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.

Current oscillations at about 24 MHz were observed during electrotransformation (ET) of the thermophilic anaerobes Clostridium thermocellum ATCC 27405, C. thermocellum DSM 1313, and Thermoanaerobacterium saccharolyticum YS 485, using a pulse gated by a square signal generated by a custom generator. In experiments in which only the field strength was varied, all three of these strains resulted in a one-to-one correspondence between the appearance of current oscillations and successful ET. Oscillations accompanied ET of both C. thermocellum strains only at field strengths of >12 kV/cm, and ET was only observed above the same threshold. Similarly, for T. saccharolyticum, oscillations were only observed at field strengths of >10 kV/cm, and ET was only observed above the same threshold. When a passive electrical filter consisting of an inductor and resistor in parallel was added to the system to prevent the development of oscillations, ET efficiencies were reduced dramatically for all three strains at all field strengths tested. The maximum tested field strength, 25 kV/cm, resulted in the maximum measured transformation efficiency for all three strains. At this field strength, the efficiency of ET in the absence of oscillations was decreased compared to that observed in the presence of oscillations by 500-fold for C. thermocellum ATCC 27405, 2,500-fold for C. thermocellum DSM 1313, and 280-fold for T. saccharolyticum. Controls using the same apparatus with Escherichia coli cells or a resistor with a value representative of the direct current resistance of typical cell samples did not develop oscillations, and ET efficiencies obtained with E. coli were the same with or without the electrical filter included in the pulse generator circuit. The results are interpreted to indicate that spontaneously arising oscillations have a large beneficial effect on transformation efficiency in the system employed here and that the development of oscillations in this system is affected by the cell species present.

Aims:  To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. Methods and Results:  The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG‐CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG‐CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l−1 ethanol and 296 mmol l−1 acetate and started producing 1·8 mol l−1 acetone in single‐stage continuous syngas fermentation. Conclusions:  The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone–butanol–ethanol fermentation of sugars. Significance and Impact of the Study:  The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl‐CoA to acetone via a non‐naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms.

Acetogen strain Clostridium sp. MT1121 produced 300 mM acetate (p<0.005) and 321 mM ethanol (p<0.005) from synthesis gas (syngas) blend 60 % CO and 40 % H(2). Clostridium sp. MT1121 was metabolically engineered to eliminate production of either acetate or acetaldehyde during syngas fermentation. We used Cre-lox66/lox71-based gene removal system to eliminate either phosphotransacetylase (pta), or acetaldehyde dehydrogenase (aldh). The resulted biocatalyst with eliminated pta increased ethanol yield to 610 mM (p<0.005). Inactivation of pta rendered only 502 mM of ethanol (p<0.005). The acetogen biocatalyst with eliminated aldh produced 450 mM acetate (p<0.005). The role of cell energy pool preservation for re-directed carbon flux is discussed. This is the first report on time- and cost-efficient gene elimination in acetogens using lox66/lox71 gene elimination system.

Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p < 0.005) and 293 mM ethanol (p < 0.005) fermenting synthesis gas blend 60% CO and 40% H₂ in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p < 0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes.