Yeast Biotechnology: Diversity and Applications

This review is an attempt in cataloguing the diversity of yeasts in Antarctica, highlight their biotechnological potential and understand the basis of adaptation to low temperature. As of now several psychrophilic and psychrotolerant yeasts from Antarctic soils and marine waters have been characterized with respect to their growth characteristics, ecological distribution and taxonomic significance. Interestingly most of these species belonged to basidiomycetous yeasts which as a group are known for their ability to circumvent and survive under stress conditions. Simultaneously their possible role as work horses in the biotechnological industry was recognized due to their ability to produce novel enzymes and biomolecules such as agents for the breakdown of xenobiotics, and novel pharmaceutical chemi cals. The high activity of psychrophilic enzymes at low and moderate temperatures offers potential economic benefits. As of now lipases from

Pseudozyma antarctica

have been extensively studied to understand their unique thermal stability at 90°C and also because of its use in the pharmaceutical, agriculture, food, cosmetics and chemical industry. A few of the other enzymes which have been studied include extracellular alpha-amylase and glucoamylase from the yeast

Pseudozyma antarctica

(

Candida antarctica

), an extra-cellular protease from

Cryptococcus humicola

, an aspartyl proteinase from

Cryptococcus humicola

, a novel extracellular subtilase from

Leucosporidium antarcticum

, and a xylanase from

Cryptococcus adeliensis

The ability of these yeasts to adapt to the low temperature conditions has also led to investigations directed towards characterizations of cold stress proteins and heat shock proteins so as to understand the role of these stress protein with respect to adaptation. Antarctic yeasts have also been used as model system to study the inter-relationship among free radicals, antioxidants and UV-induced cell damage

The approach to yeast identification has significantly changed in just a few decades due to rapid increase in basic biological knowledge, increased interest in the practical applications and biodiversity of this important microbial group, and enormous technological advances especially in the sphere of molecular tools. While some conventional methods are still tenable, many molecular techniques have been developed that allow for strain classification at all taxonomic levels. However, the oldest tool of microbiology, the microscope, is still a fundamental accessory for studies involving yeast biology, biodiversity and taxonomy

The basidiomycetous yeasts, are currently recognized, in three classes of the Basidiomycota: Ustilaginomycetes, Urediniomycetes and Hymenomycetes. These yeasts have considerable economic, agricultural and medical importance and estimates suggest that the number of known yeasts represents only about 1 to 5 % of the species that exist in nature. There is an increased interest in exploration of these species for economic exploitation and there is a need to understand their biodiversity and ecological roles

Identification and phylogenetic placement of the basidiomycetous yeasts is not always easy, partly because of their polyphyletic nature. The unifying characteristic of these fungi is a predominant unicellular growth phase. Separation of yeasts into the three classes of fungi is based on septal morphology, cell wall composition and rDNA analysis. Generic diagnosis is based on sexual and vegetative biology, in addition to physiological tests such as growth on inositol or D-glucuronic acid and formation of extracellular starch-like compounds. Species are usually differentiated by physiological attributes, particularly the utilization of carbon and nitrogen sources, and by measurement of DNA reassociations between closely related species. Currently approximately 50 genera and 250 species of basidiomycetous yeasts are known. Molecular methods used in their identification include, species-specific PCR primers, analysis of RFLPs PFGE, randomly amplified polymorphic DNA (RAPD) and single-stranded conformational polymorphisms (SSCP). Significant advances in basidiomycete systematics have resulted from sequence analysis of the large and small subunits of rDNA.

Basidiomycetous yeast species are associated with living plants,

viz., Sporobolomyces

and

Phaffia

. Several species have been found to play a prominent role in biocontrol of plant disease whereas others have application in agro based industry. For example,

Phaffia rhodozyma

produces a pigment astaxanthin that has considerable market in aquaculture industry. On the other hand several species produce polysaccharases and can store lipids in amounts reaching upto 65 % of their biomass. Some species of

Cryptococcus, Rhodotorula

and

Trichosporon

can degrade varied aromatic compounds and thus are a candidate in bioremediation. On the negative side is the pathogenic

Filobasidiella neoformans

that poses medical problem since both the varieties of this basidiomycetous yeast infect the lungs which can result in pneumococcal-type pneumonia. Lipophilic

Malassezia

spp. are associated with skin surfaces but can cause serious pulmonary and other infections.

Diversity searches in the natural environment have resulted in description of new species within the basidiomycetous yeasts at a rapid pace and the field is wide open to global exploration.

