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The determination of rock’s Capillary water absorption (CWA) requires extensive and complex empirical investigations, while forecasting algorithms could reduce the cost and time required. To achieve this goal, various rock records were collected from different rock types. For the forecasting model, two techniques named ANFIS and SVR were developed, with the model parameters determined using the Imperialist Competitive Algorithm (ICA). The proposed hybrid ICA-SVR and ICA-ANFIS models demonstrate exceptional performance in predicting CWA, with R^2 values exceeding 0.9851 and 0.9586 for the testing and training stages, respectively, indicating a strong correlation between predicted and measured CWA values. Considering all computed metrics, the SVR model optimized with ICA yielded better results than the ICA-ANFIS model in both testing and training stages. For example, the R^2 values for the ICA-SVR model were 0.9758 and 0.9747 for the testing and training datasets, respectively, with VAF values of 0.9889 and 0.9859 . The ICA-ANFIS model also produced acceptable results, though its performance was slightly weaker than the SVR model. Compared to a previous study, the proposed models show a significant improvement in efficiency, with the R^2 value increasing from 0.708 to 0.9889 . In summary, the enhanced ICA-SVR model can be considered a reliable and powerful tool for accurately determining the optimal values of the system’s key variables.

Aminoglycosides (AGs) are a class of antibiotics with a broad spectrum of activity. However, their use is limited by safety concerns associated with nephrotoxicity and ototoxicity, as well as drug resistance. To address these issues, semi-synthetic approaches for modifying natural AGs have generated new generations of AGs, however, with limited types of modification due to significant challenges in synthesis. This study explores a novel approach that harness the bacterial biosynthetic machinery of gentamicins and kanamycins to create hybrid AGs. This was achieved by glycodiversification of gentamicins via swapping the glycosyltransferase (GT) in their producer with the GT from kanamycins biosynthetic pathway and resulted in the creation of a series of novel AGs, therefore referred to as genkamicins (GKs). The manipulation of the hybrid biosynthetic pathway enabled the targeted accumulation of different GK species and the isolation and characterization of six GK components. These compounds display retained antimicrobial activity against a panel of World Health Organization (WHO) critical priority pathogens, and GK-C2a, in particular, demonstrates low ototoxicity compared to clinical drugs in zebrafish embryos. This study provides a new strategy for diversifying the structure of AGs and a potential avenue for developing less toxic AG drugs to combat infectious diseases.

Abstract Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSCBE3-NG-Hypa, tailored with both high efficiency and -fidelity for Streptomyces. Of note, eSCBE3-NG-Hypa recognizes NG PAM and exhibits high activity at challenging sites with high GC content or GC motifs, while displaying minimal off-target effects. To illustrate its practicability, we employ eSCBE3-NG-Hypa to achieve precise key amino acid conversion of the dehydratase (DH) domains within the modular polyketide synthase (PKS) responsible for the insecticide avermectins biosynthesis, achieving domains inactivation. The resulting DH-inactivated mutants, while ceasing avermectins production, produce a high yield of oligomycin, indicating competitive relationships among multiple biosynthetic gene clusters (BGCs) in Streptomyces avermitilis. Leveraging this insight, we use eSCBE3-NG-Hypa to introduce premature stop codons into competitor gene cluster of ave in an industrial S. avermitilis, with the mutant Δolm exhibiting the highest 4.45-fold increase in avermectin B1a compared to the control. This work provides a potent tool for modifying biosynthetic pathways and advancing metabolic engineering in Streptomyces.

Spinosad, a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa, has significant market potential. Despite its effectiveness, the regulatory mechanisms of spinosad biosynthesis remain unclear. Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16, located upstream of spinosad biosynthetic genes, in spinosad biosynthesis. Through reverse transcription PCR (RT-PCR) and 5′-rapid amplification of cDNA ends (5′-Race), we unveiled that the spinosad biosynthetic gene cluster (BGC) contains six transcription units and seven promoters. Electrophoretic mobility shift assays (EMSAs) demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC, indicating its involvement in regulating spinosad biosynthesis. Notably, deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L, accompanied by decreased transcription levels of spinosad biosynthetic genes, confirming its positive regulatory function. Additionally, isothermal titration calorimetry (ITC) and EMSA confirmed that spinosyn A, the main product of the spinosad BGC, served as an effector of ORF-L16. Specifically, it decreased the binding affinity between ORF-L16 and spinosad BGC promoters, thus exerting negative feedback regulation on spinosad biosynthesis. This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S. spinosa.

Abstract Polyethers play a crucial role in the development of anticancer drugs. To enhance the anticancer efficacy and reduce the toxicity of these compounds, thereby advancing their application in cancer treatment, herein, guided by the structure‐activity relationships of aglycone polyethers, novel aglycone polyethers are rationally redesigned with potentially improved efficacy and reduced toxicity against tumors. To realize the biosynthesis of the novel aglycone polyethers, the gene clusters and the post‐polyketide synthase tailoring pathways for aglycone polyethers endusamycin and lenoremycin are identified and subjected to combinatorial biosynthesis studies, resulting in the creation of a novel aglycone polyether termed End‐16, which demonstrates significant potential for treating bladder cancer (BLCA). End‐16 demonstrates the ability to suppress the proliferation, migration, invasion, and cellular protrusions formation of BLCA cells, as well as induce cell cycle arrest in the G1 phase in vitro. Notably, End‐16 exhibits superior inhibitory activity and fewer side effects against BLCA compared to the frontline anti‐BLCA drug cisplatin in vivo, thereby warranting further preclinical studies. This study highlights the significant potential of integrating combinatorial biosynthesis strategies with rational design to create unnatural products with enhanced pharmacological properties.

