Biomedical subjects

George N Bennett

Publications and source records attributed to George N Bennett.

49 records · Page 3Linked to original sources

PubMed2003

Mutagenicity of nitroaromatic degradation compounds.

The mutagenicity of 2,4-dinitrotoluene (24DNT), and 2,6-dinitrotoluene (26DNT), and their related transformation products such as hydroxylamine and amine derivatives, which are formed by Clostridium acetobutylicum, were tested in crude cell extracts using Salmonella typhimurium TA100. A previous publication already reported the mutagenic activities of 2,4,6-trinitrotoluene (TNT) and its related hydroxylamine derivatives in this test system. A time course of the mutagenicity during the anaerobic transformation of TNT, 24DNT, and 26DNT was also investigated under the same conditions to compare with the results from the pure compounds. The monohydroxylamino intermediates 2-hydroxylamino-4-nitrotoluene (2HA4NT), 4-hydroxylamino-2-nitrotoluene (4HA2NT) and 2-hydroxylamino-6-nitrotoluene (2HA6NT) formed during anaerobic transformation of dinitrotoluenes were proven to be mutagenic in the Ames test using Salmonella typhimurium TA100. This study reports that 4HA2NT is the most stable derivative, whereas 2HA4NT and 2HA6NT are less stable and these intermediates are mutagenic in the Ames test. Both 24DNT and 26DNT and their final metabolites 2,4-diaminotoluene (24DAT) and 2,6-aminotoluene (26DAT) appeared nonmutagenic. In a time-course study of TNT degradation, the temporal sample containing 85% of 2,4-dihydroxylamino-6-nitrotoluene (24HA6NT) is most mutagenic. These observations suggest that the bioremediation approach for treatment of 24DNT and 26DNT should be carried past the hydroxylamino intermediate.

Air Pollutants, Occupational↗

PubMed2002

Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli.

Applications of genetic engineering or metabolic engineering have increased in both academic and industrial institutions. Most current metabolic engineering studies have focused on enzyme levels and on the effect of the amplification, addition, or deletion of a particular pathway. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. It is conceivable that in cofactor-dependent production systems, cofactor availability and the proportion of cofactor in the active form may play an important role in dictating the overall process yield. Hence, the manipulation of these cofactor levels may be crucial in order to further increase production. We have demonstrated that manipulation of cofactors can be achieved by external and genetic means and these manipulations have the potential to be used as an additional tool to achieve desired metabolic goals. We have shown experimentally that the NADH/NAD(+) ratio can be altered by using carbon sources with different oxidation states. We have shown further that the metabolite distribution can be influenced by a change in the NADH/NAD(+) ratio as mediated by the oxidation state of the carbon source used. We have also demonstrated that the total NAD(H/(+)) levels can be increased by the overexpression of the pncB gene. The increase in the total NAD(H/(+)) levels can be achieved even in a complex medium, which is commonly used by most industrial processes. Finally, we have shown that manipulation of the CoA pool/flux can be used to increase the productivity of a model product, isoamyl acetate.

Acetyl Coenzyme A↗

PubMed2002

Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase.

Metabolic engineering studies have generally focused on manipulating enzyme levels through either the amplification, addition, or deletion of a particular pathway. However, with cofactor-dependent production systems, once the enzyme levels are no longer limiting, cofactor availability and the ratio of the reduced to oxidized form of the cofactor can become limiting. Under these situations, cofactor manipulation may become crucial in order to further increase system productivity. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. However, cofactor manipulations can potentially become a powerful tool for metabolic engineering. Nicotinamide adenine dinucleotide (NAD) functions as a cofactor in over 300 oxidation-reduction reactions and regulates various enzymes and genetic processes. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. This paper investigates a genetic means of manipulating the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase. More specifically, it explores the effect on the metabolic patterns in Escherichia coli under anaerobic and aerobic conditions of substituting the native cofactor-independent formate dehydrogenase (FDH) by and NAD(+)-dependent FDH from Candida boidinii. The over-expression of the NAD(+)-dependent FDH doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed, increased the final cell density, and provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically. Under anaerobic conditions, the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol-to-acetate ratio. Even more interesting is the observation that during aerobic growth, the increased availability of NADH induced a shift to fermentation even in the presence of oxygen by stimulating pathways that are normally inactive under these conditions.