Hansenula polymorpha (Pichia angusta)

belongs to a limited number of methylotrophic yeast species. It is able to assimilate nitrate and can grow on a range of carbon sources. Furthermore,

H. polymorpha

is a thermo-tolerant microorganism with some strains growing at temperatures up to 50° C and more. These unusual characteristics render

H. polymorpha

attractive as a model organism to study the development and functions of peroxisomes and the biochemistry of nitrate assimilation.

H. polymorpha

provides an established platform for heterologous gene expression and is distinguished by an impressive track record as producer of recom-binant proteins that include commercially available pharmaceuticals like hepatitis B vaccine, insulin and the IFN α-2a

The yeast

Debaryomyces hansenii

which was isolated from saline environments such as sea water, concentrated brines, salty food, is one of the most halotolerant species. It can grow in media containing as high as 4 M NaCl, while the growth of

Saccharomyces cerevisiae

is limited in media with more than 1.7 M NaCl. This species is very important for food industry as it is used for surface ripening of cheese and meat products. In the recent past, there is growing interest in understanding the molecular mechanisms of high halotolerance exhibited by

D. hansenii

. Availability of genome sequence of

D. hansenii

has opened up new vistas in this direction

Debaryomyces hansenii

(teleomorph of asporogenous strains known as

Candida famata

) belongs to the group of so named ‘ flavinogenic yeasts ’ capable of riboflavin oversynthesis during starvation for iron. Some strains of

C. famata

belong to the most flavinogenic organisms known (accumulate 20 mg of riboflavin in 1 ml of the medium) and were used for industrial production of riboflavin in USA for long time. Many strains of

D. hansenii

are characterized by high salt tolerance and are used for ageing of cheeses whereas some others are able to convert xylose to xylitol, anti-caries sweetener. Transformation system has been developed for

D. hansenii

. It includes collection of host recipient strains, vectors with complementation and dominant markers and several transformation protocols based on protoplasting and electroporation. Besides, methods of multicopy gene insertion and insertional mutagenesis have been developed and several strong constitutive and regulatable promoters have been cloned. All structural genes of riboflavin synthesis and some regulatory genes involved in this process have been identified. Genome of

D. hansenii

has been sequenced in the frame of French National program ‘Genolevure’ and is opened for public access

Pichia guilliermondii

(asporogenous strains of this species are designated as

Candida guilliermondii

) is the model organism of a group so named “ flavinogenic yeasts ” capable of riboflavin oversynthesis during starvation for iron. Besides, some strains of this species efficiently convert xylose to xylitol, an anti-caries sweetener. However, there are also pathogenic

C. guilliermondii

strains. This species has been used for studying enzymology of riboflavin synthesis due to overproduction of participating enzymes and intermediates under iron-limiting conditions as well as for identification of genes of negative and positive action involved in such a regulation. Besides,

P. guilliermondii

was used for identification and studying the properties of the systems for active transport of riboflavin in the cell (riboflavin permease) and out of the cell (riboflavin “ excretase ” ). The genetic line of

P. guilliermondii

with high fertility has been selected and the methods of classic genetics (hybridization and analysis of meiotic segregation) have been developed. More recently, tools for molecular genetic studies of

P. guilliermondii

have been developed which include collection of host strains, vectors with recessive and dominant markers, several transformation protocols including that for gene knock out. Recently, the genome of this yeast species was sequenced and become publicly available (

http://www.broad.mit.edu

)

Yeast taxa traditionally are distinguished by growth tests on several sugars and organic acids. During the last decades it became apparent that many yeast species assimilate a much greater variety of naturally occurring carbon compounds as sole source of carbon and energy. These abilities are indicative of a greater role of yeasts in the carbon cycle than previously assumed. Especially in acidic soils and other habitats, yeasts may play a role in the degradation of carbon compounds. Such compounds include purines like uric acid and adenine, aliphatic amines, diamines and hydroxyamines, phenolics and other benzene compounds and polysaccharides. Assimilation of purines and amines is a feature of many ascomycetes and basidiomycetes. However, benzene compounds are degraded by only a few ascomycetous yeasts (e.g. the

Stephanoascus/ Blastobotrys

clade and black yeastlike fungi) but by many basidiomycetes, e.g. Filobasidiales, Trichosporonales, red yeasts producing ballistoconidia and related species, but not by Tremellales. Assimilation of polysaccharides is wide-spread among basidiomycetes

