Israel Goldberg

l-Malic acid formation by immobilized Saccharomyces cerevisiae amplified for fumarase

The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric acid to l-malic acid at a rate of 65 mM g−1 h−1 in free cell experiments, and approximately 87% of the fumaric acid was converted to l-malic acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.

The cytosolic pathway of L-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase.

Saccharomyces cerevisiae accumulates l-malic acid but only minute amounts of fumaric acid. A 13C-nuclear magnetic resonance study following the label from glucose to l-malic acid indicates that the l-malic acid is synthesized from pyruvic acid via oxaloacetic acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to l-malic acid is responsible for the l-malic acid production in yeast. The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to l-malic acid but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards l-malic acid; the Km for l-malic acid is very high indicating that l-malic acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more l-malic acid). (c) According to 13C NMR studies there is no interconversion of cytosolic l-malic and fumaric acids.

Overexpression of cytosolic malate dehydrogenase (MDH2) causes overproduction of specific organic acids in Saccharomyces cerevisiae.

Saccharomyces cerevisiae accumulates l-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a 6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in l-malic acid accumulation in the production medium. The high apparent Km of MDH2 for l-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function of the enzyme and differs from the previously published Km of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be a limiting factor, thus providing a system for further metabolic engineering of l-malic acid production. The overexpression of MDH2 activity also causes an elevation in the accumulation of fumaric acid and citric acid. Accumulation of fumaric acid is presumably caused by high intracellular l-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing possibility that cytosolic l-malic acid is a direct precursor of citric acid in yeast.

Malic acid accumulation by Aspergillus flavus

Summary13C Nuclear magnetic resonance and fumarase and NAD-malate dehydrogenase isoenzyme studies were carried out in a strain of A. flavus which produces relatively high levels of l-malic acid from glucose. The results of the 13C NMR showed that the 13C label from [1-13C] glucose was incorporated only to C-3 (-CH2-) of l-malic acid and indicated that this acid must be synthesized from pyruvate mainly via oxaloacetate. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for fumarase and malate dehydrogenase. Changes in the isoenzyme pattern were observed for malate dehydrogenase but not for fumarase during acid production. Cycloheximide inhibited profoundly both l-malic acid production and the increase in the major isoenzyme of malate dehydrogenase, without affecting either the total activity of fumarase or its isoenzyme pattern. The results suggested that de novo protein synthesis is involved in the increase in the activity of the major isoenzyme of malate dehydrogenase and that this isoenzyme is essential for l-malic acid production and accumulation.

Involvement of Gluconic Acid and Glucose Oxidase in the Pathogenicity of Penicillium expansum in Apples

ABSTRACT The contribution of gluconic acid secretion to the colonization of apple tissue by Penicillium expansum was analyzed by modulation (increase or decrease) of gluconic acid accumulation at the infection court. P. expansum isolates that express the most gox2 transcripts and concomitant glucose oxidase (GOX) activity and that secrete the most gluconic acid cause disease of apple at the fastest rate. Cultures grown under reduced oxygen concentration generated fewer gox2 transcripts, produced less gluconic acid, and led to a 15% reduction in disease. Furthermore, the detection of significantly high levels of transcripts of gox2 and GOX activity at the edge of the decaying tissue emphasize the involvement of GOX in tissue acidification of the decaying tissue. Taken together, these results emphasize the importance of GOX in the production of the gluconic acid that leads, in turn, to host tissue acidification. This acidification enhanced the expression of pectolytic enzymes and the establishment of conditions for necrotrophic development of P. expansum.

Conversion of Fumaric Acid to l-Malic by Sol-Gel Immobilized Saccharomyces cerevisiae in a Supported Liquid Membrane Bioreactor

Conversion of fumaric acid (FA) to l‐malic acid (LMA) was carried out in a bioreactor divided by two supported liquid membranes (SLMs) into three compartments: Feed, Reaction, and Product. The Feed/Reaction SLM, made of tri‐ n‐octylphosphine oxide (vol 10%) in ethyl acetate, was selective toward the substrate, fumaric acid ( SFA/LMA = 10). The Reaction/Product SLM, made of di(2‐ethylhexyl) phosphate (vol 10%) in dichloromethane, was selective toward the product, l‐malic acid ( SLMA/FA = 680). Immobilized yeast engineered to overproduce the enzyme fumarase [E.C. 4.2.1.2] was placed in the Reaction compartment and served as the catalyst. The yeast was immobilized in small glasslike beads of alginate‐silicate sol‐gel matrix. The construction of the bioreactor ensured unidirectional flow of the substrate from the Feed to the Reaction and of the product from the Reaction to the Product compartments, with the inorganic counterion traveling in the opposite direction. The conversion of almost 100%, above the equilibrium value of ca. 84% and higher than that for the industrial process, 70%, was achieved. In contrast to the existing industrial biocatalytic process resulting in l‐malic acid salts, direct production of the free acid is described.

