Ladies and Gentlemen,

I am honoured to speak to you on this special occasion. In my lecture, I will try to link three seemingly unrelated subjects: our planet, a humble micro-organism and the fuel indicator of your car. Let’s start with our planet.

Industrial Biotechnology: Drive My Car!

Jack Pronk

Department of BiotechnologyFaculty of Applied Sciences

164th Dies NatalisJanuary 13, 2006

Over the past century, our economy and our way of life have become heavily dependent on oil. This petrochemical carbon cycle is a simple representation of our oil-based economy. At first glance, this cycle looks reassuringly sustainable. Its energy source, the sun, is virtually inexhaustible. Moreover, any oil-based product, be it a plastic bottle or car fuel, releases carbon dioxide at the end of its life cycle. In principle, this carbon dioxide can once again be fixed by plants to produce new fossil fuels. The intrinsic problem of this cycle is related to time. The geological processes responsible for oil formation take aeons, yet the existing reserves will only last us, at best, a couple of centuries. Moreover, because carbon fixation by plants cannot keep up with our rate of oil consumption, carbon dioxide levels in

the Earth’s atmosphere are steadily increasing.

τ = 106-108 y

τ = 101-102 y

The Petrochemical Carbon Cyclesunlight

mining

combustion

geologicalprocesses

plantbiomass

CO2

photosynthesis

fuelschemicals

geological reservoirs

oilrefinery

chemical industry

Ladies and gentlemen, as you are no doubt aware, this increase of atmospheric carbon dioxide levels correlates with an alarming rise in the global temperature. But global warming is not the only challenge associated with an oil-based economy: As exploration for new oil reserves struggles to keep up with oil consumption rates, ever more people are claiming their share of the world’s oil reserves. What is more, the uneven distribution of oil reserves over the planet gives rise to serious geopolitical concerns for societies that lack their own reserves. These issues provide strong incentives for seeking alternatives to our current oil-based economy.

Challenges Resulting from an Oil-Based Economy

1000 1500 2000Year

CO2 concentration

Global Temperature

A logical first step would be to eliminate fossil reserves from the cycle. If we could base industrial production on agriculture or forestry, this would enable us to balance carbon dioxide emission and carbon fixation by plants. As a result, the carbon dioxide coming from our car exhausts would be fixed again by plants in the next growing season, thereby preventing a net accumulation of carbon dioxide in the atmosphere. Industrial biotechnology tackles this problem by converting plant biomass into simple building blocks, such as sugars, using a process called biorefinery. It then uses micro-organisms to convert these building blocks into all the products that we currently make from oil. This application of micro-organisms will form the focus of my lecture today.

sunlight

plantbiomass

CO2

agriculture,forestry

fuelschemicals

combustion

Balancing the Carbon Cycle: Industrial Biotechnology

sugars

biorefinery

industrialmicrobiology

In industrial biotechnology, bacteria, yeasts and fungi are used as miniature chemical factories that convert sugar into a product of interest. This ‘cell factory’ concept is by no means novel. As I speak, micro-organisms are being put to work in the manufacture of a wide range of food products, pharmaceuticals and fine chemicals. In this role, these minute organisms, less than a few micrometres in size, are making decisive contributions to our quality of life.

Feedstock(sugar)

Product

Industrial Biotechnology:Micro-organisms as ‘Cell Factories’

Food PharmaceuticalsChemicalsFuels

BacteriaYeastsFungi

As in all living organisms, the blueprint for microbial cell factories is encrypted in their DNA. DNA is organised in discrete units of information that we call genes. Each gene contains the information that is needed to make a single, unique protein molecule. These protein molecules are the real ‘workers’ in the cell factory. Protein molecules determine the structure of cells, enhance chemical reactions inside cells and regulate a multitude of cellular processes. Recombinant-DNA technology has enabled us to introduce very precise changes in the DNA, with the aim of improving the performance of cell factories. This technological discipline is known as metabolic engineering.