Growth tests on these compounds separate

Trichosporon

species that otherwise are hardly distinguishable. Yeasts able to degrade phenolics can be applied for cresol removal from polluted soil and styrene removal from air by biofilters containing black yeast. Yeasts growing on polysaccharides may be a valuable source of hydrolytic enzymes that can be applied in food technology. Biodegradative abilities of yeasts inhabiting aerial plant surfaces and the fate of these yeasts during anaerobiosis and lactic acid fermentation are also dealt with

In the latest edition of the standard treatise of yeasts, in 1998, 700 species were described. Since then, the number of recognized yeast species has doubled, with a steep increase particularly in the number of the basidiomycetous yeasts. Of all these yeast species, only about a dozen is used at industrial scale, and some 70 – 80 species have been shown at laboratory scale to possess potential value in biotechnology; their ratio is, in the best case, 5 – 10 %. If it is accepted, that according to a modest estimate, the known yeast species represent only 5 % of the total number which may inhabit the Earth, then there is ample room to search for new species with novel potential to exploit. Where could these yeasts be discovered?

In recent years we are witnessing great progress in exploring the diverse ecological niches of yeasts, and revealing the great diversity of species living in the various habitats. Still, compared to the profusing metabolic capability of bacteria living in the soil, surprisingly less is known about the soil yeasts. Much remains to be learned on yeasts associated with insects, invertebrates and fishes in the deep ocean, inhabiting tropical forests, or striving in extreme environments. It could reasonably be expected, that among the numerous species to be discovered in specific and unusual habitats, many will be found to possess enzymes, carry out metabolic routes and show physiological properties which hold out promises to be valuable for biotechnological applications. This chapter will examine these potential values from the point of view of ecology and biodiversity of yeasts

People across the world have learnt to culture and use the essential microorganisms for production of fermented foods and alcoholic beverages. A fermented food is produced either spontaneously or by adding mixed/pure starter culture(s). Yeasts are among the essential functional microorganisms encountered in many fermented foods, and are commercially used in production of baker's yeast, breads, wine, beer, cheese, etc. In Asia, moulds are predominant followed by amylolytic and alcohol-producing yeasts in the fermentation processes, whereas in Africa, Europe, Australia and America, fermented products are prepared exclusively using bacteria or bacteria-yeasts mixed cultures. This chapter would focus on the varieties of fermented foods and alcoholic beverages produced by yeasts, their microbiology and role in food fermentation, widely used commercial starters (pilot production, molecular aspects), production technology of some common commercial fermented foods and alcoholic beverages, toxicity and food safety using yeasts cultures and socio-economy

In an agricultural environment, the native flora is replaced by a commercial crop and consequently the native microbiota also undergoes changes and, no seldom, species with antagonistic action against pathogens are eliminated. The lack of natural competitors may result in an outburst of diseases or herbivores that will feed upon the growing crop. Several strategies such as: chemical control, pathogen resistant cultivars and biological control may be used to avoid economical loses in the crop. Biological control protocols are based on the assumption that in an undisturbed environment outbursts of diseases are seldom due to the presence of naturally occurring antagonists and therefore, the introduction/augmentation of antagonism in a disturbed environment will control the disease. A successful agent for biological control has to hold several characteristics such: antagonism against pathogens, well know biology, specificity, be ease to produce and apply, be safe to the environment. Yeast may present all of those characteristics and are used in several biological control protocols. We will discuss in this chapter the basic concepts of biological control, the use of yeasts as biological control agents and describe the commercial products that use yeasts for biological control

Advances in medical research, made during the last few decades, have improved the prophylactic, diagnostic and therapeutic capabilities for variety of infections/diseases. However, many of the prophylactic and therapeutic procedures have been seen in many instances to exact a price of host-vulnerability to an expanding group of opportunistic pathogens and yeasts are one of the important members in it. Fortunately amongst the vast majority of yeasts present in nature only few are considered to have the capability to cause infections when certain opportunities predisposes and these are termed as ‘opportunistic pathogenic yeasts.’ However, the term ‘pathogenic’ is quite tricky, as it depends of various factors of the host, the ‘bug’ and the environment to manifest the clinical infection. The borderline is expanding. In the present century with unprecedented increase in number of immune-compromised host in various disciplines of health care settings, where any yeast, which has the capability to grow at 37 ° C (normal body temperature of human), can be pathogenic and cause infection in particular situation

Spectrum of infective yeasts varies in different geographical region and mainly depends upon nature of immune suppression of the patients and prevailing yeast in the environment. Opportunistic yeast pathogen mostly reported are

