The reactions and enzymes involved with nucleotide cofactors, as listed in KEGG
1. Introduction
Microorganisms are able to produce a wide range of valuable chemicals and materials, and microbial fermentation is widely used as an alternative route for the production of chemicals in industry[1]. The key elements that determine the efficiency of a fermentation process are high titer, high yield, high productivity and process robustness[2]. These parameters are highly dependent on the host microorganism. In order to enhance the metabolic capabilities of the host microorganism, early research focused on screening appropriate microorganisms that naturally overproduce target products and improving their performance by random mutagenesis and by optimizing the fermentation processes. With the advent of metabolic engineering, many different genetic or metabolic engineering strategies have been adopted to improve the metabolic capabilities of the host strains, including relief of feedback inhibition, deletion of competing pathways, up-regulation of primary biosynthetic pathways, re-direction of central metabolism towards the target pathway, over-expression of export processes and insertion of new metabolic pathways. More recently, the emergence of systems biology integrated with metabolic engineering has provided a comprehensive understanding of microbial physiology, followed by a more global-wide identification of the target genes to be manipulated[3]. Those approaches have been proven to be powerful in developing microbial strains for the commercial production of organic acids[4], amino acids, biofuels and pharmaceuticals[5,6,7]. Nevertheless, problems such as the accumulation of toxic intermediates or metabolic stress resulting in decreased cellular fitness are still far from being solved. Over-expression, deletion or introduction of heterologous genes in target metabolic pathways does not always result in the desired phenotype. A good example is the attempts to increase the glycolytic flux, which cannot be increased by individual or combinational over-expression of genes encoding the key enzymes in either a eukaryotic or prokaryotic microorganism[8]. The essence of the problems listed above lies in the fact that, in addition to the modification of key genes by metabolic engineering, the researcher needs to study the effects of the internal environment (e.g. the intracellular energy charge and the interior redox potential and intracellular pH) on the phenotype, based on an accurate analysis of the metabolic network structure. If such an approach is adopted, manipulation of the form and level of intracellular cofactors can potentially be an efficient strategy for obtaining a desired phenotype.
In 1998, Hugenholtz from Delft University of Technology introduced the
ATP | ADP | NADH | NAD+ | NADPH | NADP+ | CoA | Acetyl-CoA | |
Number of reactions | 496 | 347 | 740 | 750 | 887 | 889 | 480 | 169 |
Number of enzymes | 454 | 350 | 433 | 455 | 462 | 462 | 250 | 119 |
2. Strategies and applications of ATP manipulation
ATP, a kind of nucleotide, widely serves as substrate, product, activator or/and inhibitor in metabolic networks. Based on these four basic functions, the demand and supply of ATP could affect active transportation, peptide folding, subunit assembly, protein relocation and phosphorylation, cell morphology, signal transduction, and stress response. Through these complicated physiology process, ATP is involved in many metabolic pathways and production of almost all of the metabolites by industrial strains. Therefore, the manipulation of ATP supply and demand could be a powerful tool to increase the metabolic performance of industrial strains. Substrate-level phosphorylation (anaerobic conditions) and oxidative phosphorylation (aerobic conditions) were two different ATP regeneration pathways. It seems that manipulation of oxidative phosphorylation was a more efficient way to regulate the intracellular ATP concentration, because under aerobic conditions, most ATP production origin from oxidative phosphorylation pathway. It is conceivable that NADH availability, electron transfer chain (ETC), proton gradient, F0F1-ATPase and oxygen supply could all be regulatory candidates for manipulating the intracellular ATP availability.
2.1. Strategies for manipulation ATP availability
Intracellular NADH, produced from glycolysis, fatty acid oxidation, and the citric acid cycle, can be converted to NAD in three separate ways. Under aerobic growth, NADH oxidation occurs through ETC, in which oxygen is used as the final electron acceptor, and a large amount of ATP is produced. Under anaerobic growth and in the absence of an alternate oxidizing agent, the oxidation of NADH can occur by fermentative pathways, such as aldehyde dehydrogenase[11], or lactate dehydrogenase[12]. In this case, energy production is mainly from substrate-level phosphorylation. NADH can also be directly oxidized into water and NAD through NADH oxidase[13]. Therefore, manipulating the availability and oxidation pathway of NADH may be an efficient strategy to manipulate the intracellular ATP level.
