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1.
J Cell Mol Med ; 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32227582

RESUMO

Piwi-interacting RNAs (piRNAs) are thought to be germline-specific and to be involved in maintaining genome stability during development. Recently, piRNA expression has been identified in somatic cells in diverse organisms. However, the roles of piRNAs in pulmonary arterial smooth muscle cell (PASMC) proliferation and the molecular mechanism underlying the hypoxia-regulated pathological process of pulmonary hypertension are not well understood. Using hypoxic animal models, cell and molecular biology, we obtained the first evidence that the expression of piRNA-63076 was up-regulated in hypoxia and was positively correlated with cell proliferation. Subsequently, we showed that acyl-CoA dehydrogenase (Acadm), which is negatively regulated by piRNA-63076 and interacts with Piwi proteins, was involved in hypoxic PASMC proliferation. Finally, Acadm inhibition under hypoxia was partly attributed to DNA methylation of the Acadm promoter region mediated by piRNA-63076. Overall, these findings represent invaluable resources for better understanding the role of epigenetics in pulmonary hypertension associated with piRNAs.

2.
Artigo em Inglês | MEDLINE | ID: mdl-32215711

RESUMO

Polyketide-terpenoid hybrid compounds are one of the largest families of meroterpenoids, with great potential for drug development for resistant pathogens. Genome sequence analysis of secondary metabolite gene clusters of a phytopathogenic fungus, Bipolaris sorokiniana 11134, revealed a type I polyketide gene cluster, consisting of highly reducing polyketide synthase, non-reducing polyketide synthase, and adjacent prenyltransferase. MS- and UV-guided isolations led to the isolation of ten meroterpenoids, including two new compounds: 19-dehydroxyl-3-epi-arthripenoid A (1) and 12-keto-cochlioquinone A (2). The structures of 1-10 were elucidated by the analysis of NMR and high-resolution electrospray ionization mass spectroscopy data. Compounds 5-8 and 10 showed moderate activity against common Staphylococcus aureus and methicillin-resistant S. aureus, with minimum inhibitory concentration (MIC) values of 12.5-100 µg/mL. Compound 5 also exhibited activity against four clinical resistant S. aureus strains and synergistic antifungal activity against Candida albicans with MIC values of 12.5-25 µg/mL. The biosynthetic gene cluster of the isolated compounds and their putative biosynthetic pathway are also proposed. KEY POINTS: • Ten meroterpenoids were identified from B. sorokiniana, including two new compounds. • Cochlioquinone B (5) showed activity against MRSA and synergistic activity against C. albicans. • The biosynthetic gene cluster and biosynthetic pathway of meroterpenoids are proposed. • Genome mining provided a new direction to uncover the diversity of meroterpenoids.

3.
Ecotoxicol Environ Saf ; 194: 110402, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32151867

RESUMO

Sulfur (S) application in pakchoi (Brassica chinensis L.) cultivation is vital for reducing cadmium (Cd) accumulation in the plants. However, the mechanism of S application on Cd uptake and translocation in pakchoi is unclear. In this study, a hydroponic experiment was performed to investigate the effects of S application on Cd accumulation in pakchoi at one Cd concentration (50 µM, in comparison to the control condition, 0 µM) and three S levels (0, 2, 4 mM). The results showed that excessive S application (4 mM) reduced Cd accumulation and alleviated pakchoi growth inhibition caused by Cd stress in shoots and roots. With increased S application, the proportion of Cd in the vacuolar fraction and the proportion of NaCl-extractable Cd increased in roots. Additionally, S application increased the content of glutathione (GSH) and phytochelatins (PCs). The reduced Cd uptake and accumulation in pakchoi shoots could have been due to increased Cd chelation and vacuolar sequestration in roots. In addition, sufficient S application (2 mM) increased the expression of γ-glutamylcysteine synthetase (GSH1) and nicotinamide synthase (NAS) in roots, and excessive S application upregulated the expression of ATP sulfurylase (ATPS) and phytochelatin synthase (PCs). This study provides evidence for the mechanism of mitigating Cd toxicity in pakchoi and will be helpful for developing strategies to reduce Cd accumulation in the edible parts of pakchoi through S fertilizer application.

