Zavarzinia marina sp. nov., a novel hydrocarbon-degrading bacterium isolated from deep chlorophyll maximum layer seawater of the West Pacific Ocean and emended description of the genus Zavarzinia.

A Gram-stain-negative, motile, non-spore-forming, strictly aerobic and rod-shaped bacterial strain, Adcm-6AT, was isolated from a seawater sample collected from the deep chlorophyll maximum layer in the West Pacific Ocean. Strain Adcm-6AT grew at 20-37 °C (optimum, 28-32 °C), at pH 6-11 (pH 7) and in the presence of 0-6 % (1-2 %) NaCl (w/v). Phylogenetic analysis based on 16S rRNA gene sequences indicated that it belonged to the genus Zavarzinia and had 97.7 and 96.9 % sequence similarity to Zavarzinia compransoris DSM 1231T and Zavarzinia aquatilis JCM 32263T, respectively. Digital DNA-DNA hybridization and average nucleotide identity values between strain Adcm-6AT and the two type strains were 22.2-22.9 % and 79.7-80.4 %, respectively. The principal fatty acids were C19:0 cyclo ω8c, summed feature 8 (C18:1 ω6c and/or C18:1 ω7c) and C16:0. The predominant respiratory quinone was Q-10. The polar lipids were diphosphatidylglycerol, two phosphatidylethanolamines, two phosphatidyglycerols and an unidentified lipid. The genomic DNA G+C content of strain Adcm-6AT was 67.7 %. Based on phylogenetic analysis and genomic-based relatedness indices, as well as phenotypic and genotypic characteristics, strain Adcm-6AT represents a novel species within the genus Zavarzinia, for which the name Zavarzinia marina sp. nov. is proposed. The type strain is Adcm-6AT (=MCCC M24951T=KCTC 82849T).

Identification of a TetR family regulator and a polyketide synthase gene cluster involved in growth development and butenyl-spinosyn biosynthesis of Saccharopolyspora pogona.

Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and broad pesticidal spectrum. However, its synthetic level was low in the wild-type strain. At present, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently editing its genome to improve the butenyl-spinosyn yield. To accelerate the genetic modification of S. pogona, we conducted comparative proteomics analysis to screen differentially expressed proteins related to butenyl-spinosyn biosynthesis. A TetR family regulatory protein was selected from the 289 differentially expressed proteins, and its encoding gene (SP_1288) was successfully deleted by CRISPR/Cas9 system. We further deleted a 32-kb polyketide synthase gene cluster (cluster 28) to reduce the competition for precursors. Phenotypic analysis revealed that the deletion of the SP_1288 and cluster 28 resulted in a 3.10-fold increase and a 35.4% decrease in the butenyl-spinosyn levels compared with the wild-type strain, respectively. The deletion of cluster 28 affected the cell growth, glucose consumption, mycelium morphology, and sporulation by controlling the expression of ptsH, ptsI, amfC, and other genes related to sporulation, whereas SP_1288 did not. These findings confirmed not only that the CRISPR/Cas9 system can be applied to the S. pogona genome editing but also that SP_1288 and cluster 28 are closely related to the butenyl-spinosyn biosynthesis and growth development of S. pogona. The strategy reported here will be useful to reveal the regulatory mechanism of butenyl-spinosyn and improve antibiotic production in other actinomycetes. KEY POINTS: • SP_1288 deletion can significantly promote the butenyl-spinosyn biosynthesis. • Cluster 28 deletion showed pleiotropic effects on S. pogona. • SP_1288 and cluster 28 were deleted by CRISPR/Cas9 system in S. pogona.

Interaction of a novel Bacillus velezensis (BvL03) against Aeromonas hydrophila in vitro and in vivo in grass carp.

