Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as “overflow metabolism” or “the Crabtree effect.” The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria

Simultaneous utilization of glucose, xylose and arabinose in the presence of acetate by a consortium of Escherichia coli strains

Background: The efficient microbial utilization of lignocellulosic hydrolysates has remained challenging because this material is composed of multiple sugars and also contains growth inhibitors such as acetic acid (acetate). Using an engineered consortium of strains derived from Escherichia coli C and a synthetic medium containing acetate, glucose, xylose and arabinose, we report on both the microbial removal of acetate and the subsequent simultaneous utilization of the sugars.Results: In a first stage, a strain unable to utilize glucose, xylose and arabinose (ALS1392, strain E. coli C ptsG manZ glk crr xylA araA) removed 3 g/L acetate within 30 hours. In a subsequent second stage, three E. coli strains (ALS1370, ALS1371, ALS1391), which are each engineered to utilize only one sugar, together simultaneously utilized glucose, xylose and arabinose. The effect of non-metabolizable sugars on the metabolism of the target sugar was minimal. Additionally the deletions necessary to prevent the consumption of one sugar only minimally affected the consumption of a desired sugar. For example, the crr deletion necessary to prevent glucose consumption reduced xylose and arabinose utilization by less than 15% compared to the wild-type. Similarly, the araA deletion used to exclude arabinose consumption did not affect xylose- and glucose-consumption.Conclusions: Despite the modest reduction in the overall rate of sugar consumption due to the various deletions that were required to generate the consortium of strains, the approach constitutes a significant improvement in any single-organism approach to utilize sugars found in lignocellulosic hydrolysate in the presence of acetate

Process Design for the Biocatalysis of Value-Added Chemicals from Carbon Dioxide

This report describes results toward developing a process to sequester CO{sub 2} centered on the enzymes PEP carboxylase and pyruvate carboxylase. The process involves the use of bacteria to convert CO{sub 2} and glucose as a co-substrate and generates succinic acid as a commodity chemical product. The study reports on strain development and process development. In the area of strain development, knockouts in genes which divert carbon from the enzymatic steps involved in CO{sub 2} consumption were completed, and were shown not to affect significantly the rate of CO{sub 2} sequestration and succinic acid generation. Furthermore, the pyc gene encoding for pyruvate carboxylase proved to be unstable when integrated onto the chromosome. In the area of process development, an optimal medium, pH and base counterion were obtained, leading to a sequestration rate as great as 800 mg/Lh. Detailed studies of gas phase composition demonstrated that CO{sub 2} composition has a significant affect on CO{sub 2} sequestration, while the presence of 'toxic' compounds in the gas, including NO{sub 2}, CO and SO{sub 2} did not have a detrimental effect on sequestration. Some results on prolonging the rate of sequestration indicate that enzyme activities decrease with time, suggesting methods to prolong enzyme activity may benefit the overall process

A co-fermentation strategy to consume sugar mixtures effectively

We report a new approach for the simultaneous conversion of xylose and glucose sugar mixtures into products by fermentation. The process simultaneously uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. The xylose-selective (glucose deficient) strain E. coli ZSC113 has mutations in the glk, ptsG and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1008 has a mutation in the xylA gene. By combining these two strains in a single process, xylose and glucose are consumed more quickly than by a single-organism approach. Moreover, we demonstrate that the process is able to adapt to changing concentrations of these two sugars, and therefore holds promise for the conversion of variable sugar feed streams, such as lignocellulosic hydrolysates

Molecular communication: An acid tale of prion formation

Some bacteria use lactic acid to communicate with yeast cells

Effect of overexpressing nhaA and nhaR on sodium tolerance and lactate production in Escherichia coli

Background: Like other bacteria, Escherichia coli must carefully regulate the intracellular concentration of sodium ion (Na+). During the bacterial production of any organic acid, cations like Na+ invariably accumulate during a process which must maintain a near neutral pH. In this study, the E. coli nhaA gene encoding the Na+/H+ antiporter membrane protein and the nhaR gene encoding the NhaA regulatory protein were overexpressed in wild-type E. coli MG1655 and in MG1655 pflB (ALS1317) which lacks pyruvate formate lyase activity and thus accumulates lactate under anaerobic conditions.Results: Expression of either the nhaA or nhaR gene on the high copy inducible expression vector pTrc99A caused a significant reduction in the growth rate of MG1655. No change in growth rate was observed for MG1655 or ALS1317 for Na+ concentrations of 0.75-0.90 M when the medium copy pBR322 plasmid was used to overexpress the two genes. In a fed-batch process to produce the model acid lactate with NaOH addition for pH control, lactate accumulation ceased in MG1655, MG1655/pBR322, MG1655/pBR322-nhaR and MG1655/pBR322-nhaA when the concentration reached 55-58 g/L. In an identical process lactate accumulation in MG1655/pBR322-nhaAR did not terminate until the concentration reached over 70 g/L.Conclusions: Although overexpression the genes did not improve growth rate at high Na+ concentrations, the overexpression of nhaA and nhaR together led to a 25% increase in lactate production. Thus, the observed (absence of) impact that these genetic modifications had on growth rate is a poor indicator of their effect on acid accumulation. The overexpression of nhaAR did not cause faster lactate production, but permitted the culture to continue accumulating lactate at 10% greater Na+ concentration

