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Richard H Baltz - One of the best experts on this subject based on the ideXlab platform.

  • Development of a Genetic System for Combinatorial Biosynthesis of Lipopeptides in Streptomyces fradiae and Heterologous Expression of the A54145 Biosynthesis Gene Cluster
    Applied and environmental microbiology, 2010
    Co-Authors: Dylan C. Alexander, Vivian Miao, Paul Brian, Jessica Rock, Richard H Baltz
    Abstract:

    A54145 factors are calcium-dependent lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18160. A54145 is structurally related to the clinically important daptomycin, and as such may be a useful scaffold for the development of a novel lipopeptide antibiotic. We developed methods to genetically manipulate S. fradiae by deletion mutagenesis and conjugal transfer of plasmids from Escherichia coli. Cloning the complete pathway on a bacterial artificial chromosome (BAC) vector and the construction of ectopic trans-complementation with plasmids utilizing the φC31 or φBT1 site-specific integration system allowed manipulation of A54145 biosynthesis. The BAC clone pDA2002 was shown to harbor the complete A54145 biosynthesis gene cluster by heterologous expression in Streptomyces ambofaciens and Streptomyces Roseosporus strains in yields of >100 mg/liter. S. fradiae mutants defective in LptI methyltransferase function were constructed, and they produced only A54145 factors containing glutamic acid (Glu₁₂), at the expense of factors containing 3-methyl-glutamic acid (3mGlu₁₂). This provided a practical route to produce high levels of pure Glu₁₂-containing lipopeptides. A suite of mutant strains and plasmids was created for combinatorial biosynthesis efforts focused on modifying the A54145 peptide backbone to generate a compound with daptomycin antibacterial activity and activity in Streptococcus pneumoniae pulmonary infections.

  • Genomics and the ancient origins of the daptomycin biosynthetic gene cluster
    The Journal of antibiotics, 2010
    Co-Authors: Richard H Baltz
    Abstract:

    Daptomycin is a clinically useful lipopeptide antibiotic produced by Streptomyces Roseosporus. The antibiotic is assembled by a nonribosomal peptide synthetase (NRPS) mechanism, and the cyclized tridecapeptide contains three non-proteinogenic-amino acids: ornithine (Orn), 3-methyl-glutamic acid (3mGlu) and kynurinine (Kyn). Daptomycin also has three D-amino acids. The two genes that encode proteins involved in the formation of 3mGlu and Kyn have no known orthologs, and hence they are good probes to search for daptomycin-like gene clusters among newly sequenced actinomycete genomes. A recent search with these genes revealed a daptomycin-like gene cluster in Saccharomonospora viridis, a causative agent for farmer's lung disease. S. viridis has a genome of 4.3 Mb, which is about one half the size of Streptomyces genomes. Searches for other NRPS and polyketide synthase (PKS) genes revealed only one other NRPS gene and no PKS genes in S. viridis. The orthologous lipopeptide biosynthetic genes and gene products in S. viridis and S. Roseosporus have diverged extensively from a last common ancestor dating back to a pathway that had evolved over 1 billion years ago.

  • Chapter 20. Biosynthesis and genetic engineering of lipopeptides in Streptomyces Roseosporus.
    Methods in enzymology, 2009
    Co-Authors: Richard H Baltz
    Abstract:

    Daptomycin is an acidic cyclic lipopeptide antibiotic approved for treatment of infections caused by Gram-positive pathogens, including Staphylococcus aureus strains resistant to other antibiotics. Daptomycin biosynthesis is carried out by a giant multisubunit, multienzyme nonribosomal peptide synthetase (NRPS). The daptomycin (dpt) biosynthetic genes have been cloned in a bacterial artificial chromosome (BAC) vector, sequenced, and expressed in Streptomyces lividans. Several of the dpt genes, including the three NRPS genes, are transcribed as a lengthy polycistronic message. The daptomycin-producing strain, Streptomyces Roseosporus, can be genetically manipulated, and a number of deletion mutants encompassing one or more of the dpt genes have been constructed. Several of the dpt genes have been expressed from ectopic chromosomal loci (varphiC31 or IS117 attB sites) under the transcriptional control of the strong constitutive ermEp* promoter, and recombinant strains produced high levels of lipopeptides, thus establishing a trans-complementation system for combinatorial biosynthesis. A number of hybrid NRPS subunits have been generated by lambda-Red-mediated recombination, and combinatorial libraries of lipopeptides have been generated by NRPS subunit exchanges, module exchanges, multidomain exchanges, deletion mutagenesis, and multiple natural lipidations, using the ectopic trans-complementation system in S. Roseosporus.

