Modeling refined differences of cortical folding patterns via spatial, morphological, and temporal fusion representations.

The gyrus, a pivotal cortical folding pattern, is essential for integrating brain structure-function. This study focuses on 2-Hinge and 3-Hinge folds, characterized by the gyral convergence from various directions. Existing voxel-level studies may not adequately capture the precise spatial relationships within cortical folding patterns, especially when relying solely on local cortical characteristics due to their variable shapes and homogeneous frequency-specific features. To overcome these challenges, we introduced a novel model that combines spatial distribution, morphological structure, and functional magnetic resonance imaging data. We utilized spatio-morphological residual representations to enhance and extract subtle variations in cortical spatial distribution and morphological structure during blood oxygenation, integrating these with functional magnetic resonance imaging embeddings using self-attention for spatio-morphological-temporal representations. Testing these representations for identifying cortical folding patterns, including sulci, gyri, 2-Hinge, and 2-Hinge folds, and evaluating the impact of phenotypic data (e.g. stimulus) on recognition, our experimental results demonstrate the model's superior performance, revealing significant differences in cortical folding patterns under various stimulus. These differences are also evident in the characteristics of sulci and gyri folds between genders, with 3-Hinge showing more variations. Our findings indicate that our representations of cortical folding patterns could serve as biomarkers for understanding brain structure-function correlations.

Rational construction of a high-quality and high-efficiency biosynthetic system and fermentation optimization for A82846B based on combinatorial strategies in Amycolatopsis orientalis.

BACKGROUND: Oritavancin is a new generation of semi-synthetic glycopeptide antibiotics against Gram-positive bacteria, which served as the first and only antibiotic with a single-dose therapeutic regimen to treat ABSSSI. A naturally occurring glycopeptide A82846B is the direct precursor of oritavancin. However, its application has been hampered by low yields and homologous impurities. This study established a multi-step combinatorial strategy to rationally construct a high-quality and high-efficiency biosynthesis system for A82846B and systematically optimize its fermentation process to break through the bottleneck of microbial fermentation production. RESULTS: Firstly, based on the genome sequencing and analysis, we deleted putative competitive pathways and constructed a better A82846B-producing strain with a cleaner metabolic background, increasing A82846B production from 92 to 174 mg/L. Subsequently, the PhiC31 integrase system was introduced based on the CRISPR-Cas12a system. Then, the fermentation level of A82846B was improved to 226 mg/L by over-expressing the pathway-specific regulator StrR via the constructed PhiC31 system. Furthermore, overexpressing glycosyl-synthesis gene evaE enhanced the production to 332 mg/L due to the great conversion of the intermediate to target product. Finally, the scale-up production of A82846B reached 725 mg/L in a 15 L fermenter under fermentation optimization, which is the highest reported yield of A82846B without the generation of homologous impurities. CONCLUSION: Under approaches including blocking competitive pathways, inserting site-specific recombination system, overexpressing regulator, overexpressing glycosyl-synthesis gene and optimizing fermentation process, a multi-step combinatorial strategy for the high-level production of A82846B was developed, constructing a high-producing strain AO-6. The combinatorial strategies employed here can be widely applied to improve the fermentation level of other microbial secondary metabolites, providing a reference for constructing an efficient microbial cell factory for high-value natural products.

Production improvement of FK506 in Streptomyces tsukubaensis by metabolic engineering strategy.

AIMS: Study of the effect of isoleucine on the biosynthesis of FK506 and modification of its producing strain to improve the production of FK506. METHODS AND RESULTS: Metabolomics analysis was conducted to explore key changes in the metabolic processes of Streptomyces tsukubaensis Δ68 in medium with and without isoleucine. In-depth analysis revealed that the shikimate pathway, methylmalonyl-CoA, and pyruvate might be the rate-limiting factors in FK506 biosynthesis. Overexpression of involved gene PCCB1 in S. tsukubaensis Δ68, a high-yielding strain Δ68-PCCB1 was generated. Additionally, the amino acids supplement was further optimized to improve FK506 biosynthesis. Finally, FK506 production was increased to 929.6 mg L-1, which was 56.6% higher than that in the starter strain, when supplemented isoleucine and valine at 9 and 4 g L-1, respectively. CONCLUSIONS: Methylmalonyl-CoA might be the key rate-limiting factors in FK506 biosynthesis and overexpression of the gene PCCB1 and further addition of isoleucine and valine could increase the yield of FK506 by 56.6%.