This study evaluated the inhibition and interaction of Bacillus velezensis BvL03 as a probiotic agent against Aeromonas hydrophila. Strain BvL03 isolated from sediment samples of fish ponds had excellent antimicrobial activity against several fish pathogenic bacteria, especially Aeromonas, including A. hydrophila, A. veronii, A. caviae, and A. sobria. The successful amplification of lipopeptide antimicrobial chemical biosynthetic genes, including iturin family (ituA, ituB, and ituD), bacillomycin family (bacA, bacD, and bacAB), surfactin family (srfAB, srfC, and srfAA), and subtilosin family (albF and sunT) from the genome of BvL03 strain, confirmed its predominant antimicrobial activity. The challenge test suggested that BvL03 significantly decreased fish mortality when challenged with A. hydrophila, which had a cumulative mortality of 12.5% in the treatment group. Toxicity and hemolytic activity of A. hydrophila after co-cultured with BvL03 were relieved as confirmed by the cell experiments, when the initial inoculated concentration of BvL03 was 109 cfu/mL or higher. Moreover, the BvL03 strain labeled with GFP protein (BvL03-GFP) and AhX040 strain labeled with mCherry protein (AhX040-mCherry) were injected into grass carps. The fluorescence levels were monitored by using In Vivo Imaging System (IVIS), in which the green color was steadily increasing, whereas the red color was gradually weakening. Whole genome sequencing revealed that strain BvL03 possesses 15 gene clusters related to antibacterial compounds, including 5 NRPS gene clusters and 3 PKS gene clusters. These results suggested that B. velezensis BvL03 has the potential to be developed as a probiotic candidate against A. hydrophila infection in aquaculture.

Bacillus thuringiensis Crystal Protein Cry6Aa Triggers Caenorhabditis elegans Necrosis Pathway Mediated by Aspartic Protease (ASP-1).

Cell death plays an important role in host-pathogen interactions. Crystal proteins (toxins) are essential components of Bacillus thuringiensis (Bt) biological pesticides because of their specific toxicity against insects and nematodes. However, the mode of action by which crystal toxins to induce cell death is not completely understood. Here we show that crystal toxin triggers cell death by necrosis signaling pathway using crystal toxin Cry6Aa-Caenorhabditis elegans toxin-host interaction system, which involves an increase in concentrations of cytoplasmic calcium, lysosomal lyses, uptake of propidium iodide, and burst of death fluorescence. We find that a deficiency in the necrosis pathway confers tolerance to Cry6Aa toxin. Intriguingly, the necrosis pathway is specifically triggered by Cry6Aa, not by Cry5Ba, whose amino acid sequence is different from that of Cry6Aa. Furthermore, Cry6Aa-induced necrosis pathway requires aspartic protease (ASP-1). In addition, ASP-1 protects Cry6Aa from over-degradation in C. elegans. This is the first demonstration that deficiency in necrosis pathway confers tolerance to Bt crystal protein, and that Cry6A triggers necrosis represents a newly added necrosis paradigm in the C. elegans. Understanding this model could lead to new strategies for nematode control.

Impact on strain growth and butenyl-spinosyn biosynthesis by overexpression of polynucleotide phosphorylase gene in Saccharopolyspora pogona.

Polynucleotide phosphorylase is a highly conserved protein found in bacteria and fungi that can regulate the transcription of related enzymes involved in amino acid metabolism, organic acid metabolism, and cell biosynthesis. We studied the effect of polynucleotide phosphorylase on Saccharopolyspora pogona (S. pogona) growth and the synthesis of secondary metabolites. First, we generated the overexpression vector pOJ260-PermE-pnp via overlap extension PCR. The vector pOJ260-PermE-pnp was then introduced into S. pogona by conjugal transfer, thereby generating the recombination strain S. pogona-Pnp. Results showed that engineering strains possessed higher biomass than those of the wild-type strains. Moreover, the ability of these strains to produce spores on solid medium was stronger than that of the wild-type strains. HPLC results revealed that the butenyl-spinosyn yield in S. pogona-Pnp increased by 1.92-fold compared with that of S. pogona alone. These findings revealed that overexpression of polynucleotide phosphorylase effectively promoted butenyl-spinosyn biosynthesis in S. pogona. This result may be extended to other Streptomyces for strain improvement.

α-Pyrones with Diverse Hydroxy Substitutions from Three Marine-Derived Nocardiopsis Strains.

