Isolation of Streptomyces inhibiting multiple-phytopathogenic fungi and characterization of lucensomycin biosynthetic gene cluster.

Soil microorganisms with diverse bioactive compounds such as Streptomyces are appreciated as valuable resources for the discovery of eco-friendly fungicides. This study isolated a novel Streptomyces from soil samples collected in the organic green tea fields in South Korea. The isolation process involved antifungal activity screening around 2400 culture extracts, revealing a strain designated as S. collinus Inha504 with remarkable antifungal activity against diverse phytopathogenic fungi. S. collinus Inha504 not only inhibited seven phytopathogenic fungi including Fusarium oxysporum and Aspergillus niger in bioassays and but also showed a control effect against F. oxysporum infected red pepper, strawberry, and tomato in the in vivo pot test. Genome mining of S. collinus Inha504 revealed the presence of the biosynthetic gene cluster (BGC) in the chromosome encoding a polyene macrolide which is highly homologous to the lucensomycin (LCM), a compound known for effective in crop disease control. Through genetic confirmation and bioassays, the antifungal activity of S. collinus Inha504 was attributed to the presence of LCM BGC in the chromosome. These results could serve as an effective strategy to select novel Streptomyces strains with valuable biological activity through bioassay-based screening and identify biosynthetic gene clusters responsible for the metabolites using genome mining approach.

Mutational analysis of 23 autosomal short tandem repeats based on trio paternity testing in the Korean population.

This study aimed to estimate A-STR mutation rates in 2,317 Korean parent-child trios by examining 20 Combined DNA Index System (CODIS) core loci (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045) and three non-CODIS loci (Penta E, Penta D, and SE33). Locus-specific mutation rate estimates varied from 0.00 to 8.63 × 10-3 per generation, with an average mutation rate of 1.62 × 10-3 (95 % CI, 1.39-1.88 × 10-3). We also combined data from previous studies to obtain comprehensive genetic values for the Korean population, and the average mutation rate was 1.59 × 10-3 (95 % CI, 1.38-1.82 × 10-3). Single-step mutations (95.69 %) and double-step mutations (3.35 %) were observed in the mutation pattern analysis, and cases expected to have multi-step mutations (0.96 %) were also observed. Large-sized alleles exhibited more loss mutations than gain mutations, and paternal mutations (62.68 %) were more frequently observed than maternal mutations (19.62 %). The calculated values and features of the 23 A-STRs explored in this study are expected to play a crucial role in establishing criteria for forensic genetic interpretation.

Epigenetic age prediction using costal cartilage for the investigation of disaster victims and missing persons.

The DNA intelligence tool, DNA methylation-based age prediction, can help identify disaster victims and suspects in criminal investigations. In this study, we developed a costal cartilage-based age prediction tool that uses massive parallel sequencing (MPS) of age-associated DNA methylation markers. Costal cartilage samples were obtained from 85 deceased Koreans, aged between 26 and 89 years. An MPS library was prepared using two rounds of multiplex polymerase chain reaction of nine genes (TMEM51, MIR29B2CHG, EDARADD, FHL2, TRIM59, ELOVL2, KLF14, ASPA, and PDE4C). The DNA methylation status of 45 CpG sites was determined and used to train an age prediction model via stepwise regression analysis. Nine CpGs in MIR29B2CHG, FHL2, TRIM59, ELOVL2, KLF14, and ASPA were selected for regression model construction. A leave-one-out cross-validation analysis revealed the high performance of the age prediction model, with a mean absolute error (MAE) and root mean square error of 4.97 and 6.43 years, respectively. Additionally, our model showed good performance with a MAE of 6.06 years in the analysis of data of 181 costal cartilage samples collected from Europeans. Our model effectively estimates the age of deceased individuals using costal cartilage samples; therefore, it can be a valuable forensic tool for disaster victim and missing person investigation.