Daptomycin production enhancement by ARTP mutagenesis and fermentation optimization in Streptomyces roseosporus.

AIMS: We evaluated whether the randomness of mutation breeding can be regulated through a double-reporter system. We hope that by establishing a new precursor feeding strategy, the production capacity of industrial microorganisms after pilot scale-up can be further improved. METHODS AND RESULTS: In this study, the industrial strain Streptomyces roseosporus L2796 was used as the starter strain for daptomycin production, and a double-reporter system with the kanamycin resistance gene Neo and the chromogenic gene gusA was constructed to screen for high-yield strain L2201 through atmospheric and room temperature plasma (ARTP). Furthermore, the composition of the culture medium and the parameters of precursor replenishment were optimized, resulting in a significant enhancement of the daptomycin yield of the mutant strain L2201(752.67 mg/l). CONCLUSIONS: This study successfully screened a high-yield strain of daptomycin through a double-reporter system combined with ARTP mutation. The expression level of two reporter genes can evaluate the strength of dptEp promoter, which can stimulate the expression level of dptE in the biosynthesis of daptomycin, thus producing more daptomycin. The developed multi-stage feeding rate strategy provides a novel way to increase daptomycin in industrial fermentation.

Impact of Solid-State Properties on the Aerosolization Performance of Spray-Dried Curcumin Powders.

Amorphous and crystalline active pharmaceutical ingredients (APIs) are both widely studied for pulmonary delivery. The past research mainly studied the impact of solid-state properties on pharmacokinetic attributes; however, the influence of solid-state properties on aerosolization performance was much less studied. This study aimed to investigate the different aerosolization performances of amorphous and crystalline curcumin (Cur) stabilized with L-leucine. Cur was spray-dried with different concentrations of L-leucine (0, 5, 20, 35, and 50%, w/w) as both solution-based and suspension-based formulations to acquire amorphous and crystalline Cur powders. The physicochemical properties of the spray-dried powders, including particle size, morphology, and solid-state characteristics, were studied. The aerosolization performance as well as dissolution properties were evaluated. It was found that 35% (w/w) L-leucine or above led to the formation of amorphous Cur in the spray-dried powders, and the amorphous Cur powders exhibited higher FPF (70.8%, with 50% L-leucine, w/w) than the crystalline Cur formulations with an FPF at 56.3% (with 50% L-leucine, w/w). In conclusion, with a high concentration of L-leucine (35% or above) in the formulations, amorphous Cur would exhibit higher aerosolization efficiency than crystalline Cur. However, with a low concentration of L-leucine (20% or less) in the formulations, crystalline Cur would be preferred for more enhanced consideration.

A novel strategy of gene screen based on multi-omics in Streptomyces roseosporus.

Daptomycin is a new lipopeptide antibiotic for treatment of severe infection caused by multi-drug-resistant bacteria, but its production cost remains high currently. Thus, it is very important to improve the fermentation ability of the daptomycin producer Streptomyces roseosporus. Here, we found that the deletion of proteasome in S. roseosporus would result in the loss of ability to produce daptomycin. Therefore, transcriptome and 4D label-free proteome analyses of the proteasome mutant (Δprc) and wild type were carried out, showing 457 differential genes. Further, five genes were screened by integrated crotonylation omics analysis. Among them, two genes (orf04750/orf05959) could significantly promote the daptomycin synthesis by overexpression, and the fermentation yield in shake flask increased by 54% and 76.7%, respectively. By enhancing the crotonylation modification via lysine site mutation (K-Q), the daptomycin production in shake flask was finally increased by 98.8% and 206.3%, respectively. This result proved that the crotonylation modification of appropriate proteins could effectively modulate daptomycin biosynthesis. In summary, we established a novel strategy of gene screen for antibiotic biosynthesis process, which is more convenient than the previous screening method based on pathway-specific regulators. KEY POINTS: • Δprc strain has lost the ability of daptomycin production • Five genes were screened by multi-omics analysis • Two genes (orf04750/orf05959) could promote the daptomycin synthesis by overexpression.