BackgroundMetronidazole (MTZ) is among the first-line drugs against the human gastric pathogen Helicobacter pylori (H. pylori). MTZ is used as a prodrug that is activated by an oxygen-insensitive enzyme NADPH nitroreductase (RdxA). Loss-of-function mutations in rdxA make H. pylori MTZ resistant; however, experimental proof is lacking.MethodsWe collected 139 gastric biopsy samples from patients suspected of H. pylori infection in Shanghai, and amplified Hp-specific rdxA gene from 134 samples. All these rdxA genes were sequenced and phylogenetically compared. The effect of mutations on RdxA function was measured by expressing them in Escherichia coli DH5α by using the MTZ sensitivity test.ResultsIn total, 134 gastric biopsy samples were identified as H. pylori positive. Of the 134 samples, 74 and 6 had point mutations at the various sites or promoter region of rdxA, generating truncated and extended fused proteins, respectively. The remaining 54 were full-length with single nucleotide variation (SNV) compared with the wild-type RdxA from H. pylori, with 49 clustering with hpEastAsia, 3 with hpEurope, and 2 with hpNEAfrica. All 134 rdxA were expressed in E. coli DH5α; 22 and 112 resultant strains showed MTZ-sensitive and MTZ-resistant phenotypes, respectively. Comparative analysis of single nucleotide polymorphisms (SNPs) in the functional and inactivated RdxA revealed 14 novel mutations in RdxA, 5 of which conferred MTZ resistance: S18F, D59S, L62I, S79N, and A187V.ConclusionThe occurrence of MTZ resistance induced by site-mutation of RdxA in patients with H. pylori infection was 83.6% (112/134) in the Shanghai region. The major form of loss-of-function mutation was truncation of RdxA translation at a rate of 58/112 (51.8%). Molecular detection reliably determined the resistance of H. pylori to MTZ. Thus, the functional mutants involved in MTZ resistance facilitate clinical diagnosis and medication based on sequence analysis.

Abstract Modulation of protein function through allosteric regulation is central in biology, but biomacromolecular systems involving multiple subunits and ligands may exhibit complex regulatory mechanisms at different levels, which remain poorly understood. Here, we discover an aldo-keto reductase termed AKRtyl and present its three-level regulatory mechanism. Specifically, by combining steady-state and transient kinetics, X-ray crystallography and molecular dynamics simulation, we demonstrate that AKRtyl exhibits a positive synergy mediated by an unusual Monod-Wyman-Changeux (MWC) paradigm of allosteric regulation at low concentrations of the cofactor NADPH, but an inhibitory effect at high concentrations is observed. While the substrate tylosin binds at a remote allosteric site with positive cooperativity. We further reveal that these regulatory mechanisms are conserved in AKR12D subfamily, and that substrate cooperativity is common in AKRs across three kingdoms of life. This work provides an intriguing example for understanding complex allosteric regulatory networks.

Abstract Selenium is an essential multifunctional trace element in diverse organisms. The only Se-glycosyltransferase identified that catalyzes the incorporation of selenium in selenoneine biosynthesis is SenB from Variovorax paradoxus. Although the biochemical function of SenB has been investigated, its substrate specificity, structure, and catalytic mechanism have not been elucidated. Here, we reveal that SenB exhibits sugar donor promiscuity and can utilize six UDP-sugars to generate selenosugars. We report crystal structures of SenB complexed with different UDP-sugars. The key elements N20/T23/E231 contribute to the sugar donor selectivity of SenB. A proposed catalytic mechanism is tested by structure-guided mutagenesis, revealing that SenB yields selenosugars by forming C-Se glycosidic bonds via spontaneous deprotonation and disrupting Se-P bonds by nucleophilic water attack, which is initiated by the critical residue K158. Furthermore, we functionally and structurally characterize two other Se-glycosyltransferases, CbSenB from Comamonadaceae bacterium and RsSenB from Ramlibacter sp., which also exhibit sugar donor promiscuity.

Abstract Background Papaya (Carica papaya) is an economically important fruit cultivated in the tropical and subtropical regions of China. However, the rapid softening rate after postharvest leads to a short shelf-life and considerable economic losses. Accordingly, understanding the mechanisms underlying fruit postharvest softening will be a reasonable way to maintain fruit quality and extend its shelf-life. Results Mitogen-activated protein kinases (MAPKs) are conserved and play essential roles in response to biotic and abiotic stresses. However, the MAPK family remain poorly studied in papaya. Here, a total of nine putative CpMAPK members were identified within papaya genome, and a comprehensive genome-wide characterization of the CpMAPKs was performed, including evolutionary relationships, conserved domains, gene structures, chromosomal locations, cis-regulatory elements and expression profiles in response to phytohormone and antioxidant organic compound treatments during fruit postharvest ripening. Our findings showed that nearly all CpMAPKs harbored the conserved P-loop, C-loop and activation loop domains. Phylogenetic analysis showed that CpMAPK members could be categorized into four groups (A-D), with the members within the same groups displaying high similarity in protein domains and intron–exon organizations. Moreover, a number of cis-acting elements related to hormone signaling, circadian rhythm, or low-temperature stresses were identified in the promoters of CpMAPKs. Notably, gene expression profiles demonstrated that CpMAPKs exhibited various responses to 2-chloroethylphosphonic acid (ethephon), 1-methylcyclopropene (1-MCP) and the combined ascorbic acid (AsA) and chitosan (CTS) treatments during papaya postharvest ripening. Among them, both CpMAPK9 and CpMAPK20 displayed significant induction in papaya flesh by ethephon treatment, and were pronounced inhibition after AsA and CTS treatments at 16 d compared to those of natural ripening control, suggesting that they potentially involve in fruit postharvest ripening through ethylene signaling pathway or modulating cell wall metabolism. Conclusion This study will provide some valuable insights into future functional characterization of CpMAPKs, and hold great potential for further understanding the molecular mechanisms underlying papaya fruit postharvest ripening.

IntroductionThe unique dormancy of Mycobacterium tuberculosis plays a significant role in the major clinical treatment challenge of tuberculosis, such as its long treatment cycle, antibiotic resistance, immune escape, and high latent infection rate.MethodsTo determine the function of MtrA, the only essential response regulator, one strategy was developed to establish its regulatory network according to high-quality genome-wide binding sites.Results and discussionThe complex modulation mechanisms were implied by the strong bias distribution of MtrA binding sites in the noncoding regions, and 32.7% of the binding sites were located inside the target genes. The functions of 288 potential MtrA target genes predicted according to 294 confirmed binding sites were highly diverse, and DNA replication and damage repair, lipid metabolism, cell wall component biosynthesis, cell wall assembly, and cell division were the predominant pathways. Among the 53 pathways shared between dormancy/resuscitation and persistence, which accounted for 81.5% and 93.0% of the total number of pathways, respectively, MtrA regulatory genes were identified not only in 73.6% of their mutual pathways, but also in 75.4% of the pathways related to dormancy/resuscitation and persistence respectively. These results suggested the pivotal roles of MtrA in regulating dormancy/resuscitation and the apparent relationship between dormancy/resuscitation and persistence. Furthermore, the finding that 32.6% of the MtrA regulons were essential in vivo and/or in vitro for M. tuberculosis provided new insight into its indispensability. The findings mentioned above indicated that MtrA is a novel promising therapeutic target for tuberculosis treatment since the crucial function of MtrA may be a point of weakness for M. tuberculosis.

