Trehalose and anhydrobiosis in tardigrades--evidence for divergence in responses to dehydration

To withstand desiccation, many invertebrates such as rotifers, nematodes and tardigrades enter a state known as anhydrobiosis, which is thought to require accumulation of compatible osmolytes, such as the non-reducing disaccharide trehalose to protect against dehydration damage. The trehalose levels of eight tardigrade species comprising Heterotardigrada and Eutardigrada were observed in five different states of hydration and dehydration. Although many species accumulate trehalose during dehydration, the data revealed significant differences between the species. Although trehalose accumulation was found in species of the order Parachela (Eutardigrada), it was not possible to detect any trehalose in the species Milnesium tardigradum and no change in the trehalose level has been observed in any species of Heterotardigrada so far investigated. These results expand our current understanding of anhydrobiosis in tardigrades and, for the first time, demonstrate the accumulation of trehalose in developing tardigrade embryos, which have been shown to have a high level of desiccation tolerance.     

Carbohydrate and energy-yielding metabolism in non-conventional yeasts

Sugars are excellent carbon sources for all yeasts. Since a vast amount of information is available on the components of the pathways of sugar utilization in Saccharomyces cerevisiae it has been tacitly assumed that other yeasts use sugars in the same way. However, although the pathways of sugar utilization follow the same theme in all yeasts, important biochemical and genetic variations on it exist. Basically, in most non-conventional yeasts, in contrast to S. cerevisiae, respiration in the presence of oxygen is prominent for the use of sugars. This review provides comparative information on the different steps of the fundamental pathways of sugar utilization in non-conventional yeasts: glycolysis, fermentation, tricarboxylic acid cycle, pentose phosphate pathway and respiration. We consider also gluconeogenesis and, briefly, catabolite repression. We have centered our attention in the genera Kluyveromyces, Candida, Pichia, Yarrowia and Schizosaccharomyces, although occasional reference to other genera is made. The review shows that basic knowledge is missing on many components of these pathways and also that studies on regulation of critical steps are scarce. Information on these points would be important to generate genetically engineered yeast strains for certain industrial uses.     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.     

Biocatalysis of nitro substituted styrene oxides by non-conventional yeasts

Yeast strains (410) from more than 45 different genera were screened for the enantioselective hydrolysis of nitro substituted styrene oxides. These strains included 262 yeasts with known epoxides hydrolase activity for various other epoxides. Epoxide hydrolase activity for p-nitrostyrene oxide (pNSO) (177 strains) and m-nitrostyrene oxide (mNSO) (148 strains) was widespread in the yeasts, while activity for o-nitrostyrene oxide (oNSO) was less ubiquitous (22 strains). The strains that displayed enantioselectivity in the hydrolysis of one or more of the nitro substituted styrene oxides (35 strains) were also screened against styrene oxide (SO). Rhodosporidium toruloides UOFS Y-0471 displayed the highest enantioselectivity for pNSO (ee 55%, yield 35%) while Rhodotorula glutinis UOFS Y-0653 displayed the highest enantioselectivity for mNSO (ee >98%, yield 29%), oNSO (ee 39%, yield 19%) and SO (ee >98%, yield 19%). (R)-Styrene oxide was preferentially hydrolysed to the corresponding (R)-diol with retention of configuration at the stereogenic centre. In the case of the nitro substituted styrene oxides the absolute configurations of the remaining epoxides and the formed diols were not established.     

Nonconventional yeasts: their genetics and biotechnological applications

To date, more than 500 species of yeasts have been described. Most of the genetic and biochemical studies have, however, been carried out with Saccharomyces cerevisiae. Although a considerable amount of knowledge has been accumulated on fundamental processes and biotechnological applications of this industrially important yeast, the large variety of other yeast genera and species may offer various advantages for experimental study as well as for product formation in biotechnology. The genetic investigation of these so-called unconventional yeasts is poorly developed and information about corresponding data is dispersed. It is the aim of this review to summarize and discuss the main results of genetic studies and biotechnological applications of unconventional yeasts and to serve as a guide for scientists who wish to enter this field or are interested in only some aspects of these yeasts.     

