Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Abstract

Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticum produce ethanol with a yield of 90% of the theoretical maximum, engineered strains of C. thermocellum produce ethanol at lower yields (∼50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in their adhE genes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, the adhE genes from six strains of C. thermocellum and T. saccharolyticum were cloned and expressed in Escherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains of T. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain of C. thermocellum has acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced into C. thermocellum and T. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level.

Importance: This work describes the characterization of the AdhE enzyme from different strains of C. thermocellum and T. saccharolyticum. C. thermocellum and T. saccharolyticum are thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production by C. thermocellum and T. saccharolyticum.

FIG 1

Primary structures of AdhE proteins of wild-type C. thermocellum and T. saccharolyticum. (A) The ALDH domain is at positions 1 to 423 for C. thermocellum and 1 to 420 for T. saccharolyticum, the ADH domain is at positions 463 to 873 for C. thermocellum and 460 to 860 for T. saccharolyticum, and the linker sequence is at positions 424 to 462 for C. thermocellum and 421 to 459 for T. saccharolyticum. NADH binding site 1 is at positions 200 to 221 for C. thermocellum and 199 to 220 for T. saccharolyticum; NADH binding site 2 is at positions 551 to 553 for C. thermocellum and 543 to 545 for T. saccharolyticum. Mutated residues discussed in this study are annotated at the appropriate positions as follows: D494G in LL350; P704L and H734R in LL346; V52A, K451N, and a 13-amino-acid (a.a.) insertion in LL1040; and G544D in LL1049. All elements are drawn to scale. Panels B and C show the sequence conservation of the NADH binding motifs (highlighted in yellow in the consensus sequence) of AdhE from Thermoanaerobacter ethanolicus, Thermoanaerobacter mathranii, T. saccharolyticum, Entamoeba histolytica, E. coli, C. thermocellum, Leuconostoc mesenteroides, Lactococcus lactis, Oenococcus oeni, and Streptococcus equinus. The residues highlighted in red are the most conserved, and those highlighted in blue are the least conserved. The numbering of amino acids is based on the AdhE sequence of C. thermocellum.

FIG 2

Homology modeling and docking analysis of the phosphate in NADPH interacting with C. thermocellum wild-type AdhE (A) and the D494G mutant form (B). The dotted lines represent hydrogen bonds. The red residue is D494, the yellow residue is G494, and the blue residues are N495 and F496.

FIG 3

Average structure of the ADH domains of AdhE from C. thermocellum wild-type LL1004 (A), the ethanol-tolerant LL346 strain (B), moderate ethanol producer LL350 (C), T. saccharolyticum wild-type LL1025 (D), high ethanol producer LL1049 (E), and high ethanol producer LL1040 (F). The amino acids of interest are shown in blue and red; the NADPH cofactor is shown color coded by elements, with its 2′-phosphate group highlighted (green open circle); and the iron ions (green) are also shown. Additionally, the 39-bp insertion in the high ethanol producer LL1040 is shown in orange (F). The yellow-filled circle represents the binding pocket where the additional 2′-phosphate of NADPH is commonly found in NADPH-dependent AdhE proteins. The locations of D494 and D486 are usually the recognition sites for NADH and do not allow the 2′-phosphate group of NADPH (green circle) to access the preferred binding pocket (yellow circle). When the green open circle and the yellow filled circle overlap, that indicates that the NADPH molecule is able to access its preferred binding pocket. This is present in panels C, E, and F but not in the other panels. Panels C, E, and F correspond to enzymes that can use NADPH as a cofactor. Note that NADPH was used for modeling purposes and does not reflect the actual cofactor specificity of the enzymes but rather was used to explain the observed levels of affinity of the enzymes for NADPH.