  • Non-ribosomal peptide synthetase module fusions to produce derivatives of daptomycin in Streptomyces Roseosporus.
    Microbiology (Reading England), 2008
    Co-Authors: Sascha Doekel, Richard H Baltz, Min Chu, Marie-françoise Coëffet-le Gal, Paul Brian
    Abstract:

    Genetic engineering has been applied to reprogramme non-ribosomal peptide synthetases (NRPSs) to produce novel antibiotics, but little is known about what determines the efficiency of production. We explored module exchanges at nucleotide sequences encoding interpeptide linkers in dptD, a gene encoding a di-modular NRPS subunit that incorporates 3-methylglutamic acid (3mGlu(12)) and kynurenine (Kyn(13)) into daptomycin. Mutations causing amino acid substitutions, deletions or insertions in the inter-module linker had no negative effects on lipopeptide yields. Hybrid DptD subunits were generated by fusing the 3mGlu(12) module to terminal modules from calcium-dependent antibiotic (CDA) or A54145 NRPSs, and recombinants produced daptomycin analogues with Trp(13) or Ile(13) at high efficiencies. A recombinant expressing DptD with a hybrid Kyn(13) module containing a di-domain from a d-Asn module caused the production of a new daptomycin analogue containing Asn(13).

  • Structural characterization of daptomycin analogues A21978C1-3(d-Asn11) produced by a recombinant Streptomyces Roseosporus strain.
    Journal of natural products, 2007
    Co-Authors: Kien T. Nguyen, Richard H Baltz, Paul Brian, Chhayal Gandhi, Vineet B. Rajgarhia, Min Chu
    Abstract:

    Three daptomycin-related lipopeptides, A21978C1-3(d-Asn11) (2-4), were purified from the fermentation broth of a recombinant Streptomyces Roseosporus strain. Their chemical structures were determined by analyses of the biosynthetic pathway, chemical transformations, d,l-amino acid quantitation by enantiomer labeling, tandem LC-MS/MS, and 2D-NMR techniques. Compounds 2-4 exhibited potent antibacterial activity against Staphylococcus aureus with MIC values of 0.6, 0.3, and 0.15 microM, respectively, well correlated to the acyl tail chain length.

Xuming Mao - One of the best experts on this subject based on the ideXlab platform.

  • crotonylation of key metabolic enzymes regulates carbon catabolite repression in Streptomyces Roseosporus
    Communications Biology, 2020
    Co-Authors: Chenfan Sun, Qingwei Zhao, Shuai Luo, Xinai Chen, Xuming Mao
    Abstract:

    Due to the plethora natural products made by Streptomyces, the regulation of its metabolism are of great interest, whereas there is a lack of detailed understanding of the role of posttranslational modifications (PTM) beyond traditional transcriptional regulation. Herein with Streptomyces Roseosporus as a model, we showed that crotonylation is widespread on key enzymes for various metabolic pathways, and sufficient crotonylation in primary metabolism and timely elimination in secondary metabolism are required for proper Streptomyces metabolism. Particularly, the glucose kinase Glk, a keyplayer of carbon catabolite repression (CCR) regulating bacterial metabolism, is identified reversibly crotonylated by the decrotonylase CobB and the crotonyl-transferase Kct1 to negatively control its activity. Furthermore, crotonylation positively regulates CCR for Streptomyces metabolism through modulation of the ratio of glucose uptake/Glk activity and utilization of carbon sources. Thus, our results revealed a regulatory mechanism that crotonylation globally regulates Streptomyces metabolism at least through positive modulation of CCR. Chen-Fan Sun et al. show that key enzymes in several metabolic pathways are crotonylated in Streptomyces Roseosporus. This study suggests that crotonylation increases carbon catabolite repression by increasing glucose uptake while reducing the activity of glucose kinase.

