Biomedical subjects

J D Keasling

Publications and source records attributed to J D Keasling.

At least 19 recordsLinked to original sources

PubMed2003

A constructed microbial consortium for biodegradation of the organophosphorus insecticide parathion.

A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophenol mineralization. The co-culture effectively hydrolyzed 500 microM parathion (146 mg l(-1)) and prevented the accumulation of p-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium members. Parathion hydrolysis by E. coli SD2 followed Michaelis-Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibition kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 microM p-nitrophenol (83 mg l(-1)) and P. putida KT2440 pSB337 inhibited by 1,000 microM p-nitrophenol (139 mg l(-1)). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construction of degradative consortia may prove beneficial.

Biodegradation, Environmental↗

PubMed2002

Nickel accumulation and nickel oxalate precipitation by Aspergillus niger.

A strain of Aspergillus niger isolated from a metal-contaminated soil was able to grow in the presence of cadmium, chromium, cobalt, copper, and unusually high levels of nickel on solid (8.0 mM) and in liquid (6.5 mM) media. This fungus removed >98% of the nickel from liquid medium after 100 h of growth but did not remove the other metals, as determined by inductively coupled plasma spectroscopy. Experiments with non-growing, live fungal biomass showed that nickel removal was not due to biosorption alone, as little nickel was bound to the biomass at the pH values tested. Furthermore, when the protonophore carbonyl cyanide p-(trifluoremetoxy) phenyl hydrazone (FCCP) was added to the actively growing fungus nickel removal was inhibited, supporting the hypothesis that energy metabolism is essential for metal removal. Analytical electron microscopy of thin-sectioned fungal biomass revealed that metal removed from the broth was localized in the form of small rectangular crystals associated with the cell walls and also inside the cell. X-ray and electron diffraction analysis showed that these crystals were nickel oxalate dihydrate.

Aspergillus niger↗

PubMed2002

Polyphosphate kinase genes from activated sludge carrying out enhanced biological phosphorus removal.

The community structure and metabolic function of activated sludge carrying out enhanced biological phosphorus removal have been investigated. Laboratory-scale sequencing batch reactors were operated at several influent COD/P ratios to obtain sludges with a range of phosphorus contents. Molecular microbiological techniques based on small subunit ribosomal RNA were used to characterize the structure of these sludges. The dominant polyphosphate accumulating organism was a close relative of Rhodocyclus tenuis, a member of the beta subclass of the Proteobacteria. Fragments of genes coding for polyphosphate kinase (PPK), thought to be responsible for polyphosphate accumulation, were retrieved from one of the sludges. The relative abundance of PPK gene copies in genomic DNA extracted from sludges was determined to confirm that at least one of the PPK gene sequences was derived from the dominant polyphosphate accumulating organism.

DNA Primers↗

PubMed2001

The in vivo synthesis of plant sesquiterpenes by Escherichia coli.

Three plant genes encoding (+)-delta-cadinene, 5-epi-aristolochene, and vetispiradiene cyclases were expressed in Escherichia coli to evaluate the potential of this bacterium to synthesize sesquiterpenes in vivo. Various growth temperatures, carbon sources, and host strains were examined to optimize terpene production. The highest levels of sesquiterpene production occurred when the enzymes were expressed in strain DH5alpha from the trc promoter (Ptrc) of the high-copy plasmidpTrc99A in M9 medium supplemented with 0.2% (v/v) glycerol at 30 degrees C for 5-epi-aristolochene and vetispiradiene and 37 degrees C for (+)-delta-cadinene. The highest concentrations of sesquiterpenes observed were 10.3 microg of (+)-delta-cadinene, 0.24 microg of 5-epi-aristolochene (measured as (+)-delta-cadinene equivalents), and 6.4 microg of vetispiradiene (measured as (+)-delta-cadinene equivalents) per liter of culture. These sesquiterpene production levels are >500-fold lower than carotenoid production, both of which are synthesized from endogenous trans-farnesyl diphosphate (FDP) in E. coli. Based on these results, we conclude that the limiting factor for sesquiterpene synthesis in E. coli is the poor expression of the cyclase enzyme and not supply of the FDP precursor.

Alkyl and Aryl Transferases↗

PubMed2001

Analysis of an engineered sulfate reduction pathway and cadmium precipitation on the cell surface.