Aerobiosis↗

PubMed2002

The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures.

It is generally known that cofactors play a major role in the production of different fermentation products. This paper is part of a systematic study that investigates the potential of cofactor manipulations as a new tool for metabolic engineering. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ and producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. We have previously investigated a genetic means of increasing the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase and have demonstrated that this manipulation provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically (Berríos-Rivera et al., 2002, Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229). The current work explores further the effect of substituting the native cofactor-independent formate dehydrogenase (FDH) by an NAD(+)-dependent FDH from Candida boidinii on the NAD(H/+) levels, NADH/NAD+ ratio, metabolic fluxes and carbon-mole yields in Escherichia coli under anaerobic chemostat conditions. Overexpression of the NAD(+)-dependent FDH provoked a significant redistribution of both metabolic fluxes and carbon-mole yields. Under anaerobic chemostat conditions, NADH availability increased from 2 to 3 mol NADH/mol glucose consumed and the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol to acetate ratio and a decrease in the flux to lactate. It was also found that the NADH/NAD+ ratio should not be used as a sole indicator of the oxidation state of the cell. Instead, the metabolic distribution, like the Et/Ac ratio, should also be considered because the turnover of NADH can be fast in an effort to achieve a redox balance.

Anaerobiosis↗

PubMed1996

Molecular characterization of adiY, a regulatory gene which affects expression of the biodegradative acid-induced arginine decarboxylase gene (adiA) of Escherichia coli.

A transcriptional regulator gene, designated adiY, was found downstream of the biodegradative arginine decarboxylase (adiA) gene (previously known as adi) of Escherichia coli. The arginine decarboxylase system is maximally induced under conditions of acidic pH, anaerobiosis and rich medium, and AdiY was found to increase the expression of adiA. The DNA sequence of adiY encodes a protein of 253 amino acids. Primer extension analysis defined the promoter. The amino acid sequence of AdiY showed homology to the XylS/AraC family of transcriptional regulators, which includes EnvY and AppY. Studies suggested that sequences required for acid induction were also necessary to observe the stimulation by AdiY. An examination of the substitution of AdiY, AppY and EnvY showed that these three proteins can, to some extent, stimulate the other systems.

Amino Acid Sequence↗

PubMedDate not supplied

Enhanced isoamyl acetate production upon manipulation of the acetyl-CoA node in Escherichia coli.

Coenzyme A (CoA) and its thioester derivative acetyl-Coenzyme A (acetyl-CoA) participate in over 100 different reactions in intermediary metabolism of microorganisms. Earlier results indicated that overexpression of upstream rate-limiting enzyme pantothenate kinase with simultaneous supplementation of precursor pantothenic acid to the culture media increased intracellular CoA levels significantly ( approximately 10-fold). The acetyl-CoA levels also increased ( approximately 5-fold) but not as much as that of CoA, showing that the carbon flux from the pyruvate node is rate-limiting upon an increase in CoA levels. In this study, pyruvate dehydrogenase was overexpressed under elevated CoA levels to increase carbon flux from pyruvate to acetyl-CoA. This coexpression did not increase intracellular acetyl-CoA levels but increased the accumulation of extracellular acetate. The production of isoamyl acetate, an industrially useful compound derived from acetyl-CoA, was used as a model reporter system to signify the beneficial effects of this metabolic engineering strategy. In addition, a strain was created in which the acetate production pathway was inactivated to relieve competition at the acetyl-CoA node and to efficiently channel the enhanced carbon flux to the ester production pathway. The synergistic effect of cofactor CoA manipulation and pyruvate dehydrogenase overexpression in the acetate pathway deletion mutant led to a 5-fold increase in isoamyl acetate production. Under normal growth conditions the acetate pathway deletion mutant strains accumulate intracellular pyruvate, leading to excretion of pyruvate. However, upon enhancing the carbon flux from pyruvate to acetyl-CoA, the excretion of pyruvate was significantly reduced.

Acetyl Coenzyme A↗

PubMedDate not supplied

Efficient succinic acid production from glucose through overexpression of pyruvate carboxylase in an Escherichia coli alcohol dehydrogenase and lactate dehydrogenase mutant.

