Identification and elimination of the competing N-acetyldiaminopentane pathway for improved production of diaminopentane by Corynebacterium glutamicum

Abstract

The present work describes the development of a superior strain of Corynebacterium glutamicum for diaminopentane (cadaverine) production aimed at the identification and deletion of the underlying unknown N-acetyldiaminopentane pathway. This acetylated product variant, recently discovered, is a highly undesired by-product with respect to carbon yield and product purity. Initial studies with C. glutamicum DAP-3c, a previously derived tailor-made diaminopentane producer, showed that up to 20% of the product occurs in the unfavorable acetylated form. The strain revealed enzymatic activity for diaminopentane acetylation, requiring acetyl-coenzyme A (CoA) as a donor. Comparative transcriptome analysis of DAP-3c and its parent strain did not reveal significant differences in the expression levels of 17 potential candidates annotated as N-acetyltransferases. Targeted single deletion of several of the candidate genes showed NCgl1469 to be the responsible enzyme. NCgl1469 was functionally assigned as diaminopentane acetyltransferase. The deletion strain, designated C. glutamicum DAP-4, exhibited a complete lack of N-acetyldiaminopentane accumulation in medium. Hereby, the yield for diaminopentane increased by 11%. The mutant strain allowed the production of diaminopentane as the sole product. The deletion did not cause any negative growth effects, since the specific growth rate and glucose uptake rate remained unchanged. The identification and elimination of the responsible acetyltransferase gene, as presented here, display key contributions of a superior C. glutamicum strain producing diaminopentane as a future building block for bio-based polyamides.

FIG. 1.

Physiological characteristics of diaminopentane-producing C. glutamicum DAP-3c (A, B) and DAP-4 (C, D) in batch cultures on glucose. The linear correlation among growth, production of lysine, diaminopentane (DAP), and N-acetyldiaminopentane (N-Ac-DAP), and consumption of glucose indicates a metabolic steady state during the cultivation. The data represent the values of three biological replicates for each strain. OD₆₆₀, optical density at 660 nm.

FIG. 2.

Comparative expression analysis of selected genes, functionally annotated as N-acetyltransferases, in the lysine-producing strain C. glutamicum 11424 and the diaminopentane-producing strain C. glutamicum DAP-3c. The data are given as ratios of the expression of 11424 to that of DAP-3c. The chosen cutoffs for significantly different expression ratios are indicated by the hatched areas. The data originate from the values of at least four biological replicates for each strain.