Chromosomal DNA preparation from yeasts of biotechnological importance

Summary Yeast chromosomal DNA was prepared under different conditions. Treatment of intact cells with proteinase K (1 mg/ml) resultes in appropriate electrophoretic karyotypes; when protoplasts were formed in situ, the presence of both sodium lauroylsarcosine and EDTA was essential. Further, the duration of cell wall lysis (12 h) and the concentrations of lytic enzymes (0.5% snail enzyme and 0.25% Novozym)had to be kept at a minimum.     

Engineering of non-conventional yeasts for efficient synthesis of macromolecules: the methylotrophic genera

Methylotrophic yeasts, named after their ability to grow on methanol as the sole carbon source, have raised large interest as recombinant protein factories. In this review, we explain the basic mechanisms underlying this interest and describe the minimal requirements to transform the two genera recognized as methylotrophic, Pichia and Candida, into a powerful protein production tool. We present a comparison between this group of yeasts and the conventional yeasts used as expression system in view of productivity, level of secretion and quality of post-translational modifications. Selected examples of recombinant protein produced by methylotrophic yeast are also included.     

Proteolysis upon dehydration of yeasts saccharomyces cerevisiae

The effect of dehydration on proteolysis and activity of proteases A, B and C in the cells of baker's yeast Saccharomyces cerevisiae was investigated. It can be concluded, that under investigated conditions of yeast Saccharomyces cerevisiae drying a decrease of proteases activity takes place. In cells a limited proteolysis takes place which is indicated by an increase in amino nitrogen content and a decrease of tryptophane synthase activity. Adding the protease inhibitor to yeast suspension prevents decrease of tryptophane synthase activity upon dehydration.     

Grape marcs as unexplored source of new yeasts for future biotechnological applications

In recent years the potential of using microbes as biotechnological sources of industrially relevant enzymes has stimulated a renewed interest in the exploration of new unconventional habitats like trove of natural biodiversity. In this work, grape marcs was selected as extreme environment because of its limited nutrients, exposure to solar radiation, temperature fluctuations and ethanol. One hundred and eighty non-Saccharomyces yeasts and two hundred and twenty Saccharomyces cerevisiae strains were screened for the production of extracellular amylases, cellulases, lipases, pectinases, proteases and xylanases. Two non-Saccharomyces strains were found effective for the hydrolysis of cellulose and starch while eleven S. cerevisiae isolates were described as proficient pectinase producers. For the first time, thirteen S. cerevisiae strains, potentially able to use starch as the sole carbon source, were reported and their potential amylolytic phenotype was found to be related to a non extracellular alpha-amylase. This study encourages the selection of yeasts isolated from grape marcs as sources of unusual and industrially interesting enzymes for future biotechnological applications.     

Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: Current state and future prospects

Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts.Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives. The comparison of innovative CRISPR-Cas expression strategies in yeasts presented here may also serve as guideline to implement and refine CRISPR-Cas systems for highly efficient genome editing in other eukaryotic organisms.     

An overview of lipid metabolism in yeasts and its impact on biotechnological processes

High energy prices, depletion of crude oil supplies, and price imbalance created by the increasing demand of plant oils or animal fat for biodiesel and specific lipid derivatives such as lubricants, adhesives, and plastics have given rise to heated debates on land-use practices and to environmental concerns about oil production strategies. However, commercialization of microbial oils with similar composition and energy value to plant and animal oils could have many advantages, such as being non-competitive with food, having shorter process cycle and being independent of season and climate factors. This review focuses on the ongoing research on different oleaginous yeasts producing high added value lipids and on the prospects of such microbial oils to be used in different biotechnological processes and applications. It covers the basic biochemical mechanisms of lipid synthesis and accumulation in these organisms, along with the latest insights on the metabolic processes involved. The key elements of lipid accumulation, the mechanisms suspected to confer the oleaginous character of the cell, and the potential metabolic routes enhancing lipid production are also extensively discussed.     