  • Crotonylation of key metabolic enzymes regulates carbon catabolite repression in Streptomyces Roseosporus.
    Communications biology, 2020
    Co-Authors: Chenfan Sun, Qingwei Zhao, Shuai Luo, Xinai Chen, Xuming Mao
    Abstract:

    Due to the plethora natural products made by Streptomyces, the regulation of its metabolism are of great interest, whereas there is a lack of detailed understanding of the role of posttranslational modifications (PTM) beyond traditional transcriptional regulation. Herein with Streptomyces Roseosporus as a model, we showed that crotonylation is widespread on key enzymes for various metabolic pathways, and sufficient crotonylation in primary metabolism and timely elimination in secondary metabolism are required for proper Streptomyces metabolism. Particularly, the glucose kinase Glk, a keyplayer of carbon catabolite repression (CCR) regulating bacterial metabolism, is identified reversibly crotonylated by the decrotonylase CobB and the crotonyl-transferase Kct1 to negatively control its activity. Furthermore, crotonylation positively regulates CCR for Streptomyces metabolism through modulation of the ratio of glucose uptake/Glk activity and utilization of carbon sources. Thus, our results revealed a regulatory mechanism that crotonylation globally regulates Streptomyces metabolism at least through positive modulation of CCR.

  • Dual regulation between the two-component system PhoRP and AdpA regulates antibiotic production in Streptomyces
    Journal of industrial microbiology & biotechnology, 2019
    Co-Authors: Yang Zheng, Chenfan Sun, Xinai Chen, Xuming Mao
    Abstract:

    Antibiotic production during secondary metabolism in Streptomyces spp. is elaborately controlled by multiple environmental signals and intracellular cascades. These include the two-component system PhoRP responding to phosphate starvation and a conserved signaling pathway mediated by the pleiotropic regulator AdpA. However, little information exists about how these two pathways work together for secondary metabolite production of Streptomyces. Herein, we report the dual regulation from the phosphate starvation-responsive regulator PhoP and AdpA on atrA promoter (atrAp) for the production of daptomycin, an antibiotic produced by Streptomyces Roseosporus. We found that PhoP directly binds to atrAp, positively regulates atrA expression and thus daptomycin production. We also observed positive auto-regulation of phoRP expression during fermentation for daptomycin production. Moreover, partial overlap between PhoP- and AdpA-binding sites on atrAp was observed, which results in partial competitive binding between these two regulators. This partial overlapping and competition between PhoP and AdpA was further confirmed by mutations and binding assays. In summary, our findings have revealed dual regulation of PhoP and AdpA on the same promoter for antibiotic production in Streptomyces. This mechanism would be beneficial to further environment-responsive fermentation optimization for antibiotic production.

  • Transposon-based identification of a negative regulator for the antibiotic hyper-production in Streptomyces.
    Applied microbiology and biotechnology, 2018
    Co-Authors: Shuai Luo, Xinai Chen, Xuming Mao
    Abstract:

    Production of secondary metabolites in Streptomyces is regulated by a complex regulatory network precisely, elaborately, and hierarchically. One of the main reasons for the low yields of some high-value secondary metabolites is the repressed expression of their biosynthetic gene clusters, supposedly by some gene cluster out-situated negative regulators. Identification of these repressors and removal of the inhibitory effects based on the regulatory mechanisms will be an effective way to improve their yields. For proof of the concept, using an antibiotic daptomycin from Streptomyces Roseosporus, we introduced Himar1-based random mutagenesis combined with a reporter-guided screening strategy to identify a transcriptional regulator PhaR, whose loss-of-function deletion led to about 2.68-fold increase of the gene cluster expression and approximately 6.14-fold or 43% increased daptomycin production in the flask fermentation or in the fed-batch fermentation, respectively. Further study showed that PhaR negatively regulates the expression of daptomycin biosynthetic gene cluster by direct binding to its promoter (dptEp). Moreover, phaR expression gradually drops down during fermentation, and PhaR is positively auto-regulated by directly binding to its own promoter, which results in positive feedback regulation to persistently reduce phaR expression. Meanwhile, the declining PhaR protein remove its repressive effects during daptomycin production. All these results support that our strategy would be a powerful method for genetic screening and rational engineering for the yield improvement of antibiotics, and could be potentially used widely in other Streptomyces species.