Positive regulation of the MarR-type regulator slnO and improvement of salinomycin production by Streptomyces albus by multiple transcriptional regulation.

The purpose of this study was to explore the function of the MarR family regulator slnO. Additionally, a high-yield strain of salinomycin was constructed using combined regulation strategies. First, the slnO gene overexpression strain (GO) was constructed in Streptomyces albus. Compared to the wild-type (WT) strain, salinomycin production in the GO strain increased by approximately 28%. Electrophoretic mobility gel shift assays (EMSAs) confirmed that the SlnO protein can bind specifically to the intergenic regions of slnN-slnO, slnQ-slnA1, and slnF-slnT. qRT-PCR experiments also showed that slnA1, slnF, and slnT1 were significantly upregulated, whereas the expression level of the slnN gene was downregulated in the GO strain. Second, the slnN gene deletion strain (slnNDM) was used as the starting strain, and the pathway-specific gene slnR in the salinomycin gene cluster was overexpressed in slnNDM. This new strain was named ZJUS01. The yield of salinomycin in the ZJUS01 strain was 25% and 56% higher than those in the slnNDM and WT strains, respectively. The above results indicate that the slnO gene has a positive regulatory effect on the biosynthesis of salinomycin. Meanwhile, the yield of salinomycin can be greatly increased by manipulating multiple transcriptional regulations.

Petroleum pollution affects soil chemistry and reshapes the diversity and networks of microbial communities.

Soil is the bearing centre of terrestrial ecosystems. Oil pollution leads to changes in the physical and chemical properties of soil to varying degrees. Polluted soils form a unique microbial species composition, which provides rich materials for the bioremediation of oil-contaminated soil through biological enhancement. Understanding the microbial composition of petroleum-contaminated soil can provide a better biological method for soil remediation. Based on this, 16 S rRNA and ITS genetic markers were used to analyse the bacterial and fungal microbiota in petroleum-contaminated soil, and their physical and chemical properties (total organic carbon, alkaline hydrolysable nitrogen, total phosphorus, total potassium, available potassium, Cu, Zn, and Cd) were measured. It was found that petroleum pollution can significantly reduce the abundance and diversity of bacteria and fungi in the soil and significantly promote the relative abundance of Proteobacteria, Pseudomonas, Pseudoxanthomonas and Pseudoallescheria, which changed the dominant flora of bacteria and fungi and reshaped the co-occurrence network relationship between bacteria and fungi in oil-contaminated soil. The content of total organic carbon in petroleum-contaminated soil was significantly higher than that in uncontaminated soil, while the content of alkaline hydrolysable nitrogen and available potassium was significantly lower than that in uncontaminated soil, and the content of Cu significantly increased after pollution. Total organic carbon is the key driving factor that changes oil-contaminated soil microorganisms and plays a significant role in regulating the remodelling and composition of the microbial community in oil-contaminated soil. This study laid a solid theoretical foundation for the bioremediation of oil-contaminated soil.

Directed Evolution of a Nonheme Diiron N-oxygenase AzoC for Improving Its Catalytic Efficiency toward Nitrogen Heterocycle Substrates.

