The influence of different methods of immobilization on the activity and enantioselectivity of CAL-B applied for hydrolytic resolution of 2-
Yeasts represent a very diverse group of microorganisms, and even strains that are classified as the same species often show a high level of genetic divergence. Yeasts biodiversity is closely related to their applicability. Biotechnological importance of yeast is almost immeasurable. For centuries, people have exploited its enzymatic potential to produce fermented food as bread or alcoholic beverages. Admittedly, yeasts application was initially instinctual, but with science and technology development, these microorganisms got the object of thorough scientific investigations. It must be recognized that yeast represents an excellent scientific model because of its eukaryotic origin and knowledge of genetics of yeast cells as well as metabolism examined in detail. In 1996, the genome of baker yeast Saccharomyces cerevisiae has been elucidated, what opened the opportunity for the global study of the expression and functioning of the eukaryotic genome. Also, currently, an international team is working on the synthesis of the 16 yeast chromosomes by synthetic biology tools, and the results are expected till the end of the year. Nowadays, yeast is regarded as a versatile tool for biotechnological purposes.
- molecular biology applications—fundamentals
1.1. Yeasts: commercial applications
Yeasts have a wide range of applications mainly in food industry (wine making, brewing, distilled spirits production, and baking) and in biomass production (single-cell protein [SCP]). More recently, yeast has also been used in the biofuel industry and for the production of heterologous compounds. Obviously, their main application arises from the metabolic capacity to carry out the transformation of sugars into ethyl alcohol and carbon dioxide under anaerobic conditions. Moreover, a large number of secondary flavor compounds are created what implies on organoleptic attributes of particular food products. However, it would be misguided to trivialize their metabolic capacities only to fermentative activity. The main factors influencing yeast metabolism are the oxygen availability and the type of carbon source. Many yeast strains can function under both anaerobic as well as aerobic conditions of environment, switching their metabolism types easily . Obviously, the courses of main metabolic pathways are conserved, but some regulative mechanisms attract the attention, denoting unusual metabolism flexibility . In food industry,
Brewer’s yeasts are divided into two separate categories: top-fermenting yeast (ale) and bottom-fermenting one (lager). Both yeast types have similar cell morphology, but they differ in some physiological and metabolic features , what closely corresponds to the process conditions and type of end product. Fermentation of ale yeast is carried out at room temperature and results in beers with a characteristic fruity aroma. In the case of lager yeast, the fermentation temperature is lower, and therefore, this step takes longer than fermentation with ale strains . The share of aerobic respiration in yeast metabolism is higher in case of ale strains than in lager yeast. Both top-fermenting and bottom-fermenting yeast belong to the genus
Various yeast species constitute the predominant microbial group of natural microbiota of fruits ecosystems, what is the reason of fast and spontaneous fermentation of juices or musts resulted in wine production. Yeast colonizing various fruits belongs to the genera
Once again yeast enzymatic power is crucial for the product chemical profile—except for ethanol biosynthesis, the creation of flavor compounds and fragrances from substrates abundant in fruits implies the organoleptic features of particular wine product. During the degradation of grape sugars, amino acids, fatty acids, terpenes, and thiols, some by-products like glycerol, carboxylic acids, aldehydes, higher alcohols, esters, and sulfides are formed, and their synthesis is largely dependent on the peculiarities of the strains used [13, 14]. In 1965, the first two commercially active dried wine yeasts called Montrachet and Pasteur Champagne were produced for a large Californian winery . Nowadays, modern winegrowers routinely use selected yeast starters in practice. These microorganisms dominate native yeast species and give desired direction of chemical transformations occurring in musts and allow to obtain product of predictable quality . Presently exploited commercially available starters have been created as a result of naturally occurring phenomenon called “genome renewal” as well as planned processes of genetic improvement  followed by a careful selection for their good fermentation performance. Genome renewal hypothesis in the standard version assumes that infrequent sexual cycles, characterized by a high degree of selfing, can help to purge deleterious alleles and fix beneficial alleles, thus helping to facilitate adaptation in yeast . This hypothesis had to be re-evaluated  due to the fact that in the case of many environmental isolates, very high levels of genomic heterozygosity had been observed [20, 21]. Presently, the majority of commercial wine yeast comprises strains of
