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Journal of Bacteriology, May 2001, p. 2979-2988, Vol. 183, No. 10
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.10.2979-2988.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Engineering a Homo-Ethanol Pathway in Escherichia coli: Increased Glycolytic Flux and Levels of Expression of Glycolytic Genes during Xylose Fermentation†

Han Tao, Ramon Gonzalez, Alfredo Martinez,Dagger Maria Rodriguez,Dagger L. O. Ingram,* J. F. Preston, and K. T. Shanmugam

Institute of Food and Agricultural Sciences, Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611

Received 10 October 2000/Accepted 16 February 2001

Replacement of the native fermentation pathway in Escherichia coli B with a homo-ethanol pathway from Zymomonas mobilis (pdc and adhB genes) resulted in a 30 to 50% increase in growth rate and glycolytic flux during the anaerobic fermentation of xylose. Gene array analysis was used as a tool to investigate differences in expression levels for the 30 genes involved in xylose catabolism in the parent (strain B) and the engineered strain (KO11). Of the 4,290 total open reading frames, only 8% were expressed at a significantly higher level in KO11 (P < 0.05). In contrast, over half of the 30 genes involved in the catabolism of xylose to pyruvate were expressed at 1.5-fold- to 8-fold-higher levels in KO11. For 14 of the 30 genes, higher expression was statistically significant at the 95% confidence level (xylAB, xylE, xylFG, xylR, rpiA, rpiB, pfkA, fbaA, tpiA, gapA, pgk, and pykA) during active fermentation (6, 12, and 24 h). Values at single time points for only four of these genes (eno, fbaA, fbaB, and talA) were higher in strain B than in KO11. The relationship between changes in mRNA (cDNA) levels and changes in specific activities was verified for two genes (xylA and xylB) with good agreement. In KO11, expression levels and activities were threefold higher than in strain B for xylose isomerase (xylA) and twofold higher for xylulokinase (xylB). Increased expression of genes involved in xylose catabolism is proposed as the basis for the increase in growth rate and glycolytic flux in ethanologenic KO11.

* Corresponding author. Mailing address: Department of Microbiology and Cell Science, University of Florida, Box 110700, Gainesville, FL 32611. Phone: (352) 392-8176. Fax: (352) 846-0969. E-mail: ingram{at}ufl.edu.

dagger Florida Agricultural Experiment Station Journal Series no. R-07817.

Dagger Present address: Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Cuernavaca, Mor. 62250, Mexico.

Journal of Bacteriology, May 2001, p. 2979-2988, Vol. 183, No. 10
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.10.2979-2988.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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