  • Regulatory and biosynthetic effects of the bkd gene clusters on the production of daptomycin and its analogs A21978C1-3.
    Journal of industrial microbiology & biotechnology, 2018
    Co-Authors: Shuai Luo, Xinai Chen, Xuming Mao
    Abstract:

    Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces Roseosporus in an acidic peptide complex A21978C. In this complex, A21978C1–3 is most abundant and contains branched-chain fatty acyl groups, while daptomycin has a straight decanoic acyl group. The branched-chain α-keto acid dehydrogenase complex (BCDH complex), encoded by bkd gene clusters in Streptomyces, is responsible for the early step of converting branched-chain amino acids into branched-chain fatty acids. In a daptomycin industrial producer S. Roseosporus L30, two alleles of bkd gene clusters, bkdA1B1C1/bkdA2B2C2, and a regulatory gene bkdR located upstream of bkdA2B2C2 are identified. We show that BkdR positively regulated bkdA2B2C2 expression and was negatively auto-regulated, but is not directly involved in regulation of daptomycin gene cluster expression. However, BkdR is required for both daptomycin and A21978C1–3 production. Furthermore, deletion of bkdA2B2C2 only led to partial reduction of A21978C1–3 production, while the ΔbkdA1B1C1 mutant shows very weak production of A21978C1–3, and the double bkd mutant has a similar production profile as the single ΔbkdA1B1C1 mutant, suggesting that bkdA1B1C1 gene cluster plays a dominant role in branched-chain fatty acid biosynthesis. So we reveal a unique regulatory function of BkdR and genetic engineered a bkd null strain for daptomycin production with reduced impurities.

Pieter C Dorrestein - One of the best experts on this subject based on the ideXlab platform.

  • Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A
    Proceedings of the National Academy of Sciences, 2014
    Co-Authors: Kazuya Yamanaka, Roland D. Kersten, Katherine S. Ryan, David J. Gonzalez, Kirk A Reynolds, Victor Nizet, Pieter C Dorrestein, Bradley S. Moore
    Abstract:

    Recent developments in next-generation sequencing technologies have brought recognition of microbial genomes as a rich resource for novel natural product discovery. However, owing to the scarcity of efficient procedures to connect genes to molecules, only a small fraction of secondary metabolomes have been investigated to date. Transformation-associated recombination (TAR) cloning takes advantage of the natural in vivo homologous recombination of Saccharomyces cerevisiae to directly capture large genomic loci. Here we report a TAR-based genetic platform that allows us to directly clone, refactor, and heterologously express a silent biosynthetic pathway to yield a new antibiotic. With this method, which involves regulatory gene remodeling, we successfully expressed a 67-kb nonribosomal peptide synthetase biosynthetic gene cluster from the marine actinomycete Saccharomonospora sp. CNQ-490 and produced the dichlorinated lipopeptide antibiotic taromycin A in the model expression host Streptomyces coelicolor. The taromycin gene cluster (tar) is highly similar to the clinically approved antibiotic daptomycin from Streptomyces Roseosporus, but has notable structural differences in three amino acid residues and the lipid side chain. With the activation of the tar gene cluster and production of taromycin A, this study highlights a unique "plug-and-play" approach to efficiently gaining access to orphan pathways that may open avenues for novel natural product discoveries and drug development.