Candida

spp. (

albicans, tropicalis, krusei, parapsilosis, kefyr, glabrata, dubliensis, rugosa

and others),

Cryptococcus neoformans

(var

grubii,var neoformans

and var

gattii

),

Trichosporon

spp. and occasionally others like

Geotrichum

spp,

Pichia

spp. etc

Among these,

C. albicans

has been regarded as the most common agent of invasive yeast infection

The population of patients at risk has expanded to include those with a broad list of medical conditions, such as solid-organ and hematopoietic stem cell transplantation (HSCT), cancer, receipt of immunosuppressive therapy, HIV/AIDS, premature birth, advanced age, and major surgery. Furthermore, the etiology of these infections has changed. In the 1980s, yeasts (particularly

Candida albicans

) were the most common causative agents of invasive mycoses. However, presently non

albi-cans

species of

Candida

(NAC) account for > 50 % of infections. In addition, infections caused by other yeasts, such as

Trichosporon

species, have been reported. This chapter intends to high light important predisposing factors responsible for increase incidence of opportunistic yeast infection, its clinical significance, diagnostic approach for early detection of pathogenic yeast, guide line of therapy and epidemiology of important opportunistic yeast pathogen specially that of

Candida

and

Cryptococcus

Zinc is an essential trace element in biological systems. For example, it acts as a cellular membrane stabiliser, plays a critical role in gene expression and genome modification and activates nearly 300 enzymes, including alcohol dehydrogenase. The present chapter will be focused on the influence of zinc on cell physiology of industrial yeast strains of

Saccharomyces cerevisiae

, with special regard to the uptake and subsequent utilisation of this metal. Zinc uptake by yeast is metabolism-dependent, with most of the available zinc translocated very quickly into the vacuole. At cell division, zinc is distributed from mother to daughter cells and this effectively lowers the individual cellular zinc concentration, which may become zinc depleted at the onset of the fermentation. Zinc influences yeast fermentative performance and examples will be provided relating to brewing and wine fermentations. Industrial yeasts are subjected to several stresses that may impair fermentation performance. Such stresses may also impact on yeast cell zinc homeostasis. This chapter will discuss the practical implications for the correct management of zinc bioavailability for yeast-based biotechnologies aimed at improving yeast growth, viability, fermentation performance and resistance to environmental stresses

Glutathione, γ -glutamyl-cysteinyl-glycine, is the most abundant non-protein thiol found in almost all eukaryotic cells (and in some prokaryotes). The tripeptide, which is synthesized non-ribosomally by the consecutive action of two soluble enzymes, is needed for carrying out numerous functions in the cell, most important of which is the maintenance of the redox buffer. The cycle of glutathione biosynthesis and degradation forms part of the γ -glutamyl cycle in most organisms although the latter half of the pathway has not been demonstrated in yeasts. Our current understanding of how glutathione levels are controlled at different levels in the cell is described. Several different routes and processes have been attempted to increase commercial production of glutathione using both yeast and bacteria. In this article we discuss the history of glutathione production in yeast. The current bottlenecks for increased glutathione production are presented based on our current understanding of the regulation of glutathione homeostasis, and possible strategies for overcoming these limitations for further enhancing and improving glutathione production are discussed

Spontaneous alcoholic fermentation from grape, agave and others musts into an alcoholic beverage is usually characterized by the presence of several non-

Saccharomyces

yeasts. These genera yeasts are dominant in the early stages of the alcoholic fermentation. However the genera

Hanseniaspora

and

Kloeckera

may survive at a significant level during fermentation and can influence the chemical composition of the beverage. Several strains belonging to the species

Kloeckera api-culata

and

Hanseniaspora guilliermondii

have been extensively studied in relation to the formation of some metabolic compounds affecting the bouquet of the final product. Indeed some apiculate yeast showed positive oenological properties and their use in the alcoholic fermentations has been suggested to enhance the aroma and flavor profiles. The non-

Saccharomyces

yeasts have the capability to produce and secrete enzymes in the medium, such as β -glucosidases, which release monoterpenes derived from their glycosylated form. These compounds contribute to the higher fruit-like characteristic of final product. This chapter reviews metabolic activity of

Kloeckera

and

Hanseniaspora

yeasts in several aspects: fermentative capability, aromatic compounds production and transformation of aromatic precursor present in the must, also covers the molecular methods for identifying of the yeast

In the last decade, the yeast

Hansenula polymorpha

(syn.:

Pichia angusta

) has become an excellent experimental model for genetic and molecular investigations of nitrate assimilation, a subject traditionally investigated in plants, filamentous fungi and bacteria. Among other advantages,

H. polymorpha

offers classical and molecular genetic tools, as well as the availability of genomic sequence data.