We previously have genetically engineered an aerobic sulfate reduction pathway in Escherichia coli for the generation of hydrogen sulfide and demonstrated the pathway's utility in the precipitation of cadmium. To engineer the pathway, the assimilatory sulfate reduction pathway was modified so that cysteine was overproduced. Excess cysteine was then converted by cysteine desulfhydrase to an abundance of hydrogen sulfide, which then reacted with aqueous cadmium to form cadmium sulfide. In this study, observations of various E. coli clones were combined with an analysis of kinetic and transport phenomena. This analysis revealed that cysteine production is the rate-limiting step in the engineered pathway and provided an explanation for the phenomenon of cell surface precipitation. An analytical model showed that cadmium sulfide must form at the cell surface because the rate of cadmium sulfide formation is extremely fast and the rate of sulfide transport is relatively slow.

Cadmium↗

PubMed2001

Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production.

Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all carotenoids. IPP in Escherichia coli is synthesized through the nonmevalonate pathway, which has not been completely elucidated. The first reaction of IPP biosynthesis in E. coli is the formation of 1-deoxy-D-xylulose-5-phosphate (DXP), catalyzed by DXP synthase and encoded by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phos- phate, catalyzed by DXP reductoisomerase and encoded by dxr. To determine if one or more of the reactions in the nonmevalonate pathway controlled flux to IPP, dxs and dxr were placed on several expression vectors under the control of three different promoters and transformed into three E. coli strains (DH5alpha, XL1-Blue, and JM101) that had been engineered to produce lycopene. Lycopene production was improved significantly in strains transformed with the dxs expression vectors. When the dxs gene was expressed from the arabinose-inducible araBAD promoter (P(BAD)) on a medium-copy plasmid, lycopene production was twofold higher than when dxs was expressed from the IPTG-inducible trc and lac promoters (P(trc) and P(lac), respectively) on medium-copy and high-copy plasmids. Given the low final densities of cells expressing dxs from IPTG-inducible promoters, the low lycopene production was probably due to the metabolic burden of plasmid maintenance and an excessive drain of central metabolic intermediates. At arabinose concentrations between 0 and 1.33 mM, cells expressing both dxs and dxr from P(BAD) on a medium-copy plasmid produced 1.4-2.0 times more lycopene than cells expressing dxs only. However, at higher arabinose concentrations lycopene production in cells expressing both dxs and dxr was lower than in cells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-inducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XL1-Blue.

Aldose-Ketose Isomerases↗

PubMed2001

Metabolic engineering of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) composition in recombinant Salmonella enterica serovar typhimurium.

A recombinant strain of Salmonella enterica serovar Typhimurium (mutant in propionate-activation activity) was metabolically engineered to control the composition of poly(3-hydroxybutyrate-co-3-hydroxy- valerate) (PHBV), a polyhydroxyalkanoate copolymer with commercially desirable properties. A gene (prpE) encoding propionyl-CoA synthetase was placed under the control of the IPTG-inducible taclacUV5 promoter (P(taclacUV5)) while the polyhydroxyalkanoate synthesis operon (phaBCA) from Acinetobacter sp. RA3849 was coexpressed under the control of the arabinose-inducible araBAD promoter (P(BAD)). S. enterica, harboring both constructs, was grown in medium containing a fixed substrate concentration and the composition of the copolymer was varied between 2 mol% and 25 mol% 3-hydroxyvalerate by controlling the IPTG level in the medium. This "dial-a-composition" system should find application in cases where the substrate concentration of a feedstream for PHBV bioplastic production is not adjustable.

Acinetobacter↗

PubMed2001

Controlling the metabolic flux through the carotenoid pathway using directed mRNA processing and stabilization.

A synthetic operon containing the crtI and crtY genes, encoding the phytoene desaturase and the lycopene cyclase, respectively, was placed under the control of the araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the genes. This construct was transformed into Escherichia coli cells harboring the genes for phytoene production. By varying the mRNA secondary structures, we were able to modulate the flux through the carotenoid pathway, resulting in a 300-fold variation in the production of beta-carotene relative to lycopene. In addition, intermediates in the pathway from phytoene to beta-carotene production that are not observed in cells expressing the recombinant operon were observed when the engineered operons were used, indicating that changes in levels of the enzymes affected the formation of intermediates. These results indicate that it is possible to coordinately regulate the genes encoding the enzymes of a metabolic pathway and balance the production of the intermediates.

