scholarly journals Optimization of tetramycin production in Streptomyces ahygroscopicus S91

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Guang Chen ◽  
Mengqiu Wang ◽  
Xianpu Ni ◽  
Huanzhang Xia
Keyword(s):  
Cytochrome P450 ◽  
Pathogenic Fungi ◽  
Biosynthetic Gene Cluster ◽  
High Yield ◽  
Original Strain ◽  
Biosynthetic Gene ◽  
Yield Strain ◽  
P450 Monooxygenase ◽  
Most Significant Change ◽  
Final Strain

Abstract Background Tetramycin is a 26-member tetraene antibiotic used in agriculture. It has two components, tetramycin A and tetramycin B. Tetramycin B is obtained by the hydroxylation of tetramycin A on C4. This reaction is catalyzed by the cytochrome P450 monooxygenase TtmD. The two components of tetramycin have different antifungal activities against different pathogenic fungi. Therefore, the respective construction of high-yield strains of tetramycin A and tetramycin B is conducive to more targeted action on pathomycete and has a certain practical value. Results Streptomyces ahygroscopicus S91 was used as the original strain to construct tetramycin A high-yield strains by blocking the precursor competitive biosynthetic gene cluster, disrupting tetramycin B biosynthesis, and overexpressing the tetramycin pathway regulator. Eventually, the yield of tetramycin A in the final strain was up to 1090.49 ± 136.65 mg·L− 1. Subsequently, TtmD, which catalyzes the conversion from tetramycin A to tetramycin B, was overexpressed. Strains with 2, 3, and 4 copies of ttmD were constructed. The three strains had different drops in tetramycin A yield, with increases in tetramycin B. The strain with three copies of ttmD showed the most significant change in the ratio of the two components. Conclusions A tetramycin A single-component producing strain was obtained, and the production of tetramycin A increased 236.84% ± 38.96% compared with the original strain. In addition, the content of tetramycin B in a high-yield strain with three copies of ttmD increased from 26.64% ± 1.97 to 51.63% ± 2.06%.

scholarly journals Comparative genomics and transcriptomics analyses provide insights into the high yield and regulatory mechanism of Norvancomycin biosynthesis in Amycolatopsis orientalis NCPC 2-48

2020 ◽  
Author(s):  
Xingxing Li ◽  
Cong Zhang ◽  
Ying Zhao ◽  
Xuan Lei ◽  
Zhibo Jiang ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Regulatory Mechanism ◽  
Biosynthetic Gene Cluster ◽  
High Yield ◽  
Scale Production ◽  
Biosynthetic Gene ◽  
Yield Strain ◽  
Biosynthetic Genes ◽  
Methicillin Resistant ◽  
Amycolatopsis Orientalis

Abstract Background: Norvancomycin has been widely used in clinic to treat against MRSA (Methicillin-resistant Staphylococcus aureus) and MRSE (methicillin-resistant Staphylococcus epidermidis) in China. Amycolatopsis orientalis NCPC 2–48, a high yield strain derived from A. orientalis CPCC 200066, has been applied in industrial large-scale production of norvancomycin by North China Pharmaceutical Group. However, the potential high-yield and regulatory mechanism involved in norvancomycin biosynthetic pathway has not yet been addressed.Results: Here we sequenced and compared the genomes and transcriptomes of A. orientalis CPCC 200066 and NCPC 2–48. These two genomes are extremely similar with an identity of more than 99.9%, and no duplication and structural variation was found in the norvancomycin biosynthetic gene cluster. Comparative transcriptomic analysis indicated that biosynthetic gene cluster of norvancomycin, as well as some primary metabolite pathways for the biosynthetic precursors of norvancomycin were generally upregulated. AoStrR1 and AoLuxR1, two cluster-situated regulatory genes in norvancomycin cluster, were 23.3-fold and 5.8-fold upregulated in the high yield strain at 48 h, respectively. Over-expression of AoStrR1 and AoLuxR1 in CPCC 200066 resulted in an increase of norvancomycin production, indicating their positive role in norvancomycin biosynthesis. Furthermore, AoStrR1 can regulate the production of norvancomycin by directly interacting with at least 8 promoters of norvancomycin biosynthetic genes or operons.Conclusion: Our results suggested that the mechanism of high yield of NCPC 2–48 can be ascribed to increased expression level of norvancomycin biosynthetic genes in its cluster as well as the genes responsible for the supply of its precursors. And the norvancomycin biosynthetic genes are positively regulated by AoStrR1 and AoLuxR1, of them AoStrR1 was the ultimate pathway-specific regulator for the norvancomycin production. These results are helpful for further clarification of the holistic and pathway-specific regulatory mechanism of norvancomycin biosynthesis in the industrial production strain.

