Metabolic engineering strategies for sustainable de novo isobutylamine production by Corynebacterium glutamicum

Short-chain primary amines are a versatile group of molecules in biological systems, derived from the decarboxylation of amino acids or the transamination of carbonyl-containing molecules. Isobutylamine, the decarboxylation product of L-valine, has applications in chemical synthesis of pharmaceuticals, herbicides, fungicides and specialized chemicals, where it is especially interesting as a substitute for petrochemical compounds. Since the chemical synthesis of short-chain primary amines is hazardous and environmentally unsustainable, we established an efficient biosynthetic approach in this work.

The natural amino acid producer Corynebacterium glutamicum is an ideal chassis for the production of amino acid-derived molecules. In this study, we assessed the toxicity of several relevant short-chain primary amines on C. glutamicum. A crude extract assay demonstrated the decarboxylation of L-valine to isobutylamine by the L-valine decarboxylase VlmD_Sv from Streptomyces viridifaciens. This enzyme was instrumental to extend production of pyruvate in three engineered strains to yield L-valine and subsequently isobutylamine. To ensure intracellular L-valine supply, the extracellular L-valine accumulation was decreased up to 20-fold by deletion of the genes for the branched-chain amino acid exporter BrnFE_Cg and/or overexpression of the L-valine importer gene brnQ_Cg. Surprisingly, expression of the transhydrogenase genes pntAB_Ec from Escherichia coli did not improve but drastically reduced isobutylamine production. Biochemical analysis revealed that pntAB_Ec expression depolarized the membrane potential.

This study demonstrated the highest production of isobutylamine and the highest production of any microbial short-chain primary amine in minimal medium. In shake flask cultivation, an isobutylamine titer of 12.1 g/L and a yield of 0.74 mol isobutylamine/mol glucose were obtained. Transfer to a 3 L fed-batch bioreactor fermentation was successful and a titer of 33.2 g/L was reached within 64 h with a yield of 0.47 mol/mol and a volumetric productivity of 0.52 g/l/h. Moreover, isobutylamine production on a second-generation feedstock was shown, highlighting the potential of sustainable bio-based production of isobutylamine.