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1.
Biotechnol Lett ; 46(2): 161-172, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38279045

RESUMO

Actinomyces are gram-positive bacteria known for their valuable secondary metabolites. Redirecting metabolic flux towards desired products in actinomycetes requires precise and dynamic regulation of gene expression. In this study, we integrated the CRISPR interference (CRISPRi) system with a cumate-inducible promoter to develop an inducible gene downregulation method in Saccharopolyspora erythraea, a prominent erythromycin-producing actinobacterium. The functionality of the cumate-inducible promoter was validated using the gusA gene as a reporter, and the successful inducible expression of the dCas9 gene was confirmed. The developed inducible CRISPRi strategy was then employed to downregulate the expression of target genes rppA in the wild-type strain NRRL2338 and sucC in the high erythromycin-producing strain E3. Through dynamic control of sucC expression, a significant enhancement in erythromycin production was achieved in strain E3. This study demonstrated the effectiveness of an inducible gene downregulation approach using CRISPRi and a cumate-inducible promoter, providing valuable insights for optimizing natural product production in actinomyces.


Assuntos
Saccharopolyspora , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Eritromicina/metabolismo , Regiões Promotoras Genéticas/genética , Regulação da Expressão Gênica
2.
NPJ Biofilms Microbiomes ; 9(1): 65, 2023 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-37726290

RESUMO

Identification of the core functional microorganisms in food fermentations is necessary to understand the ecological and functional processes for making those foods. Wheat qu, which provides liquefaction and saccharifying power, and affects the flavor quality, is a key ingredient in ancient alcoholic huangjiu fermentation, while core microbiota of them still remains indistinct. In this study, metagenomics, metabolomics, microbial isolation and co-fermentation were used to investigate huangjiu. Although Aspergillus is usually regarded as core microorganism in wheat qu to initiate huangjiu fermentations, our metagenomic analysis showed that bacteria Saccharopolyspora are predominant in wheat qu and responsible for breakdown of starch and cellulose. Metabolic network and correlation analysis showed that Saccharopolyspora rectivirgula, Saccharopolyspora erythraea, and Saccharopolyspora hirsuta made the greatest contributions to huangjiu's metabolites, consisting of alcohols (phenylethanol, isoamylol and isobutanol), esters, amino acids (Pro, Arg, Glu and Ala) and organic acids (lactate, tartrate, acetate and citrate). S. hirsuta J2 isolated from wheat qu had the highest amylase, glucoamylase and protease activities. Co-fermentations of S. hirsuta J2 with S. cerevisiae HJ resulted in a higher fermentation rate and alcohol content, and huangjiu flavors were more similar to that of traditional huangjiu compared to co-fermentations of Aspergillus or Lactiplantibacillus with S. cerevisiae HJ. Genome of S. hirsuta J2 contained genes encoding biogenic amine degradation enzymes. By S. hirsuta J2 inoculation, biogenic amine content was reduced by 45%, 43% and 62% in huangjiu, sausage and soy sauce, respectively. These findings show the utility of Saccharopolyspora as a key functional organism in fermented food products.


Assuntos
Saccharopolyspora , Fermentação , Saccharopolyspora/genética , Saccharomyces cerevisiae , Aminoácidos , Celulose
3.
J Antibiot (Tokyo) ; 76(11): 658-664, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37596418