Carbazomycins (1–8) are a subgroup of carbazole derivatives that contain oxygen at the C3 and C4 positions and an unusual asymmetric substitution pattern. Several of these compounds exhibit antifungal and antioxidant activities. To date, no systematic biosynthetic studies have been conducted on carbazomycins. In this study, carbazomycins A and B (1 and 2) were isolated from Streptomyces luteosporeus NRRL 2401 using a one-strain-many-compound (OSMAC)-guided natural product mining screen. A biosynthetic gene cluster (BGC) was identified, and possible biosynthetic pathways for 1 and 2 were proposed. The in vivo genetic manipulation of the O-methyltransferase-encoding gene cbzMT proved indispensable for 1 and 2 biosynthesis. Size exclusion chromatography indicated that CbzMT was active as a dimer. In vitro biochemical assays confirmed that CbzMT could repeatedly act on the hydroxyl groups at C3 and C4, producing monomethylated 2 and dimethylated 1. Monomethylated carbazomycin B (2) is not easily methylated; however, CbzMT seemingly prefers the dimethylation of the dihydroxyl substrate (12) to 1, even with a low conversion efficiency. These findings not only improve the understanding of carbazomycin biosynthesis but also expand the inventory of OMT-catalyzing iterative methylations on different acceptor sites, paving the way for engineering biocatalysts to synthesize new active carbazomycin derivatives.

The peptidoglycan (PG) layer is a critical component of the bacterial cell wall and serves as an important target for antibiotics in both gram-negative and gram-positive bacteria. The hydrolysis of septal PG (sPG) is a crucial step of bacterial cell division, facilitated by FtsEX through an amidase activation system. In this study, we present the cryo-EM structures of Escherichia coli FtsEX and FtsEX-EnvC in the ATP-bound state at resolutions of 3.05 Å and 3.11 Å, respectively. Our PG degradation assays in E. coli reveal that the ATP-bound conformation of FtsEX activates sPG hydrolysis of EnvC-AmiB, whereas EnvC-AmiB alone exhibits autoinhibition. Structural analyses indicate that ATP binding induces conformational changes in FtsEX-EnvC, leading to significant differences from the apo state. Furthermore, PG degradation assays of AmiB mutants confirm that the regulation of AmiB by FtsEX-EnvC is achieved through the interaction between EnvC-AmiB. These findings not only provide structural insight into the mechanism of sPG hydrolysis and bacterial cell division, but also have implications for the development of novel therapeutics targeting drug-resistant bacteria.

ABSTRACTThe Clp protease system is important for maintaining proteostasis in bacteria. It consists of ClpP serine proteases and an AAA+ Clp-ATPase such as ClpC1. The hexameric ATPase ClpC1 utilizes the energy of ATP binding and hydrolysis to engage, unfold, and translocate substrates into the proteolytic chamber of homo- or hetero-tetradecameric ClpP for degradation. The assembly between the hetero-tetradecameric ClpP1P2 chamber and the Clp-ATPases containing tandem ATPase domains from the same species has not been studied in depth. Here, we present cryo-EM structures of the substrate-bound ClpC1:shClpP1P2 from Streptomyces hawaiiensis, and shClpP1P2 in complex with ADEP1, a natural compound produced by S. hawaiiensis and known to cause over-activation and dysregulation of the ClpP proteolytic core chamber. Our structures provide detailed information on the shClpP1-shClpP2, shClpP2-ClpC1, and ADEP1-shClpP1/P2 interactions, reveal conformational transition of ClpC1 during the substrate translocation, and capture a rotational ATP hydrolysis mechanism likely dominated by the D1 ATPase activity of chaperones.IMPORTANCEThe Clp-dependent proteolysis plays an important role in bacterial homeostasis and pathogenesis. The ClpP protease system is an effective drug target for antibacterial therapy. Streptomyces hawaiiensis can produce a class of potent acyldepsipeptide antibiotics such as ADEP1, which could affect the ClpP protease activity. Although S. hawaiiensis hosts one of the most intricate ClpP systems in nature, very little was known about its Clp protease mechanism and the impact of ADEP molecules on ClpP. The significance of our research is in dissecting the functional mechanism of the assembled Clp degradation machinery, as well as the interaction between ADEP1 and the ClpP proteolytic chamber, by solving high-resolution structures of the substrate-bound Clp system in S. hawaiiensis. The findings shed light on our understanding of the Clp-dependent proteolysis in bacteria, which will enhance the development of antimicrobial drugs targeting the Clp protease system, and help fighting against bacterial multidrug resistance.

Abstract Background Klebsiella pneumoniae is a notorious clinical pathogen and frequently carries various plasmids, which are the main carriers of antimicrobial resistance and virulence genes. In comparison to self-transmissible conjugative plasmids, mobilizable plasmids have received much less attention due to their defects in conjugative elements. However, the contribution of mobilizable plasmids to the horizontal transfer of antimicrobial resistance genes and virulence genes of K. pneumoniae remains unclear. In this study, the transfer, stability, and cargo genes of the mobilizable plasmids of K. pneumoniae were examined via genetic experiments and genomic analysis. Methods Carbapenem-resistant (CR) plasmid pHSKP2 and multidrug-resistant (MDR) plasmid pHSKP3 of K. pneumoniae HS11286, virulence plasmid pRJF293 of K. pneumoniae RJF293 were employed in conjugation assays to assess the transfer ability of mobilizable plasmids. Mimic mobilizable plasmids and genetically modified plasmids were constructed to confirm the cotransfer models. The plasmid morphology was evaluated through XbaI and S1 nuclease pulsed-field gel electrophoresis and/or complete genome sequencing. Mobilizable plasmid stability in transconjugants was analyzed via serial passage culture. In addition, in silico genome analysis of 3923 plasmids of 1194 completely sequenced K. pneumoniae was performed to investigate the distribution of the conjugative elements, the cargo genes, and the targets of the CRISPR-Cas system. The mobilizable MDR plasmid and virulence plasmid of K. pneumoniae were investigated, which carry oriT but lack other conjugative elements. Results Our results showed that mobilizable MDR and virulence plasmids carrying oriT but lacking the relaxase gene were able to cotransfer with a helper conjugative CR plasmid across various Klebsiella and Escherichia coli strains. The transfer and stability of mobilizable plasmids rather than conjugative plasmids were not interfered with by the CRISPR–Cas system of recipient strains. According to the in silico analysis, the mobilizable plasmids carry about twenty percent of acquired antimicrobial resistance genes and more than seventy-five percent of virulence genes in K. pneumoniae. Conclusions Our work observed that a mobilizable MDR or virulence plasmid that carries oriT but lacks the relaxase genes transferred with the helper CR conjugative plasmid and mobilizable plasmids escaped from CRISPR–Cas defence and remained stable in recipients. These results highlight the threats of mobilizable plasmids as vital vehicles in the dissemination of antibiotic resistance and virulence genes in K. pneumoniae.