Another example of
In addition to alcoholic beverages production, enzymatic power of yeasts is also essential for baking industry, where concentrated yeast biomass is used as a starter in dough fermentation in order to produce bread and other bakery products. Commercially available baker’s yeast forms include fresh compressed biomass, dehydrated cells (dry yeast), and lyophilized cells (instant). Fermentation of dough substrates leads to ethanol production as well as number of volatile and nonvolatile compounds that have an important contribution to the flavor of bread . As a result of carbohydrates (maltose mainly), fermentation carbon dioxide is generated what increases the dough volume and is responsible for crumb texture. Baker’s yeast is simply brewery yeast produced
The main ingredient of industrial production medium used in yeast production factories are beet or cane molasses, mainly because of the low cost of this waste products and high sucrose content. In most cases, the industrial production is a multistage process carried out under batch or fed-batch conditions with sequential stages differing in fermenter size, performed under controlled intense aeration . Aeration is generally considered as the most important single factor to increase yeast yield and numerous studies have been carried out to investigate the optimization of particular technological solutions . It should be underlined, the particular uniqueness of yeast metabolism—baker’s yeast must exhibit efficient respiratory metabolism during yeast manufacturing, which determines biomass yield, but at the same time, cells must possess strong fermentative potential in order to produce excellent bakery products. During the fermentation of dough yeast cells is exposed to numerous environmental stresses (
Next long-standing industrial processes involving yeast are the production of single-cell protein (SCP)—alternative source of high nutritional value proteins used as a food or feed supplements. Idea of such protein concentrate production was born in response to growing human population in the world and worldwide protein deficiency . SCP manufacture includes simply the cell mass obtaining by way of the application of cheap, waste raw materials to cultivate various nonpathogenic microorganisms (bacteria, fungi, algae) under conditions of submerged (rarely solid-state) fermentation. Besides its high protein content (about 60–82% of dry matter), SCP also contains fats, carbohydrates, nucleic acids, vitamins, and minerals . In practice, several technologies were evaluated, products commercialized and currently obtained SCP found the application primarily in animal feeding. However, baker’s yeast mentioned above can be considered also as an example of particular SCP preparation. Many fungal species are used as protein-rich food. Most popular among them are the yeast species
Yeast can also be considered as an alternative source of lipids. Some species are capable of synthesis and accumulation of over 20% of biomass in form of neutral lipids and for that reason are called “oleaginous.” Under optimal growth conditions and/or as a result of genetic improvement, the level of lipid accumulation can reach even 70%. Oleaginous yeast includes species of
2. Yeasts as whole-cell biocatalysts
Different genera of yeasts are convenient biocatalysts applied in many fields of chemistry, especially for the synthesis of chiral building blocks and fine chemicals. They are interesting catalytic tools, not only for their varied enzymatic activities but also for their microbiological features such as simplicity of cultivation, low nutritional requirements, and adaptive capacities. These capacities result from their flexible metabolism, which responds to the environmental impacts, so the direction (also stereoselectivity) and the effectiveness of the biotransformations can be driven by the physical chemical parameters of the process. What is also important, they are susceptible to the engineering of the reaction media (e.g., water, organic) and to the biocatalysts form (e.g., permeabilization, immobilization). This significantly broadens the field of their application by overcoming the limitations such as low solubility of the bioconversion substrates. It can be said that yeasts are used for decades and are one of the first whole-cell biocatalysts applied in industrial processes.
