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Multivariate curve resolution: a method of evaluating the kinetics of biotechnological reactions.

In biotechnology, strong emphasis is placed on the development of wet chemical analysis and chromatography to separate target components from a complex matrix. In bioprocessing, the development of single compound biosensors is an important activity. The advantages of these techniques are their high sensitivity and specificity. Inline or online monitoring by means of spectroscopy has the potential to be used as an "all-in-one" analysis technique for biotechnological studies, but it lacks specificity. Multivariate curve resolution (MCR) can be used to overcome this limitation. MCR is able to extract the number of components involved in a complex spectral feature, to attribute the resulting spectra to chemical compounds, to quantify the individual spectral contributions, and to use this quantification to develop kinetic models for the process with or without a priori knowledge. After a short introduction to MCR, two applications are presented. In the first example, the spectral features of hemp are monitored and analysed during growth. MCR provides unperturbed spectra on the activity of, for example, lignin and cellulose during plant development. In a second example, the kinetics of a laccase enzyme-catalysed degradation of aromatic hydrocarbons are calculated from UV/VIS spectra.

Biotechnology↗

Dereplication for biotechnology screening: PyMS analysis and PCR-RFLP-SSCP (PRS) profiling of 16S rRNA genes of marine and terrestrial actinomycetes.

The search for exploitable biology is a major task for biotechnology-based industries. In this context, discrimination between previously tested or recovered micro-organisms (dereplication) is imperative, in order to reduce screening costs by sorting large collections of isolates, which are then subjected to further detailed evaluation. Pyrolysis mass spectrometry (PyMS) is a whole-cell fingerprinting technique that enables the rapid and reproducible sorting of micro-organisms, uses small samples and has the advantage of being fully automated. In this study, we compare chemometric fingerprinting with a ribotyping fingerprinting method, in order to investigate the extent to which pyrogroups formed by PyMS analysis relate to genetic diversity, using polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS). A mixture of environmental strains of mycolic acid containing actinomycetes was used to mimic the selection of colonies from primary isolation plates. The congruence found between the clusters defined by the chemometric and molecular fingerprinting techniques was very high and demonstrated the effectiveness of PyMS as a rapid sorting and dereplicating procedure for putatively novel strains, criteria that are critical for biotechnological screens. Moreover, PyMS analysis revealed significant variation within pyrogroups that contained strains with the same genotypic (PRS) characteristics, thus emphasising its discriminatory capacity at the infraspecies level.

Actinomycetales↗

Bacterial amino acid transport proteins: occurrence, functions, and significance for biotechnological applications.

Transport processes play a pivotal role in cellular metabolism, e.g. for the uptake of nutrients or the excretion of metabolic waste products. Moreover, they are also important in biotechnological processes such as the production of various amino acids by the use of microorganisms. The focus of this review is on bacterial amino acid transport systems, in particular those of Corynebacterium glutamicum and Escherichia coli, with respect to their function and biotechnological significance.

Amino Acid Transport Systems↗

Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus.

The genus Pleurotus comprises a group of edible ligninolytic mushrooms with medicinal properties and important biotechnological and environmental applications. The cultivation of Pleurotus spp is an economically important food industry worldwide which has expanded in the past few years. P. ostreatus is the third most important cultivated mushroom for food purposes. Nutritionally, it has unique flavor and aromatic properties; and it is considered to be rich in protein, fiber, carbohydrates, vitamins and minerals. Pleurotus spp are promising as medicinal mushrooms, exhibiting hematological, antiviral, antitumor, antibiotic, antibacterial, hypocholesterolic and immunomodulation activities. The bioactive molecules isolated from the different fungi are polysaccharides. One of the most important aspects of Pleurotus spp is related to the use of their ligninolytic system for a variety of applications, such as the bioconversion of agricultural wastes into valuable products for animal feed and other food products and the use of their ligninolytic enzymes for the biodegradation of organopollutants, xenobiotics and industrial contaminants. In this Mini-Review, we describe the properties of Pleurotus spp in relation to their biotechnological applications and potential.

Biodegradation, Environmental↗

Diatom cultivation and biotechnologically relevant products. Part I: cultivation at various scales.

Biotechnological applications of diatoms are still in development. Further development at the industrial scale will depend on optimisation of the culture process with the aim of reducing costs. Because of the photoautotrophic status of the majority of diatoms, microalgal cultures suffer from the limitation of light diffusion, which requires the development of suitable photobioreactors. Thus, genetically engineered microalgae that may be cultivated in heterotrophic conditions present a new opportunity. Other limiting factors, such as nutrients (phosphate, nitrogen, silicon), pH, temperature, bioturbation and many more must be taken into account. Most of the time, metabolic stress conditions lead to an overproduction of the products of interest, with a decrease in biomass production as a consequence. Outdoor cultures in open ponds are usually devoted to aquaculture for the feeding of shrimps and bivalve molluscs (commercial production), while closed axenic indoor/outdoor photobioreactors are used for biotechnological compounds of homogeneous composition (still at the laboratory scale). In addition to the optimum culture conditions that have to be taken into account for photobioreactor design, the localisation of produced metabolites (intra- or extracellular) may also be taken into account when choosing the design. Microalgal cell immobilisation may be a suitable technique for application to benthic diatoms, which are usually sensitive to bioturbation and/or metabolites which may be overexpressed.

