Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.     

Metabolic pathway engineering for complex polyketide biosynthesis in Saccharomyces cerevisiae

Polyketides are a diverse group of natural products with significance in human and veterinary medicine. Because polyketides are structurally complex molecules and fermentation is the most commercially viable route of production, a generic heterologous host system for high-level polyketide production is desirable. Saccharomyces cerevisiae has been shown to be an excellent production host for a simple polyketide, yielding 1.7 g of 6-methylsalicylic acid per liter of culture in un-optimized shake-flask fermentations. However, a barrier to the heterologous production of more complex 'modular' polyketides in S. cerevisiae is the lack of required polyketide precursor pathways. In this work, we describe the introduction into S. cerevisiae of pathways for the production of methylmalonyl-coenzyme A (CoA), a precursor for complex polyketides, by both propionyl-CoA-dependent and propionyl-CoA-independent routes. Furthermore, we demonstrate that the methylmalonyl-CoA produced in the engineered yeast strains is used in vivo for the production of a polyketide product, a triketide lactone.     

Metabolic engineering for improved microbial pentose fermentation

Global concern over the depletion of fossil fuel reserves, and the detrimental impact that combustion of these materials has on the environment, is focusing attention on initiatives to create sustainable approaches for the production and use of biofuels from various biomass substrates. The development of a low-cost, safe and eco-friendly process for the utilization of renewable resources to generate value-added products with biotechnological potential as well as robust microorganisms capable of efficient fermentation of all types of sugars are essential to underpin the economic production of biofuels from biomass feedstocks. Saccharomyces cerevisiae, the most established fermentation yeast used in large scale bioconversion strategies, does not however metabolise the pentose sugars, xylose and arabinose and bioengineering is required for introduction of efficient pentose metabolic pathways and pentose sugar transport proteins for bioconversion of these substrates. Our approach provided a basis for future experiments that may ultimately lead to the development of industrial S. cerevisiae strains engineered to express pentose metabolising proteins from thermophilic fungi living on decaying plant material and here we expand our original article and discuss the strategies implemented to improve pentose fermentation.     

Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants

Plants and most animals can synthesize their own L-ascorbic acid (vitamin C), but a mutation in the L-gulono--lactone oxidase gene in the primate lineage makes it necessary for humans to acquire this vital compound from their diet. Despite the fact that plants and animals synthesize ascorbic acid via different pathways, transgenic tobacco and lettuce plants expressing a rat cDNA encoding L-gulono--lactone oxidase accumulated up to seven times more ascorbic acid than untransformed plants. These results demonstrate that basal levels of ascorbic acid in plants can be significantly increased by expressing a single gene from the animal pathway.     

Evidence for a pentose phosphate pathway in Helicobacter pylori

Abstract Evidence for the presence of enzymes of the pentose phosphate pathway in Helicobacter pylori was obtained using ³¹P nuclear magnetic resonance spectroscopy. Activities of enzymes which are part of the oxidative and non-oxidative phases of the pathway were observed directly in incubations of bacterial lysates with pathway intermediates. Generation of NADPH and 6-phosphogluconate from NADP⁺ and glucose 6-phosphate indicated the presence of glucose 6-phosphate dehydrogenase and 6-phosphogluconolactonase. Reduction of NADP⁺ with production of ribulose 5-phosphate from 6-phosphogluconate revealed 6-phosphogluconate dehydrogenase activity. Phosphopentose isomerase and transketolase activities were observed in incubations containing ribulose 5-phosphate and xylulose 5-phosphate, respectively. The formation of erythrose 4-phosphate from xylulose 5-phosphate and ribose 5-phosphate suggested the presence of transaldolase. The activities of this enzyme and triosephosphate isomerase were observed directly in incubations of bacterial lysates with dihydroxyacetone phosphate and sedoheptulose 7-phosphate. Glucose-6-phosphate isomerase activity was measured in incubations with fructos 6-phosphate. The presence of these enzymes in H. pylori suggested the existence of a pentose phosphate pathway in the bacterium, possibly as a mechanism to provide NADPH for reductive biosynthesis and ribose 5-phosphate for synthesis of nucleic acids.     

