Heat stable, fe dependent alcohol dehydrogenase for aldehyde detoxification

Abstract

The present invention relates to microorganisms and polypeptides for detoxifying aldehydes associated with industrial fermentations. In particular, a heat-stable, NADPH- and iron-dependent alcohol dehydrogenase was cloned from Thermoanaerobacter pseudethanolicus 39E and displayed activity against a number of aldehydes including inhibitory compounds that are produced during the dilute-acid pretreatment process of lignocellulosic biomass before fermentation to biofuels. Methods to use the microorganisms and polypeptides of the invention for improved conversion of bio mass to biofuel are provided as well as use of the enzyme in metabolic engineering strategies for producing longer-chain alcohols from sugars using thermophilic, fermentative microorganisms.

We claim: 
     
         1 . An anaerobic, thermophilic microorganism comprising a vector that expresses an exogenous heat-stable, NADPH- and iron-dependent alcohol dehydrogenase cloned from  Thermoanaerobacter pseudethanolicus  39E, or a homolog thereof. 
     
     
         2 . The microorganism of  claim 1 , wherein the NADPH- and iron-dependent alcohol dehydrogenase has an aldehyde reductase specific activity of at least about 2-20 μmol/min/mg with furfural as a substrate, of at least about 5-50 μmol/min/mg with 5-hydroxymethylfurfural as a substrate, of at least about 2-20 μmol/min/mg with acetaldehyde as a substrate, of at least about 0.3-3 μmol/min/mg with isobutyraldehyde as a substrate, and/or at least about 7-70 μmol/min/mg with butyraldehyde as a substrate. 
     
     
         3 . The microorganism of  claim 2 , wherein said aldehyde reductase specific activity is at least 4 μmol/min/mg with furfural as a substrate, at least 10 μmol/min/mg with 5-hydroxymethylfurfural as a substrate, of at least 4 μmol/min/mg with acetaldehyde as a substrate, of at least about 0.6 μmol/min/mg with isobutyraldehyde as a substrate, and/or at least 14 μmol/min/μmg with butyraldehyde as a substrate. 
     
     
         4 . The microorganism of  claim 1 , wherein the microorganism is selected from the group consisting of  Thermoanaerobacter brockii, Thermoanaerobacter ethanolicus, Thermoanaerobacter pseudethanolicus, Thermoanaerobacterium aotearoense, Thermoanaerobacterium saccharolyticum, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium xylanolyticum, Clostridium thermocellum, Clostridium straminisolvens, Clostridium thermocopriae, Caldicellulosiruptor bescii, Caldicellulosiruptor saccharolyticus, Caldicellulosiruptor hydrothermalis, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor kronotskyensis, Caldicellulosiruptor lactoaceticus, Caldicellulosiruptor owensensi, Caldicellulosiruptor acetigenus  and  Caldicellulosiruptor obsidiansis.    
     
     
         5 . A method to generate an anaerobic, thermophilic microorganism that expresses an exogenous heat-stable, NADPH- and iron-dependent alcohol dehydrogenase cloned from  Thermoanaerobacter pseudethanolicus  39E, or a homolog thereof, comprising introducing a nucleic acid vector that expresses an exogenous heat-stable, NADPH- and iron-dependent alcohol dehydrogenase cloned from  Thermoanaerobacter pseudethanolicus  39E, or a homolog thereof into an anaerobic, thermophilic microorganism. 
     
     
         6 . A nucleic acid vector capable of expressing an exogenous heat-stable, NADPH- and iron-dependent alcohol dehydrogenase cloned from  Thermoanaerobacter pseudethanolicus  39E, or a homolog thereof in an anaerobic, thermophilic microorganism. 
     
     
         7 . The nucleic acid vector of  claim 6 , wherein the exogenous heat-stable, NADPH- and iron-dependent alcohol dehydrogenase cloned from  Thermoanaerobacter pseudethanolicus  39E, or a homolog thereof has the amino acid sequence set forth in SEQ ID NO: 1. 
     
     
         8 . The nucleic acid vector of  claim 6 , wherein the exogenous heat-stable, NADPH- and iron-dependent alcohol dehydrogenase cloned from  Thermoanaerobacter pseudethanolicus  39E, or a homolog thereof has at least 70, 80, 90, 95, 98 or 99% identity to the amino acid sequence set forth in SEQ ID NO: 1. 
     
     
         9 . A lignocellulosic biomass fermentation process comprising contacting biomass with the microorganism of  claim 1  for a time and under thermophilic, anaerobic conditions to detoxify inhibitory aldehydes present in the biomass, and to allow fermentation of the biomass and conversion of the biomass to biofuel. 
     
     
         10 . The process of  claim 9 , wherein said biomass has been pretreated by acid hydrolysis, hot water, or enzymatic hydrolysis. 
     
     
         11 . A method of improving the yield and/or efficiency of lignocellulosic biomass conversion to biofuel, comprising contacting lignocellulosic biomass with the microorganism of  claim 1  for a time and under thermophilic, anaerobic conditions sufficient to detoxify inhibitory aldehydes present in said biomass, and to produce improved yields or efficiency of biomass conversion to biofuel, relative to lignocellulosic biomass that has not been so contacted. 
     
     
         12 . The method of  claim 11 , wherein said lignocellulosic biomass has been pretreated by acid hydrolysis, hot water, or enzymatic hydrolysis. 
     
     
         13 . A lignocellulosic biomass fermentation process comprising contacting lignocellulosic biomass with the microorganism of  claim 1  and an anaerobic, thermophilic microorganism for a time and under thermophilic, anaerobic conditions to detoxify inhibitory aldehydes present in the biomass and to allow fermentation of the biomass to proceed and conversion of biomass to biofuel. 
     
     
         14 . The method of  claim 13 , wherein said lignocellulosic biomass has been pretreated by acid hydrolysis, hot water, or enzymatic hydrolysis. 
     
     
         15 . An improved method of producing a bulk or platform chemical from lignocellulosic biomass, the improvement comprising contacting said lignocellulosic biomass with the anaerobic, thermophilic microorganism of  claim 1  for a time and under conditions to reduce the aldehyde content associated with said lignocellulosic biomass and to improve production efficiency or yield of said bulk or platform chemical, and recovering the bulk or platform chemical. 
     
     
         16 . The method of  claim 15 , wherein said lignocellulosic biomass has been pretreated by acid hydrolysis, hot water, or enzymatic hydrolysis. 
     
     
         17 . The method of  claim 15 , wherein the bulk or platform chemical is selected from the group consisting of ethanol, butanol, lactate, furfuryl alcohol, 1,4-dicarboxylic acids (succinic, fumaric, malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L-ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, fatty acids, fatty-derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, and isoamylacetate. 
     
     
         18 . The method o  claim 15 , wherein the lignocellulosic biomass is furan waste from pulp or paper processing.