Crystal Structural Characteristics and Electrical Properties of (Ba0.7Sr0.3-xCax)(Ti0.9Zr0.1)O3 Ceramics Prepared Using the Citrate Gelation Method.

The electrical properties of (Ba0.7Sr0.3-xCax)(Ti0.9Zr0.1)O3 (0 ≤ x ≤ 0.2) (BSCTZ) ceramics prepared using citrate gelation (CG) method were investigated by substituting Ca2+ ions for the Sr2+ sites based on the structural characteristics of the ceramics. BSCTZ was sintered for 3 h at 1300 °C, lower than the temperature (1550 °C) at which the specimens prepared using the solid-state reaction (SSR) method were sintered, which lasted for 6 h. As the amount of substituted Ca2+ ions increased, the unit cell volume of the BSCTZ decreased because of the smaller ionic radius of the Ca2+ ions compared to the Sr2+ ions. The dielectric constant of BaTiO3-based ceramics is imparted by factors such as the tetragonality and B-site bond valence of the ceramics. Although the ceramic tetragonality increased with Ca2+ ion substitution, the x = 0.05 specimens exhibited the highest dielectric constant. The decrease in the dielectric constant of the sintered x > 0.05 specimens was attributed to the increase in the B-site bond valence of the ABO3 perovskite structure. Owing to the large number of grain boundaries, the breakdown voltage (6.6839 kV/mm) of the BSCTZ prepared using the CG method was significantly improved in relation to that (2.0043 kV/mm) of the specimen prepared using the SSR method.

CRISPR-Driven Genome Engineering for Chorismate- and Anthranilate-Accumulating Corynebacterium Cell Factories.

In this study, we aimed to enhance the accumulation of chorismate (CHR) and anthranilate (ANT), key intermediates in the shikimate pathway, by modifying a shikimate over-producing recombinant strain of Corynebacterium glutamicum [19]. To achieve this, we utilized a CRISPR-driven genome engineering approach to compensate for the deletion of shikimate kinase (AroK) as well as ANT synthases (TrpEG) and ANT phosphoribosyltransferase (TrpD). In addition, we inhibited the CHR metabolic pathway to induce CHR accumulation. Further, to optimize the shikimate pathway, we overexpressed feedback inhibition-resistant Escherichia coli AroG and AroH genes, as well as C. glutamicum AroF and AroB genes. We also overexpressed QsuC and substituted shikimate dehydrogenase (AroE). In parallel, we optimized the carbon metabolism pathway by deleting the gntR family transcriptional regulator (IolR) and overexpressing polyphosphate/ATP-dependent glucokinase (PpgK) and glucose kinase (Glk). Moreover, acetate kinase (Ack) and phosphotransacetylase (Pta) were eliminated. Through our CRISPR-driven genome re-design approach, we successfully generated C. glutamicum cell factories capable of producing up to 0.48 g/l and 0.9 g/l of CHR and ANT in 1.3 ml miniature culture systems, respectively. These findings highlight the efficacy of our rational cell factory design strategy in C. glutamicum, which provides a robust platform technology for developing high-producing strains that synthesize valuable aromatic compounds, particularly those derived from the shikimate pathway metabolites.

Engineered Escherichia coli cell factory for anthranilate over-production.

Anthranilate is a key platform chemical in high demand for synthesizing food ingredients, dyes, perfumes, crop protection compounds, pharmaceuticals, and plastics. Microbial-based anthranilate production strategies have been developed to overcome the unstable and expensive supply of anthranilate via chemical synthesis from non-renewable resources. Despite the reports of anthranilate biosynthesis in several engineered cells, the anthranilate production yield is still unsatisfactory. This study designed an Escherichia coli cell factory and optimized the fed-batch culture process to achieve a high titer of anthranilate production. Using the previously constructed shikimate-overproducing E. coli strain, two genes (aroK and aroL) were complemented, and the trpD responsible for transferring the phosphoribosyl group to anthranilate was disrupted to facilitate anthranilate accumulation. The genes with negative effects on anthranilate biosynthesis, including pheA, tyrA, pabA, ubiC, entC, and trpR, were disrupted. In contrast, several shikimate biosynthetic pathway genes, including aroE and tktA, were overexpressed to maximize glucose uptake and the intermediate flux. The rationally designed anthranilate-overproducing E. coli strain grown in an optimized medium produced approximately 4 g/L of anthranilate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for microbial-based anthranilate production will play a key role in complementing traditional chemical-based anthranilate production processes.

