Abstract
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 NADH-linked 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.
Highlights
Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that naturally produces ethanol
Growth rates significantly decreased in these Δrex strains and each Δrex strain had a μMAX that was less than a third of that in wild type (Table 2)
Adaptation increased growth rate but decreased ethanol yield in Δrex strains To investigate the stability of the high ethanol phenotype in the Δrex strains, we conducted serial transfers for strains Rex-2, 4, 5 and 8
Summary
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 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].
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