There are three different strategies to manipulate the NADH availability to adjust the intracellular ATP content, based on NADH-related metabolic pathways. Firstly, manipulating NADH availability through over-expression or deletion of the key NADH related enzymes, such as
Complex I, II, III and IV are the key components of ETC and play the vital role in ATP production. Focusing on those four different complexes, three separate strategies have been used to disrupt the ETC's capacity to reduce energy production. To decrease ATP content by disrupting ETC, specific inhibitors of ETC components were added to the culture broth and a reduced ATP level was observed[15]. For yeast
In aerobic growth, when oxygen is used as the final electron acceptor of the ETC, the abundance of oxygen in culture broth is the decisive environmental factor of ATP production, especially for some fermentation processes, which are high-density, high-viscous and high-energy requiring. Many studies have demonstrated that an increased ATP supply can be achieved by increasing oxygen supply. In the past decades, the strategies of process control and genetic modification have been applied to enhance the ATP production efficiency through increasing oxygen supply. The first strategy can be further divided into two different approaches. One is controlling the aeration rate through the agitation speed in bioreactors, or aeration with pure oxygen[17,18,19]. Many complicated oxygen-supply control strategies were developed based on these simple methods. Another approach is adding oxygen vectors to the culture broth, such as
F0F1-ATPase, the final component of oxidative phosphorylation, plays the central role in ATP production. Three different methods have been performed to reduce the intracellular ATP by decreasing F0F1-ATPase activity. The first was supplementing the culture medium with an external and specific inhibitor of F0F1-ATPase, such as oligomycin, neomycin and N’,N’-dicyclohexylcarbodiimide[21]. The second was genetic manipulation and traditional mutation of F0F1-ATPase[22]. In prokaryotic microorganisms,
2.2. Applications of ATP manipulation
The strategies to enhance the concentration, the yield, and the productivity of the target metabolites with ATP-based manipulation could be divided into three groups: (1) decreasing ATP supply; (2) increasing ATP supply; (3) multi-phase ATP-supply regulation strategies. The ultimate objective of ATP manipulation is to achieve the highest product concentration, the highest yield and the highest productivity, singly or in combination. In the past decades, ATP-oriented bioprocess optimization has developed expeditiously, and has successfully extended the boundaries of metabolic engineering. Here we present some representative works to further illustrate the concept of bioprocess optimization based on the regulation of ATP availability.
A higher target metabolite concentration in the fermentation broth increases the bioreactor utility and reduces the expense for the subsequent extraction process. Regulation of the ATP availability in industrial strain could further increase the target metabolite concentration. Three examples are presented to illustrate the feasibility of further increasing target metabolite concentration by increasing ATP supply.
Studies had demonstrated that a continuous and abundant supply of ATP was essential for glutathione (GSH) synthesis and secretion. A direct, efficient, but costly method to further increase GSH production is to supplement the culture broth with pure ATP, although this is too expensive to use on an industrial scale. Since 1978, researchers have been attempting to establish a coupled system for GSH production using genetically engineered
Increasing the fermentation productivity is an efficient way to increase the economy of bioprocess, because a high productivity decreases the fermentation period, the cost of equipment and energy expenditure. For this aim, it is extremely important to increase the rate of carbon flux through central metabolic pathways,
On the other hand, an elevated intracellular ATP level can also improve the productivity of some metabolites. For hyaluronic acid (HA) production by
During fine or bulk chemicals production by industrial strain, many byproducts, such as acetic acid, lactic acid and glycerol, are secreted into the culture broth. The accumulation of byproducts results in a decrease in the yield of product on substrate and an increase in the bioprocess cost, and the environmental burden. The following examples illustrate how to decrease the byproduct formation by manipulating ATP-related metabolic pathways[29,30].