About two-thirds of small molecule drugs contain methyl group and it plays a very important role in the drug development. So, methyltransferases catalyzing the methylation have always attracted great attention. Hangtaimycin (HTM) is a potent hepatoprotective agent. Previous study showed that its biosynthetic gene cluster contained three methyltransferase domains, but their characteristics in HTM biosynthetic pathway has not been revealed. In this study, we clarified multi-methylations in HTM biosynthesis in vivo. It showed that the two S-adenosylmethionine-dependent methyltransferases (SAM-MTs) of HtmA2(-module 6)-MT domain and HtmB2(-module 18)-MT domain are responsible for the installation of methyl group at C-45 and N-12, respectively, whereas the FK506 methyltransferase (FKMT) type O-methyltransferase of HtmB1(-module 16)-MT domain take care of the methylation at O-21 of HTM. We also reported the antibacterial activities of HTM in this study, and found that it showed activities against M. luteus, B. thuringiensis and A. baumannii with MIC of 4 μg/mL, 4 μg/mL, and 64 μg/mL, respectively.

Borosins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylated backbones. Although the first mature compound of borosin was reported in 1997, the biosynthetic pathway was elucidated 20 years later. Until this work, borosins have been able to be categorized into 11 types based on the features of their protein structure and core peptides. Type III borosins were reported only in fungi initially. In order to explore the sources and potential of type III borosins, a precise genome mining work of type III borosins was conducted in bacteria and KchMA’s self-methylation activity was validated by biochemical experiment. Furthermore, a commercial protease and AI-assisted rational design was employed to engineer KchMA for the capacity to produce various N-methylated peptides. Our work demonstrates that type III borosins are abundant not only in eukaryotes but also in bacteria and have immense potential as a tool for synthetic biology.

Abstract Base editors, which enable targeted locus nucleotide conversion in genomic DNA without double‐stranded breaks, have been engineered as powerful tools for biotechnological and clinical applications. However, the application of base editors is limited by their off‐target effects. Continuously expressed deaminases used for gene editing may lead to unwanted base alterations at unpredictable genomic locations. In the present study, blue‐light‐activated base editors (BLBEs) are engineered based on the distinct photoswitches magnets that can switch from a monomer to dimerization state in response to blue light. By fusing the N‐ and C‐termini of split DNA deaminases with photoswitches Magnets, efficient A‐to‐G and C‐to‐T base editing is achieved in response to blue light in prokaryotic and eukaryotic cells. Furthermore, the results showed that BLBEs can realize precise blue light‐induced gene editing across broad genomic loci with low off‐target activity at the DNA‐ and RNA‐level. Collectively, these findings suggest that the optogenetic utilization of base editing and optical base editors may provide powerful tools to promote the development of optogenetic genome engineering.

Anisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of aniP and its homologue siAniP confirmed their indispensable role in 1 biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (3) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of 1 and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of 1 and its derivatives.

Abstract Diazo compounds are rare natural products possessing various biological activities. Kinamycin and lomaiviticin, two diazo natural products featured by the diazobenzofluorene core, exhibit exceptional potency as chemotherapeutic agents. Despite the extensive studies on their biosynthetic gene clusters and the assembly of their polyketide scaffolds, the formation of the characteristic diazo group remains elusive. l-Glutamylhydrazine was recently shown to be the hydrazine donor in kinamycin biosynthesis, however, the mechanism for the installation of the hydrazine group onto the kinamycin scaffold is still unclear. Here we describe an O-methyltransferase-like protein, AlpH, which is responsible for the hydrazine incorporation in kinamycin biosynthesis. AlpH catalyses a unique SAM-independent coupling of l-glutamylhydrazine and polyketide intermediate via a rare Mannich reaction in polyketide biosynthesis. Our discovery expands the catalytic diversity of O-methyltransferase-like enzymes and lays a strong foundation for the discovery and development of novel diazo natural products through genome mining and synthetic biology.

Tylosin is a 16-membered macrolide antibiotic widely used in veterinary medicine to control infections caused by Gram-positive pathogens and mycoplasmas. To improve the fermentation titer of tylosin in the hyperproducing Streptomyces xinghaiensis strain TL01, we sequenced its whole genome and identified the biosynthetic gene cluster therein. Overexpression of the tylosin efflux gene tlrC, the cluster-situated S-adenosyl methionine (SAM) synthetase gene metKcs, the SAM biosynthetic genes adoKcs-metFcs, or the pathway-specific activator gene tylR enhanced tylosin production by 18%, 12%, 11%, and 11% in the respective engineered strains TLPH08-2, TLPH09, TLPH10, and TLPH12. Co-overexpression of metKcs and adoKcs-metFcs as two transcripts increased tylosin production by 22% in the resultant strain TLPH11 compared to that in TL01. Furthermore, combinational overexpression of tlrC, metKcs, adoKcs-metFcs, and tylR as four transcripts increased tylosin production by 23% (10.93g/L) in the resultant strain TLPH17 compared to that in TL01. However, a negligible additive effect was displayed upon combinational overexpression in TLPH17 as suggested by the limited increment of fermentation titer compared to that in TLPH08-2. Transcription analyses indicated that the expression of tlrC and three SAM biosynthetic genes in TLPH17 was considerably lower than that of TLPH08-2 and TLPH11. Based on this observation, the five genes were rearranged into one or two operons to coordinate their overexpression, yielding two engineered strains TLPH23 and TLPH24, and leading to further enhancement of tylosin production over TLPH17. In particular, the production of TLPH23 reached 11.35 g/L. These findings indicated that the combinatorial strategy is a promising approach for enhancing tylosin production in high-yielding industrial strains.