  • imaging mass spectrometry and genome mining via short sequence tagging identified the anti infective agent arylomycin in Streptomyces Roseosporus
    Journal of the American Chemical Society, 2011
    Co-Authors: Wei Ting Liu, Roland D. Kersten, Bradley S. Moore, Yu Liang Yang, Pieter C Dorrestein
    Abstract:

    Here, we described the discovery of anti-infective agent arylomycin and its biosynthetic gene cluster in an industrial daptomycin producing strain Streptomyces Roseosporus. This was accomplished via the use of MALDI imaging mass spectrometry (IMS) along with peptidogenomic approach in which we have expanded to short sequence tagging (SST) described herein. Using IMS, we observed that prior to the production of daptomycin, a cluster of ions (1–3) was produced by S. Roseosporus and correlated well with the decreased staphylococcal cell growth. With a further adopted SST peptidogenomics approach, which relies on the generation of sequence tags from tandem mass spectrometric data and query against genomes to identify the biosynthetic genes, we were able to identify these three molecules (1–3) to arylomycins, a class of broad-spectrum antibiotics that target type I signal peptidase. The gene cluster was then identified. This highlights the strength of IMS and MS guided genome mining approaches in effectively b...

  • Imaging mass spectrometry and genome mining via short sequence tagging identified the anti-infective agent arylomycin in Streptomyces Roseosporus
    Journal of the American Chemical Society, 2011
    Co-Authors: Wei Ting Liu, Roland D. Kersten, Bradley S. Moore, Yu Liang Yang, Pieter C Dorrestein
    Abstract:

    Here, we described the discovery of anti-infective agent arylomycin and its biosynthetic gene cluster in an industrial daptomycin producing strain Streptomyces Roseosporus. This was accomplished via the use of MALDI imaging mass spectrometry (IMS) along with peptidogenomic approach in which we have expanded to short sequence tagging (SST) described herein. Using IMS, we observed that prior to the production of daptomycin, a cluster of ions (1-3) was produced by S. Roseosporus and correlated well with the decreased staphylococcal cell growth. With a further adopted SST peptidogenomics approach, which relies on the generation of sequence tags from tandem mass spectrometric data and query against genomes to identify the biosynthetic genes, we were able to identify these three molecules (1-3) to arylomycins, a class of broad-spectrum antibiotics that target type I signal peptidase. The gene cluster was then identified. This highlights the strength of IMS and MS guided genome mining approaches in effectively bridging the gap between phenotypes, chemotypes, and genotypes.

Shuai Luo - One of the best experts on this subject based on the ideXlab platform.

  • crotonylation of key metabolic enzymes regulates carbon catabolite repression in Streptomyces Roseosporus
    Communications Biology, 2020
    Co-Authors: Chenfan Sun, Qingwei Zhao, Shuai Luo, Xinai Chen, Xuming Mao
    Abstract:

    Due to the plethora natural products made by Streptomyces, the regulation of its metabolism are of great interest, whereas there is a lack of detailed understanding of the role of posttranslational modifications (PTM) beyond traditional transcriptional regulation. Herein with Streptomyces Roseosporus as a model, we showed that crotonylation is widespread on key enzymes for various metabolic pathways, and sufficient crotonylation in primary metabolism and timely elimination in secondary metabolism are required for proper Streptomyces metabolism. Particularly, the glucose kinase Glk, a keyplayer of carbon catabolite repression (CCR) regulating bacterial metabolism, is identified reversibly crotonylated by the decrotonylase CobB and the crotonyl-transferase Kct1 to negatively control its activity. Furthermore, crotonylation positively regulates CCR for Streptomyces metabolism through modulation of the ratio of glucose uptake/Glk activity and utilization of carbon sources. Thus, our results revealed a regulatory mechanism that crotonylation globally regulates Streptomyces metabolism at least through positive modulation of CCR. Chen-Fan Sun et al. show that key enzymes in several metabolic pathways are crotonylated in Streptomyces Roseosporus. This study suggests that crotonylation increases carbon catabolite repression by increasing glucose uptake while reducing the activity of glucose kinase.

