A biotechnology breakthrough :

the total biosynthesis of hydrocortisone drug by a humanized yeast

This biotechnology breakthrough, which will be published in the February 2003 issue of the review Nature Biotechnology (access to the article) is the fruit of an exemplary collaboration between the French public research institution CNRS and the Aventis-Pharma company. This work, which had also involved other academic and industrial French partners (ESTBB in Bordeaux, Transgène in Strasbourg) places itself within the framework of a long term project aiming to substitute Biotechnology to Chemistry for the total synthesis of drugs with industrial scale, involving new generation of sophisticated recombinant micro-organisms. The published article described the construction of a yeast strain capable of producing a large range of steroid hormones, and particularly the major anti-inflammatory drug hydrocortisone. The steroids are indeed largely used as drugs for anti-inflammatory, contraceptive, anti-cancer and new developing anti-aging purpose. The main current way of production of hydrocortisone, is an hemi-synthesis which uses biliary acids or phytosterols as starting point of an industrial process involving many stages of chemical conversions and a stage of bioconversion implying a natural micro-organism. However, this process which relies mainly upon fine chemistry remains relatively complex, expensive and generator of by-products.

The realization of the total biosynthesis of hydrocortisone by a micro-organism able to manufacture in a completely autonomous way this drug from simple sources of carbon (alcohol, sugar) and oxygen (air) represents a decisive achievement, as well as in regards to industrial competitiveness than for the development of new "green" industrial production processes more respectful of the environment than chemical processes. It is precisely what the teams of the Aventis company and of the Center for Molecular Genetics of CNRS realized in Vitry and Gif-sur-Yvette (France), by introducing into the baker yeast Saccharomyces cerevisiae a large set of genes originally belonging to man, bovine, plant, and microbe and by diverting or reprogramming some metabolic stages of this micro-organism.

This success was made possible by a very narrow partnership between public research, through one of its organizations the CNRS and the pharmaceutical group Aventis-Pharma, which accepted the challenge to support a long-term research implementing the most modern techniques of genomic and molecular engineering. The developed technology is potentially able to be extrapolated for the production by "green chemistry" of very large variety of molecules of interest as regards to human, animal and plant health. This major technological jump was made possible in particular by the recent accumulation of information on the sequence of the genome of many species: micro-organisms, plants and human.

© Denis POMPON, 2003

Contact CNRS :
Dr. Denis Pompon
CNRS-Centre de Génétique Moléculaire
91198 Gif-sur-Yvette Cedex FRANCE

Phone : 33 (0)1 69 82 36 80
Fax : 33 (0)1 69 82 36 82
E-Mail : pompon@cgm.cnrs-gif.fr

Artificial pathway for the biosynthesis of the hydrocortisone as rebuilt in yeast

The enzymes in red come from human, bovine and plant organisms and their genes were engineered before introduction in yeast cells.

Those in blue are original yeast enzymes whose genes were modified to change their expression levels.

The green pathway is the main pathway for hydrocortisone production, the red one corresponds to controlled side reactions and the yellow one to secondary biosynthesis pathway coexisting with the major green one.

From metabolism re-routing to the total biosynthesis of drugs

or

" How to build a factory smaller than a pinhead? "

The majority of drugs are complex organic molecules. In the frequent case they are derived for naturally occurring substances, a first mode of preparation consists in extracting them by physicochemical processes starting from natural resources (plants for example). It is what one does without the knowledge by preparing a herb tea. Nevertheless, these natural sources are often not limited, expensive and their exploitation pose serious ecological problems.

The second and more generally employed manner of reaching that point, is the chemical synthesis. This one can be total, on the basis of simple elements like water, carbon and nitrogen, or partial (one speaks about hemi-synthesis) when the starting molecule is already a complex natural molecule (a peptide, a sugar, a sterol, etc. ). The chemical syntheses involves multi-steps industrial processes often generating many by-products which should be eliminated in a suitable manner to avoid risk of pollution with obviously additional costs. These processes are often long and expensive, justifying a sometimes high price of the active molecule obtained at the end of the chain.

Conversely, living organisms realize a large number of such synthesis of an incredible complexity with a very great effectiveness within each one of their cells and this practically without by-products. They owe these remarkable properties by involving the performing nano-machines of synthesis what constitutes the enzymes. These biological catalysts are able to carry out each one of the required step of synthesis with an extreme precision and to connect these stages in order to avoid any loss of material or any drift towards non-desired products.

The association of stages of chemical synthesis to enzymatic stages is used currently more and more industrially, at least when adequate natural enzymes exist (emerging technologies like "directed evolution" will probably make it possible to create non-natural one with activity at will in a close future). Nevertheless, the problem is that these catalysts are fragile and often expensive to prepare, which generally severely limits their use for the production of industrial products. A solution making possible to reduce these costs, is the use of whole micro-organisms instead of isolated (purified) enzymes. Micro-organisms (bacteria, yeasts, mushrooms) are able to naturally produce such enzymes for their own use. Such natural micro-organisms were largely used since a very long time, for example in food industry: cheeses, yoghourts, wine and beer owe them their tastes and numbers their pleasant properties.