BAC cloning and heterologous expression of a giant biosynthetic gene cluster encoding antifungal neotetrafibricin in streptomyces rubrisoli.

Polyene natural products including nystatin A1, amphotericin B, ECO-02301, and mediomycin belong to a large family of valuable antifungal polyketide compounds typically produced by soil actinomycetes. A previous study (Park et al., Front. Bioeng. Biotechnol., 2021, 9, 692340) isolated Streptomyces rubrisoli Inha501 with strong antifungal activity and analyzed a large-sized biosynthetic gene cluster (BGC) of a linear polyene compound named Inha-neotetrafibricin (I-NTF) using whole genome sequencing and bioinformatics. In the present study, an entire I-NTF BGC (∼167 kb) was isolated through construction and screening of Streptomyces BAC library. Overexpression of the cloned I-NTF BGC in the wild-type S. rubrisoli Inha501 and its heterologous expression in S. lividans led to 2.6-fold and 2.8-fold increase in I-NTF yields, respectively. The qRT-PCR confirmed that the transcription levels of I-NTF BGC were significantly increased in both homologous and heterologous hosts containing the BAC integration of I-NTF BGC. In addition, the I-NTF aglycone-producing strains were constructed by a target-specific deletion of glycosyltransferase gene present in I-NTF BGC. A comparison of the in vitro biological activities of I-NTF and I-NTF aglycone confirmed that the rhamnose sugar motif of I-NTF plays a critical role in both antifungal and antibacterial activities. These results suggest that the Streptomyces BAC cloning of a large-sized natural product BGC is a valuable approach for natural product titer improvement and biological activity screening of natural product in actinomycetes.

Isolation and Characterization of Engineered Nucleoside Deoxyribosyltransferase with Enhanced Activity Toward 2'-Fluoro-2'-Deoxynucleoside.

Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDTL59Q, showed 1.7-fold (at 40°C) and 4.4-fold (at 50°C) higher 2FDU-to-2FDA conversions compared to the NDTWT, respectively. Subsequently, both NDTWT and NDTL59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDTL59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

A Novel Method to Inspect 3D Ball Joint Socket Products Using 2D Convolutional Neural Network with Spatial and Channel Attention.

Product defect inspections are extremely important for industrial manufacturing processes. It is necessary to develop a special inspection system for each industrial product due to their complexity and diversity. Even though high-precision 3D cameras are usually used to acquire data to inspect 3D objects, it is hard to use them in real-time defect inspection systems due to their high price and long processing time. To address these problems, we propose a product inspection system that uses five 2D cameras to capture all inspection parts of the product and a deep learning-based 2D convolutional neural network (CNN) with spatial and channel attention (SCA) mechanisms to efficiently inspect 3D ball joint socket products. Channel attention (CA) in our model detects the most relevant feature maps while spatial attention (SA) finds the most important regions in the extracted feature map of the target. To build the final SCA feature vector, we concatenated the learned feature vectors of CA and SA because they complement each other. Thus, our proposed CNN with SCA provides high inspection accuracy as well as it having the potential to detect small defects of the product. Our proposed model achieved 98% classification accuracy in the experiments and proved its efficiency on product inspection in real-time.

Streptomyces BAC Cloning of a Large-Sized Biosynthetic Gene Cluster of NPP B1, a Potential SARS-CoV-2 RdRp Inhibitor.