  • Crotonylation of key metabolic enzymes regulates carbon catabolite repression in Streptomyces Roseosporus.
    Communications biology, 2020
    Co-Authors: Chenfan Sun, Qingwei Zhao, Shuai Luo, Xinai Chen, Xuming Mao
    Abstract:

    Due to the plethora natural products made by Streptomyces, the regulation of its metabolism are of great interest, whereas there is a lack of detailed understanding of the role of posttranslational modifications (PTM) beyond traditional transcriptional regulation. Herein with Streptomyces Roseosporus as a model, we showed that crotonylation is widespread on key enzymes for various metabolic pathways, and sufficient crotonylation in primary metabolism and timely elimination in secondary metabolism are required for proper Streptomyces metabolism. Particularly, the glucose kinase Glk, a keyplayer of carbon catabolite repression (CCR) regulating bacterial metabolism, is identified reversibly crotonylated by the decrotonylase CobB and the crotonyl-transferase Kct1 to negatively control its activity. Furthermore, crotonylation positively regulates CCR for Streptomyces metabolism through modulation of the ratio of glucose uptake/Glk activity and utilization of carbon sources. Thus, our results revealed a regulatory mechanism that crotonylation globally regulates Streptomyces metabolism at least through positive modulation of CCR.

  • Transposon-based identification of a negative regulator for the antibiotic hyper-production in Streptomyces.
    Applied microbiology and biotechnology, 2018
    Co-Authors: Shuai Luo, Xinai Chen, Xuming Mao
    Abstract:

    Production of secondary metabolites in Streptomyces is regulated by a complex regulatory network precisely, elaborately, and hierarchically. One of the main reasons for the low yields of some high-value secondary metabolites is the repressed expression of their biosynthetic gene clusters, supposedly by some gene cluster out-situated negative regulators. Identification of these repressors and removal of the inhibitory effects based on the regulatory mechanisms will be an effective way to improve their yields. For proof of the concept, using an antibiotic daptomycin from Streptomyces Roseosporus, we introduced Himar1-based random mutagenesis combined with a reporter-guided screening strategy to identify a transcriptional regulator PhaR, whose loss-of-function deletion led to about 2.68-fold increase of the gene cluster expression and approximately 6.14-fold or 43% increased daptomycin production in the flask fermentation or in the fed-batch fermentation, respectively. Further study showed that PhaR negatively regulates the expression of daptomycin biosynthetic gene cluster by direct binding to its promoter (dptEp). Moreover, phaR expression gradually drops down during fermentation, and PhaR is positively auto-regulated by directly binding to its own promoter, which results in positive feedback regulation to persistently reduce phaR expression. Meanwhile, the declining PhaR protein remove its repressive effects during daptomycin production. All these results support that our strategy would be a powerful method for genetic screening and rational engineering for the yield improvement of antibiotics, and could be potentially used widely in other Streptomyces species.

  • Regulatory and biosynthetic effects of the bkd gene clusters on the production of daptomycin and its analogs A21978C1-3.
    Journal of industrial microbiology & biotechnology, 2018
    Co-Authors: Shuai Luo, Xinai Chen, Xuming Mao
    Abstract:

    Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces Roseosporus in an acidic peptide complex A21978C. In this complex, A21978C1–3 is most abundant and contains branched-chain fatty acyl groups, while daptomycin has a straight decanoic acyl group. The branched-chain α-keto acid dehydrogenase complex (BCDH complex), encoded by bkd gene clusters in Streptomyces, is responsible for the early step of converting branched-chain amino acids into branched-chain fatty acids. In a daptomycin industrial producer S. Roseosporus L30, two alleles of bkd gene clusters, bkdA1B1C1/bkdA2B2C2, and a regulatory gene bkdR located upstream of bkdA2B2C2 are identified. We show that BkdR positively regulated bkdA2B2C2 expression and was negatively auto-regulated, but is not directly involved in regulation of daptomycin gene cluster expression. However, BkdR is required for both daptomycin and A21978C1–3 production. Furthermore, deletion of bkdA2B2C2 only led to partial reduction of A21978C1–3 production, while the ΔbkdA1B1C1 mutant shows very weak production of A21978C1–3, and the double bkd mutant has a similar production profile as the single ΔbkdA1B1C1 mutant, suggesting that bkdA1B1C1 gene cluster plays a dominant role in branched-chain fatty acid biosynthesis. So we reveal a unique regulatory function of BkdR and genetic engineered a bkd null strain for daptomycin production with reduced impurities.