4.
Cell ; 180(6): 1144-1159.e20, 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32169217

RESUMO

In eukaryotic cells, organelle biogenesis is pivotal for cellular function and cell survival. Chloroplasts are unique organelles with a complex internal membrane network. The mechanisms of the migration of imported nuclear-encoded chloroplast proteins across the crowded stroma to thylakoid membranes are less understood. Here, we identified two Arabidopsis ankyrin-repeat proteins, STT1 and STT2, that specifically mediate sorting of chloroplast twin arginine translocation (cpTat) pathway proteins to thylakoid membranes. STT1 and STT2 form a unique hetero-dimer through interaction of their C-terminal ankyrin domains. Binding of cpTat substrate by N-terminal intrinsically disordered regions of STT complex induces liquid-liquid phase separation. The multivalent nature of STT oligomer is critical for phase separation. STT-Hcf106 interactions reverse phase separation and facilitate cargo targeting and translocation across thylakoid membranes. Thus, the formation of phase-separated droplets emerges as a novel mechanism of intra-chloroplast cargo sorting. Our findings highlight a conserved mechanism of phase separation in regulating organelle biogenesis.

5.
Environ Int ; 136: 105515, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32006763

RESUMO

Reactive oxygen species (ROS) are a class of substances that are of general concern in terms of human health and are used to represent the oxidation potential (OP) of the atmosphere. In this study, the ROS levels in 116 daily fine particulate matter (PM2.5) samples taken over Xi'an in 2017 were measured with the dithiothreitol (DTT) method. The sources of DTTv (volume-based DTT consumption) in PM2.5 as well as their contributions were identified by both positive matrix factorization (PMF) and multiple linear regression (MLR) based on the measured chemical species in particulate matter (PM). The results showed that the yearly average DTTv over Xi'an was 0.53 nmol/min/m3 (0.19-1.10 nmol/min/m3). The highest DTTv level occurred in winter, followed by spring, summer and autumn. DTTv was the most strongly correlated with the water-soluble organic carbon (WSOC; r = 0.85), but the effects of WSOC on DTTv were very limited. SO2, NO2, CO, elemental carbon (EC) and K+ (r > 0.64) had moderate correlations with DTTv and were moderately related to environmentally persistent free radicals (EPFRs) (r = 0.56). The linear mixed-effects model showed that pollutants originating from incomplete combustion had greater effects on DTTv than those from complete combustion. Source apportionment results from PMF showed that motor vehicle emissions (27.4%), secondary sulfates (21.6%) and coal combustion sources (18.8%) were more important contributors to the DTTv in PM2.5 than dust sources (8.4%), metal processing (4.9%), industrial emissions (11.3%) and secondary nitrates (7.5%). The PMF results for the DTTv were consistent with the MLR results, which verified that both PMF and MLR are feasible methods for source apportionment of PM2.5 as well as specific species such as ROS and EPFRs. Backward trajectory clusters showed that the dominant cluster groups were local and regional transport, while the OP of the PM2.5 over Xi'an was affected more by long-range transport than by local transport. As stated above, the improvement of atmospheric oxidation potential require not only regional efforts but also large-scale joint cooperation. Furthermore, this study on the OP of PM as well as the specific source information provides important guidance for health effect research.

