The redox-sensing protein Rex modulates ethanol production in Thermoanaerobacterium saccharolyticum
April
The redox-sensing protein Rex modulates ethanol production in Thermoanaerobacterium saccharolyticum
Tianyong Zheng 0 1
Anthony A. Lanahan 1
Lee R. Lynd 0 1
Daniel G. Olson 1
0 Department of Biological Sciences, Dartmouth College , Hanover , New Hampshire, United States of America, 2 BioEnergy Science Center, Oak Ridge, Tennessee, United States of America, 3 Thayer School of Engineering, Dartmouth College , Hanover , New Hampshire, United States of America, 4 Center for Bioenergy Innovation , Oak Ridge, Tennessee , United States of America
1 Editor: Jeffrey L Blanchard, University of Massachusetts , UNITED STATES
Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type T. saccharolyticum: a single deletion of rex resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the rex gene also had the effect of increasing expression of alcohol dehydrogenase genes, adhE and adhA. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δrex strains adapted for faster growth. In several cases, secondary mutations had appeared in the adhE gene. Furthermore, in these strains the NADHlinked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce rex into the Δrex strains: reintroducing rex decreased ethanol yield to below wild type levels in the Δrex strain without adhE mutations, but did not change the ethanol yield in the Δrex strain where an adhE mutation occurred.
Introduction
Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that naturally produces
ethanol. Wild type T. saccharolyticum produces ethanol at about 46% of the theoretical
maximum yield [
1
], it also generates other fermentation products such as lactate and acetate. T.
saccharolyticum has been engineered to produce ethanol at ~90% theoretical maximum yield and
a titer of 70 g/L [
2,3
]; in these engineered strains the lactate and acetate production pathways
have been deleted. While T. saccharolyticum is able to consume many of the sugars present in
the hemicellulose fraction of lignocellulose, it is unable to consume cellulose. The organism
has been studied both for its high levels of ethanol production and as a co-culture partner for a
cellulolytic organism (e.g. Clostridium thermocellum) [4]. Numerous studies have focused on
Funding: The BioEnergy Science Center and Center
for Bioenergy Innovation are U.S. Department of
Energy Bioenergy Research Centers supported by
the Office of Biological and Environmental
Research in the DOE Office of Science. Grant
number DE-AC05-00OR22725 was awarded to LL.
The funder had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
the roles of enzymes in T. saccharolyticum, including secreted hydrolases involved in the
degradation of hemicellulose [
5
], the bifunctional alcohol dehydrogenase AdhE [
6
], and alcohol
dehydrogenase AdhA [
1
]. Additionally, fermentation end-product analyses [
7
] and
genomescale microarray data [
8
] have been reported.
In addition to studying the central metabolic pathways in T. saccharolyticum, we are also
interested in the regulation of genes involved in the ethanol production pathway. Rex is a
global transcription factor that has been studied in many facultative [9±12] and strict [13±19]
anaerobes. It senses intracellular NADH/NAD+ levels and controls the expression of many
genes involved in energy metabolism and anaerobic fermentation. In its homodimer form,
Rex represses gene expression by binding to both the promoter region of a DNA strand
(N-terminus) and an NAD+ molecule (C-terminus) thus inhibiting transcription of the target gene.
When the molecule of NAD+ bound to its C-terminus is replaced by NADH, conformational
changes trigger the release of the Rex dimer from the DNA thereby allowing transcription to
proceed [
20
]. The Rex protein is important in regulating metabolism: when NADH/NAD+
ratios are high in the cell, it signals that energy-generating catabolic processes (such as
glycolysis) are proceeding at a sufficient rate. Therefore by releasing adhE repression under high
concentrations of NADH, AdhE can convert NADH to NAD+ and replenish the NAD+ pool.
We have recently identified key alcohol dehydrogenase genes in the T. saccharolyticum
ethanol production pathway: adhE and adhA are both necessary for high levels of ethanol
production in T. saccharolyticum [
1
]. In vitro assays have shown that Rex regulates adhE (...truncated)