Enhanced bioethanol production by Zymomonas mobilis in response to the quorum sensing molecules AI-2

Abstract

The depletion of non-renewable energy resources, the environmental concern over the burning of fossil fuels, and the recent price rises and instability in the international oil markets have all combined to stimulate interest in the use of fermentation processes for the production of alternative bio-fuels. As a fuel, ethanol is mainly of interest as a petrol additive, or substrate, because ethanol-blended fuel produces a cleaner, more complete combustion that reduces greenhouse gas and toxic emissions. As a consequence of the surge in demand for biofuels, ethanol producing microorganisms, such as the bacterium Zymomonas mobilis, are of considerable interest due to their potential for industrial-scale bioethanol production.

Although bioethanol has traditionally been produced in batch fermentation with the yeast Saccharomyces cerevisiae, there are advantages in using Z. mobilis as an alternative for bioethanol production. In comparison to yeast, Z. mobilis grows and ferments rapidly, without the requirement for the controlled supply of oxygen during fermentation, and has a significantly higher ethanol product rate and yield. Most importantly, it has a high tolerance for ethanol.

Bacteria communicate with one another using chemical signalling molecules. In general, chemical communication involves producing, releasing, detecting, and responding to small hormone-like molecules termed autoinducers (AI). This process allows bacteria to monitor the environment for other bacteria and to alter behaviour on a population-wide scale in response to changes in the number and/or species present in a community.
Currently, there are three well-defined classes of molecules that serve as the paradigms for chemical signaling in bacteria: oligopeptides, AI-1 (AHLs) and AI-2. Oligopeptide signalling is the predominant signal used by Gram-positive bacteria, and AHLs (acyl-homoserine lactones) are for species-specific communication in Gram-negative bacteria. Finally, the LuxS/AI-2 pathway is generally considered as involved in interspecies communication because the luxS gene, which is responsible for AI-2 production, is found in various bacteria.

Many physiological functions in bacteria such as toxin, virulence factor and bacteriocin production, biofilm formation, bioluminescence, type III secretion, have been shown to be under the control of AI-2 quorum sensing. In Z. mobilis, in vitro synthesized and in vivo produced AI-2 treatment enhanced ethanol production by this bacterium up to a maximum of 50% in comparison with untreated control cells. This appears attributable to the overproduction of the glycolytic enzymes, enolase and pyruvate carboxylase, which are only rarely found in bacteria and the key enzymes for ethanol production.

From the perspective of interspecies communication, enhanced ethanol production in Z. mobilis, under the control of the AI-2 signalling molecules, could represent a good example of a bacterium that does not produce AI-2, but responds to it.

Another interesting finding is that two extracellular proteins from Z. mobilis, ZMO0994 and ZMO0134 which were originally induced by AI-2, were secreted when they were cloned, transformed and expressed in E. coli strain BL21 DE3; since it is generally accepted that nonpathogenic strains of E. coli, particularly derivatives of K12, do not secrete proteins under routine growth conditions. Presumably, these proteins possess signal sequences for secretion that could be used to provide a strategy for their use as carriers of recombinant proteins produced in E. coli K12. The merit of this system is that there would be few contaminant cytoplasmic proteins, and could possibly solve problems in protein purification, such as protease activity, protein misfolding and inclusion body formation.

Finally, the discovery that the metabolic pathway leading to ethanol production is regulated by AI-2 is of considerable biotechnological importance because it will provide a basis for further engineering of strains for more efficient ethanol production. Indeed, engineering Z. mobilis by introducing the genes that encode Pfs and LuxS to produce AI-2, would be a means to stimulate increased ethanol production.