Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose

Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids -predominantly L-valine and L-alanine -were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso-and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. Conclusions: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

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“…kegg/]) [62,63]. The alcohols formed in this manner are called fusel alcohols, and the pathway in yeast is known as the Ehrlich pathway.…”

Section: Additional Fermentation Productsmentioning

confidence: 99%

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

Holwerda

Thorne

Olson

et al. 2014

Biotechnol Biofuels

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“…CAC and CAP numbers are the ORF numbers in genome and megaplasmid, respectively. (B) The pathway for isobutanol production in C. cellulolyticum [59] from cellulose. In order to achieve direct isobutanol production from pyruvate, the genes encoding B. subtilis -acetolactate synthase, E. coli acetohydroxyacid isomeroreductase, E. coli dihydroxy acid dehydratase, Lactococcus lactis ketoacid decarboxylase, and E. coli and L. lactis alcohol dehydrogenases were cloned, respectively.…”

Section: Metabolic Engineering Of Mesophilic Clostridiamentioning

confidence: 99%

“…This engineered E. coli strain was able to produce 16 mM butanol using 4% (w/v) glucose as a carbon source [58]. More recently, metabolic engineering has been used for the development of C. cellulolyticum H10 for isobutanol synthesis directly from cellulose [59] (Fig. 1B).…”

Section: Consolidated Bioprocessing By Clostridial Speciesmentioning

confidence: 99%

Lignocellulosic Biomass Utilization Toward Biorefinery Using Meshophilic Clostridial Species

Tamaru¹,

López-Contreras²

2013

Cellulose - Biomass Conversion

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“…cellulolyticum, as a potential CBP organism, similar to C. thermocellum can utilize cellulose as well as other sugars released from hemicellulose degradation, including xylose, fructose, galactose, arabinose, mannose, and ribose (Gowen and Fong, 2010;Higashide et al, 2011). Recently, investigations have been focused on engineering of these bacteria to enhance their CBP butanol production.…”

Section: Genetic Engineering For Cbp Butanol Production In Clostridiamentioning

confidence: 99%

“…Recently, investigations have been focused on engineering of these bacteria to enhance their CBP butanol production. Higashide et al (2011) by expressing different enzymes (B. subtilis α-acetolactate synthase, E. coli acetohydroxyacid isomeroreductase, E. coli dihydroxy acid dehydratase, Lactococcus lactis ketoacid decarboxylase, and E. coli and L. lactis alcohol dehydrogenases) involved in direct conversion of pyruvate to isobutanol could engineer valine biosynthesis pathway. This metabolic engineering approach resulted in production of up to 660 mg/l isobutanol from cellulose.…”

Section: Genetic Engineering For Cbp Butanol Production In Clostridiamentioning

confidence: 99%

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

2015

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HIGHLIGHTS Various CBP strategies have been discussed. Recently, lignocellulosic biomass as the most abundant renewable resource has been widely considered for bioalcohols production. However, the complex structure of lignocelluloses requires a multi-step process which is costly and time consuming. Although, several bioprocessing approaches have been developed for pretreatment, saccharification and fermentation, bioalcohols production from lignocelluloses is still limited because of the economic infeasibility of these technologies. This cost constraint could be overcome by designing and constructing robust cellulolytic and bioalcohols producing microbes and by using them in a consolidated bioprocessing (CBP) system. This paper comprehensively reviews potentials, recent advances and challenges faced in CBP systems for efficient bioalcohols (ethanol and butanol) production from lignocellulosic and starchy biomass. The CBP strategies include using native single strains with cellulytic and alcohol production activities, microbial co-cultures containing both cellulytic and ethanologenic microorganisms, and genetic engineering of cellulytic microorganisms to be alcohol-producing or alcohol producing microorganisms to be cellulytic. Moreover, high-throughput techniques, such as metagenomics, metatranscriptomics, next generation sequencing and synthetic biology developed to explore novel microorganisms and powerful enzymes with high activity, thermostability and pH stability are also discussed. Currently, the CBP technology is in its infant stage, and ideal microorganisms and/or conditions at industrial scale are yet to be introduced. So, it is essential to bring into attention all barriers faced and take advantage of all the experiences gained to achieve a high-yield and low-cost CBP process.

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Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose

Cited by 267 publications

References 33 publications

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

Lignocellulosic Biomass Utilization Toward Biorefinery Using Meshophilic Clostridial Species

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

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