Dataset Information

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

ABSTRACT: In Corynebacterium glutamicum, cyclic adenosine monophosphate (cAMP) serves as an effector of the global transcriptional regulator GlxR. Synthesis of cAMP is catalyzed by the membrane-bound adenylate cyclase CyaB. In this study, we investigated the consequences of decreased intracellular cAMP levels in a ?cyaB mutant. While no growth defect of the ?cyaB strain was observed on glucose, fructose, sucrose, or gluconate alone, the addition of acetate to these growth media resulted in a severe growth inhibition, which could be reversed by plasmid-based cyaB expression or by supplementation of the medium with cAMP. The effect was concentration- and pH-dependent, suggesting a link to the uncoupling activity of acetate. In agreement, the ?cyaB mutant had an increased sensitivity to the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The increased uncoupler sensitivity correlated with a lowered membrane potential of acetate-grown ?cyaB cells compared to wild-type cells. A reduced membrane potential affects major cellular processes, such as ATP synthesis by F1F O -ATP synthase and numerous transport processes. The impaired membrane potential of the ?cyaB mutant could be due to a decreased expression of the cytochrome bc 1-aa 3 supercomplex, which is the major contributor of proton-motive force in C. glutamicum. Expression of the supercomplex genes was previously reported to be activated by GlxR-cAMP. A suppressor mutant of the ?cyaB strain with improved growth on acetate was isolated, which carried a single mutation in the genome leading to an Ala131Thr exchange in GlxR. Introduction of this point mutation into the original ?cyaB mutant restored the growth defect on acetate. This supported the importance of GlxR for the phenotype of the ?cyaB mutant and, more generally, of the cAMP-GlxR system for the control of energy metabolism in C. glutamicum.

SUBMITTER: Wolf N

PROVIDER: S-EPMC7026483 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Wolf Natalie N Bussmann Michael M Koch-Koerfges Abigail A Katcharava Nino N Schulte Julia J Polen Tino T Hartl Johannes J Vorholt Julia A JA Baumgart Meike M Bott Michael M

Frontiers in microbiology 20200211

In Corynebacterium glutamicum, cyclic adenosine monophosphate (cAMP) serves as an effector of the global transcriptional regulator GlxR. Synthesis of cAMP is catalyzed by the membrane-bound adenylate cyclase CyaB. In this study, we investigated the consequences of decreased intracellular cAMP levels in a ΔcyaB mutant. While no growth defect of the ΔcyaB strain was observed on glucose, fructose, sucrose, or gluconate alone, the addition of acetate to these growth media result ...[more]

PMID: 32117117

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Project description:BackgroundItaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy.ResultsIn this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis-aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt) produced 3.43 ± 0.59 g itaconic acid L-1 with a product yield of 81 ± 9 mmol mol-1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L-1 and 116 ± 15 mmol mol-1, respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt), produced 3.38 ± 0.28 g itaconic acid L-1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis.ConclusionAs shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum. Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

Dataset Information

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

Publications

Molecular Basis of Growth Inhibition by Acetate of an Adenylate Cyclase-Deficient Mutant of <i>Corynebacterium glutamicum</i>.

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