Authors: Jens Schneider Dorit Eberhardt Volker F Wendisch
Publish Date: 2012/02/28
Volume: 95, Issue: 1, Pages: 169-178
Abstract
Corynebacterium glutamicum shows a great potential for the production of the polyamide monomer putrescine 14diaminobutane Previously we constructed the putrescineproducing strain PUT1 by deletion of argF the gene for ornithine transcarbamoylase OTC and argR encoding the larginine repressor combined with heterologous expression of the Escherichia coli gene for lornithine decarboxylase SpeC As a consequence of argF deletion this strain requires supplementation of larginine and shows growthdecoupled putrescine production To avoid costly supplementation with larginine and the strong feedback inhibition of the key enzyme Nacetylglutamate kinase ArgB by larginine a plasmid addiction system for lowlevel argF expression was developed By finetuning argF expression through modifications of the promoter the translational start codon and/or the ribosome binding site high productivity and titer could be obtained OTC activity varied almost thousandfold between 960 and 1 mU mg−1 resulting in putrescine yields on glucose from less than 0001 up to 026 g g−1 the highest yield in bacteria reported to date The most promising strain designated PUT21 was characterized comprehensively PUT21 strain grew with a rate of 019 h−1 in mineral salt medium without the need for larginine supplementation and produced putrescine with a yield of 016 g g−1 glucose at a volumetric productivity of 057 g L−1 h−1 and a specific productivity of 0042 g g−1 h−1 The carbon balance suggested that no major unidentified byproduct was produced Compared to the firstgeneration strain PUT1 the putrescine yield observed with PUT21 was increased by 60 In fedbatch cultivation with C glutamicum PUT21 a putrescine titer of 19 g L−1 at a volumetric productivity of 055 g L−1 h−1 and a yield of 016 g g−1 glucose could be achieved Moreover while plasmid segregation of the initial strain required antibiotic selection plasmid segregation in C glutamicum PUT21 was fully stable for more than 60 generations without antibiotic selection even in the presence of larginine The ornithine decarboxylase gene speC was expressed from this argF addiction plasmid ensuring stable putrescine production by the engineered C glutamicum strain
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