Eight new α-pyrones 1-8 and three known α-pyrones 9-11 were isolated from three marine-derived Nocardiopsis strains SCSIO 10419, SCSIO 04583, and SCSIO KS107. The structures of compounds 1-8 were elucidated by comprehensive spectral analyses. The absolute configurations of 4-deoxyphomapyrone C (1), 4-deoxy-11-hydroxyphomapyrone C (3), 4-deoxy-7R-hydroxyphomapyrone C (5), and phomapyrone C (11) were determined by TDDFT-ECD calculations for the solution conformers, which revealed that the conformation of the side chain was decisive for the sign of the characteristic high-wavelength ECD transition. (-)-4-Deoxy-8-hydroxyphomapyrone C (4) was isolated from SCSIO 10419 and was deduced as a diastereomeric mixture containing (8S)- and (8R)-4-deoxy-8-hydroxyphomapyrone C in a ratio of 2.6:1 (8R:8S), by chiral-phase HPLC analysis and Mosher's ester analysis. Interestingly, 7-hydroxymucidone (9) was isolated from both SCSIO 04583 and SCSIO KS107, as an enantiomeric mixture containing (7S)-hydroxymucidone (major in 9 from SCSIO 04583) and (7R)-hydroxymucidone (major in 9 from SCSIO KS107). α-Pyrones 3-5 were identified as three isomers of phomapyrone C (11) with diverse hydroxy substitutions. α-Pyrones 10-hydroxymucidone (6), 4-hydroxymucidone (8), and 9, differed in the position of the hydroxy group. Several α-pyrones exhibited moderate growth inhibitory activity against Micrococcus luteus and Bacillus subtilis.

The diverse nematicidal properties and biocontrol efficacy of Bacillus thuringiensis Cry6A against the root-knot nematode Meloidogyne hapla.

Cry6A toxin from Bacillus thuringiensis is a representative nematicidal crystal protein with a variety of nematicidal properties to free-living nematode Caenorhabditis elegans. Cry6A shares very low homology and different structure with Cry5B, another representative nematicidal crystal protein, and probably acts in a distinct pathway. All these strongly indicate that Cry6A toxin is likely a potent candidate for nematicide. The present study dealt with global investigation to determine the detrimental impacts of Cry6Aa2 toxin on Meloidogyne hapla, a root-knot nematode, and evaluated its biocontrol efficacy in pot experiment. Obtained results indicated that Cry6Aa2 toxin exhibits obvious toxicity to second-stage juvenile of M. hapla, and significantly inhibits egg hatch, motility, and penetration to host plant. Pot experiment suggested that soil drenching with spore-crystal mixture of Cry6Aa2 can clearly lighten the disease of root-knot nematode, including reduction of galling index and egg masses on host plant root, decreasing final population of nematode in soil. Moreover, application of Cry6Aa2 can obviously promote plant growth. These results demonstrated that Cry6Aa2 toxin is a promising nematicidal agent, and possesses great potential in plant-parasitic nematode management and construction of transgenic crop with constant resistance to nematode.

Systemic nematicidal activity and biocontrol efficacy of Bacillus firmus against the root-knot nematode Meloidogyne incognita.

A strain of marine bacterium Bacillus firmus YBf-10 with nematicidal activity was originally isolated by our group. In the present study, the systemic nematicidal activity and biocontrol efficacy in pot experiment of B. firmus YBf-10 were investigated. Our results showed that YBf-10 exhibits systemic nematicidal activity against Meloidogyne incognita, including lethal activity, inhibition of egg hatch and motility. Pot experiment suggested that soil drenching with YBf-10 efficiently reduced damage of M. incognita to tomato plants, such as reduction of galls, egg masses on roots, and final nematode population in soil; and moreover, YBf-10 significantly promoted host plant growth. In addition, our results also indicated that the systemic nematicidal activity is likely attributed to the secondary metabolites produced by YBf-10. The obtained results of the current study confirmed that B. firmus YBf-10 is a promising nematicidal agent, and has great potential in plant-parasitic nematicidal management.

Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa.

BACKGROUND: Saccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins. RESULTS: Two-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis. CONCLUSIONS: This study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance the production yield of spinosad in future studies.

Improved insecticidal toxicity by fusing Cry1Ac of Bacillus thuringiensis with Av3 of Anemonia viridis.

