title: Physiological characterization of the high malic acid-producing Aspergillus oryzae strain 2103a-68.
abstract: Malic acid is a C₄ dicarboxylic acid that is currently mainly used in the food and beverages industry as an acidulant. Because of the versatility of the group of C₄ dicarboxylic acids, the chemical industry has a growing interest in this chemical compound. As malic acid will be considered as a bulk chemical, microbial production requires organisms that sustain high rates, yields, and titers. Aspergillus oryzae is mainly known as an industrial enzyme producer, but it was also shown that it has a very competitive natural production capacity for malic acid. Recently, an engineered A. oryzae strain, 2103a-68, was presented which overexpressed pyruvate carboxylase, malate dehydrogenase, and a malic acid transporter. In this work, we report a detailed characterization of this strain including detailed rates and yields under malic acid production conditions. Furthermore, transcript levels of the genes of interest and corresponding enzyme activities were measured. On glucose as carbon source, 2103a-68 was able to secrete malic acid at a maximum specific production rate during stationary phase of 1.87 mmol (g dry weight (DW))⁻¹ h⁻¹ and with a yield of 1.49 mol mol⁻¹. Intracellular fluxes were obtained using ¹³C flux analysis during exponential growth, supporting the success of the metabolic engineering strategy of increasing flux through the reductive cytosolic tricarboxylic acid (rTCA) branch. Additional cultivations using xylose and a glucose/xylose mixture demonstrated that A. oryzae is able to efficiently metabolize pentoses and hexoses to produce malic acid at high titers, rates, and yields.
authors.affiliation: Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, 412 96, Gothenburg, Sweden.
authors.forename: Christoph
authors.initials: C
authors.lastname: Knuf
authors.forename: Intawat
authors.initials: I
authors.lastname: Nookaew
authors.forename: Ilse
authors.initials: I
authors.lastname: Remmers
authors.forename: Sakda
authors.initials: S
authors.lastname: Khoomrung
authors.forename: Stephen
authors.initials: S
authors.lastname: Brown
authors.forename: Alan
authors.initials: A
authors.lastname: Berry
authors.forename: Jens
authors.initials: J
authors.lastname: Nielsen
mesh_terms: D001236:Aspergillus oryzae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D002247:Carbon Isotopes / Q000378:metabolism; D020869:Gene Expression Profiling; D007553:Isotope Labeling; D008293:Malates / Q000378:metabolism*; D060847:Metabolic Engineering; D064688:Metabolic Flux Analysis; D053858:Metabolic Networks and Pathways / Q000235:genetics*
chemical_list: D002247:Carbon Isotopes; D008293:Malates; C030298:malic acid