Alex Praseuth - Academia.edu (original) (raw)
Papers by Alex Praseuth
Biotechnology Progress, Nov 1, 2008
Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary me... more Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary metabolite. Surprisingly, this strain also produced a trace amount of echinomycin. We sequenced the entire triostin A biosynthetic gene cluster from S. triostinicus, and found that this 36 kilobase-long gene cluster contained an ORF homologous to ecm18 that encodes a thioacetal-forming enzyme responsible for the triostin A-to-echinomycin bioconversion. These findings indicate that, unlike previously thought, S. triostinicus is capable of producing not only triostin A but also echinomycin. Our observation suggests potential value in careful reanalysis for metabolites from previously characterized natural product producers with the current technologies.
Nature Chemical Biology, Jun 25, 2006
Methods in Enzymology, 2009
Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPS... more Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPSs) that catalyze a peptide bond-forming reaction using natural amino acids as substrates. Most members of this class of natural products exhibit remarkable biological activities, but many of these valuable compounds are often difficult to obtain in sufficient quantities from their natural sources due to low production levels in the producing organisms or difficulty in culturing them. Harnessing recent progress in our genetic and biochemical understanding of the biosynthesis of these nonprimary metabolites, our laboratory has successfully developed an alternative, straightforward approach for obtaining desired natural products by placing the entire biosynthetic gene cluster in our heterologous host of choice, Escherichia coli. This effort led to the first successful de novo production of heterologous bioactive complex NRPs in E. coli. Through developing our heterologous biosynthetic system, we were able to construct a novel platform suitable for generating an NRP library through rational engineering of the natural modular assembly-line array composed of NRPSs and the auxiliary enzymes. This chapter describes the basic concept in establishing an E. coli-based plasmid-borne heterologous NRP biosynthetic system, and gives selected protocols that have been used successfully for engineering NRP biosynthesis.
Journal of the American Chemical Society, Jun 10, 2009
Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic... more Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for rational design and synthesis of potent antibiotic unnatural natural product
The Journal of Antibiotics, Nov 24, 2010
Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative... more Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative manner using a variety of acyl-CoA molecules as substrates when biosynthesizing complex polyketides. Although most members from this class of natural products exhibit notable biological activities, often they are naturally produced in trace levels or cultivation of the analyteproducing organism is less than feasible. Appropriately, to contend with the former challenge, one must identify any translational bottleneck and perform functional analysis of the associated enzymes. In recent years, many gene clusters purportedly responsible for biosynthesizing polyketides have been identified and cataloged from a variety of fungal genomes including genes coding for iterative PKSs, particulary bikaverin, zearalenone and hypothemycin biosynthetic enzymes. Mounting appreciation of these highly specific codons and their translational consequence will afford scientists the ability to anticipate the fungal metabolite by correlating an organism's genomic cluster to an appropriate biosynthetic system. It was observed in recent reports, the successful production of these recombinant enzymes using an Escherichia coli expression system which in turn conferred the anticipated metabolite in vitro. This review will focus on iterative PKSs responsible for biosynthesizing bikaverin, zearalenone and hypothemycin, and expand on befitting enzymatic reaction mechanisms and development of a highly versatile system that could potentially generate biologically active compounds.
天然有機化合物討論会講演要旨集, Sep 15, 2005
Methods in enzymology, 2009
Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPS... more Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPSs) that catalyze a peptide bond-forming reaction using natural amino acids as substrates. Most members of this class of natural products exhibit remarkable biological activities, but many of these valuable compounds are often difficult to obtain in sufficient quantities from their natural sources due to low production levels in the producing organisms or difficulty in culturing them. Harnessing recent progress in our genetic and biochemical understanding of the biosynthesis of these nonprimary metabolites, our laboratory has successfully developed an alternative, straightforward approach for obtaining desired natural products by placing the entire biosynthetic gene cluster in our heterologous host of choice, Escherichia coli. This effort led to the first successful de novo production of heterologous bioactive complex NRPs in E. coli. Through developing our heterologous biosynthetic syste...