An adhE, ldhA double mutant Escherichia coli strain, SBS110MG, has been constructed to produce succinic acid in the presence of heterologous pyruvate carboxylase (PYC). The strategic design aims at diverting maximum quantities of NADH for succinate synthesis by inactivation of NADH competing pathways to increase succinate yield and productivity. Additionally an operational PFL enzyme allows formation of acetyl-CoA for biosynthesis and formate as a potential source of reducing equivalents. Furthermore, PYC diverts pyruvate toward OAA to favor succinate generation. SBS110MG harboring plasmid pHL413, which encodes the heterologous pyruvate carboxylase from Lactococcus lactis, produced 15.6 g/L (132 mM) of succinate from 18.7 g/L (104 mM) of glucose after 24 h of culture in an atmosphere of CO(2) yielding 1.3 mol of succinate per mole of glucose. This molar yield exceeded the maximum theoretical yield of succinate that can be achieved from glucose (1 mol/mol) under anaerobic conditions in terms of NADH balance. The current work further explores the importance of the presence of formate as a source of reducing equivalents in SBS110MG(pHL413). Inactivation of the native formate dehydrogenase pathway (FDH) in this strain significantly reduced succinate yield, suggesting that reducing power was lost in the form of formate. Additionally we investigated the effect of ptsG inactivation in SBS110MG(pHL413) to evaluate the possibility of a further increase in succinate yield. Elimination of the ptsG system increased the succinate yield to 1.4 mol/mol at the expense of a reduction in glucose consumption of 33%. In the presence of PYC and an efficient conversion of glucose to products, the ptsG mutation is not indispensable since PEP converted to pyruvate as a result of glucose phosphorylation by the glucose specific PTS permease EIICB(glu) can be rediverted toward OAA favoring succinate production.

Alcohol Dehydrogenase↗

PubMedDate not supplied

Redistribution of metabolic fluxes in the central aerobic metabolic pathway of E. coli mutant strains with deletion of the ackA-pta and poxB pathways for the synthesis of isoamyl acetate.

Although the bacterium E. coli is chosen as the host in many bioprocesses, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that could have otherwise been used for the desired product. The production of the ester isoamyl acetate from acetyl-CoA by ATF2, a yeast alcohol acetyl transferase, was used as a model system to demonstrate the beneficial effects of reducing acetate production. All strains tested for ester production also overexpressed panK, a native E. coli gene that previous studies have shown to increase free intracellular CoA levels when fed with pantothenic acid. A recombinant E. coli strain with a deletion in ackA-pta produces less acetate and more isoamyl acetate than the wild-type E. coli strain. When both acetate-producing pathways were deleted, the acetate production was greatly reduced. However, pyruvate began to accumulate, so that the overall ester production remained largely unchanged. To produce more ester, a previously established strategy of increasing the flux from pyruvate to acetyl-CoA was adopted by overexpressing pyruvate dehydrogenase. The ester production was then 80% higher in the poxB, ackA-pta strain (0.18 mM) than that found in the single ackA-pta mutant (0.10 mM), which also overexpressed PDH.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Increasing the acetyl-CoA pool in the presence of overexpressed phosphoenolpyruvate carboxylase or pyruvate carboxylase enhances succinate production in Escherichia coli.

An in vivo strategy to apply the activation effect of acetyl-CoA on phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (PYC) to increase succinate production in Escherichia coli was studied. This approach relies on the increased intracellular acetyl-CoA and CoA levels by overexpressing E. coli pantothenate kinase (PANK). The results showed that coexpression of PANK and PEPC, and PANK and PYC, did improve succinate production compared to the individual expression of PEPC and PYC, respectively. The intracellular acetyl-CoA and CoA levels were also measured, and each showed a significant increase when the PANK was overexpressed. Another effect observed was a decrease in lactate production. The least amount of lactate was produced when PANK and PEPC, and PANK and PYC, were coexpressed. This result showed increased competitiveness of the succinate pathway at the phosphoenolpyruvate and pyruvate nodes for the carbon flux, as a result reducing the carbon flux toward the lactate pathway. The study also demonstrates a feasible method for metabolic engineering to modulate enzyme activity in vivo through specific activators and inhibitors.

Acetyl Coenzyme A↗

PubMedDate not supplied

Characterization of the acetate-producing pathways in Escherichia coli.