Av3, a neurotoxin of Anemonia viridis, is toxic to crustaceans and cockroaches but inactive in mammals. In the present study, Av3 was expressed in Escherichia coli Origami B (DE3) and purified by reversed-phase liquid chromatography. The purified Av3 was injected into the hemocoel of Helicoverpa armigera, rendering the worm paralyzed. Then, Av3 was expressed alone or fusion expressed with the Cry1Ac in acrystalliferous strain Cry(-)B of Bacillus thuringiensis. The shape of Cry1Ac was changed by fusion with Av3. The expressed fusion protein, Cry1AcAv3, formed irregular rhombus- or crescent-shaped crystalline inclusions, which is quite different from the shape of original Cry1Ac crystals. The toxicity of Cry1Ac was improved by fused expression. Compared with original Cry1Ac expressed in Cry(-)B, the oral toxicity of Cry1AcAv3 to H. armigera was elevated about 2.6-fold. No toxicity was detected when Av3 was expressed in Cry(-)B alone. The present study confirmed that marine toxins could be used in bio-control and implied that fused expression with other insecticidal proteins could be an efficient way for their application.

Reinforcement Learning-Based Fractional-Order Adaptive Fault-Tolerant Formation Control of Networked Fixed-Wing UAVs With Prescribed Performance.

This article investigates the fault-tolerant formation control (FTFC) problem for networked fixed-wing unmanned aerial vehicles (UAVs) against faults. To constrain the distributed tracking errors of follower UAVs with respect to neighboring UAVs in the presence of faults, finite-time prescribed performance functions (PPFs) are developed to transform the distributed tracking errors into a new set of errors by incorporating user-specified transient and steady-state requirements. Then, the critic neural networks (NNs) are developed to learn the long-term performance indices, which are used to evaluate the distributed tracking performance. Based on the generated critic NNs, actor NNs are designed to learn the unknown nonlinear terms. Moreover, to compensate for the reinforcement learning errors of actor-critic NNs, nonlinear disturbance observers (DOs) with skillfully constructed auxiliary learning errors are developed to facilitate the FTFC design. Furthermore, by using the Lyapunov stability analysis, it is shown that all follower UAVs can track the leader UAV with predesigned offsets, and the distributed tracking errors are finite-time convergent. Finally, comparative simulation results are presented to show the effectiveness of the proposed control scheme.

Hierarchical Distributed Adaptive Fault-Tolerant Control of Nonlinear Fractional-Order Multiagent Systems With Faults and Periodic Disturbances Using Event-Triggered Communication.

This article presents a distributed fault-tolerant control (FTC) scheme for nonlinear fractional-order (FO) multiagent systems (MASs) with the order lying in (0, 1, such that the proposed control architecture can be directly applied to both FO and integer-order (IO) systems without any modifications. To handle the unexpected actuator faults encountered by the FO MASs, a hierarchical FTC mechanism is developed for each system by constructing an event-triggered distributed FO estimator at the upper layer to estimate the leader system's output via conditionally triggered neighboring information, and an FTC unit at the lower layer to counteract the loss-of-effectiveness faults via Nussbaum function with FO criteria. To further address the unknown nonlinear functions involving bias faults and periodic disturbances, the Fourier series expansion technique is used to construct the input variables of fuzzy neural networks (FNNs), such that the FNNs with dynamically adjusted weight matrices, centers, and widths can be developed for each FO system to act as the learning module. It is shown by FO Lyapunov stability analysis that all follower systems can track the leader system against faults and periodic disturbances. Simulation results on FO systems and hardware-in-the-loop experiment results on IO fixed-wing unmanned aerial vehicles show the extensive feasibility of the developed scheme.

The effects of Bacillus thuringiensis Cry6A on the survival, growth, reproduction, locomotion, and behavioral response of Caenorhabditis elegans.