Animal Feed↗

Biotechnological applications of green fluorescent protein.

Since its first use as a reporter gene in 1994, green fluorescent protein (GFP) has served as the researcher's agent: slipping, virtually undetected, into unseen spaces, reporting back valuable information, and securing the delivery of precious cargo through hostile domains. GFP's strength lies in its small size, formidable stability, and relative ease of use. It requires only oxygen and an energy source to do its work, which can be supplied at low cost and high precision, respectively. With such a low threshold for use, GFP is often the first line of inquiry into an unknown space. Here is provided a brief compendium of GFP's contributions to biotechnology. They are linked by a need for a level of information that was previously inaccessible, both spatially and temporally. Protein fusions, transcriptional reporters, whole-organism visualization, and various other screening applications are reviewed with respect to biotechnological applications. Germane molecular improvements to GFP are also discussed.

Animals↗

Biotechnological advantages of laboratory-scale solid-state fermentation with fungi.

Despite the increasing number of publications dealing with solid-state (substrate) fermentation (SSF) it is very difficult to draw general conclusion from the data presented. This is due to the lack of proper standardisation that would allow objective comparison with other processes. Research work has so far focused on the general applicability of SSF for the production of enzymes, metabolites and spores, in that many different solid substrates (agricultural waste) have been combined with many different fungi and the productivity of each fermentation reported. On a gram bench-scale SSF appears to be superior to submerged fermentation technology (SmF) in several aspects. However, SSF up-scaling, necessary for use on an industrial scale, raises severe engineering problems due to the build-up of temperature, pH, O2, substrate and moisture gradients. Hence, most published reviews also focus on progress towards industrial engineering. The role of the physiological and genetic properties of the microorganisms used during growth on solid substrates compared with aqueous solutions has so far been all but neglected, despite the fact that it may be the microbiology that makes SSF advantageous against the SmF biotechnology. This review will focus on research work allowing comparison of the specific biological particulars of enzyme, metabolite and/or spore production in SSF and in SmF. In these respects, SSF appears to possess several biotechnological advantages, though at present on a laboratory scale only, such as higher fermentation productivity, higher end-concentration of products, higher product stability, lower catabolic repression, cultivation of microorganisms specialized for water-insoluble substrates or mixed cultivation of various fungi, and last but not least, lower demand on sterility due to the low water activity used in SSF.

Aspergillus oryzae↗

Micro total analysis system (micro-TAS) in biotechnology.

Nanobiotechnology raises fascinating possibilities for new analytical assays in various fields such as bioelectronic assembly, biomechanics and sampling techniques, as well as in chips or micromachined devices. Recently, nanotechnology has greatly impacted biotechnological research with its potential applications in smart devices that can operate at the level of molecular manipulation. Micro total analysis system (micro-TAS) offers the potential for highly efficient, simultaneous analysis of a large number of biologically important molecules in genomic, proteomic and metabolic studies. This review aims to describe the present state-of-the-art of microsystems for use in biotechnological research, medicine and diagnostics.

Biosensing Techniques↗

Glutathione: a review on biotechnological production.

This Mini-Review summarizes the historic developments and technological achievements in the biotechnological production of glutathione in the past 30 years. Glutathione is the most abundant non-protein thiol compound present in living organisms. It is used as a pharmaceutical compound and can be used in food additives and the cosmetic industries. Glutathione can be produced using enzymatic methods in the presence of ATP and its three precursor amino acids (L-glutamic acid, L-cysteine, glycine). Alternatively, glutathione can be produced by direct fermentative methods using sugar as a starting material. In the latter method, Saccharomyces cerevisiae and Candida utilis are currently used to produce glutathione on an industrial scale. At the molecular level, the genes gshA and gshB, which encode the enzymes gamma-glutamylcysteine synthetase and glutathione synthetase, respectively, have been cloned from Escherichia coli and over-expressed in E. coli, S. cerevisiae, and Lactococcus lactis. It is anticipated that, with the design and/or discovery of novel producers, the biotechnological production of glutathione will be further improved to expand the application range of this physiologically and medically important tripeptide.

Adenosine Triphosphate↗

Applications of zeolite inorganic composites in biotechnology: current state and perspectives.