For production of penicillin and its derivatives by
During the bioprocess, industrial strain may encounter a series of environmental stresses, such as acid, cold, oxidative and osmotic changes. As a consequence, the survival, growth, and metabolic function of industrial strain are affected by those stresses. A number of environmental stress resistance mechanisms have been identified and characterized. It was hypothesized that the supply of ATP plays significant roles in facilitating the stress response of industrial strain, through active transport and signaling pathways[35,36]. The primary mechanism by which industrial strain survive high stress is to control the intracellular environment by membrane-bound ATPases, which translocate specific ions to the environment at the expense of ATP hydrolysis. A deficiency in those ATPases greatly weakens the cells' resistance to environmental challenges, resulting in the cessation of growth and target metabolite accumulation. For instance, a mutant of
The ATP-based stress-induced signaling pathways have been widely studied in industrial strain. ATP was an essential substrate for signal pathways. Several signal transduction nodes in the high osmotic glycerol (HOG) pathway were shown to use ATP as an energy source in protecting against high osmotic stress. Similarly, ATP also facilitated signaling in other stress response networks, such as the signal of cold stress, heat stress and oxidative stress. In turn, some signaling pathways could also affect ATP synthesis under stress. In
3. Strategies and applications of NADH manipulation
As the predominant redox product of catabolism, NADH has been found to be involved in more than 700 biochemical reactions in the microbial metabolic network (Table 1). Its
Manipulation strategy | Results/conclusion | Ref. |
Feeding external electron acceptors | ||
Acetaldehyde | Decreased NADH/NAD+ ratio | [47] |
Fumarate or nitrate | Decreased NADH/NAD+ ratio | [48] |
Acetoin | Decreased NADH level | [49,50] |
Pyruvate, citrate, O2 or fructose | Decreased NAD(P)H level | [51] |
Furfural | Decreased NADH level | [52] |
Adding carbon sources with different oxidation states | ||
Sorbitol | Increased NADH availability | [44] |
Gluconate | Decreased NADH/NAD+ ratio | [53] |
Adding a NAD+ precursor | ||
Nicotinic acid | Increased NAD+ level | [47] |
Altering culture conditions | ||
Lower temperature | Increased NADH/NAD+ ratio | |
Increased dissolved oxygen level | Increased NADH availability | [54] |
Extracellular oxidoreduction potential | Decreased NAD+/NADH ratio in a relatively oxidative environment | [55] |
Over-expressing enzymes association with NADH metabolism | ||
Nicotinic acid phosphoribosyltransferase | Increased NAD+ levels and decreased NADH/NAD+ ratio | [56] |
Eliminating NADH competition pathways | ||
Inactivating aldehyde dehydrogenase | Increased NADH availability | [57] |
Deactivating | Increased NADH availability | [58] |
Introducing heterogeneous NADH metabolic pathways | ||
H2O-NADH oxidase | Decreased NADH level and NADH/NAD+ ratio | [59] |
Alternative oxidase | Decreased NADH/NAD+ ratio | [60] |
NAD+-dependent formate dehydrogenase | Increased NADH availability | [53,61] |
physiological roles can be divided into five aspects (Fig. 1): (1) regulation of energy metabolism – NADH uses oxygen as the final electron acceptor to produce a large quantity of ATP through the electron transport chain in mitochondria; (2) adjustment of the microbial intracellular redox state – NADH/NAD+ is the main index of redox potential; (3) manipulation of carbon flux – NADH can redistribute carbon flux by activating or inhibiting key enzymes in the target metabolic pathway; (4) modification of mitochondrial function – NADH can modify mitochondrial function by affecting mitochondrial permeability, controlling the mitochondrial membrane anion channel and increasing the mitochondrial membrane potential; (5) regulation of cell life cycle. Based on the above, it is conceivable that NADH/NAD+ could potentially act as an efficient tool to manipulate microbial growth and phenotype. In general, there are two different manipulation strategies for NAD(H/+) availability (Table 2): (1) biochemical engineering approaches that include feeding external electron acceptors, adding carbon sources with different oxidation potentials or NAD synthesis precursors to the fermentation broth and controlling the culture conditions, such as the dissolved oxygen content, temperature and extracellular oxidoreduction potential[44,45]; (2) metabolic engineering methods such as over-expressing enzymes associated with NAD+(NADH) metabolism, eliminating NAD+(NADH) competition pathways and introducing an NAD+(NADH) regeneration system[46]. These strategies have been proven to provide efficient control of the intracellular NAD+(NADH) content.