6.
BMC Infect Dis ; 20(1): 108, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32033541

RESUMO

BACKGROUND: Urinary tract infection (UTI) is a common cause of morbidity worldwide. Uropathogenic Escherichia coli (UPEC) bacteria are the major cause of urinary tract infections. UPEC strains derive from different phylogenetic groups and possess an arsenal of virulence factors that contribute to their ability to overcome different defense mechanisms and cause disease. The objective of this study was to identify phylogroup and virulence genes of UPEC among urinary tract infection patients. METHODS: A cross sectional study was conducted from January 1, 2017 to October 9, 2017. E. coli bacteria were isolated from UTI patients using culture and conventional biochemical tests. Identification of phylogroup and genes that encodes for virulence factors was done using multiplex polymerase chain reaction (PCR). Data was processed and analyzed with SPSS version16.0 and Epi-info version 3.4.1 software. RESULTS: The most common urologic clinical manifestation combinations in this study were dysuria, urine urgency and urgency incontinence. The frequent UPEC virulence gene identified was fimH 164 (82%), followed by aer 109 (54.5%), hly 103 (51.5%), pap 59 (29.5%), cnf 58 (29%), sfa 50 (25%) and afa 24 (12%).There was significant association between pap gene and urine urgency (p-0.016); sfa and dysuria and urine urgency (p-0.019 and p-0.043 respectively); hly and suprapubic pain (p-0.002); aer and suprapubic pain, flank pain and fever (p-0.017, p-0.040, p-0.029 respectively). Majority of E. coli isolates were phylogroup B2 60(30%) followed by D 55(27.5%), B1 48(24%) and A 37(18.5%). There was significant association between E. coli phylogroup B2 and three virulence genes namely afa, pap, and sfa (p-0.014, p-0.002, p-0.004 respectively). CONCLUSION: In this study the most frequent E. coli virulence gene was fimH, followed by aer, hly, pap, cnf, sfa and afa respectively. There was significant association between E. coli virulence genes and clinical symptoms of UTI. The phylogenetic analysis indicates majority of uropathogenic E. coli isolates were phylogroup B2 followed by phylogroup D. Phylogroup B2 carries more virulence genes. Hence, targeting major UPEC phylogroup and virulence genes for potential vaccine candidates is essential for better management of UTI and further research has to be conducted in this area.

7.
Hypertension ; 75(3): 668-679, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32008463

RESUMO

Pulmonary artery smooth muscle cell proliferation is the pathological basis of pulmonary vascular remodeling in hypoxic pulmonary hypertension. Recent studies suggest that circular RNA (circRNA) can regulate various biological processes, including cell proliferation. Therefore, it is possible that circRNA may have important roles in pulmonary artery smooth muscle cell proliferation in hypoxic pulmonary hypertension. In the present study, we aimed to identify functional circRNAs and clarify their roles and mechanisms in pulmonary artery smooth muscle cell proliferation in pulmonary hypertension. RNA sequencing identified 67 circRNAs that were differentially expressed in hypoxic lung tissues of mice. Screening by bioinformatics and quantitative polymerase chain reaction revealed significant elevation of a circRNA derived from alternative splicing of the calmodulin 4 gene (designated circ-calm4). Notably, this circRNA absorbed miR-337-3p. We further identified Myo10 (myosin 10) as a target protein of miR-337-3p. miR-337-3p bound to the 3'-untranslated region of Myo10 mRNA, thereby attenuating the translation of Myo10. Using loss-of-function and gain-of-function approaches, we found that circ-calm4 regulated cell proliferation by regulating the cell cycle. Additionally, we verified the functions of miR-337-3p and Myo10 in hypoxic pulmonary artery smooth muscle. Our results suggested that the circ-calm4/miR-337-3p/Myo10 signal transduction axis modulated the proliferation of pulmonary artery smooth muscle cells at the molecular level, thus establishing potential targets for the early diagnosis and treatment of pulmonary hypertension.

8.
Microb Biotechnol ; 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-32096925

RESUMO

The potential advantages for fermentation production of chemicals at high temperatures are attractive, such as promoting the rate of biochemical reactions, reducing the risk of contamination and the energy consumption for fermenter cooling. In this work, we de novo engineered the thermophile Geobacillus thermoglucosidasius to produce riboflavin, since this bacterium can ferment diverse carbohydrates at an optimal temperature of 60°C with a high growth rate. We first introduced a heterogeneous riboflavin biosynthetic gene cluster and enabled the strain to produce detectable riboflavin (28.7 mg l-1 ). Then, with the aid of an improved gene replacement method, we preformed metabolic engineering in this strain, including replacement of ribCGtg with a mutant allele to weaken the consumption of riboflavin, manipulation of purine pathway to enhance precursor supply, deletion of ccpNGtg to tune central carbon catabolism towards riboflavin production and elimination of the lactate dehydrogenase gene to block the dominating product lactic acid. Finally, the engineered strain could produce riboflavin with the titre of 1034.5 mg l-1 after 12-h fermentation in a mineral salt medium, indicating G. thermoglucosidasius is a promising host to develop high-temperature cell factory of riboflavin production. This is the first demonstration of riboflavin production in thermophilic bacteria at an elevated temperature.