Abstract CRISPR-Cas immunity systems safeguard prokaryotic genomes by inhibiting the invasion of mobile genetic elements. Here, we screened prokaryotic genomic sequences and identified multiple natural transpositions of insertion sequences (ISs) into cas genes, thus inactivating CRISPR-Cas defenses. We then generated an IS-trapping system, using Escherichia coli strains with various ISs and an inducible cas nuclease, to monitor IS insertions into cas genes following the induction of double-strand DNA breakage as a physiological host stress. We identified multiple events mediated by different ISs, especially IS1 and IS10, displaying substantial relaxed target specificity. IS transposition into cas was maintained in the presence of DNA repair machinery, and transposition into other host defense systems was also detected. Our findings highlight the potential of ISs to counter CRISPR activity, thus increasing bacterial susceptibility to foreign DNA invasion.

Abstract Background Streptomyces are well known for their potential to produce various pharmaceutically active compounds, the commercial development of which is often limited by the low productivity and purity of the desired compounds expressed by natural producers. Well-characterized promoters are crucial for driving the expression of target genes and improving the production of metabolites of interest. Results A strong constitutive promoter, stnYp, was identified in Streptomyces flocculus CGMCC4.1223 and was characterized by its effective activation of silent biosynthetic genes and high efficiency of heterologous gene expression. The promoter stnYp showed the highest activity in model strains of four Streptomyces species compared with the three frequently used constitutive promoters ermEp*, kasOp*, and SP44. The promoter stnYp could efficiently activate the indigoidine biosynthetic gene cluster in S. albus J1074, which is thought to be silent under routine laboratory conditions. Moreover, stnYp was found suitable for heterologous gene expression in different Streptomyces hosts. Compared with the promoters ermEp*, kasOp*, and SP44, stnYp conferred the highest production level of diverse metabolites in various heterologous hosts, including the agricultural-bactericide aureonuclemycin and the antitumor compound YM-216391, with an approximately 1.4 − 11.6-fold enhancement of the yields. Furthermore, the purity of tylosin A was greatly improved by overexpressing rate-limiting genes through stnYp in the industrial strain. Further, the yield of tylosin A was significantly elevated to 10.30 ± 0.12 g/L, approximately 1.7-fold higher than that of the original strain. Conclusions The promoter stnYp is a reliable, well-defined promoter with strong activity and broad suitability. The findings of this study can expand promoter diversity, facilitate genetic manipulation, and promote metabolic engineering in multiple Streptomyces species.

Polyene macrolactams are a special group of natural products with great diversity, unique structural features, and a wide range of biological activities. Herein, a cryptic gene cluster for the biosynthesis of putative macrolactams was disclosed from a sponge-associated bacterium, Streptomyces sp. DSS69, by genome mining. Cloning and heterologous expression of the whole biosynthetic gene cluster led to the discovery of weddellamycin, a polyene macrolactam bearing a 23/5/6 ring skeleton. A negative regulator, WdlO, and two positive regulators, WdlA and WdlB, involved in the regulation of weddellamycin production were unraveled. The fermentation titer of weddellamycin was significantly improved by overexpression of wdlA and wdlB and deletion of wdlO. Notably, weddellamycin showed remarkable antibacterial activity against various Gram-positive bacteria including MRSA, with MIC values of 0.10–0.83 μg/mL, and antifungal activity against Candida albicans, with an MIC value of 3.33 μg/mL. Weddellamycin also displayed cytotoxicity against several cancer cell lines, with IC50 values ranging from 2.07 to 11.50 µM.

Klebsiella pneumoniae is an important human pathogen known for its resistance to carbapenem antibiotics, especially the increasing carbapenem-resistant hypervirulent variants. The carbapenem resistance is mainly caused by the carbapenemase gene blaKPC which was commonly found on the IncFII transferable plasmids in K. pneumoniae ST11 isolates in regions of China. However, the mechanisms of the plasmid-carrying blaKPC regulation by the host strain are not clear. To investigate the chromosome-encoded two-component system (TCS) that regulates the carbapenem resistance of K. pneumoniae caused by blaKPC, twenty-four TCSs of a carbapenem-resistant classical K. pneumoniae ST11 clinical isolate were knocked out. The deletion mutation of the TCS regulator cpxR exhibited increased sensitivity to carbapenem, which could be restored by complementation with cpxR in trans. Electrophoretic mobility shift, isothermal titration calorimetry and DNase I footprinting results revealed that CpxR directly bound to the promoter DNA of blaKPC and the binding was abolished by disrupting the DNA-binding domain in CpxR. The subsequent in vivo assays using the lacZ reporter system and qPCR showed that CpxR upregulates the transcription of blaKPC. Notably, CpxR was also found to activate the transfer of the blaKPC-carrying IncFII plasmid between the hypervirulent K. pneumoniae and E. coli isolates, in which CpxR promoted the transcription of the tra operon via binding to its promoter region. These results provide an important insight into the regulation of the host factor CpxR in the plasmid-carrying carbapenemase gene in the classical and hypervirulent K. pneumoniae.

Abstract Assembly-line polyketide synthases (PKSs) are molecular factories that produce diverse metabolites with wide-ranging biological activities. PKSs usually work by constructing and modifying the polyketide backbone successively. Here, we present the cryo-EM structure of CalA3, a chain release PKS module without an ACP domain, and its structures with amidation or hydrolysis products. The domain organization reveals a unique “∞”-shaped dimeric architecture with five connected domains. The catalytic region tightly contacts the structural region, resulting in two stabilized chambers with nearly perfect symmetry while the N-terminal docking domain is flexible. The structures of the ketosynthase (KS) domain illustrate how the conserved key residues that canonically catalyze C–C bond formation can be tweaked to mediate C–N bond formation, revealing the engineering adaptability of assembly-line polyketide synthases for the production of novel pharmaceutical agents.

Intermodular recognition in polyketide synthase (PKS) is a key prerequisite for catalysis and assembly-line engineering. Here, the authors present a specific domain interaction between modules and an evolution-oriented strategy for PKS engineering derived from the enoylreduction module of azalomycin F.

