BEORA - Biotechnology exploitation of orotic acid production

摘要

The gnon-conventionalh yeast Kluyveromyces lactis has become increasingly attractive for the industry and has been successively used in biotechnology mostly for the production of heterologous proteins. In this study, we demonstrated that the Klura3Delta and Klura5Delta mutants of the yeast K. lactis are able to produce and secrete into the growth medium significant amounts of orotic acid, an intermediate of the de novo pyrimidines biosynthetic pathway. Using yeast extract - peptone - glucose (YPD) based media, we optimized production conditions in flask and bioreactor cultures. With cells grown in YPD 5% glucose medium, the best production in flask was obtained using a 1 : 12.5 culture volume / flask volume ratio, 180 rpm, 28 oC and 200 mM MOPS for pH stabilization at neutral values (initial culture pH at 8.0). The best production in a 2 L bioreactor was achieved at 500 rpm with 1 vvm aeration, 28 oC and pH 7.0. Under these optimum conditions, similar rates of orotic acid production were obtained and maximum concentration achieved after 96 h was 6.7 g/L in flask and bioreactor cultures. These results revealed an excellent reproducibility between both systems and provided evidence for the biotechnological potential of Klura3Delta and Klura5Delta strains since the amounts obtained are comparable to the production in flask using a similar mutant of the industrially valuable Corynebacterium glutamicum currently used in industry to produce orotic acid.;We have also established the grounds for the development of a low cost production media for industrial scale-up. A media containing 4% (v/v) corn steep liquor (CSL), 3.4% (v/v) molasse, 0.25% (w/v) urea and 200 mM MOPS for pH stabilization at neutral values (initial culture pH at 8.0), which only had a total of 2% (w/v) sugars, allowed the production of 4.5 g/L of orotic acid after 80 h. CSL proved to be very promising as the main low cost constituent of the production media. However, it contains inhibiting factors for the production of orotic acid, thus limiting the concentration of CSL that can be used in the media. Before further development of the industrial media, the inhibition caused by CSL should be first addressed to reduce or eliminate its effects in order to take full advantage of CSL benefits.;A putative link between the pyrimidines biosynthetic pathway and mitochondria led us to assess changes in the oxidative stress markers in the orotic acid producing mutants. Our results show that at latter stages of growth, catalase activity increased in pyrimidines mutants of K. lactis with the interruption at any step of the de novo pyrimidines pathway. Depending of the growth phase, the Klura3Delta mutant displayed a distinct or a more marked phenotype compared to other pyrimidine mutants, suggesting different mechanisms may be involved in this particular mutant. Notably, at latter stages of growth, the high catalase activity exhibited by Klura3Delta cells was correlated with an increased resistance to H2O2. Moreover, deletion of any of the dihydroorotate dehydrogenase genes (DHODases; Klura1Delta and Klura9Delta) in Klura3Delta was shown to alter its phenotype, indicating a possible connection of the de novo pyrimidines pathway with oxidative stress response that involves Klura3p, Klura1p and Klura9p.