Nature Chemical Biology, 2006
Journal of the American Chemical Society, 2009
Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic... more Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for rational design and synthesis of potent antibiotic unnatural natural product
The Journal of Antibiotics, 2010
Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative... more Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative manner using a variety of acyl-CoA molecules as substrates when biosynthesizing complex polyketides. Although most members from this class of natural products exhibit notable biological activities, often they are naturally produced in trace levels or cultivation of the analyteproducing organism is less than feasible. Appropriately, to contend with the former challenge, one must identify any translational bottleneck and perform functional analysis of the associated enzymes. In recent years, many gene clusters purportedly responsible for biosynthesizing polyketides have been identified and cataloged from a variety of fungal genomes including genes coding for iterative PKSs, particulary bikaverin, zearalenone and hypothemycin biosynthetic enzymes. Mounting appreciation of these highly specific codons and their translational consequence will afford scientists the ability to anticipate the fungal metabolite by correlating an organism's genomic cluster to an appropriate biosynthetic system. It was observed in recent reports, the successful production of these recombinant enzymes using an Escherichia coli expression system which in turn conferred the anticipated metabolite in vitro. This review will focus on iterative PKSs responsible for biosynthesizing bikaverin, zearalenone and hypothemycin, and expand on befitting enzymatic reaction mechanisms and development of a highly versatile system that could potentially generate biologically active compounds.
Current Opinion in Chemical Biology, 2007
Customizing biosynthesis of natural products to yield biologically active derivatives has captiva... more Customizing biosynthesis of natural products to yield biologically active derivatives has captivated scientists in the field of biosynthetic research. To substantiate this goal, there are scores of obstacles to consider. To create novel metabolites by mutating amino acid residues in wild-type enzymes, a researcher must broaden the range of the enzymes substrate tolerance and increase its turnover rate during reaction catalysis. In the past decade, numerous gene clusters responsible for the biosynthesis of notable natural products have been identified from a variety of organisms. Several genes coding for type III polyketide synthases, particularly the chalcone synthase superfamily enzymes, were recently uncovered and expressed in E. coli. Furthermore, it was observed and reported how these recombinant enzymes are capable of producing essential metabolites in vitro. Three of the type III polyketide synthases, chalcone synthase, octaketide synthase and pentaketide chromone synthase, have been characterized and their active sites subjected to rational engineering for biosynthetic production of their analogs. Because they are encoded in a single open reading frame and are post-translationally small in size, type III polyketide synthases are ideal targets for protein engineering. The relative ease with which these genes are expressed makes molecular biological manipulation to obtain mutated enzymes more procurable, ameliorating analysis of its biosynthetic pathway. In summary, time devoted to modification of biosynthetic proteins and unravelling of the detailed reaction mechanisms involved in biosynthesis will be shortened, paving the way for a much wider scope for metabolic engineers in future. This review focuses on the use of chalcone synthase, octaketide synthase and pentaketide chromone synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds.
Biotechnology Progress, 2008
Proficient production of the antitumor agent triostin A was developed using engineered Escherichi... more Proficient production of the antitumor agent triostin A was developed using engineered Escherichia coli (E. coli). The bacterium played host to 15 genes that encode integral biosynthetic proteins which were identified and cloned from Streptomyces lasaliensis. In this study, triostin A production was dramatically increased by more than 20-fold, 13 mg/L, with the introduction of exogenous quinoxaline-2-carboxylic acid (QXC), the speculative starting unit for biosynthesis of triostin A. Conversely, de noVo production of triostin A by means of high cell density fedbatch fermentation that is exclusive of exogenous QXC bore a modest amount of the antitumor agent. Noteworthy production of the biologically active molecule was achieved with smallscale cultivation and quantitative analysis of the product was accomplished with a liquid chromatography-mass spectrometer. This simple and speedy system could easily provide us with valuable information for maximizing the production titer. Our entirely heterologous production system also establishes a basis for the future use of E. coli for generation of novel bioactive compounds through tolerable precursor-directed biosynthesis.
Biotechnology Progress, 2008
Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary me... more Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary metabolite. Surprisingly, this strain also produced a trace amount of echinomycin. We sequenced the entire triostin A biosynthetic gene cluster from S. triostinicus, and found that this 36 kilobase‐long gene cluster contained an ORF homologous to ecm18 that encodes a thioacetal‐forming enzyme responsible for the triostin A‐to‐echinomycin bioconversion. These findings indicate that, unlike previously thought, S. triostinicus is capable of producing not only triostin A but also echinomycin. Our observation suggests potential value in careful re‐analysis for metabolites from previously characterized natural product producers with the current technologies.