3.1. Manipulation of NADH availability through biochemical engineering approaches
Many reports have demonstrated that aldehydes, ketones, organic acids, molecular nitrogen or nitrate can be used as internal electron acceptors to enhance NADH oxidation to maintain an optimum oxidoreduction level (using the NADH/NAD+ ratio as the index) in industrial microorganisms. For example, during the heterofermentative lactic acid fermentation by
When glucose, sorbitol and gluconate were compared as carbon sources in microbial glycolysis, sorbitol produced more NADH than glucose while gluconate was transformed directly to pyruvate with no NADH production. Therefore, the oxidation states of these three different carbon sources were -1 (sorbitol), 0 (glucose) and +1 (gluconate). It is thus conceivable that these carbon sources will have a pronounced effect on the intracellular NADH/NAD+ ratio and subsequently on the carbon flux distribution. In a series of chemostat experiments under anaerobic conditions, San et al used three different carbon sources as a simple way of manipulating the cellular NADH/NAD+ ratio from 0.51 (gluconate) to 0.75 (glucose) to 0.94 (sorbitol). The changes in the NADH/NAD+ ratio increased the ethanol to acetate ratio from 1.00 with glucose to 3.62 with sorbitol and decreased it to 0.29 with gluconate. This result provided a simple method for manipulating the distribution of metabolic flux to the desired metabolites. In the case of succinate production by an engineered strain of
In microbial cells, there are two different NAD synthesis pathways for maintaining the total NADH/NAD+ intracellular pool: the de novo pathway and the pyridine nucleotide salvage pathway. For the de novo pathway, NAD is synthesized from aspartate and dihydroxyacetone phosphate. The pyridine nucleotide salvage pathway recycles intracellular NADH breakdown products, such as nicotinamide mononucleotide (NMN), as well as other preformed pyridine compounds from the environment, such as nicotinamide and nicotinic acid (NA). As the NA concentration (8 mg/L) increased in the fermentation medium of
External environmental conditions, such as the dissolved oxygen concentration and temperature, also modulate the intracellular NADH, NAD+ and ATP levels, thus shifting the metabolic pattern. Under oxygen limited conditions, oxidation of NADH in
The extracellular oxidoreduction potential (ORP) of the fermentation medium is a comprehensive index of environmental conditions, essentially depending on the chemical composition, pH, temperature and dissolved oxygen (DO) concentration of the culture medium. Many reports have revealed that ORP plays a major role in the distribution of carbon flux through changes in the activities of key enzymes that have a metal cofactor in the active site. Du suggested that ORP manipulates the NADH level by affecting the activities of some NADH- or NAD+-related enzymes that participate in electron transcription. Another example was presented by Qin et al, when potassium ferricyanide was added to the
3.2. Manipulation of NADH availability through metabolic engineering strategies
The second strategy that can be used to manipulate NADH availability is the metabolic engineering approach. The application of genetic and metabolic engineering has the potential to considerably affect NADH availability through the amplification, addition or deletion of NAD-related metabolic pathways. Two distinct genetic engineering methods can be used: the first approach aims at increasing the total NAD(H/+) pool while the second approach focuses on changing the NADH/NAD+ ratio. It is also conceivable that a combination of these two approaches may lead to both an increased NAD(H/+) pool and an increased ratio. As previously mentioned, nicotinic acid can be used to directly synthesize nicotinate mononucleotide, a direct precursor of NAD, catalyzed by nicotinic acid phosphoribosyltransferase (NAPRTase; EC2.4.2.11). This enzyme is encoded by the
The second approach to manipulating NADH is the deletion or weakening of the NADH competition pathways (Fig.2), to redirect NADH to the target metabolic pathway to enhance the production of the desired metabolites. By inactivating aldehyde dehydrogenase (ALDH) in
The third approach for manipulating NADH is heterologous production of oxido-reduction related enzymes, to change the ways NADH is regenerated or oxidized, thus changing the ratio NADH/NAD+. In microbial cells, cytosolic NADH needs to shuttle to the mitochondria and be oxidized. It is conceivable that an increase in the efficiency and rate of NADH oxidation may be achieved through over-expression of water-forming NADH oxidase, which directly oxidizes NADH to NAD+ in the cytoplasm. In
4. Strategies and applications of CoA manipulation
As acetyl carriers, coenzyme A (CoA) (Table 2) and its derivatives, acetyl-CoA, succinyl coenzyme A (succinyl-CoA) and malonyl coenzyme A (malonyl-CoA), are involved in more than 600 biochemical reactions in microbial cell metabolism. Acetyl-CoA is an essential intermediate in many energy-yielding metabolic pathways and is a substrate in enzymatic
production of industrially useful compounds such as esters and lipid molecules. As illustrated in Fig. 3, CoA and its derivatives take part in a variety of metabolic functions, such as the citric acid cycle, fatty acid synthesis and decomposition, macromolecule fat synthesis, amino acid metabolism, ketogenesis, sterol synthesis and as regulators to control some key enzymes in specific metabolic pathway[62,63].