RESUMO

A novel actinobacterium, designated as strain WRP15-2T, was isolated from rhizosphere soil of rice plant (Oryza rufipogon). The strain was Gram-stain-positive, aerobic, and non-motile. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strain WRP15-2T fell into the genus Saccharopolyspora. The strain shared the highest 16S rRNA gene sequence similarity with the type strains Saccharopolyspora kobensis JCM 9109T (99.1%), Saccharopolyspora indica VRC122T (98.9%), and Saccharopolyspora antimicrobica DSM 45119T (98.7%). However, the digital DNA-DNA hybridization and average nucleotide identity values among these strains confirmed that the microorganism represented a novel member of the genus Saccharopolyspora. Chemotaxonomic data revealed that strain WRP15-2T possessed MK-9(H4) as the predominant menaquinone. It contained meso-diaminopimelic acid as the diagnostic diaminopimelic acid and arabinose, galactose, and ribose as predominant whole-cell sugars. The detected phospholipids were dominated by phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylmethylethanolamine, hydroxy-phosphatidylmethylethanolamine, and phosphatidylcholine. The predominant cellular fatty acids were iso-C16:0, C16:0, and iso-C15:0. The G + C content of the genomic DNA was 69.5%. Based on these genotypic and phenotypic data, it is supported that strain WRP15-2T represents a novel species of the genus Saccharopolyspora, for which the name Saccharopolyspora oryzae sp. nov. is proposed. The type strain is WRP15-2T ( = TBRC 15728T = NBRC 115560T).


Assuntos
Oryza , Saccharopolyspora , Fosfatidiletanolaminas , Saccharopolyspora/genética , Rizosfera , Filogenia , RNA Ribossômico 16S/genética , Ácido Diaminopimélico , DNA Bacteriano/genética , Ácidos Graxos , Análise de Sequência de DNA , Técnicas de Tipagem Bacteriana
4.
Bioprocess Biosyst Eng ; 46(9): 1303-1318, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37392219

RESUMO

In this study, the cellular metabolic mechanisms regarding ammonium sulfate supplementation on erythromycin production were investigated by employing targeted metabolomics and metabolic flux analysis. The results suggested that the addition of ammonium sulfate stimulates erythromycin biosynthesis. Targeted metabolomics analysis uncovered that the addition of ammonium sulfate during the late stage of fermentation resulted in an augmented intracellular amino acid metabolism pool, guaranteeing an ample supply of precursors for organic acids and coenzyme A-related compounds. Therefore, adequate precursors facilitated cellular maintenance and erythromycin biosynthesis. Subsequently, an optimal supplementation rate of 0.02 g/L/h was determined. The results exhibited that erythromycin titer (1311.1 µg/mL) and specific production rate (0.008 mmol/gDCW/h) were 101.3% and 41.0% higher than those of the process without ammonium sulfate supplementation, respectively. Moreover, the erythromycin A component proportion increased from 83.2% to 99.5%. Metabolic flux analysis revealed increased metabolic fluxes with the supplementation of three ammonium sulfate rates.


Assuntos
Saccharopolyspora , Saccharopolyspora/metabolismo , Sulfato de Amônio , Fermentação , Eritromicina/farmacologia , Suplementos Nutricionais
5.
Appl Microbiol Biotechnol ; 107(17): 5439-5451, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37428187

RESUMO

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.


Assuntos
Actinobacteria , Saccharopolyspora , Peróxido de Hidrogênio/metabolismo , Saccharopolyspora/metabolismo , Actinobacteria/metabolismo , Macrolídeos/metabolismo , Combinação de Medicamentos
6.
Nucleic Acids Res ; 51(13): 6870-6882, 2023 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-37283056

RESUMO

Actinobacteria are ubiquitous bacteria undergoing complex developmental transitions coinciding with antibiotic production in response to stress or nutrient starvation. This transition is mainly controlled by the interaction between the second messenger c-di-GMP and the master repressor BldD. To date, the upstream factors and the global signal networks that regulate these intriguing cell biological processes remain unknown. In Saccharopolyspora erythraea, we found that acetyl phosphate (AcP) accumulation resulting from environmental nitrogen stress participated in the regulation of BldD activity through cooperation with c-di-GMP. AcP-induced acetylation of BldD at K11 caused the BldD dimer to fall apart and dissociate from the target DNA and disrupted the signal transduction of c-di-GMP, thus governing both developmental transition and antibiotic production. Additionally, practical mutation of BldDK11R bypassing acetylation regulation could enhance the positive effect of BldD on antibiotic production. The study of AcP-dependent acetylation is usually confined to the control of enzyme activity. Our finding represents an entirely different role of the covalent modification caused by AcP, which integrated with c-di-GMP signal in modulating the activity of BldD for development and antibiotic production, coping with environmental stress. This coherent regulatory network might be widespread across actinobacteria, thus has broad implications.