Literature data proved that the core applications of yeasts are connected with their extraordinary reductive abilities. Since it was proven that whole-cell biocatalysis is (as enzymatic one) chemoselective, regioselective, and stereoselective and able to regenerate dehydrogenases cofactors under biocatalytic conditions, yeasts were extensively examined as reductive catalysts for chiral building blocks synthesis—especially chiral alcohols of defined absolute configuration. The activity of a number of yeasts genera has been tested toward structurally different ketones, and in the most cases desired, alcohols have been obtained as pure enantiomers . Alcohols drawn below (Figure 4) represent both, simple, and more complicated—unusual structures obtained thanks to the whole-cell biocatalysis driven by yeasts.
Reductive activity of
Entirely a different activity of yeasts as biocatalysts found some practical application, lately. Successful experiments with
Such indol motifs are crucial part of the biologically active compounds such as arbidol (influenza A and B virus treatment and prophylactic); golotimod (immunostimulating, antimicrobial, and antineoplastic agent); and panobinostat (acute myeloid leukemia treatment; Figure 9).
3. Yeast’s enzymes applications
Biocatalysis includes both biotransformations (e.g., the conversion of xenobiotics using whole cells or resting cell systems) and enzyme catalysis (e.g., the conversion of xenobiotics using cell-free extracts or purified enzymes) . Although both whole cells and isolated enzymes can be used as biocatalysts, whole cells are very often preferable because they are more stable and cheap sources than purified enzymes, without the need for purification and coenzyme addition. However, in the case of single-step biotransformation, isolated enzymes can be considered as a better choice and can be used as a free or immobilized biocatalyst either in aqueous or organic media . Yeasts, especially
3.1. Yeast’s lipases
Lipases are widely distributed in nature and are produced by plants, animals, and microorganisms. Microbial enzymes are more useful than the other ones because of the diversity of catalytic activities, simplicity of manipulations, and low cost production (extracellularly during rapid growth on inexpensive media) . Additionally, microbial enzymes are free from problems associated with contamination with hormones, viruses, and can be used in food processing and pharmaceutics productions for vegetarian or kosher diets . Microbial lipases (EC 126.96.36.199) are suitable enzymes for organic synthesis because they are active toward broad range of nonphysiological substrates and are stable in biphasic systems or pure organic media. Lipases can be applied for either of lipid modifications and synthesis of special compounds: pharmaceuticals, polymers, biodiesels, and biosurfactants . Under physiological conditions, lipases catalyze hydrolysis of ester bond in triacylglycerol to glycerol and free fatty acids. Under nonaqueous conditions, they catalyze the reverse process—esterification. The term transesterification refers to exchange the group between an ester and acid, ester and alcohol, or at least between two esters (Figure 10) .
Mentioned features make them significant biocatalyst for various applications. There are a certain number of yeast species able to produce lipases, most of them belong to
For pharmaceutical industry, lipases are used to resolve racemic mixtures of alcohols or carboxylic acids through asymmetric hydrolysis of acyl derivatives.
Another possible way to change the enantioselectivity of hydrolysis is the addition of the organic co-solvent to reaction medium. In organic media, the conformation of enzyme appears to be more rigid which may influence the enantioselectivity of the reaction. For the
Another interesting application of CAL-A is the regioselective hydrolysis of cyclic diacetates, useful building blocks for the synthesis of vitamin D3 derivatives , or hydrolysis of different sterically hindered carboxylic acids .
3.1.2. Esterification and transesterification
The most important application of lipases in organic synthesis is esterification important for the resolution of racemic mixtures of secondary alcohols and carboxylic acids. Chiral secondary alcohols serve as intermediates for pharmaceutical synthesis [85–87]. Lipase-catalyzed methods available for the preparation of enantiopure compounds are kinetic resolution (KR), dynamic kinetic resolution (DKR), and desymmetrization. Enzymatic kinetic resolution is based on the difference between the reaction rates of the enantiomers of a racemate at the presence of chiral catalyst—enzyme. Dynamic kinetic resolution combines kinetic resolution with the
Also, enantiomerically pure amines constitute a class of compounds with possible biological properties and industrial applications .