Carotenoids↗

PubMed2001

Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene.

The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.

Aerobiosis↗

PubMed2001

Effects of transcription induction homogeneity and transcript stability on expression of two genes in a constructed operon.

A synthetic operon was constructed using the reporter genes gfp and lacZ and the arabinose-inducible araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 3' and 5' ends of the genes and a putative RNase E site was placed between the genes. These mRNA control elements have been shown to affect transcript processing and decay, resulting in altered protein levels. These constructs were transformed into cells harboring the native arabinose-inducible araE gene encoding the arabinose transport protein and engineered cells harboring a constitutively expressed araE. In the strains with arabinose-dependent transport the linear response in the production of both reporter proteins to inducer concentration occurred over a narrow range of arabinose concentrations. In the strains with constitutive transport the linear range of gene expression occurred over a much larger arabinose concentration range than in strains with the arabinose-inducible transport. Strains with the arabinose-inducible transport harboring different operon constructs produced the two reporter proteins at very different levels at low arabinose concentrations; as inducer concentrations increased, differences in relative expression levels decreased. In contrast, strains with constitutive transport harboring different operon constructs produced the reporter proteins at very different levels across the entire range of inducer concentrations, pointing to the importance of optimizing gene expression control at various levels to control the production of heterologous proteins.

Arabinose↗

PubMed2001

Uranyl precipitation by biomass from an enhanced biological phosphorus removal reactor.

Heavy metal and radionuclide contamination presents a significant environmental problem worldwide. Precipitation of heavy metals on membranes of cells that secrete phosphate has been shown to be an effective method of reducing the volume of these wastes, thus reducing the cost of disposal. A consortium of organisms, some of which secrete large quantities of phosphate, was enriched in a laboratory-scale sequencing batch reactor performing Enhanced Biological Phosphorus Removal, a treatment process widely used for removing phosphorus. Organisms collected after the aerobic phase of this process secreted phosphate and precipitated greater than 98% of the uranyl from a 1.5 mM uranyl nitrate solution when supplemented with an organic acid as a carbon source under anaerobic conditions. Transmission electron microscopy, energy dispersive x-ray spectroscopy, and fluorescence spectroscopy were used to identify the precipitate as membrane-associated uranyl phosphate, UO2HPO4.

Biomass↗

PubMed2001

Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter.

Genes placed under the control of the arabinose-inducible araBAD promoter (P(BAD)) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium. Previous work showed that all-or-none gene expression from P(BAD) was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from P(BAD) in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter. Based on these results, two expression systems were developed to allow regulatable control of genes under control of P(BAD). In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths. In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid. Both systems allow regulatable expression of a plasmid-borne P(BAD)-controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations. While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration.

Arabinose↗

PubMed2000

Polyphosphate binding and chain length recognition of Escherichia coli exopolyphosphatase.

Exopolyphosphatase of Escherichia coli (PPX) is a highly processive enzyme demonstrating the ability to recognize polyphosphates of specific lengths. The mechanisms responsible for the processivity and polymer length recognition of the enzyme were investigated in relation to the manner in which polyphosphate is bound to the enzyme. Multiple polyphosphate binding sites were identified on distant portions of the enzyme and were determined to be responsible for the polymer length recognition of the enzyme. In addition, two independently folded domains were identified. The N-terminal domain contained a quasi-processive polyphosphatase active site belonging to the sugar kinase/actin/hsp70 superfamily. The C-terminal domain contained a single polyphosphate binding site and was responsible for nearly all of the PPX affinity for polyphosphate. This domain was also found to confer a highly processive mode of action to PPX. Collectively, these results were used to describe the interaction of polyphosphate with PPX.

Acid Anhydride Hydrolases↗

PubMed2000

The effect of monovalent ions on polyphosphate binding to Escherichia coli exopolyphosphatase.

The thermodynamic driving force for the interaction of Escherichia coli exopolyphosphatase (PPX) with polyphosphate was investigated by varying salt choice and concentration. This interaction was found to be cation concentration independent but weakly dependent on the concentration of certain anions. Both of these traits are very uncommon for nonspecific protein-polyelectrolyte interactions. Interpretation of these results based on theory indicated that binding was not entropy driven due to release of polyelectrolyte-condensed counterions, as is the case for nearly all protein-polyelectrolyte interactions. The thermodynamics of the PPX-polyphosphate interaction showed similarity only to the interaction of polynucleotides with single stranded binding proteins.

