No abstract

The driving force for organo- or lithotrophic growth as well as for each step in the metabolic network is the Gibbs reaction energy. For each enzymatic step it must be negative. Thermodynamics contributes therefore to the in-silico description of living systems. It may be used for assessing the feasibility of a given pathway because it provides a further constraint for those pathways which are feasible from the point of view of mass balance calculations (metabolic flux analysis) and the genetic potential of an organism. However, when this constraint was applied to lactic acid fermentation according to a method proposed by Mavrovouniotis (1993a, ISMB 93:273-283) it turned out that an unrealistically wide metabolite concentration range had to be assumed to make this well-known glycolytic pathway thermodynamically feasible. During a search for the reasons of this surprising result the insufficient consideration of the activity coefficients was identified as main cause. However, it is shown in the present contribution that the influence of the activity coefficients on Gibbs reaction energy can be easily taken into account based on the intracellular ionic strength. The uncertainty of the tabulated equilibrium constants and of the apparent standard Gibbs energies derived from them was found to be the second most important reason for the erroneous result of the feasibility analysis. Deviations of intracellular pH from the standard value and bad estimations of currency metabolites, e.g., NAD(+) and NADH, were found to be of lesser importance but not negligible. The pH dependency of Gibbs reaction enthalpy was proved to be easily taken into account. Therefore, the application of thermodynamics for a better in-silico prediction of the behavior of living cell factories calls predominantly for better equilibrium data determined under well defined conditions and also for a more detailed knowledge about the intracellular ionic strength and pH value.

The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes ectABC of the halophilic bacterium Chromohalobacter salexigens into an Escherichia coli strain using the expression vector pASK-IBA7. Under the control of a tet promoter, the transgenic E. coli synthesized 6 g liter ؊1 ectoine with a space-time yield of 40 mg liter ؊1 h ؊1 , with the vast majority of the ectoine being excreted.Halophilic microorganisms live in highly saline environments. There are two major strategies of adaptation to these hostile conditions. Extreme halophiles such as Halobacterium salinarum accumulate salt in the cytosol to maintain the osmotic balance (the salt-in strategy). Other halophiles synthesize and accumulate small organic molecules as osmotic counterweights (the organic-osmolyte strategy). Unlike intracellular salt, the small organic compounds do not affect the metabolism of the organism and are thus called compatible solutes. The enzymes of organic-osmolyte strategists do not need to be haloadapted, allowing these organisms to cope with strong salinity fluctuations. Non-salt-tolerant bacteria like Escherichia coli are usually unable to synthesize large amounts of compatible solutes but may resist halostress to a certain extent by taking up and accumulating compatible solutes (8,9,12,27).Different classes of chemical compounds, including polyols, sugars, methylamines, and linear and cyclic amino acids and betaines, have been found to act as compatible solutes. Besides functioning as osmotic counterweights, compatible solutes were shown to protect biomolecules and whole cells against denaturation caused by heating, freezing, desiccation, or chemical agents (15,19,22). This property has attracted commercial attention. Compatible solutes can be used as chemical chaperones for protein folding, enhancers of PCR performance, cryoprotectants of microorganisms, cosmeceuticals, and pharmaceuticals (20, 32). A potentially promising future application could be the enhancement of drought tolerance or salt tolerance of transgenic plants (1, 37).The best-investigated compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine), is biotechnologically produced with the halophilic bacterium Halomonas elongata (34). The physiology and genetics of ectoine biosynthesis in this bacterium have been studied in detail (7,23,24,25,26,29). The technical bioprocess for the synthesis of ectoine exploits the salt adaptation strategy of H. elongata and is called "bacterial milking" (34). When the medium has a high salt concentration, the bacterium s...

Rhodococcus opacus PD630 was investigated for physiological and morphological changes under water stress challenge. Gluconate- and hexadecane-grown cells were extremely resistant to these conditions, and survival accounted for up to 300 and 400 days; respectively, when they were subjected to slow air-drying. Results of this study suggest that strain PD630 has specific mechanisms to withstand water stress. Water-stressed cells were sensitive to the application of ethanol, high temperatures and oxidative stress, whereas they exhibited cross-protection solely against osmotic stress during the first hours of application. Results indicate that the resistance programme for water stress in R. opacus PD630 includes the following physiological and morphological changes, among others: (1) energetic adjustments with drastic reduction of the metabolic activity ( approximately 39% decrease during the first 24 h and about 90% after 190 days under dehydration), (2) endogenous metabolism using intracellular triacylglycerols for generating energy and precursors, (3) biosynthesis of different osmolytes such as trehalose, ectoine and hydroxyectoine, which may achieve a water balance through osmotic adjustment and may explain the overlap between water and osmotic stress, (4) adjustments of the cell-wall through the turnover of mycolic acid species, as preliminary experiments revealed no evident changes in the thickness of the cell envelope, (5) formation of short fragmenting-cells as probable resistance forms, (6) production of an extracellular slime covering the surface of colonies, which probably regulates internal and external changes in water potential, and (7) formation of compact masses of cells. This contributes to understanding the water stress resistance processes in the soil bacterium R. opacus PD630.

No abstract