Twenty years ago, only a handful of laboratories, some in industry and some in academia, were interested in using enzyme-catalysed reactions (biotransformations) in organic synthesis. At best those researchers were considered to be at the margin of mainstream synthetic organic chemistry; at worst they were considered to be downright odd.

In the 1980s there was an exponential increase in interest in the area of biotransformations. That worldwide increase in attention to this field of research can be assigned to several factors, including the perception and perseverance of the original researchers in the field, the increased availability of a wide variety of enzymes, and the realization that many families of enzymes will transform a wide range of unnatural compounds, as well as their natural substrates.

The utilization of enzymes in organic synthesis can be advantageous for several reasons:

1. Enzymes catalyse reactions under mild conditions with regard to temperature (ca. 37 °C), pressure (1 atm), and pH (ca. 7.0). The transformations are often remarkably energy-efficient when compared with the corresponding chemical processes.

2. Enzymes often promote highly chemoselective, regioselective, and stereoselective reactions, and being chiral catalysts, they are often able to generate optically active compounds. The increased awareness regarding the need to have optically pure compounds for such uses as Pharmaceuticals and agrichemicals (so as to avoid unnecessary toxicity and/or ecological damage) has been a significant driving force in the development and exploitation of non-natural enzyme-catalysed reactions.

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Introduction

As discussed in Chapter 1, there has been much interest, over many years, in oxidation and reduction reactions catalysed by micro-organisms. Processes for alcohol production and for the manufacture of vinegar and vitamin C have intrigued investigators for ages. Today there is great interest in discovering new whole-cell and isolated-enzyme-catalysed reactions of this type. As described in Chapter 3, the focus of the presentday work in this area is the production of compounds (often in optically active form) that might be useful as intermediates or end-products for the pharmaceutical, fragrance, flavour and agrichemical industries.

For many oxidation and reduction reactions it is often necessary to make a choice between using a whole-cell system or an isolated enzyme (with the requisite co-factors) for the biotransformation. In this area, factors that influence the choice often are much more complicated than those for hydrolysis reactions. The pros and cons of employing whole cells or partially purified oxidoreductase enzymes have been discussed in Chapter 2, and the salient features are summarized in Table 2.3. While the advantages and disadvantages will not be discussed in detail again, many of the points made earlier will recur in this discussion.

Reduction of ketones using whole cells

For example, consider one of the simplest reduction processes, namely the reduction of a ketone to a secondary alcohol. Of course, this can be accomplished chemically using sodium borohydride.

Introduction

Biological catalysis has such a pervasive influence on the industries which are closely associated with our daily lives that it is difficult to contain a short review within sensible bounds. The traditional large-scale crafts associated with agriculture, the manufacture of foods and drinks, and the preparation and cleaning of fabrics all use biological catalysis. At the other extreme, biological catalysis has an important role to play in analytical chemistry. Enzymes are used to monitor the levels of metabolites as an important feature in the diagnosis and therapy of disease. These techniques have been extended to the use of enzymes to amplify the responses in antibodybased assays, as well as to mediate the detection of an environmental pollutant at an electrode. The same catalysts also play a role in the degradation of pollutants, which is, perhaps, simply an extension of the role of biochemistry applied to the traditional craft of cleaning.

However, it is the use of enzymes in the large-scale manufacture of synthetic organic chemicals which is the main topic of this chapter. The recent interest amongst organic chemists arises from the high degrees of specificity and selectivity that characterize enzyme catalysis. That this is a valuable resource in manufacture cannot be doubted, as judged by the value of the products which it produces. Nevertheless, the methodology should not be oversold. Biocatalysis has a part to play in the modern chemical industry, but it also has limits which it is necessary to understand.

Introduction

The use of transgenic rodents, in particular transgenic mice, to address a number of diverse biological problems has been so vast that this review chapter cannot cover every facet of transgenesis. We hope that by explaining the technology associated with the generation of transgenic mice and by focusing on a few current areas of research where transgenic animals are proving invaluable, we will help the reader understand the use of transgenic animals in research, as well as the power and limitations of this approach.

In this review a transgenic animal is defined as an animal that carries a foreign piece of DNA stably integrated into the genome of every cell in that animal. Thus, all somatic cells and the germ-cells of a transgenic animal carry the same DNA fragment at the same chromosomal location, and this DNA fragment can be transmitted in a Mendelian fashion to offspring. This DNA element is termed a transgene.