Assimilative nitrate metabolism in

H. polymorpha

has an enzymological layout that is similar to other fungal species, and undergoes nitrogen metabolite repression elicited by preferred nitrogen sources such as glutamine. Genes involved in nitrate assimilation are clustered and independently transcribed. The information that puzzles is the presence of two homologous, albeit different, transcriptional activators acting upon the nitrate cluster genes, as all other known fungal nitrate assimilatory pathways have only one activator of this family. Recent work enables a first outline of the interplay between these two activators to be depicted, and suggests that one of them plays a central role in chromatin remodelling within the cluster.

The information, which has recently emerged regarding complex post-translational down-regulatory mechanism acting upon the major nitrate transporter suggests that this protein plays a central role in the regulation of nitrate assimilation.

Nitrogen metabolite repression acting upon nitrate assimilative genes is also being investigated through the isolation and characterisation of

H. polymorpha Nmr

mutants. These studies have suggested that the repression mechanisms are mediated by several interacting factors in this organism, which are also believed to participate in nitrogen metabolite repression of other metabolic pathways. All these are involved in the utilisation of secondary nitrogen sources such as arginine, meth-ylamine, urea and asparagine.

Yeast can be recognized as one of the very important groups of microorganisms on account of its extensive use in the fermentation industry and as a basic eukaryotic model cellular system. The yeast

Saccharomyces cerevisiae

has been extensively used to elucidate the genetics and regulation of several key functions in the cell such as cell mating, electron transport chain, protein trafficking, cell cycle events and others. Even before the genome sequence of the yeast was out, the structural organization and function of several of its genes was known. With the availability of the origin of replication from the 2 μm plasmid and the development of transformation system, it became the host of choice for expression of a number of important proteins. A large number of episomal and integrative shuttle vectors are available for expression of mammalian proteins. The latest developments in genomics and micro-array technology have allowed investigations of individual gene function by site-specific deletion method. The application of metabolic profiling has also assisted in understanding the cellular network operating in this yeast. This chapter is aimed at reviewing the use of this system as an experimental tool for conducting classical genetics. Various vector systems available, foreign genes expressed and the limitations as a host will be discussed. Finally, the use of various yeast enzymes in biotechnology sector will be reviewed.

No single yeast-based platform exists which is optimal for every protein. It is advisable to assess several platform candidates in parallel for optimal expression characteristics in a given case. For this approach, a wide-range yeast vector has been established that can be targeted to the various yeast host strains. The vector is built up in a modular way. In its basic form, it contains conserved rDNA-derived segments for targeting. For heterologous gene expression control, it is equipped with a promoter that is functional in all yeast species tested so far. For selection, a range of dominant and auxotrophic selection markers can be employed. Examples are presented applying vector variants with dominant or auxotrophic selection markers to the comparative simultaneous integration and expression of single or multiple foreign genes in a range of yeast platforms.

Yeast has been a favoured lower eukaryotic system for the expression and production of recombinant proteins for both basic research and practical applications, and the demand for foreign-gene expression systems is increasing rapidly. Despite the vast amount of information on the molecular biology and physiology of

Saccharomyces cerevisiae

, which has consequently been the first choice as host system for recombinant protein production in the past, several limitations have been identified in this expression system. These limitations have recently been relieved by the development of expression systems in other yeast species known as ‘ non-conventional yeasts’ or ‘non-

Saccharomyces

’ yeasts. With the increasing interest in the biotechnological applications of these yeasts in applied and fundamental studies and processes, the term ‘ non-conventional ’ yeast may well soon become redundant. As there is no universal expression system for heterologous protein production, it is necessary to recognize the merits and demerits of each system in order to make a right choice. This chapter will evaluate the competitive environment of non-conventional expression platforms represented by some of the best-known alternative yeasts systems including

Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris

and more recently,

Arxula adeninivorans

.

