Modifikationen der Atmungskette in Corynebacterium glutamicum\textit{Corynebacterium glutamicum} und Rolle des Flavohämoproteins Hmp

$\textit{Corynebacterium glutamicum}$ possesses a branched aerobic respiratory chain with two terminal oxidases, cytochrome bd oxidase with a high oxygen affinity but a low H$^{+}$/O ratio, and the proton pumping cytochrome aa$_{3}$ oxidase with a low oxygen affinity but a high H$^{+}$/O ratio. The latter forms a supercomplex with the cytochrome bc1 complex. Under aerobic conditions these two braches build up an electrochemical proton gradient, which, in the process of oxidative phosphorylation, is used for ATP synthesis by F$_{1}$F$_{O}$-ATP synthase. Within this work the respiratory chain of $\textit{C. glutamicum}$ was modified and the influences of these modifications were studied. The cytochrome bc$_{1}$-aa${3}$ supercomplex is the major proton pump in $\textit{C. glutamicum}$. To study the influence of increased cellular levels of the supercomplex, expression of its structural genes was enhanced two-fold by exchange of the native promoters with the P$_{tuf}$ promoter of elongation factor Tu. Higher levels of cytochromes a, b and c were detectable in the recombinant strain and resting cells showed a 30% increased oxygen consumption rate. However, growth was comparable to the wild type. In a similar approach, the native promoter of the atpBEFHAGDC operon was exchanged by P$_{tuf}$. Surprisingly this resulted in a 50-60% decreased expression of the $\textit{atp}$ genes, indicating that the native atp promoter is stronger than the $\textit{tuf}$ promoter and might be suitable for enhancing gene expression in producer strains of $\textit{C. glutamicum}$. In order to improve energy conservation under microaerobic conditions, attempts were made to replace the $\textit{bd}$ oxidase by a cytochrome $\textit{cbb}_{3}$-type oxidase from $\textit{Bradyrhizobium japonicum}$, a proton-pumping heme-copper type enzyme with a very high oxygen affinity. However, as the synthesis of the accessory protein FixG proved to be toxic for $\textit{C. glutamicum}$, functional expression of the heterologous $\textit{cbb}_{3}$-type oxidase could not be achieved. $\textit{C. glutamicum}$ possesses a limited potential for anaerobic growth by nitrate respiration via the nitrate reductase NarGHJI. However, due to the lack of a nitrite reductase, nitrite accumulates and inhibits growth. In order to allow conversion of nitrite to ammonia and thus the use of nitrate as a nitrogen source, attempts were made to express a dissimilatory nitrite reductase from $\textit{Wolinella succinogenes}$ and an assimilatory nitrite reductase from Mycobacterium smegmatis in $\textit{C. glutamicum}$. Unfortunately, anaerobic nitrite reduction did not work with either of these enzymes. In another aspect of this work, the influence of nitrite on growth and global gene expression was analyzed. The gene most strongly upregulated by nitrite was found to be $\textit{hmp}$ encoding a flavohaemoprotein. A $\Delta \textit{hmp}$ mutant was more sensitive towards nitrite and the NO-donating agent sodium nitroprusside under aerobic conditions. Anaerobic growth by nitrate respiration was also severely impaired. Thus flavohaemoprotein Hmp of $\textit{C. glutamicum}$ is important for coping with nitrite-derived stress under aerobic and anaerobic conditions

Conversion of Corynebacterium glutamicum from an aerobic respiring to an aerobic fermenting bacterium by inactivation of the respiratory chain

AbstractIn this study a comparative analysis of three Corynebacterium glutamicum ATCC 13032 respiratory chain mutants lacking either the cytochrome bd branch (ΔcydAB), or the cytochrome bc1–aa3 branch (Δqcr), or both branches was performed. The lack of cytochrome bd oxidase was inhibitory only under conditions of oxygen limitation, whereas the absence of a functional cytochrome bc1–aa3 supercomplex led to decreases in growth rate, biomass yield, respiration and proton-motive force (pmf) and a strongly increased maintenance coefficient under oxygen excess. These results show that the bc1–aa3 supercomplex is of major importance for aerobic respiration. For the first time, a C. glutamicum strain with a completely inactivated aerobic respiratory chain was obtained (ΔcydABΔqcr), named DOOR (devoid of oxygen respiration), which was able to grow aerobically in BHI (brain–heart infusion) glucose complex medium with a 70% reduced biomass yield compared to the wild type. Surprisingly, reasonable aerobic growth was also possible in glucose minimal medium after supplementation with peptone. Under these conditions, the DOOR strain displayed a fermentative type of catabolism with l-lactate as major and acetate and succinate as minor products. The DOOR strain had about 2% of the oxygen consumption rate of the wild type, showing the absence of additional terminal oxidases. The pmf of the DOOR mutant was reduced by about 30% compared to the wild type. Candidates for pmf generation in the DOOR strain are succinate:menaquinone oxidoreductase, which probably can generate pmf in the direction of fumarate reduction, and F1FO-ATP synthase, which can couple ATP hydrolysis to the export of protons

A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum

Kranz A, Polen T, Kotulla C, et al. A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum. Scientific Data. 2022;9: 594