DNA: the Blueprint of Cell Factories

Gene(DNA)

Protein (enzyme)

StructureCatalysisRegulation

Metabolic Engineering:targeted improvement of catalysis and regulation in living cells via recombinant-DNA technology

Even a simple micro-organism contains thousands of genes, which in turn enable it to make thousands of proteins. Together, these thousands of proteins form complicated networks of biochemical processes. In this simplified diagram of a cell factory, the dots indicate small molecules, while the lines represent chemical reactions that are enhanced by specific proteins. Before recombinant-DNA technology can be successfully applied to change these complicated networks, we need to know which genes to target: in other words, we need to understand the networks in detail. Given the huge economic importance of industrial biotechnology, research in this field is fiercely competitive. Fortunately, Europe is in a promising position to help shape developments in industrial biotechnology, thanks to a strong research tradition in this field and the existence of successful partnerships between academia and industry.

Carbohydrates Various

Nucleotides

Glycolysis

Amino acids

Lipids

Energy

Amino acids

Lipids

Understanding Industrial Micro-organisms:Simplified Scheme of a Cell Factory

Strong position

This is my favourite micro-organism, as seen through an electron microscope. These beautiful budding cells are known as baker’s yeast or by their Latin name, Saccharomyces cerevisiae. Baker’s yeast has been used in the production of bread and beer since the time of the Ancient Egyptians. A more recent milestone was reached in 1996. In that year, a large international research effort, with a decisive input from a major European consortium, culminated in the complete elucidation of the DNA sequence of baker’s yeast. Today, baker’s yeast is a truly multi-purpose cell factory, with products ranging from fine wines to life-saving pharmaceuticals. To give you just one example, half of the global production of insulin is currently made using genetically modified baker’s yeast. My focus today is on a baker’s yeast product with massive potential for reducing our carbon dioxide emissions: fuel ethanol.

Bakers’ yeast (Saccharomyces cerevisiae):A Work Horse of Industrial Biotechnology

< 2000 BC used for making alcoholic beverages

1996Complete DNA sequence (genome)

6571 genes

ProductsBread, wine, beer

FlavoursPharmaceuticals (insulin!)

ChemicalsFuel ethanol

Ethanol is a normal product of yeast – baker’s yeast produces the alcohol that we consume whenever we drink a glass of beer or wine. Surprisingly, this same simple molecule is eminently suitable for use as car fuel. This is not just theory: Brazil and the United States, in particular, have successfully implemented ethanol in an existing transport-fuel infrastructure. It can be blended with ordinary gasoline and used with minimal modifications to car engines. Most importantly: as a biotechnological product, ethanol is made from renewable agricultural resources and not from oil. Its use can therefore make a substantial contribution to reducing carbon-dioxide emissions.

Ethanol as a Car Fuel

Compatible with existing infrastructure and enginesBlends with petrol (0-100 %)

Extensive experience in Brazil, USAAgricultural feedstocks: closed carbon cycle

C2H5OH

In 2003, the European Union underlined the need to increase the contribution of biofuels to our transport-fuel budget. A specific EU target has been set to increase the contribution made by these fuels to 5.75% by the end of 2010. At current levels of transport-fuel consumption, this corresponds to a bioethanol requirement of 30 million tonnes per year. Is such a figure realistic? To answer this question, it is worth taking a look at the current global production of ethanol.

DIRECTIVE 2003/30/EC, May 8th, 2003

Member States should ensure that a minimum proportion ofbiofuels and other renewable fuels is placed on their markets,

and, to that effect, shall set national indicative targets.

Biofuels: a Growing Sense of Urgency

A reference value for these targets shall be 5,75 %,calculated on the basis on energy content, of all petrol and diesel for transport purposes placed on their markets by 31 December 2010

At current transport fuel consumption rates,this corresponds to 30 million tonnes of

fuel ethanol per year

The current worldwide production of ethanol amounts to approximately 40 million tonnes per year and it is growing rapidly, especially in the United States and Brazil. So, by expanding the current production of ethanol, it may well be feasible to meet EU targets. But is this really the way to go forward? Let’s examine the current processes for ethanol production. Without exception, current industrial production of ethanol starts with agricultural crops that are rich in starch or sucrose. After harvesting, the starch fraction is split into small sugar molecules, which are then converted to ethanol by our little friend Saccharomyces cerevisiae.

