The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains

Tauch A, Götker S, Pühler A, Kalinowski J, Thierbach G. The alanine racemase gene alr is an alternative to antibiotic resistance genes in cloning systems for industrial Corynebacterium glutamicum strains. JOURNAL OF BIOTECHNOLOGY. 2002;99(1):79-91.The potential of the alanine racemase gene alr from Corynebacterium glutamicum ATCC 13032 to substitute for antibiotic resistance determinants in cloning systems has been investigated. The alr gene was identified by a PCR technique and its nucleotide sequence was determined. The deduced protein revealed the highest amino acid sequence similarity to the Alr protein from Mycobacterium smegmatis with 45% identical and 58% similar amino acids. A defined alr deletion mutant of C. glutamicum displayed a strict dependence on the presence of D-alanine for growth on complex and minimal medium. The alr gene was placed on a novel C. glutamicum vector which is completely free of antibiotic resistance genes. In vivo complementation of the chromosomal alr deletion with alr-carrying vectors permitted growth of the mutant strain in the absence of external D-alanine and provided strong selective pressure to maintain the plasmid. The alr gene enabled the selection of C. glutamicum transformants with a similar efficiency as the tetracycline resistance gene tetA (33). These data provided experimental evidence that the alr gene can be applied as an alternative selection marker to antibiotic resistance genes in industrial C. glutamicum strains. In an application example, the novel Deltaalr host-alr(+) vector-system for C. glutamicum was used to overproduce the vitamin D-pantothenic acid. (C) 2002 Elsevier Science B.V. All rights reserved

Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1

Tauch A, Kirchner O, Löffler B, Götker S, Pühler A, Kalinowski J. Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. CURRENT MICROBIOLOGY. 2002;45(5):362-367.Efficient transformation of the human pathogen Corynebacterium diphtheriae was achieved with novel cloning vectors consisting of a mini-replicon from the cryptic C. glutamicum plasmid pGA1 as well as of the aph(3')-IIa or tetA(Z) antibiotic resistance genes. Plasmid-containing transformants of C. diphtheriae were recovered at frequencies ranging from 1.3 x 10(5) to 4.8 x 10(6) colony forming units (cfu)/mug of plasmid DNA. Vector DNA was directly transferred from Escherichia coli into C. diphtheriae with frequencies up to 5.6 x 10(5) cfu/mug of plasmid DNA. On the basis of the pGA1 mini-replicon, an expression vector system was established for C. diphtheriae by means of the P-tac promoter and the green fluorescent reporter protein. In addition, other commonly used vector systems from C. glutamicum, including the pBL1 and pHM1519 replicons, and the sacB conditionally lethal selection market from Bacillus subtilis, were shown to be functional in C. diphtheriae. Thus, the ability to apply the standard methods of C. glutamicum recombinant DNA technology will greatly facilitate the functional analysis of the recently completed C. diphtheriae genome sequence

Biotin protein ligase from Corynebacterium glutamicum: role for growth and L-lysine production

Peters-Wendisch P, Stansen KC, Götker S, Wendisch VF. Biotin protein ligase from Corynebacterium glutamicum: role for growth and L-lysine production. Applied Microbiology and Biotechnology. 2012;93(6):2493-2502

Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production

Peters-Wendisch P, Götker S, Heider S, et al. Engineering biotin prototrophic Corynebacterium glutamicum strains for amino acid, diamine and carotenoid production. Journal of Biotechnology. 2014;192:346-354