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Title: Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli

Abstract

Previous studies have shown an unexpectedly high nutrient requirement for efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. The basis for this requirement has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopy plasmids which express pdc and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: {sigma}{sup 70}, {sigma}{sup 38}, and {sigma}{sup 28}. The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pdc and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageousmore » to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Univ. of Florida, Gainesville, FL (US)
Sponsoring Org.:
USDA; USDOE
OSTI Identifier:
20001025
DOE Contract Number:  
FG02-96ER20222
Resource Type:
Journal Article
Journal Name:
Biotechnology Progress
Additional Journal Information:
Journal Volume: 15; Journal Issue: 5; Other Information: PBD: Sep-Oct 1999; Journal ID: ISSN 8756-7938
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; ETHANOL; ESCHERICHIA COLI; GENE REGULATION; BIOSYNTHESIS; PLASMIDS; ENZYME ACTIVITY; DECARBOXYLASES; ZYMOMONAS MOBILIS; FERMENTATION; YIELDS

Citation Formats

Martinez, A., York, S.W., Yomano, L.P., Pineda, V.L., Davis, F.C., Shelton, J.C., and Ingram, L.O. Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli. United States: N. p., 1999. Web. doi:10.1021/bp990103p.
Martinez, A., York, S.W., Yomano, L.P., Pineda, V.L., Davis, F.C., Shelton, J.C., & Ingram, L.O. Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli. United States. doi:10.1021/bp990103p.
Martinez, A., York, S.W., Yomano, L.P., Pineda, V.L., Davis, F.C., Shelton, J.C., and Ingram, L.O. Fri . "Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli". United States. doi:10.1021/bp990103p.
@article{osti_20001025,
title = {Biosynthetic burden and plasmid burden limit expression of chromosomally integrated heterologous genes (pdc, adhB) in Escherichia coli},
author = {Martinez, A. and York, S.W. and Yomano, L.P. and Pineda, V.L. and Davis, F.C. and Shelton, J.C. and Ingram, L.O.},
abstractNote = {Previous studies have shown an unexpectedly high nutrient requirement for efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. The basis for this requirement has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopy plasmids which express pdc and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: {sigma}{sup 70}, {sigma}{sup 38}, and {sigma}{sup 28}. The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pdc and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation.},
doi = {10.1021/bp990103p},
journal = {Biotechnology Progress},
issn = {8756-7938},
number = 5,
volume = 15,
place = {United States},
year = {1999},
month = {10}
}