jorge galazzo | California Institute of Technology (original) (raw)

Papers by jorge galazzo

Antimicrobial Agents and Chemotherapy, 2001

APE1 is the major nuclease for excising abasic (AP) sites and particular 39-obstructive termini f... more APE1 is the major nuclease for excising abasic (AP) sites and particular 39-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC 1280 ), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays -a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen -and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals. Citation: Simeonov A, Kulkarni A, Dorjsuren D, Jadhav A, Shen M, et al. (2009) Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1. PLoS ONE 4(6): e5740.

Cheminform, 2010

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Farmaco, 2001

A library of 85000 microbial fermentation extracts was screened for inhibitors of multidrug resis... more A library of 85000 microbial fermentation extracts was screened for inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa and Candida albicans. New compounds EA-371alpha and EA-371delta were isolated and demonstrated to be potent and specific inhibitors of the MexAB-OprM pump in P. aeruginosa. Two series of fungal metabolites, enniatins and beauvericins, were found to be ubiquitous and potent inhibitors of ABC transporters. Milbemycins were rediscovered as potent inhibitors of the CDRI pump in C. albicans, and demonstrated to potentiate effectively the antifungal activity of fluconazole and SCH-56592 against a wide variety of Candida clinical isolates.

Annals of The New York Academy of Sciences, 1990

Enzyme and Microbial Technology, 1990

Measurements of rates of glucose uptake and of glycerol and ethanol formation combined with knowl... more Measurements of rates of glucose uptake and of glycerol and ethanol formation combined with knowledge of the metabolic pathways involved in S. cerevisiae were employed to obtain in vivo rates of reaction catalysed by pathway enzymes for suspended and alginate-entrapped cells at pH 4.5 and 5.5. lntracellular concentrations of substrates and effectors for most key pathway enzymes were estimated from in vivo phosphorus-31 nuclear magnetic resonance measurements. These data show the validity in vivo of kinetic models previously proposed for phosphofructokinase and pyruvate kinase based on in vitro studies. Kinetic representations of hexokinase, glycogen synthetase, and glyceraldehyde 3phosphate dehydrogenase, which incorporate major regulatory properties of these enzymes, are all consistent with the in vivo data. This detailed model of pathway kinetics and these data on intracellular metabolite concentrations allow evaluation of flux-control coefficients for all key enzymes involved in glucose catabolism under the four different cell environments examined. This analysis indicates that alginate entrapment increases the glucose uptake rate and shifts the step most influencing ethanol production from glucose uptake to phosphofructokinase. The rate of ATP utilization in these nongrowing cells strongly limits ethanol production at pH 5.5 but is relatively insignificant at pH 4.5.

Annals of The New York Academy of Sciences, 1987

Biotechnology and Bioengineering, 1990

Nongrowing Saccharomyces cerevisiae cells previously grown in alginate exhibit ethanol production... more Nongrowing Saccharomyces cerevisiae cells previously grown in alginate exhibit ethanol production rates 1.5 times greater than cells previously grown in suspension. Analysis of glucose, ethanol, and glycerol formation data using quasi-steady-state pathway stoichiometry shows that alginate-grown cells possess phosphofructokinase (PFK), ATPase, and polysaccharide synthesis maximum activities which are approximately two-, two-, and ninefold larger, respectively, than in suspension-grown cells. The estimated change in PFK maximum velocity is consistent with in vitro assays of PFK activity in extracts of suspension- and alginate-grown yeast. Estimation of ethanol production flux control coefficients using in vivo nuclear magnetic resonance (NMR) spectroscopy measurements of intracellular metabolite concentrations and a previously proposed detailed kinetic model of ethanol fermentation in yeast shows that glucose uptake dominates flux control in alginate-grown cells in suspension while earlier research revealed that PFK and ATPase exert significant flux control in suspension-grown cells. When placed in a calcium alginate matrix, alginate-grown cells produced ethanol 1.8 times more rapidly and accumulated substantially more polyphosphate than suspension-grown cells placed in alginate. Cells growing in alginate elicit responses at the genetic level which substantially alter pathway rates and flux control when these cells are used as either a suspended or an immobilized biocatalyst. These responses in gene expression to growth in alginate serve to reconfigure flux controls in alginate to a pattern which is similar to that obtained for suspended-grown cells in suspension.