The azoxy compounds with an intriguing chemical bond [-N=N+(-O-)-] are known to have broad applications in many industries. Our previous work revealed that a nonheme diiron N-oxygenase AzoC catalyzed the oxidization of amino-group to its nitroso analogue in the formation of azoxy bond in azoxymycins biosynthesis. However, except for the reported pyridine alkaloid azoxy compounds, most azoxy bonds of nitrogen heterocycles have not been biosynthesized so far, and the substrate scope of AzoC is limited to p-aminobenzene-type compounds. Therefore, it is very meaningful to use AzoC to realize the biosynthesis of azoxy nitrogen heterocycles compounds. In this work, we further studied the catalytic potential of AzoC toward nitrogen heterocycle substrates including 5-aminopyrimidine and 5-aminopyridine compounds to form new azoxy compounds through directed evolution. We constructed a double mutant L101I/Q104R via molecular engineering with improved catalytic efficiency toward 2-methoxypyrimidin-5-amine. These mutations also proved to be beneficial for N-oxygenation of methyl 5-aminopyrimidine-2-carboxylate. The structural analysis showed that relatively shorter distance between the substrate and the diiron center and amino acid residues of the active center may be responsible for the improvement of catalytic efficiency in L101I/Q104R. Our results provide a molecular basis for broadening the AzoC catalytic activity and its application in the biosynthesis of azoxy six-membered nitrogen catenation compounds.

Rational engineering strategies for achieving high-yield, high-quality and high-stability of natural product production in actinomycetes.

Actinomycetes are recognized as excellent producers of microbial natural products, which have a wide range of applications, especially in medicine, agriculture and stockbreeding. The three main indexes of industrialization (titer, purity and stability) must be taken into overall consideration in the manufacturing process of natural products. Over the past decades, synthetic biology techniques have expedited the development of industrially competitive strains with excellent performances. Here, we summarize various rational engineering strategies for upgrading the performance of industrial actinomycetes, which include enhancing the yield of natural products, eliminating the by-products and improving the genetic stability of engineered strains. Furthermore, the current challenges and future perspectives for optimizing the industrial strains more systematically through combinatorial engineering strategies are also discussed.

Improvement of FK506 production via metabolic engineering-guided combinational strategies in Streptomyces tsukubaensis.

BACKGROUND: FK506, a macrolide mainly with immunosuppressive activity, can be produced by various Streptomyces strains. However, one of the major challenges in the fermentation of FK506 is its insufficient production, resulting in high fermentation costs and environmental burdens. Herein, we tried to improve its production via metabolic engineering-guided combinational strategies in Streptomyces tsukubaensis. RESULTS: First, basing on the genome sequencing and analysis, putative competitive pathways were deleted. A better parental strain L19-2 with increased FK506 production from 140.3 to 170.3 mg/L and a cleaner metabolic background was constructed. Subsequently, the FK506 biosynthetic gene cluster was refactored by in-situ promoter-substitution strategy basing on the regulatory circuits. This strategy enhanced transcription levels of the entire FK506 biosynthetic gene cluster in a fine-tuning manner and dramatically increased the FK506 production to 410.3 mg/mL, 1.41-fold higher than the parental strain L19-2 (170.3 mg/L). Finally, the FK506 production was further increased from 410.3 to 603 mg/L in shake-flask culture by adding L-isoleucine at a final concentration of 6 g/L. Moreover, the potential of FK506 production capacity was also evaluated in a 15-L fermenter, resulting in the FK506 production of 830.3 mg/L. CONCLUSION: From the aspects of competitive pathways, refactoring of the FK506 biosynthetic gene cluster and nutrients-addition, a strategy for hyper-production and potentially industrial application of FK506 was developed and a hyper-production strain L19-9 was constructed. The strategy presented here can be generally applicable to other Streptomyces for improvement of FK506 production and streamline hyper-production of other valuable secondary metabolites.

Activation and discovery of tsukubarubicin from Streptomyces tsukubaensis through overexpressing SARPs.