Generation of transgenic mice

There are essentially three ways of generating animals with the capacity to transmit a genetic element through the germ-line to their offspring. These are (a) retroviral integration into an early-developing embryo, (b) injection of DNA into the pronucleus of a newly fertilized egg or (c) genetic manipulation of embryonic stem cells (Fig. 5.1). All three routes have been employed to generate transgenic mice, while a few transgenic rat strains have been established chiefly through the pronuclear injection route.

Retroviral infection

If preimplantation embryos are exposed to retrovirus, a proportion of the embryonic cells will stably integrate proviral sequences into their genome, usually as one copy per cell.

Introduction

Birds are virtually unique among domesticated species in being bred for the nutrient value of their ova, and few organisms have been subjected to as much genetic selection as poultry. These two features encapsulate both the attractions and the frustrations of producing transgenic birds.

Current breeding practices in the poultry industry depend on natural genetic variation that is exploited by selective reproduction. New variants are introduced from mutations or translocations, but there is a progressive loss of ‘wild-type’ characteristics. By inserting foreign DNA into organisms it is possible to bypass reproductive barriers and induce gene flow between vastly different organisms from quite distinct populations. Thus, the potential benefits of genetic engineering are extremely attractive, for they could dramatically increase both the genetic repertoire and the rate of change of a particular breed. Unfortunately, one of the characteristics of birds that makes them so commercially attractive – their large eggs – is also one of the features that makes it necessary to devise new approaches to their genetic engineering. The most common way of introducing foreign DNA into mammals is microinjection into the male pronucleus (Palmiter & Brinster 1986). The large size of birds' eggs makes it virtually impossible to see these structures and, to make matters worse, polyspermy is common. There may be up to 20 male pronuclei in a fertilized fowl egg with no indication as to which will eventually fuse with the female pronucleus.

Introduction

This chapter deals with three important classes of biotransformations. Firstly, those enzymes that catalyse the stereoselective formation of carbon-carbon bonds will be examined. These enzymes, whose natural functions often are to degrade carbohydrate-like molecules, have proved to be versatile catalysts for C—C bond synthesis. Secondly, we shall look at those enzymes that mediate the formation of C—X bonds, where X = O, N, S, Hal (halogen). These enzymes are termed lyases (see Table 2.1) and often carry out very simple reactions (e.g. the addition of water to a double bond) with very high stereoselectivity and regioselectivity. Finally, the application of a range of enzymes (including C—C bond formation) to carbohydrate synthesis will be examined. This chapter will conclude with some examples of the ways in which multienzyme reactions can be constructed to enable highly complex molecules to be assembled in an efficient manner.

Enzyme–catalysed asymmetric carbon–carbon bond formation

Aldolases

The synthesis of carbon–carbon bonds, particularly with control of stereochemistry, has been an area of intense activity in recent years. Much attention has been focussed on the asymmetric aldol reaction, principally using stoichiometric quantities of a chiral auxiliary, but more recently in the catalytic mode. The use of aldolases for the asymmetric construction of carbon-carbon bonds presents a potentially useful complementary methodology.

The aldolases are a diverse class of enzymes that catalyse the coupling of a carbonyl-containing compound (nucleophile), containing one, two or three carbons, with an aldehyde (electrophile). In most cases the nucleophile is either pyruvic acid or dihydroxyacetone phosphate, whereas the electrophilic aldehyde is much more variable in structure.

Introduction

What are transgenic animals?

Since the earliest days of animal domestication, people have sought to improve their flocks and herds and companion animals by selective breeding. Although the results of such breeding are impressive, the procedure is by nature slow and to some extent imprecise. So the dream existed that perhaps one could take a more direct hand in stock improvement by recovering the genetic factors involved and adding these selectively to individual animals. The first attempts to do this took place some thirty years ago, but since the methods of DNA purification were suboptimal and methods had not been developed for gene isolation and cloning, complete genomic DNA preparations were used. These were simply mixed with animal eggs or embryos in the hope that transfer might occur, and then the resulting adults were screened for phenotypic features previously present only in the donors. Many of us can remember such experiments being undertaken with DNA from pigmented Ambystoma (the Mexican axolotl), such DNA being added to or injected into albino embryos of this species in the hope that the pigment gene would be transferred. Since that gene was likely to be swamped by the DNA for at least 1 million alternative sequences, one can see with the benefit of hindsight that the odds against success were enormous.

