title: Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction.
abstract: Using metabolic engineering, an efficient L-leucine production strain of Corynebacterium glutamicum was developed. In the wild type of C. glutamicum, the leuA-encoded 2-isopropylmalate synthase (IPMS) is inhibited by low L-leucine concentrations with a K(i) of 0.4 mM. We identified a feedback-resistant IMPS variant, which carries two amino acid exchanges (R529H, G532D). The corresponding leuA(fbr) gene devoid of the attenuator region and under control of a strong promoter was integrated in one, two or three copies into the genome and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved (i) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (ii) deletion of the gene encoding the transcriptional regulator IolR to increase glucose uptake, (iii) reduction of citrate synthase activity to increase precursor supply, and (iv) introduction of a gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of about 24 g/l. The molar product yield was 0.30 mol L-leucine per mol glucose and the volumetric productivity was 4.3 mmol l⁻¹ h⁻¹. These values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application.
authors: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany.
authors: Michael
authors: M
authors: Vogt
authors: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany.
authors: Sabine
authors: S
authors: Haas
authors: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany.
authors: Simon
authors: S
authors: Klaffl
authors: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany.
authors: Tino
authors: T
authors: Polen
authors: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany.
authors: Lothar
authors: L
authors: Eggeling
authors: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany. Electronic address: j.van.ooyen@fz-juelich.de.
authors: Jan
authors: J
authors: van Ooyen
authors: Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany. Electronic address: m.bott@fz-juelich.de.
authors: Michael
authors: M
authors: Bott
mesh_terms: D015079:2-Isopropylmalate Synthase / Q000096:biosynthesis / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D007930:Leucine / Q000096:biosynthesis* / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D020125:Mutation, Missense; D012097:Repressor Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D015079:2-Isopropylmalate Synthase; D007930:Leucine
keywords: Amino acid production; Corynebacterium glutamicum; Industrial application; Metabolic engineering; Rational strain design; l-leucine