Current Ethanol Production: ca. 40 Million Tonnes/y

starch, sucrose

glucose, fructose

hydrolysis

ethanol

S. cerevisiae(bakers’ yeast)

fermentation

A major drawback of the current technology is that it uses only a small fraction of the agricultural crop. This is exemplified by corn stover, the plant material that is left in massive amounts after the corn harvest. Similar agricultural residues are derived from many other crops. If we could only tap into these abundantly available agricultural residues, we could achieve an enormous expansion of the feedstock availability for ethanol production. At the same time, this would enable us to avoid a potential conflict of interests between using agricultural resources for food production and for fuel production. The technological challenge is that these agricultural residues do not contain starch, a compound that can be easily split into sugars and converted to ethanol. Instead, they are rich in cellulose and hemicellulose. These plant polymers also consist of sugars, but they are not as easy to convert into ethanol.

Crop residues:Abundantly available

No competition with food production(Hemi)cellulose instead of starch/sucrose

Current Technology: No Utilization of Crop Residues

‘corn stover’

The strategy envisaged for converting agricultural residues into ethanol starts by pre-treating the plants to make them accessible for further processing. Subsequently, enzymes produced by micro-organisms are used to split cellulose and hemicellulose into simple sugars. These are then converted into ethanol by baker’s yeast. Or at least, that is the plan. As is so often the case, the real-life situation is a bit more complicated. This has to do with the types of sugar that occur in agricultural residues.

Fuel Ethanol from Crop Residues: Strategy

Crop Residues

CelluloseHydrolysisPretreatment FermentationSugar

Mixture

Lignin

Ethanol

Combustion

Heat, electricity

When agricultural residues such as corn stover, wheat straw or bagasse (a residue of sugar cane) are split into simple sugars, this does indeed yield sugars that can be converted into ethanol by baker’s yeast. However, these residues also contain sugars that cannot be converted by this yeast. By far the most important of these is xylose or wood sugar. Unless we can make ethanol from xylose, producing ethanol using agricultural residues simply doesn’t make economic sense.

Non-fermentable by S. cerevisiae

Sugars in Crop Residues

23.116.917.7lignin

Other (%)2.22.23.2uronic acids

2.12.42.5arabinose

22.119.219.3xylose

0.50.81.0galactose

0.40.30.4mannose

39.032.634.6glucose

Sugars (%)BagasseWheat strawCorn stover

So, what we’d like to do is design an optimised baker’s yeast that does convert xylose to ethanol – a typical challenge for a metabolic engineer. I have been warned not to make things too complicated for you, but I cannot resist the temptation of exposing you to some biochemistry. At least the biochemistry of xylose metabolism in baker’s yeast is simple: nothing happens. However, baker’s yeast is able to convert xylulose, a sugar that not only sounds similar to xylose, but also has a very similar structure. The DNA of baker’s yeast harbours 5 genes that together enable it to convert xylulose into compounds that can be fed into the high-capacity biochemical pathway for ethanol production. The first task, then, seems very simple: introduce a gene that enables baker’s yeast to convert xylose into xylulose. A protein that does exactly this, called xylose isomerase, is found in many bacteria but not in yeast, and many attempts to introduce bacterial genes for xylose isomerase into baker’s yeast have proved unsuccessful. The bacterial genes simply refused to work in yeast. So, the hunt was on for a xylose isomerase gene that

would work in baker’s yeast. The answer came from an unexpected source.

