White biotechnology: State of the art strategies for the development of biocatalysts for biorefining.

White biotechnology is a term that is now often used to describe the implementation of biotechnology in the industrial sphere. Biocatalysts (enzymes and microorganisms) are the key tools of white biotechnology, which is considered to be one of the key technological drivers for the growing bioeconomy. Biocatalysts are already present in sectors such as the chemical and agro-food industries, and are used to manufacture products as diverse as antibiotics, paper pulp, bread or advanced polymers. This review proposes an original and global overview of highly complementary fields of biotechnology at both enzyme and microorganism level. A certain number of state of the art approaches that are now being used to improve the industrial fitness of biocatalysts particularly focused on the biorefinery sector are presented. The first part deals with the technologies that underpin the development of industrial biocatalysts, notably the discovery of new enzymes and enzyme improvement using directed evolution techniques. The second part describes the toolbox available by the cell engineer to shape the metabolism of microorganisms. And finally the last part focuses on the 'omic' technologies that are vital for understanding and guide microbial engineering toward more efficient microbial biocatalysts. Altogether, these techniques and strategies will undoubtedly help to achieve the challenging task of developing consolidated bioprocessing (i.e. CBP) readily available for industrial purpose.

A new approach for detecting and mapping sewage impacts.

Increased nitrogen loading has been implicated in eutrophication occurrences worldwide. Much of this loading is attributable to the growing human population along the world's coastlines. A significant component of this nitrogen input is from sewage effluent, and delineation of the distribution and biological impact of sewage-derived nitrogen is becoming increasingly important. Here, we show a technique that identifies the source, extent and fate of biologically available sewage nitrogen in coastal marine ecosystems. This method is based on the uptake of sewage nitrogen by marine plants and subsequent analysis of the sewage signature (elevated delta 15N) in plant tissues. Spatial analysis is used to create maps of delta 15N and establish coefficient of variation estimates of the mapped values. We show elevated delta 15N levels in marine plants near sewage outfalls in Moreton Bay, Australia, a semi-enclosed bay receiving multiple sewage inputs. These maps of sewage nitrogen distribution are being used to direct nutrient reduction strategies in the region and will assist in monitoring the effectiveness of environmental protection measures.

Genetic and biochemical characterization of a highly thermostable alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus.

The gene encoding an alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus D3, AbfD3, was isolated. Characterization of the purified recombinant alpha-L-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90 degrees C) and pH (stable in the pH range 4 to 12). On the basis of amino acid sequence similarities, this 56, 071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides.

The type II pullulanase of Thermococcus hydrothermalis: molecular characterization of the gene and expression of the catalytic domain.

The gene encoding a hyperthermostable type II pullulanase produced by Thermococcus hydrothermalis (Th-Apu) has been isolated. Analysis of a total of 5.2 kb of genomic DNA has revealed the presence of three open reading frames, one of which (apuA) encodes the pullulanase. This enzyme is composed of 1,339 amino acid residues and exhibits a multidomain structure. In addition to a typical N-terminal signal peptide, Th-Apu possesses a catalytic domain, a domain bearing S-layer homology-like motifs, a Thr-rich region, and a potential C-terminal transmembrane domain. The presence of these noncatalytic domains suggests that Th-Apu may be anchored to the cell surface and be O glycosylated.

A method for introducing site-specific mutations using oligonucleotide primers and its application to site-saturation mutagenesis.

Oligonucleotide primer-directed mutagenesis is a useful molecular biological tool, which is invaluable for the study of the structure/function relationships in proteins and for the creation of mutant proteins possessing modified or novel biological activities. Mutagenesis studies in which a site-saturation approach is employed require a high-efficiency mutagenesis procedure, which will generate a population of mutated molecules containing an even distribution of all possible amino acid changes, or a subset thereof. This article describes such a mutagenesis technique and discusses the adaptations that are necessary to perform site-saturation mutagenesis.

The roles of the prosequence of thermolysin in enzyme inhibition and folding in vitro.

The zinc endopeptidase thermolysin (EC 3.4.24.27), an extracellular enzyme from Bacillus thermoproteolyticus, is synthesized as a preproprotein, with the prosequence (204 residues) being two-thirds the size of the mature enzyme (316 residues). This prosequence, expressed in and purified from Escherichia coli, inhibited thermolysin in vitro with an IC50 value of 14 nM. It also inhibited a closely related enzyme produced by Bacillus stearothermophillus, albeit with a 16-fold higher IC50 value (220 nM). The IC50 value for thermolysin inhibition was also increased 15-fold (210 nm) by a monoclonal antibody that recognizes an epitope close to, but not forming a part of, the active site. At a prosequence concentration of 5 microM a mammalian, thermolysin-like enzyme, neutral endopeptidase 24.11, was not inhibited. The prosequence appeared to act as a mixed, noncompetitive inhibitor of thermolysin activity, with a Ki value of 6 nM for its interaction with the enzyme alone and a Ki' value of 20 nM for its interaction with the enzyme-substrate complex. In addition, when thermolysin was denatured in 6 M guanidinium hydrochloride at acid pH and then brought to neutral pH by rapid dilution, the prosequence was found to facilitate the recovery of active enzyme in a stoichiometric manner.

Overexpression in Pichia pastoris and crystallization of an elicitor protein secreted by the phytopathogenic fungus, Phytophthora cryptogea.