Biotechnology Progress, Nov 1, 2008
Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary me... more Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary metabolite. Surprisingly, this strain also produced a trace amount of echinomycin. We sequenced the entire triostin A biosynthetic gene cluster from S. triostinicus, and found that this 36 kilobase-long gene cluster contained an ORF homologous to ecm18 that encodes a thioacetal-forming enzyme responsible for the triostin A-to-echinomycin bioconversion. These findings indicate that, unlike previously thought, S. triostinicus is capable of producing not only triostin A but also echinomycin. Our observation suggests potential value in careful reanalysis for metabolites from previously characterized natural product producers with the current technologies.
Nature Chemical Biology, Jun 25, 2006
Methods in Enzymology, 2009
Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPS... more Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPSs) that catalyze a peptide bond-forming reaction using natural amino acids as substrates. Most members of this class of natural products exhibit remarkable biological activities, but many of these valuable compounds are often difficult to obtain in sufficient quantities from their natural sources due to low production levels in the producing organisms or difficulty in culturing them. Harnessing recent progress in our genetic and biochemical understanding of the biosynthesis of these nonprimary metabolites, our laboratory has successfully developed an alternative, straightforward approach for obtaining desired natural products by placing the entire biosynthetic gene cluster in our heterologous host of choice, Escherichia coli. This effort led to the first successful de novo production of heterologous bioactive complex NRPs in E. coli. Through developing our heterologous biosynthetic system, we were able to construct a novel platform suitable for generating an NRP library through rational engineering of the natural modular assembly-line array composed of NRPSs and the auxiliary enzymes. This chapter describes the basic concept in establishing an E. coli-based plasmid-borne heterologous NRP biosynthetic system, and gives selected protocols that have been used successfully for engineering NRP biosynthesis.
Journal of the American Chemical Society, Jun 10, 2009
Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic... more Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for rational design and synthesis of potent antibiotic unnatural natural product
The Journal of Antibiotics, Nov 24, 2010
Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative... more Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative manner using a variety of acyl-CoA molecules as substrates when biosynthesizing complex polyketides. Although most members from this class of natural products exhibit notable biological activities, often they are naturally produced in trace levels or cultivation of the analyteproducing organism is less than feasible. Appropriately, to contend with the former challenge, one must identify any translational bottleneck and perform functional analysis of the associated enzymes. In recent years, many gene clusters purportedly responsible for biosynthesizing polyketides have been identified and cataloged from a variety of fungal genomes including genes coding for iterative PKSs, particulary bikaverin, zearalenone and hypothemycin biosynthetic enzymes. Mounting appreciation of these highly specific codons and their translational consequence will afford scientists the ability to anticipate the fungal metabolite by correlating an organism's genomic cluster to an appropriate biosynthetic system. It was observed in recent reports, the successful production of these recombinant enzymes using an Escherichia coli expression system which in turn conferred the anticipated metabolite in vitro. This review will focus on iterative PKSs responsible for biosynthesizing bikaverin, zearalenone and hypothemycin, and expand on befitting enzymatic reaction mechanisms and development of a highly versatile system that could potentially generate biologically active compounds.
天然有機化合物討論会講演要旨集, Sep 15, 2005
Methods in enzymology, 2009
Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPS... more Nonribosomal peptides (NRPs) are synthesized by modular mega-enzymes called NRP synthetases (NRPSs) that catalyze a peptide bond-forming reaction using natural amino acids as substrates. Most members of this class of natural products exhibit remarkable biological activities, but many of these valuable compounds are often difficult to obtain in sufficient quantities from their natural sources due to low production levels in the producing organisms or difficulty in culturing them. Harnessing recent progress in our genetic and biochemical understanding of the biosynthesis of these nonprimary metabolites, our laboratory has successfully developed an alternative, straightforward approach for obtaining desired natural products by placing the entire biosynthetic gene cluster in our heterologous host of choice, Escherichia coli. This effort led to the first successful de novo production of heterologous bioactive complex NRPs in E. coli. Through developing our heterologous biosynthetic syste...
