The redox-sensing protein Rex modulates ethanol production in <i>Thermoanaerobacterium saccharolyticum</i>

<div><p><i>Thermoanaerobacterium saccharolyticum</i> 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 <i>T</i>. <i>saccharolyticum</i>: a single deletion of <i>rex</i> 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 <i>rex</i> gene also had the effect of increasing expression of alcohol dehydrogenase genes, <i>adhE</i> and <i>adhA</i>. 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 Δ<i>rex</i> strains adapted for faster growth. In several cases, secondary mutations had appeared in the <i>adhE</i> gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce <i>rex</i> into the Δ<i>rex</i> strains: reintroducing <i>rex</i> decreased ethanol yield to below wild type levels in the Δ<i>rex</i> strain without <i>adhE</i> mutations, but did not change the ethanol yield in the Δ<i>rex</i> strain where an <i>adhE</i> mutation occurred.</p></div

ADH activity in Δ<i>rex</i> strains.

<p>ADH activity in Δ<i>rex</i> strains.</p

Gene expression levels of <i>adhE</i> and <i>adhA</i>.

<p>Panel A shows <i>adhE</i> expression levels and panel B shows <i>adhA</i> expression levels. Strains were grown at 55°C to mid-log phase—OD₆₀₀ ~ 0.5 for faster growing strains (wild type, LL1356-1359), and OD₆₀₀ ~ 0.2 for slower growing strains (LL1414-1417). Wt is shown in grey, Δ<i>rex</i> strains before adaptation in blue and Δ<i>rex</i> strains after adaptation in red. Gene expression levels of <i>adhE</i> and <i>adhA</i> are normalized to <i>recA</i>. Biological duplicates were performed for each reaction; error bars represent one standard deviation.</p

Growth rate and Max OD₆₀₀ of Δ<i>rex</i> strains.

<p>Growth rate and Max OD₆₀₀ of Δ<i>rex</i> strains.</p

Strains used in this study.

<p>Strains used in this study.</p

Secondary mutations in adapted Δ<i>rex</i> strains.

<p>Secondary mutations in adapted Δ<i>rex</i> strains.</p

Ethanol yields of Δ<i>rex</i> strains before and after serial transfers.

<p>Wt is shown in grey, Δ<i>rex</i> strains before adaptation are shown in blue, Δ<i>rex</i> strains after adaptations in red, and <i>rex</i> complementation strains in green. Cultures were grown on 5 g/L cellobiose in modified MTC-6 media (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195143#sec002" target="_blank">Material and methods</a>), and theoretical ethanol yield from cellobiose is 0.54 g ethanol per gram cellobiose. Ethanol yields are calculated based on the amount of substrate consumed (initial and final concentrations of cellobiose were measured). Ethanol yield is presented in percent theoretical maximum, which assumes that one molecule of glucose (or glucose equivalent) can be converted into, at most, two molecules of ethanol.</p

Putative Rex-binding sites in <i>T</i>. <i>saccharolyticum</i>.

<p>Putative Rex-binding sites in <i>T</i>. <i>saccharolyticum</i>.</p