Effects of a Pirin-like protein on strain growth and spinosad biosynthesis in Saccharopolyspora spinosa.

Pirin family proteins perform a variety of biological functions and widely exist in all living organisms. A few studies have shown that Pirin family proteins may be involved in the biosynthesis of antibiotics in actinomycetes. However, the function of Pirin-like proteins in S. spinosa is still unclear. In this study, the inactivation of the sspirin gene led to serious growth defects and the accumulation of H2O2. Surprisingly, the overexpression and knockout of sspirin slightly accelerated the consumption and utilization of glucose, weakened the TCA cycle, delayed sporulation, and enhanced sporulation in the later stage. In addition, the overexpression of sspirin can enhance the ß-oxidation pathway and increase the yield of spinosad by 0.88 times, while the inactivation of sspirin hardly produced spinosad. After adding MnCl2, the spinosad yield of the sspirin overexpression strain was further increased to 2.5 times that of the wild-type strain. This study preliminarily revealed the effects of Pirin-like proteins on the growth development and metabolism of S. spinosa and further expanded knowledge of Pirin-like proteins in actinomycetes. KEY POINTS: • Overexpression of the sspirin gene possibly triggers carbon catabolite repression (CCR) • Overexpression of the sspirin gene can promote the synthesis of spinosad • Knockout of the sspirin gene leads to serious growth and spinosad production defects.

Screening of new Paenibacillus polymyxa S3 and its disease resistance of grass carp (Ctenopharyngodon idellus).

A new strain of Paenibacillus polymyxa S3 with antagonistic effects on 11 major fish pathogens (especially Aeromonas hydrophila), but had no toxicity to grass carp, was screened from the sediment of fishponds. In vivo colonization studies showed that strain S3 could be colonized and distributed in the gill and abdomen of the grass carp. Bioassay results showed that the weight growth rate of grass carp in the strain S3 oral group (16.01%) and strain S3 immersion group (16.44%) was significantly higher than those of the control group (8.61%). At the same time, the activities of ACP, AKP, CAT and GSH-Px in the serum of grass carp in oral and immersion groups were significantly higher than those of the control group. In addition, the treatment with strain S3 could significantly upregulate the expression of the antioxidant-related genes and immune-related genes Keap1, Nrf2, C3, LZM, IgM, TLR-4 and MyD-88 in grass carp tissues. The challenge test showed that strain S3 treatment significantly increased the survival rate of grass carp infected with Aeromonas hydrophila. Whole genome sequencing analysis showed that strain S3 had 16 active metabolite gene clusters, indicating that it had abundant gene resources, which provided important support for its development for fish microecological preparations. In summary, a new strain of Paenibacillus polymyxa S3 with antibacterial activity against a variety of fish pathogens was screened in this study and its probiotic function was evaluated, proving its potential value in fisheries.

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.

Aeromonas veronii infection remarkably increases expression of lysozymes in grass carp (Ctenopharyngodon idellus) and injection of lysozyme expression cassette along with QCDC adjuvant significantly upregulates immune factors and decreases cumulative mortality.

Aeromonas veronii AvX005 is a pathogenic bacterium with high toxicity to grass carp (Ctenopharyngodon idellus). The expression levels of g-type (goose-type lysozyme, Lys-g) and c-type lysozyme (chicken-type lysozyme, Lys-c) in the spleen of grass carp infected with AvX005 were significantly increased by approximately 4.5 times and 27 times, respectively. The recombinant proteins rLys-g and rLys-c produced in a recombinant expression system of Escherichia coli showed significant antibacterial activity against the pathogenic bacteria AvX005. A challenge test was conducted after rLys-g and rLys-c were expressed in grass carp L8824 liver cells, and compared with the survival rate of the control cells (46.3%), the survival rate of the experimental cells (77.6% for rLys-g and 68.6% for rLys-c) was significantly increased. Grass carp were infected with AvX005 on the second day after delivering pcDNA3.1-lys-g and pcDNA-lys-c with the Quil A/cholesterol/DDA/Carbopol (QCDC) adjuvant, and both pcDNA3.1-lys-g and pcDNA-lys-c provided 70% relative protection for grass carp. The activity of lysozyme and alkaline phosphatase in the serum of grass carp was significantly increased after injection of DNA. The expression of the immune factors IgM, C3 and IL8 in the kidney was upregulated to varying degrees for pcDNA3.1-lys-g and immune factors C3 and IgM was upregulated for pcDNA-lys-c. The results indicated that pcDNA3.1-lys-g and pcDNA-lys-c may be used as immunostimulants to protect grass carp from the pathogenic bacterium AvX005.

