Improving growth properties of Corynebacterium glutamicum by implementing an iron-responsive protocatechuate biosynthesis.

Corynebacterium glutamicum experiences a transient iron limitation during growth in minimal medium, which can be compensated by the external supplementation of protocatechuic acid (PCA). Although C. glutamicum is genetically equipped to form PCA from the intermediate 3-dehydroshikimate catalysed by 3-dehydroshikimate dehydratase (encoded by qsuB), PCA synthesis is not part of the native iron-responsive regulon. To obtain a strain with improved iron availability even in the absence of the expensive supplement PCA, we re-wired the transcriptional regulation of the qsuB gene and modified PCA biosynthesis and degradation. Therefore, we ushered qsuB expression into the iron-responsive DtxR regulon by replacing the native promoter of the qsuB gene by the promoter PripA and introduced a second copy of the PripA -qsuB cassette into the genome of C. glutamicum. Reduction of the degradation was achieved by mitigating expression of the pcaG and pcaH genes through a start codon exchange. The final strain C. glutamicum IRON+ showed in the absence of PCA a significantly increased intracellular Fe2+ availability, exhibited improved growth properties on glucose and acetate, retained a wild type-like biomass yield but did not accumulate PCA in the supernatant. For the cultivation in minimal medium C. glutamicum IRON+ represents a useful platform strain that reveals beneficial growth properties on different carbon sources without affecting the biomass yield and overcomes the need of PCA supplementation.

Metabolic engineering of Vibrio natriegens for anaerobic succinate production.

The biotechnological production of succinate bears serious potential to fully replace existing petrochemical approaches in the future. In order to establish an economically viable bioprocess, obtaining high titre, yield and productivity is of central importance. In this study, we present a straightforward engineering approach for anaerobic succinate production with Vibrio natriegens, consisting of essential metabolic engineering and optimization of process conditions. The final producer strain V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pycCg (Succ1) yielded 1.46 mol of succinate per mol of glucose under anaerobic conditions (85% of the theoretical maximum) and revealed a particularly high biomass-specific succinate production rate of 1.33 gSucc gCDW -1 h-1 compared with well-established production systems. By applying carbon and redox balancing, we determined the intracellular flux distribution and show that under the tested conditions the reductive TCA as well as the oxidative TCA/glyoxylate pathway contributed to succinate formation. In a zero-growth bioprocess using minimal medium devoid of complex additives and expensive supplements, we obtained a final titre of 60.4 gSucc l-1 with a maximum productivity of 20.8 gSucc l-1 h-1 and an overall volumetric productivity of 8.6 gSucc l-1 h-1 during the 7 h fermentation. The key performance indicators (titre, yield and productivity) of this first engineering approach in V. natriegens are encouraging and compete with costly tailored microbial production systems.