Cofactor Engineering In Saccharomyces Cerevisiae

Redox cofactors play a pivotal role in coupling catabolism with anabolism and energy generation in the overall process of metabolism.There exists a delicate balance in the intracellular level of these cofactors to ascertain an optimal metabolic output.Engineering the level of metabolic cofactors to induce alternate regulatory pathways is emerging to be an attractive strategy for bioprocess applications.In the present work,we targeted cofactors,not only because of its high degree of connectivity in the metabolic network,but also because product formation is the consequence of redox homeostasis.We demonstrate that altering cofactors metabolism is a powerful strategy in affecting the carbon fluxes in Saccharomyces cerevisiae.NADH is predominantly produced in the catabolism of glucose and redox homeostasis is maintained by the action of NADH dehydrogenases.Since NADH cannot traverse the mitochondrial membrane,S.cerevisiae has two external and one internal NADH dehydrogenases.Glucose cannot be oxidized as fast as it can be consumed and a portion of it is fermented to ethanol.This phenomenon which is called Crabtree effect is often attributed to the inhibition of respiration.Ethanol production is a redox-neutral process and hence,does not contribute to NADH accumulation.The additional NADH generated from concomitant biomass synthesis is oxidized via glycerol production.NADPH has a greater role in anabolism as many of the reactions involved in the biosynthesis of amino acids,lipids and nucleotides use NADPH as the reducing agent.In S.cerevisiae,a majority of the NADPH is generated in the oxidative pathway of the pentose phosphate pathway,in the conversion of glucose-6-phosphate to ribose-5-phosphate in the cytosol. Mitochondrial NADPH is synthesized mainly from the NADP⁺-dependent isocitrate dehydrogenase in the TCA cycle.Besides these,there are other reactions that might produce NADPH.such as the NADP⁺-dependent acetaldehyde dehydrogenase.NADP⁺-dependant malate dehydrogenase.S.cerevisiae also has NADH kinase mediate the ATP-driven conversion of NADH into NADPH.During growth of Saccharomyces cerevisiae on glucose,the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis,respectively.However, the metabolism of xylose by recombinant S.cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH,and creates cofactor imbalance during growth on xylose.In this study,we demonstrated two possible solutions to overcoming this imbalance.One strategy is changing the cofactor specificity of XDH.The genes XYL2 (D207A/1208R/F209S) and XYL2(S96C/S99C/Y102C/D207A/I208R/F209S) were introduced to construct xylose metabolism S.cerevisiae,respectively.The specific activities of mutated XDH in both strains showed a distinct increase in NADP⁺-dependent activity. During xylose fermentation,the strain with XDH(D207A/1208R/F209S) had a large decrease in xylitol and glycerol yield,while the xylose consumption and ethanol yield were decreased. In the strain with XDH(S96C/S99C/Y102C/D207A/1208R/F209S),the xylose consumption and ethanol yield were also decreased,and the xylitol yield was increased,due to low XDH activity.The results showed that changing xylitol dehydrogenase cofactor specificity was a sufficient method for reducing the production of xylitol,but high activity of XDH and the high XDH/XR ratio were also required for improved ethanol formation.As another possible solution to overcoming cofactor imbalance,the effect of overexpressing the native NADH kinase(encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated.The physiology of the NADH kinase containing strains was also evaluated during growth on glucose.Overexpressing NADH kinase in the cytosol redirected carbon flow from CO₂ to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose.However,cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption,by channeling carbon flow from ethanol to xylitol.In contrast,overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent.Overall,although NADH kinase did not increase the rate of xylose consumption. we believe that it can be an important strategy to engineer the redox metabolism in yeast.Xylose metabolism is a typical example which is limited by cofactors.Beside it.we also presented a detailed analysis of the impact of perturbations in redox cofactors in the cytosol or mitochondria on glucose and energy metabolism in Saccharomyces cerevisiae to aid metabolic engineering decisions that involve cofactor engineering.First,we enhanced NADH oxidation by introducing NADH oxidase or alternative oxidase,its ATP-mediated conversion to NADPH using NADH kinase as well as the interconversion of NADH and NADPH independent of ATP by the soluble,non proton-translocating bacterial transhydrogenase.Decreased cytosolic NADH level decreased glycerol production,while decreased mitochondrial NADH decreased ethanol production.However,when these reactions were coupled with NADPH production,the metabolic changes were more moderated.The direct consequence of these perturbations could be seen in the shift of the intracellular concentrations of corresponding cofactors.Besides the cofactors,the concentration of other intracellular metabolites also varied to counteract the perturbations. These changes in product formation profile and concentration of intracellular metabolites were closely linked to the ATP requirement for biomass synthesis and the efficiency of oxidative phosphorylation,as estimated from a simple stoichiometric model for glucose metabolism to different products(biomass,CO₂,ethanol and glycerol).The results presented here will provide valuable insights for a quantitative understanding and prediction of cellular response to redox-based perturbations for metabolic engineering applications.Then,we demonstrated that increasing NADH level is a powerful strategy in affecting the carbon fluxes in Saccharomyces cerevisiae.In a S.cerevisiae strain that was disabled to consume formate,we overexpressed the native NAD⁺-dependent formate dehydrogenase either in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence from the CYB2 gene.Upon exposure to formate,the mutant strains readily consumed formate and induced fermentative metabolism even under glucose de-repressed conditions.Ethanol was the main by-product when formate dehydrogenase was directed into the mitochondria while we observed glycerol and some ethanol when it was overexpressed in the cytosol.Clearly.these results point towards strong compartmental regulation of redox homeostasis.By following the intracellular metabolite profiles during the pulse,we identified phosphofructokinase,glyceraldehyde-3-phosphate dehydrogenase andα-ketoglutarate dchydrogenase as the most sensitive enzymes to NADH perturbation.As an example,we demonstrated an effective cofactor engineering strategy and expressed NADH oxidase in Saccharomyces cerevisiae under the control of the glycerol pathway by GPD2 promoter to modulate the decrease in cytosolic NADH to the right level where the heterologous enzyme does not compete with oxidative phosphorylation while at the same time,decreasing glycerol production.The metabolic design eliminated glycerol production by 57%and did not decreased ethanol production.In the meanwhile,it increased the specific growth rate of S.cerevisiae by 14%and biomass yield by 9%.It indicated that the strategy optimize the NADH/NAD⁺ ration in S.cerevisiae.Although we demonstrated the amenability of cofactors in dictating product profile and metabolism profile only in S.cerevisiae,we believe that this concept could be used in other industrially relevant microorganisms as well.