Genome sequencing has revealed that each Streptomyces contains a wide range of biosynthetic gene clusters (BGCs) and has the capability to produce more novel natural products than what is expected. However, most gene clusters for secondary metabolite biosynthesis are cryptic under normal growth conditions. In Streptomyces tsukubaensis, combining overexpression of the putative SARPs (Streptomyces antibiotic regulatory proteins) and bioactivity-guided screening, the silent gene cluster (tsu) was successfully activated and a novel bioactive anthracycline tsukubarubicin was further isolated and identified. Biological activity assays demonstrated that tsukubarubicin possessed much better antitumor bioactivities against various human cancer cell lines (especially the breast cancer cell lines) than clinically used doxorubicin. Moreover, the previously unreported gene cluster (tsu) for biosynthesis of tsukubarubicin was first characterized and detailed annotations of this gene cluster were also conducted. Our strategy presented in this work is broadly applicable in other Streptomyces and will assist in enriching the natural products for potential drug leads. KEY POINTS: • Generally scalable strategy to activate silent gene clusters by manipulating SARPs. • The novel anthracycline tsukubarubicin with potent antitumor bioactivities. • Identification and annotation of the previously uncharacterized tsu gene cluster.

Genomic and transcriptomic survey of an endophytic fungus Calcarisporium arbuscula NRRL 3705 and potential overview of its secondary metabolites.

BACKGROUND: Secondary metabolites as natural products from endophytic fungi are important sources of pharmaceuticals. However, there is currently little understanding of endophytic fungi at the omics levels about their potential in secondary metabolites. Calcarisporium arbuscula, an endophytic fungus from the fruit bodies of Russulaceae, produces a variety of secondary metabolites with anti-cancer, anti-nematode and antibiotic activities. A comprehensive survey of the genome and transcriptome of this endophytic fungus will help to understand its capacity to biosynthesize secondary metabolites and will lay the foundation for the development of this precious resource. RESULTS: In this study, we reported the high-quality genome sequence of C. arbuscula NRRL 3705 based on Single Molecule Real-Time sequencing technology. The genome of this fungus is over 45 Mb in size, larger than other typical filamentous fungi, and comprises 10,001 predicted genes, encoding at least 762 secretory-proteins, 386 carbohydrate-active enzymes and 177 P450 enzymes. 398 virulence factors and 228 genes related to pathogen-host interactions were also predicted in this fungus. Moreover, 65 secondary metabolite biosynthetic gene clusters were revealed, including the gene cluster for the mycotoxin aurovertins. In addition, several gene clusters were predicted to produce mycotoxins, including aflatoxin, alternariol, destruxin, citrinin and isoflavipucine. Notably, two independent gene clusters were shown that are potentially involved in the biosynthesis of alternariol. Furthermore, RNA-Seq assays showed that only expression of the aurovertin gene cluster is much stronger than expression of the housekeeping genes under laboratory conditions, consistent with the observation that aurovertins are the predominant metabolites. Gene expression of the remaining 64 gene clusters for compound backbone biosynthesis was all lower than expression of the housekeeping genes, which partially explained poor production of other secondary metabolites in this fungus. CONCLUSIONS: Our omics data, along with bioinformatics analysis, indicated that C. arbuscula NRRL 3705 contains a large number of biosynthetic gene clusters and has a huge potential to produce a profound number of secondary metabolites. This work also provides the basis for development of endophytic fungi as a new resource of natural products with promising biological activities.

Enhanced Triacylglycerol Metabolism Contributes to Efficient Oil Utilization and High-Level Production of Salinomycin in Streptomyces albus ZD11.

Streptomyces is well known for biosynthesis of secondary metabolites with diverse bioactivities. Although oils have been employed as carbon sources to produce polyketide antibiotics for several industrial Streptomyces strains, the intrinsic correlation between oil utilization and high production of antibiotics still remains unclear. In this study, we investigated the correlation between oil metabolism and salinomycin biosynthesis in Streptomyces albus ZD11, which employs soybean oil as the main carbon source. Comparative genomic analysis revealed the enrichment of genes related to triacylglycerol (TAG) metabolism in S. albus ZD11. Transcriptomic profiling further confirmed the enhancement of TAG metabolism and acyl coenzyme A biosynthesis in S. albus ZD11. Multiple secreted lipases, which catalyze TAG hydrolysis, were seen to be working in a synergistic and complementary manner in aiding the efficient and stable hydrolyzation of TAGs. Together, our results suggest that enhanced TAG hydrolysis and fatty acid degradation contribute to the high efficiency of oil utilization in S. albus ZD11 in order to provide abundant carbon precursors for cell growth and salinomycin biosynthesis.IMPORTANCE In order to obtain high-level production of antibiotics, oils have been used as the main carbon source for some Streptomyces strains. Based on multiomics analysis, this study provides insight into the relationship between triacylglycerol (TAG) metabolism and antibiotic biosynthesis in S. albus ZD11, an oil-preferring industrial Streptomyces strain. Our investigation into TAG hydrolysis yielded further evidence that this strain utilizes complicated strategies enabling an efficient TAG metabolism. In addition, a novel secreted lipase was identified that exhibited highly hydrolytic activity for medium- and long-chain TAGs. Our findings represent a good start toward clarifying the complicated relationship between TAG catabolism and high-level antibiotic production in the industrial strains.