4.1. Strategies of CoA manipulation
The total levels of CoA and its derivatives are dependent on the kinds and amounts of carbon sources in the fermentation medium. When
4.2. Applications of CoA manipulation
From the above discussion, it can be concluded that the ratio of acetyl-CoA/CoA is a key index that reflects the metabolic state of carbon and energy metabolism during the fermentation process. San et al over-expressed pantothenate kinase in
Apart from metabolic engineering strategies, the biochemical engineering strategy has been proven to be an effective way to manipulate CoA and acetyl-CoA levels and the acetyl-CoA/CoA ratio. An example is the manipulation of thiamine, biotin and Ca2+ levels as a tool for redistributing carbon flux from pyruvate to -ketoglutarate in
5. Concluding remarks and future directions
Recently, many studies have demonstrated that the fermentation process from sugar to the target product is not just a simple biochemical reaction but rather a comprehensive network, including the gene regulatory network, protein-protein interaction network, signal transduction network and metabolic network, dependent on the physical and chemical interactions of genes, proteins and metabolites. Therefore, interest in cofactor engineering in the future should be concerned with: (1) identification of the active site of the cofactor in the biochemical reaction, metabolic pathway and metabolic network; (2) evaluation of the effect of the cofactor on the metabolic reaction, pathway and network; (3) finding the threshold values at which the metabolic networks and regulatory networks respond to cofactor changes; (4) development of directed, precise strategies for cofactor manipulation. The increasing availability of genome sequences and accumulation of high-throughput biological data allow us to understand the physiological functions of cofactors and to propose precise strategies for cofactor manipulation of microbial physiology.
References
- 1.
Nielsen J. Otero J. M. 2010 Industrial Systems Biology .105 439 460 . - 2.
Nielsen J. Liu L. M. Agren R. Bordel S. 2010 Use of genome-scale metabolic models for understanding microbial physiology. FEBS Letters584 2556 2564 . - 3.
Lee S. Y. Park J. H. 2008 Towards systems metabolic engineering of microorganisms for amino acid production. Current Opinion in Biotechnology19 454 460 . - 4.
Lin H. Bennett G. N. San K. Y. 2005 Fed-batch culture of a metabolically engineered Escherichia coli strain designed for high-level succinate production and yield under aerobic conditions. 90 775 779 . - 5.
Liao J. C. Atsumi S. 2008 Metabolic engineering for advanced biofuels production from Escherichia coli. Current Opinion in Biotechnology19 414 419 . - 6.
Wendisch V. F. Bott M. Eikmanns B. J. 2006 Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids .9 268 274 . - 7.
Xian M. Yu C. Cao Y. J. Zou H. B. 2011 Metabolic engineering of Escherichia coli for biotechnological production of high-value organic acids and alcohols .89 573 583 . - 8.
Chen J. Liu L. M. Li Y. Li H. Z. 2006 Significant increase of glycolytic flux in Torulopsis glabrata by inhibition of oxidative phosphorylation .6 1117 1129 . - 9.
Lopez de Felipe. F. Kleerebezem M. de Vos W. M. Hugenholtz J. 1998 Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. Journal of Bacteriology180 3804 3808 . - 10.
Aoki-Kinoshita K. F. 2006 Overview of KEGG applications to omics-related research. Journal of Pesticide Science31 296 299 . - 11.
Cao Z. Zhang Y. P. Li Y. Du C. Y. Liu M. 2006 Inactivation of aldehyde dehydrogenase: A key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae. Metabolic Engineering8 578 586 . - 12.
Zhu J. Shimizu K. 2004 The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli. Applied Microbiology and Biotechnology64 367 375 . - 13.
Nielsen J. Vemuri G. N. Eiteman M. A. Mc Ewen J. E. Olsson L. 2007 Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America104 2402 2407 . - 14.
Underwood S. A. Zhou S. Causey T. B. Yomano L. P. Shanmugam K. T. et al. 2002 Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli. 68 6263 6272 . - 15.