scholarly journals Characterization of two cytochrome P450 monooxygenase genes of the pyripyropene biosynthetic gene cluster from Penicillium coprobium

2011 ◽  
Vol 64 (3) ◽  
pp. 221-227 ◽  
Author(s):  
Jie Hu ◽  
Hiroto Okawa ◽  
Kentaro Yamamoto ◽  
Kazuhiko Oyama ◽  
Masaaki Mitomi ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Cytochrome P450 Monooxygenase ◽  
Biosynthetic Gene ◽  
P450 Monooxygenase

scholarly journals Cytochrome P450 Monooxygenase CYP139 Family Involved in the Synthesis of Secondary Metabolites in 824 Mycobacterial Species

2019 ◽  
Vol 20 (11) ◽  
pp. 2690 ◽  
Author(s):  
Puleng Rosinah Syed ◽  
Wanping Chen ◽  
David R. Nelson ◽  
Abidemi Paul Kappo ◽  
Jae-Hyuk Yu ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Secondary Metabolites ◽  
Biosynthetic Gene Cluster ◽  
Cytochrome P450 Monooxygenase ◽  
Mycobacterial Species ◽  
P450 Monooxygenase ◽  
Physiological Capacity ◽  
Unique Amino Acid

Tuberculosis (TB) is one of the top infectious diseases causing numerous human deaths in the world. Despite enormous efforts, the physiology of the causative agent, Mycobacterium tuberculosis, is poorly understood. To contribute to better understanding the physiological capacity of these microbes, we have carried out extensive in silico analyses of the 1111 mycobacterial species genomes focusing on revealing the role of the orphan cytochrome P450 monooxygenase (CYP) CYP139 family. We have found that CYP139 members are present in 894 species belonging to three mycobacterial groups: M. tuberculosis complex (850-species), Mycobacterium avium complex (34-species), and non-tuberculosis mycobacteria (10-species), with all CYP139 members belonging to the subfamily “A”. CYP139 members have unique amino acid patterns at the CXG motif. Amino acid conservation analysis placed this family in the 8th among CYP families belonging to different biological domains and kingdoms. Biosynthetic gene cluster analyses have revealed that 92% of CYP139As might be associated with producing different secondary metabolites. Such enhanced secondary metabolic potentials with the involvement of CYP139A members might have provided mycobacterial species with advantageous traits in diverse niches competing with other microbial or viral agents, and might help these microbes infect hosts by interfering with the hosts’ metabolism and immune system.

scholarly journals Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Mark J. Lee ◽  
Alexander M. Geller ◽  
Natalie C. Bamford ◽  
Hong Liu ◽  
Fabrice N. Gravelat ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Fungal Pathogens ◽  
Molecular Mechanisms ◽  
Pathogenic Fungi ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Bacterial Biofilm ◽  
Invasive Infection ◽  
Biosynthetic Gene ◽  
Adhesive Properties

ABSTRACTThe moldAspergillus fumigatuscauses invasive infection in immunocompromised patients. Recently, galactosaminogalactan (GAG), an exopolysaccharide composed of galactose andN-acetylgalactosamine (GalNAc), was identified as a virulence factor required for biofilm formation. The molecular mechanisms underlying GAG biosynthesis and GAG-mediated biofilm formation were unknown. We identified a cluster of five coregulated genes that were dysregulated in GAG-deficient mutants and whose gene products share functional similarity with proteins that mediate the synthesis of the bacterial biofilm exopolysaccharide poly-(β1-6)-N-acetyl-d-glucosamine (PNAG). Bioinformatic analyses suggested that the GAG cluster geneagd3encodes a protein containing a deacetylase domain. Because deacetylation ofN-acetylglucosamine residues is critical for the function of PNAG, we investigated the role of GAG deacetylation in fungal biofilm formation. Agd3 was found to mediate deacetylation of GalNAc residues within GAG and render the polysaccharide polycationic. As with PNAG, deacetylation is required for the adherence of GAG to hyphae and for biofilm formation. Growth of the Δagd3mutant in the presence of culture supernatants of the GAG-deficient Δuge3mutant rescued the biofilm defect of the Δagd3mutant and restored the adhesive properties of GAG, suggesting that deacetylation is an extracellular process. The GAG biosynthetic gene cluster is present in the genomes of members of thePezizomycotinasubphylum of theAscomycotaincluding a number of plant-pathogenic fungi and a single basidiomycete species,Trichosporon asahii, likely a result of recent horizontal gene transfer. The current study demonstrates that the production of cationic, deacetylated exopolysaccharides is a strategy used by both fungi and bacteria for biofilm formation.IMPORTANCEThis study sheds light on the biosynthetic pathways governing the synthesis of galactosaminogalactan (GAG), which plays a key role inA. fumigatusvirulence and biofilm formation. We find that bacteria and fungi use similar strategies to synthesize adhesive biofilm exopolysaccharides. The presence of orthologs of the GAG biosynthetic gene clusters in multiple fungi suggests that this exopolysaccharide may also be important in the virulence of other fungal pathogens. Further, these studies establish a molecular mechanism of adhesion in which GAG interacts via charge-charge interactions to bind to both fungal hyphae and other substrates. Finally, the importance of deacetylation in the synthesis of functional GAG and the extracellular localization of this process suggest that inhibition of deacetylation may be an attractive target for the development of novel antifungal therapies.