Comprehensive dissection of dispensable genomic regions in Streptomyces based on comparative analysis approach.

BACKGROUND: Large-scale genome reduction has been performed to significantly improve the performance of microbial chassis. Identification of the essential or dispensable genes is pivotal for genome reduction to avoid synthetic lethality. Here, taking Streptomyces as an example, we developed a combinatorial strategy for systematic identification of large and dispensable genomic regions in Streptomyces based on multi-omics approaches. RESULTS: Phylogenetic tree analysis revealed that the model strains including S. coelicolor A3(2), S. albus J1074 and S. avermitilis MA-4680 were preferred reference for comparative analysis of candidate genomes. Multiple genome alignment suggested that the Streptomyces genomes embodied highly conserved core region and variable sub-telomeric regions, and may present symmetric or asymmetric structure. Pan-genome and functional genome analyses showed that most conserved genes responsible for the fundamental functions of cell viability were concentrated in the core region and the vast majority of abundant genes were dispersed in the sub-telomeric regions. These results suggested that large-scale deletion can be performed in sub-telomeric regions to greatly streamline the Streptomyces genomes for developing versatile chassis. CONCLUSIONS: The integrative approach of comparative genomics, functional genomics and pan-genomics can not only be applied to perform a multi-tiered dissection for Streptomyces genomes, but also work as a universal method for systematic analysis of removable regions in other microbial hosts in order to generate more miscellaneous and versatile chassis with minimized genome for drug discovery.

Medication Tracking: Design and Fabrication of a Dry Powder Inhaler with Integrated Acoustic Element by 3D Printing.

PURPOSE: Asthma is a prevalent lung disorder that cause heavy burdens globally. Inhalation medicaments can relieve symptoms, improve lung function and, thus, the quality of life. However, it is well-documented that patients often do not get the prescribed dose out of an inhaler and the deposition of drug is suboptimal, due to incorrect handling of the device and wrong inhalation technique. This study aims to design and fabricate an acoustic dry powder inhaler (ADPI) for monitoring inhalation flow and related drug administration in order to evaluate whether the patient receives the complete dose out of the inhaler. METHODS: The devices were fabricated using 3D printing and the impact of the acoustic element geometry and printing resolution on the acoustic signal was investigated. Commercial Foradil (formoterol fumarate) capsules were used to validate the availability of the ADPI for medication dose tracking. The acoustic signal was analysed with Partial-Least-Squares (PLS) regression. RESULTS: Indicate that specific acoustic signals could be generated at different air flow rates using a passive acoustic element with specific design features. This acoustic signal could be correlated with the PLS model to the air flow rate. A more distinct sound spectra could be acquired at higher printing resolution. The sound spectra from the ADPI with no capsule, a full capsule and an empty capsule are different which could be used for medication tracking. CONCLUSIONS: This study shows that it is possible to evaluate the medication quality of inhaled medicaments by monitoring the acoustic signal generated during the inhalation process.

Genome mining and homologous comparison strategy for digging exporters contributing self-resistance in natamycin-producing Streptomyces strains.