  • Negative regulation of daptomycin production by DepR2, an ArsR-family transcriptional factor
    Journal of industrial microbiology & biotechnology, 2017
    Co-Authors: Xuming Mao, Shuai Luo
    Abstract:

    Daptomycin, a lipopeptide antibiotic potently active against Gram-positive bacterial pathogens, is produced by Streptomyces Roseosporus, but the transcriptional regulation on its biosynthesis is not fully understood. Here, we report that DepR2, an ArsR-family transcriptional regulator isolated previously by DNA-affinity purification, interacts directly with dptEp, the major promoter of the daptomycin gene cluster. DepR2 binds to an imperfect palindromic sequence at the very upstream of dptEp. Meanwhile, higher dptEp activities were consistently observed in the ΔdepR2 mutant, correlating with a nearly 2.5-fold increased production of daptomycin and three structurally related secondary metabolites A21978C1–3. Thus, our data suggest that the ArsR-family transcriptional regulator DepR2 negatively regulates production of daptomycin by directly repressing the expression of its gene cluster in S. Roseosporus. To the best of our knowledge, this is the first report to show the involvement of an ArsR-family regulator in the direct regulation of secondary metabolite biosynthesis in Streptomyces.

Guojian Liao - One of the best experts on this subject based on the ideXlab platform.

  • Roseosporol A, the first isolation of a novel sesquiterpenoid from Streptomyces Roseosporus.
    Natural product research, 2018
    Co-Authors: Li-na Deng, Guojian Liao, Rui Wang, Guo-wei Wang, Mingxu Liu, Zhihua Liao, Min Chen
    Abstract:

    A novel sesquiterpenoid, roseosporol A (1), together with 12 known compounds (2-13) were isolated from the ethyl acetate extract of Lsr2-deletion mutant strain of Streptomyces Roseosporus. Their structures were determined by spectroscopic methods, including NMR, HRMS, UV, IR and ECD.

  • wblA, a pleiotropic regulatory gene modulating morphogenesis and daptomycin production in Streptomyces Roseosporus
    Journal of applied microbiology, 2017
    Co-Authors: Xingwei Huang, Jun Tian, Lan Shen, Hua Zuo, Guojian Liao
    Abstract:

    Aims The wblA gene, encoding a homolog of the WhiB family protein, was identified in the sequenced genome of daptomycin producer S.Roseosporus. To explore the function of wblA, we construct wblA disruption strains, complemented strains and overexpression strains to test whether it can affect the production of secondary metabolites and morphogenesis. Methods and results We constructed disruption strains by homologous recombination in S.Roseosporus. The disruption mutant of wblA could block aerial mycelium sporulation and enhance the production of daptomycin by 51%. In contrast, overexpression of wblA resulted in significantly decreased the yield of daptomycin. In agreement with it, the transcription of key daptomycin regulatory genes atrA, dptR2 and dptR3 and structural gene dptE remarkably increased in the wblA disruption mutant. Conclusions wblA plays a key role in control of daptomycin biosynthesis and is essential for sporulation. The disruption of wblA could accumulate the transcription level of some key genes involving in daptomycin biosynthesis. Significance and Impact of the Study Daptomycin is an important antibiotic with potent activity against a variety of Gram-positive pathogens. This study demonstrates that valuable improvement in the yield of daptomycin can be achieved through modulating the expression of wblA transcription regulator. This article is protected by copyright. All rights reserved.

  • Targeted isolation of sulfur-containing metabolites from Lsr2-deletion mutant strain of Streptomyces Roseosporus
    RSC Advances, 2017
    Co-Authors: Li-na Deng, Guojian Liao, Rui Wang, Guo-wei Wang, Mingxu Liu, Zhihua Liao, Min Chen
    Abstract:

    Deletion of the Lsr2 gene in Streptomyces Roseosporus up-regulated silent gene clusters and produced new secondary metabolites. An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS/MS) method was used to analyze metabolites of the mutant and wild-type strains, and recognize previously unreported sulfur-containing compounds based on their molecular formulas and fragmentation ions. The targeted isolation of unidentified compounds afforded six new sulfur-containing compounds, pyrismycins A–F (1–6), together with seven known analogues 7–13. Their cytotoxic effects were evaluated using four clinically relevant human cancer cell lines, gastric carcinoma SGC7901, breast carcinoma MDA-MB-231, lung carcinoma A549 and hepatocellular carcinoma HepG2. Compound 7 exhibited the most potent cytotoxicity with IC50 values of 1.7, 5.8 and 6.3 μM against the SGC7901, HepG2 and MDA-MB-231, respectively.