Assuntos
Antibacterianos , Saccharopolyspora , Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Saccharopolyspora/metabolismo
7.
Gene ; 850: 146959, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36220451

RESUMO

The rapid development of biotechnology has provided new perspectives to observe and helped to gradually understand the significance of genetic instability in Actinobacteria. High frequency deletions of extremities and abnormal methylation of chromosomes suggest there might be relevant between the two phenomena. With this suspicion, we used single molecule real-time (SMRT) sequencing to map the genome-level methylation of one branch of actinomycetes, Saccharopolyspora erythraea, which have ring-shaped chromosomes. S. erythraea used for analysis in this study shares the same highly unstable phenotypic traits, as evidenced by diverse spore morphology and fluctuating erythromycin production. Multiple amplification of genomic islands closes to the replication initiation site and 6-methyladenine (m6A) deletion in genomic islands suggest that the interaction between the restriction modification (R-M) system and transposable elements provides an explanation for the division of labor by genomic heterogeneity in actinomycetes.


Assuntos
Actinobacteria , Saccharopolyspora , Actinobacteria/genética , Elementos de DNA Transponíveis , Saccharopolyspora/genética , Eritromicina/metabolismo , Cromossomos
8.
Biotechnol Appl Biochem ; 70(3): 1035-1043, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36479705

RESUMO

Spinosad, a combination of spinosyn A and D produced by Saccharopolyspora spinosa, is a highly efficient pesticide. There has been a considerable interest in the improvement of spinosad production because of a low yield achieved by wild-type S. spinosa. In this study, we designed and constructed a pIBR-SPN vector. pIBR-SPN is an integrative vector that can be used to introduce foreign genes into the chromosome of S. spinosa. Different combinations of genes encoding forasamine and rhamnose were synthesized and used for the construction of different recombinant plasmids. The following recombinant strains were developed: S. spinosa pIBR-SPN (only the vector), S. spinosa pIBR-SPN F (forosamine genes), S. spinosa pIBR-SPN R (rhamnose genes), S. spinosa pIBR-SPN FR (forosamine and rhamnose genes), S. spinosa pIBR-SPN FRS (forosamine, rhamnose, and SAM [S-adenosyl-L-methionine synthetase] genes), and S. spinosa MUV pIBR-SPN FR. Among these recombinant strains, S. spinosa pIBR-SPN FR produced 1394 ± 163 mg/L spinosad, which was 13-fold higher than the wild-type. S. spinosa MUV pIBR-SPN FR produced 1897 (±129) mg/L spinosad, which was seven-fold higher than S. spinosa MUV and 17-fold higher than the wild-type strain.


Assuntos
Engenharia Metabólica , Saccharopolyspora , Ramnose/metabolismo , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Combinação de Medicamentos
9.
Int J Mol Sci ; 23(24)2022 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-36555813

RESUMO

Steroids are abundant molecules in nature, and various microorganisms evolved to utilize steroids. Thermophilic actinobacteria play an important role in such processes. However, very few thermophiles have so far been reported capable of degrading or modifying natural sterols. Recently, genes putatively involved in the sterol catabolic pathway have been revealed in the moderately thermophilic actinobacterium Saccharopolyspora hirsuta VKM Ac-666T, but peculiarities of strain activity toward sterols are still poorly understood. S. hirsuta catalyzed cholesterol bioconversion at a rate significantly inferior to that observed for mesophilic actinobacteria (mycobacteria and rhodococci). Several genes related to different stages of steroid catabolism increased their expression in response to cholesterol as was shown by transcriptomic studies and verified by RT-qPCR. Sequential activation of genes related to the initial step of cholesterol side chain oxidation (cyp125) and later steps of steroid core degradation (kstD3, kshA, ipdF, and fadE30) was demonstrated for the first time. The activation correlates with a low cholesterol conversion rate and intermediate accumulation by the strain. The transcriptomic analyses revealed that the genes involved in sterol catabolism are linked functionally, but not transcriptionally. The results contribute to the knowledge on steroid catabolism in thermophilic actinobacteria and could be used at the engineering of microbial catalysts.


