Physiological Function of Alcohol Dehydrogenases and Long-Chain (C ₃₀ ) Fatty Acids in Alcohol Tolerance of Thermoanaerobacter ethanolicus

Abstract

A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C 30 fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).Microorganisms such as Saccharomyces or Zymomonas strains that are used for industrial ethanol production from glucose or sucrose have high alcohol tolerance for growth (i.e., Ͼ6% [vol/vol]). Other species that produce ethanol from cheaper substrates such as cellulose or starch, like Clostridium thermocellum or Thermoanaerobacter ethanolicus, generally have a low alcohol tolerance for growth (Ͻ2% [vol/vol]). In general, alcohol-producing microbes respond to increasing solvent concentrations by increasing the percentage of unsaturated versus saturated fatty acids, long-chain fatty acids, and hopanes into their cytoplasmic membranes (2,8,9). These structural changes prevent the loss of membrane function from fluidization caused by a high solvent concentration.Thermophilic ethanol fermentations offer the potential of direct degradation of cellulose or starch and direct recovery of ethanol at fermentation temperatures under reduced pressure (5,16,17,18,21,23). This potential has not been demonstrated because of low-end product concentrations caused by bacterial ethanol inhibition. Thermophilic bacteria employ two different pathways for ethanol production, using either a primary alcohol dehydrogenase (ADH), as in C. thermocellum, or primary and secondary ADHs, as in T. ethanolicus (13,15). Herrero and coworkers (3, 4) studied ethanol tolerance in C. thermocellum and concluded that the low tolerance to ethanol (Ͻ2% [vol/vol]) was a combined result of general solvent effects on membrane fluidity and a specific inhibition of enzymes involved in sugar metabolism. Work in the labs of Ljundahl, Wiegel, Demain, Zeikus, and others has showed that thermophilic anaerobic bacteria can adapt their tolerance to about 4% (vol/vol) ethanol (for a review, see reference 16).We previously demonstrated (15) that moderate ethanol tolerance (Ͻ4% [vol/vol]) of a T. ethanolicus mutant strain was related to enzymatic prevention of metabolic inhibition caused by ethanol overreducing the pyridine nucleotide pool and inhibiting glycolysis. The ethanol-tolerant mutant 39EA lacked primary ADH...

Respiratory behaviour of a Zymomonas mobilis adhB::kanr mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions

“…Special functions for particular ADH isoenzymes might be common among ethanologenic microorganisms. An interesting scheme of two alcohol dehydrogenases catalysing opposite reactions has been postulated by Burdette and Zeikus [22,23] for anaerobically growing Thermoanaerobacter ethanolicus. They suggested that one of the two ADH isoenzymes in this anaerobe was participating both in ethanol synthesis from acetaldehyde and NADH, and in ethanol oxidation coupled to NADP + reduction, thus maintaining the redox balance between the NAD(P)(H) cofactor pools.…”

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

“…The NADH/NADϩ ratio in the T. ethanolicus wild-type strain was elevated during pulses of ethanol in continuous culture, whereas the adapted strain was largely unaffected. The NADH to NADPH shift is likely an adaptive response to overcome NADH inhibition of glycolysis at moderate ethanol concentrations (21).…”

EST-analysis of the thermo-acidophilic red microalga Galdieriasulphuraria reveals potential for lipid A biosynthesis and unveils the pathway of carbon export from rhodoplasts

“…However, G. sulphuraria possesses a very rigid protein-rich cell wall (Bailey and Staehelin, 1968;Staehelin, 1968), and it has been hypothesized, based on studies with archaebacteria, that the incorporation of sterols, saturated fatty acids, bipolar tetraether lipids, and proteins could account for this impermeability (Benz and Cros, 1978;Benz et al, 1980;Elferink et al, 1992;Komatsu and Chong, 1998). Very long chain dicarboxylic acids are thought to have similar roles in some eubacteria (Jung et al, 1993(Jung et al, , 1994Burdette et al, 2002).…”