Elsevier

Metabolic Engineering

Volume 6, Issue 4, October 2004, Pages 352-363
Metabolic Engineering

Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments

https://doi.org/10.1016/j.ymben.2004.06.002Get rights and content

Abstract

The yeast Saccharomyces cerevisiae is an attractive cell factory, but in many cases there are constraints related with balancing the formation and consumption of redox cofactors. In this work, we studied the effect of having an additional source of NADPH in the cell. In order to do this, two strains were engineered by overexpression of malic enzyme. In one of them, malic enzyme was overexpressed as its wild-type mitochondrial form, and in the other strain a short form lacking the mitochondrial targeting sequence was overexpressed. The recombinant strains were analyzed in aerobic batch and continuous cultivations, and the basic growth characteristics were generally not affected to a great extent, even though pleiotropic effects of the manipulations could be seen by the altered in vitro activities of selected enzymes of the central metabolism. Moreover, the decreased pentose-phosphate pathway flux and the ratios of redox cofactors showed that a net transhydrogenase effect was obtained, which can be directed to the cytosol or the mitochondria. This may find application in redirecting fluxes for improving specific biotechnological applications.

Introduction

The chemical industry is turning to biotechnology for the production of fine chemicals, fuels and materials. There are three main drivers for this: (1) the relative costs of crop based raw materials are decreasing; (2) biotech processes represent a green technology that generally has a lower environmental burden than classical chemical processes and this may in some cases favor biotech processes in terms of the overall process economy; and (3) the power of technologies offered by the rapid progress in the field of metabolic engineering and genomics. In the process of developing novel bioprocesses for production of chemicals it is generally desirable to develop generic cell factories that ensure a fast and efficient conversion of the raw material to the desired product. The yeast Saccharomyces cerevisiae is an attractive cell factory, due to the very well established molecular biology and fermentation techniques, together with its GRAS status (Generally Regarded As Safe, FDA, USA). Furthermore, the flexibility of this yeast's metabolism, which can be fermentative, respiratory or mixed respiro-fermentative, makes it applicable to a variety of processes. Processes run under aerobic conditions can be based on respiratory metabolism, as in the case of biomass-associated products like backer's yeast and heterologous proteins, or on mixed respiro-fermentative metabolism, important for fermentation-associated products that require oxygen. Besides being the most widely used microorganism in biotechnology, in processes ranging from bulk products like bioethanol and baker's yeast to high-value added pharmaceutical products like human insulin and vaccines, S. cerevisiae is currently being developed as a producer of a variety of chemicals from biomass sugar (by the National Renewable Energy Laboratory, USA, http://www.ntis.gov7ordering.htm).

In order to ensure efficient production of many chemicals, it is important to balance the electron fluxes via the cofactors NADH and NADPH. Several metabolic engineering strategies have focused on engineering the NADH metabolism of Escherichia coli (San et al., 2002; Berrios-Rivera et al., 2003a, Berrios-Rivera et al., 2003b, Berrios-Rivera et al., 2003c) and Lactococcus lactis (Lopez et al., 1998). In S. cerevisiae, there is an increased complexity in terms of balancing the formation and consumption of cofactors as there is no transhydrogenase activity that can convert NADH directly into NADPH. Furthermore, the metabolism is compartmentalized into, e.g., the cytosol and the mitochondria, and formation and consumption of the cofactors NADH and NADPH need to balance in each compartment, which imposes constraints on the carbon fluxes. In S. cerevisiae NADPH is generated in a few reactions only: (1) reactions catalyzed by the two dehydrogenases of the pentose-phosphate (PP) pathway (glucose-6-P dehydrogenase and 6-phosphogluconate dehydrogenase), (2) the reaction catalyzed by the NADP+-dependent isocitrate dehydrogenase, (3) the reaction catalyzed by the NADP+-dependent acetaldehyde dehydrogenase and (4) the reaction catalyzed by malic enzyme.

The PP pathway represents the main source of NADPH production in S. cerevisiae (Gancedo and Serrano, 1989; van Dijken and Scheffers, 1986). However, glucose-6-P is a flexible node (Stephanopoulos and Vallino, 1991; Gombert et al., 2001), and alteration of the flux through glucose-6-P dehydrogenase can therefore hardly be achieved via manipulation of the node's enzyme levels. Activity of malic enzyme (converting malate to pyruvate) can be regarded as part of a metabolic shunt that also includes pyruvate carboxylase and malate dehydrogenase (catalyzing a reversible reduction of oxaloacetate to malate). In this shunt, NADPH is produced at the expense of one ATP consumed by pyruvate carboxylase and one NADH consumed by malate dehydrogenase: in other words, the operation of the shunt does not have a net effect on the carbon metabolism (see Fig. 1A). It therefore appears to be a good target for engineering the redox metabolism.