Acid Anhydride Hydrolases↗

PubMed2000

Biodesulfurization of dibenzothiophene in Escherichia coli is enhanced by expression of a Vibrio harveyi oxidoreductase gene.

One possible alternative to current fuel hydrodesulfurization methods is the use of microorganisms to remove sulfur compounds. Biodesulfurization requires much milder processing conditions, gives higher specificity, and does not require molecular hydrogen. In the present work we have produced two compatible plasmids: pDSR3, which allows Escherichia coli to convert dibenzothiophene (DBT) to hydroxybiphenyl (HBP), and pDSR2, which produces a Vibrio harveyi flavin oxidoreductase. We show that the flavin oxidoreductase enhances the rate of DBT removal when co-expressed in vivo with the desulfurization enzymes. The plasmids pDSR2 and pDSR3 were co-expressed in growing cultures. The expression of oxidoreductase caused an increase in the rate of DBT removal but a decrease in the rate of HBP production. The maximum rate of DBT removal was 8 mg/h. g dry cell weight. Experiments were also conducted using resting cells with the addition of various carbon sources. It was found that the addition of glucose or glycerol to cultures with oxidoreductase expression produced the highest DBT removal rate (51 mg/h. g dry cell weight). The culture with acetate and no oxidoreductase expression had the highest level of HBP production. For all carbon sources, the DBT removal rate was faster and the HBP generation rate slower with the expression of the oxidoreductase. Analysis of desulfurization intermediates indicates that the last enzyme in the pathway may be limiting.

Base Sequence↗

PubMed2000

Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria.

Multicopy plasmids are often chosen for the expression of recombinant genes in Escherichia coli. The high copy number is generally desired for maximum gene expression; however, the metabolic burden effects that usually result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications. In this study, low-copy mini-F plasmids were compared to high-copy pMB1-based plasmids for production of two metabolites in E. coli: polyphosphate (polyP) and lycopene derived from isopentenyl diphosphate (IPP). The stationary-phase accumulation of polyP on a per cell basis was enhanced approximately 80% when either high- or low-copy plasmids were used, from 120 micromol/g DCW without augmented polyP kinase (PPK) activity to approximately 220 micromol/g DCW. The cell density of the high-copy plasmid-containing culture at stationary phase was approximately 24% lower than the low-copy culture and 30% lower than the control culture. This difference in cell density is likely a metabolic burden effect and resulted in a lower overall product concentration for the high-copy culture (approximately 130 micromol/L culture) relative to the low-copy culture (approximately 160 micromol/L culture). When the gene for DXP (1-deoxy-D-xylulose 5-phosphate) synthase, the first enzyme in the IPP mevalonate-independent biosynthetic pathway, was expressed from the tac promoter on multicopy and low-copy plasmids, lycopene production was enhanced two- to threefold over that found in cells expressing the chromosomal copy only. Cell growth and lycopene production decreased substantially when isopropyl beta-D-thiogalactosidase (IPTG) was added to the high-copy plasmid-containing culture, suggesting that overexpression of DXP synthase was a significant metabolic burden. In the low-copy plasmid-containing culture, no differences in cell growth or lycopene production were observed with any IPTG concentrations. When dxs was placed under the control of the arabinose-inducible promoter (P(BAD)) on the low-copy plasmid, the amount of lycopene produced was proportional to the arabinose concentration and no significant changes in cell growth resulted. These results suggest that low-copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism.

Acid Anhydride Hydrolases↗

PubMed2000

Dual labeling with green fluorescent proteins for confocal microscopy.

We report a dual labeling technique involving two green fluorescent protein (GFP) variants that is compatible with confocal microscopy. Two lasers were used to obtain images of (i) mixed cultures of cells, where one species contained GFPuv and another species contained GFPmut2 or GFPmut3, and (ii) a single species containing both GFPuv and GFPmut2 in the same cell. This method shows promise for monitoring gene expression and as a nondestructive and in situ technique for confocal microscopy of multispecies biofilms.

Biofilms↗

PubMed2000

Commensal interactions in a dual-species biofilm exposed to mixed organic compounds.

There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the p-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at high p-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and Escherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.

Biofilms↗