Molecular Basis of Growth Inhibition by Acetate of an Adenylate Cyclase-Deficient Mutant of Corynebacterium glutamicum

Abstract

Wolf et al. Corynebacterium glutamicum cyaB Mutant ATCC 13032, biotin-auxotrophic wild-type strain (WT) Kinoshita et al., 1957 C. glutamicum cyaB ATCC 13032 with an in frame deletion of the adenylate cyclase gene cyaB (cg0375) This study C. glutamicum cpdA ATCC 13032 with an in frame deletion of the phosphodiesterase gene cpdA (cg2761) Schulte et al., 2017b C. glutamicum cyaB cpdA ATCC 13032 with an in frame deletion of the gene cyaB and the phosphodiesterase gene cpdA (cyaB cpdA) This study C. glutamicum qcr ATCC 13032 with an in frame deletion of the qcrCAB genes (cg2405, cg2404, cg2403) encoding the three subunits of the cytochrome bc 1 complex Niebisch and Bott, 2001 C. glutamicum cyaB_sup1 C. glutamicum cyaB suppressor mutant carrying a single genomic mutation (C to T) at position 307072 in BA000036.3 leading to the amino acid exchange Ala131Thr in GlxR (cg0350) This study C. glutamicum cyaB_sup2 C. glutamicum cyaB suppressor mutant carrying two genomic mutations in BA000036.3, one at position 877553 (A to G) located in the intergenic region of serC (cg0948) and gltA (cg0949) and one at position 1548741 (G to C) located in the intergenic region of gpt (cg1659) and cg1660 This study C. glutamicum cyaB_sup3 C. glutamicum cyaB suppressor mutant carrying two genomic mutations in BA000036.3, one at position 307072 (C to T) leading to the amino acid exchange Ala131Thr in GlxR and one at position 2564086 (G to T) leading to a silent mutation of the Gly106 codon of ribose 5-phosphate isomerase (cg2658) This study C. glutamicum::glxR_A131T C. glutamicum WT in which an Ala131Thr exchange in the glxR coding sequence was introduced by double homologous recombination This study C. glutamicum cyaB::glxR_A131T C. glutamicum cyaB mutant in which an Ala131Thr exchange in the glxR coding sequence was introduced by double homologous recombination This study Plasmids pAN6 Kan R ; P tac , lacI q pBL1 oriV Cg pUC18 oriV Ec , C. glutamicum/E. coli shuttle vector, derivative of pEKEx2 Frunzke et al., 2008 pAN6-cyaB Kan R ; pAN6 derivative carrying the cyaB gene (cg0375) including 300 bp upstream of the start codon and a 3-terminal StrepTag-II-encoding sequence under the control of an IPTG-inducible tac promoter This study pAN6-glxR-Twinstrep Kan R ; pAN6 derivative carrying the glxR gene (cg0350) including a 3-terminal Twinstrep-tag encoding sequence before the stop codon (WSHPQFEKGGGSGGGSGGSAWSHPQFEK) This study pAN6-glxR-A131T-Twinstrep Kan R ; pAN6-glxR-Twinstrep derivative with a Ala131T exchange This study pK19mobsacB Kan R ; oriT oriV Ec sacB lacZα; vector for allelic exchange in C. glutamicum Schäfer et al., 1994

The ldhA Gene Encoding Fermentative l-Lactate Dehydrogenase in Corynebacterium Glutamicum Is Positively Regulated by the Global Regulator GlxR

“…GlxR belongs to a CRP-type transcriptional regulator family and binds to target binding sites in a cAMP-dependent manner in vitro [ 30 , 36 , 37 ]. In the C. glutamicum genome, a sole adenylate cyclase is encoded by cyaB [ 25 , 38 ]. To examine the effect of cAMP deficiency on ldhA expression, a cyaB deletion mutant was constructed, which grew slightly slower than the wild type ( Figure S1a ).…”

High-quality physiology of Alcanivorax borkumensis SK2 producing glycolipids enables efficient stirred-tank bioreactor cultivation

“…A prolonged lag phase and reduced biomass yield with increasing acetate concentrations, but not higher than 15 g L -1 , was also observed in this study ( Figure 5B ; Figures 6A,B ). Several other bacteria, including P. putida , E. coli , and Corynebacterium glutamicum , have demonstrated biotechnological production of different products using acetate, with the ability to grow on acetate concentrations of at least 10 g L -1 ( Noh et al, 2018 ; Arnold et al, 2019 ; Wolf et al, 2020 ; Schmollack et al, 2022 ). Acetate serves as a crucial precursor for various biotechnological products, such as itaconic acid production in E. coli and C. glutamicum , as well as rhamnolipid production in P. putida ( Noh et al, 2018 ; Arnold et al, 2019 ; Merkel et al, 2022 ).…”

Regulation of γ-Aminobutyrate (GABA) Utilization in Corynebacterium glutamicum by the PucR-Type Transcriptional Regulator GabR and by Alternative Nitrogen and Carbon Sources

“…A GlxR binding site (5 -TGTATCTCACCTCACA-3 ) has been detected in the gabR-gabT intergenic region (Toyoda et al, 2011;Jungwirth et al, 2013), which is located downstream of the gabT TSS (Figure 6A), indicating a repressor function of GlxR for gabTDP. GlxRbinding to DNA is controlled by cAMP, which is formed by the membrane-bound adenylate cyclase CyaB (Cha et al, 2010;Wolf et al, 2020). The tested additional carbon sources might lead to elevated cAMP levels and thus to enhanced binding of GlxR to the gabT promoter and repression.…”