A TetR family transcriptional regulator, SP_2854 can affect the butenyl-spinosyn biosynthesis by regulating glucose metabolism in Saccharopolyspora pogona.

BACKGROUND: Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involve in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficiently understanding its regulatory mechanism, and improving its production by metabolic engineering. RESULTS: Here, we identified a TetR family transcriptional regulator, SP_2854, that can positively regulate butenyl-spinosyn biosynthesis and affect strain growth, glucose consumption, and mycelial morphology in S. pogona. Using targeted metabolomic analyses, we found that SP_2854 overexpression enhanced glucose metabolism, while SP_2854 deletion had the opposite effect. To decipher the overproduction mechanism in detail, comparative proteomic analysis was carried out in the SP-2854 overexpressing mutant and the original strain, and we found that SP_2854 overexpression promoted the expression of proteins involved in glucose metabolism. CONCLUSION: Our findings suggest that SP_2854 can affect strain growth and development and butenyl-spinosyn biosynthesis in S. pogona by controlling glucose metabolism. The strategy reported here will be valuable in paving the way for genetic engineering of regulatory elements in actinomycetes to improve important natural products production.

Pathogenicity of fish pathogen Pseudomonas plecoglossicida and preparation of its inactivated vaccine.

Many fishes infected with Pseudomonas plecoglossicida generally suffer from "visceral white spot disease" or even die. In this study, a dominant pathogen strain was isolated from the intestinal tract of diseased crucian carp in the Wangcheng Lake area, Changsha, and it was identified as P. plecoglossicida. The selected strain was a new strain named as P. plecoglossicida LQJ06.Strain LQJ06 basically colonized the intestine and poisoned zebrafish as show by fluorescent labelling. Pathological structural analysis of tissue sections indicated that the intestinal tract was seriously damaged, epithelial cells in the intestinal tissue were necrotic, intestinal villi were sloughed, liver cells were vacuolated, nuclei were pyknotic and shifted, and lymphocytes were proliferated in the spleen. P. plecoglossicida LQJ06 strain could invade and proliferate in the grass carp liver cell line L8824, which led to a stress response, including apoptosis. Cell morphology was changed owing to the toxicity of the culture supernatant of the LQJ06 strain, which mainly manifested as aggregation between cells, pyknosisd and slow growth or even death. An inactivated vaccine derived from P. plecoglossicida LQJ06 prepared in this study was safe and nontoxic to grass carp liver cells. Compared with those after oral administration, most of the cellular immune factors were expressed earlier and at a higher level after injection immunization. The intestinal tract and liver from zebrafish mainly expressed the IFN-γ2 and IL-1ß genes, respectively, after immunization. The upregulation of these immune-related genes proved that the vaccine could strengthen the immunity of zebrafish, induce inflammation and promote resistance to pathogenic infection. The results of these preliminary tests provide a scientific basis for further research on the prevention and control of P. plecoglossicida, and an essential preliminary basis for the development of an inactivated vaccine against P. plecoglossicida.

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.

Flaviolin-Like Gene Cluster Deletion Optimized the Butenyl-Spinosyn Biosynthesis Route in Saccharopolyspora pogona.

Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes. Saccharopolyspora pogona can produce butenyl-spinosyn during aerobic fermentation, and its genome contains 32 different gene clusters. This suggests that there is a large amount of potential competitive metabolism in S. pogona, which affects the biosynthesis of butenyl-spinosyn. By analyzing the genome of S. pogona, six polyketide gene clusters were identified. From those, the complete deletion of clu13, a flaviolin-like gene cluster, generated a high butenyl-spinosyn-producing strain. Production of this strain was 4.06-fold higher than that of the wildtype strain. Transcriptome profiling revealed that butenyl-spinosyn biosynthesis was not primarily induced by the polyketide synthase RppA-like but was related to hypothetical protein Sp1764. However, the repression of sp1764 was not enough to explain the enormous enhancement of butenyl-spinosyn yields in S. pogona-Δclu13. After the comparative proteomic analysis of S. pogona-Δclu13 and S. pogona, two proteins, biotin carboxyl carrier protein (BccA) and response regulator (Reg), were investigated, whose overexpression led to great advantages of butenyl-spinosyn biosynthesis. In this way, we successfully discovered three key genes that obviously optimize the biosynthesis of butenyl-spinosyn. Gene cluster simplification performed in conjunction with multiomics analysis is of great practical significance for screening dominant chassis strains and optimizing secondary metabolism. This work provided an idea about screening key factors and efficient construction of production strains.

Comparative Proteomics Reveals the Effect of the Transcriptional Regulator Sp13016 on Butenyl-Spinosyn Biosynthesis in Saccharopolyspora pogona.

Butenyl-spinosyn is a highly effective and broad-spectrum biopesticide produced by Saccharopolyspora pogona. However, the yield of this compound is difficult to increase because the regulatory mechanism of secondary metabolism is still unknown. Here, the transcriptional regulator Sp13016 was discovered to be highly associated with butenyl-spinosyn synthesis and bacterial growth. Overexpression of sp13016 improved butenyl-spinosyn production to a level that was 2.84-fold that of the original strain, while deletion of sp13016 resulted in a significant decrease in yield and growth inhibition. Comparative proteomics revealed that these phenotypic changes were attributed to the influence of Sp13016 on the central carbon metabolism pathway to regulate the supply of precursors. Our research helps to reveal the regulatory mechanism of butenyl-spinosyn biosynthesis and provides a reference for increasing the yield of natural products of Actinomycetes.

Bacterioferritin: a key iron storage modulator that affects strain growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona.

BACKGROUND: Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. RESULTS: Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. CONCLUSION: Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes.

Effects of acuC on the growth development and spinosad biosynthesis of Saccharopolyspora spinosa.

BACKGROUND: Acetoin utilization protein (acuC) is a type I histone deacetylase which is highly conserved in bacteria. The acuC gene is related to the acetylation/deacetylation posttranslational modification (PTM) system in S. spinosa. Spinosyns, the secondary metabolites produced by Saccharopolyspora spinosa, are the active ingredients in a family of insect control agents. However, the specific functions and influences of acuC protein in S. spinosa are yet to be characterized. RESULTS: The knockout strain and overexpression strain were constructed separately with the shuttle vector pOJ260. The production of spinosyns A and D from S. spinosa-acuC were 105.02 mg/L and 20.63 mg/L, which were 1.82-fold and 1.63-fold higher than those of the wild-type strain (57.76 mg/L and 12.64 mg/L), respectively. The production of spinosyns A and D from S. spinosa-ΔacuC were 32.78 mg/L and 10.89 mg/L, respectively. The qRT-PCR results of three selected genes (bldD, ssgA and whiA) confirmed that the overexpression of acuC affected the capacities of mycelial differentiation and sporulation. Comparative proteomics analysis was performed on these strains to investigate the underlying mechanism leading to the enhancement of spinosad yield. CONCLUSIONS: This study first systematically analysed the effects of overexpression acuC on the growth of S. spinosa and the production of spinosad. The results identify the differentially expressed proteins and provide evidences to understand the acetylation metabolic mechanisms which can lead to the increase of secondary metabolites.