The control of gene expression, predominantly at the level of transcription, plays a fundamental role in biological processes determining the phenotypic changes in cells and organisms. The eukaryotes have evolved a complex and sophisticated transcription machinery to transcribe DNA into RNA. RNA polymerase II enzyme lies at the centre of the transcription apparatus that comprises nearly 60 polypeptides and is responsible for the expression and regulation of proteinencoding genes. Much of our present understanding and knowledge of the RNA polymerase II transcription apparatus in eukaryotes has been derived from studies in

Saccharomyces cerevisiae

. More recently,

Schizosaccharomyces pombe

has emerged as a better model system to study transcription because the transcription mechanism in this yeast is closer to that in higher eukaryotes. Also, studies on components of the basal transcription machinery have revealed a number of properties that are common with other eukaryotes, but have also highlighted some features unique to

S. pombe

. In fact, the fungal transcription associated protein families show greater species specificity and only 15% of these proteins contain homologues shared between both

S. cerevisiae

and

S. pombe

. In this chapter, we compare the RNA polymerase II transcription apparatus in different yeasts.

Generating novel yeast strains for industrial applications should be quite straightforward; after all, research into the genetics, biochemistry and physiology of Baker's Yeast,

Saccharomyces cerevisiae

, has paved the way for many advances in the modern biological sciences. We probably know more about this humble eukaryote than any other, and it is the most tractable of organisms for manipulation using modern genetic engineering approaches. In many countries, however, there are restrictions on the use of genetically-modified organisms (GMOs), particularly in foods and beverages, and the level of consumer acceptance of GMOs is, at best, variable. Thus, many researchers working with industrial yeasts use genetic engineering techniques primarily as research tools, and strain development continues to rely on non-GM technologies. This chapter explores the non-GM tools and strategies available to such researchers.

Yeast organisms, and specifically

Saccharomyces cerevisiae

, have become model systems for many aspects in fundamental and applied research. Consistently, many papers have been published applying proteome techniques to study these organisms. The review will give an overview on the proteome research performed on yeast systems so far; however, due to the large number of publications, only selected reports can be cited neglecting many more interesting ones in the interest of space. The review will focus on research involving mass spectrom-etry as a basic proteome technique, although many more approaches are relevant for the functional characterization of proteins in the cell, e.g. the yeast two-hybrid system. We will provide an overview on yeasts as models in the context of pro-teome analysis, and explain the basic techniques currently applied in proteome approaches. The main part of the review will deal with a survey on the current status of proteomic studies in yeasts. In a first part of this chapter, we will deal with the currently available proteome maps of yeasts, and in the following part we will discuss studies dealing with fundamental aspects, but also mention proteome studies related to applied microbiology. Finally, we will envisage future perspectives of the proteome technology for studying yeasts, and draw major conclusion on the current status reached in this field of functional genomics.

The rapid advances and scale up of projects in DNA sequencing dur ing the past two decades have produced complete genome sequences of several eukaryotic species. The versatile genetic malleability of the yeast, and the high degree of conservation between its cellular processes and those of human cells have made it a model of choice for pioneering research in molecular and cell biology. The complete sequence of yeast genome has proven to be extremely useful as a reference towards the sequences of human and for providing systems to explore key gene functions. Yeast has been a ‘legendary model’ for new technologies and gaining new biological insights into basic biological sciences and biotechnology. This chapter describes the awesome power of yeast genetics, genomics and proteomics in understanding of biological function. The applications of yeast as a screening tool to the field of drug discovery and development are highlighted and the traditional importance of yeast for bakers and brewers is discussed.

The ethanol industry of today utilizes raw materials rich in saccharides, such as sugar cane or sugar beets, and raw materials rich in starch, such as corn and wheat. The concern about supply of liquid transportation fuels, which has brought the crude oil price above 100 $ /barrel during 2006, together with the concern about global warming, have turned the interest towards large-scale ethanol production from lignocellulosic materials, such as agriculture and forestry residues. Baker's yeast

Saccharomyces cerevisiae

is the preferred fermenting microorganism for ethanol production because of its superior and well-documented industrial performance. Extensive work has been made to genetically improve

S. cerevisiae

to enable fermentation of lignocellulosic raw materials. Ethanolic fermentation processes are conducted in batch, fed-batch, or continuous mode, with or without cell recycling, the relative merit of which will be discussed.

In recent years, the use of renewable and abundantly available starchy and cellulosic materials for industrial production of ethanol is gaining importance, in view of the fact, that ethanol is one of the most prospective future motor fuels, that can be expected to replace fossil fuels, which are fast depleting in the world scenario. Although, the starch and the starchy substrates could be converted successfully to ethanol on industrial scales by the use of commercial amylolytic enzymes and yeast fermentation, the cost of production is rather very high. This is mainly due to the non-enzymatic and enzymatic conversion (gelatinization, liquefaction and saccharification) of starch to sugars, which costs around 20 % of the cost of production of ethanol from starch. In this context, the use of amylolytic yeasts, that can directly convert starch to ethanol by a single step, are potentially suited to reduce the cost of production of ethanol from starch. Research advances made in this direction have shown encouraging results, both in terms of identifying the potentially suited yeasts for the purpose and also their economic ethanol yields. This chapter focuses on the types of starch and starchy substrates and their digestion to fermentable sugars, optimization of fermentation conditions to ethanol from starch, factors that affect starch fermentation, potential amylolytic yeasts which can directly convert starch to ethanol, genetic improvement of these yeasts for better conversion efficiency and their future economic prospects in the new millennium.