scholarly journals Heterologous Biosynthesis and Genomics-Driven Derivatization of Fungal Bioactive Sesterterpenoid Variecolin

2021 ◽  
Author(s):  
Dexiu Yan ◽  
Jemma Arakelyan ◽  
Teng Wan ◽  
Tsz Ki Chan ◽  
Dohyun Ahn ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Anticancer Activity ◽  
Gene Cluster ◽  
Aspergillus Oryzae ◽  
Biosynthetic Gene Cluster ◽  
Heterologous Production ◽  
Biosynthetic Gene ◽  
Aspergillus Aculeatus ◽  
Heterologous Biosynthesis

The biosynthetic gene cluster of fungal bioactive sesterterpenoids, variecolin (1) and variecolactone (2), was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues, one of which exhibited potent anticancer activity comparable to that of 1.

scholarly journals RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Chen Li ◽  
Xinqiang Liu ◽  
Chao Lei ◽  
Han Yan ◽  
Zhihui Shao ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
Biosynthetic Gene Cluster ◽  
High Yield ◽  
Biosynthetic Gene ◽  
Amycolatopsis Mediterranei ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type

ABSTRACT Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae. Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathway-specific regulator for the rif cluster. IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.

scholarly journals The plmS2-Encoded Cytochrome P450 Monooxygenase Mediates Hydroxylation of Phoslactomycin B in Streptomyces sp. Strain HK803

2005 ◽  
Vol 187 (23) ◽  
pp. 7970-7976 ◽  
Author(s):  
Mohini S. Ghatge ◽  
Kevin A. Reynolds
Keyword(s):  
Cytochrome P450 ◽  
Biosynthetic Pathway ◽  
Sequence Similarity ◽  
Biosynthetic Gene Cluster ◽  
Conjugative Plasmid ◽  
Cytochrome P450 Monooxygenases ◽  
Phosphate Ester ◽  
High Sequence Similarity ◽  
P450 Monooxygenase ◽  
Complementation Experiment

ABSTRACT Streptomyces sp. strain HK803 produces six analogues of phoslactomycin (Plm A through Plm F). With the exception of Plm B, these analogues contain a C-18 hydroxyl substituent esterified with a range of short-alkyl-chain carboxylic acids. Deletion of the plmS 2 open reading frame (ORF), showing high sequence similarity to bacterial cytochrome P450 monooxygenases (CYPs), from the Plm biosynthetic gene cluster has previously resulted in an NP1 mutant producing only Plm B (N. Palaniappan, B. S. Kim, Y. Sekiyama, H. Osada, and K. A. Reynolds, J. Biol. Chem. 278:35552-35557, 2003). Herein, we report that a complementation experiment with an NP1 derivative (NP2), using a recombinant conjugative plasmid carrying the plmS 2 ORF downstream of the ermE* constitutive promoter (pMSG1), restored production of Plm A and Plm C through Plm F. The 1.2-kbp plmS 2 ORF was also expressed efficiently as an N-terminal polyhistidine-tagged protein in Streptomyces coelicolor. The recombinant PlmS2 converted Plm B to C-18-hydroxy Plm B (Plm G). PlmS2 was highly specific for Plm B and unable to process a series of derivatives in which either the lactone ring was hydrolyzed or the C-9 phosphate ester was converted to C-9/C-11 phosphorinane. This biochemical analysis and complementation experiment are consistent with a proposed Plm biosynthetic pathway in which the penultimate step is hydroxylation of the cyclohexanecarboxylic acid-derived side chain of Plm B by PlmS2 (the resulting Plm G is then esterified to provide Plm A and Plm C through Plm F). Kinetic parameters for Plm B hydroxylation by PlmS2 (Km of 45.3 ± 9.0 μM and k cat of 0.27 ± 0.04 s−1) are consistent with this step being a rate-limiting step in the biosynthetic pathway. The penultimate pathway intermediate Plm G has less antifungal activity than Plm A through Plm F and is not observed in fermentations of either the wild-type strain or NP2/pMSG1.

Sign in / Sign up

Export Citation Format

Share Document