Metabolic engineering of Corynebacterium glutamicum for production of L-leucine and 2-ketoisocaproate

Due to the depletion of fossil energy sources, there is an increasing demand for alternative sustainable production processes utilizing renewable resources. Biotechnological approaches using microorganisms such as Corynebacterium glutamicum as biocatalysts play an important role for the production of beneficial substances. These include the three branched-chain amino acids L-valine, L-isoleucine, and L-leucine, as well as their respective keto acid precursors, which have diverse commercial applications in food, feed, and pharmaceutical industry. In this work, metabolic engineering of the C. glutamicum wild type was employed to develop efficient strains for the production of L-leucine and 2-ketoisocaproate. The key-player enzyme in L-leucine biosynthesis is the leuA-encoded 2-isopropylmalate synthase which is feedback-inhibited by low L-leucine concentrations with a Ki of 0.4 mM. A feedback-resistant variant of the 2-isopropylmalate synthase was identified and characterized biochemically in the available weak L-leucine producer B018, which had been obtained by random mutagenesis and screening. The respective gene leuA_B018, devoid of the attenuator region and under control of a strong promoter, was integrated in up to three copies into the genome of C. glutamicum wild type and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved I) deletion of the gene ltbR encoding the repressor LtbR to increase expression of genes leuBCD, II) deletion of the gene iolR encoding the transcriptional regulator IolR to increase glucose uptake, III) reduction of citrate synthase activity to increase precursor supply, and IV) introduction of a modified ilvN gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in shake flask and bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of approximately 24 g l-1. The maximal molar product yield and volumetric productivity were 0.30 mol per mol glucose and 4.3 mmol l-1 h-1, respectively. Moreover, the achieved values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application.