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

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.

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

10.21203/rs.3.rs-42758/v3 ◽

2021 ◽

Author(s):

Xingxing Li ◽

Cong Zhang ◽

Ying Zhao ◽

Xuan Lei ◽

Zhibo Jiang ◽

...

Keyword(s):

Large Scale ◽

Regulatory Mechanism ◽

Biosynthetic Gene Cluster ◽

High Yield ◽

Scale Production ◽

Yield Strain ◽

Biosynthetic Genes ◽

Primary Metabolite ◽

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) infections 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 genes 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 roles 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 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. The norvancomycin biosynthetic genes are presumably regulated by AoStrR1 and AoLuxR1, of them AoStrR1 is possibly 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.

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

10.21203/rs.3.rs-42758/v2 ◽

2021 ◽

Author(s):

Xingxing Li ◽

Cong Zhang ◽

Ying Zhao ◽

Xuan Lei ◽

Zhibo Jiang ◽

...

Keyword(s):

Large Scale ◽

Regulatory Mechanism ◽

Biosynthetic Gene Cluster ◽

High Yield ◽

Scale Production ◽

Yield Strain ◽

Biosynthetic Genes ◽

Primary Metabolite ◽

Methicillin Resistant ◽

Amycolatopsis Orientalis

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

Microbial Cell Factories ◽

10.1186/s12934-021-01521-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Xingxing Li ◽

Cong Zhang ◽

Ying Zhao ◽

Xuan Lei ◽

Zhibo Jiang ◽

...

Keyword(s):

Large Scale ◽

Regulatory Mechanism ◽

Biosynthetic Gene Cluster ◽

High Yield ◽

Scale Production ◽

Yield Strain ◽

Biosynthetic Genes ◽

Primary Metabolite ◽

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) infections 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 genes 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 roles 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 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. The norvancomycin biosynthetic genes are presumably regulated by AoStrR1 and AoLuxR1, of them AoStrR1 is possibly 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.

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Refactoring the formicamycin biosynthetic gene cluster to make high-level producing strains and new molecules

10.1101/2020.09.15.275776 ◽

2020 ◽

Cited By ~ 1

Author(s):

Rebecca Devine ◽

Hannah McDonald ◽

Zhiwei Qin ◽

Corinne Arnold ◽

Katie Noble ◽

...

Keyword(s):

Gene Cluster ◽

Large Scale ◽

Liquid Culture ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Resistant Bacteria ◽

Drug Resistant ◽

Biosynthetic Gene ◽

Biosynthetic Genes

AbstractThe formicamycins are promising antibiotics with potent activity against Gram-positive pathogens including VRE and MRSA and display a high barrier to selection of resistant isolates. They were first identified in Streptomyces formicae KY5, which produces the formicamycins at low levels on solid agar but not in liquid culture, thus hindering further investigation of these promising antibacterial compounds. We hypothesised that by understanding the organisation and regulation of the for biosynthetic gene cluster, we could rationally refactor the cluster to increase production levels. Here we report that the for biosynthetic gene cluster consists of 24 genes expressed on nine transcripts. Seven of these transcripts, including those containing all the major biosynthetic genes, are repressed by the MarR-regulator ForJ which also controls the expression of the ForGF two-component system that initiates biosynthesis. A third cluster-situated regulator, ForZ, autoregulates and controls production of the putative MFS transporter ForAA. Consistent with these findings, deletion of forJ increased formicamycin biosynthesis 5-fold, while over-expression of forGF in the ΔforJ background increased production 10-fold compared to the wild-type. De-repression by deleting forJ also switched on biosynthesis in liquid-culture and induced the production of two novel formicamycin congeners. By combining mutations in regulatory and biosynthetic genes, six new biosynthetic precursors with antibacterial activity were also isolated. This work demonstrates the power of synthetic biology for the rational redesign of antibiotic biosynthetic gene clusters both to engineer strains suitable for fermentation in large scale bioreactors and to generate new molecules.ImportanceAntimicrobial resistance is a growing threat as existing antibiotics become increasingly ineffective against drug resistant pathogens. Here we determine the transcriptional organisation and regulation of the gene cluster encoding biosynthesis of the formicamycins, promising new antibiotics with activity against drug resistant bacteria. By exploiting this knowledge, we construct stable mutant strains which over-produce these molecules in both liquid and solid culture whilst also making some new compound variants. This will facilitate large scale purification of these molecules for further study including in vivo experiments and the elucidation of their mechanism of action. Our work demonstrates that understanding the regulation of natural product biosynthetic pathways can enable rational improvement of the producing strains.

