms of gentamicin action and resistance, its biosynthesis and structural modification were studiedin great depth. Meanwhile, its emerging novel bioactivities and potential applications are also under extensive exploration. Here we summarize the latest progresses and prospects towards the future development of gentamicin for more efficient and effective uses.