The developments which have now tipped the balance in our favour are chiefly threefold. The first, along with improvements in methods of DNA extraction and purification, was the discovery of restriction enzymes, which made it possible to dissect DNA into manageable fragments of precise length and sequence.

Introduction

Enzyme-catalysed hydrolysis and esterification reactions are the most commonly exploited biotransformations. There are two main reasons for this state of affairs. Firstly, the reactions are very easy to perform, and no special apparatus is required. Secondly, there is a wide range of hydrolase enzymes available from commercial suppliers. Furthermore, the stereoselectivities and chemoselectivities of the enzymes are well known, and the likely stereochemical outcomes of such enzyme-catalysed reactions on previously unused substrates are now becoming predictable.

There are many types of enzyme-catalysed hydrolysis reactions. In this chapter, the hydrolysis of esters and amides will be surveyed quite extensively, while the hydrolysis of nitriles and epoxides will be mentioned briefly at the end. The use of hydrolase enzymes in organic solvents will be discussed also, in connection with the preparation of esters and amides.

In a number of instances, whole-cell preparations have been preferred as catalysts for selected hydrolysis reactions; in most of these cases the micro-organisms are easy to grow and simple to handle and are utilized because of the low cost involved. These cases will be integrated into the discussion as and when appropriate.

Hydrolysis of esters

The enzyme-catalysed hydrolysis of simple esters (Scheme 3.1) takes place at temperatures around 35°C and at pH values around 7 (i.e. under very mild conditions). Such reactions have been studied in great detail, and the ways in which the enzymes catalyse the reactions are being investigated.

Introduction

There are basically two strategies for carrying out biotransformations: (1) to use pure or partially purified enzymes isolated by the investigator or purchased from a commercial supplier, or (2) to use whole cells. Enzymes are categorized by the Enzyme Commission (EC) according to their functions (Table 2.1), and each individual enzyme is given a unique code made up of four numbers, such as 2.1.2.4. These reference numbers are derived as follows:

The first number indicates the class (1 through 6; see Table 2.1).

The second number in the series indicates the subclass. For oxidoreductases, the subclass number indicates the type of group in the donor which undergoes oxidation (1 denoting a secondary alcohol group, 2 denoting an aldehyde or ketone unit, etc.); for transferases, it gives an indication of the functional group which is transferred (1 indicates the transfer of a one-carbon unit); for the hydrolases, it earmarks the functional group hydrolyzed (1 is used when an ester group is hydrolyzed); for the lyases, it indicates the group HX (3 indicates the addition of ammonia); for the isomerases, it shows the type of isomerization (2 indicates alkene cis-trans isomerization); for ligases, it indicates the type of bond formed (4 indicates carbon-carbon bond formation).

The third number in the series serves to allocate the enzyme to a sub-subclass. For oxidoreductase enzymes, this third number shows the type of acceptor involved [e.g. 1 denotes a co-enzyme, such as nicotinamideadenine dinucleotide phosphate (NADP); 2 denotes a cytochrome; 3 denotes molecular oxygen].

The range of transgenic animals

As seen in previous chapters, animal species of many kinds have been used for experiments in transgenic induction. Some offer particular benefits to the experimentalist; others have proved difficult or even impossible to use. In this book we have attempted to review all of those in which transgenic induction has been successful. The present range is as follows: protozoans such as Paramecium and Trypanosomid species, a few species of sea urchins, the nematode Caenorhabditis elegans, a few insects such as the silk moth Bombyx, the mosquito Anopheles and the fruit fly Drosophila, the predatory mite Metaseiulus, fish of many species, the amphibian Xenopus laevis, the domestic chicken and both laboratory mammals such as rats, mice and rabbits and large mammals such as cattle, sheep and pigs.

The failures and successes of applying transgenic technology to all of these experimental systems makes fascinating reading, yet clearly we are only at the threshold of this interesting study area. As well as the major transgenic systems that we have addressed here, there are a number of interesting minor ones, which will be briefly reviewed in this chapter. Of course, since this book is about transgenic animals, we have not discussed the extensive work on transgenic plants, fungi such as yeast and Aspergillus or bacteria, in which transgenism is a way of life, facilitated as it is by the extensive range of natural plasmids.