Biotechnology Techniques, 1987

31P nuclear magnetic resonance has been employed to monitor noninvasively Saccharomyces cerevisia... more 31P nuclear magnetic resonance has been employed to monitor noninvasively Saccharomyces cerevisiae anaerobic glucose metabolism in suspended and immobilized cells. Results show that cell entrapment in Ca-alginate beads alters cell metabolism compared to that in suspended cells. Assuming similar intracellular ionic strength, differences in intracellular phosphate chemical shift indicate that the internal pH of the immobilized cells is lower than the suspended cell internal pH. This result is consistent with higher ethanol production rates exhibited by immobilized yeast.

Biotechnology and Bioengineering, 1990

Estimation of intracellular intermediary metabolite levels is of fundamental importance to charac... more Estimation of intracellular intermediary metabolite levels is of fundamental importance to characterize cell metabolic processes and their regulation. Usually, intracellular intermediates are determined by stopping the cell metabolism, e.g., by immersing the cell sample in liquid nitrogen, and performing percNoric acid extracts of the cells. The metabolite levels are then obtained either by standard analytical methods'-3 or by using nuclear magnetic resonance (NMR) ~pectroscopy.~*~ Using this technique, it is possible to obtain only one point per sample because the method is destructive. Thus, transient studies are not possible on the same cell sample, and a series of aliquots are required with the assumption that all have the same dynamic behavior and are exposed to the same initial conditions. Also, information on compartmentalization within the cell is lost when extracts are prepared. For example, this prevents differentiation of compounds in the cytoplasm from those in the vacuole in yeast.

Bio/technology, 1991

Secondary metabolite production by Streptomyces is often highly sensitive to oxygen supply, which... more Secondary metabolite production by Streptomyces is often highly sensitive to oxygen supply, which can be limiting in large-scale fermentations. In an attempt to improve oxygen utilization by the cells, we expressed a heterologous bacterial hemoglobin gene in Streptomyces coelicolor and Streptomyces lividans. Hemoglobin expression was demonstrated by immunoblot analysis and carbon monoxide binding activity. In batch fermentations run under reduced aeration, the expression of hemoglobin in S. coelicolor resulted in a ten-fold increase in specific yields of the aromatic polyketide, actinorhodin. Actinorhodin yields were also much less sensitive to aeration conditions in the hemoglobin-expressing strain. In addition, hemoglobin-expressing S. lividans cells grown under reduced aeration had higher final cell densities and exhibited greater oxygen consumption rates than non-expressing cells.

Biotechnology and Bioengineering, 1989

Fermentation rates and intracellular compositions have been determined for alginate-entrapped Sac... more Fermentation rates and intracellular compositions have been determined for alginate-entrapped Saccharomyces cerevisiae and for identical cells in suspension. Glucose uptake and ethanol and glycerol production are approximately two times faster in immobilized cells than in suspended cells. Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy of fermenting immobilized and suspended cells shows differences in intermediate metabolite levels such as fructose-1,6 diphosphate, glucose-6-phosphate, and 3-phosphoglycerate and in internal pH. Carbon-13 NMR shows an increase in polysaccharide production. These data suggest that immobilization has accelerated the rate of glucose transport or of glucose phosphorylation. These effects of immobilization upon cell metabolism are observed in a very short period of time under conditions in which negligible DNA, RNA, or protein synthesis takes place.