Selection of non-conventional yeasts and their use in immobilized form for the bioremediation of olive oil mill wastewaters

The yeast population dynamics in olive wastewaters (OMW), sampled in five mills from Salento (Apulia, Southern Italy), were investigated. Three hundred yeasts were isolated in five industrial mills and identified by molecular analysis. Strains belonging to Geotrichum, Saccharomyces, Pichia, Rhodotorula and Candida were detected. Five G. candidum strains were able to grow in OMW as the sole carbon source and to reduce phenolics, chemical oxygen demand (COD) and antimicrobial compounds. One G. candidum isolate was selected for whole-cell immobilization in calcium alginate gel. The COD and phenolic reduction obtained with immobilized cells showed a 2.2- and 2-fold increase compared to the removal obtained with free cells, respectively. The immobilization system enhanced yeast oxidative activity by avoiding the presence of microbial protease in treated OMW. To our knowledge, this is the first report on G. candidum whole-cell immobilization for OMW bioremediation.     

Luminescence microscopy determination of the survival of yeasts after dehydration

The technique of luminescent microscopy can be used to determine the survival rate of yeast organisms dehydrated by the convective method. If the cells were subjected to more severe extreme actions, the technique should be combined with the direct microscopy to follow the growth of reactivated organisms and to count the number of microcolonies being formed. The authors propose to classify the state of living organisms after the action of sublethal factors basing on the ability of cells to grow after reactivation.     

Improving the politics of biotechnological innovations in food security and other sustainable development goals

The unwarranted interference of some environmental non-governmental organisations (ENGOs) in decision-making over genetically modified (GM) crops has prompted calls for politics to be removed from the regulatory governance of these products. However, regulatory systems are inevitably political because their purpose is to decide whether the use of particular products will help or hinder the delivery of public policy objectives. ENGOs are most able to interfere in regulatory decision-making when policy objectives and decision-making criteria are vague, making the process vulnerable to disruption by organisations that have a distinct agenda. Making regulatory decision-making about GM crops and other green biotechnology more resistant to interference therefore requires better politics not the removal of politics. Better politics begins with political leadership making a case for green biotechnology in achieving food security and other sustainable development goals. Such a policy must involve making political choices and cannot be outsourced to science. Other aspects of better politics include regulatory reform to set policy aims and decision-making criteria that encourage innovation as well as control risk, and engagement with civil society that discusses the values behind attitudes to the application of green biotechnology. In short, green biotechnology for sustainable development needs better politics to counter well-organised opposition, to encourage innovation, and to build the trust of civil society for these policies. Removing politics from regulatory governance would be a gift to ENGOs that are opposed to the use of biotechnology.

     

Psychrophilic yeasts from worldwide glacial habitats: diversity, adaptation strategies and biotechnological potential

Glacial habitats (cryosphere) include some of the largest unexplored and extreme biospheres on Earth. These habitats harbor a wide diversity of psychrophilic prokaryotic and eukaryotic microorganisms. These highly specialized microorganisms have developed adaptation strategies to overcome the direct and indirect life-endangering influence of low temperatures. For many years Antarctica has been the geographic area preferred by microbiologists for studying the diversity of psychrophilic microorganisms (including yeasts). However, there have been an increasing number of studies on psychrophilic yeasts sharing the non-Antarctic cryosphere. The present paper provides an overview of the distribution and adaptation strategies of psychrophilic yeasts worldwide. Attention is also focused on their biotechnological potential, especially on their exploitation as a source of cold-active enzymes and for bioremediation purposes.     

Application of metabolic engineering for the biotechnological production of l-valine

The branched chain amino acid l-valine is an essential nutrient for higher organisms, such as animals and humans. Besides the pharmaceutical application in parenteral nutrition and as synthon for the chemical synthesis of e.g. herbicides or anti-viral drugs, l-valine is now emerging into the feed market, and significant increase of sales and world production is expected. In accordance, well-known microbial production bacteria, such as Escherichia coli and Corynebacterium glutamicum strains, have recently been metabolically engineered for efficient l-valine production under aerobic or anaerobic conditions, and the respective cultivation and production conditions have been optimized. This review summarizes the state of the art in l-valine biosynthesis and its regulation in E. coli and C. glutamicum with respect to optimal metabolic network for microbial l-valine production, genetic strain engineering and bioprocess development for l-valine production, and finally, it will shed light on emerging technologies that have the potential to accelerate strain and bioprocess engineering in the near future.