A synthetic gene encoding beta-cryptogein, a member of the elicitin family, has been cloned into a vector for expression by the methylotrophic yeast, Pichia pastoris. Having first optimized the gene construction for secretion, we have overexpressed a modified beta-cryptogein in a secreted form. A purification scheme suited to this expression system has been developed and highly pure, biologically active protein has been obtained. For structural analysis of this recombinant beta-cryptogein, and new mutated forms thereof, optimal conditions for the crystallization of this protein have been determined and crystals that diffract to 2.2 A have been obtained.

The role of histidine 231 in thermolysin-like enzymes. A site-directed mutagenesis study.

In the zinc metallopeptidases produced by the genus Bacillus, an active site histidine has been proposed to either stabilize the transition state in catalysis by donating a hydrogen bond to the hydrated peptide (Matthews, B. W. (1988) Acc. Chem. Res. 21, 333-340) or to polarize a water molecule, which subsequently attacks the peptidyl bond (Mock, W. L., and Aksamawati, M. (1994) Biochem. J. 302, 57-68). Site-directed mutagenesis techniques have been used to change this residue in the zinc endopeptidase from Bacillus stearothermophillus to either phenylalanine or alanine. At pH 7.0, the kcat/Km values of the substrate leucine enkephalin for the phenylalanine and alanine mutants were reduced by factors of 430- and 500-fold, respectively, as compared with the wild-type enzyme, mostly due to changes in kcat. In addition, the enzymatic activities of the mutant enzymes showed little pH dependence in the alkaline range, unlike the wild-type enzyme. The mutations did not greatly alter the binding affinities of inhibitors containing sulfydryl groups to chelate the active site zinc, while those of inhibitors containing hydroxamate or carboxylate zinc-chelating groups were increased between 80- and 250-fold. The largest change in the binding affinity of an inhibitor (> 5 orders of magnitude) was found with the proposed transition state mimic, phosphoramidon. The results are generally in agreement with x-ray crystallography studies and favor the involvement of the active site histidine in transition state binding.

Chemical synthesis, expression and mutagenesis of a gene encoding beta-cryptogein, an elicitin produced by Phytophthora cryptogea.

Elicitins are 10 kDa holoproteins secreted by Phytophthora fungi, that elicit an incompatible hypersensitive reaction, leading to resistance against fungal and bacterial plant pathogens. Comparison of primary sequences of alpha-elicitins and beta-elicitins indicated several potential necrotic activity-determining residues. All of the highly necrotic beta-elicitins have a hydrophilic residue (usually lysine) at position 13, whereas in the less necrotic alpha-elicitins this residue is replaced by a valine. Here, we report the synthesis and expression of a gene encoding a highly necrotic elicitin, beta-cryptogein, and we show that the substitution of Lys-13 of this recombinant protein by a valine leads to a drastic alteration to the necrotic activity of the recombinant protein.

Cloning and expression in Bacillus subtilis of the npr gene from Bacillus thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin.

We report the isolation, cloning and expression, in Bacillus subtilis, of the gene coding for thermolysin, a thermostable metalloprotease which is produced by Bacillus thermoproteolyticus Rokko. The nucleotide sequence has revealed that, like neutral proteases produced by other members of the Bacillus species, thermolysin is probably produced as a preproenzyme carrying a typical N-terminal membrane signal sequence. Further, the thermolysin gene shares a strong homology with two other previously cloned genes from two different strains of Bacillus stearothermophilus. The sequence of the mature secreted protease, inferred from the DNA sequence, is, with two exceptions, identical with the previously published protein sequence of thermolysin [Titani, Hermodson, Ericsson, Walsh and Neurath (1972) Nature (London) 238, 35-37]. The exceptions are Asn37 and Gln119, originally reported to be Asp and Glu respectively. The biochemical characterization of the secreted recombinant protein shows that it is indistinguishable from the wild-type thermolysin.

Tyr26 and Phe73 are essential for full biological activity of the Fd gene 5 protein.

Using site-saturation mutagenesis, we have established all possible amino acid substitutions at Tyr26 and Phe73 that are compatible with biological activity of the gene 5 protein in vivo. No substitutions were found at either site that gave rise to a fully functional gene 5 protein, indicating that these two amino acid residues are crucial. However, partial activity was found if either residue was replaced by another aromatic amino acid (Y26F, Y26W, F73Y, F73W). The results suggest that both Tyr26 and Phe73 are important for base stacking in the nucleoprotein complex. The functional consequences of the removal of the hydroxyl group from Tyr26 argue that this residue may, in addition, be involved in hydrogen bond formation to confer greater stability on the complex. In contrast, the addition of such a group to Phe73 reduces activity.

Nitrogen fixation in sediments and the rhizosphere of the seagrassZostera capricorni.

Rates of nitrogen fixation in seagrass beds (Zostera capricorni) were determined with(15)N and reduction of acetylene in intact cores of sediment and seagrass. There was good agreement in the results from the two techniques, with a molar ratio of 3∶1.9 ethylene: ammonia produced. Fixed nitrogen was rapidly utilized by the plants, with significant amounts of(15)N found in the roots and rhizomes and 50% of fixed(15)N apparently translocated to the leaves. Rates of fixation were high in summer (25 to 40 mg N m(-2) day(-1)) and lower in winter (10 mg N m(-2) day(-1)) and were estimated to supply between one-third and one-half of the nitrogen requirements of the seagrass. Rates of nitrogen fixation were greater in the light than in the dark, and in cores of intact seagrass than in defoliated cores, indicating that the bacteria were dependent on organic compounds secreted by the plants.