The fungal biocontrol agent Coniothyrium minitans: production by solid-state fermentation, application and marketing.

Biological control agents (BCAs) are potential alternatives for the chemical fungicides presently used in agriculture to fight plant diseases. Coniothyrium minitans is an example of a promising fungal BCA. It is a naturally occurring parasite of the fungus Sclerotinia sclerotiorum, a wide-spread pathogen which substantially reduces the yield of many crops. This review describes, exemplified by C. minitans, the studies that need to be carried out before a fungal BCA is successfully introduced into the market. The main aspects considered are the biology of C. minitans, the development of a product by mass production of spores using solid-state fermentation technology, its biocontrol activity and marketing of the final product.

Defined media and inert supports: their potential as solid-state fermentation production systems.

Solid-state fermentation (SSF) using inert supports impregnated with chemically defined liquid media has several potential applications in both scientific studies and in the industrial production of high-value products, such as metabolites, biological control agents and enzymes. As a result of its more defined system, SSF on inert supports offers numerous advantages, such as improved process control and monitoring, and enhanced process consistency, compared with cultivation on natural solid substrates.

Growth and sporulation stoichiometry and kinetics of Coniothyrium minitans on agar media.

Coniothyrium minitans was cultivated on agar media with different concentrations of starch, urea, and trace elements. By means of elemental balances, the stoichiometry of growth and sporulation was established. C. minitans produced byproducts on all media, especially in the medium with high urea concentrations, where 30% of the starch was converted into byproducts. Simple empirical models were used to describe the kinetics of growth, sporulation, CO(2) production, and substrate consumption on all media. Total biomass and mycelium could be described reasonably well with the logistic law. Starch, urea, and oxygen consumption and CO(2) production could be described as a function of total biomass by the linear-growth model of Pirt. There were almost no differences between media for the estimates of yield coefficients and maintenance coefficients. Only at high urea concentrations were maintenance coefficients much higher. Similar to substrate consumption and CO(2) production, the kinetics of sporulation could be described as a function of mycelium production with the linear-growth model. It is shown that sporulation of C. minitans is growth-associated. Based on kinetics, the process costs for producing spores are roughly calculated. In addition, it is shown that fermentor costs represent the majority of production costs.

Engineering of factors determining alpha-amylase and cyclodextrin glycosyltransferase specificity in the cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1.

The starch-degrading enzymes alpha-amylase and cyclodextrin glycosyltransferase (CGTase) are functionally and structurally closely related, with CGTases containing two additional domains (called D and E) compared to the three domains of alpha-amylases (A, B and C). Amino acid residue 196 (Thermoanaerobacterium thermosulfurigenes EM1 CGTase numbering) occupies a dominant position in the active-site cleft. All alpha-amylases studied have a small residue at this position (Gly, Leu, Ser, Thr or Val), in contrast to CGTases which have a more bulky aromatic residue (Tyr or Phe) at this position, which is highly conserved. Characterization of the F196G mutant CGTase of T. thermosulfurigenes EM1 revealed that, for unknown reasons, apart from the F196G mutation, domain E as well as a part of domain D had become deleted [mutant F196G(delta'DE)]. This, nevertheless, did not prevent the purification of a stable and active mutant CGTase protein (62 kDa). The mutant protein was more similar to an alpha-amylase protein in terms of the identity of residue 196, and in the domain structure containing, however, some additional C-terminal structure. The mutant showed a strongly reduced temperature optimum. Due to a frameshift mutation in mutant F196G, a separate protein of 19 kDa with the DE domains was also produced. Mutant F196G(delta'DE) displayed a strongly reduced raw-starch-binding capacity, similar to the situation in most alpha-amylases that lack a raw-starch-binding E domain. Compared to wild-type CGTase, cyclization, coupling and disproportionation activities had become drastically reduced in the mutant F196G(delta'DE), but its saccharifying activity had doubled, reaching the highest level ever reported for a CGTase. Under industrial production process conditions, wild-type CGTase converted starch into 35% cyclodextrins and 11% linear oligosaccharides (glucose, maltose and maltotriose), whereas mutant F196G(delta'DE) converted starch into 21% cyclodextrins and 18% into linear oligosaccharides. These biochemical characteristics indicate a clear shift from CGTase to alpha-amylase specificity.

Engineering of cyclodextrin product specificity and pH optima of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EM1.