Biotechnology Techniques, 1989

Low sensitivity of nuclear magnetic resonance (NMR) measurements of living cell composition by co... more Low sensitivity of nuclear magnetic resonance (NMR) measurements of living cell composition by conventional methods requires samples with high cell density compared to that in growing cultures. Reasonably accurate intracellular concentration estimates from lower cell density samples can be obtained by treating the time-domain NMR data by linear prediction singular value decomposition (LPSVD) prior to Fourier transformation. Alternatively, application of LPSVD enables intracellular concentration estimates in less NMR acquisition time, improving time resolution in NMR measurements of intracellular transients.

Chemical Engineering Science, 1986

The flow of a viscous fluid on the outside of a partially submerged, rotating horizontal cylinder... more The flow of a viscous fluid on the outside of a partially submerged, rotating horizontal cylinder is one of the basic fundamental coating flows. The main features of this flow for a Newtonian fluid were described by Ckzmpanella and Cerro [Chem. Engng Sci. 39,1443 (1984)]. A rapid flow approximation [Cerro and Striven Ind. Engng Chem. Fun&ma. 19,40 (1980)] IS used to develop a tilm profile equation for a powerlaw type non-Newtonian fluid. The film profile equation allows to compute a unique critical flow regime for each kind of power law fluid characterized by two rheological constants, K and n. Theoretical results are shown to be in very good agreement with experimental results for a wide range of fluid properties.

Biotechnology Progress, 2004

A process for the production of erythromycin aglycone analogues has been developed by combining c... more A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1° mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production. Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity. The solubility of 15-methyl-6-dEB in water was determined to be 0.25–0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.

Journal of Biotechnology, 2004

A robust high cell-density fed-batch bioprocess was developed for the heterologous production of ... more A robust high cell-density fed-batch bioprocess was developed for the heterologous production of 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, with a recombinant Escherichia coli strain expressing the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. Initial evaluation of the E. coli strain in a 5-l bioreactor with the addition of exogenous propionate for polyketide biosynthesis resulted in a maximum cell density of 30 g l −1 (OD 600 ∼ 60) and the production of 700 mg l −1 of 6-dEB. Retention of the two plasmids harboring the heterologous genes was maintained between 90 and 100% even in the absence of antibiotic selection. However, the accumulation of excess ammonia in the culture medium was found to significantly decrease the productivity of the cells. Through optimization of the medium composition and fermentation conditions, the maximum cell density was increased by two-fold, and a final titer of 1.1 g l −1 of 6-dEB was achieved. This represents an 11-fold improvement compared to the highest reported titer of 100 mg l −1 with E. coli as the production host.

Cheminform, 2004

Isolation and Characterization of 7-Hydroxy-6-demethyl-6-deoxy-erythromycin D, a New Erythromycin... more Isolation and Characterization of 7-Hydroxy-6-demethyl-6-deoxy-erythromycin D, a New Erythromycin Analogue, from Engineered Saccharopolyspora erythraea. -(STARKS, C. M.; RODRIGUEZ, E.; CARNEY, J. R.; DESAI, R. P.; CARRERAS, C.; MCDANIEL, R.; HUTCHINSON, R.; GALAZZO, J. L.; LICARI*, P. J.; J. Antibiot. 57 (2004) 1, 64-67; Discovery Res., Kosan Biosciences, Inc., Hayward, CA 94545, USA; Eng.) -M. Schroeter 33-225

Journal of Natural Products, 2006

Three new hypothemycin analogues were isolated from the fungal strains Hypomyces subiculosus DSM ... more Three new hypothemycin analogues were isolated from the fungal strains Hypomyces subiculosus DSM 11931 and DSM 11932. The structures of these compounds were elucidated by spectroscopic methods, chemical conversion, and X-ray crystallographic analysis. One of the analogues, 4-O-demethylhypothemycin, exhibited potent and selective cytotoxic activity against cell lines with a BRAF mutation.