  • Unexpected extensive lysine acetylation in the trump-card antibiotic producer Streptomyces Roseosporus revealed by proteome-wide profiling.
    Journal of proteomics, 2014
    Co-Authors: Guojian Liao, Longxiang Xie, Zhongyi Cheng, Jianping Xie
    Abstract:

    Abstract Lysine acetylation is emerging as a ubiquitous and conserved posttranslational modification in living cells. While the role of lysine acetylation in regulating primary metabolism is well-established, its function in secondary metabolism remains largely elusive. To gain insight into the nature, extent and biological function of lysine acetylation in Streptomyces reseosporus , a producer of daptomycin, dubiously dubbed as the trump card antibiotic, we used immunoaffinity-based acetyllysine peptide enrichment integrated with high resolution mass spectrometry to comprehensively characterize lysine acetylated proteins in this microbe. We identified a total of 667 proteins with 1143 unique sites, representing the largest acetylproteome reported to date in bacteria. Acetylated proteins belong to various functional classes such as metabolism and gene expression according to the gene ontology. We demonstrated for the first time that proteins involved in the biosynthesis of diverse secondary metabolites are acetylated, such as a nonribosomal peptide synthetase, enzymes essential for hydroxamate siderophore and phosphinic acid natural products biosynthesis, implying an important role of acetylation in these processes. Taken together, this proteomic analysis revealed a surprising breadth of cellular processes affected by lysine acetylation and also furnishes some fresh intervention nodes for the rational improvement of the antibiotic producer. Biological significance Despite considerable efforts have been devoted to elucidating the mechanism underlying secondary metabolism in Streptomyces , which are prolific producers of secondary metabolites with diverse biological activities, such as bacteriocides and antitumors, the full map of regulation and corresponding network is still far from perfect. Protein lysine acetylation is an evolutionarily conserved protein post-translational modification, abundantly existing in proteins with diverse biological context. We took advantage of integrated high throughput PTM proteomics followed by intensive bioinformatic analysis to profile lysine acetylome of Streptomyces Roseosporus . In total, 1134 unique Kac sites in 667 lysine acetylated substrates were identified, representing the largest aceylomics in prokaryotes to date. Significantly, a nonribosomal peptide synthetase, an enzyme essential for hydroxamate siderophore and phosphinic acid natural products biosynthesis, was found to be acetylated. Given the conservation of these enzymes in biosynthesis of diverse secondary metabolites, lysine acetylation likely plays an important role in regulating secondary metabolism in Streptomyces .

  • Manipulation of kynurenine pathway for enhanced daptomycin production in Streptomyces Roseosporus.
    Biotechnology progress, 2013
    Co-Authors: Guojian Liao, Qing Liu, Lei Wang, Feifei Guan, Yuqi Huang
    Abstract:

    Daptomycin is a cyclic lipopeptide natural product produced by Stretptomyces Roseosporus, displaying good bactericidal activity against a wide range of gram-positive pathogens. Daptomycin contains a 13 amino acid and kynurenine (Kyn) is essential for optimal activity of daptomycin. In this study, we characterized the Kyn pathway in S. Roseosporus and investigated its role in supplying precursor for daptomycin biosynthesis. Two genes (dptJ and tdo) coding for tryptophan-2,3-dioxgenase existed in the chromosome. dptJ is located in the daptomycin biosynthetic gene cluster, while tdo is in other locus. Disruption of dptJ or tdo resulted in reduced yield by ∼50%. The introduction of an additional copy of dptJ but not tdo led to enhanced production of daptomycin by 110%. Furthermore, disruption of kyn encoding kynureninase showed improved daptomycin productivity by 30%. Our results demonstrated that the enhancement of Kyn supply through metabolic engineering approach is an efficient way to increase daptomycin production.