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

In 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.

Reduced by-product formation and modified oxygen availability improve itaconic acid production in Aspergillus niger.

Aspergillus niger has an extraordinary potential to produce organic acids as proven by its application in industrial citric acid production. Previously, it was shown that expression of the cis-aconitate decarboxylase gene (cadA) from Aspergillus terreus converted A. niger into an itaconic acid producer (Li et al., Fungal Genet Bio 48: 602-611, 2011). After some initial steps in production optimization in the previous research (Li et al., BMC biotechnol 12: 57, 2012), this research aims at modifying host strains and fermentation conditions to further improve itaconic acid production. Expression of two previously identified A. terreus genes encoding putative organic acid transporters (mttA, mfsA) increased itaconic acid production in an A. niger cis-aconitate decarboxylase expressing strain. Surprisingly, the production did not increase further when both transporters were expressed together. Meanwhile, oxalic acid was accumulated as a by-product in the culture of mfsA transformants. In order to further increase itaconic acid production and eliminate by-product formation, the non-acidifying strain D15#26 and the oxaloacetate acetylhydrolase (oahA) deletion strain AB 1.13 ∆oahA #76 have been analyzed for itaconic acid production. Whereas cadA expression in AB 1.13 ∆oahA #76 resulted in higher itaconic acid production than strain CAD 10.1, this was not the case in strain D15#26. As expected, oxalic acid production was eliminated in both strains. In a further attempt to increase itaconic acid levels, an improved basal citric acid-producing strain, N201, was used for cadA expression. A selected transformant (N201CAD) produced more itaconic acid than strain CAD 10.1, derived from A. niger strain AB1.13. Subsequently, we have focused on the influence of dissolved oxygen (D.O.) on itaconic acid production. Interestingly, reduced D.O. levels (10-25 %) increased itaconic acid production using strain N201 CAD. Similar results were obtained in strain AB 1.13 CAD + HBD2.5 (HBD 2.5) which overexpressed a fungal hemoglobin domain. Our results showed that overexpression of the hemoglobin domain increased itaconic acid production in A. niger at lower D.O. levels. Evidently, the lower levels of D.O. have a positive influence on itaconic acid production in A. niger strains.

Enhanced itaconic acid production in Aspergillus niger using genetic modification and medium optimization.

BACKGROUND: Aspergillus niger was selected as a host for producing itaconic acid due to its versatile and tolerant character in various growth environments, and its extremely high capacity of accumulating the precursor of itaconic acid: citric acid. Expressing the CAD gene from Aspergillus terreus opened the metabolic pathway towards itaconic acid in A. niger. In order to increase the production level, we continued by modifying its genome and optimizing cultivation media. RESULTS: Based on the results of previous transcriptomics studies and research from other groups, two genes : gpdA encoding the glyceraldehyde -3-dehydrogenase (GPD) and hbd1 encoding a flavohemoglobin domain (HBD) were overexpressed in A. niger. Besides, new media were designed based on a reference medium for A. terreus. To analyze large numbers of cultures, we developed an approach for screening both fungal transformants and various media in 96-well micro-titer plates. The hbd1 transformants (HBD 2.2/2.5) did not improve itaconic acid titer while the gpdA transformant (GPD 4.3) decreased the itaconic acid production. Using 20 different media, copper was discovered to have a positive influence on itaconic acid production. Effects observed in the micro-titer plate screening were confirmed in controlled batch fermentation. CONCLUSIONS: The performance of gpdA and hbd1 transformants was found not to be beneficial for itaconic acid production using the tested cultivation conditions. Medium optimization showed that, copper was positively correlated with improved itaconic acid production. Interestingly, the optimal conditions for itaconic acid clearly differ from conditions optimal for citric- and oxalic acid production.