No other sustainable option for production of transportation fuels can match ethanol made from lignocellulosic biomass with respect to its dramatic environmental, economic, strategic and infrastructure advantages. Substantial progress has been made in advancing biomass ethanol (bioethanol) production technology to the point that it now has commercial potential, and several firms are engaged in the demanding task of introducing first-of-a-kind technology into the marketplace to make bioethanol a reality in existing fuel-blending markets. In order to lower pollution India has a long-term goal to use biofuels (bioethanol and biodiesel). Ethanol may be used either in pure form, or as a blend in petrol in different proportions. Since the cost of raw materials, which can account up to 50 % of the total production cost, is one of the most significant factors affecting the economy of alcohol, nowadays efforts are more concentrated on using cheap and abundant raw materials. Several forms of biomass resources exist (starch or sugar crops, weeds, oil plants, agricultural, forestry and municipal wastes) but of all biomass cellulosic resources represent the most abundant global source. The lignocellulosic materials include agricultural residues, municipal solid wastes (MSW), pulp mill refuse, switchgrass and lawn, garden wastes. Lignocellulosic materials contain two types of polysaccharides, cellulose and hemicellulose, bound together by a third component lignin. The principal elements of the lignocellulosic research include: i) evaluation and characterization of the waste feedstock; ii) pretreatment including initial clean up or dewatering of the feedstock; and iii) development of effective direct conversion bioprocessing to generate ethanol as an end product. Pre-treatment of lignocellulosic materials is a step in which some of the hemicellulose dissolves in water, either as monomeric sugars or as oligomers and polymers. The cellulose cannot be enzymatically hydrolyzed to glucose without a physical and chemical pre-treatment. The pre-treatment processes normally applied on the different substrates are acidic hydrolysis, steam explosion and wet oxidation. A problem for most pretreatment methods is the generation of compounds that are inhibitory towards the fermenting microorganisms, primarily phenols. Degradation products that could have inhibitory action in later fermentation steps are avoided during pre-treatment by wet oxidation. Followed by pre treatment, hydrolysed with enzymes known as cellulases and hemicellulases, which hydrolyse cellulose and hemicellulose respectively. The production of bioethanol requires two steps, fermentation and distillation. Practically all ethanol fermentation is still based on

Saccharomyces cerevisiae

. The fermentation using thermotolerant yeasts has more advantageous in that they have faster fermentation rates, avoid the cooling costs, and decrease the over all fermentation costs, so that ethanol can be made available at cheaper rates. In addition they can be used for efficient simultaneous saccharification and fermentation of cellulose by cellulases because the temperature optimum of cellulase enzymes (about 40 ° C to 45 ° C) is close to the fermentation temperature of thermotolerant yeasts. Hence selection and improvement of thermotolerant yeasts for bioconversion of lignocellulosic substrates is very useful.

The yeast

Yarrowia lipolytica

is often found associated to proteinaceous or hydrophobic substrates such as alkanes or lipids. To assimilate these hydropho-bic substrates,

Y. lipolytica

has developed an adaptative strategy resulting in elaborated morphological and physiological changes leading to terminal and β-oxidation of substrates as well as to lipid storage. The completion of the

Y. lipolytica

genome greatly improved our understanding of these mechanisms. Three main applications of this metabolism will be discussed. The first class corresponds to bioconver-sion processes for the production of secondary metabolites (citric acid), of aroma ( γ - lactone, green note, epoxy geraniol) and of chemicals (dicarboxylic acids). The second class leads to fine chemical production by enantio separation of pharmaceutical compounds using

Y. lipolytica

enzymes such as epoxyde hydrolase or lipase. The third one refers to production of Single Cell Oils (SCO) from agriculture feedstock. In addition to its ability to handle hydrophobic substrates,

Y. lipolytica

has also been recognised as a strong secretor of various proteins such as proteases, lipases, RNases and others. A comprehensive review of recent developments of the

Y. lipolytica

expression/secretion system will finally be presented.