Malic enzyme is found in the mitochondria of S. cerevisiae (Boles et al., 1998). It converts malate, an intermediate of the TCA cycle, into pyruvate, which is the end product of glycolysis and a key metabolite in the split of respiratory and fermentative metabolism. Pyruvate is, furthermore, involved in the only anaplerotic reaction known to operate in this yeast during growth on glucose, namely through carboxylation leading to the formation of oxaloacetete. This reaction is catalyzed by pyruvate carboxylase that is present as two cytosolic isozymes in S. cerevisiae (Walker et al., 1991). Malate dehydrogenase exists as three isozymes: one mitochondrial, a cytosolic form susceptible to glucose catabolite inactivation (Minard and McAlister-Henn, 1992) and a peroxisomal form (Steffan and McAlister-Henn, 1992). Depending on which of these forms takes part in the metabolic shunt mentioned above, either oxaloacetate or malate have to be transported across the mitochondrial membrane. This can be made via the transporters OCA1 (Palmieri et al., 1999a) or DIC1 (Palmieri et al., 1999b), respectively (see Fig. 1A). The transport of pyruvate from the mitochondria to the cytosol is not a requirement for maintaining the flux via the shunt in question during oxidative growth on sugars, since there is a very high flux of this metabolite in the other direction. A closed metabolic cycle would of course require that pyruvate exits the mitochondria, and even though a pyruvate transporter has not yet been found, there is indirect evidence for its existence. Thus, a mutant containing a double deletion of both pyruvate kinases of S. cerevisiae can grow on ethanol, whereas a triple mutant that also contains a deletion in the gene coding for malic enzyme is not viable (Boles et al., 1998). As mentioned above malic enzyme is mitochondrial, and if this enzyme can fulfill the cytosolic requirement for pyruvate as suggested by the findings, then transport of pyruvate from the mitochondria to the cytosol must occur. Passive diffusion through the membrane of the non-dissociated form of the acid could take place, but due to the hydrophilic nature of pyruvate this is likely to occur at a very low rate.

In this work, we wanted to study the effect of having an additional source of NADPH in the cell, either in the mitochondria or in the cytosol. In order to do this, two strains were engineered involving manipulation of malic enzyme. In both strains malic enzyme was overexpressed together with pyruvate carboxylase, but while in one of them malic enzyme was overexpressed as its wild-type form, in the other strain a short form expected to be targeted to the cytosol was overexpressed instead (see Figs. 1A and B). The basic growth characteristics of the strains were analyzed in aerobic batch and continuous cultivations. Furthermore, the enzyme activities, the distribution of intracellular fluxes and the concentration of redox cofactors were determined in chemostat cultivations at a low dilution rate.

Section snippets

Strains

S. cerevisiae CEN.PK113-7D (MATaMAL2-8c SUC2) was used as the genetic reference strain. The recombinant strains used in this study, listed in Table 1, are all isogenic to this strain. All strains are prototrophic and have mating type a.

Molecular biology techniques

Overexpressions were made in all cases by genomic integration (see below). The transformation of yeast cells was carried out according to Schiestl and Gietz (1989) with minor modifications. Amplification of recombinant plasmids was made using E. coli DH5α.

Strain construction

Results

The fermentation physiology of the different strains was studied during batch and continuous cultivations with the aim of characterizing the effects of the genetic manipulations on the fermentation properties, namely the maximum specific growth rate, the biomass and product yields, the critical dilution rate and the metabolic fluxes. All cultivations were carried out at aerobic conditions, and glucose was used as carbon source while ammonium was used as nitrogen source.

The metabolism of S.

Discussion

Activity of malic enzyme can be looked at as part of a metabolic shunt where NADPH is produced at the expense of one ATP consumed by pyruvate carboxylase and one NADH consumed by malate dehydrogenase. For the recombinant strains constructed in this work, the cellular compartmentation of these reactions varied (Fig. 1).

The strategy of overexpressing pyruvate carboxylase in the strains engineered in this work was chosen because this enzyme catalyzes an irreversible reaction that is part of the

Acknowledgements

Margarida Moreira dos Santos acknowledges Fundação para a Ciência e Tecnologia, Portugal, for the award of a research fellowship. Gerda Thygesen and Clémentine Marie are gratefully acknowledged for skillful technical assistance.

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