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Biosynthetic Burden and Plasmid Burden Limit Expression of Chromosomally Integrated Heterologous Genes ( p dc, a dhB ) in Escherichia c oli
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...
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Published in: | Biotechnology progress 1999-01, Vol.15 (5), p.891-897 |
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Main Authors: | , , , , , , |
Format: | Article |
Language: | English |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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:
σ
70
,
σ
38
, and
σ
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. From this work, we propose that the activation of biosynthetic genes in nutrient‐poor media creates a biosynthetic burden that reduces the expression of chromosomal
pdc
and
adhB
by competing for transcriptional and translational machinery. This reduced expression can be viewed as analogous to the effect of plasmids (plasmid burden) on the expression of native chromosomal genes. |
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ISSN: | 8756-7938 1520-6033 |
DOI: | 10.1021/bp990103p |