It is important to improve the production of bioactive secondary products for drug development. The Escherichia coli—Streptomyces shuttle vector pSET152 and its derived vector pIB139 containing a strong constitutive promoter ermEp* are commonly used as integrative vectors in actinomycetes. Four new integrative vectors carrying the strong constitutive promoter kasOp*, hrdBp, SCO5768p, and SP44, respectively, were constructed and proven to be functional in different mangrove-derived Streptomyces host strains by using kanamycin resistance gene neo as a reporter. Some biosynthetic genes of elaiophylins, azalomycin Fs, and armeniaspirols were selected and inserted into these vectors to overexpress in their producers including Streptomyces sp. 219807, Streptomyces sp. 211726, and S. armeniacus DSM 43125, resulting in an approximately 1.1–1.4-fold enhancement of the antibiotic yields.

The interplay of various enzymes and compounds gives rise to the intricate secondary metabolic networks observed today. However, the current understanding of their formation and expansion remains limited. BAHD acyltransferases play important roles in the biosynthesis of numerous significant secondary metabolites. In plants, they are widely distributed and exhibit a diverse range of activities. Among them, rosmarinic acid synthase (RAS) and hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) have gained significant recognition and have been extensively investigated as prominent members of the BAHD acyltransferase family. Here, we conducted a comprehensive study on a unique group of RAS homologous enzymes in Mentha longifolia that display both catalytic activities and molecular features similar to HCT and Lamiaceae RAS. Subsequent phylogenetic and comparative genome analyses revealed their derivation from expansion events within the HCT gene family, indicating their potential as collateral branches along the evolutionary trajectory, leading to Lamiaceae RAS while still retaining certain ancestral vestiges. This discovery provides more detailed insights into the evolution from HCT to RAS. Our collective findings indicate that gene duplication is the driving force behind the observed evolutionary pattern in plant-specialized enzymes, which probably originated from ancestral enzyme promiscuity and were subsequently shaped by principles of biological adaptation.

Abstract Sesquiterpenes have been identified as promising ingredients for aviation fuels due to their high energy density and combustion heat properties. Despite the characterization of numerous sesquiterpene structures, studies testing their performance properties and feasibility as fuels are scarce. In this study, 122 sesquiterpenoid skeleton compounds, obtained from existing literature reports, are tested using group contribution and gaussian quantum chemistry methods to assess their potential as high‐energy aviation fuels. Seventeen sesquiterpene compounds exhibit good predictive performance and nine compounds are further selected for overproduction in yeast. Through fed‐batch fermentation, all compounds achieve the highest reported titers to date. Subsequently, three representative products, pentalenene, presilphiperfol‐1‐ene, and α‐farnesene, are selected, produced, purified in large quantities, and tested for use as potential fuels. The performance of pentalenene, presilphiperfol‐1‐ene, and their derivatives reveals favorable prospects as high‐energy aviation fuels.

SspABCD–SspE is a phosphorothioation-sensing bacterial defence system. Here, authors find that SspE exploits DNA phosphorothioation-binding preference to modulate its biochemical activities for the self/nonself discrimination and phage resistance.

Metabolomics is an essential discipline in omics technology that promotes research on the biology of microbial systems. Streptomyces albus J1074 is a model organism used in fundamental research and industrial microbiology. Nevertheless, a comprehensive and standardized method for analyzing the metabolome of S. albus J1074 is yet to be developed. Thus, we comprehensively evaluated and optimized the analytical procedure and sample preparation for profiling polar metabolites using hydrophilic interaction liquid chromatography (HILIC) coupled with high-resolution mass spectrometry (HRMS). We systematically examined the HILIC columns, quenching solutions, sample-to-quenching ratios, and extraction methods. Then, the optimal protocol was used to investigate the dynamic intracellular polar metabolite profile of the engineered S. albus J1074 strains during spinosad (spinosyn A and spinosyn D) fermentation. A total of 3648 compounds were detected, and 83 metabolites were matched to the standards. The intracellular metabolomic profiles of engineered S. albus J1074 strains (ADE-AP and OE3) were detected; furthermore, their metabolomes in different stages were analyzed to reveal the reasons for their differences in their spinosad production, as well as the current metabolic limitation of heterologous spinosad production in S. albus J1074. The HILIC-HRMS method is a valuable tool for investigating polar metabolomes, and provides a reference methodology to study other Streptomyces metabolomes.

Assembly artificial pathway in design connecting media can increase biosynthetic efficiency, but the choice of connecting media is limited. Here, the authors develop a new protein assembly strategy using a pool of docking peptides from polyketide synthase and show its application in astaxanthin biosynthesis in E. coli.

Phage contamination is a persistent problem in industrial biotechnology processes employing bacterial strains. Here, the authors report the construction of E. coli host strains with broad antiphase activities via the genomic integration of the Ssp defense system and mutations of components essential for phage infection cycles.

Synthetic lethality (SL) occurs when mutations in two genes together lead to cell or organism death, while a single mutation in either gene does not have a significant impact. This concept can also be extended to three or more genes for SL. Computational and experimental methods have been developed to predict and verify SL gene pairs, especially for yeast and Escherichia coli. However, there is currently a lack of a specialized platform to collect microbial SL gene pairs. Therefore, we designed a synthetic interaction database for microbial genetics that collects 13,313 SL and 2,994 Synthetic Rescue (SR) gene pairs that are reported in the literature, as well as 86,981 putative SL pairs got through homologous transfer method in 281 bacterial genomes. Our database website provides multiple functions such as search, browse, visualization, and Blast. Based on the SL interaction data in the S. cerevisiae, we review the issue of duplications' essentiality and observed that the duplicated genes and singletons have a similar ratio of being essential when we consider both individual and SL. The Microbial Synthetic Lethal and Rescue Database (Mslar) is expected to be a useful reference resource for researchers interested in the SL and SR genes of microorganisms. Mslar is open freely to everyone and available on the web at http://guolab.whu.edu.cn/Mslar/.