Miyoshi H. Abe M. Kubo A. Yamamoto S. Hatoh Y. et al. 2008 Dynamic function of the spacer region of acetogenins in the inhibition of bovine mitochondrial NADH-ubiquinone oxidoreductase (complex I) .47 6260 6266 . - 16.
Johnson CH, Prigge JT, Warren AD, McEwen JE 2003 Characterization of an alternative oxidase activity of Histoplasma capsulatum. Yeast20 381 388 . - 17.
Huang H. Qu L. Ji X. J. Ren L. J. Nie Z. K. et al. 2011 Enhancement of docosahexaenoic acid production by Schizochytrium sp. using a two-stage oxygen supply control strategy based on oxygen transfer coefficient. 52 22 27 . - 18.
Huang H. Peng C. Ji X. J. Liu X. Ren L. J. et al. 2010 Effects of n-Hexadecane Concentration and a Two-Stage Oxygen Supply Control Strategy on Arachidonic Acid Production by Mortierella Alpina ME-1 .33 692 697 . - 19.
Li Y. Hugenholtz J. Chen J. Lun S. Y. 2002 Enhancement of pyruvate production by Torulopsis glabrata using a two-stage oxygen supply control strategy .60 101 106 . - 20.
Tang K. X. Zhang L. Li Y. J. Wang Z. N. Xia Y. et al. 2007 Recent developments and future prospects of Vitreoscilla hemoglobin application in metabolic engineering .25 123 136 . - 21.
Johnson K. M. Cleary J. CA Fierke Opipari. A. W. Jr Glick G. D. 2006 Mechanistic basis for therapeutic targeting of the mitochondrial F1F0-ATPase. ACS Chem Biol1 304 308 . - 22.
Yokota A. Henmi M. Takaoka N. Hayashi C. Takezawa Y. et al. 1997 Enhancement of glucose metabolism in a pyruvic acid-hyperproducing Escherichia coli mutant defective in F-1-ATPase activity. Journal of Fermentation and Bioengineering83 132 138 . - 23.
Sekine H. Shimada T. Hayashi C. Ishiguro A. Tomita F. et al. 2001 H+-ATPase defect in Corynebacterium glutamicum abolishes glutamic acid production with enhancement of glucose consumption rate. Appl Microbiol Biotechnol57 534 540 . - 24.
Liu L. M. Li Y. Du G. C. Chen J. 2006 Increasing glycolytic flux in Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation. Journal of Applied Microbiology100 1043 1053 . - 25.
Garcia J. J. Morales-Rios E. Cortes-Hernandez P. Rodriguez-Zavala J. S. 2006 The inhibitor protein (IF1) promotes dimerization of the mitochondrial F1F0-ATP synthase. 45 12695 12703 . - 26.
Zhang X. Liu S. Takano T. 2008 Overexpression of a mitochondrial ATP synthase small subunit gene (AtMtATP6) confers tolerance to several abiotic stresses in Saccharomyces cerevisiae and Arabidopsis thaliana. Biotechnol Letter30 1289 1294 . - 27.
Liao X. Deng T. Zhu Y. Du G. Chen J. 2008 Enhancement of glutathione production by altering adenosine metabolism of Escherichia coli in a coupled ATP regeneration system with Saccharomyces cerevisiae. Journal of Applied Microbiology104 345 352 . - 28.
Ahn W. S. Park S. J. Lee S. Y. 2000 Production of Poly(3-hydroxybutyrate) by fed-batch culture of recombinant Escherichia coli with a highly concentrated whey solution. Applied Environmental Microbiology66 3624 3627 . - 29.
Karakashev D. Thomsen A. B. Angelidaki I. 2007 Anaerobic biotechnological approaches for production of liquid energy carriers from biomass. Biotechnology Letter29 1005 1012 . - 30.
Suwannakham S. Huang Y. Yang S. T. 2006 Construction and characterization of ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation. 94 383 395 . - 31.
Harris D. M. van der Krogt Z. A. van Gulik W. M. van Dijken J. P. Pronk J. T. 2007 Formate as an auxiliary substrate for glucose-limited cultivation of Penicillium chrysogenum: impact on penicillin G production and biomass yield. Applied Environmental Microbiology73 5020 5025 . - 32.
