Metabolic engineering of $\textit{Corynebacterium glutamicum}$ for efficient succinate production

The C$_{4}$-dicarboxylate succinate has been identified as a promising candidate to become a key building block in a future “green” chemical industry based on renewable carbon sources. In this work, metabolic engineering was applied to construct $\textit{Corynebacterium glutamicum}$ strains for the efficient production of succinate from glucose or glycerol under aerobic or anaerobic conditions. In order to make succinate to the end-product of the oxidative TCA cycle under aerobic conditions, a deletion of the succinate dehydrogenase genes was required. The resulting first aerobic succinate producer strain $\textit{C. glutamicum}$ $\Delta \textit{sdhCAB}$ accumulated 40 mM succinate in the supernatant together with high concentrations of acetate (125 mM). To reduce by-product accumulation and to enhance the anaplerotic provision of oxaloacetate (OAA), the genes for all known acetate-producing pathways were deleted and the genes for pyruvate carboxylase (PCx) and phosphoenolpyruvate carboxylase (PEPCx) were overproduced. The resulting strain BL-1/pAN6-pyc$^{P458S} \textit{ppc}$ reached a succinate titer of 82 mM and showed the highest specific productivity (1.60 mmol g (cdw)$^{-1}$ h$^{-1}$) among currently described aerobic bacterial succinate producers. Acetate accumulation was reduced by 84% to 20 mM. Optimization of the process conditions by decoupling of growth and production using strain BL-1/pAN6- pyc$^{P458S} \textit{ppc}$ resulted in an additional increase of the succinate titre to 90 mM and a yield of 0.45 mol/mol glucose. The second part of this work dealt with the use of glycerol as carbon source for the aerobic succinate producers described above. For this purpose, the $\textit{C. glutamicum strains}$ $\Delta \textit{sdhCAB}$ and BL-1 were transformed with plasmid pVWEx1-$\textit{glpFKD}$, which confers the ability for glycerol utilisation. Batch culture experiments in minimal medum showed that both strains produced succinate from glycerol and strain BL-1/pVWEx1-$\textit{glpFKD}$ reached a maximal titre of 79 mM, corresponding to 42% of the maximal theoretical yield. The volumetric productivity of 3.59 mM h$^{-1}$ respresents the highest value among currently described aerobic succinate production processes from glycerol. The third part of this work focussed on succinate production from glucose under anaerobic, non-growing conditions. In a first step, strain $\textit{C. glutamicum}$ BOL-1 was constructed, in which the genes for all known pathways for lactate and acetate formation were deleted. Subsequent chromosomal integration of the pyc$^{P458S}$ gene coding for a mutated pyruvate carboxylase (PCx) resulted in strain BOL-2, which reached the maximal expected succinate yield of 1 mol/mol glucose calculated on the basis of the redox balance and assuming minimal by-product formation. In order to provide additional reducing equivalents for succinate production, the $\textit{fdh}$ gene from $\textit{Mycobacterium vaccae}$ encoding an NAD$^{+}$- dependent formate dehydrogenase was chromosomally introduced into the BOL-2 strain. The resulting strain BOL-3 was able to metabolize glucose and formate in parallel under anaerobic conditions and achieved a 20% increased succinate yield (1.26 mol/mol glucose) compared to glucose as sole substrate. A process delay caused by a temporary production inhibition during glucose/formate co-utilization was removed by plasmid-borne overexpression of the $\textit{gap}$ gene coding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the strain BOL-3/pAN6-$\textit{gap}$. An anaerobic fed-batch process with this strain using glucose and formate as substrates resulted in the accumulation of 1134 mM succinate within 53 h, a volumetric productivity of 21 mM h$^{-1}$, a succinate yield of 1.7 mol/mol glucose and a total by-product level of only 9% (mol/mol succinate). These values compare very favourable with the best scores described for other succinate producers and underline the potential of $\textit{C. glutamicum}$ as a production platform in industrial biotechnology