The pentose phosphate pathway in Trypanosoma cruzi

The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).     

The pentose phosphate pathway in the endoplasmic reticulum

Approximately the same levels of six of the seven enzymes catalyzing reactions of the pentose phosphate pathway are in the cisternae of washed microsomes from rat heart, spleen, lung, and brain. Renal and hepatic microsomes also have detectable levels of these enzymes except ribulose-5-phosphate epimerase and ribose-5-phosphate isomerase. Their location in the cisternae is indicated by their latencies, i.e. requirement for disruption of the membrane for activity. In addition, transketolase, transaldolase, and glucose-6-phosphatase, a known cisternal enzyme, are inactivated by chymotrypsin and subtilisin only in disrupted hepatic microsomes under conditions in which NADPH-cytochrome c reductase, an enzyme on the external surface, is inactivated equally in intact and disrupted microsomes. The failure to detect the epimerase and isomerase in hepatic microsomes is due to inhibition of their assays by ketopentose-5-phosphatase. Xylulose 5-phosphate is hydrolyzed faster than ribulose 5-phosphate. A mild heat treatment destroys hepatic xylulose-5-phosphatase and glucose-6-phosphatase without affecting acid phosphatase. These results plus the established wide distribution of glucose dehydrogenase, the microsomal glucose-6-phosphate dehydrogenase, and its localization to the lumen of the endoplasmic reticulum suggest that most mammalian cells have two sets of enzymes of the pentose phosphate pathway: one is cytoplasmic and the other is in the endoplasmic reticulum. The activity of the microsomal pentose phosphate pathway is estimated to be about 1.5% that of the cytoplasmic pathway.     

The pentose phosphate pathway in developing chick cornea

Embryonic chick corneas at different stages of development were evaluated for activity of the pentose phosphate pathway. The appearance of activity was concurrent with the onset of corneal transperancy (stage 40). Highest values were found after complete transparency is achieved (stage 45 and after hatching). Phenazine methosulfate, an artificial electron acceptor, increased activity at all stages studied even before endogenous activity was measurable; however, no increase in glucose uptake was observed. Thus, the enzymes for the pathway are present at early stages (i.e., stage 38 and 40) although in latent form. The pathway probably functions in the developing cornea to generate NADPH rather than sugar moieties for macromolecular incorporation.     

Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger

Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.     

Metabolic consequences of drug-induced inhibition of the pentose phosphate pathway in neuroblastoma and glioma cells.

6-Aminonicotinamide leads to a considerable accumulation of 6-phosphogluconate, which is 3 times higher in C-6 glial cells than it is in C-1300 neuroblastoma cells. Dephosphorylation of the accumulated 6-phosphogluconate causes a rise of intracellular gluconate, which can be released from the cells. The higher dephosphorylating capacity of neuroblastoma cells leads to an intracellular gluconate content which is 4 times that found in C-6 glial cells. Although 6-phosphogluconate is a potent competitive inhibitor of glucose phosphate isomerase, no reduction of glycolytic flux and ATP content in stationary phase neuroblastoma cells was found in contrast to observations in C-6 glial cells. Morphological changes are only found in C-6 glial cells during the experimental period.     

Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli

A barrier to heterologous production of complex polyketides in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common extender substrate for the biosynthesis of complex polyketides by modular polyketide synthases. One biosynthetic route to (2S)-methylmalonyl-CoA involves the sequential actions of two enzymes, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase, which convert succinyl-CoA to (2R)- and then to (2S)-methylmalonyl-CoA. As reported [McKie, N., et al. (1990) Biochem. J. 269, 293-298; Haller, T., et al. (2000) Biochemistry 39, 4622-4629], when genes encoding coenzyme B(12)-dependent methylmalonyl-CoA mutases were expressed in E. coli, the inactive apo-enzyme was produced. However, when cells harboring the mutase genes from Propionibacterium shermanii or E. coli were treated with the B12 precursor hydroxocobalamin, active holo-enzyme was isolated, and (2R)-methylmalonyl-CoA represented approximately 10% of the intracellular CoA pool. When the E. coli BAP1 cell line [Pfeifer, B. A., et al. (2001) Science 291, 1790-1792] harboring plasmids that expressed P. shermanii methylmalonyl-CoA mutase, Streptomyces coelicolor methylmalonyl-CoA epimerase, and the polyketide synthase DEBS (6-deoxyerythronolide B synthase) was fed propionate and hydroxocobalamin, the polyketide 6-deoxyerythronolide B (6-dEB) was produced. Isotopic labeling studies using [(13)C]propionate showed that the starter unit for polyketide synthesis was derived exclusively from exogenous propionate, while the extender units stemmed from methylmalonyl-CoA via the mutase-epimerase pathway. Thus, the introduction of an engineered mutase-epimerase pathway in E. coli enabled the uncoupling of carbon sources used to produce starter and extender units of polyketides.     

The pentose phosphate pathway and parasitic protozoa

The pentose phosphate pathway plays a crucial role in the host-parasite relationship. It maintains a pool of NADPH, which serves to protect against oxidant stress and which generates carbohydrate intermediates used in nucleotide and other biosynthetic pathways. Deficiency in the first enzyme of the pathway, glucose-6-phosphate dehydrogenase, protects human erythrocytes from infection with Plasmodium falciparum for reasons that remain obscure. Loss of the third enzyme of the pathway, 6-phosphogluconate de-hydrogenase, is toxic, suggesting this enzyme might be a target for chemotherapy. Mike Barrett here summarizes the roles of the pentose phosphate pathway in various parasitic protozoa.     

Mutants of the pentose phosphate pathway in Aspergillus nidulans

Mutants of the pentose phosphate pathway have been isolated in Aspergillus nidulans. These fail to grow on a variety of carbohydrates that are catabolized through the pentose phosphate pathway. They also grow poorly on nitrate and nitrite as sole nitrogen sources. The pentose phosphate pathway mutations have been assigned to two unlinked genes. Mutants with lesions in the pppB locus have reduced activities of four enzymes of the pentose phosphate pathway, of glucose-phosphate isomerase, and of mannitol-1-phosphate dehydrogenase. pppA(-) mutants have elevated activities of these same enzymes except for transaldolase, for which they have much reduced activity. Both classes of mutants accumulate sedoheptulose-7-phosphate to an extent that is increased considerably when nitrate is present in the medium. Nitrate does not cause an increase in accumulation of sedoheptulose-7-phosphate in double mutants which, in addition to the pppA1 mutation, carry a mutation that leads to the lack of nitrate reductase activity. These last results suggest that nitrate stimulates the flux through the oxidative pentose phosphate pathway, but that this stimulation depends upon the metabolism of nitrate.     

Activity of the pentose phosphate pathway during lignification

Summary We did this work to see if there is a correlation between lignin synthesis and the activity of the pentose phosphate pathway. Excision of the third internode of the stem of Coleus blumei Benth. followed by incubation on sucrose and indoleacetic acid led to extensive formation of tracheids. During this lignification we determined the activities of glucose-6-phosphate dehydrogenase and fructose-1,6-diphosphate aldolase, and the extent to which [1-¹⁴C]-,[3,4-¹⁴C]-, and [6-¹⁴C]glucose labelled CO₂ and the major cellular components. The results indicate that the pentose phosphate pathway was active during lignification, and that the activity of this pathway relative to glycolysis increased at the onset of lignification. Explants of storage tissue of Helianthus tuberosus L. were cultured under conditions which caused extensive lignification. ¹⁴CO₂ production from [1-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose indicated activity of the pentose phosphate pathway during tracheid formation. We suggest that lignification is accompanied by appreciable activity of the pentose phosphate pathway and that this could provide the reducing power for lignin synthesis.Abbreviations NADP nicotinamide-adenine dinucleotide phosphate - IAA indoleacetic acid