Garrido J. M. van Benthum W. A. van Loosdrecht M. C. Heijnen J. J. 1997 Influence of dissolved oxygen concentration on nitrite accumulation in a biofilm airlift suspension reactor. Biotechnology and Bioengineering53 168 178 . - 33.
van Gulik W. M. de Laat W. T. Vinke J. L. Heijnen J. J. 2000 Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G. Biotechnology and Bioengineering68 602 618 . - 34.
Neves A. R. Pool W. A. Kok J. Kuipers O. P. Santos H. 2005 Overview on sugar metabolism and its control in Lactococcus lactis- the input from in vivo NMR. FEMS Microbiol Review29 531 554 . - 35.
Lokanath N. K. Matsuura Y. Kuroishi C. Takahashi N. Kunishima N. 2007 Dimeric core structure of modular stator subunit E of archaeal H+-ATPase. J Mol Biol366 933 944 . - 36.
Salinas P. Ruiz D. Cantos R. Lopez-Redondo M. L. Marina A. et al. 2007 The regulatory factor SipA provides a link between NblS and NblR signal transduction pathways in the cyanobacterium Synechococcus sp. PCC 7942. Mol Microbiol66 1607 1619 . - 37.
Shima J. Ando A. Takagi H. 2008 Possible roles of vacuolar H+-ATPase and mitochondrial function in tolerance to air-drying stress revealed by genome-wide screening of Saccharomyces cerevisiae deletion strains. Yeast25 179 190 . - 38.
Milgrom E. Diab H. Middleton F. Kane P. M. 2007 Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress .282 7125 7136 . - 39.
Stewart C. M. Cole M. B. JD Legan Slade. L. Schaffner D. W. 2005 Solute-specific effects of osmotic stress on Staphylococcus aureus .98 193 202 . - 40.
Hasona A. Crowley P. J. Levesque C. M. Mair R. W. Cvitkovitch D. G. et al. 2005 Streptococcal viability and diminished stress tolerance in mutants lacking the signal recognition particle pathway or YidC2 . Proc Natl Acad Sci U S A102 17466 17471 . - 41.
Hasona A. Zuobi-Hasona K. Crowley P. J. Abranches J. MA Ruelf et. al 2007 Membrane composition changes and physiological adaptation by Streptococcus mutans signal recognition particle pathway mutants. 189 1219 1230 . - 42.
Canovas M. Bernal V. Sevilla A. Iborra J. L. 2007 Salt stress effects on the central and carnitine metabolisms of Escherichia coli. 96 722 737 . - 43.
Sanchez C. Neves A. R. Cavalheiro J. dos Santos. MM Garcia-Quintans N. et al. 2008 Contribution of citrate metabolism to the growth of Lactococcus lactis CRL264 at low pH. Appl Environ Microbiol74 1136 1144 . - 44.
Lin H. Bennett G. N. San K. Y. 2005 Effect of carbon sources differing in oxidation state and transport route on succinate production in metabolically engineered Escherichia coli. J Ind Microbiol Biotechnol32 87 93 . - 45.
Ma Pan B. Zupancic S. J. Cormack M. L. B. P. 2007 Assimilation of NAD(+) precursors in Candida glabrata. Mol Microbiol66 14 25 . - 46.
Cordier H. Mendes F. Vasconcelos I. Francois J. M. 2007 A metabolic and genomic study of engineered Saccharomyces cerevisiae strains for high glycerol production. Metabolic Engineering9 364 378 . - 47.
Liu L. M. Li Y. Shi Z. P. Du G. C. Chen 2006 (2006) Enhancement of pyruvate productivity in Torulopsis glabrata: Increase of NAD(+) availability. J Biotechnol126 173 185 . - 48.
de Graef M. R. Alexeeva S. Snoep J. L. Teixeira de Mattos. M. J. 1999 The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli. J Bacteriol181 2351 2357 . - 49.
Panagiotou G. Christakopoulos P. 2004 NADPH-dependent D-aldose reductases and xylose fermentation in Fusarium oxysporum. J Biosci Bioeng97 299 304 . - 50.
Panagiotou G. Christakopoulos P. Villas-Boas S. G. Olsson L. 2005 Fermentation performance and intracellular metabolite profiling of Fusarium oxysporum cultivated on a glucose-xylose mixture. Enzyme Microb Technol36 100 106 . - 51.
Zaunmuller T. Eichert M. Richter H. Unden G. 2006 Variations in the energy metabolism of biotechnologically relevant heterofermentative lactic acid bacteria during growth on sugars and organic acids. Applied Microbiology Biotechnology72 421 429 . - 52.