9.
Biomed Res Int ; 2020: 5658212, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32076608

RESUMO

Dental caries is the most common oral disease. The bacteriological aetiology of dental caries promotes the use of antibiotics or antimicrobial agents to prevent this type of oral infectious disease. Antibiotics have been developed for more than 80 years since Fleming discovered penicillin in 1928, and systemic antibiotics have been used to treat dental caries for a long time. However, new types of antimicrobial agents have been developed to fight against dental caries. The purpose of this review is to focus on the application of systemic antibiotics and other antimicrobial agents with respect to their clinical use to date, including the history of their development, and their side effects, uses, structure types, and molecular mechanisms to promote a better understanding of the importance of microbial interactions in dental plaque and combinational treatments.

10.
J Phys Condens Matter ; 32(25): 255701, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32106104

RESUMO

Lithium niobate (LiNbO3, LN) plays an important role in holographic storage, and molybdenum doped LiNbO3 (LN:Mo) is an excellent candidate for holographic data storage. In this paper, the basic features of Mo doped LiNbO3, such as the site preference, electronic structure, and the lattice distortions have been explored from first-principles calculations. Mo substituting Nb with its highest charge state +6 is found to be the most stable point defect form. The energy levels formed by Mo with different charge states are distributed in the band gap, which are responsible for the absorption in the visible region. The transition of Mo in different charge states implies molybdenum can serve as a photorefractive center in LN:Mo. In addition, the interactions between Mo and intrinsic or extrinsic point defects are also investigated in this work. Intrinsic defects [Formula: see text] could cause the movement of the [Formula: see text] energy levels. The exploration of Mo, Mg co-doped LiNbO3 reveals that although Mg ion could not shift the energy level of Mo, it can change the distribution of electrons in Mo and Mg co-doped LN (LN:Mo,Mg) which help with the photorefractive phenomenon.

11.
Int J Mol Sci ; 21(4)2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-32075313

RESUMO

WRKY transcription factors play central roles in developmental processes and stress responses of wheat. Most WRKY proteins of the same group (Group III) have a similar function in abiotic stress responses in plants. TaWRKY46, a member of Group III, was up-regulated by PEG treatment. TaWRKY46-GFP fusion proteins localize to the nucleus in wheat mesophyll protoplasts. Overexpression of TaWRKY46 enhanced osmotic stress tolerance in transgenic Arabidopsis thaliana plants, which was mainly demonstrated by transgenic Arabidopsis plants forming higher germination rate and longer root length on 1/2 Murashige and Skoog (MS) medium containing mannitol. Furthermore, the expression of several stress-related genes (P5CS1, RD29B, DREB2A, ABF3, CBF2, and CBF3) was significantly increased in TaWRKY46-overexpressing transgenic Arabidopsis plants after mannitol treatment. Taken together, these findings proposed that TaWRKY46 possesses vital functions in improving drought tolerance through ABA-dependent and ABA-independent pathways when plants are exposed to adverse osmotic conditions. TaWRKY46 can be taken as a candidate gene for transgenic breeding against osmotic stress in wheat. It can further complement and improve the information of the WRKY family members of Group III.

12.
FEMS Microbiol Ecol ; 96(4)2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32105329

RESUMO

Flies are well-known vectors of bacterial pathogens, but there are little data on their role in spreading microbial community and antimicrobial resistance. In this study, we compared the bacterial community, antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in flies with those in the feces of sympatric animals. A 16S rRNA-based microbial analysis identified 23 bacterial phyla in fecal samples and 25 phyla in flies; all the phyla identified in the fecal samples were also found in the flies. Bray-Curtis dissimilarity analysis showed that the microbiota of the flies were more similar to the microbiota of the feces of their sympatric animals than those of the feces from the three other animal species studied. The qPCR array amplified 276 ARGs/MGEs in fecal samples, and 216 ARGs/MGEs in the flies, while 198 of these genes were identified in both flies and feces. Long-term studies with larger sample numbers from more geospatially distinct populations and infection trials are indicated to further evaluate the possibility of flies as sentinels for antimicrobial resistance.

