Breaking phylogenetic barriers for fine and bulk chemical products in engineered Saccharomyces cerevisiae

Industrial biotechnologies allow today to obtain both fine and bulk chemicals and yeasts as cell factories can produce many products belonging to both field (Branduardi et al., 2008, Porro and Branduardi, 2009). Among yeasts, Saccharomyces cerevisiae still represents the microorganism of election to develop such cell factories. As regard bioethanol production, yeasts utilization is well established for its natural fermentation ability, but new generation biofuels require the development of strain more and more robust, able to face conditions imposed by the process that should be as cheaper and more profitable as possible. In this direction, processes that use lignocellulosic feedstock for bioethanol production (the so called second generation biofuels) are still in development (NEMO project-Novel high performance Enzymes and Micro-Organisms for conversion of lignocellulosic biomass to ethanol, seventh framework program). This means that the cell factory has to be deeper and deeper investigated in structures and pathways trying to find good targets for improving the robustness. Among the many different functions, cell cytoplasmic membrane plays a key role in cell homeostasis and is deeply involved in facing and reacting to stressing conditions, so it can represent a good target for direct improvements of laboratory as well as industrial strains. Fishing in Arabidopsis thaliana genome, a potentially interesting gene was found, codifying for a Temperature Induced Lipocalin, named TIL. As it has positive effects in plants in unfavourable situations (Charron et al., 2008), TIL was expressed in S. cerevisiae and the recombinant strains compared with the parental counterpart under some of the stresses typical of industrial processes. The recombinant yeasts show an increased tolerance towards heat shock and to the presence of hydrogen peroxide and organic acids. In detail TIL expressing strain generate lower levels of ROS and accumulate less amounts of reactive electrophile species generated after membrane lipids fragmentation. Another industrial field that is gaining more and more importance is represented by bioplastics not coming from petrochemical sources. Vegetable oils derived fatty acids are interesting as bulk compounds for the synthesis of biopolymers, even if they have to be previously modified to possess two chemically reactive groups at molecules extremities. High catalytic activity and stability together with high versatility in reaction of performed by bacteria cytochromes answered to this need. The heterologous expressed B. subtilis cytochrome CYP102A2 in S. cerevisiae showed some activity, measured in terms of NADPH consumption towards fatty acids of different chain length, interestingly also towards short chain fatty acids. However as CYP is able to catalyze different type of reaction involving NADPH consumption (hydroxylation, oxidation and epooxidation as example), the products will be further characterized to understand what kind of modifications are carried out on the tested substrates. Considering the valuable reactions that cytochromes P450 are able to catalyze on a vast variety of substrates (fatty acids, steroids and a multitude of non natural compounds such as drugs, organic solvents and hydrocarbon products), their successful expression in yeast could open the possibility to develop sustainable processes in alternative to classical chemical synthesis. Because of the nice and positive results, S. cereviasiae potential as cell factory was deeper exploited for the expression of a whole plant biosynthetic pathway. In detail, yeast was engineered to express the pathway leadind to the formation of glucobrassicin, a nutraceutical indicated as a potential cancer chemoprotective agent. In this work we describe the construction of a recombinant S. cerevisiae strain able to produce glucobrassicin. Despite some investigation about possible strain optimization through the employment of multicopy plasmids, the final producer will exploit only integrative vectors and the described findings and the process of production were deposited as patent application [Mauro Magnani, E. Bartolucci, Danilo Porro, Paola Branduardi, Vera Codazzi, Umberto Benatti, Gianluca Damonte, Giovanni Schippa e Stefano Bianchini Sviluppo di una cell factory ricombinante per la produzione di Glucobrassicina]. Specific analysis on biosynthetic intermediates suggest steps on which focusing the attention to further improve the glucobrassicin production levels. Despite the number of biotechnological processes based on engineered microorganisms for the production of metabolites is still limited in comparison with the potentiality expressed at lab scale, the studies about strain robustness and heterologous pathway optimization are going to change that situation very soon.