Wahlbom C. F. Hahn-Hagerdal B. 2002 Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae. BiotechnologyBioengineering78 172 178 . - 53.
San K. Y. Bennett G. N. Berrios-Rivera S. J. Vadali R. V. Yang Y. T. et al. 2002 Metabolic engineering through cofactor manipulation and its effects on metabolic flux redistribution in Escherichia coli. Metabolic Engineering4 182 192 . - 54.
Qiang H. Shimuzu K. 1999 Effect of dissolved oxygen concentration on the intracellular flux distribution for pyruvate fermentation. Journal of Biotechnology68 135 147 . - 55.
Du C. Y. Yan H. Zhang Y. P. Li Y. Cao Z. A. 2006 Use of oxidoreduction potential as an indicator to regulate 1,3-propanediol fermentation by Klebsiella pneumoniae. Applied Microbiology Biotechnology69 554 563 . - 56.
Berrios-Rivera S. J. San K. Y. Bennett G. N. 2002 The effect of NAPRTase overexpression on the total levels of NAD, the NADH/NAD(+) ratio, and the distribution of metabolites in Escherichia coli. Metabolic Engineering4 238 247 . - 57.
Zhang Y. P. Li Y. Du C. Y. Liu M. Cao Z. 2006 Inactivation of aldehyde dehydrogenase: A key factor for engineering 1,3-propanediol production by Klebsiella pneumoniae. Metabolic Engineering8 578 586 . - 58.
Sanchez A. M. Bennett G. N. San K. Y. 2005 Novel pathway engineering design of the anaerobic central metabolic pathway in Escherichia coli to increase succinate yield and productivity. Metabolic Engineering7 229 239 . - 59.
Heux S. Cachon R. Dequin S. 2006 Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism. Metabolic Engineering8 303 314 . - 60.
Vemuri G. N. Eiteman M. A. Mc Ewen J. E. Olsson L. Nielsen J. 2007 Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. PNAS104 2402 2407 . - 61.
Saanchez A. M. Bennett G. N. San K. Y. 2005 Effect of different levels of NADH availability on metabolic fluxes of Escherichia coli chemostat cultures in defined medium. Journal Biotechnologyl117 395 405 . - 62.
Jackowski S. Rock C. O. 1986 Consequences of reduced intracellular coenzyme A content in Escherichia coli. Journal of Bacteriology166 866 871 . - 63.
Chohnan S. Furukawa H. Fujio T. Nishihara H. Takamura Y. 1997 Changes in the size and composition of intracellular pools of nonesterified coenzyme A and coenzyme A thioesters in aerobic and facultatively anaerobic bacteria. Applied Environmental Microbiology63 553 560 . - 64.
DS Vallari Jackowski. S. Rock C. O. 1987 Regulation of pantothenate kinase by coenzyme A and its thioesters. Journal of Biological Chemistry262 2468 2471 . - 65.
Chohnan S. Izawa H. Nishihara H. Takamura Y. 1998 Changes in size of intracellular pools of coenzyme A and its thioesters in Escherichia coli K-12 cells to various carbon sources and stresses. Bioscience Biotechnolology and Biochemical62 1122 1128 . - 66.
Lee S. Y. Chang H. N. 1995 Production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli strains: genetic and fermentation studies. Candia Journal Microbiology 41 Suppl1 207 215 . - 67.
Vadali R. V. Bennett G. N. San K. Y. 2004 Enhanced isoamyl acetate production upon manipulation of the acetyl-CoA node in Escherichia coli. Biotechnol Prog20 692 697 . - 68.
Vadali R. V. Bennett G. N. San K. Y. 2004 Applicability of CoA/acetyl-CoA manipulation system to enhance isoamyl acetate production in Escherichia coli. Metabolic Engineering6 294 299 . - 69.
Vadali R. V. Bennett G. N. San K. Y. 2004 Cofactor engineering of intracellular CoA/acetyl-CoA and its effect on metabolic flux redistribution in Escherichia coli. Metabolic Engineering6 133 139 . - 70.
Liu L. Li Y. Zhu Y. Du G. Chen J. 2007 Redistribution of carbon flux in Torulopsis glabrata by altering vitamin and calcium level. Metabolic Engineering9 21 29 .