As valuable antibiotics, microbial natural products have been in use for decades in various fields. Among them are polyene compounds including nystatin, amphotericin, and nystatin-like Pseudonocardia polyenes (NPPs). Polyene macrolides are known to possess various biological effects, such as antifungal and antiviral activities. NPP A1, which is produced by Pseudonocardia autotrophica, contains a unique disaccharide moiety in the tetraene macrolide backbone. NPP B1, with a heptane structure and improved antifungal activity, was then developed via genetic manipulation of the NPP A1 biosynthetic gene cluster (BGC). Here, we generated a Streptomyces artificial chromosomal DNA library to isolate a large-sized NPP B1 BGC. The NPP B1 BGC was successfully isolated from P. autotrophica chromosome through the construction and screening of a bacterial artificial chromosome (BAC) library, even though the isolated 140-kb BAC clone (named pNPPB1s) lacked approximately 8 kb of the right-end portion of the NPP B1 BGC. The additional introduction of the pNPPB1s as well as co-expression of the 32-kb portion including the missing 8 kb led to a 7.3-fold increase in the production level of NPP B1 in P. autotrophica. The qRT-PCR confirmed that the transcription level of NPP B1 BGC was significantly increased in the P. autotrophica strain containing two copies of the NPP B1 BGCs. Interestingly, the NPP B1 exhibited a previously unidentified SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibition activity in vitro. These results suggest that the Streptomyces BAC cloning of a large-sized, natural product BGC is a valuable approach for titer improvement and biological activity screening of natural products in actinomycetes.

Shikimate Metabolic Pathway Engineering in Corynebacterium glutamicum.

Shikimate is a key high-demand metabolite for synthesizing valuable antiviral drugs, such as the anti-influenza drug, oseltamivir (Tamiflu). Microbial-based strategies for shikimate production have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. In this study, a microbial cell factory using Corynebacterium glutamicum was designed to overproduce shikimate in a fed-batch culture system. First, the shikimate kinase gene (aroK) responsible for converting shikimate to the next step was disrupted to facilitate the accumulation of shikimate. Several genes encoding the shikimate bypass route, such as dehydroshikimate dehydratase (QsuB), pyruvate kinase (Pyk1), and quinate/shikimate dehydrogenase (QsuD), were disrupted sequentially. An artificial operon containing several shikimate pathway genes, including aroE, aroB, aroF, and aroG were overexpressed to maximize the glucose uptake and intermediate flux. The rationally designed shikimate-overproducing C. glutamicum strain grown in an optimized medium produced approximately 37.3 g/l of shikimate in 7-L fed-batch fermentation. Overall, rational cell factory design and culture process optimization for the microbial-based production of shikimate will play a key role in complementing traditional plant-derived shikimate production processes.

Screening and Isolation of a Novel Polyene-Producing Streptomyces Strain Inhibiting Phytopathogenic Fungi in the Soil Environment.

Microbial-based eco-friendly biological substances are needed to protect crops from phytopathogenic fungi and replace toxic chemical fungicides that cause serious environmental issues. This study screened for soil antifungal Streptomyces strains, which produce rich, diverse, and valuable bioactive metabolites in the soil environment. Bioassay-based antifungal screening of approximately 2,400 Streptomyces strains led to the isolation of 149 strains as tentative antifungal producers. One Streptomyces strain showing the most potent antifungal activities against Candida albicans and Fusarium oxysporum was identified as a putative anti-phytopathogenic soil isolate that is highly homologous to Streptomyces rubrisoli (named S. rubrisoli Inha 501). An in vitro antifungal assay, pot-test, and field-test against various phytopathogenic fungi confirmed that S. rubrisoli Inha 501 is a potential novel phytopathogenic fungicide producer to protect various crops in the soil environment. Whole-genome sequencing of S. rubrisoli Inha 501 and an anti-SMASH genome mining approach revealed an approximately 150-kb polyene biosynthetic gene cluster (BGC) in the chromosome. The target compound isolation and its BGC analysis confirmed that the giant linear polyene compound exhibiting the anti-phytopathogenic activity in S. rubrisoli Inha 501 was highly homologous to the previously reported compound, neotetrafibricin A. These results suggest that a bioassay-based screening of a novel antifungal Streptomyces strain followed by its genome mining for target compound BGC characterization would be an efficient approach to isolating a novel candidate phytopathogenic fungicide that can protect crops in the soil environment.

