Hubert Bahl

Metabolic engineering of Clostridium acetobutylicum: recent advances to improve butanol production.

The biosynthesis of the solvents 1-butanol and acetone is restricted to species of the genus Clostridium, a diverse group of Gram-positive, endospore forming anaerobes comprising toxin-producing strains as well as terrestrial non-pathogenic species of biotechnological impact. Among solventogenic clostridia, Clostridium acetobutylicum represents the model organism and general but yet important genetic tools were established only recently to investigate and understand the complex life cycle-accompanied physiology and its regulatory mechanisms. Since clostridial butanol production regained much interest in the past few years, different metabolic engineering approaches were conducted--although promising and in part successful strategies were employed, the major breakthrough to generate an optimum phenotype with superior butanol titer, yield and productivity still remains to be expected.

Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum

SummaryClostridium acetobutylicum cells were collected from chemostats which were run at pH 4.3 or 6.0 and which produced either acetone-butanol or acetate-butyrate; they were used to determine the level of enzymes involved either in solvent or in acid formation. The highest activity of phosphotransacetylase, phosphotransbutyrylase, acetate kinase, and butyrate kinase was found in cells which carried out an acetate-butyrate fermentation; these enzymes were present in solvent-producing cells at a level of about 10–50% as compared to acid-producing cells. Hydrogenase activity was detectable in approximately the same amounts in both cell types; however, in solvent-producing cells it was only measurable following a lag-period. Butyraldehyde and butanol dehydrogenases were found in small amounts exclusively in solvent-producing cells. It was demonstrated that the formation of acetone was initiated by the action of a coenzyme A-transferase which transferred coenzyme A from acetoacetyl-CoA to either acetate or butyrate. This coenzyme A-transferase as well as acetoacetate decarboxylase were hardly detectable in acid-producing cells, but reached high levels in solvent producing cells. Similar changes of the activity of the enzymes mentioned were observed when a batch culture was shifted from acid to solvent formation.

Effect of pH and butyrate concentration on the production of acetone and butanol by Clostridium acetobutylicum grown in continuous culture

SummaryWhen Clostridium acetobutylicum was grown in continuous culture under glucose limitation at neutral pH and varying dilution rates the only fermentation products formed were acetate, butyrate, carbon dioxide and molecular hydrogen. The Yglucosemaxand (YATPmax)glucexpvalues were 48.3 and 23.8 dry weight/mol, respectively. Acetone and butanol were produced when the pH was decreased below 5.0 (optimum at pH 4.3). The addition of butyric acid (20 to 80 mM) to the medium with a pH of 4.3 resulted in a shift of the fermentation from acid, to solvent formation.

Continuous production of acetone and butanol by Clostridium acetobutylicum in a two-stage phosphate limited chemostat

When Clostridium acetobutylicum was grown in continuous culture under phosphate limitation (0.74 mM) at a pH of 4.3, glucose was fermented to butanol, acetone and ethanol as the major products. At a dilution rate of D=0.025 h−1 and a glucose concentration of 300 mM, the maximal butanol and acetone concentrations were 130 mM and 74 mM, respectively. 20% of the glucose remained in the medium. On the basis of these results a two-stage continuous process was developed in which 87.5% of the glucose was converted into butanol, acetone and ethanol. The cells and minor amounts of acetate and butyrate accounted for the remaining 12.5% of the substrate. The first stage was run at D=0.125 h−1 and 37° C and the second stage at D=0.04 h−1 and 33° C. High yields of butanol and acetone were also obtained in batch culture under phosphate limitation.

V. Functions of S‐layers

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

Introduction to the Physiology and Biochemistry of the Genus Clostridium

The genus Clostridium was created by Prazmowski in 1880. Four criteria presently classify an organism as a Clostridium: (1) the ability to form endospores; (2) restriction to an anaerobic energy metabolism; (3) the inability to carry out a dissimilatory reduction of sulfate; and (4) the possession of a gram-positive cell wall, which may react gram-negative. These criteria are met by an otherwise diverse assembly of microorganisms, and the genus Clostridium has grown to be one of the largest genera among prokaryotes. A total of 83 species are listed in Bergey’s Manual of Systematic Bacteriology (Cato et al., 1986). Since this list was compiled, a number of new species have been described, while others, such as C. tetanomorphum and C. cylindrosporum, have been omitted (see Chapter 1). In this chapter the span of properties found among the Clostridia will be outlined. Additional information on the general taxonomy, the general properties of Clostridia, and clostridial fermentations may be found in a number of recent reviews (Barker, 1961, 1978, 1981; Wood, 1961; Thauer et al., 1977; Gottschalk and Andreesen, 1979; Gottschalk et al., 1981; Booth and Mitchell, 1987).