Assuntos
Actinobacteria , Fitosteróis , Saccharopolyspora , Esteróis/metabolismo , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Esteroides/metabolismo , Colesterol/metabolismo , Fitosteróis/metabolismo , Actinobacteria/genética , Actinobacteria/metabolismo
10.
Appl Microbiol Biotechnol ; 106(19-20): 6551-6566, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36075984

RESUMO

TetR family transcriptional regulators (TFRs) are widespread in actinomycetes, which exhibit diverse regulatory modes in antibiotic biosynthesis. Nitrogen regulators play vital roles in modulation of primary and secondary metabolism. However, crosstalk between TFR and nitrogen regulator has rarely been reported in actinomycetes. Herein, we demonstrated that a novel TFR, SACE_4839, was negatively correlated with erythromycin yield in Saccharopolyspora erythraea A226. SACE_4839 indirectly suppressed erythromycin synthetic gene eryAI and resistance gene ermE and directly inhibited its adjacent gene SACE_4838 encoding a homologue of nitrogen metabolite repression (NMR) regulator NmrA (herein named NmrR). The SACE_4839-binding sites within SACE_4839-nmrR intergenic region were identified. NmrR positively controlled erythromycin biosynthesis by indirectly stimulating eryAI and ermE and directly repressing SACE_4839. NmrR was found to affect growth viability under the nitrogen source supply. Furthermore, NmrR directly repressed glutamine and glutamate utilization-related genes SACE_1623, SACE_5070 and SACE_5979 but activated nitrate utilization-associated genes SACE_1163, SACE_4070 and SACE_4912 as well as nitrite utilization-associated genes SACE_1476 and SACE_4514. This is the first reported NmrA homolog for modulating antibiotic biosynthesis and nitrogen metabolism in actinomycetes. Moreover, combinatorial engineering of SACE_4839 and nmrR in the high-yield S. erythraea WB resulted in a 68.8% increase in erythromycin A production. This investigation deepens the understanding of complicated regulatory network for erythromycin biosynthesis. KEY POINTS: • SACE_4839 and NmrR had opposite contributions to erythromycin biosynthesis. • NmrR was first identified as a homolog of another nitrogen regulator NmrA. • Cross regulation between SACE_4839 and NmrR was revealed.


Assuntos
Actinobacteria , Saccharopolyspora , Actinobacteria/genética , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Intergênico , Eritromicina , Glutamatos/metabolismo , Glutamina/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Nitrogênio/metabolismo , Saccharopolyspora/metabolismo
11.
Am J Physiol Lung Cell Mol Physiol ; 323(3): L329-L337, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35881171

RESUMO

Previously we have shown that a gain-of-function MUC5B promoter variant (rs35705950) is the strongest risk factor for the development of idiopathic pulmonary fibrosis. We have also found that Muc5b overexpression reduces mucociliary clearance in mice, potentially leading to recurrent injury to the bronchoalveolar epithelia. Hypersensitivity pneumonitis (HP) is induced by inhalation of numerous causative antigens that may be affected by mucociliary clearance. We conducted this study to determine the role of Muc5b in a mouse model of HP induced by Saccharopolyspora rectivirgula (SR) antigen. We used Muc5b-deficient and wild-type (WT) mice to determine whether Muc5b plays a role in inflammation and fibrosis at 3 and 6 wk in an SR model of HP. We measured cell concentrations and MUC5B expression in whole lung lavage (WLL) and quantified fibrosis using hydroxyproline assay and second harmonic generation. Muc5b expression in WLL fluid was significantly increased in SR-exposed WT mice compared with saline controls. WT mice challenged with SR developed more inflammation and lung fibrosis at 6 wk compared with 3 wk postexposure. Moreover, we found that 6 wk following challenge with SR, Muc5b-deficient mice had less lung inflammation and less lung fibrosis than Muc5b WT mice. Furthermore, Muc5b-deficient mice had significantly lower concentrations of TGF-ß1 in the WLL compared with Muc5b WT mice at 6 wk of exposure. Muc5b appears to play a role in fibrosis in the animal model of HP and this may have implications for HP in humans.


Assuntos
Alveolite Alérgica Extrínseca , Fibrose Pulmonar Idiopática , Saccharopolyspora , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Humanos , Fibrose Pulmonar Idiopática/genética , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Mucina-5B/genética
12.
Appl Microbiol Biotechnol ; 106(13-16): 5153-5165, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35821431

RESUMO

As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metabolism regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3ΔsucC was identified by using a high-resolution mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metabolism, and antibiotics biosynthesis in S. erythraea. Enzymatic experiments in vivo and in vitro were further conducted to determine the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3ΔsucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermentation. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metabolism changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sg1 was increased by 54.7% compared with E3, which was 1200.5 mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea. KEY POINTS: • We reported the first systematic profiling of the S. erythraea succinylome. • We found that the succinylation regulation on the activity of aconitase. • We enhanced the production of erythromycin by using CRISPRi to regulate the transcription of gene sucC.


Assuntos
Eritromicina , Saccharopolyspora , Aconitato Hidratase/genética , Aconitato Hidratase/metabolismo , Acil Coenzima A , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ligases/genética , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Saccharopolyspora/genética , Saccharopolyspora/metabolismo
13.
Arch Microbiol ; 204(7): 371, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35670849

RESUMO

A novel Gram-stain positive, aerobic, non-motile actinobacterium, designated strain K220T, was isolated from soil collected from Cape Andreas (Zafer Burnu), Northern Cyprus, and subjected to a polyphasic taxonomic approach. The organism was shown to have phylogenetic, chemotaxonomic, cultural and morphological properties consistent with its classification in the genus Saccharopolyspora. 16S rRNA gene sequence analysis of strain K220T showed that it is closely related to the type strains of Saccharopolyspora maritima 3SS5-12 T, Saccharopolyspora kobensis JCM 9109 T and Saccharopolyspora hirsuta ATCC 27875 T with 97.6, 97.5 and 97.0% sequence similarity, respectively. In silico DNA-DNA hybridization and average nucleotide identity values between strain K220T and type strains of the genus Saccharopolyspora with publicly available genomes were 22.1-31.2% and 76.0-83.16%, respectively. The DNA G + C content of strain K220T was 68.3 mol%. The genome of strain K220T has genes associated with 24 biosynthetic gene clusters. The strain contained MK-9(H4) and iso-C16: 0 as the predominant respiratory quinone and fatty acid, respectively. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine. Based on evidence collected from the genotypic, phenotypic and phylogenetic analyses, strain K220T is considered to represent a novel species in the genus Saccharopolyspora, for which the name Saccharopolyspora soli sp. nov. is proposed. The type strain is K220T (= JCM 33912T = KCTC 49395T).


Assuntos
Saccharopolyspora , Técnicas de Tipagem Bacteriana , Chipre , DNA Bacteriano/genética , Ácidos Graxos , Fosfolipídeos , Filogenia , RNA Ribossômico 16S/genética , Saccharopolyspora/genética , Análise de Sequência de DNA , Solo , Microbiologia do Solo
14.
J Sci Food Agric ; 102(15): 7301-7312, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35757866

RESUMO

BACKGROUND: Mechanized Huangjiu is a stable product, is not subject to seasonal production restrictions, and markedly reduces labor intensity compared to traditional manual Huangjiu. However, the bitterness of mechanized Huangjiu impedes its further development. RESULTS: Based on process optimization, when the fermentation temperature was 45 °C and the fermentation time was 122 h, the inoculation amount of Saccharopolyspora was 5%, the amount of added water was 26%, and the glucoamylase and amylase activities of wheat Qu increased by 27% and 40% respectively, compared with those before optimization. Huangjiu fermented by raw wheat Qu inoculated with Saccharopolyspora rosea F2014 showed a significant (P < 0.05) decrease in bitter amino acid content (1.24 vs. 2.86 g L-1 , a decrease of 56%), which attenuated its bitterness. CONCLUSION: An innovative fermentation process of inoculating Saccharopolyspora into raw wheat Qu was developed for the first time. Such a process could be used to control bitterness based on raw wheat Qu inoculated with Saccharopolyspora rosea F2014, instead of traditional wheat Qu in Huangjiu fermentation. © 2022 Society of Chemical Industry.