Biotechnology Progress, 2004

The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analog... more The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue (R-EryA) by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution. This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter. Real-time PCR analysis of RNA confirmed higher expression of eryK in the resulting strain, S. erythraea K301–105B, compared to its parent. In shake flasks, K301–105B produced less of the shunt product 15-fluoro-erythromycin B (15F-EryB), suggesting a shift in congener distribution toward the desired product, 15-fluoro-erythromycin A (15F-EryA). In bioreactor studies, K301–105B produced 1.3 g/L of 15F-EryA with 75–80% molar yield on fed precursor, compared with 0.9 g/L 15F-EryA with 50–55% molar yield on fed precursor by the parent strain. At higher precursor feed rates, K301–105B produced 3.5 g/L of 15F-EryA while maintaining 75–80% molar yield on fed precursor.

Journal of Medicinal Chemistry, 2009

Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We descr... more Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We describe the preparation of potent non-benzoquinone ansamycins. One of these analogues, generated by feeding 3-amino-5-chlorobenzoic acid to a genetically engineered strain of Streptomyces hygroscopicus, shows high accumulation and long residence time in tumor tissue, is well-tolerated upon intravenous dosing, and is highly efficacious in the COLO205 mouse tumor xenograft model.

Antimicrobial Agents and Chemotherapy, 2001

APE1 is the major nuclease for excising abasic (AP) sites and particular 39-obstructive termini f... more APE1 is the major nuclease for excising abasic (AP) sites and particular 39-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC 1280 ), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays -a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen -and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals. Citation: Simeonov A, Kulkarni A, Dorjsuren D, Jadhav A, Shen M, et al. (2009) Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1. PLoS ONE 4(6): e5740.

Cheminform, 2010

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was e... more ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Farmaco, 2001

A library of 85000 microbial fermentation extracts was screened for inhibitors of multidrug resis... more A library of 85000 microbial fermentation extracts was screened for inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa and Candida albicans. New compounds EA-371alpha and EA-371delta were isolated and demonstrated to be potent and specific inhibitors of the MexAB-OprM pump in P. aeruginosa. Two series of fungal metabolites, enniatins and beauvericins, were found to be ubiquitous and potent inhibitors of ABC transporters. Milbemycins were rediscovered as potent inhibitors of the CDRI pump in C. albicans, and demonstrated to potentiate effectively the antifungal activity of fluconazole and SCH-56592 against a wide variety of Candida clinical isolates.

Annals of The New York Academy of Sciences, 1990

Enzyme and Microbial Technology, 1990

Measurements of rates of glucose uptake and of glycerol and ethanol formation combined with knowl... more Measurements of rates of glucose uptake and of glycerol and ethanol formation combined with knowledge of the metabolic pathways involved in S. cerevisiae were employed to obtain in vivo rates of reaction catalysed by pathway enzymes for suspended and alginate-entrapped cells at pH 4.5 and 5.5. lntracellular concentrations of substrates and effectors for most key pathway enzymes were estimated from in vivo phosphorus-31 nuclear magnetic resonance measurements. These data show the validity in vivo of kinetic models previously proposed for phosphofructokinase and pyruvate kinase based on in vitro studies. Kinetic representations of hexokinase, glycogen synthetase, and glyceraldehyde 3phosphate dehydrogenase, which incorporate major regulatory properties of these enzymes, are all consistent with the in vivo data. This detailed model of pathway kinetics and these data on intracellular metabolite concentrations allow evaluation of flux-control coefficients for all key enzymes involved in glucose catabolism under the four different cell environments examined. This analysis indicates that alginate entrapment increases the glucose uptake rate and shifts the step most influencing ethanol production from glucose uptake to phosphofructokinase. The rate of ATP utilization in these nongrowing cells strongly limits ethanol production at pH 5.5 but is relatively insignificant at pH 4.5.

