Abstract
Previously, our lab replaced the endogenous FAD-dependent pathway for glycerol catabolism in S. cerevisiae by the synthetic NAD-dependent dihydroxyacetone (DHA) pathway. The respective modifications allow the full exploitation of glycerol’s higher reducing power (compared to sugars) for the production of the platform chemical succinic acid (SA) via a reductive, carbon dioxide fixing and redox-neutral pathway in a production host robust for organic acid production. Expression cassettes for three enzymes converting oxaloacetate to SA in the cytosol (“SA module”) were integrated into the genome of UBR2CBS-DHA, an optimized CEN.PK derivative. Together with the additional expression of the heterologous dicarboxylic acid transporter DCT-02 from Aspergillus niger, a maximum SA titer of 10.7 g/L and a yield of 0.22 ± 0.01 g/g glycerol was achieved in shake flask (batch) cultures. Characterization of the constructed strain under controlled conditions in a bioreactor supplying additional carbon dioxide revealed that the carbon balance was closed to 96%. Interestingly, the results of the current study indicate that the artificial “SA module” and endogenous pathways contribute to the SA production in a highly synergistic manner.
Highlights
Succinic acid (SA) has been traditionally used as surfactant, ion chelator and additive in agriculture and food (Ahn et al, 2016)
A S. cerevisiae CEN.PK113-1A derivative in which the native FAD-dependent L-glycerol 3-phosphate (L-G3P) pathway for glycerol catabolism had been replaced by the NAD-dependent DHA pathway (Klein et al, 2016) was used as a baseline strain
Apart from the genetic modifications previously referred to as “DHA pathway module II,” this strain carried a replacement of the endogenous UBR2 allele by the respective allele from the glycerol-utilizing wildtype isolate CBS 6412-13A (UBR2CBS) as described by Swinnen et al (2016)
Summary
Succinic acid (SA) has been traditionally used as surfactant, ion chelator and additive in agriculture and food (Ahn et al, 2016). Apart from these traditional applications, SA has nowadays been considered one of the most promising platform chemicals that can be produced from renewable resources (Dusselier et al, 2014; Pinazo et al, 2015). Certain bacteria isolated from rumen such as Mannheimia succiniciproducens (Lee et al, 2002; Scholten and Dägele, 2008) and Actinobacillus succinogenes (Guettler et al, 1999) naturally secrete significant amounts of SA These wild-type organisms are auxotrophic for several vitamins and amino acids (Beauprez et al, 2010), and the use of complex media is not feasible for economic production of bulk products. Rational metabolic pathway engineering has been applied to well-established model bacteria such as Escherichia coli and Corynebacterium glutamicum
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