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.

Effects of lytS-L on the primary metabolism and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona.

The LytTR family two-component system widely exists in bacterial cells and plays an important role in metabolic regulation. The lytS-L gene that encodes for a LytTR family sensor kinase was knocked out to study its influence on the growth, phenotype, and the biosynthesis of the insecticidal polyketide butenyl-spinosyn in Saccharopolyspora pogona NRRL 30141 (S. pogona). High performance liquid chromatography (HPLC) results showed that the butenyl-spinosyn yield of the lytS-L knockout mutant decreased by 58.9% compared with that of the parental strain. This is manifested by a weak toxicity of the mutant against the insect Helicoverpa assulta (H. armigera). Comparative proteomic analysis revealed the expression characteristics of the proteins in S. pogona and S. pogona-ΔlytS-L: a total of 14 proteins involved in energy metabolism were down-regulated, 9 proteins related to carbon metabolism such as glycolysis, and tricarboxylic acid cycle (TCA) were up-regulated, while 13 proteins involved in the biosynthesis of butenyl-spinosyn were down-regulated (fold change >1.2 or< 0.83). The qRT-PCR (Quantitative Real-time PCR) analysis illustrated that the changes in the expression levels of transcription and translation of the identified genes were consistent. This study explores the function of the two-component system of the LytTR family in S. pogona and shows that the lytS-L gene has an important influence on regulating primary metabolism and butenyl-spinosyn biosynthesis of S. pogona.

Deletion of a hybrid NRPS-T1PKS biosynthetic gene cluster via Latour gene knockout system in Saccharopolyspora pogona and its effect on butenyl-spinosyn biosynthesis and growth development.

Butenyl-spinosyn, a promising biopesticide produced by Saccharopolyspora pogona, exhibits stronger insecticidal activity and a broader pesticidal spectrum. However, its titre in the wild-type S. pogona strain is too low to meet the industrial production requirements. Deletion of non-target natural product biosynthetic gene clusters resident in the genome of S. pogona could reduce the consumption of synthetic precursors, thereby promoting the biosynthesis of butenyl-spinosyn. However, it has always been a challenge for scientists to genetically engineer S. pogona. In this study, the Latour gene knockout system (linear DNA fragment recombineering system) was established in S. pogona. Using the Latour system, a hybrid NRPS-T1PKS cluster (˜20 kb) which was responsible for phthoxazolin biosynthesis was efficiently deleted in S. pogona. The resultant mutant S. pogona-Δura4-Δc14 exhibited an extended logarithmic phase, increased biomass and a lower glucose consumption rate. Importantly, the production of butenyl-spinosyn in S. pogona-Δura4-Δc14 was increased by 4.72-fold compared with that in the wild-type strain. qRT-PCR analysis revealed that phthoxazolin biosynthetic gene cluster deletion could promote the expression of the butenyl-spinosyn biosynthetic gene cluster. Furthermore, a TetR family transcriptional regulatory gene that could regulate the butenyl-spinosyn biosynthesis has been identified from the phthoxazolin biosynthetic gene cluster. Because dozens of natural product biosynthetic gene clusters exist in the genome of S. pogona, the strategy reported here will be used to further promote the production of butenyl-spinosyn by deleting other secondary metabolite synthetic gene clusters.

SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora pogona.

Butenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

Deciphering the Metabolic Pathway Difference Between Saccharopolyspora pogona and Saccharopolyspora spinosa by Comparative Proteomics and Metabonomics.