Biochemistry of Xylose Metabolism in S. cerevisiae

xylose

‘Missing Link’:Xylose Isomerase

• absent from S. cerevisiae• bacterial XI genes do not

function in yeast

xylulose

C5P

C3P + C6P

‘normal’sugar metabolism

ethanol

5Yeast genes

This, ladies and gentlemen, is an Indian elephant. Our esteemed colleagues at Nijmegen University were fascinated by micro-organisms that inhabit the elephant’s digestive tract. In elephant dung, they discovered a new fungus, capable of degrading many plant materials present in the elephant’s diet. To their surprise and ours, part of the DNA of this fungus bore a striking resemblance to bacterial xylose-isomerase genes – a gene of this kind had never before been found in a fungus. Might this novel gene be the answer to our problems? Indeed, when we introduced the fungal gene into baker’s yeast, we found a very active xylose isomerase in the genetically engineered yeast. The time had finally come to start designing and constructing a strain of baker’s yeast capable of efficiently converting xylose.

Exploring Biodiversity: a New Xylose Isomerase Gene

Research at Radboud University, Nijmegen, The Netherlands:

• Piromyces fungus from elephant dung• Fungus has gene for xylose isomerase (XylA)• Fungal gene works in yeast!

Using the recombinant-DNA toolbox, we first introduced the fungal xylose-isomerase gene into baker’s yeast. Subsequently, we strongly increased the activity of the 5 genes that link xylulose to the normal ‘highway’ of ethanol production. For good measure, we also eliminated a gene that might contribute to the formation of a small amount of byproduct. I would like to stress that experiments like these were only made possible by considerable investments in fundamental research, including the completion of the yeast genome sequence 10 years ago. The results exceeded our wildest expectations: the genetically engineered yeast converted xylose into ethanol rapidly and efficiently. However, before industrial implementation could be considered, the engineered yeast still had to be optimised a little further.

xylose

glycerol

CO2ethanol

Metabolic Engineering of Xylose Conversion Overexpression of 5 yeast genes & removal of a sixth gene

6 xylulose

6 C5P

2 C3P + 4 C6P

6 xylose

‘normal’sugar metabolism

byproduct

ethanol

For this further improvement, we turned to evolution in the laboratory. This may sound strange: the word ‘evolution’ tends to conjure up associations with gradual changes that, almost literally, take forever. However, under optimal conditions, the generation time of yeast cells is only a couple of hours. This enables us to observe evolution in the laboratory and to harness it to improve microbial processes. To improve xylose conversion, we simply provided our genetically engineered baker’s yeast cells with xylose as their only food source. This was done by taking a little bit of yeast culture that had eaten all its xylose and transferring it to a culture with fresh xylose. By repeating this procedure for a couple of months, we obtained spontaneous mutants with dramatically improved xylose conversion rates.

Further Improvement: ‘Evolution in the Lab’

Generation time~ 20 years

Generation time~ 2 hours

54

3

2

1

33 x

This combination of metabolic engineering and ‘evolution in the lab’ has now led to baker’s yeast strains that effectively convert mixtures of sugars similar to those occurring in plant biomass hydrolysates. Of course, we have taken the necessary steps to protect our findings with patent applications. Throughout this research, we have collaborated intensively with industry, and in particular with Royal Nedalco, a Netherlands-based ethanol producing company. Collaboration with industry is also crucial to the next phase, the rapid transfer of this new technology to large-scale industrial production of fuel ethanol. I am happy to report that this phase is now in full swing.

CO2

ethanol

xylose

glucose

Efficient Ethanol Production from Sugar Mixtures

Patent applicationsResearch with industry on large-scale implementation

Ladies and gentlemen, I would like to leave you with a number of conclusions. I am convinced that industrial biotechnology can and will make significant contributions to sustainable industrial production. It is a pleasure to work in a university that fully recognises the unprecedented opportunities for academia, industry and society offered by the rapid developments in the area of life science and technology. Finally, to return to the title of my presentation, industrial biotechnology will, in the very near future, drive my car … and yours too.

Conclusions

Industrial Biotechnology will make important contributions to sustainableproduction

Genomics and Metabolic Engineeringoffer unprecedented opportunitiesfor process design and optimization

Bioethanol will Drive My Car

...and Yours!

Thank you for your kind attention.