Artificial cell factory design for shikimate production in Escherichia coli.

Shikimate is a key intermediate in high demand for synthesizing valuable antiviral drugs, such as the anti-influenza drug and oseltamivir (Tamiflu®). Microbial-based shikimate production strategies have been developed to overcome the unstable and expensive supply of shikimate derived from traditional plant extraction processes. Although shikimate biosynthesis has been reported in several engineered bacterial species, the shikimate production yield is still unsatisfactory. This study designed an Escherichia coli cell factory and optimized the fed-batch culture process to achieve a high titer of shikimate production. Using the previously constructed dehydroshikimate (DHS)-overproducing E. coli strain, two genes (aroK and aroL) responsible for converting shikimate to the next step were disrupted to facilitate shikimate accumulation. The genes with negative effects on shikimate biosynthesis, including tyrR, ptsG, and pykA, were disrupted. In contrast, several shikimate biosynthetic pathway genes, including aroB, aroD, aroF, aroG, and aroE, were overexpressed to maximize the glucose uptake and intermediate flux. The shiA involved in shikimate transport was disrupted, and the tktA involved in the accumulation of both PEP and E4P was overexpressed. The rationally designed shikimate-overproducing E. coli strain grown in an optimized medium produced approximately 101 g/l of shikimate in 7-l fed-batch fermentation, which is the highest level of shikimate production reported thus far. Overall, rational cell factory design and culture process optimization for microbial-based shikimate production will play a key role in complementing traditional plant-derived shikimate production processes.

Efficacy of reduced-size short tandem repeat PCR analysis for degraded DNA samples.

BACKGROUND: Short tandem repeats (STR) typing is an essential analysis method for human identification in forensic field. When DNAs obtained from the field as evidences are severely degraded or in too small amounts, STR analysis often shows allele drop-out. OBJECTIVE: To improve STR analysis for degraded DNA or trace DNA, reduced-size STR (rSTR) polymerase chain reaction (PCR) system was devised by selecting relatively large-size STR loci. METHODS: The rSTR PCR system consisted of 8 loci (amelogenin, SE33, CSF1PO, D7S820, D13S317, D2S1338, TPOX, and FGA). The size of PCR product was reduced by designing new primers in the flanking region. The efficiency of this system was verified against existing kits through concordance study, sensitivity study, efficiency study, and casework sample study. RESULTS: The size of PCR product in the rSTR PCR system was reduced to be less than 322 bp. The amplicon of each locus was reduced by about 100 bp on average. Results of this rSTR PCR system were confirmed using 146 Korean samples and other commercial kits. The rSTR PCR system was capable of analyzing DNA samples with a minimum amount of DNA of 16 pg and a degradation index of 4.215. CONCLUSION: The rSTR PCR system was more effective than other PCR kits for obtaining genetic profiles from a small amount of DNA or degraded DNA. The combination of this new system and other commercial kits is more effective than existing systems. This combination is expected to be helpful for the identification of unidentified bodies and skeletal samples.

WblA, a global regulator of antibiotic biosynthesis in Streptomyces.

Streptomyces species are soil-dwelling bacteria that produce vast numbers of pharmaceutically valuable secondary metabolites (SMs), such as antibiotics, immunosuppressants, antiviral, and anticancer drugs. On the other hand, the biosynthesis of most SMs remains very low due to tightly controlled regulatory networks. Both global and pathway-specific regulators are involved in the regulation of a specific SM biosynthesis in various Streptomyces species. Over the past few decades, many of these regulators have been identified and new ones are still being discovered. Among them, a global regulator of SM biosynthesis named WblA was identified in several Streptomyces species. The identification and understanding of the WblAs have greatly contributed to increasing the productivity of several Streptomyces SMs. This review summarizes the characteristics and applications on WblAs reported to date, which were found in various Streptomyces species and other actinobacteria.