The product specificity and pH optimum of the thermostable cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes EM1 was engineered using a combination of x-ray crystallography and site-directed mutagenesis. Previously, a crystal soaking experiment with the Bacillus circulans strain 251 beta-CGTase had revealed a maltononaose inhibitor bound to the enzyme in an extended conformation. An identical experiment with the CGTase from T. thermosulfurigenes EM1 resulted in a 2.6-A resolution x-ray structure of a complex with a maltohexaose inhibitor, bound in a different conformation. We hypothesize that the new maltohexaose conformation is related to the enhanced alpha-cyclodextrin production of the CGTase. The detailed structural information subsequently allowed engineering of the cyclodextrin product specificity of the CGTase from T. thermosulfurigenes EM1 by site-directed mutagenesis. Mutation D371R was aimed at hindering the maltohexaose conformation and resulted in enhanced production of larger size cyclodextrins (beta- and gamma-CD). Mutation D197H was aimed at stabilization of the new maltohexaose conformation and resulted in increased production of alpha-CD. Glu258 is involved in catalysis in CGTases as well as alpha-amylases, and is the proton donor in the first step of the cyclization reaction. Amino acids close to Glu258 in the CGTase from T. thermosulfurigenes EM1 were changed. Phe284 was replaced by Lys and Asn327 by Asp. The mutants showed changes in both the high and low pH slopes of the optimum curve for cyclization and hydrolysis when compared with the wild-type enzyme. This suggests that the pH optimum curve of CGTase is determined only by residue Glu258.

Crystal structure at 2.3 A resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermonanaerobacterium thermosulfurigenes EM1.

The crystal structure of the cyclodextrin glycosyltransferase (CGTase) from the thermophilic microorganism Thermoanaerobacterium thermosulfurigenes EM1 has been elucidated at 2.3 A resolution. The final model consists of all 683 amino acid residues, two calcium ions and 343 water molecules, and has a crystallographic R-factor of 17.9% (Rfree 24.9%) with excellent stereochemistry. The overall fold of the enzyme is highly similar to that reported for mesophilic CGTases and differences are observed only at surface loop regions. Closer inspection of these loop regions and comparison with other CGTase structures reveals that especially loops 88-95, 335-339 and 534-539 possibly contribute with novel hydrogen bonds and apolar contacts to the stabilization of the enzyme. Other structural features that might confer thermostability to the T. thermosulfurigenes EM1 CGTase are the introduction of five new salt-bridges and three Gly to Ala/Pro substitutions. The abundance of Ser, Thr and Tyr residues near the active site and oligosaccharide binding sites might explain the increased thermostability of CGTase in the presence of starch, by allowing amylose chains to bind non-specifically to the protein. Additional stabilization of the A/E domain interface through apolar contacts involves residues Phe273 and Tyr187. No additional or improved calcium binding is observed in the structure, suggesting that the observed stabilization in the presence of calcium ions is caused by the reduced exchange of calcium from the protein to the solvent, rendering it less susceptible to unfolding. The 50% decrease in cyclization activity of the T. thermosulfurigenes EM1 CGTase compared with that of B. circulans strain 251 appears to be caused by the changes in the conformation and amino acid composition of the 88-95 loop. In the T. thermosulfurigenes EM1 CGTase there is no residue homologous to Tyr89, which was observed to take part in stacking interactions with bound substrate in the case of the B. circulans strain 251 CGTase. The lack of this interaction in the enzyme-substrate complex is expected to destabilize bound substrates prior to cyclization. Apparently, some catalytic functionality of CGTase has been sacrificed for the sake of structural stability by modifying loop regions near the active site.

Cyclodextrin formation by the thermostable alpha-amylase of Thermoanaerobacterium thermosulfurigenes EM1 and reclassification of the enzyme as a cyclodextrin glycosyltransferase.

Extensive characterization of the thermostable alpha-amylase of Clostridium thermosulfurogenes EM1, recently reclassified as Thermoanaerobacterium thermosulfurigenes, clearly demonstrated that the enzyme is a cyclodextrin glycosyltransferase (CGTase). Product analysis after incubation of the enzyme with starch revealed formation of alpha-, beta-, and gamma-cyclodextrins, as well as linear sugars. The specific activity for cyclization of this CGTase was similar to those of other CGTases, whereas the specific activity for hydrolysis was relatively high in comparison with other CGTases. Alignment of the amino acid sequence of the T. thermosulfurigenes enzyme with sequences from known bacterial CGTases showed high homology. The four consensus regions of carbohydrate-converting enzymes, as well as a C-terminal raw-starch binding motif, could be identified in the sequence.