Enterobactin is an archetypical catecholate siderophore that plays a key role in the acquisition of ferric iron by microorganisms. Catechol moieties have been shown to be promising siderophore cores. Variants of the conserved 2,3-dihydroxybenzoate (DHB) moiety with structural modifications expand the bioactivity. Streptomyces are characterized by metabolites with diverse structures. The genomic sequence of Streptomyces varsoviensis indicated that it possessed a biosynthetic gene cluster for DHB containing siderophores and metabolic profiling revealed metabolites correlated with catechol-type natural products. Here, we report the discovery of a series of catecholate siderophores produced by S. varsoviensis and a scale-up fermentation was performed to purify these compounds for structural elucidation. A biosynthetic route for the catecholate siderophores is also proposed. These new structural features enrich the structural diversity of the enterobactin family compounds. One of the new linear enterobactin congeners shows moderate activity against a food-borne pathogen Listeria monocytogenes. This work demonstrated that changing culture conditions is still a promising approach to explore unexplored chemical diversity. The availability of the biosynthetic machinery will enrich the genetic toolbox of catechol siderophores and facilitate such engineering efforts.

In order to deepen the understanding of the role and regulation mechanisms of prokaryotic global transcription regulators in complex processes, including virulence, the associations between the affinity and binding sequences of Mycobacterium tuberculosis MtrA have been explored extensively. Analysis of MtrA 294 diversified 26 bp binding sequences revealed that the sequence similarity of fragments was not simply associated with affinity. The unique variation patterns of GC content and periodical and sequential fluctuation of affinity contribution curves were observed along the sequence in this study. Furthermore, docking analysis demonstrated that the structure of the dimer MtrA-DNA (high affinity) was generally consistent with other OmpR family members, while Arg 219 and Gly 220 of the wing domain interacted with the minor groove. The results of the binding box replacement experiment proved that box 2 was essential for binding, which implied the differential roles of the two boxes in the binding process. Furthermore, the results of the substitution of the nucleotide at the 20th and/or 21st positions indicated that the affinity was negatively associated with the value of minor groove width precisely at the 21st position. The dimerization of the unphosphorylated MtrA facilitated by a low-affinity DNA fragment was observed for the first time. However, the proportion of the dimer was associated with the affinity of substrate DNA, which further suggested that the affinity was actually one characteristic of the stability of dimers. Based on the finding of 17 inter-molecule hydrogen bonds identified in the interface of the MtrA dimer, including 8 symmetric complementary ones in the conserved α4-β5-α5 face, we propose that hydrogen bonds should be considered just as important as salt bridges and the hydrophobic patch in the dimerization. Our comprehensive study on a large number of binding fragments with quantitative affinity values provided new insight into the molecular mechanism of dimerization, binding specificity and affinity determination of MtrA and clues for solving the puzzle of how global transcription factors regulate a large quantity of target genes.

Structural analyses of a Streptomyces viridochromogenes product template (PT) domain suggests molecular and functional similarities with known fungal PTs involved in polyketide synthase activity.

Darobactin is a ribosomally synthesized and post-translationally modified peptide featuring a unique scaffold and potent activity against Gram-negative bacteria. Here, the authors identify darobactin synthase DarE as responsible for the bicyclic scaffold formation and propose the name daropeptide for this growing class of enzymes.

Current commercialized production processes of glyphosate are generally associated with manufacturing hazards and toxic wastes. Here, to address the problems, the authors report the bioproduction of aminomethylphosphonate (AMP) and a chemical process for the conversion of AMP to glyphosate.

Proteins secreted by Gram-negative bacteria are tightly linked to the virulence and adaptability of these microbes to environmental changes. Accurate identification of such secreted proteins can facilitate the investigations of infections and diseases caused by these bacterial pathogens. However, current bioinformatic methods for predicting bacterial secreted substrate proteins have limited computational efficiency and application scope on a genome-wide scale. Here, we propose a novel deep-learning-based framework—DeepSecE—for the simultaneous inference of multiple distinct groups of secreted proteins produced by Gram-negative bacteria. DeepSecE remarkably improves their classification from nonsecreted proteins using a pretrained protein language model and transformer, achieving a macro-average accuracy of 0.883 on 5-fold cross-validation. Performance benchmarking suggests that DeepSecE achieves competitive performance with the state-of-the-art binary predictors specialized for individual types of secreted substrates. The attention mechanism corroborates salient patterns and motifs at the N or C termini of the protein sequences. Using this pipeline, we further investigate the genome-wide prediction of novel secreted proteins and their taxonomic distribution across ~1,000 Gram-negative bacterial genomes. The present analysis demonstrates that DeepSecE has major potential for the discovery of disease-associated secreted proteins in a diverse range of Gram-negative bacteria. An online web server of DeepSecE is also publicly available to predict and explore various secreted substrate proteins via the input of bacterial genome sequences.

Multiple signal strategies remarkably improve the accuracy and efficiency of electrochemiluminescence (ECL) immunoassays, but the lack of potential-resolved luminophore pairs and chemical cross talk hinders their development. In this study, we synthesized a series of gold nanoparticles (AuNPs)/reduced graphene oxide (Au/rGO) composites as adjustable oxygen reduction reaction and oxygen evolution reaction catalysts to promote and modulate tris(2,2′-bipyridine) ruthenium(II) (Ru(bpy)32+)’s multisignal luminescence. With the increase in the diameter of AuNPs (3 to 30 nm), their ability to promote Ru(bpy)32+’s anodic ECL was first impaired and then strengthened, and cathodic ECL was first enhanced and then weakened. Au/rGOs with medium-small and medium-large AuNP diameters remarkably increased Ru(bpy)32+’s cathodic and anodic luminescence, respectively. Notably, the stimulation effects of Au/rGOs were superior to those of most existing Ru(bpy)32+ co-reactants. Moreover, we proposed a novel ratiometric immunosensor construction strategy using Ru(bpy)32+’s luminescence promoter rather than luminophores as tags of antibodies to achieve signal resolution. This method avoids signal cross talk between luminophores and their respective co-reactants, which achieved a good linear range of 10−7 to 10−1 ng/ml and a limit of detection of 0.33 fg/ml for detecting carcinoembryonic antigen. This study addresses the previous scarcity of the macromolecular co-reactants of Ru(bpy)32+, broadening its application in biomaterial detection. Furthermore, the systematic clarification of the detailed mechanisms for converting the potential-resolved luminescence of Ru(bpy)32+ could facilitate an in-depth understanding of the ECL process and should inspire new designs of Ru(bpy)32+ luminescence enhancers or applications of Au/rGOs to other luminophores. This work removes some impediments to the development of multisignal ECL biodetection systems and provides vitality into their widespread applications.