Although the bacterium E. coli is chosen as the host in many bioprocesses, the accumulation of a common byproduct, acetate, is often problematic. Acetate, when present at high levels, will inhibit both cell growth and recombinant protein productivity. In addition, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that otherwise could have been used for the desired product. We have created mutant E. coli strains with a deletion of either the poxB or the ackA-pta pathway. These two strains, along with the wild-type strain, have been studied in batch reactors over a 12 h time period, at pH 7.0 and 6.0. The wild-type strain has also been studied using glucose as the carbon source. Data were collected to correlate cellular growth, extracellular metabolite production, enzyme activity, and gene expression. Results show that the ackA-pta pathway dominates in exponential phase, and the poxB pathway dominates in stationary phase. The ackA-pta pathway is repressed in acidic environments, whereas the poxB pathway is activated.

Acetate Kinase↗

PubMedDate not supplied

Enhanced lycopene productivity by manipulation of carbon flow to isopentenyl diphosphate in Escherichia coli.

Lycopene is a useful phytochemical that holds great commercial value. In our study the lycopene production pathway in E. coli originating from the precursor isopentenyl diphosphate (IPP) of the non-mevalonate pathway was reconstructed. This engineered strain of E. coli accumulated lycopene intracellularly under aerobic conditions. As a next step, the production of lycopene was enhanced through metabolic engineering methodologies. Various competing pathways at the pyruvate and acetyl-CoA nodes were inactivated to divert more carbon flux to IPP and subsequently to lycopene. It was found that the ackA-pta, nuo mutant produced a higher amount of lycopene compared to the parent strain. To further enhance lycopene production, a novel mevalonate pathway, in addition to the already existing non-mevalonate pathway, was engineered. This pathway utilizes acetyl-CoA as precursor, condensing it to form acetoacetyl-CoA and subsequently leading to formation of IPP. Upon the introduction of this new pathway, lycopene production increased by over 2-fold compared to the ackA-pta, nuo mutant strain.

Carbon↗

PubMedDate not supplied

Expression of the pfl gene and resulting metabolite flux distribution in nuo and ackA-pta E. coli mutant strains.

Our laboratory previously studied the interaction between nuo and the acetate-producing pathway encoded by ackA-pta in Escherichia coli. We examined metabolic patterns, particularly the ethanol and acetate production rates, of several mutant strains grown under anaerobic growth conditions. Since the pyruvate formate-lyase (PFL) pathway is the major route for acetyl-CoA and formate production under anaerobic conditions, we examined the effects of nuo and ackA/pta mutations on the expression of pyruvate formate-lyase (pfl) under anaerobic conditions. The ackA-pta mutant has a pfl::lacZ expression level much higher than that of the wild-type strain, and cultures also exhibit the highest ethanol production. Real-time PCR demonstrated that the adhE gene expression in the ack-pta mutant strain was approximately 100 fold that of the same gene in the ackA-pta nuo mutant strain. This result correlates with the observed ethanol production rates in cultures of the strain. However, the lack of exact correlation between the ethanol production rates and the RT-PCR data suggests additional regulation actions at the posttranslation level. In addition, the activity of the pfl gene as indicated by mRNA levels was also considerably greater in theack-pta mutant. We can conclude that deletions of nuo and ack/pta can partially affect the expression of the genes encoding adhE and pfl under anaerobic conditions.

Acetates↗

PubMedDate not supplied

Effect of overexpression of a soluble pyridine nucleotide transhydrogenase (UdhA) on the production of poly(3-hydroxybutyrate) in Escherichia coli.

A soluble pyridine nucleotide transhydrogenase (UdhA) has been used to increase the productivity and yield of PHB in vivo. By inducing a high level of UdhA, which can transfer reducing equivalents between NAD and NADP, we have increased NADPH availability, resulting in high yield and productivity of PHB in Escherichia coli. Coexpression of the phb operon from Alcaligenes eutrophus H16 and the native udhA from E. coli from high copy plasmids resulted in an increase in PHB yield from 49 to 66% g of PHB per gram of total cell dry weight and an increase in final concentration from 3.52 to 6.42 g/L; the PHB concentration of the udhA carrying strain is almost twice that of the control strain expressing only the phb operon. The results of this study demonstrate the effectiveness of cofactor manipulation and its application as a tool in metabolic engineering.

Escherichia coli↗