Annals of The New York Academy of Sciences, 1987

Biotechnology and Bioengineering, 1990

Nongrowing Saccharomyces cerevisiae cells previously grown in alginate exhibit ethanol production... more Nongrowing Saccharomyces cerevisiae cells previously grown in alginate exhibit ethanol production rates 1.5 times greater than cells previously grown in suspension. Analysis of glucose, ethanol, and glycerol formation data using quasi-steady-state pathway stoichiometry shows that alginate-grown cells possess phosphofructokinase (PFK), ATPase, and polysaccharide synthesis maximum activities which are approximately two-, two-, and ninefold larger, respectively, than in suspension-grown cells. The estimated change in PFK maximum velocity is consistent with in vitro assays of PFK activity in extracts of suspension- and alginate-grown yeast. Estimation of ethanol production flux control coefficients using in vivo nuclear magnetic resonance (NMR) spectroscopy measurements of intracellular metabolite concentrations and a previously proposed detailed kinetic model of ethanol fermentation in yeast shows that glucose uptake dominates flux control in alginate-grown cells in suspension while earlier research revealed that PFK and ATPase exert significant flux control in suspension-grown cells. When placed in a calcium alginate matrix, alginate-grown cells produced ethanol 1.8 times more rapidly and accumulated substantially more polyphosphate than suspension-grown cells placed in alginate. Cells growing in alginate elicit responses at the genetic level which substantially alter pathway rates and flux control when these cells are used as either a suspended or an immobilized biocatalyst. These responses in gene expression to growth in alginate serve to reconfigure flux controls in alginate to a pattern which is similar to that obtained for suspended-grown cells in suspension.

Biotechnology Techniques, 1987

31P nuclear magnetic resonance has been employed to monitor noninvasively Saccharomyces cerevisia... more 31P nuclear magnetic resonance has been employed to monitor noninvasively Saccharomyces cerevisiae anaerobic glucose metabolism in suspended and immobilized cells. Results show that cell entrapment in Ca-alginate beads alters cell metabolism compared to that in suspended cells. Assuming similar intracellular ionic strength, differences in intracellular phosphate chemical shift indicate that the internal pH of the immobilized cells is lower than the suspended cell internal pH. This result is consistent with higher ethanol production rates exhibited by immobilized yeast.

Biotechnology and Bioengineering, 1990

Estimation of intracellular intermediary metabolite levels is of fundamental importance to charac... more Estimation of intracellular intermediary metabolite levels is of fundamental importance to characterize cell metabolic processes and their regulation. Usually, intracellular intermediates are determined by stopping the cell metabolism, e.g., by immersing the cell sample in liquid nitrogen, and performing percNoric acid extracts of the cells. The metabolite levels are then obtained either by standard analytical methods'-3 or by using nuclear magnetic resonance (NMR) ~pectroscopy.~*~ Using this technique, it is possible to obtain only one point per sample because the method is destructive. Thus, transient studies are not possible on the same cell sample, and a series of aliquots are required with the assumption that all have the same dynamic behavior and are exposed to the same initial conditions. Also, information on compartmentalization within the cell is lost when extracts are prepared. For example, this prevents differentiation of compounds in the cytoplasm from those in the vacuole in yeast.

Bio/technology, 1991

Secondary metabolite production by Streptomyces is often highly sensitive to oxygen supply, which... more Secondary metabolite production by Streptomyces is often highly sensitive to oxygen supply, which can be limiting in large-scale fermentations. In an attempt to improve oxygen utilization by the cells, we expressed a heterologous bacterial hemoglobin gene in Streptomyces coelicolor and Streptomyces lividans. Hemoglobin expression was demonstrated by immunoblot analysis and carbon monoxide binding activity. In batch fermentations run under reduced aeration, the expression of hemoglobin in S. coelicolor resulted in a ten-fold increase in specific yields of the aromatic polyketide, actinorhodin. Actinorhodin yields were also much less sensitive to aeration conditions in the hemoglobin-expressing strain. In addition, hemoglobin-expressing S. lividans cells grown under reduced aeration had higher final cell densities and exhibited greater oxygen consumption rates than non-expressing cells.

Biotechnology and Bioengineering, 1989

Fermentation rates and intracellular compositions have been determined for alginate-entrapped Sac... more Fermentation rates and intracellular compositions have been determined for alginate-entrapped Saccharomyces cerevisiae and for identical cells in suspension. Glucose uptake and ethanol and glycerol production are approximately two times faster in immobilized cells than in suspended cells. Phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy of fermenting immobilized and suspended cells shows differences in intermediate metabolite levels such as fructose-1,6 diphosphate, glucose-6-phosphate, and 3-phosphoglycerate and in internal pH. Carbon-13 NMR shows an increase in polysaccharide production. These data suggest that immobilization has accelerated the rate of glucose transport or of glucose phosphorylation. These effects of immobilization upon cell metabolism are observed in a very short period of time under conditions in which negligible DNA, RNA, or protein synthesis takes place.

Biotechnology Techniques, 1989

Low sensitivity of nuclear magnetic resonance (NMR) measurements of living cell composition by co... more Low sensitivity of nuclear magnetic resonance (NMR) measurements of living cell composition by conventional methods requires samples with high cell density compared to that in growing cultures. Reasonably accurate intracellular concentration estimates from lower cell density samples can be obtained by treating the time-domain NMR data by linear prediction singular value decomposition (LPSVD) prior to Fourier transformation. Alternatively, application of LPSVD enables intracellular concentration estimates in less NMR acquisition time, improving time resolution in NMR measurements of intracellular transients.

Chemical Engineering Science, 1986

The flow of a viscous fluid on the outside of a partially submerged, rotating horizontal cylinder... more The flow of a viscous fluid on the outside of a partially submerged, rotating horizontal cylinder is one of the basic fundamental coating flows. The main features of this flow for a Newtonian fluid were described by Ckzmpanella and Cerro [Chem. Engng Sci. 39,1443 (1984)]. A rapid flow approximation [Cerro and Striven Ind. Engng Chem. Fun&ma. 19,40 (1980)] IS used to develop a tilm profile equation for a powerlaw type non-Newtonian fluid. The film profile equation allows to compute a unique critical flow regime for each kind of power law fluid characterized by two rheological constants, K and n. Theoretical results are shown to be in very good agreement with experimental results for a wide range of fluid properties.

Biotechnology Progress, 2004

A process for the production of erythromycin aglycone analogues has been developed by combining c... more A process for the production of erythromycin aglycone analogues has been developed by combining classical strain mutagenesis techniques with modern recombinant DNA methods and traditional process improvement strategies. A Streptomyces coelicolor strain expressing the heterologous 6-deoxyerythronolide B (6-dEB) synthase (DEBS) for the production of erythromycin aglycones was subjected to random mutagenesis and selection. Several strains exhibiting 2-fold higher productivities and reaching >3 g/L total macrolide aglycones were developed. These mutagenized strains were cured of the plasmid carrying the DEBS genes and a KS1° mutant DEBS operon was introduced for the production of novel analogues when supplemented with a synthetic diketide precursor. The strains expressing the mutant DEBS were screened for improved 15-methyl-6-dEB production, and the best clone, strain B9, was found to be 50% more productive as compared to the parent host strain used for 15-methyl-6-dEB production. Strain B9 was evaluated in 5-L fermenters to confirm productivity in a scalable process. Although peak titers of 0.85 g/L 15-methyl-6-dEB by strain B9 confirmed improved productivity, it was hypothesized that the low solubility of 15-methyl-6-dEB limited productivity. The solubility of 15-methyl-6-dEB in water was determined to be 0.25–0.40 g/L, although higher titers are possible in fermentation medium. The incorporation of the hydrophobic resin XAD-16HP resulted in both the in situ adsorption of the product and the slow release of the diketide precursor. The resin-containing fermentation achieved 1.3 g/L 15-methyl-6-dEB, 50% higher than the resin-free process. By combining classical mutagenesis, recombinant DNA techniques, and process development, 15-methyl-6-dEB productivity was increased by over 100% in a scalable fermentation process.

