Optimization Of Fission Yeast Fermentation And Application To Biotechnological Processes

Biotechnology offers a useful toolbox to industry for the production of various chemical compounds.Biotransformation and bioproduction processes are preferred to chemical one for a number of reasons,including mild operative conditions(interesting from a cost-effective point of view),the easiness to apply recombinant DNA technology to make the desired improvements in microbes involved in this process and in some specific cases the possibility to achieve regio-,stereo-and enantio?selective reactions at non?activated positions.The fission yeast Schizosaccharomyces pombe is a facultatively fermentative yeast showing both a short-and long-term Crabtree effect and was originally discovered in an East African millet beer.In recent decades,this microbe has increasingly been used for the recombinant expression of proteins with a view towards biotechnological applications.Recombinant fission yeast strains are used for whole-cell biotransformations of xenobiotics.Moreover,non-recombinant fission yeast strains can also be used for some applications such as brewing,baking,and winemaking.Invariably,biotechnological applicability of promising strains requires scale-up from benchtop shaking flasks to fermentors.Thus,the availability of robust and reproducible fed-batch fermentation protocols for S.pombe is of interest.It was previously reported on the fed-batch fermentation of recombinant fission yeast strains for the production of human lysosomal acid lipase,ubiquinone,and antigens for in vitro diagnostic testing,as well as for the production of metabolites of drugs,doping compounds,and steroid hormones.This research work is divided into two major parts:the design of a reproducible fermentation fission yeast protocol and the synthesis of a long-term doping compound metabolite.The first aim of this project was to develop an open-loop fed-batch protocol for highly reproducible fermentation of fission yeast that starts from batch cultures instead of glucose-limited aerobic chemostat cultures.Reproducibility is an important aspect of process optimization.A new strategy was employed in this research consisting of an exponential feeding phase followed by a starvation period and then a linear feeding phase.A comparison of three independent fed-batch fermentation using JMN8 recombinant fission yeast strain showed that while during the initial phase process parameters such as glucose consumption and CO₂evolution varied considerably as expected,they were much more uniform during the third phase.For instance,the normalized standard deviation of glucose consumption was thirty times higher during the exponential feeding phase of the fermentation during the linear feeding phase.These data demonstrated the usefulness of the proposed strategy.Besides,it was shown that by variation of only two parameters(the total amount of glucose fed in the initial phase and the time frame of the starvation phase)the protocol was easily adapted to other strains.The second investigation line in this project was focused on the metabolization of a popular doping compound.Doping compounds are frequently used in sports competitions to enhance athletes'performance.One of the most abused substances in sports competitions since 1960 is Cl MT(4-chloro-17amethyl-androst-4-en-17bol-3-one,brand name Methyl Clostebol).Long-term doping metabolites are incontestable proof of doping abuse in sports competitions.It was previously reported that DHCMT's metabolite,a doping compound with a similar chemical structure to Cl MT,can be detectable for more than 22 days in urine samples.The synthesis of Cl MT long-term metabolite(17?OH-Nor Cl MT)was then the aim of this second part.An initial chemical step(Wagner-Meerwin rearrangement)and a second biotechnological step(whole-cell biotransformation)were employed.Whole-cell biotransformation was carried out using CAD75 fission yeast strain to express CYP21A2human enzyme using the reproducible fermentation protocol developed in the first phase of the project.The whole-cell biotransformation was also performed at lower p H during the constant feeding phase achieving higher yield.In summary,a new fission yeast fermentation protocol was designed to achieve a reproducible glucose consumption rate during the fermentation process.Also,the synthesis of the long-term metabolite 17?OH-Nor Cl MT was achieved and its production was successfully improved in this study.