Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of l-malic acid

Abstract

Malic acid, a petroleum-derived C4-dicarboxylic acid that is used in the food and beverage industries, is also produced by a number of microorganisms that follow a variety of metabolic routes. Several members of the genus Aspergillus utilize a two-step cytosolic pathway from pyruvate to malate known as the reductive tricarboxylic acid (rTCA) pathway. This simple and efficient pathway has a maximum theoretical yield of 2 mol malate/mol glucose when the starting pyruvate originates from glycolysis. Production of malic acid by Aspergillus oryzae NRRL 3488 was first improved by overexpression of a native C4-dicarboxylate transporter, leading to a greater than twofold increase in the rate of malate production. Overexpression of the native cytosolic alleles of pyruvate carboxylase and malate dehydrogenase, comprising the rTCA pathway, in conjunction with the transporter resulted in an additional 27 % increase in malate production rate. A strain overexpressing all three genes achieved a malate titer of 154 g/L in 164 h, corresponding to a production rate of 0.94 g/L/h, with an associated yield on glucose of 1.38 mol/mol (69 % of the theoretical maximum). This rate of malate production is the highest reported for any microbial system.

Evaluation of Pyrolysis Oil as Carbon Source for Fungal Fermentation

“…The possibility of an enhanced release of malate and fumarate in the culture medium could be explained by a potentially occurring permeabilization of the cell membrane by the presence of phenol. It is known that one bottleneck of the malic acid production is the export from cytoplasm to medium (Brown et al, 2013) and therefore the permeabilization of the membrane could lead to an accelerated transport by the concentration gradient. With increasing concentrations of phenol, malic acid production drops off quickly due to the toxic effects of increasing cell membrane permeabilization.…”

Production of valuable compounds by molds and yeasts

“…Additional applications include medical uses. Metabolic engineering of Aspergillus oryzae NRRL 3488 has been used to overproduce malic acid at 154 g l − 1 , 116 with a selling price of $2-3 kg − 1 . The result was achieved by overexpressing (1) the C4-dicarboxylate transporter and (2) the cytosolic alleles of pyruvate carboxylase and malate dehydrogenase.…”

Overexpression of a C4-dicarboxylate transporter is the key for rerouting citric acid to C4-dicarboxylic acid production in Aspergillus carbonarius

“…In recent years, bio-based production of C 4 -dicarboxylic acids has received increasing research attention and achieved an important status in bio-economy. So far, bio-based succinic acid production has succeeded with a number of commercialized processes using bacterial strains ( Escherichia coli and Actinobacillus succinogenes ) and yeast strains ( Saccharomyces cerevisiae ) [7], and biotechnological processes for malic acid and fumaric acid are under research development [8, 9]. The bottlenecks in the current biotechnologies for production of C 4 -dicarboxylic acids are relatively low productivity in production processes and high production cost due to the choice of substrates (glucose) and downstream product purification [10].…”

“…In addition to central carbon metabolic pathways, export of C 4 -dicarboxylic acids is an essential step to consider in metabolic engineering of microbial strains for production of C 4 -dicarboxylic acids. In Aspergillus oryzae and S. cerevisiae , synergistic impacts on malic acid production were obtained when C 4 -dicarboxylate transporters were overexpressed in combination with other genetic modifications [9, 21]. In A. carbonarius , there is not yet any report regarding the C 4 -dicarboxylate transporter.…”