Butenyl-spinosyn, a secondary metabolite produced by Saccharopolyspora pogona, exhibits strong insecticidal activity than spinosyn. However, the low synthesis capacity and unknown metabolic characteristics of butenyl-spinosyn in wild-type S. pogona limit its broad application and metabolic engineering. Here, we showed that S. pogona exhibited increased glucose consumption ability and growth rate compared with S. spinosa, but the production of butenyl-spinosyn was much lower than that of spinosyn. To further elucidate the metabolic mechanism of these different phenotypes, we performed a comparative proteomic and metabolomic study on S. pogona and S. spinosa to identify the change in the abundance levels of proteins and metabolites. We found that the abundance of most proteins and metabolites associated with glucose transport, fatty acid metabolism, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine and pyrimidine metabolism, and target product biosynthesis in S. pogona was higher than that in S. spinosa. However, the overall abundance of proteins involved in butenyl-spinosyn biosynthesis was much lower than that of the high-abundance protein chaperonin GroEL, such as the enzymes related to rhamnose synthesis. We speculated that these protein and metabolite abundance changes may be directly responsible for the above phenotypic changes in S. pogona and S. spinosa, especially affecting butenyl-spinosyn biosynthesis. Further studies revealed that the over-expression of the rhamnose synthetic genes and methionine adenosyltransferase gene could effectively improve the production of butenyl-spinosyn by 2.69- and 3.03-fold, respectively, confirming the reliability of this conjecture. This work presents the first comparative proteomics and metabolomics study of S. pogona and S. spinosa, providing new insights into the novel links of phenotypic change and metabolic difference between two strains. The result will be valuable in designing strategies to promote the biosynthesis of butenyl-spinosyn by metabolic engineering.

Effect of the TetR family transcriptional regulator Sp1418 on the global metabolic network of Saccharopolyspora pogona.

BACKGROUND: Saccharopolyspora pogona is a prominent industrial strain due to its production of butenyl-spinosyn, a high-quality insecticide against a broad spectrum of insect pests. TetR family proteins are diverse in a tremendous number of microorganisms and some are been researched to have a key role in metabolic regulation. However, specific functions of TetR family proteins in S. pogona are yet to characterize. RESULTS: In the present study, the overexpression of the tetR-like gene sp1418 in S. pogona resulted in marked effects on vegetative growth, sporulation, butenyl-spinosyn biosynthesis, and oxidative stress. By using qRT-PCR analysis, mass spectrometry, enzyme activity detection, and sp1418 knockout verification, we showed that most of these effects could be attributed to the overexpression of Sp1418, which modulated enzymes related to the primary metabolism, oxidative stress and secondary metabolism, and thereby resulted in distinct growth characteristics and an unbalanced supply of precursor monomers for butenyl-spinosyn biosynthesis. CONCLUSION: This study revealed the function of Sp1418 and enhanced the understanding of the metabolic network in S. pogona, and provided insights into the improvement of secondary metabolite production.

[Effect of fcl gene for butenyl-spinosyn biosynthesis and growth of Saccharopolyspora pogona].

The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.

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.

[Effect of ribosome engineering on butenyl-spinosyns synthesis of Saccharopolyspora pogona].

Through introducing mutations into ribosomes by obtaining spontaneous drug resistance of microorganisms, ribosome engineering technology is an effective approach to develop mutant strains that overproduce secondary metabolites. In this study, ribosome engineering was used to improve the yield of butenyl-spinosyns produced by Saccharopolyspora pogona by screening streptomycin resistant mutants. The yields of butenyl-spinosyns were then analyzed and compared with the parent strain. Among the mutants, S13 displayed the greatest increase in the yield of butenyl-spinosyns, which was 1.79 fold higher than that in the parent strain. Further analysis of the metabolite profile of S13 by mass spectrometry lead to the discovery of Spinosyn α1, which was absent from the parent strain. DNA sequencing showed that there existed two point mutations in the conserved regions of rpsL gene which encodes ribosomal protein S12 in S13. The mutations occurred a C to A and a C to T transversion mutations occurred at nucleotide pair 314 and 320 respectively, which resulted in the mutations of Proline (105) to Gultamine and Alanine (107) to Valine. It also demonstrated that S13 exhibited genetic stability even after five passages.