Biological control of fungal strawberry diseases by Serratia plymuthica HRO-C48

To develop a biological control product for commercial strawberry production, the chitinolytic rhizobacterium Serratia plymuthica strain HRO-C48 was evaluated for plant growth promotion of strawberries and biological control of the fungal pathogens Verticillium dahliae and Phytophthora cactorum. In phytochamber experiments, treatment with S. plymuthica HRO-C48 resulted in a statistically significant enhancement of plant growth dependent on the concentration of the bacterium that was applied. In greenhouse trials, bacterial treatment reduced the percentage of Verticillium wilt (18.5%) and Phytophthora root rot (33.4%). In three consecutive vegetation periods, field trials were carried out in soil naturally infested by both soilborne pathogens on commercial strawberry farms located in various regions of Germany. Dipping plants in a suspension of S. plymuthica prior to planting reduced Verticillium wilt compared with the nontreated control by 0 to 37.7%, with an average of 24.2%, whereas the increase of yield ranged from 156 to 394%, with an average of 296%. Bacterial treatment reduced Phy-tophthora root rot by 1.3 to 17.9%, with an average of 9.6%, and increased strawberry yield by 60% compared with the nontreated control. Under field conditions, strain HRO-C48 survived at approximately log10 3 to 7 CFU/g of root in the strawberry rhizosphere at 14 months after root application. Although results of the field trials were influenced by pathogen inoculum density, cropping history of the field site, and weather conditions, S. plymuthica HRO-C48 successfully controlled wilt and root rot of strawberry.

PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum

Clostridia belong to those bacteria which are considered as obligate anaerobe, e.g. oxygen is harmful or lethal to these bacteria. Nevertheless, it is known that they can survive limited exposure to air, and often eliminate oxygen or reactive derivatives via NAD(P)H‐dependent reduction. This system does apparently contribute to survival after oxidative stress, but is insufficient to establish long‐term tolerance of aerobic conditions. Here we show that manipulation of the regulatory mechanism of this defence mechanism can trigger aerotolerance in the obligate anaerobe Clostridium acetobutylicum. Deletion of a peroxide repressor (PerR)‐homologous protein resulted in prolonged aerotolerance, limited growth under aerobic conditions and rapid consumption of oxygen from an aerobic environment. The mutant strain also revealed higher resistance to H2O2 and activities of NADH‐dependent scavenging of H2O2 and organic peroxides in cell‐free extracts increased by at least one order of magnitude. Several genes encoding the putative enzymes were upregulated and identified as members of the clostridial PerR regulon, including the heat shock protein Hsp21, a reverse rubrerythrin which was massively produced and became the most abundant protein in the absence of PerR. This multifunctional protein is proposed to play the crucial role in the oxidative stress defence.

Modifying the product pattern of Clostridium acetobutylicum Physiological effects of disrupting the acetate and acetone formation pathways

Clostridial acetone–butanol–ethanol (ABE) fermentation is a natural source for microbial n-butanol production and regained much interest in academia and industry in the past years. Due to the difficult genetic accessibility of Clostridium acetobutylicum and other solventogenic clostridia, successful metabolic engineering approaches are still rare. In this study, a set of five knock-out mutants with defects in the central fermentative metabolism were generated using the ClosTron technology, including the construction of targeted double knock-out mutants of C. acetobtuylicum ATCC 824. While disruption of the acetate biosynthetic pathway had no significant impact on the metabolite distribution, mutants with defects in the acetone pathway, including both acetoacetate decarboxylase (Adc)-negative and acetoacetyl-CoA:acyl-CoA transferase (CtfAB)-negative mutants, exhibited high amounts of acetate in the fermentation broth. Distinct butyrate increase and decrease patterns during the course of fermentations provided experimental evidence that butyrate, but not acetate, is re-assimilated via an Adc/CtfAB-independent pathway in C. acetobutylicum. Interestingly, combining the adc and ctfA mutations with a knock-out of the phosphotransacetylase (Pta)-encoding gene, acetate production was drastically reduced, resulting in an increased flux towards butyrate. Except for the Pta-negative single mutant, all mutants exhibited a significantly reduced solvent production.

The redox-sensing protein Rex, a transcriptional regulator of solventogenesis in Clostridium acetobutylicum

Solventogenic clostridia are characterised by their biphasic fermentative metabolism, and the main final product n-butanol is of particular industrial interest because it can be used as a superior biofuel. During exponential growth, Clostridium acetobutylicum synthesises acetic and butyric acids which are accompanied by the formation of molecular hydrogen and carbon dioxide. During the stationary phase, the solvents acetone, butanol and ethanol are produced. However, the molecular mechanisms of this metabolic switch are largely unknown so far. In this study, in silico, in vitro and in vivo analyses were performed to elucidate the function of the CAC2713-encoded redox-sensing transcriptional repressor Rex and its role in the solventogenic shift of C. acetobutylicum ATCC 824. Electrophoretic mobility shift assays showed that Rex controls the expression of butanol biosynthetic genes as a response to the cellular NADH/NAD+ ratio. Interestingly, the Rex-negative mutant C. acetobutylicum rex::int(95) produced high amounts of ethanol and butanol, while hydrogen and acetone production were significantly reduced. Both ethanol and butanol (but not acetone) formation started clearly earlier than in the wild type. In addition, the rex mutant showed a de-repression of the bifunctional aldehyde/alcohol dehydrogenase 2 encoded by the adhE2 gene (CAP0035) as demonstrated by increased adhE2 expression as well as high NADH-dependent alcohol dehydrogenase activities. The results presented here clearly indicated that Rex is involved in the redox-dependent solventogenic shift of C. acetobutylicum.