Elimination of polyamine N-acetylation and regulatory engineering improved putrescine production by Corynebacterium glutamicum

Corynebacterium glutamicum has been engineered for production of the polyamide monomer putrescine or 1,4-diaminobutane. Here, N-acetylputrescine was shown to be a significant by-product of putrescine production by recombinant putrescine producing C. glutamicum strains. A systematic gene deletion approach of 18 (putative) N-acetyltransferase genes revealed that the cgl 722 gene product was responsible for putrescine acetylation. The encoded enzyme was purified and characterized as polyamine N-acetyltransferase. The enzyme accepted acetyl-CoA and propionyl-CoA as donors for acetylation of putrescine and other diamines as acceptors, but showed highest catalytic efficiency with the triamine spermidine and the tetraamine spermine and, hence, was named SnaA. Upon deletion of snaA in the putrescine producing strain PUT21, no acteylputrescine accumulated, but about 41% more putrescine as compared to the parent strain. Moreover, a transcriptome approach identified increased expression of the cgmAR operon encoding a putative permease and a transcriptional TetR-family repressor upon induction of putrescine production in C. glutamicum PUT21. CgmR is known to bind to cgm0 upstream of cgmAR and gel mobility shift experiments with purified CgmR revealed that putrescine and other diamines perturbed CgmR-cgm0 complex formation, but not migration of free cgm0 DNA. Deletion of the repressor gene cgmR resulted in expression changes of a number of genes and increased putrescine production of C. glutamicum PUT21 by 19% as compared to the parent strain. Overexpression of the putative transport gene cgmA increased putrescine production by 24% as compared to the control strain. However, cgmA overexpression in PUT21 Delta snaA did not further improve putrescine production, hence, the beneficial effects of both targets were not synergistic at the highest described yield of 0.21 g g(-1). (C) 2014 Elsevier B.V. All rights reserved.