The catalytic domains in nonribosomal peptide synthetases (NRPSs) are responsible for a choreography of events that elongates substrates into natural products. Here, the authors present cryo-EM structures of a siderophore-producing dimeric NRPS elongation module in multiple distinct conformations, which provides insight into the mechanisms of catalytic trajectory.

Abstract Epidemic diseases and antibiotic resistance are urgent threats to global health, and human is confronted with an unprecedented dilemma to conquer them by expediting development of new natural product related drugs. C-nucleoside antibiotics, a remarkable group of microbial natural products with diverse biological activities, feature a heterocycle base linked with a ribosyl moiety via an unusual C-glycosidic bond, and have played significant roles in healthcare and for plant protection. Elucidating how nature biosynthesizes such a group of antibiotics has provided the basis for engineered biosynthesis as well as targeted genome mining of more C-nucleoside antibiotics towards improved properties. In this review, we mainly summarize the recent advances on the biosynthesis of C-nucleoside antibiotics, and we also tentatively discuss the future developments on rationally accessing C-nucleoside diversities in a more efficient and economical way via synthetic biology strategies.

Abstract Bacterial integrative and conjugative elements (ICEs) are highly modular mobile genetic elements critical to the horizontal transfer of antibiotic resistance and virulence factor genes. To better understand and analyze the ongoing increase of ICEs, we developed an Integrative and Conjugative Element Ontology (ICEO) to represent the gene components, functional modules, and other information of experimentally verified ICEs. ICEO is aligned with the upper-level Basic Formal Ontology and reuses existing reliable ontologies. There are 31,081 terms, including 26,814 classes from 14 ontologies and 4128 ICEO-specific classes, representing the information of 271 known experimentally verified ICEs from 235 bacterial strains in ICEO currently and 311 predicted ICEs of 272 completely sequenced Klebsiella pneumoniae strains. Three ICEO use cases were illustrated to investigate complex joins of ICEs and their harboring antibiotic resistance or virulence factor genes by using SPARQL or DL query. ICEO has been approved as an Open Biomedical Ontology library ontology. It may be dedicated to facilitating systematical ICE knowledge representation, integration, and computer-assisted queries.

The dietary composition has been approved to be strongly associated with the risk of colorectal cancer (CRC), one of the most serious malignancies worldwide, through regulating the gut microbiota structure, thereby influencing the homeostasis of colonic epithelial cells by producing carcinogens, i.e., ammonia or antitumor metabolites, like butyrate. Though butyrate-producing Fusobacterium nucleatum has been considered a potential tumor driver associated with chemotherapy resistance and poor prognosis in CRC, it was more frequently identified in the gut microbiota of healthy individuals rather than CRC tumor tissues. First, within the concentration range tested, the fermentation broth of F. nucleatum exhibited no significant effects on Caco-2 and NCM460 cells viability except for a notable up-regulation of the expression of TLR4 (30.70%, p < 0.0001) and Myc (47.67%, p = 0.021) and genes encoding proinflammatory cytokines including IL1B (197.57%, p < 0.0001), IL6 (1704.51%, p < 0.0001), and IL8 (897.05%, p < 0.0001) in Caco-2 cells exclusively. Although no marked effects of polydextrose or fibersol-2 on the growth of F. nucleatum, Caco-2 and NCM460 cells were observed, once culture media supplemented with polydextrose or fibersol-2, the corresponding fermentation broths of F. nucleatum significantly inhibited the growth of Caco-2 cells up to 48.90% (p = 0.0003, 72 h, 10%) and 52.96% (p = 0.0002, 72 h, 10%), respectively in a dose-dependent manner. These two kinds of fibers considerably promoted butyrate production of F. nucleatum up to 205.67% (p < 0.0001, 6% polydextrose at 24 h) and 153.46% (p = 0.0002, 6% fibersol-2 at 12 h), which explained why and how the fermentation broths of F. nucleatum cultured with fibers suppressing the growth of Caco-2 cells. Above findings indicated that dietary fiber determined F. nucleatum to be a carcinogenic or antitumor bacterium, and F. nucleatum played an important role in the association between the dietary composition, primarily the content of dietary fibers, and the risk of CRC.

Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete, Saccharopolyspora spinosa. However, their large-scale overproduction is hindered by poorly understood bottlenecks in optimizing the original strain, and poor adaptability of the heterologous strain to the production of spinosyn. In this study, we genetically engineered heterologous spinosyn-producer Streptomyces albus J1074 and optimized the fermentation to improve the production of spinosad (spinosyn A and spinosyn D) based on our previous work. We systematically investigated the result of overexpressing polyketide synthase genes (spnA, B, C, D, E) using a constitutive promoter on the spinosad titer in S. albus J1074. The supply of polyketide synthase precursors was then increased to further improve spinosad production. Finally, increasing or replacing the carbon source of the culture medium resulted in a final spinosad titer of ∼70 mg/L, which is the highest titer of spinosad achieved in heterologous Streptomyces species. This research provides useful strategies for efficient heterologous production of natural products.

The Lianhua Qingwen (LHQW) capsule is a popular traditional Chinese medicine for the treatment of viral respiratory diseases. In particular, it has been recently prescribed to treat infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, due to its complex composition, little attention has been directed toward the analysis of chemical constituents present in the LHQW capsule. This study presents a reliable and comprehensive approach to characterizing the chemical constituents present in LHQW by high-performance liquid chromatography-Q Exactive-Orbitrap mass spectrometry (HPLC-Q Exactive-Orbitrap-MS) coupled with gas chromatography-mass spectrometry (GC-MS). An automated library alignment method with a high mass accuracy (within 5 ppm) was used for the rapid identification of compounds. A total of 104 compounds, consisting of alkaloids, flavonoids, phenols, phenolic acids, phenylpropanoids, quinones, terpenoids, and other phytochemicals, were successfully characterized. In addition, the fragmentation pathways and characteristic fragments of some representative compounds were elucidated. GC-MS analysis was conducted to characterize the volatile compounds present in LHQW. In total, 17 compounds were putatively characterized by comparing the acquired data with that from the NIST library. The major constituent was menthol, and all the other compounds were terpenoids. This is the first comprehensive report on the identification of the major chemical constituents present in the LHQW capsule by HPLC-Q Exactive-Orbitrap-MS, coupled with GC-MS, and the results of this study can be used for the quality control and standardization of LHQW capsules.