Kinetic and Nuclear Magnetic Resonance Studies of Xylose Metabolism by Recombinant Zymomonas mobilis ZM4(pZB5)

Abstract

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucosexylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by 13 Cnuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo 31 P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.Zymomonas mobilis has attracted widespread interest for fuel ethanol production because of its higher specific rates of sugar uptake and ethanol production, higher ethanol tolerance, and higher ethanol conversion efficiencies when compared to the traditionally used yeasts (10,14,21,22,26). However, wild-type strains of Z. mobilis can only utilize glucose, fructose, and sucrose, and they lack the pentose metabolism pathway necessary to ferment such sugars as xylose or arabinose. The cloning of enzymes for xylose assimilation and metabolism in Z. mobilis has now been reported (30), and a subsequent study has resulted in the successful integration of the requisite genes into the Zymomonas genome (M. Zhang, Y. C. Chou, X. K. Lai, S. Milstrey, N. Danielson, K. Evans, A. Mohagheghi, and M. Finkelstein, Abstr. 21st Symp. Biotechnol. Fuels Chem., abstr. 2-16, 1999). These genetically engineered strains can now convert xylose to ethanol by the combined use of the Entner-Doudoroff and pentose pathways facilitated by the cloned enzymes xylose isomerase and xylulokinase for xylose assimilation and by transketolase and transaldolase for pentose metabolism. In a further study, the cloning of three additional enzymes for arabinose utilization has been reported (4). However, when xylose is the sole carbon source, lower biomass yields and slower growth rates as well as lower ethanol yields for recombinant strains have been reported (9, 11, 12, 23; H. G. Lawford and J. D. Rousseau, Abstr. 21st Symp. Biotechnol. Fuels Chem., abstr. 2-24, 1999).In this study, the fermentation characteristics of the recombinant Z. mobilis ZM4(pZB5) on xylose or glucose alone, or on xylose-glucose mixtures, have been investigated to determine possib...

Adaptation yields a highly efficient xylose‐fermenting Zymomonas mobilis strain

“…At a metabolite level, it was observed that A3 produced much less xylitol during xylose fermentation. As previously suggested by other researchers (Feldmann et al, 1992;Jeon et al, 2005;Kim et al, 2000;Scangos and Reiner, 1979), xylitol formation and its toxicity may be an important factor. This was investigated as a potential reason for the improvement in A3.…”

“…Thus, xylose fermentation at high concentration is a better measure for a strain's ability to use xylose than mixed sugar fermentation. Previously, the highest xylose concentration that supports cell growth was $6% (Kim et al, 2000;, and in some cases, the fermentation was incomplete with significant amount of residual xylose (>1%; .…”

“…An important breakthrough came when Zhang et al (1995) successfully applied metabolic engineering to expand its substrate range to xylose. In the ensuing decade, however, xylose metabolism continued to be a focal point of research as it was found that the ability of engineered strains to metabolize xylose was considerably lower than that of glucose and improvement was needed (De Graaf et al, 1999;Gao et al, 2002;Jeon et al, 2005;Kim et al, 2000).…”

“…Much research over the past decade has been devoted to identify potential bottlenecks in xylose fermentation. Studies using NMR techniques suggested that xylosemetabolizing cells were in a less energized state compared to glucose-metabolizing cells (Kim et al, 2000). A 1992 study by Feldmann et al (1992) was the first to identify that xylitol formed during xylose metabolism was a significant limiting factor.…”

Fermentasi Etanol Sari Buah Semu Jambu Mete (Anacardium occidentale) oleh Zymomonas mobilis dengan Penambahan Urea

“…Hal itu menyebabkan penambahan kadar gula reduksi ke dalam media fermentasi. Menurut Kim et al (2000) Z. mobilis cenderung akan mengkonsumsi monosakarida terlebih dahulu ketika tumbuh dalam media campuran antara monosakarida dan disakarida.…”