Mutant alcohol dehydrogenase leads to improved ethanol tolerance in Clostridium thermocellum

Abstract

Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene ( adhE ), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assays confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.

Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii

“…The phylogenetically related, thermophilic Firmicute C. thermocellum, however, encodes an NADH-dependent AdhE (Cthe0423), which is one of the most highly expressed genes (24) and abundant proteins (25) in C. thermocellum and is the key enzyme in ethanol production in this organism. Based on its known thermostability, coenzyme specificity (NADH-dependent), similarity in codon use, and favorable catalytic stoichiometry (26), this adhE was a promising candidate to generate an ethanol production pathway in C. bescii. The adhE gene from C. thermocellum was expressed in the C. bescii ldh mutant strain (JWCB018), resulting in strain C. bescii JWCB032 (Table 1).…”

“…‡ Cthe-adhE (Cthe0423; Bifunctional acetaldehyde/alcohol dehydrogenase derived from Clostridium thermocellum ATCC27405). § Cthe-adhE*(EA) (Cthe0423-P704L,H734R: Bifunctional acetaldehyde-CoA/alcohol dehydrogenase derived from ethanol tolerant Clostridium thermocellum strain EA) (26).…”

“…S2). Plasmid pDCW145 is identical to pDCW144 except for the cloning of Cthe0423 adhE*(EA) (26), which contains two point mutations in coding sequences, into pDCW142. To make this change, a 2.62-kb DNA fragment containing the coding sequence of Cthe0423* was amplified by PCR using DC469 (with BamHI site) and DC470 (with SphI site) using C. thermocellum EtOH (26) genomic DNA as a template.…”

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

“…In addition, recent studies have confirmed the feasibility to manipulate some cellulytic microorganisms e.g. Clostridium (Brown et al, 2011) and Thermobifida spp. (Deng and Fong, 2011).…”

Genetic engineering and enzyme research in lignocellulosic ethanol production

“…A high concentration of ethanol itself is an inhibiting factor to microorganisms, and therefore ethanol tolerant strains are being investigated. Brown et al (2011) resequenced the genome of an ethanol-tolerant Clostridium thermocellum mutant, and showed that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE). Biochemical assays confirmed a complete loss of NADHdependent activity with concomitant acquisition of NADPHdependent activity, which, they conclude, likely affects electron flow in the mutant.…”