Abstract
(Englisch)
|
The contribution of our group to this project focuses on the short term flux effects at the pyruvate branch of carbon metabolism in S. cerevisiae. In particular we want to channel metabolite flux by over expression of pyruvate carboxylase and to isolate mutant strains showing instant availability of higher oxidative capacity.
Research on the metabolism at the pyruvate branch point is of academic as well as industrial interest This is to prevent or at least minimize the formation of undesired 'by-products' ethanol, acetate and acetaidehyde in processes for baker's yeast production, but also for 'modem' heterologous protein expression. The three main enzymes operating at this branching point are pyruvate decarboxytase, the pyruvatedehydrogenase complex, and pyruvate carboxylase. While the first two are considered 'catabolic' enzymes, the latter is an 'anabolic' enzyme, replenishing the TCA cycle for biosynthetic precursors. What renders the task particularly difficult is the fact that pyruvate directly or via other metabolites needs to be transported into the mitochondrial compartment to be metabolites further in the TCA cycle. Since these transporters are not known so far their control on metabolic flux is not assessable.
To channel the metabolic flux away from pyruvate decarboxylase into the 'pyruvate bypass' we studied the effect of the over-expression of pyruvate carboxylase and of a heterologous phosphoenolpyruvate carboxylase from E. coli (obtained from C. Gancedo, Madrid).
For this approach the Yeast PYC2 gene and the E. coli PPC gene were over-expressed under the control of the constitutive Yeast ADH1-promoter in a multicopy 2 my vector in CEN.PK113-7D ura3 'wildtype' and a CEN.PK113-7D ura3Dpyc1 Dpyc2 mutant, respectively. Stable 4-fold increases were observed for pyruvate carboxylase and 400 mU/mg protein for phosphoenolpyruvate carboxylase. These constructs turned out to be more efficient than the earlier reported constructs with the single copy integrated GAL1-promoter constructs, which were not considered for further studies. As reported earlier, the clear but limited over-expression of the corresponding genes is probably also due to posttranslational factors, since Northern analysis showed significantly higher increases of transcription.
In extension of the accelerostat experiments that failed to show higher maximal oxidative growth rates in the over-expression strains as compared to the wild type, a new type of experimental set-up was chosen.
In a first experimental senes, steady-state carbon-limited chemostat cultures growing at a dilution rate of D=0.1 (in 'Verduyn'-medium with 12.5g/l of glucose) were pulsed 5,20 and 50mM glucose, respectively. The transient accumulation of ethanol and acetate in the medium were measured. The results unequivocally showed that none of the glucose-pulse concentrations led to a difference between wild type cells and the mutants over-expressing pyruvate carboxylase and phoshoenolepyruvate carboxylase, respectively.
To investigate this further, the same experimental set-up was used to test also internal metabolite accumulation. Glucose pulses of 5mM were given steady-state carbon-limited chemostat cultures growing at a dilution rate of D=0.1 and cells were quenched and extracted according to a method as developed by Rizzi and Reuss in the frame of this project. The results obtained showed again that the metabolic flux into the 'pyruvate bypass' was not influenced by the over-expression of pyruvate carboxylase and phoshoenolepyruvate carboxylase, respectively. In addition to that it was found that neither the fluxes via cytoplasmic toward asparaginelaspartate, nor via mitochondrial oxaloacetate toward citrate, were enhanced. However it was found that flux via cytoplasmic oxaloacetate toward malate was increased as indicated by significantly increased internal malate levels in both mutant strains. These data, in conclusion, indicate that the 'overflow reaction' at the pymvate branch point could not be significantly influenced by up modulating pyruvate carboxylase and phoshoenolepyruvate caboxylase, respectively. Flux increases appear to take place exclusively in the cytoplasm toward malate.
As a secondary part of the project, mutants showing instant availability of higher oxidative capacity were searched for. The mutant screen for suppressors of the growth defect of the Dadh1 mutant on high glucose concentrations led to the isolation of six recessive suppressor mutants showing increased oxidative activity under these conditions under the same conditions, the maximal growth rate at all the mutants was reduced to about 10-20 % at the wild-type level, however, making the direct usefulness questionable.
To investigate the role of glucose repression for the oxidative capacity of Seccharomyces cereVisiae further, targeted deletions of genes involved in glucose repression, MIG1, HXK2, ROX1 and REG1, respectively, were made. Study of oxygen consumption rates in these mutants at repressing glucose concentrations showed that whereas MIGI deletion had no effect, HXK2 and REGI deletions, respectively, led to an increase of specific oxygen consumption. The ROXI deletion mutant showed reduced oxygen consumption rate compared to the wild-type strain. These results indicate that oxidative capacity in Sacchammyces cerevisiae is controlled by glucose repression and confirm previous findings, showing that HXK2 and REG1 gene products play a role in glucose repression of genes involved in respiratory metabolism. The results as elaborated so far do not allow making the prediction that these mutations are of general use for the construction of industrially interesting strains.
|