More recently, the development of the genetic engineering (set of methods allowing to extract, modify and transfer genes) allowed the construction of genetically modified organisms. Human being thus realizes on their time scale and on their ends what nature does at the slow rhythm of evolution to meet its own needs. The genetic engineering made possible to isolate new genes of interest and to produce in great quantity with micro-organisms the precise enzymes which the chemist needs to carry out his syntheses. This approach allowed significantly to reduce the cost of isolated biotechnological stages of synthesis. Nevertheless, the tool remained imperfect, much of stages having still to be realized by traditional chemistry.

The idea was then born to massively reprogram genetic information of organisms (generally a micro-organism) in order to transform it into a true chemical factory, able to carry out not one or two stages of a synthesis but the entire process without any external intervention. It was the roof of refinement to require also from the biological factory to assembly from elementary carbon , oxygen, nitrogen and oxygen sources, the starting molecules for the synthesis and to produce also the energy required. This reprogramming implies the use of the genetic engineering to extract, alter and transfer the useful genes, therefore corresponding to required assembly of enzymatic functions in the same micro-organism.

Nevertheless, the total biosynthesis of the complex structures of drugs starting from simple molecules, like a sugar, requires a considerable number of enzymatic catalysts. This very high number made the task of genetic engineering excessively complex. The idea then was to select an host micro-organism naturally knowing how to make natural molecules resembling as much as possible some of the intermediaries of synthesis of the drug of interest. These drug precursors are in general for the host cells simple intermediary in the manufacture of their natural molecules which are useful for its but who, in general, do not have pharmaceutical interest. One then will try to divert his metabolism, i.e. to prevent host from using these intermediaries normally so that they accumulate and that one can make use of them as products of departure for manufacture of the drug of interest. This first stage of the diversion is done in general by selectively destroying a certain number of natural genes of the host micro-organism. Once that made, begins the second stage, which consists in introducing into the host a whole series of new genes, coding the extra requested enzymes. Those (in fact corresponding genes), which can belong to very different species (microbes, fungi, plant, fish, animal, even man) are selected by the biotechnologist in order to allow the biotransformation of the natural molecule diverted in the first stage, into the expected drug which one seeks to manufacture..

The baker yeast Saccharomyces cerevisiae, an eukaryote micro-organism, which is equipped with intracellular structures close to those of the ones of mammal cells, is well-known to allow the stable production at low prices of foreign proteins. This host thus appeared as an interesting solution for the reconstruction of the complex artificial pathways of biosynthesis which mimic the ones naturally present only in higher organisms. Other advantage, one knows perfectly the genome of yeast, which was entirely sequenced a few years ago within the framework of a European program. One also knows that the "domestic yeast" Saccharomyces cerevisiae does not present any potential risk in terms of biosecurity, particularly as in addition to it natural absence of pathogenicity, the genetic engineering process was designed to make impossible viability the engineered micro-organism in natural environment. On the other hand, the yeast had a heavy handicap: it did not carry out any the enzymatic step naturally leading to the synthesis of the human steroid hormones !!

It was thus necessary to entirely reconstitute in yeast the human chain of biosynthesis of hydrocortisone, "to humanize it" in another words, by introducing there the whole of genes necessary. In fact, the problem was even more complex than to simply introduce human missing genes: Substitution of human genes by equivalent (coding for enzyme with similar activity) but most performing genes coming from other species could be useful to optimize the process; it thus was necessary to make the best choices by addressing all the richness of natural biodiversity. In addition, certain natural genes of the yeast host can be deleterious for the artificial biosynthesis by creating non-desired side reactions, which are absent in the human cells but unfortunately present in yeast. It was thus necessary to identify these "parasitic genes" and to destroy them in an intelligent way, i.e. in a way allowing to prevent or to limit the non-desired reactions, while at the same time, preserving them sufficiently to do not kill "goose that lays the golden eggs", i.e. the production host itself. Another major difficulty was the problem of the "compartimentation". Indeed in human, several different bodies (the liver, the glands suprarenals, …) are implied in the synthesis of hydrocortisone. The yeast, a unicellular fungi measuring only few thousandth millimetre, does not have bodies. It thus was necessary to rebuild the things a little differently from nature, to imagine new solutions, new subcellular localizations for the necessary enzymes (catalysts), in order not to "mix" together incompatible reactions (particularly to avoid short-cut in electron transfer steps) within the micro-organism).