Several families of crystal proteins from Bacillus thuringiensis exhibit nematicidal activity. Cry5B protein, a pore-forming toxin, has been intensively studied yielding many insights into the mode of action of crystal protein at molecular level and pathogenesis of pore-forming toxins. However, little attention was paid to Cry6A, another representative nematicidal crystal protein. Cry6A shares very low homology with Cry5B at amino acid sequence and probably acts in a distinct pathway from Cry5B and even the other main commercial crystal proteins. In the current study, we comprehensively investigated the nematicidal properties of Cry6Aa2 against the free-living soil nematode Caenorhabditis elegans and examined the physical response of C. elegans to Cry6Aa2 attack. Our results indicate that Cry6Aa2 exhibits high lethal activity to C. elegans and could cause detrimental effects on C. elegans, including obviously suppressed growth, decreased brood size, and even abnormal motility. Meanwhile, our study additionally shows that C. elegans could defend against the Cry6Aa2 toxin harmful threat through behavioral defense responses, such as reduced oral uptake and physical avoidance. In general, this study suggests that Cry6Aa2 possesses diverse nematicidal properties, which strongly indicates that Cry6Aa2 is a promising potential candidate of nematicidal agent. Moreover, this study highlights the importance of behavioral responses in defense of C. elegans for survival and demonstrates the key role of crystal protein in the interaction of B. thuringiensis-C. elegans. These findings could shed light on understanding the interaction of C. elegans with B. thuringiensis and provide a perfect model to study the role of pathogenic factor in the interaction of pathogen-host.

Construction of gene library of 20 kb DNAs from parasporal crystal in Bacillus thuringiensis Strain 4.0718: phylogenetic analysis and molecular docking.

The 20 kb DNAs are associated with crystals in many subspecies of Bacillus thuringiensis. We isolated 20 kb DNAs from crystals of B. thuringiensis strain 4.0718, then constructed a gene library using DNA fragments of Sau3AI partial digestion and pbluescriptIISK(+) vector. We screened out 440 recombinants, yielding a genomic coverage of ten and including 99% sequence of DNA which achieved the required theoretical value to construct the gene library. Through NCBI Blast and homology analysis, the sequencing results proved that the DNA came from the chromosome of B. thuringiensis. Moreover, we have completed the multiple alignment of homologous ropB protein sequences and phylogenetic analysis using bioinformatic software. For further investigation of the interactions between 20 kb DNAs and protoxins, molecular docking has also been done.

Distributed prescribed performance containment control for unmanned surface vehicles based on disturbance observer.

This paper introduces a distributed containment control strategy for multiple unmanned surface vehicles (USVs) under the unknown external disturbances. The communication network of the USVs is a fixed, directed topology and only a part of the follower USVs can read the states of leader USVs. To guarantee the transient and steady-state performance of the system, the tracking errors are converted into new error functions. By utilizing the disturbance observer, the external disturbances are effectively estimated. According to the new group of errors, a distributed containment controller is proposed with the estimated external disturbances. Furthermore, using graph theory and Lyapunov approach, it is proved that all variables of the multiple USV systems are bounded and the tracking errors can be confined within the specified performance ranges. Finally, simulation results illustrate the effectiveness of the designed distributed containment controller.

Distributed Fractional-Order Intelligent Adaptive Fault-Tolerant Formation-Containment Control of Two-Layer Networked Unmanned Airships for Safe Observation of a Smart City.

This article investigates a distributed fractional-order fault-tolerant formation-containment control (FOFTFCC) scheme for networked unmanned airships (UAs) to achieve safe observation of a smart city. In the proposed control method, an interval type-2 fuzzy neural network (IT2FNN) is first developed for each UA to approximate the unknown term associated with the loss-of-effectiveness faults in the distributed error dynamics, and then a disturbance observer (DO) is proposed to compensate for the approximation error and bias fault encountered by each UA, such that the composite learning strategy composed of the IT2FNN and the DO is obtained for each UA. Moreover, fractional-order (FO) calculus is incorporated into the control scheme to provide an extra degree of freedom for the parameter adjustments. The salient feature of the proposed control scheme is that the composite learning algorithm and FO calculus are integrated to achieve a satisfactory fault-tolerant formation-containment control performance even when a portion of leader/follower UAs is subjected to the actuator faults in a distributed communication network. Furthermore, it is shown by Lyapunov stability analysis that all leader UAs can track the virtual leader UA with time-varying offset vectors, and all follower UAs can converge into the convex hull spanned by the leader UAs. Finally, comparative hardware-in-the-loop (HIL) experimental results are presented to show the effectiveness and superiority of the proposed method.

Distributed Adaptive Fault-Tolerant Time-Varying Formation Control of Unmanned Airships With Limited Communication Ranges Against Input Saturation for Smart City Observation.

