The Bifunctional Alcohol and Aldehyde Dehydrogenase Gene, adhE , Is Necessary for Ethanol Production in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Abstract

Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are anaerobic thermophilic bacteria being investigated for their ability to produce biofuels from plant biomass. The bifunctional alcohol and aldehyde dehydrogenase gene, adhE, is present in these bacteria and has been known to be important for ethanol formation in other anaerobic alcohol producers. This study explores the inactivation of the adhE gene in C. thermocellum and T. saccharolyticum. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In both adhE deletion strains, fermentation products shifted from ethanol to lactate production and resulted in lower cell density and longer time to reach maximal cell density. In T. saccharolyticum, the adhE deletion strain lost >85% of alcohol dehydrogenase (ADH) activity. Aldehyde dehydrogenase (ALDH) activity did not appear to be affected, although ALDH activity was low in cell extracts. Adding ubiquinone-0 to the ALDH assay increased activity in the T. saccharolyticum parent strain but did not increase activity in the adhE deletion strain, suggesting that ALDH activity was inhibited. In C. thermocellum, the adhE deletion strain lost >90% of ALDH and ADH activity in cell extracts. The C. thermocellum adhE deletion strain contained a point mutation in the lactate dehydrogenase gene, which appears to deregulate its activation by fructose 1,6-bisphosphate, leading to constitutive activation of lactate dehydrogenase. IMPORTANCEThermoanaerobacterium saccharolyticum and Clostridium thermocellum are bacteria that have been investigated for their ability to produce biofuels from plant biomass. They have been engineered to produce higher yields of ethanol, yet questions remain about the enzymes responsible for ethanol formation in these bacteria. The genomes of these bacteria encode multiple predicted aldehyde and alcohol dehydrogenases which could be responsible for alcohol formation. This study explores the inactivation of adhE, a gene encoding a bifunctional alcohol and aldehyde dehydrogenase. Deletion of adhE reduced ethanol production by >95% in both T. saccharolyticum and C. thermocellum, confirming that adhE is necessary for ethanol formation in both organisms. In strains without adhE, we note changes in biochemical activity, product formation, and growth.

Deletion of nfnAB in Thermoanaerobacterium saccharolyticum and Its Effect on Metabolism

“…It has been shown that in a T. saccharolyticum adhE deletion strain, there were still significant levels of NADPH-linked ADH activity, suggesting that there may be other functional NADPH-linked alcohol dehydrogenases (5). Interestingly, this predicted NADPHlinked alcohol dehydrogenase, adhA, is located directly upstream of nfnAB (Fig.…”

“…Bacteria for transformations and biochemical characterization were grown in modified DSMZ M122 rich medium containing 5 g/liter cellobiose and 4.5 g/liter yeast extract as previously described (10). To prepare cell extracts, cells were grown to an optical density at 600 nm (OD 600 ) of 0.5 to 0.8, separated from the medium by centrifugation, and used immediately or stored anaerobically in serum vials at Ϫ80°C as previously described (4,5). For quantification of fermentation products on cellobiose, strains were grown with shaking in 150-ml glass bottles with a 50-ml working volume in MTC defined medium on 5 g/liter (14.4 mM or 0.72 mmol) cellobiose, as previously described (11), with the following modifications for T. saccharolyticum: urea was replaced with ammonium chloride, and thiamine hydrochloride was added to a final concentration of 4 mg/liter.…”

“…This ethanologen strain of T. saccharolyticum was created by deleting lactate dehydrogenase (ldh), phosphotransacetylase (pta), and acetate kinase (ack). In T. saccharolyticum, glycolysis, pyruvate oxidation, and ethanol formation are believed to proceed by reactions 1, 2, and 3, respectively (4,5) (where Fd ox is oxidized ferredoxin, Fd red is reduced ferredoxin, and acetyl-CoA is acetyl coenzyme A):…”

“…Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activities were measured as previously described (5). ADH and ALDH were ).…”

Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application

“…They typically comprise two main domains, an N-terminal aldehyde dehydrogenase domain and an iron-dependent alcohol dehydrogenase C-terminal domain. The two domains are connected by a small linker [97,98]. It is unclear whether CoA-dependent aldehyde dehydrogenases gained CoA ester formation activity during evolution, or if this feature was originally present in primitive aldehyde dehydrogenases but it was later lost in most of them [99].…”