Advances in Development of a Genetic System for
            <i>Thermoanaerobacterium</i>
            spp.: Expression of Genes Encoding Hydrolytic Enzymes, Development of a Second Shuttle Vector, and Integration of Genes into the Chromosome

Mai, Volker; Wiegel, Juergen

doi:10.1128/aem.66.11.4817-4821.2000

Cited by 42 publications

(17 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We focus on the body of work involving these three organisms because this encompasses the most advanced embodiment of the recombinant cellulolytic organism development strategy to date. Some work on heterologous cellulase expression has been undertaken with additional hosts (328,419,520), whose potential utility should not be dismissed. The possibility of restoring functional expression of cryptic cellulase genes in C. acetobutylicum (606) is also intriguing.…”

Section: Recombinant Cellulolytic Strategymentioning

confidence: 99%

“…Electrotransformation is increasingly common and is usually viewed as a preferred gene transfer method (440,771). Electrotransformation systems were developed during the 1980s and 1990s in the case of noncellulolytic mesophilic clostridia including C. acetobutylicum, C. beijerinckii, C. perfringens, and at least five additional species (771), as well as T. thermosaccharolyticum (341) and Thermoanaerobacterium saccharolyticum (418,419).…”

Section: Native Cellulolytic Strategymentioning

confidence: 99%

See 1 more Smart Citation

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,019

2,868

View full text Add to dashboard Cite

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts

show abstract

Section: Recombinant Cellulolytic Strategymentioning

confidence: 99%

Section: Native Cellulolytic Strategymentioning

confidence: 99%

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Lynd

Weimer

Zyl³

et al. 2002

Microbiol Mol Biol Rev

4,019

2,868

View full text Add to dashboard Cite

show abstract

“…Extensive efforts using classical mutagenesis techniques to obtain stable strains exhibiting high-ethanol yields over a range of conditions have not been successful (13). Genetic systems suitable for engineering thermophiles have long limited strain development, but have started to emerge (14)(15)(16) along with the first reports of metabolic engineering in thermophilic, saccharolytic hosts (17,18). Here, we report engineering T. saccharolyticum JW/SL-YS485 to produce ethanol as the only significant organic product.…”

mentioning

confidence: 99%

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

Shaw

Podkaminer²,

Desai³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

329

242

View full text Add to dashboard Cite

We report engineering Thermoanaerobacterium saccharolyticum, a thermophilic anaerobic bacterium that ferments xylan and biomass-derived sugars, to produce ethanol at high yield. Knockout of genes involved in organic acid formation (acetate kinase, phosphate acetyltransferase, and L-lactate dehydrogenase) resulted in a strain able to produce ethanol as the only detectable organic product and substantial changes in electron flow relative to the wild type. Ethanol formation in the engineered strain (ALK2) utilizes pyruvate:ferredoxin oxidoreductase with electrons transferred from ferredoxin to NAD(P), a pathway different from that in previously described microbes with a homoethanol fermentation. The homoethanologenic phenotype was stable for >150 generations in continuous culture. The growth rate of strain ALK2 was similar to the wild-type strain, with a reduction in cell yield proportional to the decreased ATP availability resulting from acetate kinase inactivation. Glucose and xylose are co-utilized and utilization of mannose and arabinose commences before glucose and xylose are exhausted. Using strain ALK2 in simultaneous hydrolysis and fermentation experiments at 50°C allows a 2.5-fold reduction in cellulase loading compared with using Saccharomyces cerevisiae at 37°C. The maximum ethanol titer produced by strain ALK2, 37 g/liter, is the highest reported thus far for a thermophilic anaerobe, although further improvements are desired and likely possible. Our results extend the frontier of metabolic engineering in thermophilic hosts, have the potential to significantly lower the cost of cellulosic ethanol production, and support the feasibility of further cost reductions through engineering a diversity of host organisms.bioenergy ͉ cellulosic ethanol ͉ thermophile

show abstract

“…An ET protocol developed for the cellulolytic mesophile Clostridium cellulolyticum (8,38) has been used to express pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis (6). Examples of gene transfer to gram-positive obligate anaerobic thermophiles are limited to Thermoanaerobacterium species (10,15,17) and a preliminary report of protoplast transformation of C. thermocellum (40). In these studies, the highest efficiencies of ET (ϳ10 3 /g of DNA) were obtained by for Thermoanaerobacterium sp.…”

mentioning

confidence: 99%

Electrotransformation of Clostridium thermocellum

Tyurin¹,

Desai²,

Lynd

2004

Appl Environ Microbiol

101

View full text Add to dashboard Cite

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 ؎ 0.5) ؋ 10 5 transformants per g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 ؎ 1.8) ؋ 10 4 transformants per g of plasmid DNA for strain ATCC 27405 and ϳ1 ؋ 10 3 transformants per g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was ϳ50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific.

show abstract

Advances in Development of a Genetic System for Thermoanaerobacterium spp.: Expression of Genes Encoding Hydrolytic Enzymes, Development of a Second Shuttle Vector, and Integration of Genes into the Chromosome

Cited by 42 publications

References 25 publications

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Microbial Cellulose Utilization: Fundamentals and Biotechnology

Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield

Electrotransformation of Clostridium thermocellum

Contact Info

Product

Resources

About