Assuntos
Saccharopolyspora , Saccharopolyspora/metabolismo , Fermentação , Glucana 1,4-alfa-Glucosidase/metabolismo , Paladar
15.
Microb Cell Fact ; 21(1): 120, 2022 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-35717184

RESUMO

BACKGROUND: Erythromycin A (Er A) has a broad antibacterial effect and is a source of erythromycin derivatives. Methylation of erythromycin C (Er C), catalyzed by S-adenosyl-methionine (SAM)-dependent O-methyltransferase EryG, is the key final step in Er A biosynthesis. Er A biosynthesis, including EryG production, is regulated by the phosphate response factor PhoP and the nitrogen response factor GlnR. However, the regulatory effect of these proteins upon S-adenosyl-methionine synthetase (MetK) production is unknown. RESULTS: In this study, we used bioinformatics approaches to identify metK (SACE_3900), which codes for S-adenosyl-methionine synthetase (MetK). Electrophoretic mobility shift assays (EMSAs) revealed that PhoP and GlnR directly interact with the promoter of metK, and quantitative PCR (RT-qPCR) confirmed that each protein positively regulated metK transcription. Moreover, intracellular SAM was increased upon overexpression of either phoP or glnR under phosphate or nitrogen limited conditions, respectively. Finally, both the production of Er A and the transformation ratio from Er C to Er A increased upon phoP overexpression, but surprisingly, not upon glnR overexpression. CONCLUSIONS: Manipulating the phosphate and nitrogen response factors, PhoP and GlnR provides a novel strategy for increasing the yield of SAM and the production of Er A in Saccharopolyspora erythraea .


Assuntos
Saccharopolyspora , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eritromicina , Regulação Bacteriana da Expressão Gênica , Metionina/metabolismo , Nitrogênio/metabolismo , Fosfatos/metabolismo , S-Adenosilmetionina/metabolismo , Saccharopolyspora/genética , Saccharopolyspora/metabolismo
16.
Microb Cell Fact ; 21(1): 83, 2022 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-35568948

RESUMO

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.


Assuntos
Proteômica , Saccharopolyspora , Transativadores/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Glucose/metabolismo , Macrolídeos/metabolismo
17.
ACS Synth Biol ; 11(8): 2697-2708, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35561342

RESUMO

Actinomycetes are versatile secondary metabolite producers with great application potential in industries. However, industrial strain engineering has long been limited by the inefficient and labor-consuming plate/flask-based screening process, resulting in an urgent need for product-driven high-throughput screening methods for actinomycetes. Here, we combine a whole-cell biosensor and microfluidic platform to establish the whole-cell biosensor and producer co-cultivation-based microfluidic platform for screening actinomycetes (WELCOME). In WELCOME, we develop an MphR-based Escherichia coli whole-cell biosensor sensitive to erythromycin and co-cultivate it with Saccharopolyspora erythraea in droplets for high-throughput screening. Using WELCOME, we successfully screen out six erythromycin hyper-producing S. erythraea strains starting from an already high-producing industrial strain within 3 months, and the best one represents a 50% improved yield. WELCOME completely circumvents a major problem of industrial actinomycetes, which is usually genetic-intractable, and this method will revolutionize the field of industrial actinomycete engineering.