But the things were even more complicated if one get interested now in the manner of practically carrying out these tasks: contrary to the bacteria, in which all the genes ensuring the various stages of production of a given molecule are laid out on the chromosome in a kind of assembly line called operon, in higher organisms (eukaryotes) they are dispersed and interrupted by portions of non-coding genetic information (named "introns"). Because of the complexity of the structure of the eukaryotes genes, the gene transfers between distant eukaryotes (animals, plants, fungi) is a complex operation requiring an elaborated molecular engineering. It was thus necessary to rebuild artificial genes by combining yeast transcription control sequences and preliminary spliced mammalian or plant "open reading frames" (the part of genetic information coding for enzymes) and to integrate them into the yeast genome, to finally allow the expression of the whole of the functions leading to the synthesis of the human steroid hormones and hydrocortisone. On the whole, about fifteen genes have been thus engineered in a suitable manner in a single cell, so that each enzymatic stage is carried out at the right speed and is connected harmoniously with the preceding and the following ones, so as to avoid the accumulation of intermediate or the formation of non-desired products. Gene expression tuning was carried out by adjusting the fine structure of each artificial gene and as well as the number of copy of each one of these genes upon introducing into yeast. Very many combination had to be tested before finding "good dosages" leading to a drug production free from awkward contaminant.

Thus our engineered micro-organism is now able to manufacture only the drug which interests us without the least help of the chemist. The chemical factory will have been replaced by a microscopic organism to which it will be enough to give for example sugar or alcohol so that it manufactures in our place the molecule of interest and release it in the culture medium where crystallisation of the drug occurs. The whole of the production chain having been integrated into new permanent genes, the micro-organism will recopy it for ever without effort with each one of its cell divisions. This genetically modified micro-organism could be long-term preserved by simple freezing and be awaked by simple culture in adapted conditions. The surplus of micro-organism, if necessary, could be easily destroyed by a simple chemical treatment, like bleach, or by incineration, without giving place to pollution. The biological factories thus open large potentialities by allowing the synthesis of a large range of molecules of interests of a great complexity, at a lower cost, without environment alteration and with a great purity.

In addition to the remarkable ingeniousness of this biotechnological approach, this work is also characterized by the audacity which was required, in particular on behalf of the industrial partner who initiated this project a dozen years ago, at one time when few biotechnological tools were available and genome sequences not yet available. This adventure, which now takes profit of the very last fundamental knowledge available, is also a remarkable example in the manner whose the French public research organization CNRS, that is primary directed towards basic research, can join in partnership with industry in order to allow the work of its researchers to be beneficial also the economic and the human health domains. The genetic engineering work published in the Nature Biotechnology February issue is currently the most complex pathway engineering work ever reported in the world and illustrate the place that France can hold in this highly competitive field of biotechnologies. It shows also how much strong commitment to support and develop public research organisms could be significant for the competitiveness of our economy, while making it possible to our companies to have access to teams of high levels in the fields of biotechnology and health.

References :

Florence Ménard Szczebara ,Cathy Chandelier, Coralie Villeret, Amélie Masurel, Stéphane Bourot, Catherine Duport, Sophie Blanchard, Agnès Groisillier, Eric Testet, Patricia Costaglioli, Gilles Cauet, Eric Degryse, David Balbuena, Jacques Winter, Tilman Achstetter, Roberto Spagnoli, Denis Pompon and Bruno Dumas. Biosynthesis of hydrocortisone from a simple carbon source in yeast. Nature Biotechnology à paraître dans le numéro de février 2003.
Link to the article

Duport C., Spagnoli R., Degryse E. et D. Pompon. 1998. Self-sufficient biosynthesis of pregnenolone and progesterone in engineered yeast. Nature Biotechnology, 16 (2), 186-189.

Authors Contacts :

Florence Ménard Szczebara, Cathy Chandelier, Coralie Villeret, Amélie Masurel, Stéphane Bourot, Catherine Duport, Denis Pompon completed the work presented in the Laboratoire d'Ingénierie des Protéines Membranaires du Centre de Génétique Moléculaire, au CNRS, à Gif-sur-Yvette, France (F-91198).

Sophie Blanchard, Agnes Groisillier, Eric Testet Patricia Costaglioli completed the work presented at the Higher School of Technology of Biomolecules of Bordeaux 2 (ESTBB), Université Victor Segalen, Bordeaux 2, 146 rue Léo Saignat, F-33076 Bordeaux, France.

Gilles Cauet, Eric Degryse, Tilman Achstetter completed the work presented at the company Transgene, 11 rue de Molsheim, F-67082 Strasbourg, France.

Jacques Winter, Roberto Spagnoli and Bruno Dumas completed the work presented at Aventis Pharma, Lead Discovery Technologies, 102 Route de Noisy, F-93235. Romainville, France and in Aventis-Pharma, Functional Genomics, 13 Quai J. Guesdes, F-93400 Vitry-sur-Seine, Cedex, France.

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