13.
Nucleic Acids Res ; 48(6): 3195-3210, 2020 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-32095829

RESUMO

Methylation of nucleotides in ribosomal RNAs (rRNAs) is a ubiquitous feature that occurs in all living organisms. The formation of methylated nucleotides is performed by a variety of RNA-methyltransferases. Chloroplasts of plant cells result from an endosymbiotic event and possess their own genome and ribosomes. However, enzymes responsible for rRNA methylation and the function of modified nucleotides in chloroplasts remain to be determined. Here, we identified an rRNA methyltransferase, CMAL (Chloroplast MraW-Like), in the Arabidopsis chloroplast and investigated its function. CMAL is the Arabidopsis ortholog of bacterial MraW/ RsmH proteins and accounts to the N4-methylation of C1352 in chloroplast 16S rRNA, indicating that CMAL orthologs and this methyl-modification nucleotide is conserved between bacteria and the endosymbiont-derived eukaryotic organelle. The knockout of CMAL in Arabidopsis impairs the chloroplast ribosome accumulation and accordingly reduced the efficiency of mRNA translation. Interestingly, the loss of CMAL leads not only to defects in chloroplast function, but also to abnormal leaf and root development and overall plant morphology. Further investigation showed that CMAL is involved in the plant development probably by modulating auxin derived signaling pathways. This study uncovered the important role of 16S rRNA methylation mediated by CMAL in chloroplast ribosome biogenesis and plant development.

14.
Ultramicroscopy ; 211: 112925, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31981842

RESUMO

Electrostatic beam blankers are an alternative to photo-emission sources for generating pulsed electron beams for Time-resolved Cathodoluminescence and Ultrafast Electron Microscopy. While the properties of beam blankers have been extensively investigated in the past for applications in lithography, characteristics such as the influence of blanking on imaging resolution have not been fully addressed. We derive general analytical expressions for the spot displacement and loss in resolution induced by deflecting the electron beam in a blanker. In particular, we analyze the sensitivity of both measures to how precise the conjugate focus is aligned in between the deflector plates. We then work out the specific case of a beam blanker driven by a linear voltage ramp as was used in recent studies by others and by us. The result shows that the spot displacement and focus blur can be reduced to the same order as the electron beam probe size, even when using a beam blanker of millimeter or larger scale dimensions. An interesting result is that, by the right choice of the focus position in the deflector, either the spot displacement from the stationary position can be minimized, or the blur can be made zero but not both at the same time. Our results can be used both to characterize existing beam blanker setups and to design novel blankers. This can further develop the field of time-resolved electron microscopy by making it easier to generate pulses with a typical duration of tens of picoseconds in a regular scanning electron microscope at high spatial resolution.

15.
Appl Microbiol Biotechnol ; 104(6): 2575-2587, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31993701

RESUMO

Leucine-responsive regulatory proteins (Lrps) are a family of transcription factors involved in diverse biological processes in bacteria. So far, molecular mechanism of Lrps for regulating antibiotics biosynthesis in actinomycetes remains largely unexplored. This study, for the first time in Streptomyces lincolnensis, identified an Lrp (named as SLCG_Lrp) associated with lincomycin production. SLCG_Lrp was validated to be a positive regulator for lincomycin biosynthesis by directly stimulating transcription of two structural genes (lmbA and lmbV), three resistance genes (lmrA, lmrB and lmrC), and a regulatory gene (lmbU) within the lincomycin biosynthetic gene (lin) cluster. SLCG_Lrp was transcriptionally self-inhibited and triggered the expression of its adjacent gene SLCG_3127 encoding a LysE superfamily protein. Further, the binding site of SLCG_Lrp in the intergenic region of SLCG_3127 and SLCG_Lrp was precisely identified. Inactivation of SLCG_3127 in S. lincolnensis resulted in yield improvement of lincomycin, which was caused by intracellular accumulation of proline and cysteine. Arginine and phenylalanine were identified as specific regulatory ligands, respectively, to reduce and promote DNA-binding affinity of SLCG_Lrp. We further found that SLCG_Lrp was directly repressed by SLCG_2919, the first identified transcription factor outside lin cluster for lincomycin production. Therefore, our findings revealed SLCG_Lrp-mediated transcriptional regulation of lincomycin biosynthesis. This study extends the understanding of molecular mechanisms underlying lincomycin biosynthetic regulation.