Recent Advances of Actinomycetes.

The discovery and development of actinomycete secondary metabolites (ASMs) have played pivotal roles in the fields of human medicine and its related biotechnology sectors over the past several decades [...].

Recent advances in heterologous expression of natural product biosynthetic gene clusters in Streptomyces hosts.

The heterologous expression of natural product biosynthetic gene clusters (BGCs) has traditionally been used as a genetic platform to link various natural product chemotypes to their corresponding genotypes. In recent years, heterologous expression has played an increasing role in natural products research with the advances in sequencing technologies and bioinformatics tools that allow for the rapid and systematic identification of known and cryptic BGCs from a large number of microbial genome sequences. The advances in synthetic biology have also facilitated the process of heterologous expression by providing tools for rapid cloning and engineering of BGCs to improve production yield or to activate silent BGCs. This paper summarizes the recent progress in the cloning and engineering of natural product BGCs and highlights recent examples of the heterologous expression of both known and cryptic BGCs in Streptomyces hosts, which will continue to play a pivotal role in genomics-driven natural product research.

Massively parallel sequencing of 25 short tandem repeat loci including the SE33 marker in Koreans.

BACKGROUND: Massively parallel sequencing (MPS) technology has recently been introduced in research, clinical diagnostics, and forensics. MPS enables determination of the genotypes of multiple short tandem repeat (STR) markers and to determine nucleotide sequence variations, additionally. OBJECTIVE: To improve STR analysis and a paternity index, a new, smaller-sized STR panel was designed that includes the SE33 locus. METHODS: This study performed MPS using an STR panel including the SE33 marker in 101 Koreans. The concordance study was conducted by comparing the data obtained from the MPS assay with the results of a capillary electrophoresis (CE)-based method. RESULTS: In this study, an in-house MPS panel is designed that incorporates the 20 Combined DNA Index System (CODIS) loci and the Penta D, Penta E, and SE33 markers for enhanced discriminatory ability. The data obtained via MPS analysis were compared with CE data to confirm concordance. Fifty previously unreported alleles were detected through the MPS analysis. Three new SNP variations in the flanking region were also identified. Statistical analysis demonstrated that the SE33 marker was most effectively determined the match probability (PM) and typical paternity index (TPI). In the sensitivity study, concentrations as low as 80 pg could be used to obtain full and concordant profiles. CONCLUSIONS: We designed a new, smaller-sized STR panel that includes the SE33 locus to improve STR analysis and the paternity index. Various new alleles were identified in SE33, indicating a high degree of polymorphism. The panel is expected to provide valid data for discrimination of unidentified bodies.

Recent Advances in Microbial Production of cis,cis-Muconic Acid.

cis,cis-Muconic acid (MA) is a valuable C6 dicarboxylic acid platform chemical that is used as a starting material for the production of various valuable polymers and drugs, including adipic acid and terephthalic acid. As an alternative to traditional chemical processes, bio-based MA production has progressed to the establishment of de novo MA pathways in several microorganisms, such as Escherichia coli, Corynebacterium glutamicum, Pseudomonas putida, and Saccharomyces cerevisiae. Redesign of the metabolic pathway, intermediate flux control, and culture process optimization were all pursued to maximize the microbial MA production yield. Recently, MA production from biomass, such as the aromatic polymer lignin, has also attracted attention from researchers focusing on microbes that are tolerant to aromatic compounds. This paper summarizes recent microbial MA production strategies that involve engineering the metabolic pathway genes as well as the heterologous expression of some foreign genes involved in MA biosynthesis. Microbial MA production will continue to play a vital role in the field of bio-refineries and a feasible way to complement various petrochemical-based chemical processes.