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Hierarchical control of virginiamycin production in Streptomyces virginiae by three pathway-specific regulators: VmsS, VmsT and VmsR

Microbiology ◽

10.1099/mic.0.022467-0 ◽

2009 ◽

Vol 155 (4) ◽

pp. 1250-1259 ◽

Cited By ~ 23

Author(s):

Nattika Pulsawat ◽

Shigeru Kitani ◽

Eriko Fukushima ◽

Takuya Nihira

Keyword(s):

Gene Cluster ◽

Response Regulator ◽

Expression Profiles ◽

Regulatory Protein ◽

Regulatory Region ◽

Biosynthetic Gene Cluster ◽

Transcriptional Analysis ◽

Biosynthetic Gene ◽

Biosynthetic Genes ◽

Streptomyces Virginiae

Two regulatory genes encoding a Streptomyces antibiotic regulatory protein (vmsS) and a response regulator (vmsT) of a bacterial two-component signal transduction system are present in the left-hand region of the biosynthetic gene cluster of the antibiotic virginiamycin, which is composed of virginiamycin M (VM) and virginiamycin S (VS), in Streptomyces virginiae. Disruption of vmsS abolished both VM and VS biosynthesis, with drastic alteration of the transcriptional profile for virginiamycin biosynthetic genes, whereas disruption of vmsT resulted in only a loss of VM biosynthesis, suggesting that vmsS is a pathway-specific regulator for both VM and VS biosynthesis, and that vmsT is a pathway-specific regulator for VM biosynthesis alone. Gene expression profiles determined by semiquantitative RT-PCR on the virginiamycin biosynthetic gene cluster demonstrated that vmsS controls the biosynthetic genes for VM and VS, and vmsT controls unidentified gene(s) of VM biosynthesis located outside the biosynthetic gene cluster. In addition, transcriptional analysis of a deletion mutant of vmsR located in the clustered regulatory region in the virginiamycin cluster (and which also acts as a SARP-family activator for both VM and VS biosynthesis) indicated that the expression of vmsS and vmsT is under the control of vmsR, and vmsR also contributes to the expression of VM and VS biosynthetic genes, independent of vmsS and vmsT. Therefore, coordinated virginiamycin biosynthesis is controlled by three pathway-specific regulators which hierarchically control the expression of the biosynthetic gene cluster.

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

Applied and Environmental Microbiology ◽

10.1128/aem.03201-16 ◽

2017 ◽

Vol 83 (8) ◽

Cited By ~ 4

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.

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Characterization of a Gene Cluster Responsible for the Biosynthesis of Anticancer Agent FK228 in Chromobacterium violaceum No. 968

Applied and Environmental Microbiology ◽

10.1128/aem.01751-06 ◽

2007 ◽

Vol 73 (11) ◽

pp. 3460-3469 ◽

Cited By ~ 38

Author(s):

Yi-Qiang Cheng ◽

Min Yang ◽

Andrea M. Matter

Keyword(s):

Gene Cluster ◽

Genomic Dna ◽

Polyketide Synthase ◽

Anticancer Agent ◽

Biosynthetic Gene Cluster ◽

Nonribosomal Peptide Synthetase ◽

Biosynthetic Gene ◽

Biosynthetic Genes ◽

Nonribosomal Peptide ◽

Peptide Synthetase

ABSTRACT A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotide-dependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates.

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