CAL-A isoform of
CAL-A is also active towards sterically hindered tertiary alcohols. This feature is quite unique among hydrolases. The first example of enantioselective kinetic resolution of racemic mixture of tertiary alcohol was acylation of 2-phenylbut-3-yn-2-ol. The reaction was quite enantioselective, but the yield was rather moderate (25%) because of the steric hindrance. Another interesting application of CAL-A is selective acylation of sterols , furyl substituted allyl alcohol , or cyanohydrins [100, 101].
3.2. Yeast’s invertase
3.3. Yeast’s oxidoreductases
Enantiometrically pure alcohols including
Other example of reductase is selective carbonyl reductase from
4. Yeast’s applications in molecular biology
Yeasts of the
Although yeast and humans have been evolving along separate paths for 1 billion years, still a substantial amount of yeast genes exhibit high homology to mammalian ones. Since the basic cellular mechanics of replication, recombination, cell division, and metabolism are generally conserved between yeast and larger eukaryotes, they constitute a good model for studying different processes such as aging, regulation of gene expression, signal transduction, cell cycle, metabolism, apoptosis, neurodegenerative disorders, and many more . Furthermore, its protein expression systems have more in common with higher organisms than with prokaryotic ones, mainly due to the posttranscriptional and posttranslational processing, which makes it a great candidate for acquiring a number of industrially or medically significant biomolecules, such as recombinant proteins for pharmaceutical purposes .
Life cycle of
Techniques used for yeast transformation and specific selection have been well described. For this purpose, shuttle vectors are commonly used due to the fact that they can transform both yeast and bacteria, such as
4.1. Yeast’s plasmid vectors
Most of the bacterial vectors are provided with genes-encoding resistance to various kinds of antibiotics, such as ampicillin (
Cloning techniques with yeast differ mostly in the strategic approach of the selective markers. In this case, usually a special kind of organism is required as the host, namely an auxotrophic mutant that is unable to obtain or synthesize a pivotal compound of one of its metabolic pathways. A good example is
There are few kinds of yeast cloning vectors, but all of them are so-called shuttle vectors, which means that they can replicate and be selected in both bacteria and yeast. Shuttle vectors were developed mostly because plasmid preparation from yeast only is highly ineffective; hence, the large-scale DNA propagation and convenient genetic manipulation are performed in bacterial organism, such as
Yeast cloning vectors based on 2-μm plasmid are called yeast episomal plasmids (YEps) (Figure 23(a)). Depending on the kind of YEp, they can either contain most of the 2-μm plasmid or just the origin of replication, their backbone is usually constructed from
The other important type of yeast vectors are yeast integrative plasmids (YIps) (Figure 23(b)). They mainly consist of
Yeast replicative plasmids (YRps) (Figure 23(c)) are another type of yeast cloning vectors. They contain a backbone from
Along with the time, there was a growing demand for much larger pieces of DNA to be manipulated through the techniques of genetic engineering. It was at this point that the last type of yeast cloning vectors was developed, namely the yeast artificial chromosomes (YACs). The general idea behind those constructs was that yeast chromosomes usually carry several hundred kilobases of genetic material, so why not imitate the native DNA? YACs were thought to contain the three key elements of a chromosome:
centromeres required for the proper chromosome positioning during the cell division;
origins of replication, which are the places on chromosome where the replication of genetic material starts;
telomers as the defenders of the chromosomes against exonucleases.
Several types of YACs have been developed over the years, they usually consist mostly of a
4.2. Yeast expression system
Recombinant proteins are the biomolecules of great importance, because among other things, they are able to mimic the functions of native proteins; hence, they are extensively studied in biotechnology and biopharmaceutical research. The critical point of target protein production is the choice of efficient expression system which enables obtaining functional product with high yield.
