“The domestication of the Saccharomyces cerevisiae yeast is the result of a long evolution that takes us back to the origin of plant evolution, more than 10,000 years ago,” explains Renaud Toussaint. “With the development of agriculture, humans became more sedentary, developing fermentation techniques to transform grains into bread and also into fermented drinks. Slowly, and in an empirical way, they succeeded in improving the use of these microorganisms.” The active selection of interesting new strains remained, for a long time, empirical and based on observation.
Progressively, the intuitive approach made way for a scientific approach to enable the isolation of increasingly efficient strains. During the first part of the 20th century, the discovery of the laws of heredity and of deoxyribonucleic acid, or DNA, as a medium for this heredity, enabled the development of a new science, genetics. “In 1996, Saccharomyces cerevisiae became the first eukaryotic organism to have its genome sequenced,” recalls Didier Colavizza, Doctor es sciences and head of Biotech Center in the Lesaffre R&D department. At the time, 20 laboratories and 150 researchers had joined forces for 10 years to achieve this advance. Lesaffre invested heavily in this by co-participating the Yeast Industrial Platform. The genome of a yeast extends across 12 to more than 50 million base pairs. “Enormous calculating power was required to assemble all the data produced by the sequencing, to annotate the genomes, and to compare them to each other,” stresses Dr. Colavizza. “Today, with the evolution of techniques, we sequence yeast genomes in our R&D center in less than one week and for less than 75 euros.”
Thus, all industrial strains used at Lesaffre have been sequenced. However, in nature, “a very small number of microorganism species have been identified,” reveals Renaud Toussaint. At Lesaffre, the strain library contains a collection of 12,250 microorganisms (yeasts, bacteria, and filamentous fungi) which is continually being enriched. While 90% of this yeast collection features yeasts of biotechnological interest close to Saccharomyces cerevisiae, our microbiology team is now keen to explore other ecosystems to expand and diversify this collection. “Selecting unconventional yeasts constitutes an important part of our work,” states Renaud Toussaint. “These are the yeasts present in the environment, the potential of which remains very poorly realized, and which remain a source of biodiversity and new functionality.” Using genetic techniques, these strains can in fact transmit specific supplementary characteristics to our industrial yeasts and thus generate future products.
From classic genetic techniques to genetic engineering
In bread-making, classic genetic techniques enable the selection of yeasts giving new aromas or enhanced efficiency. “The main techniques used is based on the natural ability of the yeasts to be cross-bred. This techniques, also called hybridization, consists of creating offspring from two individuals with complementary characteristics, in order to obtain yeasts that combine these two characteristics. The exposure of yeasts to mutagenic agents (such as ultra-violet rays) generates randomized mutations in the genome. Mutated strains are then selected according to the characteristic that we wish to prioritize,” outlines the microbiology R&D manager. Another technique uses the yeast’s ability to accumulate mutations and select yeasts with improved properties.
Genetic strategies have evolved at the same pace as the evolution of understanding our strains’ genomes and have thus enabled new approaches similar to quantitative genetics. “The principles of these improvement techniques remain the same, but these techniques are now used on a much larger scale,” describes Renaud Toussaint. “Previously conducted from a few hundred of strains, the selection programs are now carried out on several tens or hundreds of thousands of strains. The use of robotics screening platforms make it possible to rapidly isolate the best individuals from a large starting population and thus constantly feed our flow of new and interesting candidates that will make the products of tomorrow.” These high-speed screening programs, for which read very high-speed, would not be possible without the support of new key skills, such as biostatistics or bioinformatics.
Nonetheless, all these ‘classic’ genetic techniques present limitations. The mutations are random, do not always involve the desired characteristics and are sometimes tricky to reproduce. The polygenic character of technological characteristics makes it difficult to improve industrial yeasts.
To remedy this, genetic engineering offers several advantages. “It makes it possible to closely modify the DNA structure, to overexpress certain genes or to replace them with other genes, with new functions or that are drawn from other species.” Gene transfer techniques make it possible to intervene in a precise way on certain sequences of the genome. It therefore becomes possible to focus solely on certain improvement criteria, such as the increase in fermenting power, increased yield, the production of aromas or the resistance to inhibitors.
In our Leaf business unit, which specializes in the biofuel sector, the introduction of eight genetic modifications, some of which are derived from species other than yeast, has enabled the latter to produce ethanol from plant biomass waste. “Ordinarily, yeasts are not able to hydrolyze plant materials such as xylose, a sugar that contains five carbon atoms,” describes Renaud Toussaint. “With these genetic modifications, the yeasts become capable of producing enzymes hyrolyzing C5 sugars.”
Towards synthetic biology
However, sometimes these molecular genetic techniques require the insertion of selection markers, often genes resistant to antibiotics. The risk is that these pass to other organisms and spread into the environment.
In addition, more precise molecular techniques have been developed. This is the case with genome editing techniques such as Crispr-Cas9. “By managing the Cas9 protein, it is possible to induce a double-strand break in yeast’s genome DNA, at very precise points, to introduce genes favoring the production and yield of amino acids or specific proteins.” With this level of precision, it is possible to create new microorganisms producing metabolites such as S-adenosylmethionine or glutathione; new yeasts that can be used as probiotics for human, animal and plant health.
Better still, synthetic biology now offers the possibility of building genomes using computer-aided DNA synthesis techniques. This will soon mean it is possible to make genetic engineering simpler, faster and less costly thanks to the use of engineering principles such as standardization, automatization and bioinformatics. “This was the stuff of science fiction 30 years ago,” notes Dr. Colavizza, “However, in 2010 the first bacteria genome was recreated in an entirely synthetic manner by the Craig Venter team in the United States. And, in 2014, the same was achieved with the chromosome 3 in S. cerevisiae yeast.”
Moreover, a global project called ‘Yeast 2.0’ is ongoing. Its aim is to reconstitute the complete genome of the S. cerevisiae yeast. “By synthesizing in this way the genome of this yeast, it will now be possible to model it so that it produces aromas, enzymes or new antibiotics, according to the needs of populations,” explains Dr. Colavizza. “Such synthetic organisms could also be used as models for human diseases in order to identify therapeutic targets for new treatments.” In summary, concludes Renaud Toussaint, “Synthetic biology is part of the continued development of genetic engineering and is the start of a new era at Lesaffre, with new opportunities for all our activity sectors.”