Abstract
BackgroundZymomonas mobilis ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. Z. mobilis performs best under anaerobic conditions, but is an aerotolerant organism. However, the genetic and physiological basis of ZM4's response to various stresses is understood poorly.ResultsIn this study, transcriptomic and metabolomic profiles for ZM4 aerobic and anaerobic fermentations were elucidated by microarray analysis and by high-performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analyses. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Greater amounts of other end-products such as acetate, lactate, and acetoin were detected under aerobic conditions and at 26 h there was only 1.7% of the amount of ethanol present aerobically as there was anaerobically. In the early exponential growth phase, significant differences in gene expression were not observed between aerobic and anaerobic conditions via microarray analysis. HPLC and GC analyses revealed minor differences in extracellular metabolite profiles at the corresponding early exponential phase time point.Differences in extracellular metabolite profiles between conditions became greater as the fermentations progressed. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 contained lower levels of amino acids such as alanine, valine and lysine, and other metabolites like lactate, ribitol, and 4-hydroxybutanoate under anaerobic conditions relative to aerobic conditions. Stationary phase microarray analysis revealed that 166 genes were significantly differentially expressed by more than two-fold. Transcripts for Entner-Doudoroff (ED) pathway genes (glk, zwf, pgl, pgk, and eno) and gene pdc, encoding a key enzyme leading to ethanol production, were at least 30-fold more abundant under anaerobic conditions in the stationary phase based on quantitative-PCR results. We also identified differentially expressed ZM4 genes predicted by The Institute for Genomic Research (TIGR) that were not predicted in the primary annotation.ConclusionHigh oxygen concentrations present during Z. mobilis fermentations negatively influence fermentation performance. The maximum specific growth rates were not dramatically different between aerobic and anaerobic conditions, yet oxygen did affect the physiology of the cells leading to the buildup of metabolic byproducts that ultimately led to greater differences in transcriptomic profiles in stationary phase.
Highlights
Zymomonas mobilis ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars
Anaerobic fermentation led to a maximal culture density of 7.0 OD600 units approximately 9 h post-inoculation, while Z. mobilis did not reach its highest culture density of 6.5 OD600 units until 13 h post-inoculation under aerobic conditions despite initial inocula concentrations being slightly greater than the former condition
Under anaerobic conditions 99.5% of the glucose had been utilized at this time point
Summary
Zymomonas mobilis ZM4 (ZM4) produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. Essential traits for an industrial microorganism include high ethanol yield, tolerance, and productivity (> 90% of theoretical, > 40 g L-1, > 1 g L-1 h-1, respectively); robust growth with simple, inexpensive growth requirements in conditions that retard contaminants (eg higher temperatures); and inhibitor tolerance, as reviewed previously [5]. Saccharomyces yeasts have been the preferred industrial biocatalyst for fuel ethanol production, genetically engineered bacterial species such as Gram-negative bacteria Escherichia coli, Zymomonas mobilis, and Klebsiella oxytoca as well as Grampositive bacteria Bacillus subtilis and Corynebacterium glutamicum are in development to address commercially important inoculum requirements [5,7,8]. A newly formed partnership between the DuPont and Broin companies will utilize recombinant strains of Z. mobilis for bio-ethanol fermentation from the lignocellulosic residues such as corn stover [9]
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