The dimorphic ascomycetous yeast

Arxula adeninivorans

exhibits some unusual properties. Being a thermo- and halotolerant species it is able to assimilate and ferment many compounds as sole carbon and/or nitrogen source. It utilises n-alkanes and is capable of degrading starch. Due to these unusual biochemical properties

A. adeninivorans

can be exploited as a gene donor for the production of enzymes with attractive biotechnological characteristics. Examples of

A. adeninivorans

-derived genes that are overexpressed include the

ALIP1

gene encoding a secretory lipase, the

AINV

encoding invertase, the

AXDH

encoding xylitol dehydrogenase and the

APHY

encoding a secretory phosphatase with phytase activity.

The thermo- and halotolerance as well as differential morphology-dependent glycosylation and the secretion characteristics render

A. adeninivorans

attractive as host for heterologous gene expression. A transformation system has been established based on homologous integration of linearised DNA fragments. Successful expression examples like that of the

E. coli

-derived

lacZ

gene,

GFP

and human

HSA

and

IL6

genes add to the attraction of

A. adeninivorans

as host for hetero logous gene expression.

The dimorphic yeasts have the equilibrium between spherical growth (budding) and polarized (hyphal or pseudohyphal tip elongation) which can be triggered by change in the environmental conditions. The reversible growth phenomenon has made dimorphic yeasts as an useful model to understand fungal evolution and fungal differentiation, in general. In nature dimorphism is clearly evident in plant and animal fungal pathogens, which survive and most importantly proliferate in the respective hosts. However, number of organisms with no known pathogenic behaviour also show such a transition, which can be exploited for the technological applications due to their different biochemical make up under different morphologies. For instance, chitin and chitosan production using dimorphic

Saccharomyces, Mucor, Rhizopus

and

Benjaminiella

, oil degradation and biotransformation with yeast-form of

Yarrowia

species, bioremediation of organic pollutants, exopolysac-charide production by yeast-phase of

Aureobasidium pullulans

, to name a few.

Myrothecium verrucaria

can be used for seed dressing in its yeast form and it produces a mycolytic enzyme complex in its hyphal-form for the biocontrol of fungal pathogens, while

Beauveria bassiana

and other entomopathogens kill the insect pest by producing yeast- like cells in the insect body. The form-specific expression of protease, chitinase, lipase, ornithine decarboxylase, glutamate dehydrogenases, etc. make

Benjaminiella poitrasii, Basidiobolus

sp., and

Mucor rouxii

strains important in bioremediation, nanobiotechnology, fungal evolution and other areas.

Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

Polysaccharide degrading enzymes are hydrolytic enzymes, which have a lot of industrial potential and also play a crucial role in carbon recycling. Pectinases, chitinases and glucanases are the three major polysaccharide degrading enzymes found abundantly in nature and these enzymes are mainly produced by fungal strains. Production of these enzymes by yeasts is advantageous over fungi, because the former are easily amenable to genetic manipulations and time required for growth and production is less than that of the latter. Several yeasts belonging to

Saccharomyces, Pichia, Rhodotorula and Cryptococcus

produce extracellular pectinases, glucanases and chitinases. This chapter emphasizes on the biological significance of these enzymes, their production and their industrial applications.

The element phosphorus is critical to all life forms as it forms the basic component of nucleic acids and ATP and has a number of indispensable biochemical roles. Unlike C or N, the biogeochemical cycling of phosphorus is very slow, and thus making it the growth-limiting element in most soils and aquatic systems. Phosphohydrolases (e.g. acid phosphatases and phytases) are enzymes that break the C-O-P ester bonds and provide available inorganic phosphorus from various inassimilable organic forms of phosphorus like phytates. These enzymes are of significant value in effectively combating phosphorus pollution. Although phytases and acid phosphatases are produced by various plants, animals and micro organisms, microbial sources are more promising for the production on a commercial scale. Yeasts being the simplest eukaryotes are ideal candidates for phytase and phos-phatase research due to their mostly non-pathogenic and GRAS status. They have not, however, been utilized to their full potential. This chapter focuses attention on the present state of knowledge on the production, characterization and potential commercial prospects of yeast phytases and acid phosphatases.

Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes,

etc

>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of

Torulopsis candida

>and

Exophiala oligosperma

>R1 are enantioselec-tive and regiospecific respectively.

Geotrichum

>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of

E. oligosperma

>R1 being active at low pH (3–6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing enzymes of yeasts.