Metabolic engineering of Thermoanaerobacterium saccharolyticum for n-butanol production

Bhandiwad, Ashwini; Shaw, Aubie; Guss, Adam M.; Guseva, Anna; Bahl, Hubert; Lynd, Lee R.

doi:10.1016/j.ymben.2013.10.012

Cited by 69 publications

(45 citation statements)

References 49 publications

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“…1) (12). AdhE is present in a variety of mesophilic and thermophilic anaerobic bacteria capable of producing ethanol as a fermentation product (13)(14)(15)(16). AdhE has also been found in parasitic eukaryotes (17), anaerobic fungi (18), and algae (19).…”

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

et al. 2015

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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 (ϳ50% 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. IMPORTANCEThis 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. T hermophilic organisms, Clostridium thermocellum in particular, hold great promise for the production of ethanol from lignocellulosic feedstocks (1, 2). C. thermocellum is a thermophilic, Gram-positive obligate anaerobe that rapidly consumes cellulose. Engineered strains of Thermoanaerobacterium saccharolyticum (3), a thermophilic anaerobe that ferments xylan and other sugars derived from biomass, have been shown to produce ethanol at Ͼ50 g/liter, a near-theoretical yield (4). While comparable concentrations of ethanol are tolerated by selected strains of C. thermocellum (5-7), the maximum reported concentration of ethanol produced by this organism is 23.6 g/liter (8) and th...

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

et al. 2015

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“…and Thermoanaerobacter spp. (18)(19)(20)(21). For each of these species, one or more strains have been shown to be genetically accessible, and engineering tools have been developed (7,22).…”

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Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

Bosma

Weijer

Daas

et al. 2015

Appl Environ Microbiol

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b Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C 5 and C 6 sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216 T and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 ؋ 10 3 colonies per g plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C 5 and C 6 sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals. Green chemicals are sustainable bio-based alternatives for chemicals based on fossil resources. Biomass is considered an attractive renewable resource for the production of such green chemicals. Major challenges for using biomass are to develop costeffective production processes and to convert substrates that go beyond first-generation pure sugars. From this perspective, the use of microbial fermentation processes that convert lignocellulosic sugars is gaining considerable attention. For particular products natural producers can be used, but often Escherichia coli and Saccharomyces cerevisiae are used as platform organisms because of their well-known physiology and ease of engineering for the production of many different products (1, 2).In general, most organisms used for the production of green chemicals and fuels are mesophiles, and current processes still are based mainly on first-generation feedstocks, i.e., sucrose from sugar beet or sugarcane or glucose derived from starches from corn or tapioca. Although the engineering of platform organisms broadens the spectrum of possible substrates (3), the efficiency of the process could be increased by the use of organisms naturally capable of degrading lignocellulose-derived sugars (4). To further increase the efficiency and reduce the costs of the microbial production of green chemicals, modera...

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“…etabolic engineering for biomass-based fuel or chemical production has focused almost exclusively on mesophilic host organisms, although now thermophilic hosts are also being considered, as molecular genetic tools become available (1)(2)(3)(4). Thermal bioprocesses can be advantageous for a variety of reasons (5).…”

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“…This effect is likely due to the increased selectivity of C. acetobutylicum AdhE, Bad, and Bdh for four-carbon substrates (30,31). Additionally, increased production of reducing equivalents or knocking out genes encoding enzymes that produce more oxidized fermentation products, both of which change the host's redox balance, can increase n-butanol production (1,24,32). Thus, the ratio of ethanol to n-butanol production seems to depend upon the specificity of the aldehyde dehydrogenase (Bad or AdhE) and alcohol dehydrogenase (Bdh or AdhE) for four-carbon substrates, although redox balancing of substrates and fermentation products also plays a role.…”

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Alcohol Selectivity in a Synthetic Thermophilic n -Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes

Loder

Zeldes

Garrison

et al. 2015

Appl Environ Microbiol

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b n-Butanol is generated as a natural product of metabolism by several microorganisms, but almost all grow at mesophilic temperatures. A synthetic pathway for n-butanol production from acetyl coenzyme A (acetyl-CoA) that functioned at 70°C was assembled in vitro from enzymes recruited from thermophilic bacteria to inform efforts for engineering butanol production into thermophilic hosts. Thermoanaerobacter sp. strain X514) were utilized to examine three possible pathways for n-butanol. These pathways differed in the two steps required to convert butyryl-CoA to n-butanol: Thl-Hbd-Crt-Ter-AdhE (C. thermocellum), Thl-Hbd-Crt-Ter-AdhE (Thermoanaerobacter X514), and Thl-Hbd-Crt-Ter-Bad-Bdh. n-Butanol was produced at 70°C, but with different amounts of ethanol as a coproduct, because of the broad substrate specificities of AdhE, Bad, and Bdh. A reaction kinetics model, validated via comparison to in vitro experiments, was used to determine relative enzyme ratios needed to maximize n-butanol production. By using large relative amounts of Thl and Hbd and small amounts of Bad and Bdh, >70% conversion to n-butanol was observed in vitro, but with a 60% decrease in the predicted pathway flux. With more-selective hypothetical versions of Bad and Bdh, >70% conversion to n-butanol is predicted, with a 19% increase in pathway flux. Thus, more-selective thermophilic versions of Bad, Bdh, and AdhE are needed to fully exploit biocatalytic n-butanol production at elevated temperatures. Metabolic engineering for biomass-based fuel or chemical production has focused almost exclusively on mesophilic host organisms, although now thermophilic hosts are also being considered, as molecular genetic tools become available (1-4). Thermal bioprocesses can be advantageous for a variety of reasons (5). Extreme thermophiles (optimum temperature [T opt ] of Ն70°C), in particular, could be especially strategic for industrial processes, due to lower risk of contamination, facilitated product recovery, and reduced cooling costs, factors which must be weighed against energy requirements to maintain bioprocesses at elevated temperatures (6, 7).In principle, thermophilic metabolic engineering platforms can potentially draw from an enzyme inventory encompassing a broad temperature range (8-10). However, one must take into account potential issues with synthetic pathways comprised of heterologous enzymes with variable levels of thermoactivity and thermostability. This factor can be exacerbated by the relative scarcity of biochemically and biophysically characterized versions of specific thermophilic enzymes of interest. As such, biocatalysts may need to be recruited from sources with functional temperature ranges that are inconsistent with the thermophilic host, leading to incompatibility between the activity and stability among enzymes selected for use in an engineered pathway. For example, metabolic engineering of Caldicellulosiruptor bescii (T opt ϭ 78°C) for increased ethanol production utilized an enzyme from Clostridium thermocellum (T opt ϭ 60°C),...

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Metabolic engineering of Thermoanaerobacterium saccharolyticum for n-butanol production

Cited by 69 publications

References 49 publications

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

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

Isolation and Screening of Thermophilic Bacilli from Compost for Electrotransformation and Fermentation: Characterization of Bacillus smithii ET 138 as a New Biocatalyst

Alcohol Selectivity in a Synthetic Thermophilic n -Butanol Pathway Is Driven by Biocatalytic and Thermostability Characteristics of Constituent Enzymes

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