Assuntos
Técnicas Biossensoriais , Saccharopolyspora , Proteínas de Bactérias/metabolismo , Eritromicina , Escherichia coli/genética , Escherichia coli/metabolismo , Saccharopolyspora/genética , Saccharopolyspora/metabolismo
18.
Appl Microbiol Biotechnol ; 106(8): 3081-3091, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35376972

RESUMO

PII signal transduction proteins are widely found in bacteria and plant chloroplast, and play a central role in nitrogen metabolism regulation, which interact with many key proteins in metabolic pathways to regulate carbon/nitrogen balance by sensing changes in concentrations of cell-mediated indicators such as α-ketoglutarate. In this study, the knockout strain Saccharopolyspora pogona-ΔpII and overexpression strain S. pogona-pII were constructed using CRISPR/Cas9 technology and the shuttle vector POJ260, respectively, to investigate the effects on the growth and secondary metabolite biosynthesis of S. pogona. Growth curve, electron microscopy, and spore germination experiments were performed, and it was found that the deletion of the pII gene inhibited the growth to a certain extent in the mutant. HPLC analysis showed that the yield of butenyl-spinosyn in the S. pogona-pII strain increased to 245% than that in the wild-type strain while that in S. pogona-ΔpII decreased by approximately 51%. This result showed that the pII gene can promote the growth and butenyl-spinosyn biosynthesis of S. pogona. This research first investigated PII nitrogen metabolism regulators in S. pogona, providing significant scientific evidence and a research basis for elucidating the mechanism by which these factors regulate the growth of S. pogona, optimizing the synthesis network of butenyl-spinosyn and constructing a strain with a high butenyl-spinosyn yield. KEY POINTS: • pII key nitrogen regulatory gene deletion can inhibit the growth and development of S. pogona. • Overexpressed pII gene can significantly promote the butenyl-spinosyn biosynthesis. • pII gene can affect the amino acid circulation and the accumulation of butenyl-spinosyn precursors in S. pogona.


Assuntos
Nitrogênio , Saccharopolyspora , Proteínas de Bactérias/genética , Genes Reguladores , Macrolídeos/metabolismo , Nitrogênio/metabolismo , Saccharopolyspora/metabolismo
19.
Immunohorizons ; 6(3): 224-242, 2022 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-35273098

RESUMO

Protein kinase D1 (PKD1), a ubiquitously expressed serine/threonine kinase, regulates diverse cellular processes such as oxidative stress, gene expression, cell survival, vesicle trafficking, Ag receptor signaling, and pattern recognition receptor signaling. We found previously that exposure to hypersensitivity pneumonitis (HP) inciting Ag Saccharopolyspora rectivirgula leads to the activation of PKD1 in a MyD88-dependent manner in various types of murine cells in vitro and in the mouse lung in vivo. However, it is currently unknown whether PKD1 plays a role in the S. rectivirgula-induced HP. In this study, we investigated contributions of PKD1 on the S. rectivirgula-induced HP using conditional PKD1-insufficient mice. Compared to control PKD1-sufficient mice, PKD1-insufficient mice showed substantially suppressed activation of MAPKs and NF-κB, expression of cytokines and chemokines, and neutrophilic alveolitis after single intranasal exposure to S. rectivirgula The significantly reduced levels of alveolitis, MHC class II surface expression on neutrophils and macrophages, and IL-17A and CXCL9 expression in lung tissue were observed in the PKD1-insufficient mice repeatedly exposed to S. rectivirgula for 5 wk. PKD1-insuficient mice exposed to S. rectivirgula for 5 wk also showed reduced granuloma formation. Our results demonstrate that PKD1 plays an essential role in the initial proinflammatory responses and neutrophil influx in the lung after exposure to S. rectivirgula and substantially contribute to the development of HP caused by repeated exposure to S. rectivirgula Our findings suggest that PKD1 can be an attractive new molecular target for therapy of S. rectivirgula-induced HP.


Assuntos
Alveolite Alérgica Extrínseca , Pneumonia , Proteína Quinase C/metabolismo , Alveolite Alérgica Extrínseca/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/induzido quimicamente , Proteínas Quinases , Saccharopolyspora
20.
J Agric Food Chem ; 70(11): 3557-3567, 2022 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-35245059

RESUMO

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.


Assuntos
Macrolídeos , Saccharopolyspora , Proteínas de Bactérias/metabolismo , Macrolídeos/metabolismo , Redes e Vias Metabólicas/genética , Saccharopolyspora/genética , Saccharopolyspora/metabolismo
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