Yeast expression system constitutes a good alternative for widely used bacterial and higher eukaryote expression systems. They are genetically well defined and are known to perform many posttranslational modifications, including proper protein folding, disulfide bond formation, and glycosylation . The culturing of yeast is also easy, rapid, and cheap, which is their big advantage over the insect or mammalian cells. They easily undergo genetical manipulation and adapt to fermentation processes; therefore, using yeasts as a cell factory is convenient and enables to obtain a fair amount of target protein. In contrast to bacteria, recombinant proteins obtained in yeast expression systems are free of endotoxins that make this system safer, especially in terms of medical and food application . In fact, about 20% of all biopharmaceuticals are produced by
However, yeast cells are limited in the production of human-like glycoproteins by their inability to produce complex
There is no ultimate procedure for yeast expression system that could work equally well for the production of all kinds of proteins. Optimization of whole process is the critical step to obtain sufficient amounts of pure, properly folded and secreted protein of interest. While small and simple in structure proteins are easy to obtain, the big and multi-domain protein could require certain chaperones to facilitate the folding process . The advantage of yeast expression system is that it allows extracellular secretion of produced protein when proper signaling sequence has been attached to the structural gene . It significantly facilitates the recombinant protein purification process from the culturing medium and allows to optimize the culturing conditions. In order to increase protein secretion level, a few strategies have been developed. One of them is protein engineering of a desired product, for instance by modifying protein coding sequences and signaling sequences [128–130]. Since this methodology is highly specific against each protein, the conditions optimized for one protein do not always work for another. Different approach is to engineer the host strains and tune-up folding and secretory machinery by overexpression or deletion genes that are critical for the protein secretion [131, 132]. Additionally, it has been shown that expression in low temperatures enhances the level of secretion .
There are numerous varieties of expression vectors available for producing heterologous proteins in yeast, and these are the derivatives of YIp, YEp, and YRp plasmids described previously. The DNA coding for the protein of interest is inserted into the vector. The type of selective marker and promoter strength are key factors that determine the plasmid copy number and the mRNA level of the recombinant protein. Varieties of inducible and constitutive promotors have been applied for gene expression in yeasts in the past. The first of these allow the controllable gene expression. Most of inducible promoters are responsive to catabolite repression or react to other environmental conditions, like in-cell iron concentration, stress, or lack of essential amino acids.
Additionally, yeasts are recognized as a generally recognized as safe (GRAS) organism, which only strengthens its position as the most frequently used microbial eukaryote for recombinant protein synthesis.
4.3. Yeast’s two hybrid system
Ever since the Field and Song discovery described in 1989, a new approach toward the examination of protein-protein interactions emerged, it was named as the yeast two-hybrid system. It allows to detect the interaction of two proteins in the yeast cell, and it can be used to select an interacting partner of a known protein. This technique takes the advantage of the fact that majority of eukaryotic transcriptional factors, such as Gal4p, consist of two independent, functional DNA domains: binding domain (BD) and transcription activation domain (AD). While the two domains are normally on the same polypeptide chain, the transcription factor also functions when these two domains are brought together by noncovalent protein-protein interactions. In yeast hybrid system, each of these domains is connected to the one from the studied protein. As a result, two fusion proteins are created: one combined to DNA-binding domain (BD) and the other joined to activation domain (AD) . The genes coding for both fusion proteins are carried by different plasmids, but each plasmid undergoes expression in the same yeast cell. If the interaction between studied proteins occurs, BD and AD domains are close enough to activate transcription of a reporter gene that is regulated by the transcription factors.
General idea of the yeast two-hybrid system can be represented by an example of transcriptional factors and a gene coding for
Discussed unique yeasts features, which are fundamental for their versatile applications are still examined and after finishing the “Synthetic Yeast Genome Project (Sc2.0)” the new perspectives of the applying them will be opened as well as in the molecular biology and in the industrial applications.
The project was supported by the Wroclaw Center of Biotechnology program, the Leading National Research Center (KNOW) for the years 2014–2018.