This article investigates the distributed fault-tolerant time-varying formation control problem for multiple unmanned airships (UAs) against limited communication ranges and input saturation to achieve the safe observation of a smart city. To address the strongly nonlinear functions caused by the time-varying formation flight with limited communication ranges and bias faults, intelligent adaptive learning mechanisms are proposed by incorporating fuzzy neural networks. Moreover, Nussbaum functions are introduced to handle the input saturation and loss-of-effectiveness faults. The distinct features of the proposed control scheme are that time-varying formation flight, actuator faults including bias and loss-of-effectiveness faults, limited communication ranges, and input saturation are simultaneously considered. It is proven by Lyapunov stability analysis that all UAs can achieve a safe formation flight for the smart city observation even in the presence of actuator faults. Hardware-in-the-loop experiments with open-source Pixhawk autopilots are conducted to show the effectiveness of the proposed control scheme.

Refined Fractional-Order Fault-Tolerant Coordinated Tracking Control of Networked Fixed-Wing UAVs Against Faults and Communication Delays via Double Recurrent Perturbation FNNs.

This article investigates the fault-tolerant coordinated tracking control problem for networked fixed-wing unmanned aerial vehicles (UAVs) against faults and communication delays. By supplementing the commonly used Gaussian functions in the fuzzy neural networks (FNNs) with sine-cosine functions and constructing two kinds of recurrent loops within the FNN architecture, double recurrent perturbation FNNs are cleverly designed to learn the unknown terms containing faults and uncertainties. Then, adaptive laws are designed for double recurrent perturbation FNNs. Moreover, by assimilating fractional-order calculus into the sliding-mode surfaces and the control signals, refined transient-state and steady-state adjustment performances can be obtained. It is shown by Lyapunov stability analysis that all fixed-wing UAVs can coordinately track their desired trajectories and the tracking errors are uniformly ultimately bounded. Comparative simulation results are provided to show the effectiveness of the proposed control strategy.

Promoting Butenyl-spinosyn Production Based on Omics Research and Metabolic Network Construction in Saccharopolyspora pogona.

Understanding the metabolism of Saccharopolyspora pogona on a global scale is essential for manipulating its metabolic capabilities to improve butenyl-spinosyn biosynthesis. Here, we combined multiomics analysis to parse S. pogona genomic information, construct a metabolic network, and mine important functional genes that affect the butenyl-spinosyn biosynthesis. This research not only elucidated the relationship between butenyl-spinosyn biosynthesis and the primary metabolic pathway but also showed that the low expression level and continuous downregulation of the bus cluster and the competitive utilization of acetyl-CoA were the main reasons for reduced butenyl-spinosyn production. Our framework identified 148 genes related to butenyl-spinosyn biosynthesis that were significantly differentially expressed, confirming that butenyl-spinosyn polyketide synthase (PKS) and succinic semialdehyde dehydrogenase (GabD) play an important role in regulating butenyl-spinosyn biosynthesis. Combined modification of these genes increased overall butenyl-spinosyn production by 6.38-fold to 154.1 ± 10.98 mg/L. Our results provide an important strategy for further promoting the butenyl-spinosyn titer.

The Global Regulator PhoU Positively Controls Growth and Butenyl-Spinosyn Biosynthesis in Saccharopolyspora pogona.

Butenyl-spinosyn, a highly effective biological insecticide, is produced by Saccharopolyspora pogona. However, its application has been severely hampered by its low yield. Recent studies have shown that PhoU plays a pivotal role in regulating cell growth, secondary metabolite biosynthesis and intracellular phosphate levels. Nevertheless, the function of PhoU remains ambiguous in S. pogona. In this study, we investigated the effects of PhoU on the growth and the butenyl-spinosyn biosynthesis of S. pogona by constructing the mutants. Overexpression of phoU increased the production of butenyl-spinosyn to 2.2-fold that of the wild-type strain. However, the phoU deletion resulted in a severe imbalance of intracellular phosphate levels, and suppression of the growth and butenyl-spinosyn biosynthesis. Quantitative Real-time PCR (qRT-PCR) analysis, distinctive protein detection and mass spectrometry revealed that PhoU widely regulated primary metabolism, energy metabolism and DNA repair, which implied that PhoU influences the growth and butenyl-spinosyn biosynthesis of S. pogona as a global regulator.