Physicochemical characterization of the PEG8000-Na2SO4 aqueous two-phase system

The polyethylene glycol-sodium sulfate aqueous two-phase system has been characterized at 23 °C. Tielines for the phase diagram were obtained experimentally. Phases in equilibrium were characterized by means of the solvatochromic parameters π*, α, and β, which provide a measurement of the polarity/polarizability and the H-bond donor and acceptor abilities, respectively. The ability of the phases to participate in hydrophobic interactions was characterized by means of the free energy of transfer of a methylene group between the conjugated phases, using the partition of a homologous series of dinitrophenylated amino acids. The results show the effect of the presence of polymer and salt in the aqueous phase, and a comparison of both phases with pure water is made.LSRE-PortoUniversidade Católica PortuguesaEscola Superior de Biotecnologia do PortoFundação para a Ciência e a Tecnologia (FCT

Multidisciplinary Graduate Education in Bioprocess Engineering

This report describes the accomplishments of the University of Georgia in establishing an academic program geared toward the emerging biobased products industry. By virtue of its strengths and structure, the University of Georgia is particularly well-suited for developing a program focused on plant- and microbial-based bioproducts, and it was in this general area that this program was developed. The program had several unique characteristics. First, we implemented a distinguished lecture series that brought outstanding scientists and engineers to our University to interact with students and share their vision of the biobased economy. Second, we offered industrially-oriented and multidisciplinary courses that provided students with a broad background on various facets of biobased business and technology. Third, we provided the students with opportunities to expand beyond the classroom by engaging in research lab rotations and industrial internships. Fourth, each student was engaged in a creative research project as led by a multidisciplinary faculty team. Throughout the implementation of these activities, we maintained a student-centered, mentoring approach to education. The most tangible outcome of this project was the graduation of two students who participated in a variety of scholarly activities, culminating in research toward the completion of a thesis and dissertation. Both research projects involved the use of microorganisms to produce industrial products from agricultural substrates via fermentation processes. The research advanced our understanding of microorganisms as used for industrial processes and products, as described in several articles published in scholarly journals and presentations made at scientific conferences (see information on pp. 14-15). Another outcome is one graduate course, Fermentation Engineering Laboratory, which is a unique experiential and multidisciplinary course. This course will be offered in the future as an elective to graduate students in several engineering and science degree programs. Other significant developments have arisen as direct or indirect consequences of this project. The University of Georgia has established a B.S. Biochemical Engineering degree and an M.S. Biochemical Engineering degree. A strong component of these degree programs is education toward a biobased economy. We will integrate particularly positive components of this project (such as the distinguished lecture series) into these degree programs. The University of Georgia is establishing a Center for Biorefining and Carbon Cycling. This multidisciplinary Center houses a pilot scale biorefinery, comprising a pyrolysis unit and an ethanol plant. Together with new faculty positions that are currently being advertised, this project has encouraged the University of Georgia to assume a leadership role in the preparation of students in the biobased industries of the future

Process Design for the Biocatalysis of Value-Added Chemicals from Carbon Dioxide

This report describes results toward developing a process to sequester CO{sub 2} centered on the enzyme pyruvate carboxylase. The process involves the use of bacteria to convert CO{sub 2} and glucose as a co-substrate and generates succinic acid as a commodity chemical product. The first phase of this research has focused on strain development and on process development. Progress in strain development has been made in three areas. The gene encoding for alcohol dehydrogenase has been ''knocked out'' of the bacteria, and thereby eliminating the synthesis of the by-product ethanol. The gene for glucokinase has been overexpressed in the production strain with the goal of faster utilization of glucose (and hence CO{sub 2}). Efforts have continued toward integrating pyruvate carboxylase gene (pyc) onto the E. coli chromosome. Progress in process development has come in conducting several dozen fermentation experiments to find a defined medium that would be successful for the growth of the bacteria, while permitting a high rate of CO{sub 2} utilization in a subsequent prolonged production phase. Using this defined medium, the strains that continue to be constructed are being compared for CO{sub 2} utilization, so that we may understand the factors that govern the biological sequestration process