The purpose of this short review is to introduce applications of inorganic composites, zeolites, in biotechnology. Although inorganic chemistry is generally considered distant from biotechnology, the two could be harmoniously integrated for biopolymer chromatography. New chromatographic carriers have been developed based on principles differing from those underlying conventional chromatography. Some can be used for the purification of proteins according to novel physicochemical principles, according to their isoelectric point (pI), molecular weight and shape. The amount of protein adsorbed is related to the pore size of the composites, which can recognize biomolecules with reference to these three parameters. Proteins adsorbed at their pI have been found to be desorbed at the pI by polyethylene glycol, but not by high ionic medium (NaCl), SDS, non-ionic detergents, ATP or urea. Therefore, inorganic composites synthesized in consideration of pore size and three-dimensional structure are suitable as new chromatographic carriers. Selective fractionation of biomaterials including proteins and nucleic acids should provide useful information regarding whether conjugated proteins in a precipitated state can be separated on net charge and whether cells can be directly fractionated in future.

Adsorption↗

Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology.

Gibberellins (GAs) constitute a large family of tetracyclic diterpenoid carboxylic acids, some members of which function as growth hormones in higher plants. As well as being phytohormones, GAs are also present in some fungi and bacteria. In recent years, GA biosynthetic genes from Fusarium fujikuroi and Arabidopsis thaliana have been cloned and well characterised. Although higher plants and the fungus both produce structurally identical GAs, there are important differences indicating that GA biosynthetic pathways have evolved independently in higher plants and fungi. The fact that horizontal gene transfer of GA genes from the plant to the fungus can be excluded, and that GA genes are obviously missing in closely related Fusarium species, raises the question of the origin of fungal GA biosynthetic genes. Besides characterisation of F. fujikuroi GA pathway genes, much progress has been made in the molecular analysis of regulatory mechanisms, especially the nitrogen metabolite repression controlling fungal GA biosynthesis. Basic research in this field has been shown to have an impact on biotechnology. Cloning of genes, construction of knock-out mutants, gene amplification, and regulation studies at the molecular level are powerful tools for improvement of production strains. Besides increased yields of the final product, GA3, it is now possible to produce intermediates of the GA biosynthetic pathway, such as ent-kaurene, ent-kaurenoic acid, and GA14, in high amounts using different knock-out mutants. This review concentrates mainly on the fungal biosynthetic pathway, the genes and enzymes involved, the regulation network, the biotechnological relevance of recent studies, and on evolutionary aspects of GA biosynthetic genes.

Biotechnology↗

Current state and perspectives of truffle genetics and sustainable biotechnology.

Mycorrhizal fungi belonging to the genus Tuber produce, after the establishment of a productive interaction with a plant host, hypogeous fruitbodies of great economic value known as ''truffles''. This review summarizes the state of art on life cycle, genetic, and biotechnological investigations of Tuber spp. The ascocarp formation in truffles is a consequence of the activation of the sexual phase of the biological cycle. The formation of a dikaryotic secondary mycelium and the karyogamy in the ascal cell (followed by meiosis with ascospores formation) have been hypothesized by several authors but some doubts yet arise from the Tuber cycle by considering that a series of abnormalities have been pointed out in respect to other Ascomycetes. It is unclear if binucleated hyphal cells are derived from the fusion of mononucleated cells belonging to mycelia from different mating types or from one only. According to the karyotypes of Tuber melanosporum, Tuber magnatum, and Tuber borchii, the numbers of hyphal chromosomes suggest a chromosome number of eight (2n); these values are in the range of those of several Ascomycetes and observed for Tuber aestivum (2n=10). The importance and growth in interest during the last years in the fungi protoplasts isolation and transformation techniques can be related to current developments in Tuber genetics and biotechnology. T. borchii could be transformed through liposome-mediated delivery of genetic material as mycelial protoplasts isolation and fusion with liposomes has already been established. On the other hand, Agrobacterium-mediated transformation has been successfully established for T. borchii.

Ascomycota↗

A rare sugar xylitol. Part II: biotechnological production and future applications of xylitol.

Xylitol is the first rare sugar that has global markets. It has beneficial health properties and represents an alternative to current conventional sweeteners. Industrially, xylitol is produced by chemical hydrogenation of D-xylose into xylitol. The biotechnological method of producing xylitol by metabolically engineered yeasts, Saccharomyces cerevisiae or Candida, has been studied as an alternative to the chemical method. Due to the industrial scale of production, xylitol serves as an inexpensive starting material for the production of other rare sugars. The second part of this mini-review on xylitol will look more closely at the biotechnological production and future applications of the rare sugar, xylitol.

Biotechnology↗

South African Myxococcota: an untapped resource for microbial ecolo gy and biotechnology.