16.
Phys Chem Chem Phys ; 22(3): 1784, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31915762

RESUMO

Correction for 'p-Type conductivity mechanism and defect structure of nitrogen-doped LiNbO3 from first-principles calculations' by Weiwei Wang et al., Phys. Chem. Chem. Phys., 2020, 22, 20-27.

17.
Appl Microbiol Biotechnol ; 104(4): 1533-1543, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894364

RESUMO

Marine microorganisms live in dramatically different environments and have attracted much attention for their structurally unique natural products with potential strong biological activity. Based on the one strain-many compounds (OSMAC) strategy and liquid chromatography mass spectrometry (LC-MS) methods, our continuing efforts on the investigation of novel active compounds from marine Verrucosispora sp. MS100137 has led to the identification of a new polycyclic metabolite, abyssomicin Y (1), together with six known abyssomicin and proximicin analogs (2-7). Abyssomicin Y is a type I abyssomicin with an epoxide group at C-8 and C-9. Compounds 1-3 showed potent inhibitory effects against the influenza A virus; their observed inhibition rates were 97.9%, 98.3%, and 95.9%, respectively, at a concentration of 10 µM, and they displayed lower cytotoxicity than 4. The structures were determined by different NMR techniques and HRMS experiments. This investigation revealed that OSMAC could serve as a useful method for enabling the activation of the silent genes in the microorganism and for the formation of previously unreported active secondary metabolites.

18.
Appl Microbiol Biotechnol ; 104(4): 1545-1553, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31897521

RESUMO

Increasing attention has recently been focused on complex symbiotic associations, for instance coral and its symbionts. Sea cucumber, harboring diverse fungi, has also attracted more and more attention for their functional diversity. Here, secondary metabolites produced by Chaetomium globosum associated with sea cucumber, Apostichopus japonicus, were investigated using gene mining with third-generation sequencing technology (PacBio SMRT). Nine compounds, including one new compound cytoglobosin X (1), were isolated from cultures of Chaetomium globosum. Compound 1 was identified based on NMR data, HRESIMS, and ECD, and the absolute configurations were identified as 3S, 4R, 7S, 8R, 9R, 16S, 19S, 20S, and 23S. In an antimicrobial assay, compound 4 showed moderate activity against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus with MICs of 47.3 and 94.6 µM, respectively. Our results suggest that the microbiomes associated with sea cucumber could be an important resource for biodiversity and structural novelty, and the bioactive compounds may protect the host from pathogen microbial.

19.
J Exp Clin Cancer Res ; 39(1): 19, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31959225

RESUMO

In the original publication of this manuscript [1], Fig. 6 contains a repeated image in error (the left image of 'Migration' and the left image of 'Invasion').

20.
Appl Microbiol Biotechnol ; 104(1): 225-239, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31788711

RESUMO

Actinobacteria are one of the most important sources of pharmaceutically valuable and industrially relevant secondary metabolites. Modern genome mining reveals that the potential for secondary metabolite production of actinomycetes has been underestimated. Recently, the establishment of CRISPR/Cas9-based genetic manipulation approaches in actinomycetes opened a new era for genome engineering of this type of organism. Compared with the traditional methods, the application of CRISPR/Cas9 shows several advantages in actinomycetes including higher efficiency and ease of operation. However, the screening process for the correctly edited mutants and the plasmid curing are still time- and labor-intensive. To address this problem, we developed an updated version of the CRISPR/Cas9 genome editing system for actinomycetes, based on two chromogenic reporter systems (GusA and IdgS). Our system facilitates both processes of positive clone screening and plasmid curing. Here, we demonstrate by three case studies in both model actinomycetes and non-model actinomycetes that this system is faster and more efficient. We performed the deletion of one single gene, actIORFI (SCO5087 of the actinorhodin gene cluster) in Streptomyces coelicolor M145, one small-size (5.5 kb) gene cluster (orange-pigmented carotenoid gene cluster), and one relatively large-size (61 kb) gene cluster (abyssomicin gene cluster) in Verrucosispora sp. MS100137. The results presented in this study indicate that this updated CRISPR/Cas9 system employing chromogenic reporters is versatile and broadly applicable in genome engineering of actinomycetes, not only for the largest genus Streptomyces.

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