## 3 Pathway Metabolic Engineering Strategies to Improve Glycerol-Based Bioprocesses
### TCA Cycle Intermediates  
The TCA cycle is the principal pathway of the central carbon metabolism that enables aerobic organisms to oxidize organic compounds and provide precursors for biomass formation, energy, and redox equivalents to the cell. Apart from diverting the TCA carbon flux towards the target biosynthetic route and exploiting the produced redox equivalents and metabolic precursors through pathway engineering for strain improvement [121], TCA intermediates _per se_--such as succinic acid (SA) and _a_-ketoglutic acid (_a_-KG)--are of high biotechnological interest (Table 4). SA is formed from _a_-KG through _a_-KG dehydrogenase and from isocitric acid through isocitrate lyase (glyoxylate cycle), whereas, succinate dehydrogenase complex (SDH) oxidizes SA to fumaric acid reducing ubiquinone to ubiquinol. _Y. lipolytica_ strains with reduced or lost SDH activity, by impairing the _SDH1_/_SDH2_ gene or exchanging the native _SDH2_ promoter with a weak promoter, accumulated about 4-5 g L\({}^{-1}\) SA using glycerol as substrate [122; 123]. [PERSON] et al., deleted the _Ylsdh5_ gene, encoding a subunit of SDH (SDH5) in _Y. lipolytica_ Po1f, and evaluated the _\(\Delta\)Ylsdh5_ strain for SA production using crude glycerol as the sole carbon source with respect to its tolerance and productivity. The strain exhibited excellent tolerance to environmental stress using 200 g L\({}^{-1}\) crude glycerol and produced 43 g L\({}^{-1}\) SA. Using fed-batch fermentation, highest fermentative SA titer was achieved (160.2 g L\({}^{-1}\)) with a SA productivity of 0.40 g L\({}^{-1}\) h\({}^{-1}\), spotlighting _Y. lipolytica_ as a potent industrial SA producer [124]. Using the _\(\Delta\)Ylsdh5_ mutant strain, [PERSON] et al., developed a bioprocess for efficient SA production by _Y. lipolytica_, using an in situ fibrous bed bioreactor (isFBB). They evaluated different agricultural residues such as wheat straw, corn stalk, and sugarcane bagasse to improve the performance of isFBB, with sugarcane bagasse being the best immobilization material. Under the optimal conditions of 20 g sugarcane bagasse, 120 g L\({}^{-1}\) crude glycerol as carbon source and 4 L min\({}^{-1}\) of aeration rate, the SA concentration was 53.6 g L\({}^{-1}\) with an average productivity of 1.45 g L\({}^{-1}\) h\({}^{-1}\) and a SA yield of 0.45 g g\({}^{-1}\). SA titer increased up to 209.7 g L\({}^{-1}\) by feeding crude glycerol, which was the highest value ever reported [125]. Although the strain achieved high SA titer at neutral pH from glycerol, it produced acetate, impeding cell growth and SA production yield. For that, the pH had to be adjusted during the fermentation to ensure high succinate titer. At low pH, SA titer was relatively low (5.2 g L\({}^{-1}\)). [PERSON] et al., further improved the strain phenotype (production and cell growth) by deleting the _Ylach_ gene responsible for acetic acid overflow (acetyl-CoA hydrolase) and overexpressing the genes which improve SA formation via reductive carboxylation and oxidative TCA pathway. They identified that the CoA-transfer from acetyl-CoA to SA in mitochondria leads to acetic acid overflow, rather than the pyruvate decarboxylation reaction in SDH negative _Y. lipolytica_. When the _Ylach_ gene was knocked out, acetic acid formation was eliminated and both SA production and cell growth improved. Furthermore, they studied how the overexpression of genes encoding key enzymes of oxidative TCA, reductive carboxylation, and glyoxylate bypass affects the SA yield and by-products formation. They observed that the overexpression of the phosphoenolpyruvate carboxykinase (_ScPCK_) gene from _S. cerevisiae_ and the endogenous succinyl-CoA synthase beta subunit (_YlSCS2_) gene yielded the best performing strain, with a SA titer improved by 4.3-fold. In fed-batch fermentation, this strain produced 110.7 g L\({}^{-1}\) with a yield of 0.53 g g\({}^{-1}\) glycerol without pH control. This is the highest SA titer achieved at low pH by yeast reported to date, using defined media [126].