As antibiotics are always toxic to the antibiotic-producing strains themselves, most Streptomyces strains have evolved several self-resistance mechanisms, among which the antibiotic efflux system is understood best and is commonly found. Among the efflux systems, the ATP-binding cassette (ABC) transporter superfamily and the major facilitator superfamily (MFS) are two important transporter families. In this work, the ABC transporters and the MFS transporters from the four reported natamycin-producing Streptomyces strains have been investigated in order to clarify whether these Streptomyces strains share similar efflux strategies for natamycin metabolism. Fifty-one groups of homologous exporter genes were identified as shared by four strains. Differential transcriptional analysis between the natamycin-producing strain Streptomyces chattanoogensis L10 and its ΔscnS0 mutant, which produces no natamycin, reveals that the expression levels of 25 of the above groups of genes were observably changed. The production of natamycin declined over 30% after solely knocking out several of these 25 groups of genes in S. chattanoogensis L10. This indicates that these transporters participate in the efflux of molecules related to natamycin biosynthesis. Our study is the first to demonstrate that the exporters participating in a particular antibiotic metabolism can be excavated and identified quickly by the strategy of genome mining and homologous comparison in the antibiotic-producing strains, leading to deeper understanding of the complex self-resistance mechanisms in Streptomycetes.

Highly efficient genome editing in N. gerenzanensis using an inducible CRISPR/Cas9-RecA system.

OBJECTIVE: To develop an inducible CRISPR/Cas9-Recombinase A (RecA) system to manipulate genes in Nonomuraea gerenzanensis effectively. RESULTS: Compared with traditional homologous recombination, the inducible CRISPR/Cas9 system achieved 68.8% editing efficiency, whereas, with both the inducible Cas9 and the overexpressed RecA, the efficiency of the combined genome editing system reached 100%. The dbv23-deleted mutant obtained by the inducible CRISPR/Cas9-RecA system was confirmed to produce more A40926 with an approximate yield of 200 mg L-1 than that of around 150 mg L-1 produced by the wild-type strain. CONCLUSIONS: This inducible CRISPR/Cas9-RecA system was successfully constructed and can be utilized as an efficient genome editing tool for Actinomyces able to shorten editing time simultaneously.

The regulatory cascades of antibiotic production in Streptomyces.

Streptomyces is famous for its capability to produce the most abundant antibiotics in all kingdoms. All Streptomyces antibiotics are natural products, whose biosynthesis from the so-called gene clusters are elaborately regulated by pyramidal transcriptional regulatory cascades. In the past decades, scientists have striven to unveil the regulatory mechanisms involved in antibiotic production in Streptomyces. Here we mainly focus on three aspects of the regulation on antibiotic production. 1. The onset of antibiotic production triggered by hormones and their coupled receptors as regulators; 2. The cascades of global and pathway-specific regulators governing antibiotic production; 3. The feedback regulation of antibiotics and/or intermediates on the gene cluster expression for their coordinated production. This review will summarize how the antibiotic production is stringently regulated in Streptomyces based on the signaling, and lay a theoretical foundation for improvement of antibiotic production and potentially drug discovery.

Identification of a secondary metabolism-responsive promoter by proteomics for over-production of natamycin in Streptomyces.

Streptomyces is currently the main producer of microbial pharmaceuticals from its secondary metabolites as natural products. It will be more beneficial if the promoters, which are particularly strong during the secondary metabolism of Streptomyces, are used to drive the efficient production of desired natural products with the coordination of bacterial growth. Here, in an industrial natamycin producer Streptomyces chattanoogensis L10, a strong promoter groESp was identified for this purpose based on the comparative proteomic analysis of the primary and secondary metabolism. With a constitutive promoter ermEp* as a control, the activity of groESp was weak in the primary metabolism, but about sixfold higher than ermEp* in the secondary metabolism, when the representative antibiotic natamycin was highly produced. Furthermore, when ScnRII, a pathway-specific positive regulator in natamycin biosynthesis, was expressed under groESp, the productivity of natamycin was about 20% higher in the secondary metabolism than that from ermEp*, but had no discrimination in the early 2 days. Thus, we showed that proteomics is an effective alternative way to identify promoters for the high yield of natamycin in S. chattanoogensis, and this strategy can be widely adaptable to other Streptomyces species for the full development of secondary metabolites with promising bioactivities.