Toward Homosuccinate Fermentation: Metabolic Engineering of Corynebacterium glutamicum for Anaerobic Production of Succinate from Glucose and Formate

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Previous studies have demonstrated the capability of<jats:named-content content-type="genus-species">Corynebacterium glutamicum</jats:named-content>for anaerobic succinate production from glucose under nongrowing conditions. In this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (Δ<jats:italic>cat</jats:italic>Δ<jats:italic>pqo</jats:italic>Δ<jats:italic>pta-ackA</jats:italic>Δ<jats:italic>ldhA</jats:italic>), was constructed. Chromosomal integration of the pyruvate carboxylase gene<jats:italic>pyc</jats:italic><jats:sup>P458S</jats:sup>into BOL-1 resulted in strain BOL-2, which catalyzed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the cosubstrate formate, the<jats:italic>fdh</jats:italic>gene from<jats:named-content content-type="genus-species">Mycobacterium vaccae</jats:named-content>, coding for an NAD<jats:sup>+</jats:sup>-coupled formate dehydrogenase (FDH), was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase gene<jats:italic>gapA</jats:italic>. In an anaerobic fed-batch process with glucose and formate, strain BOL-3/pAN6-<jats:italic>gap</jats:italic>accumulated 1,134 mM succinate in 53 h with an average succinate production rate of 1.59 mmol per g cells (dry weight) (cdw) per h. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose).</jats:p>

Complex Regulation of the Phosphoenolpyruvate Carboxykinase Gene pck and Characterization of Its GntR-Type Regulator IolR as a Repressor of myo-Inositol Utilization Genes in Corynebacterium glutamicum

“…Recent metabolic engineering studies have shown that C. glutamicum is also capable of producing a variety of other commercially interesting compounds, e.g., other L-amino acids (4), D-amino acids (5), organic acids such as succinate (6)(7)(8)(9), diamines such as cadaverine (10,11) or putrescine (12), biofuels such as ethanol or isobutanol (13)(14)(15), and proteins (16)(17)(18).…”

Corynebacterium glutamicum harbours a molybdenum cofactor-dependent formate dehydrogenase which alleviates growth inhibition in the presence of formate

“…Carboxylic acids such as formate, acetate and propionate can inhibit bacterial cell growth resulting in cellular responses including expression changes (Lee et al, 2006;Litsanov et al, 2012a;Polen et al, 2003). C. glutamicum ATCC 13032 also showed reduced growth rates with increasing formate concentrations (Fig.…”

“…In enzyme assays with cell-free extracts, we could not detect formate dehydrogenase activity using NAD + or NADP + as cofactors (data not shown). In whole cell biotransformation processes, FDHs are frequently used for cofactor regeneration, like the NAD + -dependent FDH from Mycobacterium vaccae (Bäumchen et al, 2007;Litsanov et al, 2012a). However, FDH from C. glutamicum ATCC 13032 is most likely not applicable for such processes and the oxidation of formate led to the generation of reducing equivalents, which are transferred to a currently unknown electron acceptor.…”

“…FDHs can be found in prokaryotic micro-organisms such as aerobic methylotrophs and chemoautotrophs, anaerobic or facultative anaerobic bacteria, as well as methanogenic archaea (Friedebold & Bowien, 1993;Jormakka et al, 2002;Karzanov et al, 1991;Schauer et al, 1986). Depending on the type of FDH, the electrons are transferred to an acceptor such as NAD Wendisch et al, 2006a, b) and has also been engineered for production of amino-acid-derived products, alcohols and organic acids from glucose and other carbon sources (Blombach et al, 2011;Buschke et al, 2011;Gopinath et al, 2011;Litsanov et al, 2012a, b;Niimi et al, 2011;Rittmann et al, 2008;Sasaki et al, 2009;Schneider et al, 2011Schneider et al, , 2012Stäbler et al, 2011). In initial experiments on the use of different carbon sources by this organism, we observed that C. glutamicum ATCC 13032 could consume formate.…”

1H NMR-based Plasma Metabolic Profiling of Dairy Cows with Type I and Type II Ketosis

“…Formate provides a carbon source for nucleic acid biosynthesis. Inflammatory diseases often occur as a result of excessive formate consumption [46]. Even though cows with ketosis are more vulnerable to mastitis and endometriosis [47], it is crucial to understand the relationship between formate and ketosis in dairy cows.…”