An extraordinary multicellular life cycle, ecological versatility, and prolific production of bioactive secondary metabolites characterise the phylum Myxococcota. While research has predominantly focused on Myxococcota in Asia, Europe, and North America, their potential occurrence in Sub-Saharan Africa remains largely unexplored. To date, only one study has isolated Myxococcota in South Africa, with additional findings limited to incidental detection through metagenomic studies. Considering South Africa's ecological diversity, its biomes may represent promising but under-examined environments for systematic bioprospecting aimed at discovering novel Myxococcota with ecological or biotechnological potential. The recent reclassification of Myxococcota from the former Deltaproteobacteria has provided a more coherent taxonomic framework to guide future ecological and systematic studies. This review presents an overview of the taxonomic revision and explores the potential occurrence of Myxococcota in South African biomes. It covers the challenges associated with conventional culture-based isolation methods and highlights potential genome- and metagenome-based approaches, including the use of metagenome-assembled genomes (MAGs) to identify cryptic biosynthetic gene clusters (BGCs), while acknowledging current limitations. Considering the increasing resistance to chemical fungicides in South African agriculture, this review further explores the potential of Myxococcota-derived secondary metabolites as candidate bioprotective alternatives. By identifying current research gaps, it aims to support future efforts towards systematic bioprospecting to investigate the ecological and biotechnological potential of Myxococcota in South Africa. KEY POINTS: • South African biomes may harbour novel Myxococcota with biosynthetic potential. • Genome mining could reveal cryptic biosynthetic gene clusters (BGCs). • Myxococcota metabolites may help control resistant fungal phytopathogens.

South Africa↗

Genetic transformation and biotechnological application of the yeast Arxula adeninivorans.

The relatively unknown, non-pathogenic, dimorphic, haploid, ascomycetous yeast Arxula adeninivorans exhibits some unusual properties which are of biotechnological interest. The yeast is able to assimilate and ferment many compounds as sole source of carbon and/or nitrogen, it utilises n-alkanes and degrades starch efficiently. A. adeninivorans features such as thermo- and haloresistance as well as the yeast's uncommon growth and secretion behaviour should be especially emphasised. In media containing up to 20% NaCl, A. adeninivorans is able to grow at cultivation temperatures up to 48 degrees C. Additionally, the dimorphism of the yeast is unusual. Arxula grows at up temperatures of up to 42 degrees C as budding cells, which turn into mycelia at higher temperatures. This environmentally conditioned dimorphism is reversible and budding is reestablished when the cultivation temperature is decreased below 42 degrees C. Alteration of morphology correlates with changes in secretion behaviour. Mycelium cultures accumulate two-fold higher protein concentrations and contain two- to five-fold higher glucoamylase and invertase activities in the medium than budding cells. Based on these unusual properties, Arxula adeninivorans is used for heterologous gene expression and as a gene donor to construct more suitable yeasts for biotechnology. For example the Arxula glucoamylase gene was successfully expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both transformed yeasts are able to assimilate and ferment starch as carbon source. A transformation system is used for heterologous gene expression which is based on integration of linearised DNA fragments in two to ten copies, e.g. into the 25S rDNA of A. adeninivorans by homologous recombination. The obtained transformants are mitotically stable. The expression of the lacZ gene from E. coli as well as the XylE gene from Pseudomonas putida indicates the suitability of A. adeninivorans as host for heterologous gene expression.

Biotechnology↗

Application of rRNA-targeted oligonucleotide probes in biotechnology.

Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.

Bioreactors↗

Flow cytometry in biotechnology.

Flow cytometry is a general method for rapidly analyzing large numbers of cells individually using light-scattering, fluorescence, and absorbence measurements. The power of this method lies both in the wide range of cellular parameters that can be determined and in the ability to obtain information on how these parameters are distributed in the cell population. Flow cytometric assays have been developed to determine both cellular characteristics such as size, membrane potential, and intracellular pH, and the levels of cellular components such as DNA, protein, surface receptors, and calcium. Measurements that reveal the distribution of these parameters in cell populations are important for biotechnology, because they better describe the population than the average values obtained from traditional techniques. This Mini-Review provides an overview of the principles of flow cytometry, with descriptions of methods used to measure various cellular parameters and examples of the application of flow cytometry in biotechnology. Finally, a discussion of the challenges and limitations of the method is presented along with a future outlook.

Animals↗

Biotechnological production of vanillin.

Vanillin is one of the most important aromatic flavor compounds used in foods, beverages, perfumes, and pharmaceuticals and is produced on a scale of more than 10 thousand tons per year by the industry through chemical synthesis. Alternative biotechnology-based approaches for the production are based on bioconversion of lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino acids, and on de novo biosynthesis, applying fungi, bacteria, plant cells, or genetically engineered microorganisms. Here, the different biosynthesis routes involved in biotechnological vanillin production are discussed.

Bacteria↗