Isoenzyme pattern and subcellular localisation of enzymes involved in fumaric acid accumulation by Rhizopus oryzae

SummaryElectrophoretic studies of fumarase and nicotine adenine dinucleotide (NAD)-malate dehydrogenase were carried out in the fumaric acid-accumulating fungus Rhizopus oryzae. The analyses revealed two fumarase isoenzymes, one localised solely in the cytosol and the other found both in the cytosol and in the mitochondrial fraction. The activity of the cytosolic isoenzyme of fumarase was higher during the acid production stage than during growth. Addition of cycloheximide inhibited fumaric acid production and decreased the activity of the cytosolic isoenzyme of fumarase. These results suggested that de novo protein synthesis is required for increase in the activity of the cytosolic isoenzyme and that such an increase in activity is essential for fumaric acid accumulation. Three distinct isoenzymes of NAD-malate dehydrogenase could be detected in R. oryzae. No changes were observed in the isoenzyme pattern of malate dehydrogenase during fumaric acid production.

Dual localization of fumarase is dependent on the integrity of the glyoxylate shunt

Fumarase and aconitase in yeast are dual localized to the cytosol and mitochondria by a similar targeting mechanism. These two tricarboxylic acid cycle enzymes are single translation products that are targeted to and processed by mitochondrial processing peptidase in mitochondria prior to distribution. The mechanism includes reverse translocation of a subset of processed molecules back into the cytosol. Here, we show that either depletion or overexpression of Cit2 (cytosolic citrate synthase) causes the vast majority of fumarase to be fully imported into mitochondria with a tiny amount or no fumarase in the cytosol. Normal dual distribution of fumarase (similar amounts in the cytosol and mitochondria) depends on an enzymatically active Cit2. Glyoxylate shunt deletion mutations (Δmls1, Δaco1 and Δicl1) exhibit an altered fumarase dual distribution (like in Δcit2). Finally, when succinic acid, a product of the glyoxylate shunt, is added to the growth medium, fumarase dual distribution is altered such that there are lower levels of fumarase in the cytosol. This study suggests that the cytosolic localization of a distributed mitochondrial protein is governed by intracellular metabolite cues. Specifically, we suggest that metabolites of the glyoxylate shunt act as ‘nanosensors’ for fumarase subcellular targeting and distribution. The possible mechanisms involved are discussed.

Purification and properties of glucose-6-phosphate dehydrogenase (NADP+/NAD+) and 6-phosphogluconate dehydrogenase (NADP+/NAD+) from methanol-grown Pseudomonas C

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADPH+ 1-oxidoreductase, EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP+ 2-oxidoreductase, EC 1.1.1943) have been purified from methanol-grown Pseudomonas C. Glucose-6-phosphate dehydrogenase exhibits activity with either NADP+ or NAD+ as coenzymes, V NADP+ = 0.96 V NAD+.Km values of 22, 290, and 250 microns are obtained for NADP+, NAD+ and glucose 6-phosphate (NADP+ as the coenzyme), respectively. ATP inhibits Glc-6P dehydrogenase activity with NAD+ as coenzyme and to a less extent the activity with DANP+. In the presence of MgCl2, ATP inhibition of Blc-6P dehydrogeanse activity is abolished. 6-Phosphogluconate dehydrogenase has a dual specificity for both NADP+ or NAD+ as coenzymes, V NADP+ = 1.66 V NAD+.Km values of 20, 500 and 100 microns are obtained for NADP+, NAD+ and 6-phosphogluconate (NADP+ as the coenzyme), respectively. With NAD+ as the coenzyme ATP inhibits 6-phosphogluconate dehydrogeanse activity, while with NADP+ as the coenzyme, activity was less affected. The possible role of these enzymes in the metabolism of one-carbon (C1)-compounds in Pseudomonas C is discussed and compared with other methylotrophic microorganisms.

The growth of Pseudomonas putida on m-toluic acid and on toluene in batch and in chemostat cultures

SummaryThe present study describes the growth of Pseudomonas putida cells (ATCC 33015) in batch and continuous cultures on two toxic substrates; toluene and m-toluic acid as sole carbon and energy sources. In fed-batch cultures on m-toluic acid up to 3.55 g cell dry weight/1 were achieved with a maximal specific growth rate (μmax) of 0.1 h-1. The average cellular yield was 1.42 g cell dry weight/g m-toluic acid utilized. When liquid toluene was added to shake-flask cultures in the presence of 0.7 g/1 m-toluic acid, the average cellular yield obtained was 1.3 g cell dry weight/g toluene utilized and the μmax was 0.13 h-1. Growth on toluene vapour in the presence of 0.7 g/l m-toluic acid in batch cultures resulted in a cellular yield of 1.28 g cell dry weight/g toluene utilized, with growth kinetics almost identical to those with liquid toluene (μmax liquid=0.13 h-1, μmax vapour=0.12 h-1). The maximal biomass concentration was 3.8 g cell dry weight/l, obtained in both cases after 100 h of incubation. Pseudomonas putida was grown in a chemostat initially on 0.7 g/l m-toluic acid and vapour toluene and then in the steady state on toluene as the sole source of carbon and energy. Toluene was added continuously to the culture as vapour with the inflowing airstream. Chemostat cultures could be maintained at steady state for several months on toluene. The maximal biomass concentration obtained in the chemostat culture was 3.2 g cell dry weight/l. The maximum specific growth rate was 0.13 h-1, with a cellular yield of 1.05 g cell dry weight/g toluene utilized. Approximately 70% of the toluene consumed was converted into biomass, and the remainder was converted to CO2 and unidentified byproducts.