Journal of Biotechnology, 2004

A robust high cell-density fed-batch bioprocess was developed for the heterologous production of ... more A robust high cell-density fed-batch bioprocess was developed for the heterologous production of 6-deoxyerythronolide B (6-dEB), the macrocyclic core of the antibiotic erythromycin, with a recombinant Escherichia coli strain expressing the 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora erythraea. Initial evaluation of the E. coli strain in a 5-l bioreactor with the addition of exogenous propionate for polyketide biosynthesis resulted in a maximum cell density of 30 g l −1 (OD 600 ∼ 60) and the production of 700 mg l −1 of 6-dEB. Retention of the two plasmids harboring the heterologous genes was maintained between 90 and 100% even in the absence of antibiotic selection. However, the accumulation of excess ammonia in the culture medium was found to significantly decrease the productivity of the cells. Through optimization of the medium composition and fermentation conditions, the maximum cell density was increased by two-fold, and a final titer of 1.1 g l −1 of 6-dEB was achieved. This represents an 11-fold improvement compared to the highest reported titer of 100 mg l −1 with E. coli as the production host.

Cheminform, 2004

Isolation and Characterization of 7-Hydroxy-6-demethyl-6-deoxy-erythromycin D, a New Erythromycin... more Isolation and Characterization of 7-Hydroxy-6-demethyl-6-deoxy-erythromycin D, a New Erythromycin Analogue, from Engineered Saccharopolyspora erythraea. -(STARKS, C. M.; RODRIGUEZ, E.; CARNEY, J. R.; DESAI, R. P.; CARRERAS, C.; MCDANIEL, R.; HUTCHINSON, R.; GALAZZO, J. L.; LICARI*, P. J.; J. Antibiot. 57 (2004) 1, 64-67; Discovery Res., Kosan Biosciences, Inc., Hayward, CA 94545, USA; Eng.) -M. Schroeter 33-225

Journal of Natural Products, 2006

Three new hypothemycin analogues were isolated from the fungal strains Hypomyces subiculosus DSM ... more Three new hypothemycin analogues were isolated from the fungal strains Hypomyces subiculosus DSM 11931 and DSM 11932. The structures of these compounds were elucidated by spectroscopic methods, chemical conversion, and X-ray crystallographic analysis. One of the analogues, 4-O-demethylhypothemycin, exhibited potent and selective cytotoxic activity against cell lines with a BRAF mutation.

Biotechnology Progress, 2004

The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analog... more The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue (R-EryA) by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution. This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter. Real-time PCR analysis of RNA confirmed higher expression of eryK in the resulting strain, S. erythraea K301–105B, compared to its parent. In shake flasks, K301–105B produced less of the shunt product 15-fluoro-erythromycin B (15F-EryB), suggesting a shift in congener distribution toward the desired product, 15-fluoro-erythromycin A (15F-EryA). In bioreactor studies, K301–105B produced 1.3 g/L of 15F-EryA with 75–80% molar yield on fed precursor, compared with 0.9 g/L 15F-EryA with 50–55% molar yield on fed precursor by the parent strain. At higher precursor feed rates, K301–105B produced 3.5 g/L of 15F-EryA while maintaining 75–80% molar yield on fed precursor.

Journal of Medicinal Chemistry, 2009

Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We descr... more Inhibition of the protein chaperone Hsp90 is a promising new approach to cancer therapy. We describe the preparation of potent non-benzoquinone ansamycins. One of these analogues, generated by feeding 3-amino-5-chlorobenzoic acid to a genetically engineered strain of Streptomyces hygroscopicus, shows high accumulation and long residence time in tumor tissue, is well-tolerated upon intravenous dosing, and is highly efficacious in the COLO205 mouse tumor xenograft model.