Nature Chemical Biology, 2006
Journal of the American Chemical Society, 2009
Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic... more Escherichia coli allows efficient modular incorporation of newly isolated quinomycin biosynthetic enzyme into echinomycin biosynthetic pathway for rational design and synthesis of potent antibiotic unnatural natural product
The Journal of Antibiotics, 2010
Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative... more Fungal polyketide synthases (PKSs) catalyze a carbon-carbon bond forming reaction in an iterative manner using a variety of acyl-CoA molecules as substrates when biosynthesizing complex polyketides. Although most members from this class of natural products exhibit notable biological activities, often they are naturally produced in trace levels or cultivation of the analyteproducing organism is less than feasible. Appropriately, to contend with the former challenge, one must identify any translational bottleneck and perform functional analysis of the associated enzymes. In recent years, many gene clusters purportedly responsible for biosynthesizing polyketides have been identified and cataloged from a variety of fungal genomes including genes coding for iterative PKSs, particulary bikaverin, zearalenone and hypothemycin biosynthetic enzymes. Mounting appreciation of these highly specific codons and their translational consequence will afford scientists the ability to anticipate the fungal metabolite by correlating an organism's genomic cluster to an appropriate biosynthetic system. It was observed in recent reports, the successful production of these recombinant enzymes using an Escherichia coli expression system which in turn conferred the anticipated metabolite in vitro. This review will focus on iterative PKSs responsible for biosynthesizing bikaverin, zearalenone and hypothemycin, and expand on befitting enzymatic reaction mechanisms and development of a highly versatile system that could potentially generate biologically active compounds.
Current Opinion in Chemical Biology, 2007
Customizing biosynthesis of natural products to yield biologically active derivatives has captiva... more Customizing biosynthesis of natural products to yield biologically active derivatives has captivated scientists in the field of biosynthetic research. To substantiate this goal, there are scores of obstacles to consider. To create novel metabolites by mutating amino acid residues in wild-type enzymes, a researcher must broaden the range of the enzymes substrate tolerance and increase its turnover rate during reaction catalysis. In the past decade, numerous gene clusters responsible for the biosynthesis of notable natural products have been identified from a variety of organisms. Several genes coding for type III polyketide synthases, particularly the chalcone synthase superfamily enzymes, were recently uncovered and expressed in E. coli. Furthermore, it was observed and reported how these recombinant enzymes are capable of producing essential metabolites in vitro. Three of the type III polyketide synthases, chalcone synthase, octaketide synthase and pentaketide chromone synthase, have been characterized and their active sites subjected to rational engineering for biosynthetic production of their analogs. Because they are encoded in a single open reading frame and are post-translationally small in size, type III polyketide synthases are ideal targets for protein engineering. The relative ease with which these genes are expressed makes molecular biological manipulation to obtain mutated enzymes more procurable, ameliorating analysis of its biosynthetic pathway. In summary, time devoted to modification of biosynthetic proteins and unravelling of the detailed reaction mechanisms involved in biosynthesis will be shortened, paving the way for a much wider scope for metabolic engineers in future. This review focuses on the use of chalcone synthase, octaketide synthase and pentaketide chromone synthase for rational biosynthetic engineering to generate molecular diversity and pursue innovative, biologically potent compounds.
Biotechnology Progress, 2008
Proficient production of the antitumor agent triostin A was developed using engineered Escherichi... more Proficient production of the antitumor agent triostin A was developed using engineered Escherichia coli (E. coli). The bacterium played host to 15 genes that encode integral biosynthetic proteins which were identified and cloned from Streptomyces lasaliensis. In this study, triostin A production was dramatically increased by more than 20-fold, 13 mg/L, with the introduction of exogenous quinoxaline-2-carboxylic acid (QXC), the speculative starting unit for biosynthesis of triostin A. Conversely, de noVo production of triostin A by means of high cell density fedbatch fermentation that is exclusive of exogenous QXC bore a modest amount of the antitumor agent. Noteworthy production of the biologically active molecule was achieved with smallscale cultivation and quantitative analysis of the product was accomplished with a liquid chromatography-mass spectrometer. This simple and speedy system could easily provide us with valuable information for maximizing the production titer. Our entirely heterologous production system also establishes a basis for the future use of E. coli for generation of novel bioactive compounds through tolerable precursor-directed biosynthesis.
Biotechnology Progress, 2008
Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary me... more Streptomyces triostinicus produces triostin A, an antitumor antibiotic, as its major secondary metabolite. Surprisingly, this strain also produced a trace amount of echinomycin. We sequenced the entire triostin A biosynthetic gene cluster from S. triostinicus, and found that this 36 kilobase‐long gene cluster contained an ORF homologous to ecm18 that encodes a thioacetal‐forming enzyme responsible for the triostin A‐to‐echinomycin bioconversion. These findings indicate that, unlike previously thought, S. triostinicus is capable of producing not only triostin A but also echinomycin. Our observation suggests potential value in careful re‐analysis for metabolites from previously characterized natural product producers with the current technologies.