Introduction

Following the discoveries of vaccines, anti-toxins and antibiotics, microbial biotechnology over the past few decades has proven itself as a creatable resource. With the advent and use of new research skills, it is giving rise to further new industries and ventures. All pervasive on the commercial front and defying monopolization by any one group or society for extended periods of time, the products of microbial technology are used by all from presidents and royalty to the poor and the underprivileged.

The potential of the microorganism for socio-industrial advancement and growth is legion and legend. Solvents, chemicals and bioenergy result from anaerobic fermentation, i.e. acetone, butanol, and methane. Food additives, dairy products and enhanced-oil-recovery result from the use of microbial polysaccharides. The potential for food, feed and ethanol production from waste cellulosic materials via microbial action in bioconversion technologies exists. Textured and flavoured mycelial food products that simulate veal and chicken have been produced. Microbial production of psychoactive and immunoactive components is a reality. Microbial synthesis of interferons, rennet and growth hormones is no longer a remote possibility. Underpinning all these processes in their transition from the laboratory-scale to the industrial stage are three indisputable determinants – society, culture and economics.

The past decade, has also been witness to fundamental changes in the biosciences. New vistas in molecular biology and human health have been opened by the techniques of genetic engineering using restriction endonucleases resulting in the development of bioactive entities now being produced in quantity using microorganisms as the producing agencies.

Introduction

Everyone wishes to remain healthy so as to work cheerfully and enjoy life to its fullest extent. A primary source for health support comes from food. For a long period of time, many fungi have been highly valued as food to fight against hunger, as a condiment to enrich the flavour of dishes and also as supplements to help maintain good health. These properties have made fungi even more popular in recent years, and this can be witnessed by the increasing demands for higher production volumes of many edible fungi in the local and international markets.

General biology of a mushroom

Life cycles A mushroom is the fruiting organ of a filamentous fungus which in most cases is a basidiomycete bearing the sexual cells, basidia, to carry out meiosis and dispersal of spores (Fig. 6.1). This filamentous fungus has a haploid form whose cells can be multinucleate (coenocytic), e.g. Agaricus bisporus (syn. A. brunnescens) and Volvariella volvacea, or uninucleate, e.g. Lentinula edodes (syn. Lentinus edodes) and Pleurotus ostreatus. Some mushrooms are self-fertile, e.g. A. bisporus and V. volvacea, while others are self-sterile but cross-fertile, e.g. L. edodes and P. ostreatus, and only after mating, fruit bodies called mushrooms are produced (Figs 6.2–6.5). For the latter species, there is usually a characteristic dikaryotic stage featured by a hyphal form with clamp connections at septa formed after mating (Fig. 6.1).

Fruiting Aggregation of the aerial hyphae by coiling and multiple forms of hyphal fusions turns a fungus into a three-dimensional ordered fruit body primordium (Figs 6.6 and 6.7).

Introduction

Biotechnology is many things to many people. Traditionalists would tell us that biotechnology is one of the oldest activities of man, beginning with the production of wines and alcoholic beverages and now encompassing all of the current fermentation industries. Modernists would suggest that biotechnology came of age with the advent of recombinant DNA technologies and it is this ‘new’ biotechnology which will have the greatest impact upon our societies and their economic structures. The reality, of course, is that both these views, the traditional and modern, are inter-dependent. The new biotechnology could not have gained such a rapid place in the industrial fabric of society had there not been the traditional roots already in place. Developments and indeed applications of genetic modifications of living cells have taken place because of the traditional view that microorganisms have much to offer in their exploitation to man's well-being. A coherent framework, thus, was already in place which then allowed genetic engineering principles to be speedily translated from laboratory concepts into industrial practices.

The implications for biotechnology in a developed and developing world are tremendous. However, it must always be appreciated that biotechnology is what it says it is: it is a technology. That is, it is an enabling discipline. It allows the exploitation of microorganisms, plant and animal cells to take place within an economic framework. Biotechnology is not then a science: it is a means of applying science for the benefit of man and society. In practice, this means that biotechnology is used to make money – or in certain instances – to save money.